Title of Invention

"A METHOD OF FACILITATING PRODUCTION OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS"

Abstract A method of facilitating production of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) from a culture of vertebrate cells, comprising the steps of: (a) providing a recombinant vertebrate cell transfected with an exogenous polynucleotide that encodes a mammalian CD 163 polypeptide having a transmembrane domain and at least 70% sequence identity to SEQ It) NO: 14, so that expression qf CD 163 polypeptide in said cell is increased; (b) contacting a culture of said cpll with PRRSV virus under conditions which permit infection of the cells and growth of the virus; and (c) recovering virus from said culture.
Full Text Cellular PermisiMtf .factorfw "viruses-ami Uses Thereof
Atf&rvirideie mAAftertvirMoe,
OF THE INVENTION
Asfmviridae Is a fatsuly of icosobedteal enveloped vbiases whose|^oiae«>ttslsts of a 'MMsteeate of fineor dotibteHBtnofal DMA of about 15QOCXM90Q0Q nnetotiies
long. Tie nas» of fte family is derived from African Swine Fever And Releted Viruses. AMem Swine Fever Virus Viroses of ftie famffly of Arteriviridae Includes ^juine artoitis was (EAV),
dehydrogenase-elevating vims (fJDV) and staa.an hemonhagic fever virus (SHFV), The Arterfviros having the greatest economic importance is Porcine Reproductive and Respiratory Syndrome Virus {PRRSV). PRESV
Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the most economically important diseases of swine. "Hie syndrome appeased almost simultaneously in North American and iri Western Braope in the late 1980s, and has since spread to become endemic in the major swine producing nations of Europe, Asia, and the Americas, The etiologic agent of PRRS is a virus that has been designated PRRS virus or PRRSV, For bath European and North American PRRS, the disease is characterized by reproductive failure in sows and gilts (late term abortions, still births, and mummies), high mortality among nursery pigs, and respiratory disease in swine of all ages. The disease has been the subject of recent reviews (Mengelmg and Lager, 2QQ0; Murtaugh et al., 2002; NodeBjk, 2002; Plagemann, 2003),
In the pig, PRRS V infection is limited to a subset of cells of the monocyte/macrophage lineage. Fully differentiated porcine alveolar macrophage (PAM) cells are the primary target «ells for viral replication (Duan et al., 1997a; Duan

et at, 1997b)« of PAM-*els is technically diaiengm|, an.d when
sttccessfitl, has to osll that -am not peraaissiw for PR!S vtes growth
(WetagartI et aL» 2002), PIES aw bmtad by i»ae3rapha§&s and
interaalii»J in claferin-coited pits by mdoeytesls, from endocytk vesicles
requires aeJdie pH (Hauwyttek et at, 199?). MM binding of various is mediated by
(Delfratte et id., 2002). InteailiE^on caa be facilitated by a 210 or 220 idDa membrane |py0of»oteJjat as incubation of PAM eels with monoclonal antibodies to this pfly/peptide block infection wift PURS vims {Dean et at, 1998; Wfesink-et at, 2(W3). Tie 210 KDa flycopts^n has wcentty been identified as sialoadbesin, a member of iigtee feniy of sidle binding inmrano^lobulfitt-Ite tetins ^Pensaert et at,, ^)03). Traasfection of the lon-peroassrve PE-1S ^sidne Mdaey) cell line with porcine sialbadhesin conferred flbe abffity to toternal.iie PRRSV pmrticles, tat -there remained an ajsptreat block at the raicoating stage, as virions entefed into cellular vesicles tat did not undeifo aueleocapsid disintegration, and vesicle membrane fusion. Viral genes were not expressed, and the traos-fected PK-1S cells were not rendered permissive for the PRES vims (Vandeiheijden et al.» 2QQ3), To owr tooMedge, transaction with sialoadbesia has not been shown to be sufficient to convert any PRRSV non-permissive cell line to a PRRSV-permissive phenotype, Apart from primary porcine cells of the monocyte/maoopha^ Hnesge, the only other cell type known to be permissive for the growth of PRRSV in cell culture is the immortalized monkey kidney cell line MA*lQ4(Oiladek et at, 1998) and derivatives such as MARC-14S (Kim et ai, 1993) and CL-2621. 1 is not known why this one particular cell line is permissive, yet other mammalian«11 lines are not. The PRRS virus binds specifically to a number of different-cell types, but does not initiate infection (Kreutz, 1998; Therrien et aL, 2000). to MARC-145-ceUs, the interaalizaiion of the virus by endocytosis and subsequent uncoating in low pH vesicles seems to mimic similar events in PAM cells (Kieute and Ackermann, 1996). However, a number of monoclonal antibodies fibat bind to porcine sialoadhesinfail to detect a homologous protein on the surface of MARC-145^ells {Duan et at, 1998; Wissink et at, 2003), suggesting tbat MARC-145 cells may use a divergent member of the same protein family or a different receptor altogether.

QntentFRRSyvaocte
potentially He as© for vaccine production las
the to of toto swiae Hues for
xenotiraspliBt am laciearingly explored as a son&e of
for ft© iatooittction of primate «eH lines to swioe
nsiay risk to tomans ncefoiqg xenoteasplanted
Urns, it would be prudent to avoid the use of simitn ceE lam in swine vteeine
prepress, 1 woulil be desirable to iienify car fenemte ncra-sinaion cells or
cell capable of s»f PliB V septt^ion. lowaiste fliis ^ oal, it ii cssentM to identify tibe prodac^s) whtt aay be asponsiMe fer-coafeocaig ttoe penalssitfty for PRiRSV n^plieifeft it ia^ixrtato simiam^is Unes as wel as • • • PAM cells, Oace sw.£4» frodi«» ne a»ffied, aoa-peiraisswB One lab has reported that &e«tr»panin protein GD151 from MARC-14S cells, when transfected into oon-psratissive B:HK~21 cells, confers penmssivity to the -PRRS virus (Kapil .aad.ShanmBkhappa, 2CX)3; Shanmikfaappa and Kapil, 2(X)1). This observation has yet to be confinaed by aa independent lab. We describe here an unrelated polypeptide, which when transfected into non-permissive cells, confers permissivity to flie PKRS vims.
References Cited
Qdadek, D. W,, Harris, L. L,, aad: Dea,S.» Gagnon»C.A,, MardaisiJH., Piixadeh3.» »d RoganJD. (2000). Current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (PRRS) virus; comparison of the North American and European isolates {Review]. Arch. Virol. 145, 659-688.
Delputte,P.L,, Vanderbeijden»N.f NauwynckJH J., and Pensaert^^.
, Neuwyncfc,HJ., and.PensaerttM.B» (1997a)» Effects of origin and state of
differentiation and activation of monocytes/macrophages on their smeeptibiMty to porcine reproductive and respiratory syndrome was (PRRSV). Arch, Vkol, 142,
2483-2497.
Daanjt, Nauwptck,H .14 and PettsaertsM.B. (1997b). Virus quantification and identification of cellular targets in the tangs and lymphoid tissues of pip at different time ffitervals after inoculation with porcine reproductive and respiratory syndrome vims ^RRSV). Vet. MicroMol. 56, 9-19,
» Nauwynek$X,Favo*eel,B.WM and PensaerOWR. (1998). Identification of a putative receptor for porcine reproductive and respiratory syndrome virus on porcine alveolar miuarophages-. I. Virot 72, 4520-4S23.
Gravereen,J.H., Mad$en»M.f and Moestrup»S.K. Gronltmd J, Vitved L. Lansen M. Slgodt K. Hotoskov U. Cloning of a novel scavenger receptor cystetae-rich type I transmembmie molecule 0*4160) expressed by human fflacrophages. Journal of tomunology 16501)*X>406-641S,
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of porcine reproductive and respiratory syndrome {PKRS) virus to a homogenous subp)palation of MA-104 cell line. Arch. Virol. 133, 477-4S3.
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reproductive and respiratory spdrome vires tropism. Virus Ses. 51, 121-128,
Kreat2;»L,C. and Actermann»M.^- (19%). Porcine reproductive and respiratory syndrome virus enters cells fferougb a low pH-dependent endocytie padiway. Virus Res. 42, 137-147.
MengeUng,WJL. and Lafer,K,M, MeulenbergJJ,M. (2000). FRRSV, the virus. Veterinary Research 31, 11-21.
Mtataugh»M.P., XiaoJLG,, and ZucfeermannJP. (2002). Immunological responses of swine to porcine reproductive and respiratory syndrome virus infection (Review]. Viral Immunology 75, S33-547.
Nauwynck,HJ., Duan»X., FavoreelJHLW., Van GostveldtJ*., and PensaertJVl.B. (1999). Entry of porcine reproductive and respiratory 'syndrome virus into porcine alveolar macrophages via receptor-mediated endocytosis. J. Gen. Virol. 50, 297-305.

(2002), Porcine Reproductive and Respirat
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Pensaert* M.» Nauwyeck, H.t and Vanderheijden, N. Nucleic acid encoding polypeptlde involved in cellular entrance of the PRRS vims. Akzo Nobel fiV. and Universiteit Gent WO 03/010200 A2» 1-24, 2-6-2003, 7-18-2002. Ref Type: Patent
PhilippidisJP., MasonJ,C.» Evans,B J., NadraJ,, TaylorJLM., HaskardJP.O., and Landis»RXI. (2004). Hemoglobin scavenger receptor QD163 mediates intrfenkto-lO release and heme oxygenase-1 synthesis - Antiinfiarajpoatory monocyte-iaaa»ont|e responses in vitro, in resolving skin blisters in vivo, and alter cardiopuliBonary byp« surgery. Qroalatiott Research 94t 119-126,
Plagemann,P.G.W. (2003). Porcine reproductive and respiratory syndrome
Origin hypoUiesis. Emrargiiig Mectious Disease? 9, 903-908.
Mtter»M»» Baecnter»C., Langmai»»f ., and SdimteyQ, (1999). scavenger ceeeptor cysMne-rich saperfamfly. Biocherafoal & Biophysical itoeafcli 260, 464474.
., Eiqiuerra^i., and Doraun^ie2;,I. (2003). Espiession of porcine QD163 on monocytes/inacfophages coirelates -wife pennissiveness to Afifcan swine fever . . infection, Aich. Virol. 148, 2307-2323.
and K^ilsS. (2001). Qoning and identification of MARC- 145 respiratory syndrome vires. Adv. Bxp. Me4 Biol. 494, 641-646,
F. and Menlenberg^J.M. (2001), .Aiteiviruses. la Reids Virology, DMXx&pe, PM.Howley,.D.E. TfaemenJD., St Pierre, Y,, and. JDea,S, ., DelputteJP.L., FavoreelJH.W., VandekerckhoveJ., Van van Woensel^.A.s and Nauwynck JJJ. f 2003). jfavolvejnent of sialoadhesin in entry of porcine reproductive and respiratory Q«dronae vires into porcine alveolar macrophages. J. Vlrol. 77, S207-8215.
WeingartlJH.M,» SabaraJML, Pasick,J.» van MooriehemJE.» and Babiuk^,. (2002), Continuous porcine cell lines developed from alveolar macropbages - Partial characterization and virus susceptibility. J. Virol. Methods 104, 203-216.
Wissink3.HJ., van Wijk,H.A.R., pol,JM.A., Godeke^J., van Rijn,P,A., RottierJP J.M., and Meu!enberg4.J.M.
iqgodactive virus. Vtool. W8,H7-IS7,
The iflveotioB t of infection of one or mam cells
by t vims tot is from the group consisting of AmrMridae and A^arviridm,
which composes the step of of a C3DM3 polypepilde
witfaiii said cell. In a preferred embodiment the C3DI63 is membrane bound. In one
embodiment the vlxm is ftom tine poip consisting of Arterivir&lae. In E
prefenred effibodinisttt toe vfros is PSJKSV, to spotter embodiment said vtes is
equine arteritte was 0BAV). In yet Mofter embodisBieot s»d. vtos is Aftiean Swine
•Fever-i?i»s (ASFV). ••• •• -
liTOsased expfesslou of a GD163 polypeptfde naay be accomplished by methods suen iB'tofiroduction of otogenous nwcfeic acids encoding OH63 pol^jeptides sa.ch methods Include bit ane not limitel to tensfection,flec1roporation and fttsion witib a cantier of a polynudeotideisomplsiiig a polynwcleotide encoding a OD163 polypeptide. Increased expression may also be accomplished by induction of expression^ of endogenous GD163 by chemical treatment
The method may render previously non IWRSV-pormissiw cells PRRS penuissive. The method may also include lendering one or more cells that previously did not express a CD163 polypeptide into cells that are induced to express a CD 163
The cells in a preferaed embodiment are animal cells. They may be vertebrate or invertebrate cells. The eeis may be ittammalian. The cells or cell Hue may be an insect cell line. The eels may be BHK21 cells. The cells may be derived from porcine Mctey cells. The cells or cell line may be derived from feline kidney cells. The cdls or cell line may be tat are not limited to BHK-21, NLST-1, NUFK-1, Veto or RL cells, The PRRSV may be or the Buropean.or Horth American genotype,
As noted above, increased expression of a CD1€3 polypeptide may be accomplished by methods which include but are not limited to: transfection, electroporation and fusion with a carrier of a polynucleofide comprising a polynucleotide encoding a CD163 polypeptide. Any CD W3 polypeptides are contemplated. Those containing a transmembrane region are preferred. Exemplary

.x „ ^ 58
listed Wow.
One sttch polpwdwtide t polpeeleoiide eiKJodtag a poljf eftide
having at feast 70% 71%, TO*. 73%, 74%, 75%, 76%, 77& 78%, 79%, S0%» 81%,
82%, 83%, 84%, 15%, 86%, 87%, 88%, 89%, 90%, 91%» 92%, 93%, 93%, 94%,
95%, 96% 97% 98%» 99%, with SBQ ff> NO: 2.
One poIpnwjle0Mde«»npris« ap3l|roeleotide encoding a polypeptide differing fern SEQ B5 HO: 2 by no mae titan -20 conservative amino acid
Oae such polpwclcotide compete! a polynucleotide One such polyaacleotlde comprises § polynucteotide encoding a polypeptide coinprMng SBQ H> MO; 2,
One snch polynucleotide comprises a polynucleotide wifli tite sequence set forth in SEQ DNO:1
One sttdh polynacleotide compriset a polynucleotide encoding a polypeptide that has at least 99% identity to a polypeptide set forth ia SEQ ID NO: 14
One such polynncleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 14 by no more than 15 conservaiive anoino acid substitutions.
One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 14 by no more than 10 conservative amno acid substitutions,
Ooe such polynncleotide comprises a polymideotide encoding a polypeptide comprising SEQ ID NO: 14.
One such polynucleotide comprises a polynucleolide with the sequence set fcmhinSEQIDNO:13.
One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 24 by no more than 2 conservative amino acid substitutions.
One such polynucleotide comprises a polynuckotide encoding a polypeptide comprising SBQ ID NO: 24.

. .One .a polpwcleoti.de wife, the sequence set ..
One melt palynndeotkfo a polpacleoti^te encoding a iwlypeptide
haraig at toast 96% 97% 98%, or 9W»» Identity to a polypeptide set forth in SBQfD NO: 27,
One such pofyoncfeotide a polpwdeotide encoding a pogypeptide
fttwn SEQ H> NO; 2? toy no more than 20 eonsovrtive aintoo acid substitutions.
differing from SEQ ID NO: 27 by no mote than 10 coaservatrve amino add substftatioas, ' ' "
One swell polynwcleotide compis® a polpoieJeotide encoding a polypqptfde comprising SEQ'lD NO; 27.
One such polynucleotide compiles a polyaucleotjde with tite sequence set forth in SEQ ID NO: 26.
One such polynncleotide comprises a polpudeotide encoding a polypeptide that- has at least 98%, 99% identity to a polypeptide set forth in SEQ ID. NO: 32
One such polpwdeotide comprises a polynacleotide encoding a polypeptide differing from SEQ ID NO; 32 by no moie than 15 conservative axnino acid
One such polpwcleotide comprises a polynncleotide encoding a polypeptide differing from SEQ 3D NO: 32 by no more than 10 conservative amino acid substitutions.
One such polynncleotide comprises a poJynndeotide encoding a polypeptide comprising SEQ H> NO: 32.
One soeh polyoucleotide comprises a polynacleotide witit tiie sequence set forth in SEQ ID NO: 31.
One such polynacleotide comprises a polpucleotide encoding a polypeptide that has at least 95%, 96%, 97%, 98%, 99% identity to a polypeptide set forth to SEQ ID NO: 34
One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 34 by no more than 15 conservative amino acid substitutions.

One neb j»lp«iekotide a ptiyoacleotide encoding a jpolygejitide
3D NO: 34 by ao more than 10 conservative amteo acid robetkntkiBs.
One polpiicteotlde comprises a polipoeleotide encoding a poJypeptide
One p01ynwdeotMfe comprises a polynadeotlde with the sequence set forth to SBQ ID NO; 33,
One polynwcleotide comprises a polynactootide encoding a polypeptide that to at test 95%, 96%, 97%, 98%, 99% identity to a pQlypepttde«t forfli inSEQ ID HO: 36
. . . -One such pt>iyroicleotfde-comprises^a polpnicieotide ooffiodiag a-pcdype|>t»le dlffeiing from SBQ E? NO: 36 by no mote than 15 eoaservafhre amino acid substitutions,
One such polymicleotide comprises a polynucteotide-eoGodinig a polypeptide differing from SEQ ID NO: 36 by no HKTO than 10 conservative amino acid substitutions,
One such polymicleotide comprises a polyiiucleotide^n0odiiig a polypeptide comprising SEQ ID NO: 36,
One such polyottdeotide comprises a polyoucleotide witih the sequence set forth in SEQ ID NO: 35.
One such polynncleotide comprises a polymicleotide encodiag a polypeptide that has at least 90%. 91%, 92%, 93%, 94%, 9S%» 96%, 97%, 98%, 99% identity to a polypeptide set forth in SEQ ID NO: 42
One sncfa polyraicteotide comprises a polynncleotide encoding a polypeptide differing from SEQ H> NO: 42 by no more than 15 conservative amino acid substitutions.
One such polynucleotide comprises a polynwcleotide encoding a polypeptide differing from SEQ ID NO: 42 by no more than 10 conservative amino acid substitutions.
One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ED NO: 42.
One such polynueleotide comprises a polynucleotide with the sequence set forth in SEQ ID NO: 41.

fltatbu it test 90%. 91%, 92%, 96%, 97%, 9B%, 99% identity to a
pofypqptide set forth to S1Q ff> HCH44
Oae pofy&iuleatide a pdyiiucleotMe encoding a polypeptifito
SEQ13 NO: 44 fey than. IS oiiservative amino acid
Ctee swell polpwclcotide apdpucleotWe raiding a polypeptide
from SBQ E) NO: 44 by no note than 10 cftawrrative wmm acid
• One such polptticleotide mfflprtses a polynucleotide eacoding a polypeptide
ooopiBiflg S1Q S>" "HCfc 44.
One swell f ofytmciwtide a polyiaicleoliile with &e seqaenoe «et
fbrttiiBSBQ]DNO:43
O»e such po!ynt3cleotide co«pi^ a polynucleotade encodings polypeptide that has at least 95%, 96%, 97%., 98%, 99% identity to a polypeptide set forth in SEQ ]DNQ:46
One sttdh polyaBcleotide comprises apolynucleotide encoding a polypeptide diffeii&g from S1Q 3D NO: 46 by no mom than IS conservative amino acid substitutions.
One such polynocleodde comprises a polynwcleotide encoding a polypeptide differing from. SEQ ID NO: 46 by no mam than 10 conservative ammo acid substitutions.
One such polynucleotide comprises a polyuucleotide encoding a polypeptide comprising SEQ ID NO: 46,
One such polynucleotide comprises a polynucleotide with the sequence set forth in SEQ ID NO: 45.
One such polynucleotide comprises apolynucleotide encoding a polypeptide that has at least 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identity to a polypeptide set forth in SEQ D NO: 48
Oae such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 48 by no more than 15 conservative amino acid substitutions.

IDffO; 48 by no tium 10 oonexvattve ammo acid
One sncb, polynielrotide a polynuclwtide encoding a fmlypeptide
compistog 5BQ II) MO: 48,
One polpwdeotide eotajsrises a pl;p«eleotide with the sequence set fixlhfaiSBQlDNO:-47.
i
Tie taefiiod of facffitettng infection described above may farther comprise the
The invention ferfber comprise the cultee isolated, by the method described
above.
Any of fee iwttwdi described above may f tether comprise the step of protodiii a PURS or otter -viral vaccine. The vaccine may be kffled.or live attenuated,
He invention also oonaprises a cell or all line wherein the ability of one or mom cells to be infected by a virus selected from the group consisting of Arteriviridae -and Asfatvividm has bead, modified by directing .increased expression of a CD163 polypeptide within said cells.
fa a preferred embodiment the CD163 polypeptide comprises a transmembraue region, fa one embodiment the virus is selected from t»e -group consisting of Arteriviridtm. la- a preferred embodiment tihe virus is PRRSV. In another embodiment said vims is equine arteritis vims (BAV), In yet anotibter embodiment said virus is AMcan Swine Fever virus (ASFV).
Tine cell or ceB line of the invention may have been previously PRRSV non-permissive and is rendered PRES V permissive by directing increased expression of a CD163 polypeptide within said cell or ceU line.
The cell or cell line of te invention includes ceife or«ell Unes mat did not express a CD163 polypeptide and is induced to express a CD 163 polypeptide.
The cells in a preferred embodiment are animal cells. They may be vertebrate or invertebrate cells, The cells may be mammalian. The cell or cell line may be an insect cell or cell line. The cells may be BHK21 cells. The cells may be derived from porcine kidney cells. The cell or cell line may be derived from feline kidney cells.

._ Ttewite l»tm fffit ftt^T-l, NXf&l* VaopjKjgL.
the fcventioa a for the pra^nsify of 8«testuell or
cell line to allow fey i virus fern the group consisting of
a) pfovidiftf t nucleic., from fi» test-cell or eel Hue;
an aaouttt of polyaieleotide encoding a GD!€3 polvpeptide
relative to t control derived from a control t?el or cell line known mot to
1 virti§-indica&s apeopeasity ofite test-cell creel! line'to-
one e»ibodii»at tite vims is selected from the prop -caonsisit^g of la a fweferod eaatediraent tlie vims is PRRSV3n, anotterembodimBiit said virus is etpiine artedtis viros ^BA¥), la yet another embodiinent smd vires is Afieieaa Swine Fever virus (ASFV).
Hje amount of polynucleotide encoding a CD163 polypeptide may be detennined t^ hyfeddiEation,
tmownt of polynacleotide encoding a C3>163 polypeptide may be
The teventkra dso includes a n^bod for measuring the propensity of a test cell or eel line to alow infection by a viros selected from fee group consisting of ArtenvirMae m&A^arvirMme comprising;
(a) providing a sample containing polypeptides from the test cell or cell line;
(b) detennining ifie amount of CD163 polypeptide k Said sample;
wherein an increased amount of a CD 163 polypeptide relative to a control sample derived from a control cell or cell line known sot to support the growth of said virus indicates a propensity of the test cell or cell line to support the replication of «aid virus.
to one embodiment the virus is selected from the group consisting of Arteriviridae. In a preferred embodiment the virus is PRRSV.fa another embodiment said virus is equine arteritis vims
polgpepiide with m for the OD163 polypeptide, ander^adMons
whendn the antibody the CD163 polypeptide,
He invention includes a method for messming the propensity of a pig to be infected by i vims 'selected from tf» group consisting ofArterivindae and AifffirvirMae
a) pwvidteg a sample cotttainiag nucleic acids front the pig to be tested;
b) deteffiittittg fee amoral of polynaeleotide encoding a OD163 polypeptide or its
complement msiM sample; , .
vsteeta an increased amount of polynueleotide encodtog a OD163 polypeptide - relative to- a control simple deaved from-a pig kaowa to be resistaatto-saidvtais - -infection indicates a popeosity of the pig to be tested to be Infected by said vires.
to one embodiment the vires is selected from the group consisting of ArterivirMm; In a preferred embodiment the vims is PKfeS V, In another embodiment said vims is eopine arteritis vims (EAV). In yet another embodiment said vires is African Swtoe Fevef vims (ASFV).
In one embodiment the detaining is accomplished by hybridization. In another embodiment the determining is accomplished by PGR, ,
The invention also includes a metitod for measuring the propensity of a pig to be infected by a virus selected from the group consisting of Arterivirtdae and AsfarvirMm comprising;.
(a) providing a sample containing polypeptides from pig to be tested;
(b) determining the amount of CD163 polypeptide in said sample;
wherein to. increased amount of a GDI63 polypeptide relative to a control sample derived from a pig known to be resistant to said viras infection indicates a propensity of the pig to be tested to be infected by said viras,
In one embodiment the viras is selected from the group consisting of ArterivMdae, In a preferred embodiment the viras is PRRSV, In another embodiment said virus is equine arteritis virus (EAV). In yet another embodiment said viras is African Swine Fever virus (ASFV).
In one embodiment the deterermining is accomplished by contacting a CDttJS polypeptide with an antibody specific for the CD163 polypeptide, under conditions wherein the antibody binds the CD M53 polypeptide.

seieete
the abo an i
70% 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
' 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 95%, 96% 97%
98%, 99%, with SEQ m NO; 2.
One such poljpeftide is a pjlypeptidb differing from SBQ' ID NO: 2 by no more than 20 conservative aoarfio add fubstitutions.
One mch polypeptide Is a polfpepttdfe differing ten SEQ ID NO: 2 by no mow ffaaa 10 c«sefvatlve aosko acid substitutions.
One such polypeptide -compcws SEQ DD NO: 2«" • ~ ~
Theiefwe &e mvention dso include! an iaslated polypeptide having at jfoast 99%, identity to apolypepllde-s«t forth fa SBQ ID NO: 14
One such polypeptide is a polypeptide differing from SEQ ID NO: 14 by no more than 15 OTiseiYStive amino acid substitutions.
One such polypeptide is a polypeptide differing from SEQ ID NO: 14 by no more than 10 conservative amino acid substitutions. _ One such polypeptide comprises SEQ JD NO: 14. Therefore the invention also includes an isolated polypeptide differing from SBQ DD NO: 24 by no more than 2 conservative amino acid substitutions. One stich polypeptide comprises SEQ ID NO: 24. Therefore tihe invention also includes an isolated polypeptide having at least 96% 97% 98%, or 99%, identity to a polypeptide set forth in SEQ ID NO: 27.
One such poiypeptide is a polypeptide differing from S1Q ID NO: 27 by no more than 20 conservative anaino acid substitutions,
One such polypeptide is a polypeptide differing from SEQ ID NO: 27 by no more flian 10 conservative amino acid substitutions.
One such polypeptide is a polypeptide comprising SEQ ED NO: 27. Therefore the invention also includes an isolated polypeptide having at least 98%, 99% identity to a polypeptide set forth in SEQ E5 NO: 32
One such polypeptide is a polypeptide differing from SEQ ID NO: 32 by no more than 15 conservative amino acid substitutions.

mote than 10 toutao sdimibitilwtiQas,
One polypepttde is a polypeptide comprising SEQ ID NO: 32.
Therefore the invention also Includes an isolated polypeplide having at least
95%, 96%, 97%, 9S%, 99% to § polypeptitte set forth in SEQ ID NO: 34
One polypeptide is a j»lypeptidie differing loom SEQ 3D NO: 34 by no mom than IS conservative amteo acid substitutions.
One such, polypeptide is i polypeptMe differing from SPQ ID HO: 34 by no mom than 10 conservative amino add substitutions.
• One such poiypepttde is a polypeptide comprising SEQ 3D NO: 34.
• Heiefofle tihe invention also includes m isolated poiypeptide having at least
95%, 97%, 98%, 99% identity to a poiypeptide set forth in SBQ ID NO: 36
One such poiypeptide is a poiypeptide dffi-fering from SEQ 3D NO: 36 by no more than 15 conservative amino add substitutions.
One such poiypeptide is a poiypeptide differing from SEQ E> NO: 36 by no more than 10 conservative .amino acid substitutions.
One mch poiypeptide is a poJypeptide comprising SEQ ED NO: 36. The invention also includes an isolated poiypeptide having at least 95%, 96%, 97%, 98%, 99% identity to a poiypeptide set forth in SBQ ID NO: 38
One such poiypeptide is a poiypeptide differing from SEQ E> NO: 38 by no more than 15 conservative aminc acid substitutions.
One such poiypeptide is a poiypeptide differing from SEQ E> NO: 38 by no more than 10 conservative amino acid substitutions.
One such poiypeptide is a poiypeptide comprising SEQ H> NO: 40 Therefore the invention also includes an isolated poiypeptide having at least 95%, 96%, 97%, 98%, 99% identity to a poiypeptide set forth in SEQ ID NO: 40
One such poiypeptide is a poiypeptide differing from SEQ ID NO: 40 by no more tiban 15 conservative amino acid substitutions.
One such poiypeptide is a poiypeptide differing from SEQ ID NO: 40 by no more than 10 conservative amino acid substitutions.
One such poiypeptide is a poiypeptide comprising SEQ ID NO: 40.

the ia«aflpai.al80 »isolated polyfeptide -having at 90%.
92%, f3%, 94%, 95%, 9C%, 97%. 96%, 99% to a pblypeptide set fix* in
SEQ ID NO: 42
Owe polypeptide Is a pol^ptifcdlffemg Item SBQ. ID NO: 42 by no moi© tban IS eoiwiraiw adt
One such poljfeptide Is a pol^^lie.llffering from SEQ H> NO: 42 by no
Oae f^lypepttte Is a j»lfpeptide comprising SEQ ID MO: 42. Tlerefore the invention also includes an isolated polypeptide having at least 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 09% identity to a polypeptide ^t
Oae sich polypeptidte Is a po!|fqptide.dMerittg from SEQ ID NO: 44 by no mca» tliaik 15 conservative wid sabstitntions,
One sodi polypeptide is a polypeptide diffeiiag from SEQ ID MO: 44 by no mcwe ttoao 10 eomervatiw amino Kid sabstitutions, •
One such polypeptide Is a polypeptMe comprising SKJ E> MO; 44. Therefore the mventloa also includes an Isolated polypeptide having at least 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to a polypeptide set forth m SEQ E> NO: 46
One such polypeptide is a polypeptide differing from -SEQ tt> NO: 46 by no more than 15 conserrative ammo acid sabstitutifoas.
One such polypeptide is a polypeptide differing from SEQ' ID MO: 46 by no more than 10 conservative amino acid substitutions,
One such poJypeptide is a polypeptide comprising SBQ. ID NO: 46. Therefore the invention also includes an isolated polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to a polypeptide set forth .in SBQ ID NO: 48
One such polypeptide is a polypeptide differing from SEQ ID NO: 48 by no more than 15 conservative amino acid substitutions,
One such polypeptide is a polypeptide differing from SEQ ID NO: 48 by no more than 10 conservative amino acid substitutions.
One such poiynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ IP NO: 48.

. . _ ... .IMinwQtlQB also CD 163 polpucleotide wherein said.. ,.
polpacleotife is fteffi tie of tie polpwteotides
an jwynueoie compisng:
(a) t polpocteotide set fa SfiQ ID t?Os: 1 or 5
74%, 75%, 76%, 77%, 71%, 79%, 80%, 81%, 82%, 8S%» 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 95%, 96% 97% 9S%, or 99%, identity
and or to a poljpeptlfciset forth in SBQ ID HO: 2
(c) a j»lyflideotWe encoding a pol.ypeptide.of SBQ ID: 2, (d)*poliyanciebtidev^^
Tteefoire lite invention also tedtodes aa Isolated polyaocteotide comprising:
(a) a polptteleotide sepenee set forth in SBQ ID NOs: 12 or 13 '
(b) a polynucleotide that a fMdjfeptide tfamt has at temt 99%, identity and or
siaoiari^ to a polj>peptide set forth in SEQ D> NO: 14
(c) a po!;pneleoti«le encoding a polypeptide of SEQ D>: 14,
(d) a polynitideotide wMcb is $ie complmient of any of TbeMom tbe inventton also inclitdes an isolated polynwcleotide comprising:
(a) a polpracleotide sequence set forth in SBQ ID HOs; 22 or 23
(b) a polynacleotide encoding a polypeptMe of SEQ D: 24,
(c) a polyfflideotide wMcb is flie complement of any of Therefore fee invention also includes an isolated polyiweleotide comprising:
(a) a polyancleotide sequence set forth in SBQ JD NOs: 25 (b) a polpacleotide tot encodes a pdypeptide that has at feast 96% 97% 98%, or
99%>s identity and or similarity to a polypeptide set ibrfi .in SEQ ID NO: 27
(c) a polyiweleotide encoding a polypeptide of SEQ ID; 27,
(d) a polyraicleotide which is the complement of any of Therefore the invention also includes an isolated polynucleotide comprising:
(a) polyrmeleotide sequence set forth in SEQ H> NOs: 30 or 31
(b) a polytkucleotide Aat encodes a polypeptide Aat has at least 98% or 99%,, identity
and or similarity to a polypeptide set forth in SEQ ID NO: 32
(c) a polynucleotide encoding a polypeptide of SEQ ID: 32,
(d) a polynucleotide which is the complement of any of^a),
Ibe feweatioa
(b) m t pol^p^tide'-lhitte at tost 95% 96%, 97%, 98% or
99%»» tod or in SEQ 3D NO; 34
(c) m i-pl^pide of SSQ 3D* 34,
(d) a-poljswdtetide witilh is fee of any of (a), Tteefors the iayeatioa also ineWes as polyancleotlde comprising:
(a) polymaeleotld® sefB«o» tet forth in. SfiQ.BB tfOr, 35
(b) a polpiucteotidt fliit a pol^pef tide tjat has at least 95% 96%, 97%, 9S% or
99%,, Jdeitiry and or «j « pol|fe|^fc wt forft in SKJ 1> MO: 36
(e)ap01pMeoMaie^^ • ~
(d) « polpBdi««i:(te wMdi is thecoajplemeirt of any of (a), 0) cr (c).
Hereford the iaventica also iactedes an isolated polyouctotide«oaiprlsfflg:
(a) polynucleotiile sequence set forA In S»3 ID JNOs: 37
(b) a polppcleotide flat escodes a polypeptide tttat has at least 95% 96%, 97%, 98% or
99%» idefltiQf and or simiarity to t polypeptide wt forA to SEQ ID NO: 38
(-c)«polpracIeotldeeiieodingapo!ypepti.deofSEQlD;38, ....
(d) a polynmcleotide -vM&h Is the compleaent of any of^a), (b) or Itotfore the invention also i»c!ides an isolated polpudeotide comprising:
(a) polpucleotide sequence set forth fa SBQ I> NOs: 39
(b) a polpwdeotide ftat encodes a polypeptide ftat has & least 95% 96%, 97%, 98% or
99%,, identity and or similarity to a poljfpeptide set forth to SBQ ID NO: 40
(c) a polynneleotMe encoding a polypeptide of SEQ ID: 40,
(d) a 3polp»cleotide which is tije aamptemeat of any of (a), (b) or (c),
Therefore the invention also mdtades an isolated poiynwdeotide comprisittg:
(a) polynudeotide seguence set forth in SEQ 15 NOs: 41 _
(b) a polynucleotide that encodes a polypeptide that has at least 95% 96%, 97%, 98% or
99%M identity and or similarity to a polypeptide set forth in SEQ ID NO: 42
(c) a polynucleotide encoding a polypeptide of SEQ ID: 42,
(d) a polynwcleotide which is the complement of any of (a), (b) or (c).
Therefore the invention also includes an isolated polynucteotide comprising: (a) polynncleotide sequence set forth in SEQ ID NOs; 43

(b) i fog ea tot has at least 95% 96%, 97%, 98% or
99%» ant! or to a pofypeptide set forth .in SBQ ID NO: 44
(c) t jpofymdteotide a lolypeptlde of SBQ ID: 44,
(d) apolyaocleotide wMcliis ttoecomflei^jt of a»yof Therefore the invention also an isolated polynneleotide comprising:
(a) pelpaeteerfide set forth In B> NOs; 45
(b) a polyiffideottde tihat a plypejitide that has at least 90%, 91%, 92%, 93%,
94%, 95% 97%, 98% or 99%M identity and 0r starikritjr to a
to SBQ ID MO: 46.
(c) a polpwcleotide encoding a polfpeptide of SiQ ID: 46,
(d) a polynweteotide which Is the ccmipleineat of aoy of He inv«ti.on. also iaeludes an isolated poiynncleotide comprising:

(a) polpracleotide se^pence set ftwtib is SEQ ID lOs: 47
(b) a polyniicleotide that encodes a polypepiide ftat has at least 90%, 91%, 92%, 93%,
94%, 95% 96%, 97%, 98% or 99%,, identity arid or similarity to a polypeptide Bet forth
in SBQ UNO: 48
(c) a polynacleotide encoding a polypeptide of SEQ E): 49,
(d) a polpucleotide which is the complement of any of Therefore the invention also includes a CD163 polypeptide in which the tirnsmembrane repon is deleted,
Hefefofe the invention also includes a polynucleotide encoding a CD163 polypeptide in which the transn»nibraue region is deleted.
In addition to Ae foregoing, tite invention inctodes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations specifically mentioned above..
Brief Description of the Sequence Listings SEQ ID N0j 1—cDNA sequence encoding porcine $usCD163vl SEQ ID MO: 2—predicted amino acid sequence of porcine sosGD163vl SEQ ED NO; 3-- cBNA sequence, Gettbank accession number AJ311716 SEQ ID NO:4—predicted amino acid sequence derived from Geabank accession number AJ311716
SEQ ID NO: 5^—cDNA sequence of susCD163vl containmg flanking (non-coding) sequence.

-SfflQID WOi
ID MOtl2 - SEQ ED N0J13 «e&NA«eqiMnce encoding porcine susCD163v2 SEQ B0: N0sl4 —predicted amlao geld sequence of porcine susCDI€3v2 S1Q ID NO:1S-W—primer S1Q IB NQ*17—cBNA sequence encoding huittan CB163v2' confcaming
JTO NOslS— eDNA
SKI HP NOJ19-- predictdt »mino aeid sequence of toman I^Q ID HO:.2W-2l-»pOTa®r sequences
IB NOtS^«-cDNA sequence eacodtog marine CD163v2 containing flanMng
SEQ KD NO&3-*- dDNA seqoeooe encoding miirtae O>163v2
S1Q H> MO:24— predicted amtoo osid sequeace of marine CD163V2
SEQ H> NO:2S»-cDNA seqyeace eacoding mraine CD163v3 coatainiiig flaaking
(notSNSodlag) sequence. • -
SEQ .ID NQj2&— dDNA sequence encoding murine CDl63v3
SEQ ID NOd57— predicted ainino acid sequence'of mtirine OD163v3
S1Q TO NO:28-2^-»prinira> sequences
SBQ ID MO:30—€|)NA sequence eacodkg MARC-145 CD163v3
flanidflg (aon-coding) sequence.
SEQ ID NOJ31— cDNA sequeoce encoding MARC-145 CB I63v3
SEQ ID ^O:32— predicted amino acid sequence of MARC-145 CB163v3
SEQ ID NOj33-cDNA sequeace encodkg Vero cell CD163v2 transcript
S1Q ID H0J34-- predicted aimno acid sequence of Vero cell GDM53v2
SEQ ID NO:35~ cDNA sequence encoding Veto cell CDl€3v3 transcript
SEQ ID N0:M~ predicted amino acid sequeace of Vero cell CD163v3
SEQ ID NO:37~ cDNA sequence encoding Vero cell CDlt>3v4 transcript
SEQ W N0:38- predicted amino acid sequence of Vero cell CD163v4
SEQ ID NO;39- cDNA sequence encoding Vero cell CD163v5 transcript
SEQ ID NO:40~ predicted amino acid sequence of Vero-eell SEQ ID NO:41-- cDNA sequence encoding Vero cell CD163v6 transcript

ID NO^—pndicted mid .of Vero cell CDl€3v6
SEQ ID NO(43- ePNA Vero eel CDltiSv? teaseript
SEQ ID N0s44- aeM of Vero cell CDJ63v7
SiQ H> NOt4S- eDMA canine OD163-V2 transcript
S1Q IB NCh4 S1Q DD NCM7- eDNA CDl€3v3 transcript,
SEQ ID NCH48- predicted acid sequence of camae C3D163v3
Brief Description of the Drawings
Ftgwre 1 Sdbemtfe-«««par!son of su$CD163vl with AI311716
Hgiii« 2 Amino acid 'Sequence al^nment of swstD163vl (SEQ JOD NO: 2) with"
AJ3im-6CSBQJDNO:4)
Flgnn 3 Nndeotide Sequence aHgrameBt of susCBMSvl witib AJ311716
Kgnre 4 Generation of DMA fragments and Hgation to place OD163 duectly behtod
the RSV promoter, Pksmids were digested with either 0raIH or JD*rdf, followed by a
blunting reaction with Ktenow enzyme. After clean up, the plasmMs were dieted
with Nott Gel pttriflcation yielded DNA fragments- that were stibseqnently Jigated
ntilizhig fte cohesive M?rl termini Promote! from RSV (pRSV) and SV40 ore indicated witii awows,
figure S Map of pCDNAS.l Directional VS/His/TOPOcloning -vector
Figure 6 Three BHK/CMVM all lines, *3, #5, and #12 and a non-permissive BUK
cell line were infected with PRRSV isolate P129 and stained with SDOW17-HTC.
Panel A shows a non-permissive BHK21 cell clone. Panel B shows BHK/CMVM
clone #3. Panel C shows BHK/CMVM clone #5, Panel D shows BHK/CMVM
clone #12,
Figure 7 Three BHK/RSV/vl cell lines, f2, #3, and *4 were infected with PRRSV
isolate P129 and stained with SDOW17-ETTC. Pane! A shows BHK/R5VM clone
n. Panel B shows BHK/RSV/vl clone #3. Panel C shows BHK/RSV/vl clone #4.
Figure 8 Feline kidney cell lines stably expressing porcine CD163vl, showing
PRRSV plaques. Cell lines NLFK-CMV-snsCDM3vl-G4F and NLFK-CMV-
susCD163vl-G4L, both at passage 4, were infected with the P129 isolate of North
American PRRSV and incubated for 6 days. Monolayers were fixed with 80%
acetone and stained with monoclonal antibody SDOW17-FITC. Phase contrast

, of vied CPE (plaqties), wfaite.PA detection
(tefi) flnl
H§»ft f Bow MMiVM ceil #|» #2, #3, and #4 we® infected with
P129 and with monoclonal SDOW17-HTC, Panel A shows
#1 cell elone. Panel B clone #2, 'Panel C shows
FE&SVM done #3. D HDRSVM #4,
Fljpure 10 re42MV**naCD163vl-A10 cells at 19, infected witti PRRSV
isolate P129. Left; Hie m-onoltyar was feed wife S0% acetone and stained wMi
jagBed itaibody SDOW17 {Bond T«hno!opes fac)t wlutoh is

Saipt-sisCD16Si^A9 it passage 17 iniected wi.th PERSV
isolate P129, Hie raoaolayer was feed witti §0% -aotone and stained, wifti F1TC-
conjw^ted monoclonal, antibody SD0W1? (Earal T«ehnolo^es foe), nWch is
specific for PRESY nacleoe^sid. - • •
Flgttrel.2 Tbn«icprasaDrtathree)MinpteoftD^ The cells
were infected with JPRRSV isolateJP129 and subsefuently stained witii SDOW17-HTC. Panel A shows cell line BHK/RSY/V2 f 1, panel B shows ceil line BH1MSV/V2 #34, aod panel C shows cell line EEK/RSV/V2 #47. Fipore 13 FK-cDNA3.1D-bttmCD163v2-A6 at passage IS infected witti PRRSV ' isolate P129. The monolayer was Aen fixed with 80% acetone and itained witto F1TC~ conjugated monoclonal antibody SDOW17 (Rtiml Technologies 3tac), which is specific for PRKSV nucleocapsid,
Ftgwre 14 The amoont of progeny PRRSV produced by four recombinant cell lines stably expressing susCDl63vl* and by MARC-145 cells, was detomined in a growth carve experiment using the JNTVSL 94-3 isolate of PRRSV, Samples harvested at 12-feotir intervals were titrated on MARC-145 eel monolayers. Pp^re IS How cytometry analysis of PRRSV infection io the presence of CD163 specific antibody. BHKr21 cells expressing MARC-145 CD163 from transient transfection were incubated with either GD163 specific antibody or normal goat JgG (NGS) and infected with a GFP-expressing PRRSV. Each data point represents the results of triplicate wells.

. Figure W How efteaetry t»^^
specific antibody. MURK human CD 163 were incubated with
eititer CD 163 specific antibody of normal goat IgG (N6S) and Mated with a GFP
expressing PRRSV, At 24 bonus post Infection the percentage of GEP expressing
infected was Each data point presents the result from a single well
ofcelb
Figure 17. Graphical deletion of six alternative splicing variants of QD163 raRNA
recovered from. Vero cells. The sk variants differ in the presence or absence of teee
exons, E6» BIOS, a»i S3, Ikons E6 and BIOS have lengths that are
multiples of fee, and titeefote do not result in a change in reading frame when absent.
* * " *** iO .-»-w-— T™
In contrast.fte absence of ESS results in a shifted reading franie and an alternative
amtoo acid sequence at tihe csrboxy teotbios of the protein (indicated by a hatched
pattern in the figure). The hydropfaoMc ttansmesibrane 0M) region is encoded witMn
BIOS.
figure 18. PK-RSVScript-suCD163v2 #9 eells infected witii PRRSV isola^ P129.
Undiluted supernatant from PKRSV isolate P201 infected PAMs was ased to Meet
• PK-RS¥Seript-8«sCD163v2 f9 cells.. After two days of incubation. th.e..cels weie
&c«sd and stained with monoclonal antibody SDQW17 as described in Example 11, Figure 19. FK-RSVScript-8ittCbl63T2 #51 cells infected with PBHSV isolate P129. Undiluted supernatant from PRRSV isolate P201 infectaed PAMs was used to infect FK-RSVScript-snsC!D163v2 #51 cells. Two days post infection the cells were acetone fixed and stained with monoclonal antibody SDOW17 as described in Bxsxnplell.
Figure 20, Infection of PK-RSV$cript-sasCDl€3v2 clone #9eeHs with PRRSV isolate P201. Panel A shows a monolayer of cells infected wira PRRSV P201 at passage 1, twenty-four hours post infection. Panel B shows a monolayer of*eells 2 days post infection with cell free supernatant PRRSV P201 at passage 10. Figure 21. NLFK parent cells and one subclone of FK-cDNA3.lD-humd>163v2-A6 were examined for the CD163 expression. Cells were fixed in 80% acetone and reacted with Goat anti-human CD 163 (R&D System at 1:200) for one hour following by washing with PBS. For visualization, donkey anti-Goat IgG conjugated with HTC (Biodesign Inc at 1:100) were used. No specific fluorescence was detected in the

MURK shown to 21 A. The majority of the F1LA6.A2 subelone
OF THE General DeflnSttons
Gels and eel can t» either virus permissive** or "virus non-permissive**. For example, a cell or cell line that is virus peimtesive is capable of allowing virus infection, sutoseepeat fffMestioti and vims frodtetion, A cell or eeU line that is virus noa-permissi w is teeajptbie of aDowimg virus Infection, subsequent itflieation and vims production. A eel line that is already somewhat permissive may be rendered. "' 'most penoiisive by the ra^hods of the toveation, -
An&rMfidae refers to a family of enveloped* positive-steaded RNA viruses belonging to the order ffidovbvtes. The family includes lactate dehydrogenase-elevating virus QjyV) of mice, equme arteritis vims Atf&vmdae is a family of Icosohedral, enveloped vtoses whose genomes consist of single molecules of linear double-steaded DNA about 15CK300-190000. nucleotides long. Hie name of the family is derived from African Swine Fever And Releted Viruses, African Swine Fever Vims (ASFV) is the type species of the Asfivinis gemis and is the sole member of fl»e family. •
The term, '1PKJRSV*' or PRRS virus refers to both European and North American PRRS virus, genotypes. Within each genotype, isolates typically share 85% or higher nucleoti.de identity. Between genotypes, however, the level of sequence identity is only about 60%,
The PRRS virus is a member of the family Artermridm, The genome of the arteriviruses is single-stranded RNA of positive polarity between 12 and 16 kb in length, capped at the 5* end and polyadenylated at the 3* end. Over«two-thirds of the genome is dedicated to open reading frames (ORFs) la and Ib, which encode the non-structural functions of die virus. ORFlb is an extension of ORFla, and is the result of a ribosomal frameshift, ORFs la and Ib are translated directly from the genomic RNA. These large polypeptide products are cleaved by viral proteases to yield 12 or 13 discrete smaller peptides. The remaining ORFs, which encode viral structural proteins, are expressed from a series of 3s co-terminal subgenomic RNAs {sgRNAs).

OMBorpi o
suett that a coiwaoa 5* lender fused to each fimscript, "Hie major
structural proteins arc -the nueleoeapsld (N, encoded by. ORF7), the matrix protein -CM*
encoded by ORF6), and the major envelope glycoprotein {GPS, encoded by QIUFS).
The remaining proteins, GP4 (ORF4), GPS (ORES), GP2 |ORF2a), and 1 CQRKb)
are minor structural components of the virion, whose functions haw not yet been
eluddated. The wdeoitar Motefy of PERSY has been the subject of recent review
articles (Dea «*«!.»2000; 2000; and Meitfenberg, 2O)1).
' As esei fce«in, tite "O3163 jpoiypeftfdB** means a prutefe eiw^led by a maniiaallM OD16S ^ue, teeloding al^lie •varitots contaMttgoHiservativeor noa.»
has ten repsrted (Genbank a»inber AJ311716), A mwiae CB163
polfpeptide has also been reporteri (Oenbant mimber AF274S83), as well ai EttoMple temia vad.aats, exemplffied by 'Genbaafc access Bombers A^EI51281attd. CAA80543. We report be»in polyaucleotides that eaoode poicine, haman, mmne, canine, and aflicttt green ffloakey QD163 polypeptides and wMch comprise tine • sequences'Set forth fe-SEQ ID-NO: 1,5,12 13,17,18,22,23,25,26,30,31 33,35,. .. 37,39,41,42,43,45 and 47. A "CD 163 polypeptide** is a member of the scavenger receptor cysteine-rich (SRCR) family of ttansmetnbrane gSycoproteins, and is thought to be expressed exclusively on mcmocytes and macropha§es. One identified role of CD163 is to inhibit oxidttive tis:sae damage following beaolysis by consmntog herooglobia:haptoglobin complexes by endocytosis, He subsequent release of inteileuMo-10 and synthesis of heme- oxygenase-1 resulfe in antiinflanoEistary and cytoprotective effects OPhiUppidis et a!., 2004; Graversen et al>, 2002). Hie human CD163 gene spans 35 Kb on chromosome 12, and consiste of 17 exons and 16 introns, A number of isoforms of tihe CD163 polypeptide, including merobmne bound, cytoplasmic and secreted types, are known to be generated by alternative splicing (litter et at, 1999). Isoforms that comprise a txausmembrane domain are particularly preferred.
A transmembrane domain is characterized by a polypeptide segment of a larger sequence that is exposed on both sides of a membrane. The cytoplasmic and extracellular domains are separated by at least one membrane-spanning segment tihat traverses the hydrophobic environment of the llpid bilayer. The membrane- spanning

segment is composed of amino acid residues wimjnonpolar side chains, usually in the
form of an alpha helix. Segments that contain about 20-30 hydrophot>ie residues arc
long enough to spaa a membrane as an alpha heli%, and they can often be identified by
means of a hydropathy plot The predicted transmembratte domain of SEQ E> NO:2
and 14 are indicated by holding in the specification. To deteimine whether other
CD 163 sequences contain a similar sequence feature is easily determined by
inspection of the sequence or hydropathy plots. SEQ ID HOS; 37-40 are
repf«senteti?e of wiairt C3D163 which do not coatein a transttteoibrane
domain and their encoding nucleic acids.
As aasi heififaafter, "polyaideotide" generally refers to any polpll»irad€Otl.ide or pofydeoxrJM«adeotiie, wMch may be unmodified RNA or DNA or modified KNA or DMA. "Polysaieleotides*1 taetode, wiHBDotlJmi.tatton» single- and1 double-steaded DNAf SNA feat is a mixture of single- and double-strauded regions, single- and dooble-straadisd. RNA, RNA tot is mixtare of singfe-and double-stranded regions, and hybrid molecules eomprlsiag DMA and KNA that may be sagle-slxanded or, fflore typically, double-stranded or a mixture of single- and - double-stranded regions* to addition, "polynacleotide" refers..to triple-stranded regions , comprising RNA or DNA or both ENA and DNA. Tie term "polymieleotide" also inctodes DMAs or RNAs containiEg one or more modified bases and DNAs or RNAs with: backbones modified for stability or for other reasons. "Modified" bases include, for example, ttitylated bases and unusual bases such as inosine. A variety of modifications may be made to DNA and RNA; thus, "polynucleotide*' embraces chemically, enzymaticaDy or naetabolieally modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. Tolynucleotide" also embraces relatively short polyOTcleotides, often referred to as cdigo&udeotides.
As used hereinafter, "polypeptide" refers to any peptide or protein comprising amino acids joined to each other by peptide bonds or modified peptide bonds. "Polypeptide" refers to both short chains, commonly referred to as peptides, oBgopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. "Polypepta'des" include amino acid sequences modified either by natural processes, such as post-translationa! processing,-or by chemical modification techniques which

. ..ass well ia ffetltt. Jieh am well described in basic fextn.iOid.in
pore as wen as ift ft Uteratom.
Modifications aisy occur is « polyp eptide, ineWifig the peptide bactttane,
fee utnito add sad the amtuo or earteyl termini, t will be appreciated
that-ibe same type of modlicfttJOB may be to the same or varying at
sevesral to a pe||p|^de.'Also, polypeptide may contem many types
of ffiodffimtions. Poljfeptikfes may be teaaehed as a malt of ubiqoittoation, «ttd they may be cycle, with or wittat bnnaM«§. Q«c&, branched and branched cyclic
spthetie methods, Modificatioas or modified fowns include acetyMon, aeylation, AUP-iftosylation, aiaidatioa, covalent-tftectewjt of flavin, covalent attachnwat of a
attachment of a Iipid orlipid derivative, covaioat attachment of phosptetidylinositol, cross-lInMng, cydfeation, disujtfide bond fonaation, demefliylatioii, formation' of eovalent csross-linfe, formation of cystine, formation of pyroglutamatts, formylatioa, gamma-carboxylMioit, glycosylttion, As used hereinafter,"isolated'f means altered by the hand of man from the natural state. If an "isolated" composition or substance occurs in nature, it has been changed or removed from its original environment, or bom. For example, a polymicleotide or a polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. Therefore "isolated" as

. used herein and in the art, j^jbether lefefing: to "isolated'*
polytaicteotietes or polypeptf des, is to mean separated from the original celalar environment fa which tibe polypeplide or nucleic acid is noimaBy found. As used hensis therefore, by way of example only, a transgenic animal or a reoombinant ceH line abstracted wfth a plynticieotide of the taxation use of fee "isolated" ' nacleic add. Specifically excluded from the defioition-of isolated polynucleotides of the invention are entire isolated chromosonMS from native host csels from which the pQlyawcleotide was orifinally derived,
In the disclosure to follow we will often, make use of the term, "identity** or siauterity as applied to the amino acid sequences of polypeptides. Percent amino acid sequence "identity" with respect to polypeptides is defined herein as fee percentage of amino acid residues to &e candidate sequence that are identical with the residues in tibe target sequences after aligning both sequences and introducing gaps, if necessary, to achieve the npxlraum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Percent sequence identity is'determined by e»«ventional methods. For example, BLASTP 2.2,6 ITatasova TA
- and -TL Madden,. "BLAST.2.§equenees~ a new toolfor comparing proteinuand nucleotide sequences." (1999) FEM.S Microbiol Lett. 174:247-230.]
Briefly, as noted above, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a-gap extension penalty of 0.1, and the *1>losum62** scoring matrix of Hemteff and Henikoff (Pmc. Nat. Acad. Sci. USA 89:10915-1090.1992).
The percent identity is then calculated as:
£ [length, of &e longer sequence + number of gaps introduced into the longer sequence to align the two sequences]
Percent sequence "similarity" (often referred to as "homology") with respect to a polypeptide of the invention is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the target sequences after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity -28-

andjeffln§@¥atiw nbst
of tie -f-romte of fatrodoced tote
Amino can be aajordtaf to physical properties and
cooMbwion to and tetiary proieato st»etaie. A conservative substitution is
recogplzed to the art as a s«bstitoti0» of one aarfao aoid for aaotihrar acaino acid that
Bxefflplirj conservatfw substitutloas ans set out in Table, 1, 2, and 3 below.
Table 1 Conservative Substitutions I
SIDE CHAM
CHABACTERISTIC - AMMO ACID
Non-polar ' . . GAP
ILV
Polar - uncharged . . CSTM
NQ
Polar -charged DE • •
KR
Aromatic . HFWY
Other NQDE
Altemadvely, conservative ammo acids can be grouped as described in Lehninger, Piochemistry, Second Edition; Worft Publishers, Inc. NY:HY

SIDE CHAIN
....................................... AMNQ.AOOD


AXIVP
FW
M

A., HydioxYl: STY
B, Amides: NQ
C, SttUhydiyl: C
D, Borieriine; O
....... - - Positively Chafed Oasic): . .. ..... KRH
Negatively darged (Acidic): DB
As still anotiber alternative, exemplary conservative sobstitutiom are set out in Table 3, immediately below.
Tables Conservative Substitutions DJ
Original Residue Ikeiaplary. Substitution
Ala (A) Val,Leu>Be
Arg (R) Lys, Ota, Asn
Asn (N) Gin, His, Lys, Arg
Asp CD) Ota
Cys (C) Ser
Gin (Q) Asn
Glu (E) Asp

HP CD Lea, Val, Met, Ala,' Phe,
Lys PQ Args Gin, Asn
Met (M) Leji»Phe,Ile
Pro

Ser(S) Thr
H*r(T) Star
Trp{W) %
Tyr (Y) T rp» Fhe, Hir» Ser
Val(V) ' Be,Lea,Met,Pbe,Aia
Methods' Directed to th« I¥eNteetat of Virus and Host Cells of the Invention
The invention provides a method of modifying production a virus that is a member of the family Arterivirtdae mdA^farviridae to a cell comprising the step of directing said eel to express a QD163 polypepti.de. This may njclwde rendering a . . virus floa-pennissive cell into a vims permissive cell, or may involve rendering a cell more permissive to the viras.
ID one embodiment, the vims that is a member of the family Arteriviridae or AtfarrirMae is selected from the group consisting of IDV of mice, equine arteiitis virus (EAV), simian hemorrhagic fever viras The invention further provides a mettod of preparing a culture of a virus that is a member of the family Arteriviridae oxAsfarviridae comprising the steps of: providing a cell line; directing said cell line to express a CD 163 polypeptio!e; infecting said cell line with viras; and causing said cell line to produce viral progeny.
to one embodiment, the virus that is a member of the family Arteriviridae is selected from the group consisting of LDV of mice, equine arteritis virus (EAV), simian benaorrhagic fever virus (SHFV), PRRSV of swine and ASFV of swine. In a preferred embodiment the virus is PRRSV.

All 'of fee above and cell. expressing a CD163
inttoduction of add iito the cell Such a cell may comprise a
polynwcleotide or vector in a manner Aat expression of an encoded CD 163
as
part of a circular plasmd, or as linear JDNA comprising an isolated protein-coding
region, or to a vis! vector. Mefeods for introducing exogenous nucleic acid into the
host cell well known tod rorttaely in the art tecfade transfiramtion,
trtnsfection, eleetropoimtion, nucleir injection, or fusion with carriers seen as liposomes, micelles, ghost eels, m& protoplasts. Host eeU systems of the mventfoii include invertebrate and vertebrate cells systems. Hosts may include, but ace not limited to, the Mowing: insect cells, porcine Mdney (PK) cells, feline kidney Hie choice of a suitable expression vector for expression of me CD163polypeptides will of course depend upon the specific host cell to be used, and is witifaan tiae skill of the .ordinary artisan. Examples of suitable -expression vectors include pSport and pcDNAS (Jtovitro§en), pCMV-Script (Siratagene), and pSVL (Pharmacia Biotech), Expression vectors for use in mammalian host -cells may indtade ttanscriptionai and translational control sequences derived from viral genomes. Commonly used promoter sequences and modifier sequences which may be used in the present invention include, but are not limited to, those derived from human cytomegalovirus (CMV), Rous sarcoma virus (RSV), Adenovims 2, Polyoma virus, and Simian virus 40 (SV40). Methods for the construction of mammalian expression vectors are disclosed, for example, in Okayama and Berg {Mol Cell Biol 5:280 (1983)); Cosman et al (Mol Jmmunol 25:935 (1986)); Cosman et al (Nature 512:768 (1984)); EP-A-0367S66; and WO 91/18982.

^
t
the may be to permit, or increase, expession of the CP163-
polypeptide. Cells cm be modified increased by In whole or in part, toe naturally 'occaning CD 163
promoter wife al or part of a heterologois promoter, so that the cells express CD163
polfpeftide at levels, Thefaeteiafogaas promoter is inserted In such a manner
tat it is Mated to endopnom ODM3 encoding sequences. {See, fer
example, PCT ttemattonal Pttblfciiioa No, WO 94/12650, PCT foteational Publication No. WO 92/20808* and PCT fttemitional Publication No, -WO 91/TOSS.] It is also contemplated' tbat, in atfditiott to heterologous piranoter DNA» ifflopifiable noafer DNA (e.g,, aste, «5^p, aad tie laulttfanetioiial cad @s»ef ^erfiich encodes for earbaniyj phosphate synthase, aspartate transcarbamylase, and dih|«troorota«) aad/or inton JDNA may be inserted along wMi the beterologous promoter DNA, ff tofeed to the CD163 coding sequence, amplfficatlon of the marte1 DNA by standaBJ selection methods results in co-ampHfieation of the CD163 coding sequences in-the cells.
. GDI 63 ejqpanession.may also be indaced by -chemical treatment, Phorbor
esters, especially phorbol myristyl acsetate (PMA), activate one or more isozymes of the ubiquitous membrane receptor, protein kmase C (PKQ and are particularly preferred means of increasing CD163 expresssion. Other methods of intracellular calcium mobilization are also contemplated. Vaccine..Broduction
The methods described above may be used to produce any virus tibat is a member of the fw&yArteriviridae or Asfarviridae for the purpose of vaccine production or diagnostics.
M one embodiment the virus that is a member of the family Arteriviridae is selected from the group consisting of LDV of mice, equine arteritis virus In a preferred embodiment the virus is PRRSV. Vaccine Production
The methods described above may be used to produce virus for the purpose of vaccine production or diagnostics.

• •-,- Bied|iia0^vated)orMw?^i«escsttbepc«lti«d, Hereforo, to mates a
live ¥*elna, a viral Isolate, ear in or variant fteeof, is -grown to
cell oultere. The TOTS is barvestedt-accwding to methods well known in the art The
vires may be and stored at -70°C, or feeeje-dried and stored
at 4*C» Prior to vscdaatilaa fte vires-is mixed at m app-opriate dosage, (which is
from about 10s to 101 ctftae per ml (T0Dso&al})» wim a
l&aimaeetttically acceptable carrier such as a saline solution, and optionally an adjuvant
Hie vaccine produced might also comprise an inactivated or Idled vaccine comprising a virus grown by the methods of tile invention. Hie inactivated vaccine is made by methods well teown. in ifae art. For example, once the virus is propagated to Mgn tilers, it would be readily apparent by those skilled in the art that the virus aBttgeoie mass could be obtained by metibods well known to the art. For example, tite vims antigeaie mass may be obtained by ditotioa, concentration, or extraction. AU of these methods have been employed to obtain appropriate viral antigemc mass to produce vaccines. Hie vims is then inactivated by tjeateent with formaim,, - betapropriolactone (BPL)f binary ethylenetatoe (BM)» or otihermetijods kooviTa- to tfiose skated to the art. The inactivated vims is then mixed with a pharmaceuticaly acceptable earner such as a saline solution, and optionally an adjuvant Examples of adjuvants include, bat not limited to, alunaaum hydroxide, oil-in-water and water-in-o2 eroalsions, AMPHZOEN, saponias sech as QuilA, and pjlypeptide adjuvants including intedeoltins, interferons, and other cytoktoes.
feactivation by formalin is performed by mixing the viral suspension with 37% formaldehyde to a final formaldehyde concentration of 0.05%. The virus-formaldehyde mixture is mixed by constant stirring for approximately 24 hours at room temperature. The inactivated virus mixture is men tested for fesidual live virus by assaying for growth on a suitable eel line.
Inactivation by BEI is performed by mixing the viral suspension of fee present invention with 0.1 M BEI (2-bromo-ethylamine in 0.17S N NaOH) to a final BEI concentration of 1 mM. The virus-BEI mixture is mixed by constant stirring for approximately 48 hours at room temperature, followed by the addition of 1.0 M sodium thipsulfate to a final concentration of 0.1 mM. Mixing is continued for an

two .tents,
for. growth on a «! Itee.
Vires gut taws to express GD163 can afeo be
used to live vims. Two coorooa methods, which am well known to titose
In the art, ate the wad the dilution assay,
CB163-expi«ssteg cell of the jtavmtiottcan be used to grow virus
for the pwposs of producing viral far diagnostic Mts. For'«xample lysates
from Mected eeDs (with optional pwrifaifioB of vital particles or extinction of selected viral proteins) may be coated on SJSA plates in order to detect and quantify antibodies to the virus in swine
Uve or inactivated virus frown in CD163-eKpr«ssinfc«lls can be tused alter
optional of the viral proteins to ioratotme animals in order to federate
'polycloial, fflonospeeiie or monoclonal mtibodies. These in tarn can be used as the basis of diagnostic assays for the detection and quantification of virus in swine serum
Assays of the Invention
The invention provides metiteds for detennjj^g (toe propensity ctf an animal to be Mected by a virus that is a member of the family Arteriviridm or Asfarviridae or of a cell line to support tiie replicatJoa of a virus that is a member of tfje family Arteriviridae or Asfarviridae, Samples from edthersource are obtained and a^ayed for expression of GD163. The level of CP163 gene-expression cam. be compared with levels of controls known not to support replication, of tfce virus.
In the case of an animal, samples can be any sample comprising sample nucleic acid molecules or proteins, and obtained from any bodily tissue expressing CD163, including, but not Emited to, alveolar macrophages, cultured cells, biopsies, or other tissue preparations. The level of expression can be assessed at either or bo Methods of determining CD 163 levels may be nucleic acid based as noted above, CD163-derived nucleic acids may be in solution or on a solid -support. In

• some may-be employed m elements in.iniefcan-ays alone m .
in with array moleralei, Nucleic acid toasedmethods
gently the of DMA or IMA from tie sample and subsequent
hybrldisttjoii or FOR amplification "using primeis derived from any known.
*
GD163 encoding la the art or those specifically disclosed as SBQ ID NO: 1,
3,5,12,13,17t 18,22, '23,25,26,30,3133,35,37,39,41,43,45, and 47. DMA or
RMA can be isolated from the sample teeordiag to my of a number of memods well
known to those of stffl in the at For example, methods of purification of nucleic
acids are described in Tij§seat P. (199$) Laboratory TeebMfuei in Biochemistry and
Molecular Biology: Hybridisation With Nucleic Acid Probes, Part I Itieory Md
Nucleic Add Preparation, Bfeevier* Mew York, N.Y. fc one prefecied-cmbodiiiient,
totd IMA is using the TRBSOL total RNA isolation rea^at 0Jfe
Technologies, toe., Gaiftersbuig Md,) and raitHA is isolated using oligo d(T) column cbromatography or glass beads. When sample nucleic acid molecules are amplified it is desirable to amplify the sample awdeie acid molecules and maintain the relative abundances of fee original simple,rmetading low abundance transcripts. RNA can be .amplified in vitro* i&'Situ,- or in vivo (See Eberwine U.S. Pat. No. 5,514,545),
It is also advantageous to include controls witMn the sample to assure (hat amplification and labeling procedures do not change the trae distribution of nucleic acid molecules in a sample, For this purpose, a sample is spiked with an amount of st control nucleic acid molecule predetermined to be detectable upon nybridkation to its complementary arrayed nucleic acid molecule and the composition of nucleic acid molecules includes reference nucleic acid molecules which specifically hybridke witib the control arrayed nucleic acid molecules. After hybridization and processing, the hybridization signals obtained should reflect accurately the amounts of control arrayed nucleic acid molecules added to the sample.
Prior to hybridization, it may be desirable to fragment the sample nucleic acid molecules, fragmentation improves hybridization by minimizing secondary structure and cross-hybridization to other sample nucleic acid motecules in the sample or noncomplementaiy oucleic acid molecules. Fragmentation-can be performed by mechanical or chemical means. Labeling The sample nucleic acid molecules or probes may be labeled with one or more

labeling to for of oiayed/san^le iitadteic.aeM -,',...
molecule The era .iaetede coffl|>osMoES Art can be
fey Moehetaieal, bioelectronie,
insmtttt«h«i»ical, electrical, optical, or«braaieii.3ffica0s. Hie labeling moieties
iaeJMte such as :C32)P» . labeled heavy metal sjs^fisfcopie marfeii, -such as
fluorescent w& dyes, mtpetie labels, Hated emymes, mass
tags, spin labels, electron teansffaf donors and *eepfars, and the Bfce,
fiBOi»(»iit ladwde C^ Md CyS fl««efhc^ss (AnasmbMn Pharmacia
Bi0tedi» flsesttawty If X),
He nocleic acid moleoilf seqw»ce of SEQ ID NO: 1,3,5,12,13,17, IS, 22, 23,25,26,30,31,33,35,37,39,41,43,45 and 47 or other OD163 encoding sequences in fhe art and fragments thereof can fee used in various hybridisation technologies for various purposes. HyWdization probes may be designed or derived from any maHjmalim CD163 ^qwenw but .may make use of those sequences -disclosed in SEQ ID NO; 1,3,5,12,13,17,18,22,23,25,26^30,.31,33,35,.37,39,. 41,43,45 and 47, Such probes may be made from a highly specific region or from a conserved motif, and used in protocols to quantify CD163 message, allelic valiants, or related sequences. The hybridizaion probes of the subject inwntian may be DM A'or RHA and may be derived from my mammalian OD163 sequence known in the art or from those sequences disclosed herein as SEQ ID NO; 1,3,5,12,'13,17,18,22,23, 25,26,30 or from genome sequences including promoters, enhancers, and introns of the mammalian gene. Hybridization or PCR probes may be produced using oligolabeling, nick translation, end-labeling, or PCR amplification in the presence of the labeled nucleotide. A vector containing the nucleic acid sequence may be used to produce an inRNA probe in vitro by addition of an RNA polymerase and labeled nucleic acid molecules. These procedures may be conducted using commercially available kits such as those provided by Amersham Pharmacia Biotech.
The stringency of hybridization is determined by the G-fC content of the probe, salt concentration, and temperature, to particular, stringency can be increased by reducing the concentration of salt or raising the hybridization temperature. In solutions used for some membrane-based hybridizations, additions of an organic solvent such as

" tih§ to-oecOT Et a lower tenperatu«e.-HyMdtealioncan-be • -
perltomed at low wiffa buflots, as SxSSC with 1% sodium dofteeyl
(5DS) at 60eC, which peoaite &e fofmatitoa of a hybridisation cefmplex
between accleotide &at irfsmatehes. Subsequent washes are
performed at higher with as Q,2x$SC with 0.1% SDS at either
45*C Cmedjtoa stoingenq?) or 68CC (high stringency). At high stringency, IryMdiaflon complexes wffl remain only wbeae fee nucleic acid sequen-fles are
s
almost completely complementary, la membrane-based hybridisations, preferably 35% or most pmftttbly 50%, fonaami.de ran be addolto tih© hybridkatiom solution to Ksiwce tih© temperatofe at ^Mdh hybridiiatfon is performed, and badkgriwnd signals can be redoced by ttie use of other determents mieti as Satkosyl or Tritoa X-100 i«d a blocking such as salmon sperm DMA. Selection of ccmpoaents and coMdMofli for hybridization aie well kaiown to ttioBe stilled ia tfse art and are reviewed in Ausubel (supra) and Sambrook et al. Exemplary M^bly stringent hybridfefttioE conditions are as follows: hybridiMfi0» at 420C in a .toybridlzation solution comprising 50% formami.de, 1 % SDS, 1M Nad, 10% &5xtran sulfate, and washiag twice for 30 minutes at 60°C in a wash solatioB comprising OJ X SSC and 1% SDS. It is understood in the art that conditions of equivalent stringency can be achieved ttnrott.gh variation of temperature and toffee, or salt concentration as described Awsnbel, et al. (Bds.)» Protocols in Molecular Biology, Joha Wiley & Sons (1994), pp. 6,0.3 to 6.4.10. Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and the percentage of fmanosine/cytosine Hybridization specificity can be evaluated by comparing the hybridization of specificity-control nucleic acid molecules to specificity-control sample nucleic acid molecules that are added to a sample in a known amount The specificity-control arrayed nucleic acid molecules may have one or more sequence mismatches compared with the corresponding arrayed nucleic acid molecules. In mis manner, it is possible to determine whether only complementary arrayed nucleic acid molecules are

hybridizing to the sample nucleic-acid molecules or ^'hct
duplexes are forming is determined, |
Hybridization reactions can be performed in absolute cs*dflfeetttial
hybridfeatioft formats. In me absolute hybridization format, mcteic add molecules
from one sample are hybridised to the molecules in A micioaaty fonnat and signals
detected after hybridization complex formation correlate to nweleic acid moleeole
levels in a sample. In the differential hybridization format, the diffiwtti
of a set of genes in two biological samples is analyzed. Fck differential
mieteie add from both biological samples are pWpied and labeled wffla
diffarent moieties, A mixture of the two labeled aneteteicid aiolecates is
i.
added to a Merotrfay, The miefoarray is then examined tinder conditions ia whkh the' emission fam fee two different labels are individually detectable. Moleedes in tbe
I
mitatJSEray ttat ire hybridized to substantially equal number of nucleic acid molecules derived from both biological samples give a distinct «>mbiaed flwo»e^»nc« (Shalon et at; PCX poUksatitm WO95/35505). In a prefeimd embodiment, te labels ..
arje flnorescent markers with distinguishable emission spectra, such as Cy3 and CyS
Alter hybridizadoa, the microarray is washed to remove nonhyteidizBd raacteUj
i-.
acid molecules and complex formation between the bybridizable away elejaenfe and. tbe nucleic acid molecules is detected. Methods for detecting consptex formation aie
j
well known to those skilled in tiae art. In a preferred embodimeiit, the nucleic acid molecules ai» labeled with a fluorescent label and measdremeat of levels and pattens of fluorescence indicative of complex formation is aceoioplished by fluorescence microsoopy, psreferably confocal fluorescence microscopy.
In a differential hybridization experiment, nucleic acid molecules fkan two or more different biological samples are labeled with two or mote different fluorescent labels with different emission wavelengths. Fluorescent signals are detected separately with different photomultipliers set to detect specific wavelengtfis. The
i.
relative abundances/ expression levels of the nucleic acid molecules in two or more samples are obtained.
Typically, microarray fluorescence intensities cari be normalized to take into
i1,
account variations in hybridization intensities when monb than one microarray is used
i
under similar test conditions. In a preferred embuduneu% individual arrayed-sample
nucleic acid moUmh complex
intensities derived fan itfemaj aonrtfetiott controls contained on each
PgtegtMe Based Agggyj
The present iawntten {norths methods and jeagents for detecting and quantifying CDI63 polyp^tiebs. Hbese mefbtib indiide€nal|tical Uocbemkad methods such as electophoim^mass speetroseapy, cfeomatogi^Mc methods and the like, or various fattOTtioiogIfcal«eftods sach as radidtoiiKino^sayl^AX en* ymc-Iinked i««»inowrtetf assays |EUSAs), tentm0flaoim^it assays , blotting, affinity captro^ass spe^eiry, biological «*&* below and apparent to tto« erf«kffl1 jinniunoassays
The present invention, ato provides «hods for deieetiw of O>163 polypeptides employing one or mom AbV CB163 antibody agents 0*,, iaimmoassays). As used herein, an immunoass^r |s m assay tel mffim an antibody {as broadly defined hoe and specifically includes fet0neirts, cWmeras and^fiiertfcdkg agenfe) tet specifically biods a OD163 polypeptide orepltope,
A number of well-established immanolo^cal Wading assayforaoats for the practice oftheinve^onwBteown Usually the CDia gene product being assayed is detected directly or indirectly using a detectable label. The particular label or detectable group used in the assay is usually not a critical aspect of the invention, so long as it does not significantly interfere with the specific binding of the antibody or antibodies used in the assay. The label may be covalently attached to the capture agcni (e.g., an anti-CD 163 antibody), or may be attached to a third moiety, such as another antibody, that specifically binds to the CD 163 polypeptide.

The present invention provides methods and reagents for competitive and noncompetitive immunoassays for detecting CD163 polypeptides, Noncompetitive immunoassays are assays in which die amount of captured analyte (in this case CD 163) is directly measured. One such assay is a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on the CD163 polypeptide. See, e.g., Maddox et al., 1983, J. Exp. Med., 158:1211 for background information. In one "sandwich" assay, the capture agent (e.g., an anti-CD 163 antibody) is bound directly to a solid substrate where it is immobilized. These
i
immobilized antibodies then capture any CD 163 polypeptide present in the test sample. The CD163 polypeptide thus immobilized can then be labeled, i.e., by binding to a second anti-CD 163 antibody bearing a label. Alternatively, the secondCD163 antibody may lack a label, but be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived. The second antibody alternatively can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme- labeled streptavidin.
In competitive assays, the amount of CD 163 polypeptide present in the sample is measured indirectly by measuring the amount of an added (exogenous) CD163 polypeptide displaced (or competed away) from a capture agent (e.g., CD 163 antibody) by the CD 163 polypeptide present in the sample. A hapten inhibition assay is another example of a competitive assay. In mis assay CD163 polypeptide is immobilized on a solid substrate. A known amount of CD 163 antibody is added to the sample, and the sample is then contacted with the immobilized CD 163 polypeptide. in this case, the amount of anti- CD 163 antibody bound to the immobilized CD163 polypeptide is inversely proportional to the amount of CD163 polypeptide present in the sample. The amount of immobilized antibody may be detected by detecting either the immobilized fraction of antibody or the fraction of the antibody that remains in solution. In this aspect, detection may be direct, where the antibody is labeled, or indirect where the label is bound to a molecule that specifically binds to the antibody as described above. Other Antibody-based Assay Formats
The invention also provides reagents and methods for detecting and quantifying the presence of CD 163 polypeptide in the sample by using an immunoblot (Western blot)

format. Another immunoassay is the so-called "lateral flow chromatography," In a non-competitive version of lateral flow chromatography, a sample moves across a substrate by, e.g., capillary action, and encounters a mobile-labeled antibody that binds die analyte forming a conjugate. The conjugate then moves across the substrate and encounters an immobilized second antibody that binds the analyte. Thus, immobilized analyte is detected by detecting the labeled antibody. In a competitive version of lateral flow chromatography a labeled version of the analyte moves across the carrier and competes with unlabeled analyte for binding with the immobilized antibody. The greater the amount of the analyte hi the sample, the less the binding by labeled analyte and, therefore, the weaker the signal. See, e.g., May et al., U.S. Pat. No. 5,322,871 and Rosenstein, U.S. Pat. No. 5,591,645.
Depending upon the assay, various components, including the antigen, target antibody, or anti-cathepsin S antibody, may be bound to a solid surface or support (i.e., a substrate, membrane, or filter paper). Many methods for immobilizing biomolecules to a variety of solid surfaces are known in the art. Fbr instance, the solid surface may be a membrane (e.g., nitrocellulose), a microtiter dish (e.g., PVC, polypropylene, or polystyrene), a test tube (glass or plastic), a dipstick (e.g. glass, PVC, polypropylene, polystyrene, latex, and the like), a microcentrifuge tube, or a glass or plastic bead. The desired component may be covalently bound or noncovalently attached through nonspecific bonding.
; A wide variety of organic and inorganic polymers, both natural and synthetic may be employed as the material for die solid surface. Illustrative polymersinclude polyethylene, polypropylene, poly (4- methylbutene), polystyrene,polymethacrylate, polyethylene terephthalate), rayon, nylon, poly(vinylbutyrate), polyvinylidene difiuoride (PVDF), silicones, polyformaldehyde.cellulose, cellulose acetate, nitrocellulose, and the like. Other materials which may be employed, include paper, glasses, ceramics, metals, metalloids, semkonductive materials, cements or the like. In addition, substances that form gels, such as proteins (e. g., gelatins), lipopolysaccharides, silicates, agarose and polyacrylamides can be used. Polymers that form several aqueous phases, such as dextrans, polyalkylene glycols or surfactants, such as phospholipids, long chain (12-24 carbon atoms) alkyl ammonium salts and the like are also suitable. Where the solid surface is porous, various pore sizes may be employed depending upon the nature of the system.

The mass of a molecule frequently can be used as an identifier of the molecule. Therefore, methods of mass spectrometry can be used to identify a protein analyte. Mass spectrometers can measure mass by determining the time required for an ionized analyte to travel down a flight tube and to be detected by an ion detector. One method of mass spectrometry for proteins is matrix-assisted laser desorption ionization mass spectrometry ("MALDI"). In MALDI the analyte is mixed with an energy absorbing matrix material that absorbs energy of the wavelength of a laser and placed on the surface of a probe. Upon striking the matrix with the laser, the analyte is, desorbed from the probe surface, ionized, and detected by the ion detector. See, for example, Hillenkamp et al., U.S. Pat No. 5,118,937.
Other methods of mass spectrometry for proteins are described in Hutchens and Yip, U.S. Pat. No. 5,719,060. In one such method referred to as Surfaces Enhanced for Affinity Capture ("SEAC") a solid phase affinity reagent that binds the analyte specifically or non-specifically, such as an antibody or a metal ion, is used to separate the analyte from other materials in a sample. Then Hie captured analyte is
desorbed from the solid phase by, e.g., laser energy, ionized, and detected by the
detector.
Nucleic Acids of the Invention
The examples disclose our discovery of several novel CD163 polynucleotides. The invention includes these novel CD163 polynucleotides. The present invention provides several isolated novel polynucleotides (e.g., DNA sequences and RNA transcripts, both sense and complementary antisense strands, both single and double-stranded, including splice variants thereof, which encode novel CD 163 polypeptides. We report herein isolated novel polynucleotides which encode porcine, murine, human, canine, and african green monkey CD 163 polypeptides and which comprise the sequences set forth in SEQ ID NO: 1,5,12 13,22,23,25,26,30 31,33,35, 37, 39,41,43,45, and 47.
It should be recognized that by disclosing SEQ ID NOs: 1,5,12 13,22,23,25, 26,30 31,33,35,37,39,41,43,45, and 47 it provides one skilled in the art a multitude of methods of obtaining these sequences. By way of example, it would be possible to generate probes from the sequence disclosed in SEQ ID NOs: 1,5,12 13, 22,23,25,26,30 31,33,35,37,39,41,43,45, and 47 and screen porcine, murine,

human, canine, and afiicattfreenmoakeycDNAor^enomic libraries and.thereby, obtain the entire SEQ ID NO: 1,3,5,12,13,17,18,22,23,25,26,30,31,33,35,37, 39,41,43,45 and 47, or its genomic equivalent Sambrook, et al., (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989). Also by way of example, one skilled in the art would immediately recognize that given the sequence disclosed in SEQ ID NO: 5,12 13,22, 23,25,26,30 31,33,35,37,39,41,43,45, and 47it is men possible to generate the
• i
appropriate primers for PCR amplification to obtain the entire sequence represented by these sequences. (See e.g., PCR Technology, H. A. Erlich, ed., Stockton Press, New York, 1989; PCR Protocols: A Guide to Methods and Applications, M. A. Innis, David H. Gelfand, John J. Sninsky, and Thomas J. White, eds., Academic Press, Inc., New York, 1990.)
DNA polynucleotides of the invention include cDNA, and DNA that has been chemically synthesized in whole or in part and is also intended to include allelic variants thereof. Allelic variants are modified forms of a wild type gene sequence, the modification resulting from recombination during chromosomal segregation, or exposure to conditions which give rise to genetic mutation. Allelic variants, like wild type genes, are naturally occurring sequences (as opposed to non-naturally occurring variants which arise from in vitro manipulation).
DNA sequences encoding the novel CD 163 polypeptides are set forth in SEQ ID NOs: 5,12 13,22,23,25,26,30 31,33,35,37,39,41,43,45, and 47. The worker of skill in the art will readily appreciate that the DNA of the invention comprises a double stranded molecule, for example the molecule having the sequence set forth in SEQ ID NO: 5,12 13,22,23,25,26,30 31,33,35,37,39,41,43,45, and'
47 along with the complementary molecule (the "non-coding strand" or
"complement*') having a sequence deducible from the sequence of SEQ ID NOs: 5,12
13,22,23,25,26,30 31,33,35,37,39,41,43,45, and 47 according to Watson-
Crick base pairing rules for DNA. Also contemplated by the invention are other
polynucleotides encoding for the porcine, murine and african green monkey
CD163polypeptides of SEQ ID NO: 2,14,24,27 and 32,34,36,38,40,42,44,46,
48 which differ in sequence from the polynucleotide of SEQ ID NOs: 1,3,5,12,13,
17,18,22,23,25,26, 30,31,33,35,37,39,41,43,45 and 47 by virtue of the well-
known degeneracy of the universal genetic code, as is well known in the art. The

present invention, therefore, contemplates mpse,Qttier DNA aud.RNA molecules that, on expression, encode the polypeptides of SEQ ID NO: 2,14,24,27 and 32. Having identified the amino acid residue sequence encoded the porcine CD163 polypeptide, and with the knowledge of all triplet codons for each particular amino acid residue, it is possible to describe all such encoding RNA and DNA sequences. DNA and RNA molecules other than those specifically disclosed herein characterized simply by a change in a codon for a particular amino acid, are, therefore, within the scope of this
i
invention.
A table of amino acids and their representative abbreviations, symbols and codons is set forth below in the following Table 4.
Table 4

taninoacid Abbrev. Symbol CodonCs)
Alanine Ala A OCA GCC SCO CX^J
rysteine Cvs C [JGC UGU
Aspartic acid Asp D SAC 3AU
jlutamic acid Gin E 3AA SAG
ftenylalanine Phe F UUC uuu
jlycine Gly G 3GA 3GC 3GG 3GU
iistidine His H :AC ^U
[soleucine He I AUA AUC AUU
-ysine - Lys ' K AAA AAG ...
.eucine Leu L UUA . JUG ^JA :uc :uo suu
tlethionine Met M AUG
Asparagine Asn ' N AAC AAU
'roline Pro P CCA ccc CCG ecu
jlutamine Gin Q CAA , CAG
Arginine Arg R AGA AGG CGA CGC CXK3 OGU
Serine Ser S AGC AGU , UCA ucc UCG ircu
rhreonine Thr T ACA ACC ACG ACU
Valine Val V 3UA QUC GUG 3UU
Ityptdphan Tip W JGG '
Fyrosine Tyr y [JAC LIAU (
As is well known in the art, codons constitute triplet sequences of nucteotides in mRNA and their corresponding cDNA molecules. Codons are characterized by the base uracil (U) when present in an mRNA molecule but are characterized by thebase thymidine (T) when present in DNA. A simple change in a codon for the same amino acid residue within a polynucfeotide will not change the sequence or structure of the encoded polypeptide. It is apparent that when a phrase stating that a particular 3 nucleotide sequence "encode(s)" any particular amino acid, the ordinarily skilled artisan would recognize that the table above provides a means of identifying the particular nucleotides at issue. By way of example, if a particular three-nudeotide

are ACA, ACG, ACC and ACU {ACT if in DNA).
The invention includes therefore, an isolated polynudeotide comprising:
(a) a susCD163vl polynucleotide sequence set forth in SEQ ID NOs: 1, and 5
(b) a polynudeotide that encodes a polypeptide that has at least 70%, 71%, 72%, 73%,
74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 95%, 96% 97% 98%, or 99%, identity
and/or similarity to a polypeptide set forth in SEQ ID NO: 2
(c) a polynudeotide encoding a polypeptide of SEQ ID: 2, ,
(d) a polynucleotide which is the complement of any of (a), (b) or (c).
The invention also includes an isolated polynucleotide comprising:
(a) a susCD163v2 polynucleotide sequence set forth in SEQ ID NOs: 12 or 13
(b) a polynucleotide mat encodes a polypeptide that has at least 99%, identity and/or
similarity to a polypeptide set forth hi SEQ ID NO: 14
(c) a polynucleotide encoding a polypeptide of SEQ ID: 14,
(d) a polynucleotide which is the complement of any of (a), (b) or (c).
The invention also includes an isolated polynucleotide comprising:
(a) a murine CD63v2 polynucleotide sequence set forth in SEQ ID NOs: 22 or 23
(b) a polynucleotide encoding a polypeptide of SEQ ID: 24,
(c) a polynucleotide which is the complement of any of (a) or (b).
The invention also includes an isolated polynucleotide comprising:
(a) a murine CD163v3 polynucleotide sequence set forth in SEQ ID NOs: 25 or 26
(b) a polynudeotide that encodes a polypeptide that has at least 96% 97% 98%, or 99%
identity and/or similarity to a polypeptide set forth in SEQ ID NO: 27
(c) a polynucleotide encoding a polypeptide of SEQ ID: 27,
(d) a polynudeotide which is the complement of any of (a), The invention also includes an isolated polynucleotide comprising:
(a) an african green monkey CD163v2 polynudeotide sequence set forth in SEQ ID
NOs: 30 or 31
(b) a polynucleotide that encodes a polypeptide that has at least 98% or 99% identity
and/or similarity to a polypeptide set forth in SEQ ID NO: 32
(c) a polynucleotide encoding a polypeptide of SEQ ID: 32,
(d) a polynucleotide which is the complement of any of (a), (b) or(c).

... The invention also Includes an isolated polynucleotide comprising:
(a) a polynucleotide sequence set forth in SEQ ID NO: 33
(b) a polynucleotide that encodes a polypeptide that has at least 95% 96%, 97%, 98% or
99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 34
(c) a polynucleotide encoding a polypeptide of SEQ ID: 34,
(d) a polynucleotide which is the complement of any of (a), (b) or {c).
The invention also includes an isolated polynucleotide comprising:
(a) a polynucleotide sequence set forth in SEQ ID NO: 35
(b) a polynucleotide mat encodes a polypeptide that has at feast 95% 96%,, 97%, 98% or
99% identity and/or similarity to a polypeptide set forth in SEQ 3D NO: 36
(c) a polynucleotide encoding a polypeptide of SEQ ID: 36,
(d) a polynucleotide which is the complement of any of (a), (b) or The invention also includes an isolated polynucleotide comprising:
(a) a polynucleotide sequence set forth in SEQ ID NO: 37
(b) a polynucleotide that encodes a polypeptide that has at feast 95% 96%, 97%, 98% or
99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 38
(c) a polynucleotide encoding a polypeptide of SEQ ID: 38,
(d) a polynucleotide which is the complement of any of The invention also includes an isolated polynudebtide comprising:
(a) a polynucleotide sequence set forth in SEQ ID NO: 39
(b) a polynucleotide that encodes a polypeptide that has at feast 95% 96%, 97%, 98% or
99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 40
(c) a polynucleotide encoding a polypeptide of SEQ ID: 40,
(d) a polynucleotide which is the complement of any of (a), (b) or (c).
The invention also includes an isolated polynucleotide comprising:
(a) a polynucleotide sequence set forth in SEQ ID NO: 41
(b) a polynucleotide that encodes a polypeptide that has at feast 95% 96%, 97%, 98% or
99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 42
(c) a polynucleotide encoding a polypeptide of SEQ ID: 42,
(d) a polynucleotide which is the complement of any of (a), (b) or (c).
The invention also includes an isolated polynucleotide comprising: (a) a polynucleotide sequence set forth in SEQ ID NO: 43

, (b) a polynucleotide that encodes a polypeptide that has at least 95% 96%, 97%, 98% 01 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 44
(c) a polymicleotide encoding a polypeptide of SEQ ID: 44,
(d) a polynucleotide which is the complement of any of (a), (b) or (c).
The invention also includes an isolated polynucleotide comprising:
(a) a polynucleotide sequence set forth in SEQ ID NO: 45
(b) a polynucleotide that encodes a polypeptide that has at least 90%f 91%, 92%, 93%,
94%, 95% 96%, 97%, 98% or 99% identity and/or similarity to a polypeptide set form
in SEQ ID NO: 46
(c) a polynucleotide encoding a polypeptide of SEQ ID: 46,
(d) a polynucleotide which is the complement of any of (a), (b) or The invention also includes an isolated polynucleotide comprising:
(a) a polynucleotide sequence set forth in SEQ ID NO: 47
(b) a polynucleotide that encodes a polypeptide that has at least 90%, 91%, 92%, 93%,
94%, 95% 96%, 97%, 98% or 99% identity and/or similarity to a polypeptide set form
in SEQ ID NO: 48
(c) a polynucleotide encoding a polypeptide of SEQ ID: 49,
(d) a polynucleotide which is the complement of any of (a), (b) or (c).
The polynucleotide sequence information provided by the invention makes possible large-scale expression of the encoded polypeptide by techniques well known and routinely practiced in the art Polynucteotides of the invention also permit identification and isolation of polynucfeotides encoding related porcine CD163vl polypeptides, such as human allelic variants and species homologs, by well-known techniques including Southern and/or Northern hybridization, and the polymerase chain reaction (PCR).
Knowledge of the sequence of a any of the CD 163 sequences disclosed herein also makes possible through use of Southern hybridization or polymerase chain reaction (PCR), the identification of geeomic DNA sequences encoding CD 163 regulatory sequences, such as promoters, operators, enhancers, repressers, and the like.
As noted in the section above entitled "Assays of the Invention" polynucteotides of the invention are also useful in hybridization assays to detect the capacity of cells to express CD 163, or to measure levels of CD 163 expression.

Polynucjeptides of the ^invention may,also be the bajris:fbr diagnostic methods useful for determining the susceptibility of an animal to virus infection 'as described above.
The disclosure herein of the full-length polynucleotides encoding a CD163polypeptide makes readily available to the worker of ordinary skill in the art fragments of the full length polynucleotide. The invention therefore provides unique fragments of the CD163 encoding polynucleotides comprising at least 15 through the length of the full-length sequence (including each and every integer value between) consecutive nucleotides of a polvmicleotide encoding a CD163 disclosed herein. Because polynucleotides of the invention (including fragments) comprise sequences unique tothe particular CD163-encoding polynudeotide sequence, they therefore would hybridize under highly stringent or moderately stringent nditions only (i.e., "specifically") to polynucleotides encoding the various CDI63 polypeptides. Sequences unique to polynucleotides of the invention are recognizable through sequence comparison to other known polynucleotides, and can be identified through use of alignment programs routinely utilized in the art, e.g., those made available in public sequence databases. Such sequences also are recognizable from Southern hybridization analyses to determine the number of fragments of genomic DNA to which a polynudeotide will hybridize. Polynucleotides of the invention can be labeled in a manner that permits their detection, including radioactive, fluorescent, and enzymatic labeling.
One or more unique fragment polynucleotides (or other CD 163 polynucleotides as discussed above) can be included in kits that are used to detect the presence of a polynudeotide encoding for CD 163, or used to detect variations in a polynudeotide sequence encoding for CD 163.Also made available by the invention are anti-sense polynucleotides that recognize and hybridize to polynucleotides encoding CD163. Full length and fragment anti-sense polynucleotides are provided. Fragment anti-sense molecules of the invention include (i) those that specifically recognize and hybridize to the CD163 variants disclosed herein (as determined by sequence comparison of DNA encoding CD 163s to DNA encoding other known molecules). Identification of sequences unique to the novel CD163-encoding polynucleotides can be deduced through the use of any publicly available sequence database, and/or through the use of commercially available sequence comparison programs. The uniqueness of selected sequences in an entire genome can be further

verified by hybridization analyses. After identification of thedesired sequences, isolation through restriction digestion or amplification using any of the various polymerase chain reaction techniques well known in the art can be performed. Anti-sense polynucleotides are particularly relevant to regulating expression of CD163 by those cells expressing CD163 mRNA.
Antisense nucleic acids (preferably 10 to 20 base pair oligonucleotides) capable of specifically binding to CD163 expression control sequences or CD 163 RNA are introduced into cells (e.g., by a viral vector or colloidal dispersion system such as a liposome). The antisense nucleic acid binds to the porcine CD163 target nucleotide sequence in the cell and prevents transcription or translation of the target sequence. Fhosphorbthioate and methylphosphonate antisense oligonucleotides are specifically contemplated for therapeutic use by the invention. Hie antisense oligonucleotides may be further modified by poly-L-lysine, transferrin polylysine, or cholesterol moieties at their 5' end. Suppression of porcine CD163 expression at either the transcriptional or translational level is useful to generate cellular or animal models for diseases characterized by aberrant porcine CD163 expression or as a
therapeutic modality.
As noted above in more detail, the nucleic acids of the invention include vectors comprising a polynucleotide of the invention. Such vectors are useful, e.g., for amplifying the polynucleotides in host cells to create useful quantities thereof. In other embodiments, the vector is an expression vector wherein the polynucleotide of the invention is operatively linked to a polynucleotide comprising an expression control sequence. Such vectors are useful for recombinant production of polypeptides of the invention.
I Also as noted above the invention provides host cells that are transformed or transfected (stably or transiently) with polynucleotides of the invention or vectors of the invention. As stated above, such host cells are useful for the production of virus and the production of vaccines.
The invention also provides isolated GDI 63 polypeptides encoded by a novel polynucleotide of the invention.

Polypeptides of the Invention
The examples disclose our discovery of several novel CDi63 polypeptides. The invention includes these novel CD163 polypeptide which are set forth in SEQ ID NOs: 2,14,19,24.27,32,34,36,38,40,42,44,46, and 48.
The invention includes therefore, an isolated polynucleotide comprising a susCD163vl polypetide with the sequence set forth in SEQ ID NO: 2.
The invention also includes a polypeptide that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 95%, 96% 97% 98%, or 99%, identity and/or similarity to a polypeptide set forth in SEQ ID NO:- 2.
The invention includes therefore, an isolated polynucleotide comprising a susCD163v2 polypetide with the sequence set forth in SEQ ID NO: 14.
The invention also includes a polypeptide that has at feast 99%, identity and or similarity to a sus CD163v2 polypeptide set forth in SEQ ID NO: 14.
The invention also includes a murine CD163v2 polypeptide having the sequence set forth in SEQ ID: 24.
The invention also includes a murine CD163v3 polypeptide having the sequence set forth hi SEQ ID: 27.
The invention also includes at least a polypeptide having 96% 97% 98%, or 99%, identity and/or similarity to a polypeptide set forth in SEQ ID NO: 27.
The invention also includes a polypeptide having the sequence set forth in SEQ ID: 32.
The invention also includes a polypeptide that has at least 98% or 99%, identity and/or similarity to a polypeptide set forth in SEQ ID NO: 32. The invention also includes a polypeptide having the sequence set forth in SEQ ID: 34.
The invention also includes a polypeptide mat has at least 95%, 96%, 97%, 98%, 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 34. The invention also includes a polypeptide having the sequence set forth in SEQ ID: 36.
The invention also includes a polypeptide that has at feast 95%, 96%, 97%, 98%, 99% identity and/or similarity to a polypeptide set form in SEQ ID NO: 36.

JThe invention also includes a polypeptide haying the sequence set forth in SEQ ID:
38.
The invention also includes a polypeptide that has at least 95%, 96%, 97%, 98%, 99%
identity and/or similarity to a polypeptide set forth in SEQ ID NO: 39.
The invention also 'includes a polypeptide having the sequence set forth in SEQ ID:
40.
The invention also includes a polypeptide that has at least 95%, 96%, 97%, 98%, 99%
identity and/or similarity to a polypeptide set forth in SEQ ID NO: 40.
The invention also includes a polypeptide having the sequence set forth hi SEQ ID:
42.
The invention also includes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%
95%, 96%, 97%, 98%, 99% identity and/or similarity to a polypeptide set forth in SEQ
ID NO: 42.
The invention also includes a polypeptide having the sequence set forth hi SEQ ID:
44.
The invention also includes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%
95%, 96%, 97%, 98%, 99% identity and/or similarity to a polypeptide set forth in SEQ
ID NO: 44.
The invention also includes a polypeptide having the sequence set forth hi SEQ ID:
46.
The invention also includes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%
95%, 96%, 97%, 98%, 99% identity and/or similarity to a polypeptide set forth hi SEQ
ID NO: 46.
The invention also includes a polypeptide having the sequence set forth in SEQ ID:
48.
The invention also includes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%
95%, 96%, 97%, 98%, 99% identity and/or similarity to a polypeptide set forth hi SEQ
ID NO: 48.
Polypeptides of the invention may be isolated from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving host cells of the invention. Use of mammalian host cells is expected to provide for such post-translational modifications (e.g., glycosylation, truncation,

lipidation, and phosphorylation) as may bejieededto confer optimal biological activity on recombinant expression products of the invention. Glycosylated and non-glycosylated forms of the novel CD163 polypeptides are embraced.
Overexpression in eukaryotic and prokaryotic hosts as described above facilitates the isolation of CD 163 polypeptides. The invention therefore includes isolated CD163 polypeptides as set out in SBQ ID NOs: 2,14,19,24,27 32,34,36, 38,40,42,44,46,48 and variants and conservative amino acid substitutions therein including labeled and tagged polypeptides.
The invention includes novel CD163 polypeptides that are "labeled". The term "labeled" is used herein to refer to the conjugating or covalent bonding of any suitable detectable group, including enzymes (e.g., horseradish peroxidase, beta -glucuronidase, alkaline phosphatase, and beta-D-galactosidase), fluorescent labels (e.g., fluorescein, luciferase), and radiolabels (e.g., 14C, 125I, *H, 32P, and ^S) to the compound being labeled. Techniques for labeling various compounds, including proteins, peptides, and antibodies, are well known. See, e.g., Morrison, Methods in Enzymology 32b, 103 (1974); Syvanen«t al., J. Biol. Chem. 284,3762 (1973); Bolton and Hunter, Biochem. J. 133, S29 (1973). The termed labeled may also , encompass a polypeptide which has covalently attached an amino acid tag as discussed below.
In addition, the novel CD 163 polypeptides of the invention may be indirectly labeled. This involves the covalent addition of a moiety to the polypeptide and subsequent coupling of the added moiety to a label or labeled compound that exhibits specific binding to the added moiety. Possibilities for indirect labeling include biotinylation of the peptide followed by binding to avidin coupled to one of the above label groups. Another example would be incubating a radiolabekd antibody specific for a histidine tag with a GDI 63s polypeptide comprising a polyhistidine tag. The net effect is to bind the radioactive antibody to the polypeptide because of the considerable affinity of the antibody for the tag.
The invention also embraces variants (or analogs) of the novel CD 163 protein. In one example, insertion variants are provided wherein one or more amino acid residues supplement a novel CD 163 amino acid sequence. Insertions may be located at either or both tennini of the protein, or may be positioned within internal regions of the novel CD 163 protein amino acid sequence. Lasertional variants with additional

residues at either or both termini can include for example, fusion proteins and proteins including ammo acid tags or labels. Insertion variants include novel CD163 polypeptides wherein one or more amino acid residues are added to a CD163 acid sequence, or to a biologically active fragment thereof.
msertional variants therfore can also include fusion proteins wherein the amino and/or carboxy termini of the novel CD163 polypeptide is fused to another polypeptide. Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the influenza HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. CeU. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9,3C7,6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and CellularBiology, 5:3610-3616 (1985)], and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include the Hag -peptide {Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)]; an alpha -tubulin epitope peptide {Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the 17 gene 10 protein peptide tag [Lutz-Freyennuth et al., Proc. Nafl. Acad. Sci. USA, 87:6393-6397(1990)]. m addition, the CD 163 polypeptide can be tagged with enzymatic proteins such as peroxidase and alkaline phosphatase.
In another aspect, the invention provides deletion variants wherein one or more amino acid residues in a novel CD163 polypeptide is removed. Deletions can be effected at one or both termini of the novel CD163 polypeptide, or with removal of one or more residues within the novel CD163 amino acid sequence. Deletion variants, therefore, include all fragments of the novel CD163 polypeptide.
CD163 polypeptides contain a transmembrane or membrane anchor region. It should be recognized that such transmembrane domains are useful when expressed in the context of a heterologous protein to aid in the targeting of the heterologous protein to membranes. It should also be recognized that it may be advantageous to delete some transmembrane domains to enhance the purification or solubility of the protein. Transmembrane deleted variants of CD 163 and polynucleotides encoding them are of potential value as antiviral therapeutics. Such variants are specifically disclosed here as SEQIDNOs: 37-40.

polypetides, that is, polypeptides that vary from the reference sequence by conservative amino acid substitutions,
Exemplary conservative substitutions are set out in Tables 1,2 and 3 in the section above entitled "Definitions".
In those situations where it is preferable to partially or completely isolate die novel CD163 polypeptides, purification can be accomplished using standard methods well known to the skilled artisan. Such methods include, without limitation,
i
separation by electrophoresis followed by electroelution, various types of chromatography (immunoaffinity, molecular sieve, and/or ion exchange), and/or high pressure liquid chromatography. In some cases, it may be preferable to use more than one of these methods for complete purification.
Purification of novel CD163 polypeptides can be accomplished using a variety of techniques. If the polypeptide has been synthesized such mat it contains a tag such as Hexahistidine (CD163 /hexaffis) or other small peptide such as FLAG (Eastman Kodak Co., New Haven, Conn.) or myc {Invitrogen, Carlsbad, Calif.) at either its carboxyl or amino terminus, it may essentially be purified in a one-step process by passing the solution through an affinity column where the column matrix has a high affinity for the tag or for the polypeptide directly (i.e., a monoclonal antibody specifically recognizing CD163). For example, polyhistidine binds with great affinity and specificity to nickel, thus an affinity column of nickel (such as the Qiagen Registered TM nickel columns) can be used for purification of CD163 /polyHis. (See for example, Ausubel et al., eds., Current Protocols in Molecular Biology, Section 10.11.8, John Wiley & Sons, New York [1993]).
Even if the novel CD163 polypeptide is prepared without a label or tag to facilitate purification, the novel CD163 of the invention may be purified by immunoaffinity chromatography. To accomplish this, antibodies specific for CD 163 polypeptides must be prepared by means well known in the ait
Antibodies generated against the novel CD 163 polypeptides of the invention can be obtained by administering the polypeptides or epitope-bearing fragments, analogues, or cells to an animal, preferably a non-human, using routine protocols. For preparation of monoclonal antibodies, any technique known in the art that provides antibodies produced by continuous cell line cultures can be used. Examples include

various techniqqes^suchas thpse ijj.Kohler,.G. and Milstein, C.,, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., pg. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985).
Where the novel GDI 63 polypeptides are prepared without a tag attached, and no antibodies are available, other well-known procedures for purification can be used. Such procedures include, without limitation, ion exchange chromatography, molecular sieve chromatography, HPLC, native gel electrophoresis in combination
i
with gel elution, and preparative isoelectric focusing ("fcoprime"machine/technique, Hoefer Scientific). In some cases, two or move of these techniques may be combined to achieve increased purity.
ft should beunderstood that the definition of polypeptides of the invention is intended to include polypeptides bearing modifications other than insertion; deletion, or substitution of amino acid residues. By way of example, the modifications may be covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic, and inorganic moieties. Antibodies
Also comprehended by the present invention are antibodies (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds which include CDR sequences which specifically recognize a polypeptide of the invention) specific for novel CD 163 or fragments thereof.
The term "specific for," when used to describe antibodies of the invention, indicates that the variable regions of the antibodies of the invention recognize and bind CD163s polypeptides exclusively {i.e., able to distinguish CD 163s polypeptides from other known polypeptides by virtue of measurable differences in binding affinity, despite the possible existence of localized sequence identity, homology, or similarity between the novel CD 163 and such polypeptides). It will be understood that specific antibodies may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the molecule. Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive

discussion of such assays, see Harlow et al. (Eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1988), Chapter 6. Antibodies mat recognize and bind fragments of the CD163s polypeptides of the invention are also contemplated, provided mat the antibodies are, first and foremost, specific for novel CD163 polypeptides. Antibodies of the invention can be produced using any method well known and routinely practiced in the art Non-human antibodies may be humanized by any methods known in the art. In one method, the
i
non-human CDRs are inserted into a human antibody or consensus antibody framework sequence. Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity.
Antibodies of the invention are useful for, diagnostic purposes to detect or quantitate GDI 63s, as well as purification of CD163s. Kits comprising an antibody of the invention for any of the purposes described herein are also^omprehended. In general, a kit of the invention also includes a control antigen for which the antibody is immunospecific
The present invention is further illustrated, but not limited, by the following
examples. :...„..
Example 1: Transient transfection with porcine CD163 confers permissivity to PRRS virus infection to a non-permissive cell line. Total mRNA from primary porcine alveolar macrophage cells was used to construct a cDNA library in me plasmid pCMV-Sport6.1 (Invitrogen), with the cDNA cloned between the EcoKV and Notl sites. A member of this library, when isolated and transiently transfected into the BHK-21 (baby hamster kidney) cell line, conferred a PRRS-permissive phenotype. Cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 5% fetal bovine serum (FBS) in a 5% CO2 atmosphere at 37°C. Cell cultures were transiently transfected using 10.0 uL of Lipofectamine 2000 (Invitrogen) and 2.0ugof plasmid. A duplicate monolayer was transfected with negative control plasmid pPAMB. This plasmid is pCMV-Sport6.1 lacking an insert Transfection efficiency was monitored with a plasmid expressing green fluorescent protein (GFP). Approximately 24 hours post-transfection, monolayers were infected with-either North American (isolate P129) or European (isolate 96V198) genotypes of PRRS virus. For detection of PRRS replication, the monolayers were fixed using 80% acetone approximately 24 hours post-infection and incubated for approximately 1 hour with

FITC-conjugated monoclonal antibody SDOW17 (Rural Technologies toe.). This monoclonal antibody is specific for PRRS viral nucleocapsid expressed from open reading frame 7. A Nikon TE 300 inverted fluorescent microscope with a lOx objective was used to photograph a monolayer containing FTTC positive cells and a negative control monolayer.
It was confirmed that transfected cells became permissive to bom the North American (isolate P129) and European (isolate 96V198) genotypes of PRRS V. Expression of viral genes could be detected in many of the transfected BHK cells, and progeny virus was readily detectable in the supernatant Control transfections using vector without insert or irrelevant plasmids did not confer pennissivity.
Sequencing of the insert in the functional plasmid, using the Big Dye Terminator Version 1.0 Sequence Reaction kit (Applied Biosystems, Foster City, CA) and the Applied Biosystems 3730 DNA Analyzer (Applied Biosystems), revealed a gene that was highly homologous to the published porcine CD163 gene^DNA (Oenbank accession number AJ311716). The cDNA we identified contained additional 5* and 3' untranslated regions relative to AJ311716, and the open reading .frame differed in three ways: (1) a 738 bp internal deletion near the 5' end, (2) a 15 bp extension of the 5* end to an upstream ATG codon, and (3) sixteen nucleotide changes predicted to cause 10 amino acid changes. Nucleotide sequence identity between the sequences was 99.4%. Alignments of the newly discovered porcine CD 163 sequence with the previously reported sequence AJ311716 are shown in figures 1 and 2. The novel porcine CD 163 variant was designated "susCD163vl".



SEQUENCE
gtaataatac aagaagattt aaatgggcat aaaaccttgg aatggacaaa ctcagaatgg €0
tgctaeatga aaactctgga tetgcagaec tgaaactgag agtggtagat ggagtcactg 120
aatgtteagg aagattggaa gtgaaattcc aaggagaatg gggmacaatc tgtgatgatg 180
gctgggatag tgatgatgcc gctgtggcat gtaagcaact gggatgtcca actgctgtca 240
ctgccattgg tcgagttaac gccagtgagg gaactggaca eatttggctt gacagtgttt 300
ettgccatgg acacgagtct gctctctggc agtgtagaca ccatgaatgg ggaaagcatt 360
attgcaatca taatgaagat gctggtgtga catgttctga tggatcagat ctggaactga 420
gacttaaagg tggaggcagc cactgtgctg ggacagtgga ggtggaaatt cagaaactgg 460
taggaaaagt gtgtgataga agctggggac tgaaagaagc tgatgtggtt tgcaggeagc S40
tgggatgtgg atctgcactc aaaacatcat atcaagttta ttccaaaacc aaggcaacaa 600
acacatggcb gtttgtaagc agctgtaatg gaaatgaaac ttctctttgg gactgcaaga fi€0
attggcagtg gggtggactt agttgtgatc actatgacga agocaaaatt acctgctcag 720
cccacaggaa acccaggctg gttggagggg acattccctg ctctggtogt gttgaagtac 760

ID NO
SEQID NO:5

aacatggaga cacgtggggc accgtctgtg attetgaott etetctggag gcggccagcg eio"
tgcegtgcag ggaaetaoag tgcggoaotg tggtttooot octgggggga gotcactttg 900
gagaaggaag tggacagate tgggctgaag aattccagtg tgaggggeae sagtcccacc 960
tttoaototg ccoagtagoa ceeegeoetg aegggaeatg tagcoaoago agggacgtcg ioao
gegtagtetg cteaagatao aoapaaatee gcttggtgaa tggeaagacc ocatgtgaag 1080
gaagagtgga gctcaacatt cttgggteet gggggtecet ctgcaactet cactgggaca 1140
tggaagatgc ecatgtttta tgccagoage ttaaatgtgg agttgocott tctatcccgg 1200
gaggagcaoc ttttgggaaa ggaagtgagc aggtctggag goacatgttt caetgeaetg 1260
ggactgagaa goaeatggga gattgttccg bcactgctet gggegeatoa ctctgttctt 1320
eagggeaagt ggoetctgta atetgctcag ggaaccagag teagacaeta tocccgtgca 1380
atteatcate ctoggaccca tcaagcteta ttatttcaga agaaaatggt gttgeetgea 1440
tagggagtgg tcaaettcgc ctggtegatg gaggtggtcg ttgtgetggg agagtagagg 1SOO
tctateatga gggetectgg ggcaeeatct gtgatgacag ctgggacctg aatgatgeec 1560
atgtggtgtg caaacagetg agetgtggat gggecattaa tgccactggt tctgctcatt 1€20
ttggggaagg aacagggeec atttggetgg atgagataaa otgtaatgga aaagaatctc 1660
atatttggca atgceactca catggttggg ggcggeaeaa ttgeaggcat aaggaggatg 1740
eaggagtcat etgctcggag ttcatgtcte tcagactgat cagtgaaaac agcagagaga 1600
cctgtgcagg gegcctggaa gttttttaea aeggagcttg gggcagcgtt ggcaagaata 1860
gcatgtetcc agccacagtg ggggtggtat gcaggeagct gggctgtgca gaeagagggg 1920
acatcagccc tgcatcttca gacaagacag tgtccaggca catgtgggtg gacaatgttc 1980
agtgtcctaa aggacctgae accctatggc agtgcccatc atctceatgg aagaagagac 2040
tggccagccc ctcagaggag acatggatca catgtgccaa tsaaaataaga ctecaagaag 2100
gaaacactaa ttgttctgga cgtgtggaga tctggtaegg aggttcctgg ggcactgtgt 21-60
gtgacgaetc ctgggacctt gaagatgctc aggtggtgtg ccgacagctg ggctgtggct 2220
cagctttgga ggcaggaaaa gaggccgcat ttggecaggg gactgggocc atatggctca 2280
atgaagtgaa gtgcaagggg aatgaaacct ccttgtggga ttgtcctgcc agateetggg 2340
gccacagtga ctgtggacac aaggaggatg ctgctgtgac gtgtteagaa attgcaaaga . • 2400
gcegagaatc cctacatgcc acaggtcgct catcttttgt tgcacttgca atctttgggg 2460
tcattctgtt ggcctgtctc atcgcattce teatttggae tcegaagoga agaeagaggc 2520
agcggctcte agttttctca ggaggagaga attctgtcca tcaaatfccaa taccgggaga 2580
tgaattcttg cctgaaagca gatgaaacgg atatgctaaa tccctcagga gaocactetg 2640
aagtacaatg aaaaggaaaa tgggaattat aacctggtga gttcagcctt taagataoct 2700
tgatgaagac ctggactatt gaatgagcaa gaatctgcct ettacactga agattacaat 2760
aeagtcctct gtctcctggt attccaaaga ctgctgttga atttctaaaa aatagattgg 2620
tgaatgtgac tactcaaagt tgtatgtaag actttcaagg gcattaaeta aaaaagaata 2880
ttgctgaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2930

1MDKLEMVLHEHSGSADLKLR
1 atggacaaactcagaatggtgctacatgaaaactctggatctgcagacctgaaactgaga 21VVDGVTECSGRLEVKFQGEW 61 gtggtagatggagt«actgaatgttcaggaagattggaagtgaaattccaaggagaatgg 41GTICDDGWDSDDAAVACKQL 121 ggaacaatctgtgatgatggctgggatagtgatgatgecgctgtggcatgtaagcaactg
61GCPTAVTAIGRVNASEGTGH 181 ggatgtccaaetgctgtcactgccattggtcgagttaacgccagtgagggaactggacac
SlIHt-DSySCHGHSSALW-QCRH

SEQID
NO:1 and 2

241 atttggcttgacagtgtttcttgccatggacacgagtctgetctctggcagtgtagacac 101 H B W 0 K Ry C B H H B D A 0 V-T C 8 D 301 eatgaatggggaaagcattattgcaatcataatgaagatgctggtgtgacatgttctgat UIGBDIiELRIilCOaaSHCAOTVE 361 gflatcagatctggaactgagacttaaaggtggaggcagccactgtgctgggacagtogaQ lUVBXQX&vaKVCDKBWQliK B A 421 gtggaBatteagaaactggtaggaaaagtgtgtgatagaagctggggactgaaagaagct 161DVVCRQLOCOSA1.KTBYQ V if 481 gatgtggtttgcaggcagctgggatgtggatctgcactoaaaacatcatatcaagtttat 181 BKTICATNTWIiPVSSCSaWBT 541 tccaaaaccaaggoaacaaaeacatggctgtttgtaagcagctgtaatggaaatgaaact aOlSLKDCKKKQUGOLSCDHyDE 601 tctetttgggactgcaagaattggcagtggggtggaottagttgtgatcactatgacgaa 221AKXTCBAHRJCPRIiVQODX PC 661 gecaaaattacctgctcagcccacaggaaaoocaggctggttggaggggacatfcccctgc 241BaRVBVOHOPTirOTVCD8D P 721 tctggtcgtgttgaagtaeaacatggagacacgtggggcacogtctgtgattctgaettc 261 eLBAASVLCRBZiQCQTVvSIi 781 tctctggaggeggeoagegtgotgtgeagggaaotaeagtgoggcactgtggttteeato 281LOaAHFQBaSQQIIfABSPQC 841 ctggggggagetcactttggagaaggaagtggacagatctgggetgaagaattccagtgt 301BOHBSHLeLCPVAPRPDOTC 901 gaggggcacgagtcecaccttccactctgcecagtagcacccogccctgacgggacatgt 3218RBRDVGVVC6RYTQZRI.VH 961 agccacagcagggaogtcggcgtagtctgctcaagatacacacaaatcogcttggtgaat 3410 K T P C 10 R V B Ii H I L 0 S NO B I> 1021 ggcaagaccccatgtgaaggaagagtggagctcaacattcttgggtcotgggggtocctc
361CN6HWDMBDAHVLCQQLKCQ 1081 tgcaactctcactgggacatggBagatgcccatgttttatgccagcagcttaaatgtgga
381 V A L B I P OOA P FO K 0 B B Q VW R 1141 gttgccctttctatcccgggaggageaecttttgggaaaggaagtgagcaggtctggagg
401HHPHCTOTBKHMODC6VTAZi 1201 eacatgtttcactgcactgggactgagaageacatgggagattgttccgtcactgobetg
4210A8IiC8 SQQVASVICSONQB 1261 ggcgcatcactctgttcfctcagggeaagtggcctctgtaatctgctcagggaaccagagc
441QTItBFCRSS86DPS68ZI6B
1321 eagacactateoccgtgcaattcatcatcctcggaeceateaagetetattatttcagaa
461BNOVACZ06GQLRLVDOGGR
1381 gaaaatggtgttgcctgcatagggagtggtcaaettcgcctggtegatggaggtggtcgt
481CAORVBVYHBOBKGTICD08 1441 tgtgctgggagagtagaggtctatcatgagggctoctggggeaccatctgtgatgacagc
501 H D LNDAHVVCKO L S CGWA I N 1S01 tgggaectgaBtgatgeccatgtggtgtgcaaacagctgagctgtggaegggocattaat
521ATOSARPOBGTGPIWLDBIN 1561 gocactggttctgetcattttggggaaggaacagggcccatttggctggatgagataaac
S41CNGKESHIWCCHSHQHGRHN 1621 tgtaatggaaaagaatctcatatttggcaatgccactcacatggttgggggcggcaeaat
561CRHKEDAGVICS E F M 6 L RI. I 1681 tgcaggcataaggaggatgcaggagtcatctgctoggagttcatgtctctcagactgatc
SB18SN8RBTCAGRLBVFYNGAN 1741 agtgaaaacagcagagagacctgtgcagggcgcctggaagttttttacaacggagottgg
601G6VGKKSM8PATVGVVCROL 1801 ggcagcgttggcaagaatagcatgtctecagtwacagtgggggtggtatgcaggcagctg
621GCAORQOZBPA88DKTVSRH 1861 ggctgtgcagaeagaggggacatcagccctgcatctteagaeaagacagtgtccaggcac
641MWVDKVQCPKGPDTtWQCPfi 1921 atgtgggtggacaatgttcagtgtcotaaaggacetgacacectatggeagtgcecatca
6618PMXKRIiA8P8EETWZTCAN 1981 tcteeatggaagaagagactggccagecccteagaggagaeatggatcacatgtgocaac
681KIRLQBGHTNC6GRVBIKYG 2041 Baaataagacttcaagaaggaaaeaetaattgttctggacgtgtggagatctggtacgga
701GSWGTVCDDSWDLEDAQVVC 2101 ggttectggggcactgtgtgtgaogactcctgggaccttgaagatgctcBggtggtgtgc
721RQLGCGSALEAGKEAAFGQG 2161 cgacagctgggctgtggcteagctttggaggcaggaaaagaggccgcatttggccagggg
741TGPZNLNBVKCXGHET8LirD 2221 actgggcccatatggctcaatgaagtgaagtgcaaggggaatgaaaoctccttgtgggat
761CPAR6HGR6DCGBXEDAAVT 2281 tgtcctgccagatcctggggccacagtgactgtggacacaaggaggaegctgctgtgaag
781CSEZAK8RESIiBAT6RSSFV 2341 tgttcagaaattgcaaagagccgagaatcectacatgccacaggtcgctcatcttttgtt
eOlAIiAZFGVZIiIiACIiZAFIiZHT 2401 gcacttgcaatctttggggtcattctgttggcctgtctcatcgcattccccatttggact
821QKRRQRQRLSVFSGGENSVH 2461 cagaagcgaagaeagaggcagcggctctcagttttctcaggaggagagaattctgtccat
841QIQYRBMKSCI.KADETDMLN 2521 caaattcaataccgggagatgaattettgcetgaaagcagatgaaacggatatgctaaat
861 PSGDH8EVQ 2581 ccctcaggagaccactetgaagtacaa

foi BEtfaionrcKK NEDASVTCSD GSDLBLRLJCO OQSHCAOTVB VBZQKLVSXV'
151 CDRSHSLREA DWCRQWOO EALKTSyQW 6KTKMWTTO FVBSCNGHST
201 SLMDcnmQN tsascDBXDE AKITCSAHRK FKLVOODXPC SURVKVUHUD
251 W8TVCDSDF SLBAASVLCR SLCC3TWSI. MOAHPOEOB OQIWftEBPQC
301 EOHBSHLSLC PVAPRPWTC 8HSRDWBWC 6RWQIRLVN GKTFCBQRVB
351 UOXiQ0M98& CMBKHCMED* HVIiCOOLKOO VRLSIPOOAP FOKOSCOVMR
401 HMFRCTOTIK HMSDCSVTAL SASLCS80QV ASVXCSONQS QXIiSPC3l888
451 SDP886XX6B ENOVACHWQ OLUiVMOQR CMRVBVYRB OSHSTrCDPS
501 NDIADMIWC JCQLSCGWUS ATC8ARFSBO TOPIULDEIW CNSKB0RXNQ
551 CBSHONQRBir qtHXBCASVI CSBPM8LRII SBKBRETCM RLBVPVHSMf
601 3BVGKHSMBP ATVOWCRQIi CCM5RODISP ASBDKTV8RR MHVDHVQCPK
651 OPDTLUOCPS SPWOOUASP SBBTIflTCAK KlRLQBOJirH CBORVBINXO
701 SSNOTVCDDS NDLBMQWC RQLOOOSALB AGXBAATOQG TGPIWLNB71C
751 CXSHEISIMD CPARSWOHSD 06HKBDAXVT CSZIAKBRES LBHT8R8SIT
801 JX&zravzuj ACLXAnxNT QKRRQRQRI^ VPSOOENSVH QZQXRBNMSC
851 UCAPglPMLtf PSODHBEVO

N0:2

Example!: Construction of plasmid pCMVsusCD163vl
Construction of the plasmid pCMVsusCD163vl was perfonned as follows. The functional clone identified in the primary porcine macrophage cDNA library as conferring PRRS V permissivity served as template for PCR amplification of the CD163 insert, including the 5' and 3' untranslated regions, using the primers 5'DS-CD163 (SEQJD NO:6) (5'-CGGAATT£eseQQATGTAATAATACAAGAAGA-3') and 3'CD163 (SEQ ID NO:7)
(5'CC(3CT^AGTAGTCC^GGTOTCATCAA
1116 ncomyein/kanamyein resistance gene under the control of botheukaryotic and prokaryotic promoters.
Example 3: Construction of the pRSV-Script expression vector and pRSVsusCD163vl
The plasmid pRc/RS V (Ihvitrogen) was used as a template for PCR amplification of the RS V promoter. RS V promoter sequence was contained within nucleotides 209 through 604 of pRc/RSV. Forward primer PCIRSVLTR (SEQ ID N0:8) (5^ACACTCGAC^3n!^GATGTACGGGCCAGATATAOGCGT-3') and reverse primer VSRRTLSAC (SEQ ID NO: 9)
rS'TTCCITACAGAGCTCGAGGTGCACACCAATGTGGTGAA -3') were synthesized. Restriction endonuclease Pci I and Sac I recognition sites (underlined) were incorporated into the 5' and 3' primers, respectively, for future cloning. PCR was performed using the HotMaster Taq DNA Polymerase kit (Eppendorf) following the manufacturer's instructions. The reactions contained 0.9ng of pRc/RSV plasmid template and 0.3jiM of each primer described above. The reactions were heated to 94° for 2 minutes then cycled 30 times through 94° for 20 seconds, 52° for 10 seconds, and 65° for 1 minute. The resulting PCR fragment was digested with restriction enzymes Pci I and Sac I, gel purified, and cloned into the plasmid pCMV-Script (Stratagene) that had been similarly digested to remove (he CMV promoter sequence. The final construct placed the RS V promoter immediately upstream of the multiple cloning site, and was named "pRS V-Script".
The susCD163vl insert was cloned behind the RSV promoter as follows. The susCD163vl sequence was excised from plasmid pCMVsusCD163vl by restriction digestion (Kpn I and SacE) and gel purified. This fragment was ligated into pRSV-Script which had also been digested with the same enzymes and gel purified. The ligation mixture was transformed into DHSoc E. coli and transformants selected using kanamycin at 5Qjjg/mI. The clone contained the correct insert was designed "pRSVsusCD163vl".
Example 4: Cloning and characterization of a longer variant of porcine CD163 cDNA
Based on the porcine CD163vl sequence, a forward primer 5"CD163NotHong (SEQIDNO:10)
CGGTCGGGAGCGGCCGCGATGTAATAATACAAGAAGATTTAAATGG-3') and a reverse primer 3 'CD163KpnI (SEQ ID NO: 11)

using the Lasergene PrimerSelect program (DNASTAR Inc., Madison WI) for amplification of a foil-length porcine CD163 gene. Restriction endonuclease sites for Not I and Kpn I (underlined) were included in 5' and 3' primers, respectively, to allow for convenient cloning. Total cellular UNA was prepared from primary alveolar macrophages (PAM) harvested from lung lavages of healthy pigs. RNA preparation was done using the RNcasy mini kit (Qiagen, Valencia, CA). RT-PCR reactions were prepared using the Superscript one-step RT-PCR for Long Templates kit (Invitrogen, Carlsbad, CA) and RT-PCR parameters were set as follows: 50°C for 30 min, 94°C for 2 min, (94°C 30 sec, 55°C 30 sec and 68°C 4 min) for 35 cycles, 72°C for 10 min. PCR products were analyzed on 0.8% SeaKem GTG agarose gels. RT-PCR products of various sizes were cut from agarose gels and DNA was extracted using the GeneClean kit (QBiogene). These RT-PCR products were cloned into the pCR2.1-TOPO cloning vector (Invitrogen). Clones were analyzed by restriction enzyme digestion for the presence of an insert Colonies containing inserts were sequenced using Big Dye Terminator Version 1.0 Sequence Reaction kit (Applied Biosystems, Foster City, CA) and Applied Biosystems 3730 DNA Analyzer (Applied Biosystems) to confirm sequence authenticity. Sequences were edited and assembled using the Lasergene EditSeq and SeqMan programs (DNASTAR Lie., Madison WT). One plasmid with a large insert was designated "pCRsusCD163v2" (pCR2.1 containing porcine CD163 variant 2 which we have designated SEQ ID NO: 12). The coding sequence contained within SEQ ID NO: 12 is reproduced below and is designated SEQ ID NO: 13. Sequence analysis showed that this porcine CD163 encodes an amino acid sequence of 1115 amino acids which we have designated SEQ ID NO: 14. When compared to the porcine CD163 sequence hi GenBank
and bolding respectively. To determine whether other CD163 sequences contain similar sequence features is easily determined by inspection of the sequence.

SEQUENCE
gtaataatac aagaagattt aaatggcata aaaocttgga atggacaaac teagaatggt So"
gctacatgaa aaetctggat 'Ctgcagactt tagaagatgt tctgcecatt taagttbctt lao
cacttttgoe gtagtogctg ttctoagtgo ctgettggtc aetagttete ttggaggaaa 180
agaeaaggag ctgaggetaa cgggtggtga .aaaoaagtgc tetggaagag tggaggtgaa 340
agtgcaggag gagtggggaa ctgtgtgtaa taaeggctgg gacatggatg tggtetetgt 300
tgtttgtagg cagetgggat gtecaactgc tatcaaagcc actggatggg ctaattttag 360
tgeaggttet ggaegcattt ggatggatea tgtttcttgt egagggaatg agteagctet 480
ctgggactgc aaaeatgatg gatggggaaa geataactgt aetcaeeaae aggatgetgg 480
agtaacctge tcagatggat ctgatttaga gatggggetg gtgaatggag gaaaecggtg 540
ettaggaaga atagaagtea aatttcaagg acggtgggga aeagtgtgtg atgataaott 600
caaeataaat eatgottotg tggtttgtaa acaaettgaa tgtggaagtg ctgtcmgtttt ««o
ctctggttca getaattttg gagaaggttc tggaecaatc tggtttgatg atcttgtatg 720
caatggaaat gagtcagcte totggaactg eaaaeatgaa ggatggggaa ageacaattg 780
cgatcatgct gaggatgctg gagtgatttg cttaaatgga geagaectga aaetgagagt 840
ggtagatgga gtcactgaat gttcaggaag attggaagtg aaattccaag gagaatgggg 900
aacaatctgt gatgatggct gggatagtga tgatgccgct gtggcatgta agcaactggg 960
atgtccaact gctgtcactg ccattggtcg agtcaaegcc agtgagggaa ctggacacat 1020
ttggcttgac agtgtttett gccatggaca cgagtctgct ctetggcagt gtagacaeea 1080
tgaatgggga aagcattatt geaatcatga tgaagatgct ggtgtgacat gttctgatgg 1140
atcagatetg gaactgagae ttaaaggtgg'aggcagccac tgtgctggga cagtggaggt 1200
ggaaattcag aaactggtag gaaaagtgtg tgatagaagc tggggactga aagaagctga 1260
tgtggtttge aggcagctgg gatgtggate tgcactcaaa acatcatatc aagtttatte 1320
caaaaceaag geaacaaaca catggctgtt tgtaagcage tgtaatggaa atgaaactec 1380
tctttgggac tgcaagaatt ggeagtgggg tggacttagt tgtgatcact atgacgaagc 1440
caaaattacc tgetcagcec acaggaaacc caggctggtt ggaggggaeattccctgdtc ISOO
tggtcgtgtt gaagtacaac atggagacae gtggggcacc gtctgtgatt ctgacttctc 1S60
tctggaggeg gccagcgtgc tgtgcaggga actacagtgc ggcactgtgg tttccctcct 1620
ggggggaget eactttggag aaggaagtgg acagatctgg getgaagaat tocagtgtga 1-680
ggggcaegag tcceaccttt cactctgccc agtagcaccc egcectgaeg ggacatgtag 1740
ccacagcagg gacgtcggcg tagtctgetc aagatacaea -caaatccgct tggtgaatgg 1800
caagacccca tgtgaaggaa gagtggagct caacattctt gggtcctggg ggtccctctg 1860
caactctcac tgggaeatgg aagatgccca tgttttatgc cagcagctta aatgtggagt 1920
tgccctttct atcccgggag gagcaccttt tgggaaagga agtgagcagg tctggaggca 1980
catgtttcac tgcactggga ctgagaagea eatgggagat tgttecgtca etgctetggg 2040
cgcatcactc tgttcttcag ggcaagtggc ctetgtaatc tgctcaggga accagagtca 2100
gacactatct ccgtgcaatt cateatectc ggacccatca agctctatta ttbcagaaga 2160
aaatggtgtt gcctgcatag ggagtggtca acttcgcctg gtcgatggag gtggtcgttg 2220
toetqtwaga gtagaggtct atcatgaggg etcctggggc accatctgtg atgacagctg 2280

ID NO
SEQID N0:12

ggaootgaat gatgeocatg tggtgtgcas acagctgagc tgtggetggg-.ccatta«tge. 2340, cactggttct geteattttg gggaaggaac agggecoatt tggotggatg agataaactg 2400 taatggaaaa gaatcteata tttggcaatg ceaeteaeat ggttgggggc ggcaeaattg ,2460 caggcataag gaggatgcag gagteatctg etcagagttc atgtctctga gactgatcag 2520 tgaaaacagc agagagacct gtgcagggcg cctggaagtt ttttacaacg gagcttgggg 2580 cagcgttggc aggaatagca tgtotccagc oacagtgggg gtggtatgca ggcagetggg 2640 ctgtgcagac agaggggaca tcagocctgc atcttcagac aagacagtgt ccaggcacat 2700 gtgggtggac aatgtteagt gtcctaaagg aeetgacaea ctatggeagt geecatcatc 2760
tceatggaag aagagactgg ecagccectc agaggagaea tggatcacat gtgecaacaa 2820
aataagactt caagaaggaa aeactaattg ttctggacgt gtggagatct ggtaeggagg 2880
ttectggggc actgtgtgtg acgactcctg ggaccttgaa gatgctcagg tggtgtgccg 2940
acagctgggc tgtggctcag ctttggaggc aggaaaagag gccgcatttg gocaggggac 30.00
tgggeccata tggctcaatg aagtgaagtg caaggggaat gaaacctcct tgtgggattg 3060
tcctgccaga tectggggee acagtgactg tggacacaag gaggatgctg ctgtgacgtg 3120
ctcagaaatt gcaaagagcc gagaatocct acatgccaca ggtogctcat cttttgttgc 3180
acttgcaatc tttggggtca ttctgttggc ctgtcteatc gcattcctca tttggacfcca 3240
gaagcgaaga cagaggcagc ggetctcagt tttctcagga ggagagaatt ctgtccatca 3300
aattcaatac cgggagatga attcttgcct gaaagcagat gaaaeggata tgctaaatcc 3360
ctcaggagae cactctgaag tacaaegaaa aggaaaafcgg gaattataac -ctggtgagtt 3420
cagcctttaa gataccttga tgaagacctg gactattgaa tgagcaagaa tctgoctctt 3480
acactgaaga ttacaataca gtcctetgtc tcctggtatt ccaaagactg ctgctgaatt 3540
tctaaagaat agattggtga atgtgactac tcaaagttgt atgtaagact ttcaagggca 3«oo
ttaaataaaa aagaatattg ctg 3623

IMDKLRMVLHENSGSADFRRC 1 atggacaaactcagaatggtgctacatgaaaactctggatetgcagactttagaagatgt 216ABL6 SFTFAVVAVIiSACIiV 61 tctgcccatttaagttccttcacttttgctgtagtcgctgttctcagtgcctgcttggtc
41TSSLGGKDKELRLTQGENKC
121 actagttctcttggaggaaaagacaaggagetgaggctaacgggtggtgaaaacaagtgc
61SORVEVKVQEEWGTVCKNQW 181 tctggaagagtggaggtgaaagtgcaggaggagtggggaactgtgtgtaataatiggctgg
81DMBVVS V V CRQ L G C P TA I KA 241 gacatggatgtggtctctgttgtttgtaggcagctgggatgbccaactgctatcaaagcc 101TGWANPSAGSGRIWMDHVSC 301 actggatgggctaattttagtgcaggttctggacgcatttggatggBtcatgtttcttgt 121R6NE8AL WDCKHDGWGKHNC 361 cgagggaatgagtcagctctctgggactgcaaacatgatggatggggaaagcataactgt 141THQQDAGVTCSDGSDLEMGL 421 actcaccaacaggatgctggagtaacctgctcagatggatctgatttagagatggggotg 161 VNGGHRCLGRIEVKFQGRMG 481 gtgaatggaggaaaccggtgcttaggaagaatagaagtcaaat ttcaaggacggtgggga 1B1TVCDDNFNINHASVVCKQLE 541 acagtgtgtgatgataacttcaacataaatcatgcttctgtggtttgtaaacaacttgaa 201 CGSAVSFSGBANFGE6SGPI 601 tgtggaBgtgctgtcagtttctctggttcagctaattttggagaaggttctggaceaatc 221WFDDLVCN6NE6ALHNCKHE 661 tggtttgatgatcttgtatgcaatggaaatgagtcagctctctggaactgcaaacatgaa 241GH6KHNCDHAEDAGVZCLMG 721 ggatggggaaagcacaattgcgatcatgctgaggatgctggagtgatttgcttaaatgga
261 ADLKIiRVVDGVTECSGRLEV
781 gcagacctgaaactgagagtggtagatggagtcactgaatgttcaggaagattggaagtg 281 KFQGEWGTICDDGWD6DDAA 841 aaattccaaggagaatggggaacaatctgtgatgatggctgggatagtgatgatgccgct 301VACKQLGCPTAVTAIGRVHA 901 gtggcatgtaagcaactgggatgtccaactgctgtcactgccafctggtcgagttaacgcc 321 S E G T G H I WLDEVSCHGHESA 961 agtgagggaactggacacatttggcttgacagtgtttcttgccatggacacgagtctgct

SEQID
NO:13
and!4

- 'I, 341 Z. If Q C R H 8 E W G X B Y C N B D E D A

401 K Q i X E A D V V C R Q £ G C 0 8 A L X
1201 tggggBctgBaagaaactgatgtBgtttgcaggcagctgggatgtggatctgcaetcBBB 421 TSYQVY, eXTXATHTHtPVSS
1261 acatcatatoaagtttattocaaaaccaaggcaacaaacacatggctgtttgtaagcagc 441C»G»ETBI,lfDCX»ll Q » 0 G I? 8
1321 tgtBatggBaatgaaBGttcecftttgggaetgeaagaattggcagtggggtggacttagt 461CDBYDBAXXTC8ABRXPRI.V
1381 tgtgatcactatgacgaagccaaaattacetgctcBgcecacaggaaacccaggctggtt 481GGDIPCSGRVBVQH CD I HOT
1441 ggaggggacattccctgctctggtcgtgttgaagtacaacatggagacacgtggggoaco SOiVCP8DFSiBAAflVI,CRB !• Q C
1501 gtctgtgattctgaettctctctgseggcggccagcgtgctgtgcagggaactacagtgc 5210TVV8LIiGGABFGEG8GQXK
1561 ggcactgtggtttccotoctggggggagctoactttggagaaggaagtggacagatctgg 541AEBFQCXGHB8BIi8IiCPVAP
1621 gctgaagaattccagtgtgaggggcacgagtcccaoctttcactctgcccagtagcacoc SClRPDGTCSHSRDVGVVCSRyT
1681 egccetgacgggacatgtagecacageagggacgtcggcgtagtctgetcaagatacaca
SSiQIRIiVNGKTPCBGRVELNXL
1741 caaatcogcttggtgaatggcaagaceccatgtgaaggaagagtggagctcaacattctt 601G8WG8IiCM8BNOMBDAHVLC
1801 gggtcotgggggtccctctgcaactctcactgggacatggaagatgcccatgttttatgo
621QQLXCGVAIi8 X PGGAPFGKG
1861 cagcagcttaaatgtggagttgocctttetataecgggaggagcaecttttgggaaagga
6418EQVHRHMFHCTGTBKHMGD 1921 BgtgBgcaggtctggaggcacatgtttcactgcactgggactgagaagcaeatgggagat
661CSVTALOASI.CS6GQVASVI 1981 tgttecgtcactgctcbgggcgcatcaotetgttcttcagggcaagtggcctctgtaate
681C8GHQ8QTIi8PCN8Se8DPS 2041 tgctcagggaaccagagtcagaeactatctcegtgcaattcateatcctcggaoceatca
70168XX8EEKGVACXG8GOLRL 2101 agctetattatttcBgaagaaaatggtgttgcetgeatagggagtggtcaaetccgectg
721VDGGGRCAGRVBVYHE 741 TICDDSWDLNDAHVVCKQIiE 2221 accatetgtgatgacagetgggacetgaatgatgcccatgtggtgtgcaaacagctgagc
761 CGWAINATGSAHFGEGTGPI 2281 tgtggatgggecattaatgecaetggttctgctcattttggggaaggaacagggcceatt
781 WliDBINCNGKEBHIWQCHSH 2341 tggctggatgagataaactgtaatggaaaagaatcteatatttggcaatgceactcacat
801GHGRHNCRHKBDAGVICSEF 2401 ggttgggggcggcaeaattgcaggcBtaaggaggatgcaggagtcatctgctcagagttc
821 M 6 !• R L I SENSRBTCACRLEV 2461 atgtctctgagactgatcagtgaaaacagcagagagacctgtgcagggcgectggaagtt
841FyNGAKGSVGRNSMS PATVG 2521 ttttacaacggBgettggggeagcgttggeaggaatagcatgtetccagccacagcgggg
861VVCRQLGCADRGDX6 PASS.D, 2581 gtggeatgcaggcagctgggetgtgcagacagaggggacafccagccctgcatcttcagac
8B1KTV8 RKMNVDNVOC P XGPD T 2641 aagacagtgtccaggcacatgtgggtggacaatgtecagtgtcctaaaggacctgacaca
901LHQCPSSPKXXRLA8 P8EBT 2701 ctatggcagtgcccateatctccatggaagaagagactggecagcccctcagaggagaea
921 NX TCANKIRLQEGHTNCSGR 2761 tggateacBtgtgecaacaaaataagacttcaagaaggaaacactaattgttctggaegt
941VEXWYGG6HGTVCD08HPIiB 2821 gtggagatctggtaeggaggttcctggggcactgtgtgtgaegactectgggaccttgaa
961DAQVVCRQLGCGSALBAGKB 2881 gatgctcaggtggtgtgecgaeagetgggctgtggctcagctttggaggcaggaaaagag
981AAFGQGTGPXHIiNEVXCXGN 2941 gccgeatttggccaggggactgggcccatatggetcaatgaagtgaagtgcaaggggaat 1001 ETSLWDCPARSWGHSDCGHK 3001 gaaacctccttgtgggattgtectgccagatcctggggccacagtgaotgtggacacaag 1021 EDAAVTC8E XAXSRE SIiRAT 3061 gaggatgetgctgtgaegtgcteagaaattgeaaagagoogagaatccctacatgccaca 1041 GR8SFVALAXFGVXLLACLX 3121 ggtcgctcatcttttgttgcacttgcaatctttggggtcattctgttggoctgtctcatc 1061 AFLXKTQKRRQRQRIiSVFSG 3181 gcattcctcatttggactcagaagcgaagaeagaggeagcggctctcagttttctcagga 1081 GENSVHQIQyREMNSCLKAD 3241 ggagagaattctgtccatcaaattcaataecgggagatgaattct'tgcctgaaageagat 1101 ETDMLNPBGDHSEVQ 3301 gaaacggatatgctaaatccctcaggagaccactctgaagtacaa
^J MPKLBMVIjHp MSGSAPPRRC SAHL5BPTFA VgAVIiSftCLV TSS^QGKDKE
51 IiRLTGGBNKC SGRVEVKVOE EWSTWPWMfSB iwmnrewirnj ,,TJ!.«~.. —

-151- SDGSDLEMOL -VKG3NRCLOR 2EVKFQGRMQ WOOTOTHX1T H&8VVCIOQLB --- JfO:14
251 aoi BDAOVXCUia ADLKLRWDO VTBCB3RLBV KFQOBWQTIC ODGND8DDAA
351 VAWtt|MWV»8r
1ST mwHWDvyc KQUfCQfiALX TSVQfWSKTK AtUTNtiFVSS CHGOTTSIiMD
501 VCD8DFSLEA ASVLCROQC OTWBLLOOA HPQEaSOQIK ASBFQCBSBB
551 601 8HLSLCPVAP RPDQTC8B8R OVQWCBXYT QIRLVKORTP CBSRVBtdOZi O8NGSLCM8B iftWBDAHVLC OQLKaSVALS IPOOAPPQICO 8BQVHRHMPH
701 751 CTOTEKHMOD C8VTALGA8L C880QV»SV1 CSGNQ8QTLS PCMBBBSOP8 88XX8BBNOV ACXQSOQUiL VDGflaRCAOR VBVSRB08N6 TICDDSHDLH nAHWOCQliS OOWkXHATOB AHPOEOTOPI NLPKXMCRQK B8HXNQCBBB
801 5WSRHNCRHK IDAQVXC8BP MSLRLISRH6 RBTCAORLEV PVKSAWSffTO

901
QSl KNInUiJnJKl VU VVUKUlMCAU KUUlBrASBO Ki'VOKiOmVD MVQCPXOPDT LWQCPS8PWC KRZA6P8BBT HITCAJOCIRL QBONnJCSGR VEXIROOSNO

1051 1101 FOVILIACLI AFIiUfTQKRR QRQRLSVPSO G8N8VHQZQY RBMNSCLKAD BTOMLNFSGD H8BVQ
Sus CD163 v2 in pCRsusCD163v2 was liberated from pCR2.1 vector after restriction enzymes Kpn I and Not I digestion and gel purification. Recipient vector pCMV-script was also cut with the same restriction enzyme pair and allowed for directional cloning of susCD163v2 into the pCMV-script. After ligation of susCD163 v2 with pCMV-script, the ligated mixture was used to transform STBL 2 E. coti cells (Invitrogen). One transformant was found to contain the CD 163 gene by restriction enzyme digestion analysis and was designed pCMV-script susCD163v2 clone#3.
Example 5: Preparation of a RSV promoter based expression system by direct ligation and transfection method.
A non-cloning based procedure to generate microgram quantities of linear DNA suitable for use in generating stable cell lines expressing CD 163 from an RSV promoter was developed1 (figure 4). The procedure involves the isolation and ligation of two pieces of DNA, one containing the neomycin gene and RSV promoter cassette derived from pRSV-script, and the other containing the susCD163v2 coding sequence from pCMVsusCD163v2. Vector plasmid pRSV-Script was linearized with Drain. upstream of the neomycin gene, and bunted with the Klenow fragment off. coli DNA polymerase. This plasmid was then digested with Notl immediately downstream of the RSV promoter. The pCMVsusGD163v2 clone was digested in the vector sequence downstream of the CD163 insert with DrdL, and blunted with Klenow fragment of DNA polymerase. The CD163 coding sequence was liberated from the vector with a Notl located immediately upstream of the CD 163 coding sequence. Fbr each plasmid digestion the appropriate fragments were purified from agarose gels. A large-scale ligation reaction was performed as follows. Approximately 20 {4-g of each

DNA fragment was incubated in a volume of 600 jiL wito 15 unite of T4 DNA ligase. The reaction was incubated at room temperature for 20 minutes, at which time an aliquot was removed and the reaction frozen on dry ice. Agarose gel analysis of the aliquot revealed that a significant amount of non-ligated DNA remained, so another 15 units of ligase Was added and incubated for another 10 minutes at room temperature. Following ligation, a linear piece of DNA containing all of the appropriate elements was purified by agarose gel electrophoresis. Ligation of die two DNA fragments via the cohesive Not I termini resulted in the placement of the 5' sequences of the CD163 gene downstream of the RSV promoter, allowing for directed expression of CD163 in mammalian cells. Once isolated, the purified DNA was used to transfect various rpflrnrnplfan cell lines. Example 6: Cloning and characterization of human CD163 cDNA
Based on a known human CD163 cDNA sequence (GenBank Accession No. BC051281), a forward primer HuS'Not (SEQ JD NO: 15)
(5'CACCGCGGCCGCGAAGTTATAAATCX3CCACCATGAGCAAACTCAGAAT GG-3') and a reverse primer HuS'Kpn (SEQ ID NO: 16) Y5^TGCTCCGGTACCTAGTCCAGGTCTTCATCAAGGTATCTTA--3>) were designed using the PrimerSelect program. Restriction sites forNotl and Kpnl (underlined) were incorporated into the 5* and 3' primers, respectively, to facilitate cloning into expression vectors. The sequence CACC was added to the 5' end of the 5' primer to allow directional cloning into the pCDNA3.1D/V5/His/rOPO vector (Cat. No. K49001, Ihvitrogen, see figure 6). Human CD 163 cDNAs were amplified from RNA extracted from the U937 cell line after stimulated with phorbol 12-myristate 13-acetate (lOOng/ml) for 3 days. Total cellular RNA was prepared using the RNeasy kit (Qiagen). RT-PCR reactions and sequencing methods were the same as described in Example 4. PCR products were separated on 0.8% SeaKem agarose gel and extracted from the gel using the GeneClean kit. PCR products were cloned directionally into the pCDNA3.1D/V5/His/TOPO vector following the manufacturer's instructions. Two clones with large inserts were sequenced. Sequencing and sequence analysis methods were described in Example 4. A clone with a correct insert was designed "pcDNA3.1D-humCD163v2" and we have designated the sequence of the insert SEQ ID NO: 17

The CD163 open reading frame in pCDNA3.1D-humCD163v2 is 1121 residues in length (designated SEQ ID NO: 18 which encodes SEQ ID NO: 19 disclosed below), and is 100% identical to Oenbank Z22968 (a human CD 163 cDNA of the same length). Our human CD163v2 sequence is also 100% identical to Genbank BC051281 and Z22969 (splice variants of human CD163) except that 42 nonbomologous residues in the two Genbank sequences replace the seven carboxy-tenninal residues of our sequence. This difference is due to the presence of an 83-nucleotide exon in BC051281 and Z22969, and the resulting frame shift at die 3' end of the exon. (Law, S.K., Micklem, KJ., Shaw, J.M., Zhang, XJ>., Dong, Y., Willis, A.C. and Mason, D.Y. (1993) A new macrophage differentiation antigen which is a member of the scavenger receptor superfamily. European Journal of Immunology 23 (9), 2320-2325).



SEQUENCE
atgagcaaac tcagaatggt gctacttgaa gactctggat ctgctgactt cagaagacat 60
tttgtcaacc tgagtccctt caccattact gtggtcttac ttctcagtgc ctgttttgtc 120
accagttcto ttggaggaac agacaaggag ctgaggctag tggatggtga aaacaagtgt 180
agcgggagag tggaagtgaa agtccaggag gagtggggaa cggtgtgtaa taatggctgg 240
agcatggaag cggtctctgt gatttgtaac cagctgggat gtecaactgc tatcaaagcc 300
cctggatggg ctaattccag tgcaggttct ggacgcattt ggatggatca tgtttcttgt 360
cgtgggaatg agteagctct ttgggattgc aaacatgatg gatggggaaa gcatagtaac 420
tgtactcacc aacaagatgc tggagtgacc tgotcagatg •gatccaattt ggaaatgagg 480
ctgaogcgtg gagggaatat gtgttctgga agaatagaga ccaaattcca aggacggtgg 540
ggaacagtgt gtgatgataa etteaacata gatcatgcat ctgtcatttg tagacaactt 600
gaatgtggaa gtgctgtcag tttctctggt tcatctaatt ttggagaagg ctctggacca 660
atctggtttg atgatcttat atgcaacgga aatgagtcag ctctctggaa etgeaaacat 720
caaggatggg gaaagcataa ctgtgaccat gctgaggatg ctggagtgat ttgctcaaag 780
ggageagatc tgagectgag actggtagat ggagtcactg aatgttcagg aagattagaa 840
gtgagattcc aaggggaatg ggggacaata tgtgatgacg gctgggacag ttacgatgct 900
gctgtggcat gcaageaaet gggatgtcea actgccgtca cagccattgg tcgagttaac 960
gccagtaagg gatttggaca catctggctt gacagegttt cttgccaggg acatgaaect 1020
gctgtctggc aatgtaaaca ccatgaatgg ggaaagcatt attgcaatca caatgaagat 1080
getggcgtga catgttctga tggatcagat ctggagetaa gacttagagg tggaggcagc 1140
cgctgtgctg ggacagttga ggtggagatt cagagactgt tagggaaggt gtgtgacaga 1200
ggctggggac tgaaagaagc tgatgtggtt tgcaggcagc tgggatgtgg atctgcaotc 1260
aaaacatctt atcaagtgta ctccaaaate caggcaaeaa acacatgget gtttctaagt 1320
agctgtaacg gaaatgaaac ttctctttgg gactgcaaga actggcaatg -gggtggactt 1380
acctgtgate actatgaaga agccaaaatt acctgctcag cccacaggga aeocagaetg 1440
gttggagggg acattccctg ttctggacgt gttgaagtga agcatggtga cacgtggggc 1500
tccatctgtg attcggactt ctctctggaa gctgccagcg ttctatocag tweattacao iseo

ID NO
SEQ ID NO:17

tgtggeaeag ttgtctctat cctgggggge gctcactttg gagagggaaa tggacagate- 1*20
tgggetgaag aattceagtg tgagggaeat gagteccate ttteaototg «ecagtagca 1680
eoeogoceag aaggaacttg tageoacage agggatgttg gagtagtctg etcaagatac 1740
acagaoatto gettggtgaa tggeaagaee eegtgtgagg geagagtgga getcaaaacg ieoo
cttggtgcct ggggatocet otgtaactct cactgggaoa tagaagatgc ccatgttctt i860
tgecagcagc ttaaatgtgg agttgocctt tetaecccag gaggageacg ttttggaaaa 1920
ggaaatggte agatctggag gcatatgttt eactgcactg ggactgagoa goacatggga 1980
gattgtcetg taactgctct aggtgottca ttatgtectt cagageaagt ggoctctgta , 2040
atctgctcag gaaaceagto ecaaaeaetg tcetogtgea attcatcgtc tttgggccca 2100
acaaggceta ccattccaga agaaagtgct gtggcetgca tagagagtgg teaaettegc 2160
ctggtaaatg gaggaggteg ctgtgctggg agagtagaga tetateatga gggcteetgg 2220
ggcaeeatet gtgatgacag ctgggaeetg agtgatgcec acgtggtttg cagacagctg 2280
ggctgtggag aggeeattaa tgceactggt tctgetcatt tfcggggaagg aacagggcee 2340
atctggctgg atgagatgaa atgeaatgga aaagaatcce gcatttggca gtgccattea 2400
caeggctggg ggcagcaaaa ttgcaggcac aaggaggatg cgggagttat ctgctcagaa 2460
ttcatgtctc tgagaetgac cagtgaagcc agcagagagg ectgtgeagg gegfcctggaa 2S20
gttttttaca atggagcttg gggcaetgtt ggcaagagta geatgtctga aaocactgtg 2560
ggtgtggtgt gcaggcagct gggctgtgca gaeaaaggga aaatcaaocc tgcatcttta 2640
gacaaggcca tgtccattcc catgtgggtg gacaatgtte agtgtccaaa aggaectgac 2700
acgctgtggc agtgcccatc atctccatgg gagaagagac tggceagccc ctcggaggag 2760
acctggatca catgtgacaa caagataaga ctteaggaag gacocacutc ctgttctgga 2620
cgtgtggaga tctggcatgg aggttcctgg gggacagtgt gtgatgaetc ttgggacttg 2880
gacgatgctc aggtggtgtg teaacaactt ggctgtggtc cagctttgaa agcattcaaa 2940
gaagcagagt ttggteaggg gactggaccg atatggctoca atgaagtgaa gtgcaaaggg , 3000
aatgagtctt ccttgtggga ttgteetgcc agacgctggg gccatagtga gtgtgggeac 3060
aaggaagacg ctgcagtgaa ttgeacagat atttcagtgc agaaaaccec acaaaaagcc 3120
acaacaggtc gctcatcccg tcagtcatcc tttattgcag tcgggatcet tggggttgtt 3180
ctgttggcca ttttcgtcgc attattcttc ttgactaaaa agcgaagaca gagacagcgg 3240
ettgcagttt cctcaagagg agagaactta gtccaccaaa ttcaataccg ggagatgaat 3300
tcttgcctga atgcagatga tctggaccta atgaattcct caggaggcca ttctgagcca 3360
cactgaaaag gaaaatggga atttataacc cagtgagttc agcctttaag ataccttgat 3420
gaagacctgg acta 3434

1MSKLRMVLLEDSGSADFRRH 1 atgagcaaactcagaatggtgctacttgaagactctggatctgctgactteagaagacat 21PVNLSPFTITVVL,I,I.SACFV 61 tttgtcaacctgagteccttcaccattactgtggtct'tacttctcagtgcctgttttgte 41TSSIiGGTDKELRIiVDGEKKC 121 accagttctcttggaggaacagacaaggagctgaggctagtggatggtgaaaacaagtgt
61SGRVEVKVQEEWSTVCNNGH 181 agcgggagagtggaagtgaaagtccaggaggagtggggaacggtgtgtaataatggctgg
81SMEAVSVICNQLGCPTAIKA 241 agcatggaagcggtctctgtgatttgtaaacagctgggatgtecaactgctatcaaagcc 101PGWANS6AGSGRIWMDHVSC 301 cctggatgggctaat'tccagtgcaggttctggacgcatttggatggatcatgtttcttgt IZlRGNEgALHDCKHDGWGKHSN

SEQID NO: 18
and!9

361 cgtgggaatgagtcagctctttgggattgcaaacatgatggatggggaaagcatagtaac 1*1 C T H Q Q D A 0 V r-C S D G 8 N I, .S M R ••
161 LT R 00 KMC8 ORI K I X, F Q G R 481 otgaogcgtggagggaatatgtgttctggaagaatagagatcaaattooaaggacggtgg lelflTVCDDKFBIDHASVICR Q i 541 ggaacagtgtgtgatgataacttcaacatagatcatgcatctgtcatttgtagacaactt 201 ECGBAVBFSaSBKPOBGSGP £01 gaatgtggaagtgctgtcagtttctctggttcatctaattttggagaaggctctggacca 221 I KFDDIiZCHGHBBAIilfKC. XB 661 atctggtttgatgatcttatatgcaacggaaatgagtcagctetetggaactgcaaacat 241 Q 0 N O XBNCDBABDAOVZCBK 721 caaggatggggaaagcataactgtgaccatgctgaggatgctggagtgatttgotcaaag aClOADIiBLRLVDeVTBCBOR I, IB 781 ggagcagatetgagcetgagaetggtagatggagtcactgaatgtteaggaagattagaa 281VRFQOIHOTICDDOKDSyDA 841 gtgagatteeaaggggaatgggggaeaatatgtgatgacggctgggaoagttaogatgct 301AVACKQLOCPTAVTAia>RVR 901 gctgtggeatgeaagoaaetgggatgteeaaetgeegteacagceattggtegagttaac 321 ABXOFOBZNIiDBVSCQOHBP 961 gccagtaagggatttggacaeatctggcttgaeagegcttcttgecagggacatgaaoet 341AVNQCXBBCirOXHyCirBirBO 1021 gctgtctggeaatgtaaaeaceatgaatggggaaagoattattgcaatcacaatgaagat
1081 gctggogtgaeatgttctgatggatcagatctggagctaagaettagaggtggaggeage
38141 C A O T V E V B I Q R L L O K V C O R 1141 ogctgtgc±gggaeagttgaggtggagattcagagactgttagggaaggtgtgtgacaga
401 OWQlilCBADVVCRQiGCGBAL 1201 ggetggggactgaaagaagctgatgtggtttgcaggcagctgggatgtggatetgeacte
421 XTfiYOVYBKIQATHTNIiFIiB 12(1 aaaacatcttateaagtgtaetccaaaatocaggeaacaaaeaeatggctgettctaagt
441 BCHGHBTSIiWDCKHHQIfGOL 1321 agetgtaaoggaaatgaaacttctetttgggaetgeaagaactggcaatggggtggactt
461 ICDHYEBAKITCSAHREPRL 13 81 acctgtgatcactatgaagaageeaaaattaectgctcagoccacagggaacccagaetg
481 V G G D I PC6GRVEVKHGOTN6 1441 gttggaggggaoattocetgttctggaegtgttgaagtgaagcatggtgacaegtgggge
SOlBICDBDFSLBAABVLCRBIiQ 1501 tccatctgtgattoggacttctctctggaagctgccagcgtt«tatgcagggaafctacag
521 CGTVV8ILQGAHPGSONGQI 1561 tgtggcacagttgbctctatcctggggggagctcactttggagagggaaatggacagatc
541 W ABB F 0 CEGHBSHLBIiCPVA 1621 tgggctgaagaattccagtgtgagggacatgagtcccatctttcactctgcccagtagca
561 PRPBGTCSB8RDVOVVC8RT 1681 ccocgcccagaaggaacttgtagccacagcagggatgttggagtagtctgctcaagatac
581 TBIRLVMGKTPCEGRVELICT 1741 acagaaattcgcttggtgaatggcaagaccccgcgtgagggcagagtggagetcaaaacg
601 LGAWGSIiCNSHWDIEDAHVI, 18 01 cttggtgcctggggatccctctgtaactctcactgggacatBgaagatgoccatgttctt
621 CQQLKCGVALSTPGGARFGK 1861 tgccagcagettaaatgtggagttgccetttctaccccaggaggagcacgttttggaaaa
641 GHGQIWRHMFHCTGTBQHMG 1921 ggaaatggtcagatctggaggcatatgtttcactgoactgggactgagcagcacatggga
661 DCPVTAXiQASIiCFSEQVASY 1961 gattgtcetgtaactgctctaggtgctc«attatgtccttcagageaagtggcetctgta
681 ICSSNQSQTIiSSCHSESLOP 2041 atctgctcaggaaaceagteceaaaeactgtectcgtgcaattcatcgtctttgggctwa
701 TRPTIPBEBAVACIEBGQLR 2101 acaaggcctaecattccagaagaaagtgetgtggcctgcatagagagtggtcaaettege
721LVHO, OGRCAGRVBI YBBGBK 2161 etggtaaatggaggaggtcgctgtgctgggagagtagagatetatcatgagggctcetgg
741 G T I CDDSWDLSDAHVVCRQL 2221 ggcaccatctgtgatgacagctgggacctgagtgatgoccacgtggtttgcagacagctg
761 GCGEAIMATGSAHF«BGTGP 2281 ggctgtggagaggccattaatgccactggttctgctcattttggggaaggaaeagggccc
781ZHIiDBHKCKGKB BRXWQCHB 2341 atctggctggatgagatgaaatgeaatggaaaagaaecccgcattcggcagtgocattca
801BGWGQQNCRBKEDAGVICBE 2401 cacggctgggggeagcaaaattgeaggcacaaggaggatgcgggagttatctgetcagaa
821FMSLRLTBEASREACAGRLE 2461 ttcatgtctctgagaetgaccagtgaageeagcagagaggectgtgcagggcgtctggaa
B41VFYHGAWGTVGXB8M8ETTV 2521 gttttttacaatggagcttggggcactgttggcaagagtageatgtctgaaaccactgtg
861GVVCRQliGCADK 881 OK A MS I PMKVDNVQ CPKGPD 2641 gacaaggccatgtecatteccatgtgggtggacaacgttcagtgt«caaaaggaecegac
901TIiHQCP8BPHEXRIiASP8BB 2701 acgctgtggcagtgcccatcatctccatgggagaagagactggccagcccctcggaggag
921 T W I TCDNKIRLQEGPTSCSG 2761 acctggateacatgtgacaacaagataagacttcaggaaggacccacttectgttctgga
941 R V K I W H G G S H G T V C D D 8 H D Zi 2821 egtgtggagatctggeatggaggttcctgggggacagtgtgtgatgactcttgggacttg

2m gaGgatgcteaggtggtgtgteaacaaettggctgtggeecagetttgaaagcattcaaa . ..,.
?««? BM^faStttggtoaggggaetggacegatatggetcaatgaagtgaagtgcaMaga 1001 HEBSLMDCPARRWOBBECGII 3001 «atgagtcttccttgtgggattgtcctgccagacgctggggcc»tagtgagtgtgggcac 1021 XSDAAVNCTDISVQKTP QIC A 3061 aaggaagaogetgeagtgaategcBoagatatttcagtgcagaaaacaooacaaaaaacc 1041 TTORBBRQBBFIAVOIIiGVV 3121 •caacaggtcgctcatecQgteagtcatcctttategcagtcgggatcattggggttgtt
3181 ctgttggccattttogtegoattattcttcttgaotaaaaagcgaagacagagaeagcgg 1081 X>AV86ROEXLVKQXOYRBMir 3241 cttgaagtttcetcaagaggagagaaattagtooaccaaattcaatacagggagatgaat 1101 BCXiffADDItDLMHSBOQRSBP 3 3 01 tottgeatgaatgeagatgatetggaoetaatgaattccteaggaggoeattetgagooa 1121 B 3361 oao
1 KSKLRMVLLE D8G8ADPRRB PVNL6PFTIT WLIiSACPV T88MSIDKB SEOID

iOi vuiuuiBHJHm uiUHHUuvlK. JOUIBBIUJHUU Kauvmtftuai 151 CSDOBHLBNR IiTROQNMCBO RZSXKPQQRM OTVCDDNPNI DHASVICRQb NO: 19
201 B08BAV8FBO 08NFOBQ88P XNPDDLXCN3 MBBAIiNNCXB QGHVOKKRCDH
..251 AlpMIVZCBIC OADIiBIiRLVD OVTBCSSRIiE VRFQ01WSTI COTCIIDSW*
301 AVACKQMCP TAVTA1QRVN ASKSFGHINL OSVSCQQHEP AVNQCXUtOnf •

401 GN8LKXADW CRQLOOSBAlj XTBXUVYHKI Qfc'lWTMLPliS BQU3KSTBUK
451 DCKKKQMOOli TCDKYBZAK1 TC8AHRBPRL VOODIPCSffll VEVXHODTNO 501 BICDSOPSIiB AABVLCRBLQ OOrVVSIMQ AHFOBGN3QI HABBPQCBOH
551 BSHIiSliCPVA PRPBGTC6HS RDVOWC6RY TBZRliVMSKT PCB6RVBLKT 601 L8AMQ8LCN8 BNDIBOARVL CQQUCCGfVUi STFGOARFQK GNQQIHRHMF

651 HCTQTBOHMO DCFVTAZiOAB LCP8BQVASV ICSSNQSQTIi SSCNSS&LOP 701 TRPTIPKEBA VACZEBOQIiR LVKSGQRCAS RVEIVHBGSH 6TICDDSNDL 751 SDAHWCRQL OOGBAIKATa BAHFQBGITGP IKLDBMTCHG KBSRXWQCHB
801 HBHOQQNCRH KKDAGVICSB FMSLRLT8BA SREACAORIiE VPUKUMOTV 851 QKSSMSBTTV OWCRQtiOCA PRQKZMPABIi DKAMSIPMWV DMVQCPXQPD 901 TLHQCPSSPK KXRLA6PBBB TWITCDNKIR LQBOPTBCBG RVBIWHOOSW
951 QTVCDDSMDL ODAQVVOQOIi OCGPALKAPK EREFGQOTGP ZHUIEVKCRS 1001 RB8BLHOCPA RRWSHSBCOH KBDAKVNCID ISVQXTPQXA TTGRSSRQSS
1051 F1AVGILGW L1AIFVALFF LTKKRRQRQR IiAVSSHGENIi VHQIQYREMN 1101 SOMADDLDL MNSSGSHSEP B
Example?: Cloning and characterization of marine CD163
Based on the murine CD163 sequence in GenBank CACCGCGGXXXK:CACACGGA GGTA
clones with large inserts were identified for rurther analysis. A plasmid containing an insert (SEQ ID NO: 22) with a murine CD163 mat encodes a protein of the same length as (1121 amino acids SEQ ID NO:24 ) and differs from Genbank AF274883 by only two amino acids (99.8% identity) was designated 't>O>NA3.1D-murCD163v2".
Another plasmid, "pODNAS.lD-murCDldSvS was generated which contained an insert (SEQ ID NO: 25) containing a murine CD163 coding sequence (SEQ ID NO: 26) which encodes a protein of 1159 amino acids in lengra (SEQ ID NO: 27). ft differs from AF274883 by only 3 amino acids within the first 1107 residues (99.7% identity), but the sequences diverge completely beginning at residue 1108. This is due to an insertion of 82 nucleotides in the cDNA, and a concomitant shift in reading frame downstream of the insertion. As a result, murine CD163v3 contains 52 amino acids at its carboxy-terminus that are not homologous to the 14 carboxy-terminal residues of murine CD163v2. These two alternative versions of "full length" murine CD163 are most likely splice variant of the same gene, as has been described for human CD 163 (Law, S.K., Micklem, K.J., Shaw, J.M., Zhang, X.P., Dong, Y., Willis, A.C. and Mason, D.Y. (1993) A new macrophage differentiation antigen which is a member of the scavenger receptor superfamily. European Journal of Immunology 23 (9), 2320-2325).



SEQUENCE

ID NO



gctttggaat gggtggacac agaatggttc ttettggagg tgctggatct cctggttgta 60
aaaggtttgt ceatetaggt ttetttgttg tggctgtgag ctcacttctc agtgccfcctg 120
ctgtcactaa cgctcctgga gaaatgaaga aggaactgag actggcgggt ggtgaaaaca 1BO
actgtagtgg gagagtggaa cttaagatcc atgacaagtg gggcacagtg tgcagtaacg , 240
gctggagcat gaatgaagtg tccgtggttt gccagcagct gggatgceca acttctatta 300
aagccettgg atgggetaac tccagcgccg gctctggata tatctggatg gacaaagttt 3«0
cttgtacagg gaatgagtca gctctttggg actgcaaaca tgatgggtgg ggaaagcata 420
actgtaccca tgaaaaagat gctggagtga cctgctcaga tggatctaat ttggagatga 480
gactggtgaa cagtgcgggc cacegatgct taggaagagt agaaataaag ttocagggaa 540
agtgggggac ggtgtgtgac gacaacttca gcaaagatea cgcttctgtg atttgtaaae €00
agcttggatg tggaagtgce attagtttct ctggctcagc taaattggga gctggttctg €60
gaceaatctg getcgatgac etggcatgca atggaaatga gtcagctctc tgggaetgca 720
aacaccgggg atggggcaag cataactgtg accatgctga ggatgtcggt gtgatttgct 760
tagagggagc agatctgagc ctgagactag tggatggagt gfcocagatgt tcaggaagat 640
tggaagtgag attccaagga gaatggggga ccgtgtgtga tgataactgg gatctccggg 900
atgcttctgt ggtgtgcaag caactgggat gtccaactgc catcagtgcc attggtcgag 9GO
tcaatgccag tgagggatct ggacagattt ggcttgacaa catttcatgc gaaggacatg 1020
aggcaactct ttgggagtgt aaacaecaag agtggggaaa gcattactgt caceatagag 1080
aagacgctgg cgtgacatgt tctgatggag cagatctgga acttagactt gtaggtggag 1140

SEQ ID
NO.-22

gcagtcgctg tgctggoatt gtggeggtgg agatteagaa getgeetggg aagatgtgta isoo
googaggctg gacaetggca gatgeggatg tggtttgcag aeagottgga -tgtggatotg iseo
cgettcaaac ccaggctaag atctactcta aaactggggo aaoaaataog tggotettte 1330
ctggatcttg taatggaaat gaaactactt tttggcaatg caaaaaetgg oagtggggcg 1380
gccttteetg tgataattte gaagaagcca aagttacotg eteaggceac agggaaccca 1440
gactggttgg aggagaaate coatgctctg gtegtgtgga agtgaaaeac ggagaegtgt isoo
ggggctocgt ctgtgatttt gaettgtete tggaagotge eagtgtggtg tgeagggaat 1S60
tacaatgtgg aacagtogtc tctatcctag ggggagoaca ttttggagaa ggaagtggae 1620
agatctgggg tgaagaattc oagtgtagtg gggatgagtc ecatetttea etatgeteag l«80
tggegeecec gctagaeaga aettgtacec acagcaggga tgtcagegta gtetgotcae 1740
gataeataga tattogtctg gcaggoggeg agteotcctg tgagggaaga gtggagctoa 1800
agaeaetegg agectggggt cccctctgca gtteteattg ggacatggaa gatgeteatg i860
tcttatgtca geagetgaag tgtggggttg oecaatetat tecagaagga gcacattttg 1920
ggaaaggagc tggteaggte tggagteaea tgttccaetg cactggaact gaggaacata I960
taggagattg eetcatgaet getctgggtg cgeegacgtg ttcegaagga caggtggcct 2040
ctgtcatetg cteaggaaae caatoceaga cactattgcc atgtagttca ttgtetecag 3100
tccaaacaac aagctctaea attccaaagg agagtgaagt fcccctgcata gcaagtggce 216O
agcttcgett ggtaggtgga ggtggtcgct gcgctggaag agtggaggte taceacgagg 2320
gctcttgggg caccgtetgt gatgacaatt gggatatgac tgatgccaat gtggtgtgca 2280
agcagetgga ctgtggcgtg gcaattaacg ccactggctc tgcttacttc ggggaaggag 2340
caggagctat ctggetagac gaagtcatct gcactgggaa agagtctcat atttggcagt 2400
gccattcaca tggctgggga cgccataact gcaggcacaa agaagatgca ggtgttatct 2460
gctccgegtt catgtctctg aggctgaeca acgaagccca caaagaaaac tgcacaggte 2520
gccttgaagt gttttacaat ggtacatggg gcagtattgg cagtageaat atgtetccaa 2580
ccactgtggg ggtggtgtge cgteagctgg gctgtgcaga caacgggact gtgaaaeoca 3640
taecttcaga caagacaeca tecaggeeca tgtgggtaga tcgtgtgcag tgtccaaaag 3700
gagttgacac tttgtggcag tgceeetcgt caccttggaa acagagacag gceagccect 2760
cctcccagga gtcctggatc atctgtgaca acaaaataag actecaggaa gggcatacag 2820
actgttctgg acgtgtggag atctggcaca. aaggttcctg gggaacagtg tgtgatgact 2880
cctgggatet taatgatgct aaggttgtat gtaagcagtt gggctgtgge -caagctgtga 3940
aggcactaaa agaagcagca tttggtccag gaactgggcc catatggctc aatgaaatta 3000
agtgtagagg gaatgagtct tccctgtggg attgtcctgc caaaccgtgg agbcacagcg 3060
actgtgggca caaagaagat gctteeatcc agtgcctccc aaaaatgact tcagaatcac 3120
atcatggcac aggtcacccc accctcacgg cactcttggt ttgtggagec attctattgg 3180
tcctccteat tgtcttcctc ctgtggacte tgaagcgacg acagatbcag cgacttacag 3240
tttcctcaag aggagaggte ttgataeatc aagttcagta ccaagagatg gat«caaagg 3300
cggatgatet ggacttgctg aaabcctcgg gggtcattca gaggcacact gagaaggaaa 3360
atgataattt ataatccact gaggttggag tttaagaagc cttgacagga cagccagcta 3420
aatggaacaa gagcccaggc aacgcacgga tgaccacagc tgcatcttca tgcagtcctt 3480
tgtttectgg aactctgctg aacctgeaaa aaccatattt gtgaatgtga ccacttaata 3540
gagatgggag actttt 3556

IMOaHRMVLIiOaABBPOCKRP 1 atgggtggaeecagaatggtecttcttggaggtgatggatatactggttgtaaaaggttt 21VHLGFFVVAV8BLZi8ASAVT 61 gtecatetaggtttetttgttgtggotgtgagctoaottcteagtgcctctgctgtcact 41VAFOEMKXEZ»RZiAOGEHBCS 181. aaegetcotggagaaatgaagaaggaaotgagactggegggtggtgaaaacaactgtagt
610 R VIL X Z B D X N O T V C S B 0 ff 8 181 gggagagtggaaettaagateeBtgaeaagtggggcacagtgtgcagtaaoggotggagc
81MNEVBVVCQQLGCPT6 I K A t 341 atgaatgaagtgtocgtggtetgecagcagotgggatgoeeaaettetattaaageccet 1010WAH68AOBOYIIfMDKV8CT 3 01 ggatgggctaactccagcgecggctctggatatatctggatggacaaagtttcttgtaea laiaWBBALMDCKHDOHQJCHNCT 361 gggaatgagtcagctctttgggactgeaaaeatgatgggtggggaaagcataactgtace 141HBKDAOVTCBDOSNliBMRI.V 421 eatgaaaaagatgetggagtgaeetgctcagatggatetaatttggagatgagaetggeg lElNBAOBRCXiORVBXXFQOXWQ 481 aaeagtgcgggocaocgatgcttaggaagagtagaaataaagtteeagggaaagtggggg laiTVCOONFSXOBASVXCKQIiO 541 acggtgtgtgaegacaaettcagcaaagatcaogettetgtgatttgtaaaeagottgga 201 C 0 8 A I 8 t B O S A K I. 0 A S S O PI 601 tgtggaagtgecattagtbtetetggcteagetaaategggagetggttctggaocaatc 221NIiOOLACHONKBAIilfDCXHR 661 tggctcgatgacetggeatgcaatggaaatgagtcagctetctgggaetgcaaacacegg 241QMOXHNCDHABDVGVICI.BO 721 ggatggggoaageataactgtgaccatgctgaggatgtcggtgtgatttgcttagaggga 261 A D !• 8LRLVDOV8RC8GR&BV 781 geagatctgagcctgagaetagtggatggagtgtceagatgttcaggaagattggaagtg 2eiRFQGBN6TVCOONHDLROA8 841 agattccaaggagaatgggggaccgtgtgtgatgataactgggatctcogggatgettet 301VVCXQLGCPTAI SAI GRVHA • 901 gtggtgtgcaagcaactgggatgtceaactgccatcagtgccattggtcgagttaatgoc 321SEGBGQIMI.DNISCBGHBAT 961 agtgagggatctggacagatttggcttgacaacatttcatgogaaggacatgaggcaact 341 1MBCKHQBKGXHYCHHRBDA
1021 ctttgggagtgtaaacaooaagagtggggaaagsattactgtcatcatagagaagacgct 361 O V T C S D O A D 1 B I» R 1 V 0 0 « 8 R
1081 ggewtgaoatgttctgatggagcagatctggaacttagaettgtaggtggaggeagtcgc 381CAGIVBVB1QXI.TGKMCSRG
1141 tgtgctggcattgtggaggtggagBttcagaagctgactgggaagatgtgtagcogaggc 401WTLADADVVCRQI.GCGSAIiQ
1201 tggaeactggcagatgcggatgtggtttgoagacagottggatgtggatctgcgcttcaa 421 TO AXiyBKTGATHTWtFPGB
1261 aecoaggctaagatctactctaaaactggggcaacaaatacgtggetctttxsetggatct 441CHGHETTFWQCXNWOWQOL8
1381 tgtgataatttcgaagaagecaaagttacctgctcaggocacagggaacccagactggtt
481GOBIPCSGRVBVKHGDVWGS 1441 ggaggagaaatcccatgctetggtcgtgtggaagtgaaacaeggagaegtgtggggctec
501VCDFDLSLBAASVVCRBLQC 1601 gtetgtgattttgacttgtetctggaagctgccagtgtggtgtgcagggaattacaatgt
S21GTVVS ILGGAHFGBGSGQIW 1561 ggaaeagtegtctctatoctagggggagoaeattttggBgaaggaagtggacagatctgg
541GBBFQCSGDB8HI.SLC8VAP 1621 ggtgaagaattccagtgtagtggggatgagtcccatottteaataegctcagtggcgccc
561PI.DRTCTHSRDV8VVCSRyi 1681 ccgctagacagaacttgtacocaeagcagggatgccagcgtagtetgctcacgataeaea
S81DIRLAGGBS6CBGRVBI.XTI1 1741 gatattcgtctggcaggcggcgagteetcctgtgagggaagagtggagotcaagacactc
601GAHGPLCSBHHDMEDAHVI.C 1801 ggagectggggtcccetetgcagttctcattgggacatggaagatgctcatgtettatgt
621QQl.XCGVAQeiPBGAHFGX-G 1861 cagcagetgaagtgtggggttgcccaatctattccagaaggageacattttgggaaagga
641AGQVHBHMFHCT-GTEBHIGD 1521 gctggtcaggtctggagtcacatgttecactgcactggaactgaggaacatataggagat
661CLMTALGAPTCSBGQVA8V1 1981 tgcetcatgactgetctgggtgcgccgacgtgttccgaaggacaggtggcctetgteatc
681CSGH08QTl.l.PC8StSPVQT 2041 tgetcaggaaaccaatcceagecaetattgccatgtagtfecattgtctccagtccaaaca
701TSSTI PKBSEVPCIABGQLR 2101 acaagctctBcaattccaaaggagagtgaagttccctgcetagcaagtggceagcttogc
'«iWVI'1'/''"
1321 tgtaatggaaatgaaactaetttttggcaatgeaaaaactggcagtggggcggcctfctcc 461CDHFEBAXVTCSGHRBPRLV

SEQID
NO:23and
24

2281 gactgtggogtggeaattaaegeeaetggatetgettaetteggggaaggagcaggaget " ' - - • 7B1-I- If IT B -8-*V M C 1-0- K Z S--H-- 1 --H--Q .-C.--H-.-S ••- •• •-• 2341 atctggctagacgBagtcatotgcactgggaaBgagtctcatBtttggcagtgceattca 801HGKORHMCRHICBDAGVXC8 B 2401 eatggetggggaegooataaotgcaggeaeaaagaagatgcaggtgttaectgetcogag •••••
2461 ttcatgtctetgaggctgaocaacgaagcecacaaagaaaaetgcacaggtcgeettgaa
2521 gtgttttacaatggtacatggggcagtattggcegtagcaatatgtctccaaocactgtg
2581 ggggtggtgtgecgtcagctgggctgtgeagaeaaogggactgtgaaacccataocttca
2641 gaeaagaeaeeateoaggeceatgtgggtagategtgtgcagtgtceaaaaggagttgae 901TLWQCPSSPKKQRQA8P8SQ 2701 actttgtggcagtgccectcgtcaccttggaaacagagacaggccagcocctectcceag
2761 gagtcctggatcatctgtgaoaacaaBBtaagactccaggaagggcatacagactgttct
941 0 R V B ZVHKOSNGTVCOD'BND '2821 ggaegtgtggagateeggeaeaaaggttoetggggaacagtgtgtgatgaoteotgggat
2881 cttaBtgatgctaaggttgtatgtaagcagttgggctgtggccaagctgtgaaggcacta
2 94 1 BaagaagcagcatttggtccaggaactgggcccatatggctcaatgaaattaagtgtBgB
1001 G N B 8 6LNDCPAKPNSRBDCG
1021 B K B 0 AS X Q C L P K M T 8 B 8 H H G


1061 IVPLLKTLKRRQIQRLTV8 8 3181 attgtcttcctcctgtggactctgaagcgacgacagattoagcgacttaoagtttectca 1081 RGBVXiXHQVQYQBMDSKADO 3341 agaggagaggtcttgatacatcaagttcagtaccaagagatggattcaaaggcggatgat 1101 LDLLX880VXQRBT. BKBRDB 3301 ctggacttgctgaaatcetogggggtcatteagaggcacactgagaaggaaaatgataat 1121 L 3361 tta
1 MQGHRMVtiLG OAGSPOCKRF VHLGPFWAV 6SLLSA6AVT NAPGEKKKBXi SEOID
51 RIiAOGENNCe ORVELKIHDK WQTVC8NGWS MNEVBWCQQ IX3CPTSIKM.
151 DGBMLBMRLV NSAGHRCLGR VEZXPQ8KMG TVCDDNFSKD HASVlCKQIiO NO:24
251 KDVSVICLEG ADLSLRLVDO VSRCSORIiBV RFQGBMGTVC DDNWDIiRDAS 301 WCKQbSCPT AISAIORVHA BEOSOQIWU) NZ8CBGHEAT IMBOKBQBNO 351 KHYCHHRBDA QVTCSDGADL BLRLVOOGSR CAGXVBVEIQ KLTGKMCSRG
401 HTLAOADWC RQLGOGSAIiO TQAXIYBXTG ATNITHLPPGB CKGNETTPWQ
451 CKNNQNGGIiS CDMFBSAKVT C80HREPRLV OGEIPCSORV EVKKGDVHQ8 501 VCDFDLSLKA ASWC31ELQC OTW8XL8SA HFQBGSGQIH GEEPQCSGDB
551 SHLSLCSVAP VIiDRTClllBR DV6WCSRYI DIRLAflGBEB CEORVfiuKTL 601 GAWGPLCSBH WDMEDAHVLC QOUCOSVAQS IPBQAHFQKO AOCJfVKSHMFH
G51 CTQTEEHIOD dMTALdAlT CfiBOQVABVX CBGHQSOITIiIi PCSSL8PVQT 701 T8STXPXB8B VPCIA8OQLR LVOOOGRCA6 KVBVXEBG8H QTVCDDNWDM 751 TOAHWCKQL DCGVAIHATO 8ATPGBSASA ZHLDBVXCTG KE6HIHQCHS
801 HQMGRHHCRH KEDAOVIC8E FHSUtLTNBA BKBNCTORLB VFyUOTHQSI
651 QSSHMBPTTV QWCRQL3CA OHGTVXPIP8 DKTPSRPMMV DRVUCPKSVD 901 TLWQCPSBPW KQRQA6PSSQ EBNXXCDNXX RLQEOHTDCS GRVEIUHKOS 951 NOTVCDD8HD XiNDAKWCKQ 1OOOQAVKAL KEAAPQPOTO PIWLREIKCR 1001 GNESSIiWDCP AKPH8HSDCG BKE&ASXQCL PKMTSEBHHG TGHPTLTALL 1051 VCXSA1LLVLL IVFLLWTLKR RQIQRLTV6S ROBVLIHQVQ YQBMDBKADD -
1101 LDLLX888VX QRHTEKENDH L
gctttggaat gggtggacae agaatggttc ttettggagg tgctggatct cctggttgta 60 aaaggtttgt ccatctaggt ttctttgttg tggctgtgag ctcacttctc agtgcetetg 130 ctgtcactaa cgctcctgga gaaatgaaga aggaactgag actggcgggt ggtgaaaaea 180 actgtagtgg gagagtggaa ettaagatcc atgacaagtg gggeacagtg tgcagtaacg 240 gctggagcat gaatgaagtg tccgtggttt goeagcagct gggatgceca acttctatta 300 aagcecttgg atgggctaac tecagcgccg gctctggata tatctggatg gaeaaagttt 360 cttgtacagg gaatgagtca getctttggg actgcaaaca tgatgggtgg ggaaageata 420 actgtaccca tgaaaaagat gctggagtga cctgctcega tggatctaat ttggagatga 460 gactggtgaa cagtgcgggc caccgatgct taggaagagt agaaataaag ttocagggaa 540 IDNO:25

:agtgggggac ggtgtgtgae gacaaettoa goaaagatea cgettetgtg atttgtaaae «oo
agcttggatg tggaagfcgoe attagtttet ctggctcago taaattggga getggttetg 660
gaeoaatctg gctegatgac etggcatgea atggaaatga gteagetete tgggaetgoa 720
aacaccgggg Btggggcaag eataaetgtg aecatgctga ggatgteggt gtgatttget 780
tagagggage agatctgagc etgagaetag tggatggagt gtceagatgt teaggaagat 840
I
tggaagtgag attecaagga gaatggggga eogtgtgtga tgataactgg gatctceggg 900
atgcttetgt ggtgtgeaag caactgggat gtecaaetge eateagtgcc attggtcgag 960
ttaatgoeag tgagggatct ggaeagattt ggettgaeaa eatttcatge gaaggacatg 1020
aggcaactet ttgggagtgt aaacaccaag agtggggaaa geattactgt eateatagag 1080
aagaegetgg ogtgacatgt tetgatggag cagatotgga acttagactt gtaggtggag 1140
gcagtegctg tgetggeatt gtggaggtgg agatteagaa getgactggg aagatgtgta 1200
gccgaggetg gaeactggca gatgeggatg tggtttgcag acagcttgga tgtggatctg 1260
egcttcaaae eeaggetaag atetaetcta aaaetggggc aaeaaatacg tggctetttc 1320 ctggatettg taatggaaat gaaactaett tttggeaatg caaaaaccgg cagtggggcg 1380 gccttteetg tgataatttc gaagaagcca aagttacetg etcaggccac agggaaecea 1440 gactggttgg aggagaaate ecatgctetg gtegtgtgga aatgaaacac ggagaegtgt 1500
ggggetecgt etgtgatttt gacttgtctc tggaagctgc eagtgtggtg tgeagggaat 1S«0
tacaatgtgg aaeagtegte tetatcetag ggggageaca ttttggagaa ggaagtggae 1620
agatetgggg tgaagaatte cagtgtagtg gggatgagtc ecatctttea etatgeteag 1680
tggegcccce gctagaeaga acttgtacce acagcaggga tgtcagcgta gtctgcteac 1740
gatacataga tattcgtctg geaggeggeg agtcctcctg tgagggaaga gtggagctca 1800
agacaetcgg ageetggggt cccctetgca gttetcattg ggacatggaa gatgctcatg i860
tcttatgtea gcagctgaag tgtggggttg eceaatetat tccagaagga gcaeattttg 1920
ggaaaggage tggtcaggte tggagtcaca tgttceactg cactggaaet gaggaacata I960
taggagattg ccteatgaet gctetgggtg cgccgacgtg ttccgaagga caggtggect 2040
ctgtcatctg eteaggaaac eaatcecaga cactattgcc atgtagttca ttgtctccag 2100
teeaaacaac aagctctaca attccaaagg agagtgaagt tocctgcata gcaagtggcc 2160
agettegctt ggtaggtgga ggtggtcgct gcgetggaag agtggaggtc taccacgagg 2220
gctettgggg eaccgtctgt gatgacaatt gggatatgac tgatgccaat gtggtgtgca 2280
agcagctgga ctgtggcgtg geaatteacg ccactggctc tgcttacttc ggggaaggag 2340
caggagctat ctggctagac gaagtcatct gcactgggaa agagtctcat atttggeagt 2400
gccattcaca tggctgggga cgccataact gcaggcacaa agaagatgca ggtgttatct 2460
gctccgagtt catgtctctg aggctgaoca acgaagccca caaagaaaac tgcacaggtc 2520
gccttgaagt gttttacaat ggtacatggg gcagtattgg cagtagcaat atgtcteeaa 2S80
ccactgtggg ggtggtgtgc cgteagctgg gctgtgcaga caacgggaet gtgaaaecca 2540
taccttcaga caagacacca tccaggecca tgtgggtaga tcgtgtgcag tgtccaaaag 2700
gagttgacac tttgtggcag tgcccctegt caocttggaa acagagacag gccagceect 2760
cctcccagga gtcctggatc atctgtgaca acaaaataag actecaggaa gggcatacag 2820
actgttctgg acgtgtggag atctggcaca aaggttcctg gggaacagtg tgtgatgact 2880
ectgggatet taatgatgct aaggttgtat gtaagcagtt gggctgtggc caagctgtga 2940
aggcactaaa agaagcagca tttggtecag gaactgggcc catatggctc aatgaaatta 3000
agtgtagagg gaatgagtct tccctgtggg attgtcctgc caaaocgtgg agtcacageg 3060

actgtgggca caaagaagat gcttceatcc agtgectcoo caaaatgact tcagaateac ,-,., 312.0
atOBtggeac aggtcaeece aeeotcaegg cactcttggt ttgtggagoc attctattgg 3180
tcctcctcat tgtcttcotc ctgtggactc tgaagegacg acagattcag ogacttaeag 3840
tttectoaag aggagaggtc etgataeato aagttcagta ceaagagatg gattcaaagg 3300
cggatgatct ggacttgctg aaateetegg aaaattoeaa eaattcatat gattttaatg 33SO
atgatggact gacatctttg tctaaatatc ttectatttc tggaattaaa aaggggtcat 3420
tcagaggeac actgagaagg aaaatgataa tttataatee aotgaggttg gagtttaaga 3460
ageettgaea ggaeagacag ctaaatggaa caagagceea ggeaacgcac ggatgaceac • 3540
agetgcatet tcatgcagto etttgtttec tggaactctg ctgaacebgc aaaaaccata 3600
tttgtgaatg tgaceaetta atagagatgg gagaetttt 3639

IMGGHRMVXiLGGAGSPGCXRP
1 atgggtggacacagaatggttettcttggaggtgctggatctcctggttgtaaaaggttt 21 V B LGPFVVAV8 S LLSA6AVT 61 gtceatctaggtttotttgttgtggctgtgagetcacttctcagtgcctctgctgtcact 41NAPOBMKKBLRLAOQBNNCS 121 aacgctectggagaaatgaagaaggaactgagactggegggtggtgaaaacaaetgtagt
616RVELKZRDKWGTVC8NON8 161 gggagagtggaacttaagatccatgacaagtggggcaeagtgtgcagtaacggctggagc
81HHBV8VVCQQLQCPTSIKAI, 241 atgaatgaagtgtccgtggtttgccagcagctgggatgoccaacttetattaaagoectt 101 GWANSeA 221 HIiDOIiACNGNBSAIiKDCKHR
661 tggctcgatgacctggcatgcaatggaaatgagtcagctctctgggactgcaaacaccgg 241 GKGKHNCDBABDVGVZCLBG 721 ggatggggcaagcataactgtgaccatgctgaggatgteggtgtgatttgcttagaggga 261 ADLBIiRIiVOGVeRCSGRLEV 761 gcagatctgagcctgagactagtggBtggagtgtceagatgttcaggaagattggaagtg 281 RFQGEWGTVCDDHWDLRDAS 841 agattccaaggagaatgggggacegtgtgtgatgataactgggatctccgggatgctt^t 301 VVCKQLGCPTAI SAZGRVNA 901 gtggtgtgcaagcaactgggatgtccaactgocatcagtgccattggtegagttaatgec 321 6BGSGQZWLDHI 8CEGBEAT 961 agtgajgggatctggacagatttggcttgacaacattbcatgogaaggacatgaggcaaet 341 L H B C K B Q EHGKBYCBBREDA
1021 ctttgggagtgtaaaeaceaagagtggggaaagcateactgtcateatagagaagacgct 361 GVTCSDQADXiELRIiVGGGBR
1081 ggcgtgacatgttctgatggagcagatetggaactfcagaettgtaggtggaggcagtcgc 361CAGZVEVEZQKIiTGKHC8RG
1141 tgtgctggcattgtggaggtggagatecagaagctgactgggaagatgtgtagccgaggc 401 KTLADADVVCRQLGCGSALQ
12 01 tggacactggcagatgcggatgtggtttgcagaeagcttggatgtggatctgegettcaa 421 TQAKZYSKTGATNTHIiF PG8
1261 acccaggctaagatctactctaaaactggggcaacaaatacgtggctctttcctggatct 441CNGNETTFHQCXNHQHGGI.8
1321 tgtaatggaaatgaaactactttttggcaatgcaaaaactggcagtggggeggcctttec 461CDHFEEAKVTCSGBREPRtiV
1381 tgtgataatttcgaagaagceaaagttaectgctcaggocacagggaacccagactggtt 481 GGEZPC8GRVEHKBGDVWG8
1441 ggaggagaaatcccatgctctggtcgtgtggaaatgaaacaeggagaegtgtggggctcc 501 VCOFDL8LEAA8VVCRBLQC
1501 gtctgtgattttgacttgtctctggaagctgccagtgtggtgtgcagggaattacaatgt
521GTVVSZXiGGABFGEG8GQZH
1S61 ggaacagtcgtetctatcctagggggagcacattttggagaaggaagtggacagatctgg 541 GEEFQC8GDB8BXiSLCSVAF
1621 ggtgaagaattccagtgtagtggggatgagtcccatctttcactatgctcagtggcgccc 561 PLDRTCTHSRDVSVVCSRYZ
1681 ccgctagacagaacttgtacocacagcagggatgtcagcgtagtctgctcacgatacata
581 DZ RIiAGGE 8 8 CEGRVELKTL 1741 gatattcgtctggcaggcggcgagtcetcetgtgagggaagagtggagetcaagacactc

E)NO:26 and 27

6010 A W 0 P L C 8 BHKDMEDAHVLC
1801 ggagcctggggtceectetgcagttctcattgggacatggaasratgetcatgtcfctatgt
CaiQQZiKCQVAQSXPEQAHPaiC, 0 1861 cagcagctgaagtgtggggttgcccaatctattccagaeggagcacattttgggaaagga
641 A Q Q V W 8 H M F H C T G T B E H Z G D 1921 gctggtcaggtctggagtcBoatgttcoactgcaotggaaetgaggaaoatataggagat
66lCtiMTAI,GAPTC8BOQVASVX 1981 tgcctcatgectgctctgggtgcgccgacgtgttcegaaggacaggtggcctctgtcatc
681CSOHQB,QTLLPC88LfiPVQT 2041 tgctcaggaaaocaatcocagacactattgeeatgtagttcattgtctccagtooaaaca
701 T S 8 T X P K E 8 IVPC X A S G Q L R 2101 aceagetctaeaattccaaaggagagtgeagttccctgcatagcaagtggccagottcgc
721LVGGGaRCAGRVEVYHEGSK 2161 ttggtaggtggaggtggtcgctgcgctggaegagtggaggtctaccacgagggetcttgg
7410TVCDDHMDMTDAKVVCICQI, 2321 ggcaccgtctgtgatgacaattgggatatgactgatgccaatgtggtgtgcaagcagctg
761DCGVAINATG8AYFGKGAOA 2281 gactgtggcgtggeaattaacgecaetggctctgcttacttcggggaaggagcaggagct
781ZWLDBVXCTOK88BZNQCH8 2341 atctggctagacgaagtcatctgeactgggaaagagtctcatatttggcagtgccattca
801 BQNORBRCXBKB.DAOVXCSB 2401 catggctggggacgccataactgcaggcaeaaagaagatgcaggtgttatctgctccgag
821 FMSLRZiTHEAHKENCTGRLE 2461 tteatgtctctgaggctgaccaacgaagcccacaaagaaaactgcacaggtcgccttgaa
841VPYHGTWGBIGB8NHSPTTV 2521 gtgttttacaatggtacatggggcagtattggcagtagcaatatgtctccaaccactgtff
861 GVVCRQXiGCADNGTVKP X t 8 2581 ggggtggtgtgccgtcagctgggetgtgcagacaacgggactgtgaaacccataccttca
881DKTP8RPMWVDRVQCPKGVD 2641 gacaagacaccatccaggcccatgtgggtagategtgtgcagtgtccaaaaggagttgac
901 T1>N'QCPS6PNKQRQA8PS8Q 2701 actttgtggcagtgeecetcgteaocttggaaacagagaeaggceagaocctccccacag
921 E 8 W I X CDNKIRLQEGHTDC8 2761 gagtcctggatcatctgtgacaaeaaaataagactocaggaagggcatacagactgttct
941GRVEXNHKG8MGTVCDD8WD ' 2821 ggaegtgtggagatctggcacaaaggttcctggggaacagtgtgtgatgactcctgggat
961 IiHrDAKVVCXQLGCGQAVXAL 2881 cttaatgatgctaaggttgtatgtaageagttgggctgtggccaagctgtgaaggcacta
981 KBAAFGPGTGPXNLNEXKCR 2941 aaagaagcagcatttggtccaggaaetgggcccatatggcbeaatgaaactaagtgtaga .1001 G NESS LWDC PAKPHSHS DCG 3001 gggaatgagtcttccctgtgggattgtcctgccaaaccgtggagtcacagegactgtggg 1021 BKEDABtQCLPKKTBESHEG 3061 cacaaagaagafcgcttccatccagtgcctccccaaaatgacttcagaatcacatcatggo 1041 TGHPTLTALLVCQAILLVLL 3121 acaggtcaceccaecctcaeggcactcttggtttgtggagceattctattggteetcetc
1061 IVFLLWTLKRRQIQRLTVES
3181 attgtcttcctcctgtggactetgaagegacgacagatteagcgacttacagttteetea 1081 RGEVLIHQV
1 MGGHRMVLLG GAGSPGCXRF VBLGFFWAV S6LLSASAVT
51 RLAGGBNNCB GRVBLK1HDK WGTVCSNGWS MNEV6VVCQQ
101 GHANSSASSB YIWMDKVSCT GHESAUTOCK HDGMGKHNCT
151 DGSKLEMRliV NSAGBRCLGR VBXKPQGKNG
ESAUIDCKHR RPQGBWSTVC HISCBGHEAT CAGIVEVEIQ ATNTOLFPGS OGEXPC8GRV HFGBGSGQIW DIRLAGGESS IPEGAHFGKG CSGKQSQTLL RVEVYHBOSH
201 CGSAISFSGS AKUUG8GPZ WLDKACNGN
251 EDVGVZdiEG ADLSLRLVDG VBRCSGRLEV
301 WCKQWCPT AXBAZGRVNA SBBBOQZKU)
351 KKYCBHRBDA GVTCSDGADIi ELRLV3GGSR
401 HTLADADWC RQIiGCGSALQ TQAKZY8KTG
451 CKNWQW33LS CDNFEEA3CVT CSGHREPRLV
501 VCDFDLBLBA ASWCRBLQC GTW6ZLGGA
551 SHLSIiCSVAP PLDRTCTHSR OVSWCBRYZ
601 GAKGPLCSSH KDHEDARVIiC OQLKCGVAQS
651 CTGTEEHIGD CLMTALGAPT CSBGQVASVZ
701 TSSTIPraSB VPCZASGQLR ZiVGGGGRCAG
751 TDANWCXQIt DOGVAIHATG BAYPGBGAGA
601 HGMGRHNCRH KBDAGVXC8E FMSLRLTOEA HKENCTGRLE
BS1 GS6KMSPTTV GWCRQIiGCA DNGTVKPIPS DKTPSRPMKV
901 TLWQCPSSPM KQRQA6F68Q eSVfZZCDHKZ RLQEGHTDCS
951 WGTVCDDSWD LNDAKWCKQ LGCGQAVKAL KEAAFGPGTG
1001 GNBSSLWDCP AKPM8BBDCG HKEDASIQCL PKMTSESBKG
1051 VCGAZLLVLL ZVFLIiNTZiKR RQZQRIiTVSS RGEVLIHQVQ
1101 LDLLKSSENS NNSYDFNDDG LTSLSKYLPI SGIKKGSFRG
1151 PIiRLEPKKP

NAPGEMKKEL LGCPT8ZKAL HEKDAGVTCS HASVICKQLG GHGXHKCDHA BDHWMiRDAS LHBCKHQEH8 KLTGKMCERG CNGNETTPHQ EMKHGDVWGS GEBFQCSGOE CEGRVELKTL AGQVK8KHFH PCSSLSPVQT GTVC3DDNWDM KESHIWQCHS VFYNGTHG8Z DRVQCPK8VD GRVEIWHKGS PIWLNEIKCR TGHPTLTAIiL YQEMDSKADD TLRRKMIITO

IDNO:27

Examples: Cloning and characterization of MARC-145 CD163
Aforwardprimer5'simianCD163(SEQIDNO:28)(5'-
CACCGGAATGAGCAAACTCAGAATGG-3' based on human CD163) and a reverse primer HuCD163-3'Kpn (SEQ ID NO:29) (5F-
TG^CGGTACXTAGTCCAGCiTCT^ were used to
amplify CD163 cDNA from MARC-145 Aftican Green Monkey kidney cells. Total cellular RNA was prepared from MARC-145 cells using me KNeasy kit RT-PCR parameters were the same as described in Example 4. RT-PCR products were cloned directionally into the pO>NA3.1D/V5/His/TOPO vector according to the manufacturer's instruction. Several clones containing large inserts were analyzed. Clone #25 was designated "pCDNA3.1D-MARC-CD163v2". This novel CD163 cDNA from MARC-145 cells is 1116 ammo acids in length. When compared to the sequences in GenBank database, the MARC-145 CD 163 amino acid sequence is 96.3% identical to human CD163 (Genbank Z22968), 84.7% identical to pig CD163 (Genbank AJ311716), and 73.9% identical to mouse CD 163 (Genbank AF274883).



SEQUENCE
atgageaaac tcagaatggt gctacttgaa gactctggat ctgctgacgt cagaagacat «0
tttgtcaact tgagtccctt cactattget gtggtcttac ttctccgtgc ctgttttgtc 120
accagttctc ttggaggaac aaccaaggag ctgaggctag tggatggtga aaacaagtgt 180
agtgggagag tggaagtgaa aatccaggag gagtggggaa cggtgtgtaa taatggctgg 240
agcatggaag cagtctctgt gatttgtaac cagctgggat gtccaactgc tatcaaagcc 300
actggatggg ctaattccag tgcaggttet ggacgcattt ggatggatca tgtttcttgt 360
cgtgggaatg agtcagctct ttgggactgc aaacatgatg gatggggaaa gcatagtaac 420
tgtactcace aacaagatgc tggagtgact tgctcagatg gatccgattt ggaaatgagg 480
ctgacgaatg gagggaatat gtgttctgga agaatagaga tcaaattcca aggacagtgg S40
ggaacagtgt gtgatgataa cttcaacatc aatcatgcat ctgtggtttg taaacaactt BOO
gaatgtggaa gtgctgtcag tttctctggt tcagctaatt ttggagaagg ctctggacca 660
atctggtttg atgatcttat atgcaacgga aatgagtcag ctctctggaa ctgcaaacat 720
caaggatggg gaaageataa ctgtgatcat gctgaggatg ctggagtgat ttgeteaaag 7BO
ggagcagatc tgagcctgag actggtagat ggagtcactg aatgttcagg aagattagaa 640
gtgagattcc aaggagaatg ggggacaata tgtgatgacg gctgggacag tcatgatgct 900
gctgtggcat gcaagcaact gggatgtcca actgctatca •cegocattgg tegagttaac 960
gccagtgagg gatttggaca catetggctt gacagtgttt cttgccaggg aeatgaacct 1020
gcggtctggc aatgtaaaea ocatgaatgg ggaaagcatt attgcaatca caatgaagat 1080
gctggcgtaa catgttctga tggateagat ctggagctaa gacttagagg tggaggcagc 1140
cgctgtgctg ggacagttga ggtggagatt cagagactgt tagggaaggt gtgtgacaga 1200
aoetoooaac toaaaaaacjc tqatgtggtt tgcacgcagc tgggatgtgg atctgcactc 1260

ID NO
SEQ ID NO: 30

- . .. -. -M-^-.»»..- -•--% nir-ftf |ji aaaacatcct ateaagtata ctocaaaato caggeaacaa acatgtggct gtttctaagt
agotgtaacg gaaatgaaae ttctotttgg gaetgeaaga aetggoaatg gggtggactt acctgtgate actatgaaga ageeaaaatt acctgoteag ooeaoaggga aeocagactg gttggaggag aeatteoctg ttetggacge gttgaagtga agcatggtga oacatggggc tcegtetgtg atteggattt ototetggaa getgeoagcg ttetatgeag ggaattacag tgtggeacag tegtetetat 'eatgggggga getcaetttg gagagggaaa tggacagatc tgggetgaag aattoeagtg tgagggacat gagteeeato ttteaetctg cocagtagca eceegeeeag aaggaaettg tagceacago agggatgttg gagtagtetg eteaagatao acagaaattc gcttggtgaa tggaaagaeo ceatgtgagg geagagtgga gcteaaaaog cttaatgeet ggggateect etgcaaetet cactgggaca tagaagatgc ceacgttctt tgccaaeaae ttaeatgtgg agttgccett tetaeoeeag gaggageaea ttttggaaaa ggaaatggtc aggtctggag geatatgttt caotgeactg ggaetgagea goacatggga gattgtoctg taactgetet gggtgettca ctatgtcctt cagggcaagt ggcctctgta atttgctcag gaaaecagte eeaaaeaotg tcctcgtgca atteateate tctgggccca aeaaggceta eeatteeaga agaaagtgct gtggeetgea tagagagtgg teaacttege ttggtaaatg gaggaggteg ctgtgctggg agagtagaga tttatcatga gggctectgg ggcaceatot gtgatgaeag ctgggacctg agegatgccc aogtggtgtg cagacagotg ggctgtggag aggecattaa tgccaetggt tctgctcatt ttggagaagg aacagggooc atetggctgg atgagatgaa atgeaatgga aaagaatccc gcatttggca gtgccattca catggctggg ggcagcaaaa ctgcaggeac aaggaggatg caggagttat otgeteagag ttcatgtctc tgagactgac cagtgaagcc agcagagagg oetgtgcagg gcgtctagaa gttttttaca aeggagcttg gggeagtgtt ggeaggagta acatgbctga aaccactgtg ggtgtggtgt gcaggcagct gggctgtgca gacaaaggga aaatcaaoec tgcatcttta gaeaaggcca tgtccattcc catgtgggtg gacaatgttc agtgtccaaa aggaectgae acgctgtgge agtgcccatc atctccatgg gagaagagac tggccaggec ctcggaggag acctggatea catgtgacaa caagatgaga ctacaagaag gacccacttc ctgttotgga cgtgtggaga tctggcaegg aggttcctgg gggacagtgt gtgatgaetc ctgggacttg aaegatgcte aggtggtgtg tcaacaactt ggctgtggtc cagetttgaa ageatfccaaa gaagcagagt ttggtcaggg gactggaccc atatggetca atgaagtgaa gtgcaaaggg aatgagtctt cettgtggga ttgtcctgcc agacgctggg gccacagtga gtgtggacac aaggaagacg ctgcagtgaa ttgcacagat atttcaacga acaaaaeecc acaaaaagcc aeaacaggtc agtcatccct tattgcagtc ggaatecttg gagttgttct cttggtcatt ttcgtcgcat tattettgac tcaaaagcga agacagagac agcggcttac agtttcctca agaggagaga aettagtcca ccaaattcaa taccgggaga tgaattettg cotgaatgca gatgatctgg aectaatgaa ttcctcagga ggccattctg aggcacactg aaaaggaaaa tgggaattta taacccagtg agccttgaag ataccttgat gaagacctgg acta

1320 1380 1440 1SOO 1S60
i«ao
1660
1740
1800
18*0
1920
1980
2040
2100
2160
2220
2280
2340
2400
2460
2S20
2560
2640
2700
2760
2820
2880
2940
3000
3060
3120
3180
3240
3300
3360
3414



1MSKLRMVLLBDSGSADVRRH 1 atgagcaaaetcagaatggtgctacttgaagactctggatctgctgacgkcagaagacat 21FVNLSPPTIAVVLLIiRACFV 61 tttgtcaacttgagtcocttcactattgctgtggtcttacttctccgtgectgttttgtc 41TSSLGGTTKKLRLVDGEHKC 121 accagttotcttggaggaacaaccaaggagctgaggotagtggatggtgaaaacaagtgt 61 S 6 R V E V K I Q B B WGTVCKKSW

SEQID NO: 31
and 32

••'•"•' •'"" "•"•"— —"• -"••
181 Bgtgggagagtggaagtgaaaatccaggaggagtggggaacggtgtgtaataatggetgg
81 S M E A - V B V 1C K Q X> Q C F T A IK A 241 agcatggaagcagtctctgtgatttgtaaccagotgggatgtcoaactgctatcaftagce 101TGNAVS8AGSGRXWMDBV6C 301 actggatgggctaattccagtgcaggttetggaegaatttggatggatcatgtttcttgt 121RGNESALWDCKHDOMGICH6N 361 cgtgggaatgagtcagctctttgggactgcaaacatgatggatggggaaagcatagtaac 141CTBQQDAGVTCSDOSDLBMR 421 tgtact caccaacaagatgctggagtgacttgctcagatggatccgatttggaaatgagg 161LTNGGNMCBGRIBIKFQGQW 4 Bl ctgacgaatggagggaatatgtgttctggaagaatagagatcaaattccaaggBcagtgg 1810TVCDDHFHXKHA8VVCKQL 541 ggaacegtgtgtgatgataacttcaacatcaatcatgoatctgtggtttgtaaacaactt 201 BCG6AVSF6GBAWFOBGSOP 601 gaatgtggaagtgctgtcagtttctctggttcagctaattttggagaaggctetggacca 221INFDOX.ICNONB8ALWNCXB 661 atctggtttgatgatcttatatgcaacggaaatgagbcagctctctggaactgcaucat 241QGWOKHNCOHABDAGVICSX 721 caaggatggggaaagcBtaactgtgatcatgctgaggatgetggagtgatttgctcaaag 261 QADLBIiRLVDOVTBCBSRIiC 781 ggagcagatctgagcctgagactggtagatggagteactgaatgtteaggaagattagaa 281 VRPQOBKGT1CDDOHD8HBA 841 gtgagattccaaggagaatgggggacaatatgtgatgacggctgggacagteatgatgct 301AVACKQLOCPTAITAIORVK 901 gctgtggcatgcaagcaactgggatgtccaactgctatcaccgocattggtcgagttaac 321A8EGFGHIHI,D8V6COOBBV 961 gccagtgagggatttggacacatctggcttgacagtgtttcttgecagggacatgaaoct 341AVWQCKHHEWGKHYCHHHBD 1021 gcggtctggcaatgtaaacaccatgaatggggaaagcattattgcaatcacaatgaagat
361 AGVTCSDGBDLBLRLROOGS 1081 gctggcgt aacatgt t ctgatggat cagat ctggagct aagacttagaggtggaggoagc
3B1 RCAGTVEVBZQRLLQKVCDR 1141 cgctgtgctgggacagttgaggtggagattcagagactgttagggaaggtgtgtgacaga
401 GWOLKEADVVCRQL6COSAL 1201 ggctggggactgaaagaagctgatgtggtttgcaggeagctgggatgtggatctgcactc
421 KTSyQVYSKIQATNMHLFLB 1261 aaaacatcctatcaagtatactccaaaatccaggcaacaaacatgtggetgtttetaagt '
441 8CNGNETSLWDCJCNWQWGOL 1321 agctgtaacggaaatgaaacttctctctgggactgcaagaactggcaatggggtggactt
461 TCDHYEKAKITCSAHREPRL 1381 acctgtgatcactatgaagaagccaaaattacctgctcagoocacagggaacccagaotg
481 VQGDIPCSGRVEVKHGDTWO 1441 gtt ggaggagacat t ccctgtt c t ggacgcgttgaagtgaagcatggtgacacacggggc
501SVCDSDF6IiEAASVI>CRELO 1501 tccgtctgtgattcggatttctctctggaagctgccagcgttctatgcagggaattacag
521 CGTVVE ILGGAHFGEGNGQX 1561 tgtggcacagtcgtctctatcctggggggagctcactttggagagggaaatggacagatc
541 HAKEFQCEGHESHLSLCPVA 1621 tgggctgaagaattccagtgtgagggacatgagtcccatctttcaetctgcccagtagca
561PRPEGTCSHSRDVGVVCSRY 1681 ccccgcccagaaggaacttgtagccacagcagggatgttggagtagtctgcteaagatac
581TEIRLVNGKTPCEGRVELXT 1741 acegaaattcgcttggtgaatggcaagaccccatgtgagggcagagtggagctcaaaacg
601 LNAWGSLCNSHHDIBDAHVL 1801 cttaatgcctggggatccctctgcaactctcactgggacatagaagatgcccacgttctt
621 CQQLKCGVAL6T PGGAHFGK 1861 tgccaacaacttaaatgtggagttgccctttctaccccaggaggagcacattttggaaaa
641 GNGQVWRHMFHCTGTEQHMG 1921 ggaaatggtcaggtctggaggcatatgtttcactgcactgggactgagcageacatggga
661DCPVTALGASLCPSGQVASV 1981 gattgtcctgtaactgctctgggtgcttcactatgtccttcagggcaagtggcctctgta
681 ICSGNQSQTL66CNSSSL«P 2041 atttgctcaggaaaccagtcccaaacactgtcctcgtgcaattcatcatctctgggceca
701 TRPTIPBBEAVACIEBGOLR 2101 acaaggcctaccattccagaagaaagtgctgtggcctgcatagagagtggteaacttcgc
721 LVNGGGRCAGRVEIYHEGEW 2161 ttggtaaatggaggaggtcgctgtgctgggagagtagagatttatcatgagggctcctgg
741 GTXCDDSHDL8DABVVCRQL 2221 ggcaccatctgtgatgacagctgggacctgagcgatgcccacgtggtgtgcagacagctg
761 GCGEAINATGSAHFGEGTGP 2281 ggctgtggagaggccattaatgccactggttctgctcattttggagaaggaacagggccc
781 IWLDEMKCNGKESRIWQCHS
2341 atctggctggatgagatgaaatgcaatggaaaagaatcccgcatttggcagtgccattca
B01 HGWGQQNCRHKEDAGVICSE 2401 catggctgggggcagcaaaactgcaggcacaaggaggatgcaggagttatctgctcagag
821 FMSLRLTS EASREACAGRLE 2461 ttcatgtctctgagactgaccagtgaagocagcagagaggcctgtgcagggcgtetagaa
841 VFYNGAWGSVGRSNMSETTV 2521 gttttetacaacggagcttggggcagtgttggcaggagtaacatgtctgaaaccactgtg
861GVVCRQLG CADKGKINPASL 2581 ggtgtggtgtgcaggcagctgggctgtgcagacaaagggaaaatcaaocctgcatcttta
861 DKAMSI PMWVDNVQCPKGPD
2641 gacaaggccatgtccattcccatgtgggtggacaatgttcagtgtccaaaaggacctgac

2701 eegctgtg0CB3tgcocatcat«tcc»tgggagBagagaetggocaaoecctcggagg«g 921 T W I TCDNKMRLQBO P T S C6 fl 3761 aoetsgatcacatgtgaeaacaagatgagactacaagaaggaeceacttcotgttctgga 941 R V B IKH008HOTVCDD6WDL 2821 cgtgtggagatetggcacggaggttoctgggggacagtgtgtgatgacteetgggacttg 961 H D A Q V V C Q QLOCOPALKAFX 2681 aaogatgctcaggtggtgtgteaacaaettggctgtggtccagctttgaaagcattcaaa 981BAEFGQGTOPIHLHBV1CC1CG 2941 gaagcagagtttggtcaggggactggaoocatatggotoaatgaagtgaagtgcaaaggg 1001 H B B 8. CMDCPARRWOHSBCGH 3001 aatgagtettccttgtgggattgtcotgecagacgctggggceaaagtgagtgtggacac
3061 aaggaagacgctgcagtgaattgeacagatatttcaaogaacaaaaoocoacaaaaagca

3121 aeaaaaggtcagtoatocottattgoagtoggaateettggagttgttetcttggtcatt



1081 R 0 BHX.VHQXQYRBHN6CLNA 3241 agaggagagaacttagtecaooaaattcaataoegggagatgaattottgcctgaatgea 1101 DDLDXiHHSSGGHSBAK 3301 gatgatetggacetaatgaattcctcaggaggocattctgaggcacac

1 MSKLRMVUjB D8GBADVRRH PVNLSPPTIA WLLLRACFV TSBLQGTTKE SEQID NO: 32
51 LRLVDOBHKC SQRVBVKIQE BMGTVCtlNGM 8MSAVBVXCN QLOCPTAIK&



151 CSDOBDLBMR X/ENSGNMCSG RIBXKFQ3QH wiVCDDNFNI NHASWCKQL

201 BOGSAVSF6O 8ANFQEGSQP IHFDDUCHG HBSAIiHNCKH QGHGKHNCDH 251 ABDA8VXC8K OADL6LRLVD aVTBCSGRLE VRPQQBNQT1 CDDGHDSBDA

301 AVACKQLGCP TAl*AIORVH ASBSPCQUHIi DfiVflCOdHttt AVHQCKHHKH

351 GKHVOfflNED AOVTCSDSSD liBfiHTiROGGS RCA9TVBVEI QRLIiQKVCDR 401 OWQLKKADW CRQLOCXJSAL KT6YQVY6KI QATNHHLFLS 8GNGNBT8UI

451 DCKHHQNSSIi TCDHYBBAKI TCBAHRBPRIi VOGDIKJiiUk VEVKHODTHO 501 SVCDEOPSLB AA6VLCRBLQ CGTWSILOG AHPOBGHOQI HABBFQCE^ 551 ESHLBLCPVA PRPBGTC8R8 ROVSWCSRY TEIRLVMOKT PCEGRVBUtT 601 LNAWOSIiCNB HHDIEDAHVL CQQLKOOVAL 8TPGGAHPGK ONOQVHRHMF 651 HCTQTBQHMG DCPVTALQAS LCPSGQVASV ICSQNQBQTL SSCHSSSLGP 701 TRPTIPEESA VACIKSOQLR LVNOOQRCAG RVEIYHBOSW GTZCDDSWDb 751 8DABWCRQL OCGEAZKATS SAHFGBQTGP IWLDEMKCHO KESRIWQCHS 801 HGWQQQNCRH KBDASVZC8B PH8LRLTSBA BREACMRLE VFYNQAWQSV 851 6R8MM6BTTV SWCRQIiOCA DRGKINPASL DKAMSZPMHV DNVQCPKBPD 901 TLWQCPS8PW BKRLARPSEE TNITCDNXMR U3BQPTSCSG RVEIWHQQSW 951 OTVGDDBHDIi NDAQWCQQL OC3PA1.KRFK EABFQQ6TGP IWtHBVKCKO

1001 NESS1.WDCPA RRHGHSBC6B KEDAAVNCTD ISTHKTPQKA TTGQSEL1AV 1051 QILOWLLVI FVAbFLTQXR RQRQRLTVSS RGENLVKQIQ TOBMNBCLHA 1101 DDLDLMNS86 GHSEAH

Example 9. Cloning and characterization of simian CD163 from Vero cells.
A forward primer 5'sunianCD163 (SEQ ID NO: 28) CACCGGAATGAGCAAACTCAGAATGG-3' based on human €01=63) and a reverse primer HuCD163-3'Kpn (SEQ ID NO:29) (5'-
IXKTCCGGTACCTAGTCCAGGTCTTCATCAAGGTATCTTA-3') were used to amplify CD 163 cDNA from Vero cells. Total cellular RNA was prepared from Vero cells using the RNeasy kit RT-PCR parameters were the same as described hi Example 4. RT-PCR products were cloned directionally into the pCDNA3.1D/V5/His/TOPO vector according to the manufacturer's instruction. Eight clones containing large inserts were sequenced, and six discreet splicing patterns were found. These patterns are depicted graphically hi Figure 17.
The six splicing variants differ in the presence or absence of three exons, designated E6, E105, and E83. Omissions of E6 or E105 do not change the reading

frame, whereas omission of E83 does. Patterns similar to v2 and/or v3 were also seen in porcine, murine, human, and MARC-145 monkey cells. Patterns v4 and v5 lack the 105-nucleotide exon that encodes the hydiophobic transmembrane region. These cDNAs were unable to render BHK cells permissive to PRRS V infection in a transient transfection assay, probably because the CD163 is secreted rather than remaining membrane bound. Although CD163 molecules lacking a transmembrane region appear to be non-functional as cellular pennissivity factors, it is possible that they may have utility either in direct virus neutralization (similar to neutralizing antibodies), or as an immunogen for the induction of anti-CD163 antibodies which would block viral infection in the host animal.
The longest spli


SEQUENCE

ID NO



IMSKXiRMVXiXiBDSGSADVRRH 1 ATQAGCAAACTCAGJATOGTGCTACTTGAAGACTCTQGATCTGCTQXCGTasOAMJ^
21 F V N L 8 P FT I A V V Ii Ii LRAC F V 61. TTTOTCAX
41 T S S LGGTTKEI.RLVDGENKC 121 ACCAI3TTCTCTW3BMQUiCMXXy^
61SGRVEVKIQBBHGTVCHH,GH 1B1 A(nXH3GAGAGTGGAAGTCUUUATCXaQGAQGAOTGG 81SMEAVSVI CNQLGCPTAIKA 241 AGKaTGGAASCJMmsrCTaT^TTTGTAACCAGCTGQGAIC^^ 101TGWAN66AGSGRIHMDHVBC 301 ACTGGATGGULTAATTCCAGTGCAGtflTC'JW^OGCAlTiUGAl'QGATCA'lViTT'l'Oi-iiji 121 RONS S. ALWDCKHDGKGKHBR 361 OGTGGGAATGAaTaWCTCTTTOGGACTGCAl^^
141CTHQQDAQVTCSDGSDI.EHR 421 TGTACTCACCAAC*AGATG
SEQ ID NO:33 and 34

241 Q G V OK~F~H"cDHABDAGVXCSi: -721 CMSCRTGfSaQ&AAQCATAAC^^
261GADXiBXiRLVDQVTECSORXiB 781 281VRPQaBWOTXCDDGICD6BDA 841
301AVACXQX,acPTAXTAXG 321 A 8 X G 7 G B X M Xi D S V 6 C Q G B B P 961 CK!CA(H 341AVWQCKHHEHGKHYCNHNBD 1021 OC«mTOOaU«OTAAACaCCAT(»ATO 361AOVTCSDG8QXi8LRIiRGGa8 1081
381RCAGTVBVBXQRLIiaKVCDR 1141 GKTQWKTOaQUSXtTIQAaOTQQJkSATTClW^
401QWaLKBADVVCRQIi6C06AIi 1201 QBCH3Q(3 421KTSYQVYSK3QATNMHLFLS 1261 AAAACATCCTrATCAMTATACTCXaiAAATOCaOOCAACAAftCa^^
4418CHGNBT6IiMDCKNWQKGaii 1321 taCKWiaBQMAVSUWCtfK^^
461TCDHYBBAKITCfiAHRKPRL 1381 ACCTClXUTCACTArr6AA€AASCaUVAATTACCRXnxaaCCC^^
481VGGDX PCSGRVBVXHGDTW43 1441
SOlSVCOBDFSLEAASVIiCREIiO 1501
521 CO T V V B I LGGAH FGSGKGQ X 1561 TO^RXiCAaumKTarTATtCCTOGaaGGAGCIXACTTI^^
541WTEEFQCEGBE6HIi6LCPVA 1621 TGGACTGAAGAATTCCAGTm'GAGGGACATGAGTCCCATCTTTCACTaX^^
561PRPBGTCBH8RDVGVVC8RY 1681 CXXXXX:CC&GAAG»AACrTCTAGOatt»GCAtXm^
581 TE XRLVNGKTPCBGRVBLKT 1741 AC3kSAMTTCGCnWnX»ATaaCAAGAaXXATGTGAG93C3^^
601LNAHGSLCNSHWDIEDAHVL 1801 CTTAATGCCTGGGOATCrCTCTGCAACTCl^aCTOQGACATAGAAGATC
621 COQLKCGVALSTPGQAHFOK 1861 TQCCAACAACCTAAATOTGOAarTGCCCrTTCTACCCXaOGAOGAGCACATTTT^^
641GKGQVNRHMFRCTGTEQHMG 1921 OOAAATGGTOUKITCTGGAGQCATATGTTTCACTaCACTOGGACTGftOCAG^
€61 DC PVTALGASLCPSGQVABV 1981 GATTGTCCIGTAACIGCTCIGGGIIGCTraUn-ATGTCCITC^^
681ICSGNQBQTLSSRNSSSLGP 2041 kTTTGCTCK3GM&CCHQ'?CCC}AkCk 701 TRPTX PEESAVACXBSGQLR 2101 ACAAGGCCTACCATTCCAGAAGAAAGnXTGrGGCCIGCATAGAGAGTGGnaUKnraCGC
721LVNGGGRCAGRVEIYHBGSH 2161 TTGGTAAATXXUUSGAGGTCGCTGTGCTGGGAGAGTAGAGATTrATCATGAGGGCTCCTGG
741 G T X CDDSWDLSDAHVVCRQL 2221 GGC»CCATCTGTGATGACAOCTGGGACCTGAGKXaTGCCC^^
761GCGEAXNATGSAHFGEGTGP 2281 GQCiqTGGAGAGQCCACTAATGCCftCTGGTTCTGCTXaTTTTgGAgAAQGA^
781IWLDEMKCNGKESRIWOCHS

r

'ATCTOGCTGaATOiilUTQAAATGa^
L H OH 8' Q 2461 TTCiVTQTCTCTflAflJV^TflAOO ftfilT^BR Aiflfi^^'^^^^^^^^^^y*lPm^lPimHQQGrPC*PJfcf3B B
861GVVCRQLGCADKGKZN8A6L 2581 GGTOTAGTGTGCAGGCAGCTGOGCTGnrGaWJACAAAGOQAAAATCAACT^^
881 DXAMSZPMWVDHVQCPKGPD 2641 GACAAGOCCATGTCCATTCCCATGTGOGTGGAa^^
901TX>NQCPB8PNBlCRIiARP8BB 2701 ACGCTGTOGCAGTOCCCATCATCTCCATGGeMAAaAaACI^^
921TWZTCDHKMRLQBGPTBC8G 2761 ^CCl\^>TCACBTOT^CAKCAAMTOABACTACAAliftV'K33>CCCT^
941RVEZWHG08WGTVCDDSWDI, 2821 CUUX^l\JGAOAu^U\X3CACQGAOGTTCCi^UUGGACA>HtfA^l!BATGACt^JL"lXjUUACTTO
961 R D A Q V V C Q Q L 0 C G P A L K A P K
981BAEPGQGTGPZKLNBVKCBG 2941 GAAGCAGAGTTTGGTCAOCK3aACTGGACCCATATGGCTCAATaAAGTGAAGTGCaAAGaa 1001 HEB6I.WDCPARRHGHSECGH 3001 AATGAuUViui!Cu"ii\*a\sGGATI^Uv^Ji\jCCAGAOGC'i\*«GGCCACAGTGAGi\4'i\»GACAC 1021 KEDAAVKCTDISTRKTPQKA 3061 AAGGAAGA 1061 F V A L P t -T - Q K R R Q R Q R L T V 8 S
3 !L fi i TTOflTcsc!flTTJftTTC^J^x^^c!T^^ukftA^GKfQ3uvlSRCJ^fl^lGftC'^^^G(yy
1081 ROEHLVHQIQYREMNSCLNA
1101 DDLDLMH8SGGHSEAH 3301



1 MSKLRMVLLE D8G8ADVRRH FVHLSPFTIA WLLLRACFV TSSLGGTTKE LRLVDGENKC 61 8GRVBVXZQE EWOTVCNHGW BMEAVEVZCN QLGCPTAIKA TGHAN86AGS GRIWMDHVSC 121 RGMBSALNDC XHDGNGKH8K CTHQQDAGVT CSOGBDLEHR LTHGONMC8G RZEZKPQGQN 181 GTVCDDNPNZ HHA6WCXQL B098AVBP8G 8AKFGBBSGP ZWPTOLICNG NESAtMNCKH 241 QGHGKHKCDH AEDAGVICSK GADLSLRLVD GVTEC8GRLE VRFQGEWGTI CDDGMD8HDA 301 AVACKQLGCP TAZTAZGRVN ASBGPSRZMIi DSVSCQGHEP AVHQCXHHSH GKHYCKBNED 361 AGVTCSDGSG LELRLRGGGS RCAGTVBVBZ QRLLGKVCDR OWSLKEADW CRQLGCGSAL 421 KTSYQVY6KI QATNMHLPLS 8CHQNET8IiH DCXNWOMGGL TCDHYK1AKI TC6AHRBPRL 481 VG90ZPCSGR VEVKHGDTWG SVC33SDPSLB AASVIiCRELQ CGTWSILGG AHFGBGHGQZ 541 NTEEPQCBGH ESHLSLCPVA PRPBGTCSHS RDVGWCSRY TEIRLVNGKT PCEGRVELKT 601 UWWGSLCNS HHDZBDABVZi OQOUGOGVAL STPGGAHFGK GNGQVHRRMF HCTGTBfflJMG 661 DCPVTALGAS IiCPSGQVASV ZCSGNQSQTIi SSRHSSSLGP TRPTIPEBSA VACIESGQLR 721 LVNQGGRCAG RVEZYHBOSH GTZCDD8WDL EDAHWCRQL GCGBAINATG EAHPGBGTGP 781 ZKZiDBNKCNG KESRZNQCHS H6NQQQNCRH KEDAGVICSB FMSLRLTSBA SREACAGRLE 641 VFYNGAWGSV GRSNMSETTV GWCRQbGCA DXGKZNSA8L DKAH6ZPMHV DNVQCPKGPD 901 TXiMQCPSSPN EXRIARP88E THITCDNKMR {«EGPTSCSG RVEIWHGGSW GTVCDDSHDL 961 NDAQWCQQL GOGPAIiKAPK BAEPGQGTGP ZNUiEVKCEG NBSSLHDCPA RRHGH8BCGH 1021 KBDAAVNCTD ZSTRKTPQKA TTGQSSLIAV GILGWLLA1 PVALPLTQKR RQRQRLTVSS 1081 RGENLVHQIQ YREHHSCLNA DDLDIJWSSG GHSEAH

SEQ ID NO: 34



IMEKLRMVLLEDeOBADVRRH
1 ATGAGCAAACTCAGAATGGTGCTACnXSAAGACTCTCK^TCTOCTGACGTCAGAAGACAT 21PVHLS PPT ZAVVIiItZiR A C F V
61

SBQ ID NO: 35 and 36

CHQliGCPTAIKA
I
MEAV
BlB 241
101 T O W__X N8SA06GR I H M D H V B, C 301 ACTGQATGGGCTAATTCCAinGCAGUTUOyGACGCATTTGGATGQATCATtfriTCTTOT 121 RaVEBALMDCKBDaNQKBSR 361 a3TGGGAATGAGTa«KnXrirrGOGACIGCAAACATaATOaA*^^ 141CTHQQDAOVTCBDQ8DLKMR 421 TOTACTX»CaUUajyt»T 161IiTlfao»KCSaRISIKPQQQ1l 481 eKUCBAATGGAGGaAMATGTGTTCTGGAMAATAaAGATCAAATTCC^^ 181 0 TVCDDHFKXNBA8VVCKQ.il 541 Q
781
DA
QGB
HaTZCDDOWDSR
A O
281VRF
RVN
841
PT
301AVACKQIiQC
901
B V S C Q 6 B B P
321 A 8 B O F 0 B IK ED
ffaXByCKBNBD
U*^ DS8DIiBIiRLRQGSB
-961 OCCAOTGBUXiOAWTgSACACK 341 AVWQCXBB
1021 361AOVTC8
XiLOKVCDR OLXBADVVCRQLGC66Ab
1081 OCTQaCOTAWaTOTT 381RCA.GTVBV
OR
BVBI 1141
40iaW 1201 OOCTOaoaACTaJULAaAMK^rGATOTOGTTTOCAaOCAOCIX3OaXTG7^^
421 KTSYQVYSKIQATNMWLFIiS 1261 AAAACATCCTATC3UfflTATACTCCaAAATC(3>3GC3UlCAAACaTBlX3OC^
441 BCNGNETSIiWDCKNNQWGGL 1321
461 TCDBYEBAXITCBABRBPRIi 1381 ACCTGTOATCACTATOAAGAASCCAAhACTAOCTGCTatO^
481 VGGDXPC6GRVBVKHGDTNG 1441 GTTGGAGGAC»CATTCCCTaTTaroGACOCGTTGAACTR^
rrTCTATG^ HPOEGNQQI
SOlSVCDSDFSLBAASVIiCRBLQ 1501 TCCS3TCTSTGATTCGGATT
IiGGA
521 CGTVVSI 1561 TGTGGOU»GTtSTCT
541 NTBBFQCEGBB6B L 8 L C P V A 1621 TGGACTGAAGAATTCCAGTGTGAGGGACATGAGTCCCATC1TTCMnlCTGCCCA6XIIlGCA
561 PRPBGTC8BBRDVGVVC8RY 1681 CCXCGCCOUSAAQGAACTTGTAGaaCAGCAQGGATGTTGaM^
581 TE IRliVNGXTPCBGRVELKT 1741 ACAGUUACTCGO-TGGIGAAIGGCAAGACXXXJiaGTGAGGGCAaAGTaGAGCTCaVAIlAO^
601LNAWGSLCHSHWDI EOABVL, 1801 CTTAATGCCTQGG 621CQQLKCGVALSTPGGAHFCK 1861 TGCCAACAACTTAAATGTGGAGTTOCXrcrTTCTACC«aK3GAS^^
641GHGQVHRBMFBCTGTBQBNG 1921 GGAAATGGTCaGOTCTGGAGOCaTATGITTCAiCTGCACKSGGACrGAOCAGC^^
661 D C P V T A_L_ G A B Xi C_P S G Q V_A6 V 1981 OATTQTCClTiTAACTGCTCTGGGTOCTTCftCTAUVyi'CC'lTCAGGGCAfcGTGGC'C'l'C'I'yiA
eeilCSGNQSQTLSBCHBSSLGP 2041 ATTTQCTCAGGAAACCAGTCCrAAACACTGTCCTCX3T(KSlATTCATCATCTCT^
701TRPTZPBE6AVACZ BSGQLR 2101 ACAAGGCCTACCATTCCAGAAGAAJVSTGCTGTGGCCTGCATAGAGASTOGTCAACTTt!^
VSlIiVRGGGRCAORVEI YBBGSW 2161 TTGGTAAATOGAGGAGGTC!GCTGTGCTOaGIU3AGTAGAGATTTATCATGAGGGCTCCTGG
741GTICDDEWDLBDAHVVCRQL 2221 GGOtCCATCXCTQATGACASCTO^CCTGAGCmTGGOCftCG^^
761GCGEAIHATG6ABFGBGTGP
2281
WLDEMKCNGKESR I H Q C H S
781I 2341
B01HGWGQQHCRHKBDAGVICSE 2401 CATGGCTGGGGGC»GaUMACIGCAGaCACAIU3GAGGATGCAGGAGTTATCTGCrCAGAG
821FMBLRLT8BASREACAGRI.E 2461 TT(ATGTCTCTGAGACTGACCAGTGAAGCCAGC»GAGAGGGCn»TGCAGGGCBTCTAGAA
841VFYKGANGSVGR8NM8ETTV 2521 GTTTTITACAAOaGAGCTTGGGGCAGTGTTaGahaGAGTAACATGTCIGAAACObCTGTG
B61GVVCRQIiGCADKGK IHSA8L 2581 GGTOTAGTGTGCAGGCAGCTGGGClXnBCAGACAAAaGGAAAATCAACTCn^
8B1DKAMSIPMWVDNVQCPKGPD 2641 GACAAGOCCATGT(XArTaXATGT 901TLWOCP5SPHE1CRLARPSEE

!.«.....„
.,,..-• 921- T . If Z . T C D 19 KM R Ii Q E 0 P T 8 C 8.

2761 ACCTOGATCJlCATOTraCAACAAtaTQAGACTACAAfflUMa^^



961VOAQVVCQQ&OCOPAZ.KAFK

2681 AACGATOCTCAGGTGGTGTOTCA&CAACTltSGCTQTaGTCCAOCITTOAAAGiJlTTCAAA 981IABFGQaTOPX1IIiHBVKCKG 2941 CUlAGCAfUVGTTTQGTCAGGGGACTOTACCCATATGKTCTX^^



3061 AAGOAAOACSKSraeaVGTaAATTaaiCAGATATTTCAAOaOOCAAAACCCawauU^^

1081 ROBNLVHQIQYRBMHSC'LHA

3301 aATOATCTOOACCTAATGAATTCCTakSAAAATTeCAATa^^





3421 CTGAaGCACACTGAAAAGGAAAATGOGAATTTA

1 MSKLRMVLLB D8G6ADVRRH FVHL6PFTIA WLLLRACFV TSSLGGTTXK UUiVDGBMKC SEQ ID NO:3€
61 8QRVXVXZQE BWQTVCNNGW 6NXAV6VICN QLGCPTAIKA TGHANB8AGS ORXMMDHV8C



181 GTVCDDNFKI KHASWCKQL BCGSAVBFBG BAKFGBG8GP ZWFODLZCNQ NEBAUOKKH

241 QGKGKHNCDH AEDAOVIC8X GADL£UUiVD OVTBCSQRIiB VRi^UitHtfTJ. CDDGMD6UDA

301 AVACKQIiOCP TAZTAZORVN ASEGFGHXHL 08V8OQGKEP AVNQCKUHBH GKHXCHIblftb 361 AGVTCBDGSB LEIiRLRGGGS RCaWJTVBVEI QRLLGICVCDR GMSbkEADW C3JQWJCQSAL



481 VOGDZPCSGR VBVKHQDTWG EVCDSDFSLH AASVIiCRBIiQ' OGTWBIIiGG AHFGSGNGQI

541 KTEEFQCBQB BSHLSLCPVA PRPBGTCSHS KLVGWCSRS XfiXkdiVHSXT PCBORVBLKT

601 IiNAWSBLCNS HHDIEDAHVL CQQLKCGVAL STPGGAHFGK GNGQVHRHMF HCTQZBQHM8 661 DCPVTALGAS LCP8GOVASV ZC8ONQ8QTL 6SCNBSSLGP TRPTIPEKSA VACIBSOQLR

721 LVKGGGRCAG RVEIYHBGBH QTZCSDDBNDL SHAHWCRQli uOUKAlNATG BUUfOUffSQif 781 ZMIiOBMKCNS XEBRZWQCHB HGWGQQNCRH KBDA6VIC8B PMSLRLTSEA SREACAGRLE 841 VFYNGAWGSV GRSNMSBTTV GWCRQLGCA DKGKZN8A6L DKAM6IPMHV DNVQCPKGPD 901 TLKQCPSSPW EKRLARP8BE TWITCDNKMR LQEGPTSCSG RVKIKH3GSW GTVCDDSNDL 961 HDAQWCQQL OGBPAUCAPK HAEFG5X3TGP IMIiNEVKCKG HESSLWDCPA RRWGHSBCGH 1021 XBDAAVHCTD ZSTRXTPQKA TTGQSELIAV GILGWLLAI FVALFLTQXR RQRQRLTVSS 1081 RGENLVHQIQ YREMNSCLNA DDLDLHN68E KSNESADFNA AELISVBKFL PISGMEKEAI

1141 UUITBXEH8H Xi

1MSKLRMVLLEDSGSADVRRH 1 ATGAGCAAACTCJVGAATGGTOCTACTTGAAGACrrcTGGATCTGCrKSACXna^^ 21PVNL8 PFTIAVVIiLIiRACFV SEQ ID N0:37 and 38
61 nTGTCAACTTGAGTCCCITCACrATTGCTOTGGTCTTACfTCrCOGVSCCl'Ql'TTTOTC 41 T 8 SLGGTTKELRLVDGENKC



181 AGTGGGAGAGTGGAAGTGAAAATrcAGGAGGAGTGGGGAACGGTGTGTAATAATGGCTGG



101TGWANSBAGSGRIWMDHVSC

121R6KE8AIiNDCKBDOH6KB6H

• KICTHQQDAGVTCSDGSDLEMR 421 TOTACTCACCAACAAGATGCTGGAGTAACITGCTCA
541 GGAACAGTGTGTGATGATAACTTCRACATCAATCATGCATCTOTGGl'rrGTAAACAACTT 201 BCGSAVBFBGBANFGE68SP

601 GAATGTOGAAGTGCTGTCAGni'CinXXSlTCACCTAATXlTtikaASAAGGCTCTSGACCA 221IKFDDLICNGKEEALWNCKH 661 ATCTGGTTTGATGATCTTATATGC&AOGGAAAK>C


^".RJ3 w °,K H,* C BBABDAGVZC 8 X
721 CAAgGATGSqQAAASCATAACTBMiaC^^ '
261GADL8LRLVDGVTEC8GRLB 781 3OASCAGATCI(SWIOCXGAQACraSTA0AT8GA 281VRFQGINaTZCODGirD8BOA 641 flM>O^-Jtk^A>flaAl^^j 301AVACXQI.OCPTAZTAZORVB 9Q1 GCT3TOGCA3nCAAGCAACIGSGATOTpC3UkCiaci%TC^^ 321ABBOr.GBZWIiO8V8CQOBBP
341AVWQCKHHBKGXHYCNHHED 1021 OCGOrCTGGCAAIOTAAACACCArGAHqGQGAAAQCACTATTG^
361AGVTC8DG8DLBLRIiRGG08 1081 G
KK»TCl«»lCnMJ«XTOU^
381 RCAOTVBVBZQRIiLOKVCDR 1141 C!QCTOTGKaqQOACaMTTa«aSMaMllWeM»Q>C^^
401 ONGLXBADVVCRQIiOCQSAZt 1201 OaCTaCPCaCTCAAMM>aCTgATGTgGmT 421 XTSYQVYSKZQATirMlfbFIiS 1261
OOLAM!^^
441SCHONBTSLHDCKHHQHOGI, 1321
461TCDHYBBAKITCBAHREPRL 1381 ACCTGT(»TCACTATX»AaAAQCCAAAArrACCTGKrrCAOCCCACAOa^
481 V G G D I P C8GRVEVKH8DTWO 1441 GTTg8ACKaGACATTCCCTOTTCTQGAqKKTTGAagTGAAQCATaGI^
SOISVCDSDPSLBAABVLCRELQ 1501 TCCy'lVIVJUATTOaQAU'l"lXJTC"lOUGAA(rciG 521 C G T V V 6 ILGGAHKGEGNGQI 1561 TOTGKK%CKGTOGTCTCTATCXrraGaSGGAGCTCaUTnG6A8J^^
541 WTBBPQCBGHB6HL8LCPVA 1621 TCIGACTGAAGAATTCCAGTGTaAGGGUKa7«A 561PRPBGTC8H6RDVGVVCSRY 1681 CCCaSCCCAGAAGGAACTTGTAGCCACaGCAGGGATGTTGGAGTAGTCTGCTC^^
581TEIRLVMGKTPCBGRVELKT 1741 AWGAAATTCGCrrroGTGAATGGCAAGACCCCATGTGAGGG
601LHAWGSLCNSHWDI BDAHVL 1801 CTTAATGCCTGGGGATCCCTCTCKaACTCT(»CrOOGACATAClAAGATGGC^
621CQQLKCGVALSTPGGAHFGK
641GHGQVWRBKFKCTGTBQHHG 1921 OIGAAATGGTOVGaiCTGGAGGCATATaTTTCACTGCACTG^
661DCPVTALGABLCPSOQVA8V 1981 GATTGTOCTOTAACTGCTCTOGGTGCTTaK!TAWUlXVl%»GGGa^
681ICSGNQSOTLBSCNSE SLGP 2041 ATTTGCTCAGGAAACCAGTCSXJUUVCACTGTCCTXXSTGaUVTTCATCATCIC^^
701 TR P T. I PEE8AVACZ BSGQXiR 2101 ACAAGGCCTACOiaTCCAGAAGAAAGTGCTGTGfXrraCATAGJ^^
721LVNGGGRCAGRVEIYHEGSN 2161 TTGGTAAATGGAaGAGGTCGCTGTGCTGGGAGAGTAQAGATTTATCAnaUJGGCXCC^
741GTICDDSWDLSDAHVVCRQL 2221 GGCACCATCTGTGATGACAGCTOGGACCTtaGCGATGCCCACGTGGTGTGCAGACAGCTG
761GCGSAINATGSAHFGEGTGP S281 GGCTXnXXaGAGGCrATTAATGCCACTQaTTCTGCTCATTTTGGAGAAGCSAACAQGGOCC
781IHI.DBHKCNGKEBRZNQCHS

801 R O N 0 Q g N C R B K 2401

A a v"""":i""e" s ' s~

B21PMSI,RLTSBA8RBACAORI,B
2461 TTCATOTciicraMACTgjtgcaflMAMf^^
841vrYff 2581 amminmaaacKaac^^
fleiDXAMBIPMKVDHVQCPKQPD 2641 OACAAOGCCATOTCCATTCCCATQTGa
901TXiNQCI>S8PlfBKRIiARPBEB 2701 AOXrraro«yUWGCCCATCATCTCC»TaaaA^
921TWZTCDHXMRI.QBG P T S C 8 941RVBIKHOa8HOTVCDDSKDL 2821
961NDAQVVCQQLOCOPALKAFK 2881 AACXanKTTCftaQTQQTOTQTCAACJUlCTTOOCIXnaQTCC^^
981BABFaQ6T6PIWLK.BVXCXG 2941 OAMCAaAGTTTOmytfWOaftCT«AaXATATO
1001 NBSeiiHDCPARRKQHSECOH 3001 A
1021 XEDAAVNCTDISTRXTPQKA 3061
1041 TTVSSRGENLVHQIQYREMN 3121 ACM -1061 8 CLNADDLKLMNSSGGHS B A 3181 TCTTCK
1081 B*K6KNBPITQ 3241 CACTGAAAAOQAAAATOOGAATTTATAftCCCAG

1 MSKLRMVLLK D8SSADVRRH FVKLSPFTIA WLLfcRACFV TSSLOGTTKE LRLVDOBNKC 61 SORVEVKIQB BHOTVCNNaK BMEAVSVICH QJjQCPTAIKA TOWAKSBAG8 GRIWMDHVSC 121 RONBSAIMDC KHDOWOKHSH CTHOQDASVT CSDQSDLBMR LTNOGNHCSG RnZKFQSQW 181 GTVCDDfflPNZ HRASWCXQIi BOSSAV8FSO SAHPGB68OP ZHPDDLXCNG NESALHNCKH 241 QGM6XHHCDH ABDAOVZC8X GADLSLRLVD QVTBC8GRLE VRFQOBN8TZ CDD9HDSHDA 301 AVACXQLGCP TAITA1QRVN ASEGPGHIKL DSVSCQGHEP AVWQCKHHBW GKHYCNHNBD 361 ASVTCSD6SD LELRLRGOOS RCAGTVEVEZ QRLliGKVCDR GWGLKEADW CRQDOdGSM. 421 KTSyOVYSKI QATNMNIiPXiS ECKONETSLW DCKHHQWOGL TCDHYEBAKZ TCEAHREPRL 481 V8GDZPC8QR VEVKHODTWG BVCDSDFSLE AA8VLCRELQ CQTWEILOO AHFGEGNOQI 541 NTBEPOCEGH ESHLSLCPVA PRPB6TCSB8 RDVGWCSRY KZRLVMOKT PCBORVELKT
601 LNAWGSLCNS HWDIKDAHVL CQQLKCGVM, STPOGAHPQK OJOQVWRHMF HCT6TEQHMG 661 DCPVTALQAS LCP80QVA8V ICSGKQ9QTL 6SCNSS6LGP TRPITPEEBA VACIESOQLR 721 LVHQOGRCAG RVEIYHBGSW GTICDD8MDL SDAHWCRQL OCQEAINATS SAHPOEGT6P 781 ZHXiDBMXCNG KB6RZHQCH8 HGMGQWCIRH KEDA0VZC8E FMSLRLTSEA 6REACAORLE 841 VFSNOAHS8V GR8KHSBTTV 6WC31QLQCA DKQKZNSASii DKAMSIPMWV DKVQCPW3PD 901 TXiMQCPSSPW EKRLARP6EE THZTCDNXMR LQBQPT8C8Q RVEIWHOSSK OtVCDDSMDL 961 NDAQWCQQL eOSPALXAFX EAEFGQGTGP ZNLHBVKCKO HBSSLKDCPA RRMBBG
1021 KEDAAVNCTD ISTRKTPQKR TTVSBRGENL VBQZQSRBMN SCLNADDLNL MNS8S6BSEA 1081 H

SEQ ID K0:38



IMSKLRMVLliEDSGSADVRRH 1 ATGAGCAAACTCaGAATOGTOCTACTTGAftSACTCiaGATCTSCntaKS
21FVNLBPFTIAVVI.LLRACFV
61 TTUVTCAACT
IiRIiVD6BNKC
41TSBIiG6TTK 121 ACCAffTTCT
61SGRVEVKIQEEHGTVCNNGW 181 AOTGGGAGAGTOGAAGTGARAAKXJkGGAQGACnQGGGAACaGrGTCTAATAATGOC^
818MEAVSVZCNQtiCCPTAZKA 241 AOCATGGAAOatGICTCTGTSATTTGTAACCASCTGSGATGTCOMCnS^

SEQ ID NO: 39 and 40

- • •— \-rrmrr in uiriimir
• « * *H8SAGSGRIHMDHVSC
301 ACWGA«KKXTAATTCOUWTKa«MTTCTGGACSCaT^
121 R 0»S SAIiWDCK-HDONOKR S'K
141CTHQQDAOVTC8DQ8DLBMR «1 MTACTCACOVACaitaiCTBCTGtaOTOJ^^
161LTHOOIf'MC80RI8IKFQOQK 481
181 0 T V C D D H F R V N H AS V V C K Q L
541
mV^
201 BCG8AV8P6O8AtrFQgaSGE> 601 ajATOTaSMaTGCI^fCMl'TtL'lVl^
231XWFODIiZCNORB6ALNKCKH
€61 lacxamttwaQiacxTtaiaaauuBaauu:^^
241 Q 0 K O KHHCDHABDA6VI C S X
721 CAAGQATO3GGAAAOCATAA 261 QADLSLRLVDOVTECSGRLB
781 GGAGCAGArCTGUkGCCTaWJACTGGTAGATOGAGTCACTGAATGTTCS^^
281 VRPQGBKGTICDDGKDSHDA
841
301 AVA'CKQIiGCPTAXTAZGRVH
901 GCTOTGGCATQCWkGCARCTGGGATGTCCAACIXXrrATCACXSXXAT^^
321 ASBGFGHIHIiDSVSCQGHEP
961
341 AVMQCKHHEHGKHYCNHNED 1021 GCGGTCTGGaULTGTAAACACCATGAATGGGKUUUlGCATTATnX^
361 A OVTCSDaeDLEIiRIiRGGGS 1081 QCTGSCGTAACATaTTCTaATG 381 RCAGTVBVEZQRLLGKVCDR 1141 CGCTGTGCTGGGACAGTTGAGGTGGAGAKCAGAtaCrGTTASa^
401 GHGLKBADVVCRQLGCGSAIi 1201 GGCTaGGSACTGAAAGAASCTGATGTG 421 KT6YQVy8KZQAXNHWLFL8 1261 MJACKKCaaCM^ATACKaAtJ^^^
441 BCNGNETSLWDCKNWQWGGIi 1321 AGCTGTAACGGAAATGAAACTTCTCTTTGKJGACIXKaMGAACIOGaUlTG^
461 TCDHYBEAICITCSAHREPR3J 1381 ACCTGTGATaCTATGAAGAAGCCAAAATTACCrSCTCACKXOCAG^
481 VG GDI PC SGRVEVKEGDTWG 1441
501 6 VCD SDF BLEAASVXiCRELQ
1501
521 CGTVVSILGGAKFGEGirGCZ 1561 TGTGGCACAGTC 541HAESFQCEGBESBIi8IiCPVA 1621 TSGGCTOAAGAATT£aiGTGTaAOO 561 FRPEGTC8H6RDVGVVCSRY 1681 CCCOXXX&GAAQGAACTTGTAGCCACAGaiGG 581 TE IRLVNGKTPCEGRVELKT 1741 ACAGAAATTOGC^T8GTGAATGG 601 LHAHGSIi CNS BHD I EDAHVL 1801 CTTAATGCCTGGGGATCCCTCTGCAACTCTCACT 621 CQQLKCGVALSTPGGAHFGK 1861 TGOCAACAACrTAAATGTOGAGITOCCCTTTCTA«X: €41 GNGQVWRHMFHCTGTEQHMG

1921 GaAAATaOTCAGOTCTOGAOOt •«1-D C P V T A L S A Si C P 8 3 OVA S v 1981 OATOmMTwOACTKiTCroscmreri^^ 681 I C 8 O » Q BQTIiSSCHSSBIiOP
701TRPTIPBBSAVACXBBaQI,R 2101 ACAAGGCCTAOCATTCCAGAAG

721 li V H G G 0 R C A G R V 2161 TTGOTAAATQaAGGA«nW3CTOTGCTGGGAGAOT
X T H B 0 S W 3ATTTATCAraAGGGCTCCTOG 741 G T I COO SWDL8DAHVVCRQL 2221 BOCACCAl^Jl\«itGATGAOtf?Cro3QACCTQAQOGAT6CCCACUlUU'JWgCAGACAGCTO
761GCaBAX»ATG8AHFGBQTGP 2261
UMA«K!CATTAAroCCACTQGTTCroCTC^ 781XWIiOBHXCHOKK8llINQCHfi 2341
lATQAGATGAAATGCAATGOAAAAGAATCXXXn'ATrrGGCAGTOOCATTCA 801HGWOQQNCRHKBDAGVXC8E 2401 CATGGC3XXSGaGC3kGCAAAACTGCAaGCACAAOaA«»TOCaoaAGTTATCT(
R BAG A G R L B
^
821 F M 8 L R L T 8 2461

ftGTAAC»T^^
8610VVCRQL6CADKGKINSASL
MWVDNVQC-PKGPD
881D 2641

901 TLWQCPSSPHBXRIiARPSBE 2701 AOH^TGGCAGTGCCC*TC*TCTCCATG3GAQAAGA 921 T H 1TCDHKMRLQEG P T 8 C S 6 2761 ACCTGGATCACaTGTGACaACAftGRTGAGACTACAAGAAGaACCCACTKXr^
941 RVBIHHGGSKGTVCDDSWDL 2821 961NDAQVVCQQXiGCOPALKAFK 2861 AAO»TGClty«WTGGTCriOTlCAACaACriWKnGTOGTOCA^^
981 B AEFGQGTGPIWLNEVKC1CG 2941

1001 NESeLKDCPARRWQHSECGH
^
CTAQXXSTHKT'PQ
3001
1021 KBOAAV
3061
1041 KATTVSERGEHLVHQIQYRB
3121 UOMCe*CMCKGTrKCKMeUISXaaM^^
1061 MHSCLNADDLDLMNSSGGHS

3181
1081 BAH*KGKWEFITQ



1 MSKLRMVIjLE DSGSADVRRH FVNLEPFTIA WUiLRACFV T86L6GTTKB LRLVDGENKC 61 8GRVBVXXQE EWC3TVCNNOW SMBAVSVXCff QL8CPTAXKA TGWANSSAOS GR1HMDHVSC 121 RGNBfiALHDC KHDGWGKHSN CTH(XX3AGVT CSOOSOLEMR LTNCWNMCSG RIEIKPQGQW SEQ ID NO .-40
181 GTVCDONFNV NHASWCKQL EOGSAVSFSG SANFGBGSGl* INFUUliICNU NEisALWNCKH



301 AVACKQIiGCP TAXTA1GRVM AS8GPGHJWL DSV8CQGHBP AVMQCXHHBta
481 VSSDXPCSGR VBVKHGDTWG SVCDSOFBliE AASVLCRELO CQTWSIU3G AHFGBONGQI

541 KAEEFQCBGB ESHLSLCPVA PRPB15TCSHS RDVGWCSRY TBIRIiVMSKT PCKikVuLiKr

601 LNAWGSLCHS HHDIEDAHVL CQQLKCGVAL STPQUAHFGR utUOVWkHMF HCTQTBQWKS 661 DCPVTALGAS IiCPSGQVA6V ICSGHQ9CJTL SSCNSSSLGP TRPTIPEBSA VACIESGQIiR 721 LVW3GGRCAG RVEIYHEGSW GTICDDSWDL SDAHWCRQL GC6BAXHATG SAHPGEGTGP 781 IWLDEMRCNG KESRIMQCHS HSMSOQNCRH KEOAGVXC8S FHSLRLT8BA GRBACAGRLE 841 VFYNGAWG6V GRSNMBETTV GWCRQLGCA DKGKXNSASIi DKAHSIPHWV DNVQCPKGPD




41 T
£160RVEVKIQBBNQTVCVHrOir 161 AOTXWGUkGAOTOGAWOTAAAATCXJ^^
81BMBAV8 VICNQIiQCPTAIJCA 241 AGCATO(2AASC»OTCTCTOnUTTiaTA»CCIAOCTGKKAT(RXX3U^ 101TOWAN8SA080RIWMDHVBC 301 A
T T K B I, R I, V D O B K K C
121 R 8KB 8A L KDCKHDQHGJCH8 V 361 C3T93aUOUMK»acrcrrT 141CTHQQDAOVTC8De8DIiXHR 421 TOTACTKaCCaACAAGA«XrraaA(naj«^rGCTCAQATOa^ leiliTKOONMCBORIEIlCPQOQW 481 CTOACGAATGGAGQQAATATOT ISlOTVCOONFRZHRASVVCXQIi 541 GGAftakGTOTGITUTahTAACirKaUlCATCaUlTCftTOCATaSTOGTI^^
201KCQ6AVePSOSANPOBG8 221 IMP DDL! CNGHESALWNCKH 661 ATCTGOTTTOATGATCTTATArGCAA 721
261GADL6LRIiVD6VrBCS6RXiB 781 GOAGCaVQATCTGAG 301AVACKQLGCPTAITAICRVN 901 GCTGTGGCATGC3UGaUVCTGGGAT 1021
361 AGVTCSDGSDLELRLRGGGS 1081 GC7GGOGrTAACATGTTCCGAIX3GATC»SATCTaGAOCTAA6ACTTA 381RCAGTVB VB Z QRLLGKVCDR 1141 aKrrGTGCTaGGAakGTTGAOGTGGAGATTCftfiAGACTWTTAGGGAftflffn^
40IGHGliKBADTVCRQL6C«6AZ> 1201 GGCT«3GGACreAAAGAAGCTGATGTGGTTTGCAGIG^^
421KTSYQVY8KIQATNMWIiFLS 1261 AAAACATCCTATCAAGTATACTCCAA^TCCAG 44ISCNGHETfiI,WDCKKWQWGGL 1321 AGCTGTAACX3GJUlATGAAACTTCTCTT7«GGACTGCAAGAftCTOGau
461 TCDHYEEAKITCSAHREPRL 1361 ACCTGTGAT 481 V G 501 8 VCD S D F E L E AA 6 V ZiCR EL Q
'IT" "" "TTV »
9TTCTASGCAGGGAATXftCA0 y y—y fl j j, g a A H r.,a E 8 H- ^,-Q r—
1561 'IVffUtfCfcCftGTCO'il.'i^'iAyffgTnaatMn^yTp^^^'p'i^fflft^iy^ft&^T^^^^g^ip^ M1WABBFQCB4BB8BL8&CPVA
1621 TOGGCTGAA 561 PRPBGTCSHSRDVGVVCSRy 1681 CCXCSCCCACaAOGAftCTIOTAecaKaaaWWG^^
581TEIRLV»aKTPC10RV«I,lCT 1741 AC*aAAATTCOCW'(WiXttAT(WCAAQACaxaiXnX2*G(SOCA^^
eOlIiMANOflLCHBHWOZIOARVI, 1801 CTTAATOC 621CQQLKCOVAI.STPGOAHPGK 1861 TOCOACAAClTAAATOTGOA 6410HGQVHRHMFHCTGT1QHHO 1921
661 DCPVTAIiGASLCPSGQVABV 1981 QAli.uiuv^
' 681 £ C S G W Q 6 Q T It 6 8 C H 8 6 6 Ii"8 2041 ATTTGC
701 T R P T I PKESAVACIESGQLR 2101 ACAAOOCeTACC*TTCCa«»AAOAA»agaCTOro8CCTOCaTAGMM
721LVNGGGRCAGRVBiyHEGSK 2161
741GTICDDBWDL6DAHVVCRQL 2221 GG«CCATCTGTGATaACAGCT33GACCTGAaOt»TOCCCaCGTOG7X3TGC^^
761 GCGEAINATGSAHFGEGTGP 2281 GGCTGTGGAGAGGCCATTAATOCCACnXKTTCTGCTCATTriiGGAGA^
-7811 WLDEMKCNGKKSRIWQC H S 2341 ATCTGGCTGGATGAtATGAAATGaATOGMU»GAATCX!OGTATTTOGCAGnX!C%
801 HGWGQQNCRHKBDAGVICSE 2401 CATGGCTQGOGGCAGCAAAACTGCAGGaU3^Q3AGGATGCRGGAGTTATCrGCTCAGAG
B21 FMSLRLTSEA6REACAGRLS 2461 TTCATGTCTCTGAGACrGACCaGTGAAGCCAGCAGAGAGG(KTGTGCAGOGCGTCTA^
841 VPyNGAWGSVGRSNMSETTV 2521 GTTTTTTACA&CGGAGCTTGK**XVUHBTTS^^
861GVVCRQIiGCADKGKZKEA61i' 2581 GGTGTGGTOTGCAaGCAGCTGGGCTGTOCA 881DKAMSIPMWVDNVQCPKGPD
2641 GAQftAQGCCATOTCCATTOOCATOTaGQTOGACAATSTTakCTQ '
901TLHQCP6 S PKEKRLARPS BE 2701 AraCTGTGGCAOTGCCCATCATCTCXATCGGAGAAG^CXGKJCa
921 TWIT CDHKMRIiQEGPTSCSG 2761 ACCTGGATC%CATGTGACAACAA6ATOJtfakCTAC»ft6AAGG»^^
941 RVBIWHGGSWGTVCDDSWDL 2821
961HDAQVVCQQLGCGPALKAFK 2881 ARCGATGCTCAGGTGGTGrGrCAACAACrTGtKrraTGGrCCaGCTTTGAAAGCATTCARA
981EABFGQGTGPIWLNXVKCKG 2941 GAAGCAGAGTTTGKSTCASGGGACTOGACCCATATOGCTCAArGAAGTGAMnX}^^ 1001 NEEBLWDCPARRHGHSECGH 3001 AAT6AGll!TTOCTlCTX»3GATT6TCCT6 1041 KATTGRSFLIAFGILGVVLL 3121 AAAGCCACAACAGGTCGGTCAnXKrTATIOCATTCGGAATCCTTGGAGTTGTTCTCTTG

1061 AI F 3161

ALFiTQICRRQRQRLT'V -^^



3241



1101 HADDLDLMHSBGGHSEAH 3301

121 RGHBSALWDC 161 Gk
24X QGFMOXHNCDH 301 AVACKQLGCP 361 AGVTCSDG8D
421 rrsYovysKi
481 VOGDIPCSGR 541 KAEBFQCBGR 601 LNAMSS&CN8 661 DCFVTAIiGAfl 721 LVNOGGRCAG 781 XWLDBMKOra 841 VPWGAWGSV 901 TUIQCPSSPH 961 MDAQWCQQL 1021 KBDAAVNCTA 1081 8SRGENLVHQ
1 M8KLRMVLLB OSS8ADVRRK 8SRVEVKXQB
ABEA0VZCSK TAITAI6RVN
FVML8PFTIA WLLLRACPV TSBLGGTTJUt UUiVIXIBNRC 8MBAV8VZCK QMCFXAZXA VGNAK86AO8 ORXHMDHV8C KHDGWOKHBM CTMQQDA8VT CSDGSDLBMR LTOOGMMCSG RZEZXFQGQK BCGSAVSFSG SAMFOSGSGP 1WFCDMCNG NESAUWCKH GADLSIiRLVD OVTBCSQRIiB VRFQQBtfOTI CDDGNDSHDA A6EGFGKINL D8VBCQQBBP AVWQCKHHBW GItBIFCHRBSO
QATWMWLPfcS VEVJUlOUTIfU
HNDIBOARVIi IiCPSOQVASV RVKYRBGBW KBSRIKQCHS OR8NM6BTTV BKRLAHPSEB GCGPALKAFK QXZ6TBXTPQ IQyRKMHSCL
RCAGTVEVBI QKLLOKVCIHl OHOLXBADW CRQLOCQSAL BCNBNBTSIM DCKHNQMSOb TCDKXEBAKZ TCBAHRHPRL 8VCDSDF6LB AABV6CRBLQ CQTWSIMG AHPSBOHGQI PRPBGTCSHS RDVGWCSRY TBZRLVNGKT CQQLKCGVAL STPOGAHFGK QBSQVMRHNF ICSGNQSQTL SSCNSSSLGP TRPTIPEESA VACIBSGQLR OTICDDSNDL SUAHWCRQL OCOBAXNAXG BAHFQBGTGP uaiiaflQMr^gp '""3AOVICBB PMSIiRLTSEA SRBACASRZiB GWCRQLGCA DJU3K1HSASL OXAMSZPHNV IWVQCPKSPD TKITCDHKMR LQBGPT8C8G RVBZNHGS8W GTVCDDSNDL BABPGQGTGP IHLKBVKCRG NBSSXMDCPA RRHGHBBCGH XATZORSFU AFGZLGWLL AIFVALPLTQ KRRQRQRLTV NADDLDUdS SGGH8BAH

SBQ ID NO.>42



1M8KLRMVLLHDSGSADVRRH
1 ATOASaUACTCASAATSGTOCZACTTGUUlCUUnrCTOaATnXX^ 21FVNL8 PFTXAVVLIiXiRACFV
iJX\rii\ii~iiA~i%;i' 41TSSL6OTTKBLRLVDGENKC 121 A 61 S 6 R V B V .K I Q B_,«.W, O T V C K H G K 181 AGTGGGAGMTGSAAGTt»AAATCCAOGAGGAGTQGaGAACC»TGTCniAATAATa3C^^
81SMEAV6VICNQLGCPTAIKA 241 AGCaTGGAAGCABTCIOWX^TTTGTAACCAGCTK3GGATGTCCAAI-"l'UCi'ATCAAAGCC SAGSGRIWHOBVeC
lOlTGWAN
301 ACTO 121RGHEBALKDCKHDGKGKHSH
361 CGTGGGAATGAGTCAGCTCTTTGGGACTGaUVACATGATGGATOGGGAAAGCRTAaTAAC
141CTHQQOAGVTC6DG8DLEHR'
421 TOTACTCACXAAOAGATGCTGGAGTGACTTGCTCAGATOGATCC^
leiliTKGGNMCS G R IBIKFQGQW
481 C^aUVATOGAGGGAATATOTGlTCTOGAAGAATAGASA^^
leiOTVCDDNFHINHASVVCKQL
541 OGMC*l3TGTGTGKKiaiWZTTCM^V 201ECGEA'VSPSGBANPGEGSGP
601 GAATCTGGAAGTGCreTCAGTTTCTCTOGTT 221IKFDDLICNGNEBALKNCKH
661 MCVKTfTSMXSlOClTkTKIOCMCaBiaATt^
241 QGWGKHKCDHABOAGVZC6K
721 OASGATGGOGAAAGCATAACTGTGATCATGCTGAGGATGCTOGAGTGATTTG 261GADLSLRLVDGVTEC6GRLE
781 GGAGCAGATCTCAGCCRSAGACTGGTAGATGGAGICACTGAATGTTCAG^
2B1VRFQGEKGTICDDGWDSHDA
841 GTOAGATTCCAAGGAGAATGGGGGAOUlTAnnx3ATOACX3aCTSGGAaVG1X^
301 AVACKQLGCPTAITAIGRVN
901 GCTGTGIGCATGCAftSaACTGGGATGTCC»A 321 A G ,-B. G F 961 GOAGTGAGGGATTTGGACACATCTGGCTTGACAGICTTrcTTGCC^

SEQ ID NO: 43 and 44

3*1 A VNQCKHBENOKHYCVHN—E~~5
"1021 «:slS 361AOVTCBDaSDI«EIiRX,RGG06 1081 O
TGGATCJroTO8GA(CTAAG^
SBlRCAGTVBVlXORIiLaiCVCDR 1141
MfcrTqWMB^^ 4010KOLJCBADVVCRQLOCOSAL
i2^i_QCKrareQQACTUAAGAAGCTGAiwwm 421 K T.S Y Q V Y 8 K I Q A T H M K I, F li 8
1261
441 8 CHOKITSLKDCKNWQHOai,
1321 , AOCTOTAMOGAAATGAAACTTCTCTTTXXWACIXKSUVaAACTOa
461 T CDBYE IAKI TC8AHRI P R L 13 Bl * 481VQOOXPC80RVBVKHGDTWO 1441 GnrZOOACKSAaACATTCCCTOtTCiaCACaOSTTSAASTCIAAOCATOCmi^ S018VCD8DF6LKAABVLCRBLQ
aAAQgPSCCftQOT
521COTVV8 ILOaAHPGBQNOQI 1561 TOT«GCACASTCCmSCTATCCT 541 WABZFQCBOBB8HL8LCPVA 1621
561 PRPEQTCSHSRDVGVVCSRy 1661 CCCCGCCCA6AAOOAACTTGTAOCCACAGC*OQGATGTTQaftGTAGTCKKTCAAGATJ^
561 TBIRLVNGKTPCHGRVELKT 1741
601 tiHAWaSLCNEBKOIEOAHVL 1601
621CQQLKCGVALSTPGQAHFGK
Cr^
1861 6410NGQVWRHMFHCTGTEQHMQ
1921 aaujaoGTCjaGmaxaauiaaaATerir
661 DCPVTAtiGABLCPSGQVASV 1961 (»TTGTCCTGTAACTGCTCTC!aniGCTTCACTAIXn«rrTaU3OOa^^
661 ICSGNQSQTLE6CNSBSLGP 2041 ATTTOCTCAGGAAACCAGTCCCAAACACTGTCCICGTGCAATT^
701TRPTIPEE8AVACZES6QLR 2101
721 LVRGGGRCAGRVEIYHEGS W 2161
741 GTICDDSHDIiBDAHVVCRQIi
761 GCGEAINATGSAHFGEQTGP 22 Bl GGCTGTGGAGAGGCCATTAAT B01HGWGQQNCRHKEDAGVICSE
821 FMSIiRLTSEABREACAGRZiE 2461 TTCATGTCTCTGAGACTGACaVOTGAAGCCAGCAGAGAGOCCTCSTGCAGOaCGTCTAGAA
BllVFYNGAWGSVGRSNMfi ETTV 2521 GTTTTTTACAACGGAGCTTGOXXXAGTGTTGGC&GGAGTAAC^
861GVVCRQ'LGCADKGKINSASL 2581 GGTGTGGTGTGOkOGCAGCTGGtK^raTGCAGACAAAOGGAAA^^


H"~ " "901-T I, K Q C P 8 8 t W B K R - 1i A- R P-S- «-«"—- —
1 J
1 SalTWITCDNKMRLQEGPTSCSG
941RVBXKHGQBiraTVCDDSWDI, 1


2801 AACX^WW%JiQGTTOTOTOTCA\CAACTTGGCT^

2941 GAAOCAGAGTTTGOTCACMGGACTOGJIOC^

1 3001 AA3H3A0I^TCCTlYffl6hjHO&TT^
3061 AAaGUUU&QQCTGCAOTGM^

1061 A I P V A Zi F !• T Q K R R Q R g R LTV
1081 88RGKNLVBQZQYRBMK8CL
1101 HADDLDLMHSSBNSNESADF 3301 AATGCAGATGATCTGGACCTAATGAATTCCTCAGAAAATTCC^ 1121 N AABLXSVBKFIiPXSGMBKB
1141 AZIiRHTElCENGNL 3421 GCOirrCTGAGGCACACTGAAAAGaAAAATOGGARTTTA
1 MSKLRMVLLE DSGSADVRRH FVNLSPFTIA VVLLLRACFV TSSIX3GTTKB LRIiVDGENKC SEQ ID
El BeRVSVKIftB; EHGTVCHNGW SMEAVBVICN QLQCPTA1KA TGWMJS6AGS GRIMMDHVSC 121 RGNBSALWDC KHDGWGKHSN CTHQQDAGVT CSDGBDIiEMR LTNGGNHCSO R1EIKPQGQW 181 GTVCDDNFNI NHASWCKQL BOGSAV878G SANFGBGBGP IWTODLICNG NESALVTOCKH 110:44
241 QGMGXKNCDH AEDAGVXCSK GADIiSIiRLVD GVTBC6GRLB VRFQ68NSXX CXO8ND8HDA
301 AVACKQLGCP TAITAIGRVN ASEGFGHIKIi DSVSCQGHBP AVWQCKHHEW GKHYCNUNED 361 AGVTC8DG8D LEUUiRGGGB RCAGTVEVE1 QRLLGKVCDR GWGLKEADW CRQLGOG8AL 421 XTSYCVYSKX OATNMWLFLS SCKGNETSLW DCKNHQHOGL TCDHYBEAXX TCSAHRBPRL
481 VGGDIPCSGR VEVKHSDTHG SVCDSDFBliE AA6VLCRELQ 541 NABBFQCEGH ESHLSLCPVA PRPBGTCSH8 kUVtiVVCSRl TtElRliVHSKr i-jjiuRVtuKT
601 UDUIQBIiCNB HWDIEURHVli CQObK^OVMi STi«sAiiKjjC GNGQVHRHMF HCTGTEQHM3 661 DCPVTALGAS LCPSGQVASV ICBGNQSQTL 6SCKSBSLGP TRPTZPBB6A VACXB9GOJ^R 721 LVNGGGRCAG RVEIVHBGSW GTZCDDSMDI. SDAHWCRQL GCGEAINATO BAHFGEGTGP 781 IWLDEMKCKG KESRIWQCES HGWGCX»»CRH KEDAGVICBE FMSLRLTSBA BRKACftGRLK 841 VFYNGAMGSV GRSNMSBTTV GWCRQLGCA DKGKtNSASL DKAMSIPMWV DNVQCPKGPD
901 TLWQCPSSPW EXRLARPSBE TW1TCDNKMR LOEGPTSCSG RVBZHHGG8W GTVCDD6WDL
1021 KEDAAVNCTA QKX8TBKTPQ KRTTGQSFLJ APGILGWLL AIFVALFLTQ XRRQRQRLTV 1081 SSRGKNLVHQ IQYREMNSO. NADDLDLMNS SENSNEBADF KAAELISVSK FLPISGMEKE 1141 AILRHTEKEN GNL
Example 10 Cloning and characterization of canine CD163 from DH82 cells.
A forward primer 5'simianCD163 (SEQ ID NO: 28) CACCGGAATGAGCAAACTCAGAATGG-3' based on human CD163) and a reverse primer HuCD163-3'Kpn (SEQ ID NO: 29)
I. RT-PCR producte were cloned directionally into the pO}NA3.1D/V5/ffis/TOPO vector according to the manufacturer's instruction. Several clones containing large inserts were analyzed. Several clones with large inserts were analyzed, and these fell into either the v2 or v3 splicing patterns seen in other species. The v2 variant is missing an 81-nucteotide exon (E81) relative to the v3 variant, which results in a reading frame shift and alternative carboxy terminal amino acid sequences. The canine CD163v2 cDNA from DH82 cells encodes a peptide of 1115 amino acids. When compared to the sequences in Genbank database, it is 83.9% identical to human CD163 {Genbank Z22968), 85.1% identical to pig CD163 (Genbank AJ311716), and 74.3% identical to mouse CD163 (Genbank AF274883). The nucleotide and aminc acid sequences of the two splice variants found in DH82 cells are provided below (SEQ ID NOS: 45-48).



SEQUENCE

ID NO



1M6KLRMVPHGNSGSADFRRC 1 ATGAOOUttCTCAGUUlTOGTCa»C»TGGAAA^ 21FALLCPSAVAVVSILSTCLM
61

SEQ ID NO: 45 and 4€

41TN6XiGRADKEMRLTDGEDHC
121 AccMTOCTaraas&SAaauj&TAMGA^
61EGRVEVKVQEEWGTVCNNGW 181
81GMDEVSVICRQLGCPTAIKA 241 GGCATGGATGAAGTCTCTCTGATTTGCAGGCAaCTaGGATGTCCCACTOCTATCAM^ 101 A G M A N S R A 121RGNESALWDCKHDGMGKHHC 361 CGAGGGAATGAATCaXJCTCTCTGGGACTGCAAACATGATOQATGOGGAAAGCACAftCTGC 1416HQQDAGVTCBDGSSLEMRL 421 ACTC%raACAGGAfOCTQ^
161MHGGNQCSGRIEVKFQGQWG 461 ATGAACGGOJGAAACCAGTGTTCTaGCAGAATAGAMTCIJkaTTCCAaGGACAaTOQOGA 181TVCDDNFNIDHABVVCKQLE 541 ACaU3'I 281KFQGBHGTVCDDGK06MDAA 841 AAATTCCAAGGGGAA7X3GGGGACAGTGTGTGATGATGOCTGG
561 AGTGAGSGAAGTC
361 OVTCSDOeDLEIiRLVOOGSR 1081 aAgCTBa»«?i^
381 CAGTVEVEIQJCLI.QicvCDRG 1141 «H«CrOOaACA8TCK3Aa3TTSAAATTCAQAJUlC3tKSAOOGAAAOT
401 WGZiXIAOVVCXQLaCOBALK 1201
4aiT8YQRYSKVKATBTIILPLSR 1261
441CBOMBT8LWDClCirirQNal 501 V C fi 8 D F 6 I. E A A 8 V t C R B fc Q C 1501 gTCrGTOATTCOGACTJir:'aVX 521 OTVISILaaAHFaKONOQIK 1561 OOCROUn^aTCraaTC
541AIIFQCB6QBSRIi8ZiC8VA8 1621
561 RPDGTCBHSRDVOVVC6RVT 1681 OOCCCtUUTGGGACCTGTASCCACAGC&SgSATaTTGSAiinCGTa^
581 BXRLVN6QSPCB 6010 NWGSLCNB H H D I E D AH V PC 1801 OGGJ^CTQGGGATCXX^CTGCRACTCTakCTOOGACRTAGAAGATOCIXATOT
621 QQtKCGVALE IPGGAHPGKG 1861
641 SOQIWRHMFHCTGTEQHMGD 1921
661 CPVTALGATLCSAGCVA8VZ 1981 TGCCCnQTAACTOCTCTOGGCGOGACGMJ'iUimUOTX:i'UIGCK3UieTOaOCT
68IC6GNQSQTLBPCN6TSLDPT 2041 T(XrraUXUVA&TaU3AG 701 RSTTSBESAVACIASGQLRL 2101
721 VNGGGRCAGRIEVYHEGSWG 2161 CTAAATGGJU3GCGGlCGCTClX3CTOGGAGAATAGA3GTCTACCATGAGGGCTCCTGGGaC
741 TICDDSKDLBDAHVVCRQLG 2221 ACCATCTGQYI&TG»CAGCTOGGAC£TGAGTGATGKXCAnn!G631STGC^
761 CGVAINATGSAHFGEGTGPI 2281
781HLDEVNCNGKESHIWQCRSH 2341 TGSCTGGACKAGGTGAACTGTAATGGAAAGGAATCTCATATCTGGaUlTGCCBC^^
801 GWGQHKCRHKEDAGVZCSEF 2401 (KKrrOQGGGCAftCACAftCrGCAGACATAAGGft.GGATiKaaGAGTTATCTGC^^
821MSLRLIDETSRDICAGRLEV 2461 ATGTCTCl^GACTC^TTGATGAAACCA 841 FYN-GAWGSVGKSNMSATTVE 2521 TTTTAOATGGAGCTTOGGGC3W3CGTT3Ga«GAGrAATATGTCTGC^
861VVCRQ I.G CADKG S I NPA « B n

881 X P H B" R~H""M"N VDKVQCPJCOPDT
2641 J»«3CXa!COTC«»G3e«aTOTGeSTOG»CAATC
SOlXiWQCFBBFVKQ'BVASBSBB'v 2701 TTATaQCAMxi^^^WMCToeM 921 NX TCASKZRLQBOTSHCSOR 2761 yQQJ^*p^j^*^^|^jB^Pio^oj^j^QifcyiMkQi|jn^*ypK|LffiKiM
941 V EL W H 00 8*0 T VCD D 8 HO Ii B
2821 am^ocTCiogcAcaaMiam
961 D A Q V V C RQLOC0PALBALXE 2881 OAHXACAAOTOOTOTaTCUCaOCTOSQCnmiQCCCAOCATT^^
981 AAFOQOTQPZNZiVDVKCKOK 2941 CKaC8CAT3rrOOTCakOaOOACTCIOOCCTATATGOCK3UTOACGMAAff 1001 B6BLMDCPARFMQH8DCQHK
1021 BDAAVRCSBIAMAQR8SHP R
3061 GM^TQCT 1041 OH 8 8 Ii V A L Q ZFOVZ L L A F L Z
1061 ALLLHTQRRRQQQRLTVSL R 3181 GCTCTCCTCTT
1081 OBHBVHQZQYR8MN88LKAO 3241 CK»U3AOAATTCTSTC»KCOUUinCMTAO0968AAAn»^ 1101 DXiDVXiTSSBDKFBVH 3301

1 MSKLRMVPHG NSG6ADPRRC FAUiCPSAVA WSIIiSTCLM TNSLGRADKE MRLTDGEMIC
61 SGRVBVKVQB EWGTVCNHGW GMDEVSV1CR QbOCPTAZKA AGHAKSRA68 ORIHMDHVSC 121 RGNEEALWDC KHDGWGXHHC SHQQDAGVTC SDGSSLEMRL MNGGNQCSGR ZBVKgQGQMS 181 TVCDDNFNID HASWCKOLE CGSAVEPSGS ANFOEG8GPI WFDDLVC8GN ESMMSCXBB 241 ONOKRNCDBA EDVCVICLDG APLSTjRIiVPS VTBC8GRLBV KFQGBMGTVC DDOHD8NDAA 301 WCXOLOCPT AVTAIGRVKA SEGSGHIKLD KLSOQGDESA LWQCRHHEWG KHYCHHNEDA 361 OVTCSDOSOIi ELRLVGOG8R CAOTVBVBZO XUiGKVCDRO WGLKEADWC KQbOOSSAIiK 421 TSYORYSICVK ATNTMLFIiSR CSSBBTSLNP CKNWQWQGIiE CDHyEEAKVT CSAHRBPRLV 481 GGDIPCSGRV BVKHODTHGT VCD8DP6LEA ASVLCRELQC GTVISIWOA HFGBGMOQZW 541 ABEFQCBOQE SHLBI^SVAS RPDGTCSHSR DVGWCBRYT BIRLVHGQSP CBGRVBLJCIL 601 GNWGSliCNeH HDZBDAHVFC QQLRCX3VALS ZPGGAMFOKO SGQIWRHMFH CTGRI
661 CPVTALGATL CSAGOWA6VI CSQHQSQIIiS PCNSTSUJPT RSTTSEESAV ACIASGQLRL 721 VNGOGRCAQR IEVYHBGBWQ TICDDSMDLS DAHWCRQLG OOVAZNAT08 AHPGBGTGPI 781 MLDBVNCNGK ESHUfQCRSH ONOffffiCRHX EDAOVZCBEF MSLRLZDETS RD1CAGRLBV 841 FYNGAWGSVG KSNMSATTVB WCRQ1X3CAD KSSZNPASSP KPMSRHMVfVD NVQCTK3PDT 901 ItMQCPSBPWX QRVASS8BET WZTCAMKZRIi QBGTSNCSGR VELMHGG8M6 TVCDDSMDIiE 961 DAQWCRQLG COPAIiEALKB AAPGQGTCPI WIiNDVKCKSK ES6LWDCPAR
1021 EDAAV8CSBZ AMAQRS8NPR GHSSLVMiGI POVZtLAPLZ ALLLWTQRRR {X2Q8I-TVEIA 1081 GENSVHQICY RBMN6SLKAD DLDVLTSSED HFEVH

8BQ ID NO:46



IMS XL RMVPHGHSGSADFRRC
1 ATGAGCAAACTCAGAATGGnrCACATSGAAaCTCTaGATCTGCTt^CTTTAGAA^ 21FALLC P8AVAVV8ZLSTCLH 61 aTl 61SGRVEVKVQEEKGTVCNNGK
81GHDEV8VICRQLGCPTAZKA
101AGWANSRAGEGRIWMDHVSC
121RGNESALWDCXHDOW6XBRC 361 CGAOOGAATGAAlCTtRrrCTCTGGGACTGCAAACATQATG
SEQ ID NO: 47

421 AGTCATCAACAOaAKKTCGAGTA*CCTOTTCA isi M N •o^fl---r^-'t"-v-crrR--'---r'-«---ir--iC"---F---i'Q'-'«-Q-'-ii--ar----
481
BTCAAGTTCCAagGACfcOTBGSaA 181TVCDDHFKXDHASVVCXQI.B
TCKnttOTTTGTAAACftGCTCSAA
201CGSAV8PSG8ANFOBG8GPI 601 'Jwil«GAAUTOCSXJTCJU»iii^iu-i\»urTCAOCTAAUVriXXlAaAA 221 H FDDLVCSGKBSAIiWKCKHE 661 'l«^U"i«ATGAT4TlVlVlX«»GTGaAAATaAGTCAGCTCTCTGaAACTGCAAOCaTaAA 241 aWGXHNCDHAEDVGVICLDG
781 QCMaaCTQMCCTaiVMC^KTK^VJ^
281 X FQOBNOTVCDOOWDSNDAA
841 AAATTCaULOOOOAATOaKWACAOTQTGTOATQATGKSCT^^
301 VVCXQLOCPTAVTAIORVNA
901 OT3OTATOTAAACAACTCKXaTOCCCAACTOCTOTCAaSKXaiTTCWTa3AGl^
32Y8 I OS O H I W !• OH L 8 C Q G D B 8 A
961 jaTaiaaaMicnaaKCMATnaacivaau^^
341IiNQCKH8BNGXHyCNHHBDA 1021 CTCroGCAGICTJUSAakCCATGAATOGGKaAAG^
361 OVTCSDQSDLKLRLVOGOSR 1081 CnnnaAeATOTICTGAiaGATC»8ACCTOGAGK3X3AQACTTO
381 CAGTVBVEIQKLLQKVCDRG 1141
401 WGLXBADVVCKCIiGCGSAIiK 1201 TGOGQACTGAARGAAOCCX^TGTCWUTfQCSlAGOiGTTGOGATflTOGATCrflCl'CTCAAA
421 T 8 Y..-Q R Y S X VKATNTWLFLS R _ 1261 ACST«JATCagaSTTATTCXaVAMrrTAAqBC3JOUaCACaTOGCTX?n^^
441 CSGNETBLWDCKNtffiHGGXiS 1321 TGTAGTaaCAATGAAACTTCCCTTTGGGACTGOUlCaACTOGCAGTOOOGTGGACT^^
461 CDHYBBAXVTCSAHRBPRLV 1381 tGTSATCACTATQAAGAAGKrrAAAGTTACCI^nxakGOCCAakGGGA^^
481 G GDIPC6GRVBVKHGDTWGT 1441 GGAGC^QATATTCCCTGCTCTaGTCCICTTGAAGTGAAAC^^
501 VCOSOFSIiEAASVIiCRBLQC 1501 GTCT(^X»rTCCGAC"lTL'lX:i"lUWJAAGCTGCCAGTGTOCTGIX3CAGAGAGTTACAGUX:rf
521GTVX6IIi6GAHFGBGNGQ'lM 1561 GGCAWOTt^TCTCCATCCTAOOOGGAGCTCACTTTOGAGAAOGAAATQGACAGAT^^
541AEBFQCBGQB6HIi6 tiCSVAS 1621 GCTGAAGAATTCXAGTGIGAaGGGCAGGAGTCXKATCTTTCACriCimTCAGTAaOCTCr
561 RPDOTCSHeRDVOVVCSRYT 1661
SBlEIRLVNGQSFCEGRVBIiKZL 1741 GAAATCCX3CTTSGTGARiaGCCAGTCXXCCrit?rGAAOGAAGAGTOGAGCT^
601GNWGSLCNSHWDIEDAHVFC 1601 GGGAACTGGG 621 QQLKCGVALSIPGGAHFGKG 1861
6416GCIHRBHFHCTGTEQHHGD 1921 AGTGGTCAGATCTGGAGGOlCAT 1OB1

J 701 R 8 I T S ETir 8 X^V A C I A"8 Q Q b R . & ]
eWeSSMWai^M^^ -r ~---










2281 TOTO8ASM(MCAOTAATOCC^CTOCru'iVJl\»u"f






821M8IiRXiZDBTBRDZCAGRIiEV









S81KPNSRBMMVOKVQCFKGPDT

901LWQCPSSPWKQRVASSSEBT

921WITCANKIRLQKQTSNCSGR

941VBLWHGGBWGTVCDDSWDDE 2821 GTGGAGCliri'GGCACaGAG


2881 GATGCACAAGTGGTGTGTCGACAGCK3QGCTGTGGCCCAQCATTAQAAGCACTAAAAGAG 9B1AAFGQGTGPIWLNDVKCKGN
1001 BB6LWDCPARPWGHSDCGHK

3001 GAGTCTTCClTGTGGGAnxjTCCTGCTAGACCCTOGGGGCACAGTGACTSTGGCCACAAG 1021 BDAAVRCSEIAMAQR6SNPR





1061 ALLLHTQRRRQQQRLTV,SIiR

3181 GCTCTCCTCTTGTGGACTOUMGGCGAAIUCAGCAACAaOGGCTTACAGn'rCCrTGAaA 1081 G B H 8 V H Q Z Q Y R E M IT SSL K A D

3241 GGAGAGAATTCTGTC(»CCAAATTCAATACCGGGAAATG*ATTXnTCCCTGAAAGCAGAT 1101 DLDVLTBSEYPHEBDDFNDA

3301 ^I'CjTiGaiCG^TSCl^CTIXXVCAGAATATCCC^ 1121 GLZ6V6KSLPISG



1 HSKLRMVPHG NSGSAOFRRC FAIibCPSAVA WSILSTCLM TNSLGRADKE MRLTOGEDNC SEQ ID NO: 48
61 SGRVEVXVQE EHGTVCHNGN GMDEV8VZCK QWCPTAIKA AGWAHSRAGS
181 TVCDDNFNID HAEWCKQLE CGSAVSB8GS AHFGEGSGPZ WFDDLVCSGN ESALHw
481 GGDIPCSGRV EVKHGOTNGT VOJBDFSliEA ABVLCRBLQC GTVIBILGGA UFGEGNGQIN 541 AEEFQCEGQE SHLSbCSVAS RPDGTCSHSR DVOWCBRYT EIRLVNGQSP CBGRVEUCIL 601 GNWGSLCNEH MDIEDAHVFC OQtlHTnvaT.C Ttonauufsirr! ean-neammi . JILMXU.II-J.,I. 1


901 LWQCPBSFWJC QRVAB6BBBT HITCAKKIM, QBOT6UCSSR VBLKH3SSMO TVCDDlmJE
~,
1021 EDAAVRCSEI AMAQRBSKPR OHSBLVALGI FSVXL&WM: ALLMTPORRR.QQCWMVBUl 1081 GBreVHQIQY XENNBSIiKAD DUJVliTSSBY PHEflDDgHM. QLISVBKSLP IBS
Example 11. Various cell lines are rendered permissive to North American PRRSV Infection following transient transfection with pCMV-susCD163vl
Porcine Kidney (PK032495), Norden Labs Swine Testicular (NLST-1), Norden Labs
Dog Kidney (NLDK-1) were obtained from Pfizer Inc. and were grown at 37° C and 5% COz in growth media consisting of Dulbecco's Modified Eagle Medium (DMEM, Invitrogen catalog number 11965) supplemented with 5% fetal bovine serum (FBS), ImM sodium pyruvate, 2mM L-glutamine and antibiotics. Ceil lines Baby Hamster Kidney (BHK21), Norden Labs Feline Kidney (NLFK-1), and Rabbit Lung (RL) were obtained from Pfizer IDC. and were grown at 37° C and 5% CCfe in growth media consisting of Dulbecco's Modified Eagle Medium (DMEM, Invitrogen catalog number 11965) supplemented with 10% fetal bovine serum (FBS), ImM sodium pyruvate, 2mM L-glutamine and antibiotics. Veto cells were obtained from Pfizer Inc. and were grown at 37° C and 5% CO2 in growth media consisting of Minimum Essential Medium Alpha (MEM, Pfizer IDC. formulation) supplemented with 10% fetal bovine serum {FBS), 2mM L-glutamine and Gentamicin at 20 micrograms per mL. Cell culture wells (35 mm) containing approximately IxlO6 cells were transfected with 2 micrograms per well of plasmid pCMV-susCD163vl, in DMEM without FBS or antibiotics, using Lipofectamine 2000 (Invitrogen catalog number 11668-027) according to the manufacturer's instructions. Cell line RL was transfected with 1.0 micrograms per well of plasmid pCMV-susCD163vl. A member of the PAM cell cDNA library without an insert, designated pPAMB (essentially and empty pSport plasmid vector), was used as a negative control plasmid. At 24 hours post transfection wells were aspirated and washed twice with DMEM/5% FBS followed by infection with North American PRRSV isolate P129. Virus was allowed to adsorb in 0.5 ml growth media for a minimum of two hours, after which additional media was added to a final volume of 2.0ml and incubated overnight. The virus was then removed, wells washed twice with growth media, and fresh growth media added (2.0 ml per well). A time zero sample of culture fluid was immediately taken in order to determine die background level of infectious virus from the inoculum. At a

monoJayer were detected by fluorescent antibody assay (FA). The FA was completed by fixing the monolayer with 80% acetone and stained with FTC-conjugated monoclonal antibody SDOW17 (Rural Technologies Ihc), which is specific for the PRRSV nucleocapsid protein. Viable virus was titrated by inoculating dilutions of culture fluids onto MARC-145 cells. Table 5 shows the resulte of virus infection by FA and the presence of progeny virus for each cell line tested.
Failure to detect progeny virus from some cell lines may be the result of low virus titer in the cell culture fluids, below the assay's limit of detection. Pennissivity of Vero cells to PRRSV infection was augmented by the expression of susCD163vl. Compared to the time zero measurement of background virus, there was nearly a two-log increase in virus titers in Vero cells transfected with pCMV-susCD163vl, whereas there was less than a one-log in titer increase in cells transfected with negative control plasmid pPAMB. All cell lines except NLDK-1 were positive by FA fpr pennissivity to North American PRRSV isolate P129 infection after transfection with pCMV-susCD163vl.
Tables
Screening of various cell lines for pennissivity to NA PRRSV isolate P129 -following transient transfection with pCMV-susCD163vl or pPAMB
Transfected cell line Fluorescent Antibody assay Progeny virus produced

pCMV-susCD163vl pPAMB pCMV-susCD163vl pPAMB
BHK21 •f-H- - +++ -
PK03249S + - + -
NKFK-1 + - + -
NLST-1 •f - - -
JVLDK-1 - NT NT
RL + - - -
Vero •H- + ++ +
; Highly positive ++ = Moderately positive + = Slightly positive
- = Not detectible
NT - Not tested
Example 12. BHK21 cells are rendered permissive to European PRRSV infection following transient transfection with pCMV-susCD163vl
Cell line Baby Hamster Kidney (BHK21) was obtained from Pfizer IDC, and grown at 37° C and 5% COz in growth media consisting of Dulbecco's Modified Eagle Medium

^ JP?M^ ;;| nlicrograms per well of plasmid pCMV-susCD163vl, in DMEM without FBS or
""{*• 1 v V antibiotics, using Lipofectamine 2000 (Invitrogen catalog number 11668-027)
i |r according to the manufacturer's instructions. At 24 hours post transfection wells were } jf aspirated and washed twice with DMEM/3% FBS followed by infection with
| ^European PRRSV isolate 96V198. Virus was allowed to adsorb for a minimum of 2 jV* I hours. The virus was then removed, wells washed twice with growth media, and fresh
• growth media added (2.0 ml per well). A time zero sample of culture fluid was
IT
"immediately taken in order to determine the background level of infectious virus from the inoculum. At a minimum of 48 hours post infection cultures were screened for
i i"f
permissivity by removing culture fluids in order to assay viable virus, and permissive jJcells in the monolayer were detected by fluorescent antibody assay (FA). The FA was
• ^ompleted by fixing the monolayer with 80% acetone and stained with FTTC-
' conjugated monoclonal antibody SDOW17 (Rural Technologies Lie), which is specific for the PRRSV nucleocapsid protein. Viable virus was titrated by inoculating dilutions of culture fluids onto MARC-145 cells. As a result of the transient
1s" '
transfection of BKH21 with pCMV-susCD163vl, cells were rendered permissive to 'European PRRSV isolate 96V198 infection and yielded progeny virus.
Example 13: CD163 genes from multiple animal species render BHK21 cells permissive to PRRS virus infection
TtplJK21 cells grown in DMEM (Invitrogen catalog number 11965) supplemented with 10% fetal bovine serum, ImM sodium pyruvate, and antibiotics, were used in
j-Jfansient transfection experiments. Before transfection cells were washed once with OptiMEM (Invitrogen) without serum or other additives. Lipofectamine 2000
! 0hvitrogen) was used in all transfection experiments according to the protocol provided by the manufacturer. The transfection mixture consisted of 10 microliters of fjpofectamine 2000 and 2-3 micrograms of DNA per 35 mm well. After overnight incubation, transfection medium was removed and cells were infected with PRRSV isolate P129. Infection was allowed to progress for 24-48 hours, when cells were fixed with 80% acetone and stained with monoclonal antibodvSDOW17'Conhn»ataH

with ETTC (Rural Technology Inc., Brpokings, SD). Staining of the micteocapsid protein was visualized under a fluorescence microscope. Table 6. Transient transfection of BHK21 cells with various CD163 genes lenders them permissive to PRRS, virus infection
Table 6

Plasmid backbone CD163gene PRRSV infection (FA)
pCMV-Script Swine CD163vl +++'
pRSV-Script Swine CD163vl -t-H-
pcDNA3.1D Swine CD163v2 •H-
pcDNA3.1D Human CD163v2 •H-
pcDNA3.1D Mouse CD163v3 +
pcDNA3.1D African green monkey +-H-
(MARC-145 cell) CD163v2
pcDNA3.1D Vero cells CD163v7 +++
OCDNA3.1D DH82cellCD163v2 +++
Highly positive •H- = Moderately positive + « Slightly positive
Example 14. Generation of PRRSV-pennissive BHK21 stable cell lines using pCMV-susCD163vl
BHK-21 cells were grown in Dulbecco's Modified Eagle Media supplemented with 10% fetal bovine serum, ImM sodium pyruvate, and antibiotics. For transfection, cells were seeded at approximately 90% confluency in 6 well plates and incubated over night at 37°C in 5% CQj. Cells were transfected with pCMV-susCD163vl DNA using IJpofectamine 2000 (Ihvitrogen) according to the manufacturer's instructions. One day after transfection the cells were trypsinized and re-seeded in 96 well plates in a dilution series. To select for stable transfectants, the media was supplemented with 1 rag/ml Geneticin(G418 sulfate, Livitrogen catalog number 10131-027) from this point forward. Medium was changed every 3-5 days. Plates were cultured until those wells with colonies derived from single cells reached confluency, at which point the plates were trypsinized and seeded into duplicate 96 well plates. One of the duplicate 96 well plates was infected with PRRSV isolate P129 and clones permissive to infection were identified by staining with HTC conjugated monoclonal antibody SDOW17. Positive clones were then-expanded from the second duplicate plate. To ensure homogeneity the positive cultures were single-

siyity were chosen for expansion. Three clones
designated BHK/GMV/vl #3, BHK/CMV/vl #5, and BHK/CMV/vl #12
i
Example 15. Generation of PRRSV-permissive BHK21 stable ceO lines using PRSV-susCD163vl
BHK-21 cells were cultured as described in Example 14. BHK-21 cells were
transfected with pRSVsusCD163vl using Lipofectamine 2000 as described in Example 14. Cloning of transfected cells and screening for permissive clones was performed essentially as described in Example 14. From the original cloning 3 single cell clones were identified as permissive and were subsequently recloned two more times to ensure homogeneity and to attempt to isolate subclones of higher pennissivity (see Figure 7). The resulting cell lines were named BHK/RSV/vl, #2, #3, and #4. All of these clones have maintained the permissive phenotype through the highest passage tested (passage 1 1 for clone #2, passage 7 for clone #3, and passage 5 for clone #4). Example 16. Generation of PRRSV-permissfve. feline kidney stable cell lines using pCMV-susCD163vl
Parental Norden Labs Feline Kidney
(calculated number of resistant cells / well is 0.3, using the Poisson distribution). These 26 wells were split into duplicate wells and allowed to settle overnight. One set of wells was infected with PRRSV isolate P129, incubated for 24 houre, then fixed with 80% acetone and stained with ElTC-conjugated monoclonal antibody SDOW17 (Rural Technologies foe), which is specific for PRRSV nucleocapsid. Of the 26 clones, 8 contained some cells that were infected by PRRSV. One of these, designated "NLFK-CMV-susGD163vl-G4", was clearly more permissive than the others with nearly 100% of the cells staining positive for viral antigen.
By cell passage number 5, there was some evidence of phenotypic heterogeneity in the NLFK-CMV~susCD163vl-O4 cell line. Therefore, the cells were single-cell cloned by limiting dilution in Norden Labs Feline Kidney (NLFK) cells were grown at 37° C and 5% CO2 in Minimal Essential Medium Alpha Medium (Invitrogen catalog number 12571-071) supplemented with 10% fetal bovine serum and antibiotics. NLFK cells were seeded in 6 well plates at approximately 90% confluency and allowed to attach overnight The cells were then transfected with plasmid pRSV-susCD163vl using Upofectamine 2000 (Invitrogen) following (he manufacturer's instructions. After 24 hours the -cells were cloned as described in Example 14. Screening for PRRSV permissive cell clones was performed as described in Example 14. Four clones were selected from the screening and were single cell cloned by limiting dilution two more times. Four clones named FK/RSV/vl #1, FK/RSVM #2, FK/RSV/vl #3, and FK/RSV/vl #4 were selected. These cell lines have maintained the PRRSV permissive phenotype through at least 8 passages (see Figure 9).
Example 18. Generation of PRRSV-permissive porcine kidney stable cell lines using pCMV-susCD163vl
Parental Porcine Kidney (PK032495) cells were obtained from Pfizer Inc.

Modified Eagle Medhim (DMEM, Invitrogen catalog number 11965) supplemented with 5% fetal bovine serum (FBS), ImM sodium pyruvate, 2mM L-glutamine and antibiotics. Tissue culture wells (35 mm) containing approximately IxlO6 ccUs each were transfected with 2 micrograms per well of plasmid pCMV-susCD163vl, in DMEM without FBS or antibiotics, using Upofectamine 2000 (Invitrogen catalog number 11668-027) according to the manufacturer's instructions. After overnight incubation, cells were washed with FBS and removed from the substrate using Accutase (Innovative Cells Technologies, catalog number AT104) and diluted in growth medium containing Geneticin (G418 sulfate, Invitrogen catalog number 10131-027) at 1.0 milligram per ml and seeded into 96-well plates at various densities
to ensure recovery of single cell clones after Geneticin selection. Throughout __.
Geneticin selection, media was changed approximately every 3 to 5 days. After selection, wells containing single cell clones were expanded into duplicate 96-well plates and allowed to incubate until 100% confluency was achieved. One set of wells was screened for PRRSV-permissivity by infecting with PRRSV isolate P129 for a minimum of 48 hours. Eleven clones were found to be permissive for PRRSV. One of these, designated "PK-CMV-susCD163vl-A10", clearly retained the permissive phenotype after numerous passages (see Figure 10).
Example 19. Generation of PRRSV-permissive BHK21 stable cell lines using pCMVScript-susCD163v2
Parental Baby Hamster Kidney (BHK21) cells were obtained from Pfizer Inc. and were grown at 37 degrees C and 5% COa in growth media consisting of Dulbecco's Modified Eagle Medium (DMEM, Invitrogen catalog number 11965) supplemented with 10% fetal bovine serum (FBS), ImM sodium pyruvate, 2mM L-glutamine and antibiotics. Tissue culture wells (35 mm) containing approximately IxlO6 cells each were transfected with 2 micrograms per well of pCMVScript-susCD163v2, in DMEM without FBS or antibiotics, using Upofectamine 2000 (Invitrogen catalog number 11668-027) according to the manufacturer's instructions. After overnight incubation, cells were washed with PBS and removed from the substrate using Accutase (Innovative Cells Technologies, catalog number AT104) and diluted in growth medium containing Geneticin (G418 sulfate, Invitrogen catalog number 10131-027) at 1.0 milligram per ml and seeded into 96-well plates at various

TCirougtonrt Geneticin selection, media was changed approximately every 3 to 5 days. After selection, wells containing single cell clones were expanded into duplicate 96-well plates and incubated until 100% confluency was achieved. One set of wells was screened for pennissivity by infecting with PRRS V isolate P129 and incubating for a minimum of 48 hours. Three clones were found to be PRRSV-permissive, and one of these, designated "BHK-CMVScript-susCD163v2-A9", was chosen for further study (see Figure 11).
Example 20. Generation of PRRSV-pennissive BHK-21 stable cell lines using pRSV-snsCD163v2
BHK-21 cells were cultured as described in Example 14,. BHK-21 cells were transfected with the ligated pRSV-susCD163v2 DNA construct described in Example 5 using Lipofectamine 2000 Example 21. Generation of PRRSV-pennissive porcine kidney stable cell lines using pCMVScript-susCD163v2
Parental Porcine Kidney (PK032495) cells were obtained from Pfizer Inc. and were grown at 37 degrees C and 5% COj in growth media consisting of Dulbecco's Modified Eagle Medium (DMEM, Ihvitrogen catalog number 11965) supplemented with 5% fetal bovine serum (FBS), ImM sodium pyruvate, 2mM L-glutamine and antibiotics. Tissue culture wells (35 mm) containing approximately IxlO6 cells each were transfected with 2 micrograms per well of pCMVScript-susCD163v2 in DMEM without FBS or antibiotics, using Lipofectamine 2000 (Ihvitrogen catalog number 11668-027) according to the manufacturer's instructions. After overnight incubation, cells were washed with PBS and removed from the substrate using Accutase (Innovative Cells Technologies, catalog number AT104) and diluted in growth medium containing Geneticin (G418 sulfate, Ihvitrogen catalog number 10131-027) at 1.0 milligram per ml and seeded into 96-well plates at various densities to ensure recovery of single cell clones after Geneticin selection. Throushout Geneticin

containing single cell clones were expanded into duplicate 96-well plates and incubated until 100% confluency was achieved. One set of wells was screened for permissivity by infecting with PRRS V isolate P129 and incubating for a minimum 48 hours. One clone designated "PK-CMVScript-susCD163v2-Dl" showed the PRRSV-permissive phenotype.
Example 22. Generation of PRRSV-permissfo BHK21 stable ceU lines using pcDNA3.1D-humCD163v2
Parental Baby Hamster Kidney (BHK21) cells were obtained from Pfizer Inc. and were grown at 37 degrees C and 5% CO? in growth media consisting of Dulbecco's Modified Eagle Medium (DMEM, Invitrogen catalog number 11965) supplemented with 10% fetal bovine serum (FBS), ImM sodium pyruvate, 2mM L-glutamine and antibiotics. Tissue culture wells (35 mm) containing approximately IxlO6 cells each were transfected with 2 micrograms per well of pcDNA3.1D-humCD163v2, in DMEM without E3S or antibiotics, using Lipofectamine 2000 (Invitrogen catalog number 11668-027) according to the manufacturer's instructions. After overnight incubation, cells were washed with PBS and removed from the substrate using Accutase (Innovative Cells Technologies, catalog number AT104) and diluted in growth medium containing Geneticin(G418 sulfate, Invitrogen catalog number 10131-027) at 1.0 milligram per ml and seeded into 96-well plates at various densities to ensure recovery of single cell clones after Geneticin selection. Throughout Geneticin selection, media was changed approximately every 3 to 5 days. After selection, wells containing single cell clones were expanded into duplicate 96-well plates and incubated until 100% confluency was achieved. One set of wells was screened for permissivity by infecting with PRRSV isolate P129, incubated for a minimum 48 hours. Seven candidate clones were found to be PRRS V-permissive. There was some evidence of phenotypic heterogeneity in each of the seven candidate clones, likely because they were not clonal. Therefore, the candidate clones were single-cell cloned by limiting dilution in G418 containing medium. One single cell clone with clear PRRS-permissivity was obtained and designated BHK-cDNA3.1D-humCD163v2-H9.
Example 23. Generation of PRRS V-permissive feline kidney stable cell lines using pcDNA3.1D-humCD163v2

Parental Norden Labs Feline Kidney (NLPK) cells were grown at 37 degrees C and 5% Cp2 in growth media consisting of Dulbecco's Modified Eagle Medium (DMEM, Ihvitrogen catalog number 11965) supplemented with 10% fetal bovine serum (FBS), ImM sodium pyruvate, 2mM L-glutamine and antibiotics. Tissue culture wells {35 mm) containing approximately IxlO6 cells each were transfected with 2 micrograms per well of pcDNAS. !D-humCD163v2 in DMEM without FBS or antibiotics, using Lipofectamine 2000 (Invitrogen catalog number 11668-027) according to the manufacturer's instructions. After overnight incubation, cells were washed with PBS, removed from the substrate using Accutase (Innovative Cells Technologies,.cataJog number AT104), diluted in growth medium containing Geneticin (G41S sulfate, Invitrogen catalog number 10131-027) at 500 micrograms per ml, and seeded into 96-well plates at various densities to ensure recovery of single cell clones after Geneticin selection. Throughout Geneticin selection, media was changed approximately every 3 to 5 days. After selection, wells containing single cell clones were expanded into duplicate 96-well plates and incubated until 100% confluency was achieved. One set of wells was screened for PRRSV-pennissivity by infecting with PRRSV isolate P129 for a minimum 48 hours. Five clones were found to be permissive. One of these, designated "FK-cDNA3.1D-humCD163v2-A6", clearly displayed the permissive phenotype (see Figure 1-3).
NLFK parent cells and one subclone of FK-cDNA3.1D-humCD163v2-A6 were examined for the CD163 expression. Cells were fixed in 80% acetone and reacted with Goat anti-human CD163 (R&D System at 1:200) for one hour following by washing with PBS. For visualization, donkey anti-Goat JgG conjugated with FJTC (Biodesign Ihc at 1:100) were used. No specific fluorescence was detected in the NLFK parent cells as shown in Figure 21A. The majority of the FFLA6.A2 subclone showed good fluorescent staining indicating the presence of CD163 (Figure 2 IB). Example 24. Generation of PRRSV-permissive porcine kidney stable cell lines using pcDNA3.1D-bumCD163v2
Parental Porcine Kidney (PK032495) cells were obtained from Pfizer Inc. and were grown at 37 degrees C and 5% CO2 in growth media consisting of Dulbecco's Modified Eagle Medium (DMEM, Ihvitrogen catalog number 11965) supplemented with 5% fetal bovine serum -*-:-: ' • •

jwere transfected with 2 micrograms per well of pcDNA3.lD-humCD163v2, in DMEM without FBS or antibiotics, using Lipofectamine 2000 (Ihvitrogen catalog number 11668-027) according to the manufacturer's instructions. After overnight incubation, cells were washed with PBS, removed from the substrate using Accutase (Innovative Cells Technologies, catalog number AT104), diluted in growth medium containing Geneticin (G418 sulfate, Ihvitrogen catalog number 10131-027) at 1.0 milligram per ml, and seeded into 96-well plates at various densities to ensure recovery of single cell clones after Geneticin selection. Throughout Geneticin selection, media was changed approximately every 3 to 5 days. After selection, wells containing single cell clones were expanded into duplicate 96-well plates and incubated until 100% confluency was achieved. One set of wells was screened for PRRSV-permissivity by infecting with PRRSV isolate P129 for a minimum 48 hours. Two clones were found to be permissive. One of these, designated "PK-cDNA3.1D-humCD163v2-Bl 1" clearly showed the PRRSV-permissive phenotype. Example 25 Generation of PRRSV-permissive feline kidney stable cell line using ligated pRSV-Script MARC CD163v2
A non-cloning based procedure to generate microgram quantities of linear DNA suitable for use in generating stable cell lines expressing CD163 from an RSV promoter was developed (Figure 4). A similar process was adapted to place simian CD163v2 from MARC-145 cells behind the RSV promoter. The procedure involves the isolation and ligation of two pieces of DNA, one containing the neomycin gene and RSV promoter cassette derived from pRSV^script, and the other containing the MARC CD163v2 coding sequence from pCDNA3.1D MARC CD163v2. Vector plasmid pRSV-Script was linearized with Hind JR. and Kpn L Plasmid was first digested with Kpn I and was bunted with the Klenow fragment off1, coli DNA polymerase. This plasmid was then digested with Hind ID immediately downstream f'of the RSV promoter. The pCDNAS.lD MARC CD163v2 clone was digested in the vector sequence downstream of the CD163 insert with EcoKV, and Hind ffl upstream of CD163. The CD163 coding sequence was liberated from the vector. For each plasmid digestion the appropriate fragments were purified from agarose gels. A large-scale ligation reaction was performed as follows. Approximately 20 jig of each DNA fragment was incubated in a volume of 600 JlL with 15 units of T4 DNA ligase. The

^.piece of DNA containing all of the appropriate dements was purified by agarose gel felectrophoresis. Restriction enzyme digestion analysis was performed to confirm the authenticity of each ligated fragment. Ugation of the two DNA fragments via the cohesive Hind UL termini resulted in the placement of the 5' sequences of the MARC CD 163 gene downstream of the RS V promoter, allowing for directed expression of CD 163 in mammalian cells. Once isolated, the purified DNA was used to transfect selected mammalian cell lines.
*, NordenLabs Feline Kidney (NLFK) cells were grown at 37° Cand 5% CQz in DMEM supplemented with 5% fetal bovine serum and antibiotics. NLFK cells were seeded in 6 well plates at approximately 90% confluency and allowed to attach overnight. The cells were then transfected with ligated plasmid pRSV-MARC CD163v2 using Lipofectamine 2000 following the manufacturer's instructions. After > 24 hours the cells were cloned as described in Example 12. Screening for PRRSV Hie amounts of progeny virus produced by PRRSV-infected BHK-21 or , NtFK cells stably engineered to express susCD163vl were quantitated. Four cell
1 '
Jiines expressing susCD163vl, BHK/CMV/susvl #3, BHK/CMV/stisvl #5, ;BHK/CMV/susvl #12, and FK/CMV/susvl G4 were seeded at sub confluency in 6 well plates and, after overnight incubation, were infected with the NVSL 94-3 isolate of PRRSV. MARC-145 cells were included in the experiment for comparison. Hie cells were infected with virus at an m.o.i. of approximately 0.1. Virus was adsorbed for 60-90 minutes and was removed. Cells were washed three times with PBS to remove residual virus. One-milJiliter aliquots were harvested from the cultures at 12-hour intervals starting immediately after infection and continued through 96 hrs. Fresh culture media was added to the cells at various time points to maintain a culture ^blume sufficient to prevent the ceU monolayer from drying out Culture supernatants i stored at -80° until all samples were collected. The amount of PRRSV «»»«»,«•;-

produce progeny PRRSV.
Example 27. Blocking PRRSV infection with anti-CD163 antibody: Transiently

BHK-21 cells, seeded in 24 well plates, were transiently transacted with the imid pO5NA3.1D-MARC-CD 163v2 described in example 8, using Lipofectamine as described in example 14. After overnight incubation to allow expression of 163, a titration of goat polyclonal antibody specific for human CD163 (R&D
Systems, cat # API 607) in PBS was added to the cells in a volume of lOOul. As a
fe •
lontrol, equivalent amounts of normal goat IgG (R&D Systems, cat # AB-108-C)
%ere used. Following a one-hour incubation at 37°C, the monolayers were infected with approximately IxlO7 pfu of a recombinant PI 29 strain of PRRSV mat expresses
ti^
•iGFP. The cell monolayers, with anti-CD163 antibody and PRRSV, were incubated at
l
37°C for one hour at which time the virus inoculum/antibody mixture was aspirated, le cell monolayer washed once with PBS, and 1ml of growth medium added to the The cells were incubated for 24 hours at 37°C to allow PRRSV directed GFP expression. For analysis, the cells were trypsinized, resuspended in SOOjuJ of PBS and analyzed by flow cytometry to innumerate the PRRSV infected cells via GFP
Expression. For flow cytometry, uninfected BHK-21 cells were used to set the
»,
baseline for fluorescence detection, and approximately 100,000 cells were analyzed
, from each subsequent sample. The results of this analysis, shown in Figure 15, show
!i ,
•? that the CD163 specific antibody was able to significantly reduce the number of
infected cells when compared to cells incubated with normal goat IgG.
t i • -
Example 28: Blocking PRRSV infection by anti CD163 antibody: Stably /• , transfected cells.
The NLFK cells that stably express human CD163 si , '
allowing the cells to attach overnight, a titration of goat polyclonal antibody specific human CD163 (R&D Systems, cat # AF1-607) in PBS was added to the cells in a
ume of 1 OOjJl. As a control, equivalent amounts of normal goat IgG (R&D
tils
fsystems, cat # AB-108-C) were used. Following a one-hour incubation at 37°C, the
4
itnbnolavers were infected with

PRRSV, were incubated at 37°C for one hour at which time the virus inoculum/antibody mixture was aspirated, the cell monolayer washed once with PBS, and Imlof growth medium added to the wells. The cells were incubated for 24 hours at 37°C to allow PRRSV directed GPP expression. For analysis, the cells were
it , '
trypsinized, reSuspended in 500^1 of PBS, and analyzed by flow cytometry to innumerate the PRRSV infected cells via GFP expression. Approximately 100,000 cells were analyzed from each sample. The results of this analysis, shown in Figure 16, show that the CD163 specific antibody was able to significantly reduce the number of infected cells when compared to cells incubated with normal goat IgG.
, Example 29. Generation of PRRSV-permissive porcine kidney stable cell lines using pRSV-susCD163v2.
Porcine kidney cells (PK032495) were cultured as described in Example 21. For
transfection, cells were seeded in a 24 well plate at 80% confluency and allowed to ,tecover overnight. Transfection of ligated pRSV-susCDU>3v2 DNA described in Example 5 was performed using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Subsequent cloning and selection of PRRSV permissive cells was performed essentially as described in Example 14. The initial cloning by 'limiting dilution failed to yield single cell derived clones, so 5 wells with PRRSV permissive cells were recloned by limiting dilution to yield clonal cell lines. 10 clones
3
Were selected for further study and one of these clones, PK-RSVScript-susCDl€3v2
u
#9 showed the ability to support foci growth of PRRSV early after infection (see
Figure 18).
Example 30. Generation of PRRSV-permissive feline kidney stable cell lines : using pRSV-susCD163v2. : NLFK feline kidney cells were cultured as described in Examplel?. For
5' s
transfection, cells were seeded at approximately 80% of maximal density in 24 well plates. After overnight incubation the monolayers were transfected with ligation derived RS V/susCD163v2 (see example 5) using Lipofectamine following the manufacturer's instructions. Cloning of the transfected cells and selection of PRRSV .permissive cell clones was performed essentially as described in Example 14. Of the 67 cell clones tested for PRRSV permissiviry, 20 were found to be positive. An example of the staining observed is shown in Figure 19. Example 31. Passage of PRRSV isolate P201 in PK-RSVScript-susCD163v2 cells
Ik «••*•..*• . ._ ,*• ... T«^ v»--*«nr ri"»-»-

dish using OptiMEM media supplemented with 2% EBS. After 6 hours the media £ was aspirated and a 2 ml aliquot of serum harvested from a PRRSV infected pig was *added to the cells. Following a 90 minute adsorption, the serum inoculum was removed and replaced with OptiMEM. At approximately 40 post infection the Supernatant was harvested and clarified with a 10 minute centrifugation. The supernatant was directly used to infect PK-RSVScript-susCD163v2 clone #9 cells * using a 6 hour adsorption. After removal of the inoculum the cells were refcd with D-. MEM. The P201 virus was serially passaged on the PK-RSVScript-susCD163v2 #9 cell line using alternating infected cell and cell free supernatant passes. We observed that for efficient spread of the virus, the cells should be seeded at 50-70 % confluency .the day before infection, using flasks of cells that were kept at sub-confluency. To follow the progression of infection, each passage was replicated in multiple wells of identically infected cells and at each day one of the wells was acetone fixed and stained with the FTTC labeled monoclonal antibody SDOW17. If the percentage of infected cells was not greater than 50% and significant progression of foci development over the prior days observations were not seen, the cells in an equivalent Well were trypsinized and passed to multiple fresh wells. These infected-cell passages were typically at a 1:4 split and sometimes included the addition of an equivalent number of cells from an unlnfected culture. Alternatively, if the SDOW17 staining revealed that the infected cell foci had spread sufficiently to account for greater than 50% of the total cells, cell free supernatant was harvested and used to infect multiple i of freshly seeded cells (Figure20). After 1.1 passages the intervening cell
sages were not necessary as the virus was able to grow to sufficient titer to allow
isecutive cell free supernatant passaging of the virus.
jnple 32. Screening various CD163 cell lines forpennissivity to various
, European and North American PRRSV isolates
5! Various CD 163 transgenic cell lines were assessed for permissivity to low passage
fe , • '•' i
iBuropean and North American PRRSV isolates (see Table 7). Transgenic CD163
|ell lines as described in earlier examples included NLFK-MARC CD163 D4, PK-ksVScript-susCD163v2clone #9 and PK-CMV-susCD163vl-A10. Each GD163 cell iline along with cell lines MARC-145, parental feline kidney, parental porcine kidney teell lines (serving as controls) were planted onto 96-well tissue culture plates. Growth ioiedia was removed finm

stained with FTTC-conjugated monoclonal antibody SDOW17 (Rural Technologies Inc.) which is specific for the nucleocapsid. Results of the fluorescent antibody (FA) assay are in Table 7. Table?

FArenilttof screening, of Tattoos CD163 cell Hues for peranMvUy to European and North American PRRSV isolate
CD163 Cell Line PRRSV Isolate1

EU98V226 P129 KOI 1151 94-3 XND5
NLFK-MARCCD163D4 ++ + +++ *++ •H- •H-H-
PK-RSVScriDt-8U«CD163v2done #9 + + • ++ + + ++
PK-CMV-«uiCD163vl-A10 + + ++ +* ++ •H-
MARC-145 ++ + +++ + 4+++ +++
Porcine Kidney (parental) . - — -• ,.,.,. .. . •- • •
Feline Kidney (parental) • - - - - •
* All PRRSV isolates an North American except EU98V226 is a European isolate.
Example 33. Phorbol 12-myristate 13-acetate (PMA) induction of CD163 renders human 17937 cells permissive to PRRSV infection.
Human U937 cells obtained from ATCC RPMI medium containing serum and additives according to ATCC specifications. : These cells are known to express CD163 when activated by PMA treatment I (Gronlund et al., 2000). U937 cells were seeded in duplicate in wells of a 6-well if plate. One set of wells was treated with 100 ng/ml of PMA and the other set was left f untreated. Three days after PMA stimulation, one well from each set was infected with the P129 isolate of PRRSV. The other well from each set was fixed and stained for expression of CD163 in an indirect immunofluorescent antibody assay using goat-anti human CD163 (R&D System) and donkey anti-goat IgG conjugated with FTTC (BioDesign International).
Untreated U937 cells continued propagation to high density 3 days after initial planting. PMA-treated U937 cells stopped propagating, became enlarged, and , attached to the surface of the culture wells. A small fraction of untreated U937 were * positive for CD163 staining, whereas almost all PMA-treated U937 were positive for
)163 staining. In untreated U937 no PRRSV infected cells were observed, fowever, hundreds of PMA treated U937 cells became infected by PRRSV. This demonstrates that non-permissive cells can be rendered permissive for PRRSV , infection following chemical induction of CD1€3 expression.

Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, including the detailed description, and all such features are intended as aspects of the invention. Likewise, features of the invention described herein can be re-combined into additional embodiments that also are intended as aspects of the invention, irrespective of whether the combination of features is specifically mentioned above as an aspect or embodiment of the invention. Also, only such limitations mat are described herein as critical to the invention should be viewed as such; variations of the invention lacking limitations that have not been described herein as critical are intended as aspects of the invention.
It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples.
Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the invention.
The entire disclosure of all publications cited herein are hereby incorporated by reference to the extent they are not inconsistent to the disclosure herein.







We claim
1. A method of facilitating production of Porcine Reproductive and Respiratory Syndrome Virus
(PRRSV) from a culture of vertebrate cells, comprising the steps of:
(a) providing a recombinant vertebrate cell transfected with an exogenous polynucleotide that encodes a mammalian CD 163 polypeptide having a transmembrane domain and at least 70% sequence identity to SEQ ID NO: 14, so that expression of CD 163 polypeptide in said cell is increased;
(b) contacting a culture of said cell with PRRSV virus under conditions which permit infection of the cells and growth of the virus; and
(c) recovering virus from said culture.
2. The method as claimed in claim 1, wherein the cell was previously permissive PRRSV
permissive and is rendered more PRRSV permissive.
3.The method as claimed claim 1, wherein the cell did not previously express a CD 163 polypeptide and is induced to express CD 163.
4. The method as claimed in claim 1, wherein the cell is selected from the group consisting of baby hamster kidney cells (BHK2 1), porcine kidney cells, feline kidney cells, avian cells, and swine testicular cells.
5. The method as claimed in claim 1, wherein the PRRSV is of the European genotype.
6. The method as claimed in claim 1, wherein the PRRSV is of the North American genotype.
7.The method as claimed in claim 1, wherein the polynucleotide encodes a polypeptide having a transmembrane domain and at least 90% sequence identity to SEQ ID NO: 14.

8. The method as claimed in claim 1, wherein the polynucleotide encodes a polypeptide having a transmembrane domain and at least 90% sequence identity to SEQ ID NO: 19.
9. The method as claimed in claim 1, wherein the polynucleotide encodes a polypeptide having a transmembrane domain and at least 90% sequence identity to SEQ ID NO:24.

10. The method as claimed in claim 1, wherein the polynucleotide encodes a polypeptide having a transmembrane domain and at least 90% sequence identity to SEQ ID NO:32.
11. The method as claimed in claim 1, wherein the polynucleotide encodes a polypeptide having a transmembrane domain and at least 90% sequence identity to SEQ ID NO:46.
12. The method as claimed in claim 1, optionally comprising the step of producing a vaccine from the recovered virus.
13. The method as claimed in claim 1 wherein transfection with an exogenous polynucleotide that encodes a mammalian CD 163 polypeptide is accomplished by electroporation.

Documents:

5603-DELNP-2006-Abstract-(24-09-2010).pdf

5603-delnp-2006-abstract.pdf

5603-DELNP-2006-Claims-(11-05-2011).pdf

5603-DELNP-2006-Claims-(24-09-2010).pdf

5603-delnp-2006-claims.pdf

5603-DELNP-2006-Correspondence Others-(11-05-2011).pdf

5603-DELNP-2006-Correspondence-Others-(24-09-2010).pdf

5603-delnp-2006-correspondence-others.pdf

5603-delnp-2006-description (complete).pdf

5603-DELNP-2006-Drawings-(24-09-2010).pdf

5603-DELNP-2006-Form-1-(24-09-2010).pdf

5603-delnp-2006-form-1.pdf

5603-delnp-2006-form-18.pdf

5603-DELNP-2006-Form-2-(24-09-2010).pdf

5603-delnp-2006-form-2.pdf

5603-DELNP-2006-Form-3-(24-09-2010).pdf

5603-delnp-2006-form-3.pdf

5603-delnp-2006-form-5.pdf

5603-DELNP-2006-GPA-(24-09-2010).pdf

5603-delnp-2006-pct-304.pdf

5603-DELNP-2006-Petition 137-(24-09-2010).pdf


Patent Number 248617
Indian Patent Application Number 5603/DELNP/2006
PG Journal Number 30/2011
Publication Date 29-Jul-2011
Grant Date 28-Jul-2011
Date of Filing 26-Sep-2006
Name of Patentee PHARMACIA & UPJOHN COMPANY LLC.
Applicant Address 7000 PORTAGE ROAD, KALAMAZOO, MICHIGAN 49001, UNITED STATES OF AMERICA USA
Inventors:
# Inventor's Name Inventor's Address
1 JAY GREGORY CALVERT 03400 22ND STREET, OTSEGO , MICHIGAN 49078 USA;
2 SHELLY LYNN SHIELDS 12200 PINE LAKE ROAD, PLAINWELL, MICHIGAN 49080 USA
3 DAVID EWELL SLADE 7426 MARFIELD STREET, PORTAGE, MICHIGAN 49024,USA
4 SIAO-KUN WAN WELCH 9087 SOUTH 6TH STREET, KALAMAZOO, MICHIGAN 49009, USA
PCT International Classification Number A61K39/12; C12N5/06; C12N7/00
PCT International Application Number PCT/US2005/011502
PCT International Filing date 2006-04-05
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/565.214 2004-04-23 U.S.A.
2 60/634,736 2004-12-09 U.S.A.