Title of Invention	ANTIVIRAL COMPOUNDS AND METHODS
Abstract	The invention relates to compounds having antiviral activity and methods utilizing the compounds to treat viral infections.

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ANTIVIRAL COMPOUNDS AND METHODS FIELD OF INVENTION The present invention rallies to methods for retarding, reducing or otherwise inhibiting viral growth and/or functional activity. The invention also relates to compounds and compositions fuitable for use in the methods. BACKGROUND OF THE INVENTION Currently, there is a great need for the development of new treatments that are effective against viral infections, particularly against viral infections which are associated with high morbidity and mortality, and which impact on sizable . populations. Treatments currently available are inadequate or ineffective in large proportions of infected patients. For example, in ameliorating AIDS symptoms and prolonging life expectancy, a measure of success has been achieved with drags targeting the viral reverse transcriptase and protease enzymes (Miller and Sarver, 1997; Mitsuya, 1992; Moore, 1997; and Thomas and Brady, 1997). However, no single treatment method is completely effective against HIV infection. (Barry et al, 1998; Deeks, 1998; Miles, 1997; Miles, 1998; Moyle et al, 1998; Rachlis and Zarowny. 1998; Veil et al, 1997; Volberding and Deeks, 1998; and Volberdin, 1998). PCT application PCT/AU99/00872 describes the use of compounds 5-(N,N- hexamethylene)-amiloride and 6-(N,N-imethyl)-amiloride in the treatment of HIV infection. Another virus considered to be a significant human pathogen is the Hepatitis C virus (HCV). This is a significant human pathogen in terms of both cost to human health and associated economic costs. HCV causes chronic hepatitis and cirrhosis and is the leading indicator for liver replacement surgery. In 2002 the Centre for Disease Control and Prevention estimated that more-than 4 million people were infected in the USA alone and that approximately 8,000 to 10,000 die as a result of chronic HCV infection yearly. There is no known cure or vaccine. More effective pharmacological agents are urgently required. A further well-known family of pathogenic viruses are the Coronaviruses. Coronaviruscs (Order Nidovirctes. family Coronaviridae, Genus Coronavirus) are enveloped positive-stranded RNA viruses that bud from the endoplasmic reticulum- Goigi intermediate compartment or the cis-Golgi network (Fischer, Stegen et al. 1998; Maeda, Maeda et al. 1999; Corse and Machamer 2000; Maeda, Repass et al. 2001; Kuo and Masters 2003). Coronaviruses infect humans and animals and it is thought that there could be a coronavirus that infects every animal. The two human coronaviruses, 229E and OC43, are known to be the major causes of the common cold and can occasionally cause pneumonia in older adult, neonates, or immunocompromised patients (Peiris, Lai et al. 2003). Animal coronaviruses can cause respiratory, gastrointestinal, neurological, or hepatic diseases in their host (Peiris, Lai et al. 2003). Several animal coronavirus are significant veteinary pathogens (Rota, Obcrste ct al. 2003). Severe acute respiratory syndrome (SARS) is caused by a newly identified virus. SARS is a respiratory illness that has recently been reported in Asia, North America, and Europe (Peiris, Lai et al. 2003). The causative agent of SARS was identified as a coronavirus. (Dnosten, Gunther et al. 2003; Ksiazek, Erdman et al. 2003; Peiri3, Lai et al. 2003). The World Health Organization reports that the cumulative number of reported probable cases of SARS from 1 November 2002 to the 11 July 2003 is 8,437 with 813 deaths, nearly a 10% death rate. It is believed that SARS will not be eradicatec, but will cause seasonal epidemics like the.cold or influenza viruses (Vogel 2003). To improve the prospect of treating and preventing viral infections, there is an on-going need to identify molecules capable of inhibiting various aspects of the viral life cycle. It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art or to provide a useful alternative. Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in ft e field. SUMMARY OF THE INVENTION The inventors have surprisingly found that certain compounds that fell under the classification of substitufrid acylguaniduies have antiviral activity against viruses fiora a range of different vim s families. Without intending to be bound by any particular theory or mechanism of action, and despite current dogma, it appears possible that viral replication can be retarded by inhibiting or otherwise down- regulating the activity of ion channels expressed in the host cell. Thus, the negative impact of the compounds of the present invention on viral replication may be mediated by the inhibition or otherwise down-regulation of a membrane ion channel relied upon by the virus for replication. This membrane ion channel may be a viral membrane ion channel (exogenous to the host cell) or a host cell ion channel induced as a result of viral infection (endogenous to the host cell). As an example, the compounds Of the present invention may inhibit Vpu or p7 function and thereby inhibit the continuation of the respective HTV or HCV life cycle. The S ARS virus encodes an E protein which is shown for the first time, by the present inventors, to act as an ion channel. As similar E proteins are present in other coronaviruses, the compound;, compositions and metiiods of the present invention would have utility in the inhibition and/or treatment of infections by other coronaviruses. The present invention is concerned with novel antiviral compounds that fall under, the classification of sutstituted acylguanidines. It does not include in its scope the use of compounds 5-(N,N-hexamethylene)amiloride and 5-(N,N-dimethyl)- amiloride ibr retarding, reduc ng or otherwise inhibiting viral growth and/or functional activity of HIV. Accordingly, a first aspect of the present invention provides an acylguanidine with antiviral activity. According to a second aspect, the present invention provides an antiviral compound of Formula I X = hydrogen, hydroxy, nitro, halo, C1-6alky], C1-6lkyloxy, C3-6cycloalky, halo-substituted C1-6alkyl, halo-substituted C1- 6alkyloxy, phenyl, C1-6alkensyl, C3-6Cycloalkeneyl, C1-6alkeneoxy, or benzo; Ra, Rb, Rc, Rd, Re , Rf, Rh, Rk, RL, Rm, Rn, Ro, Rp independently = hydrogen, amino, halo, C1-5alkyl, C1-5alkyloxy, hydroxy, aryl, substituted aryl, substituted amino, mono or dialkyl-substituted amino, cycloalkyl-substituted amino, aryl-substituted amino, Preferably, the compounds of the invention are capable of reducing, retarding or otherwise inbibiting viral growth and/or replication. Preferably, the antiviral activity of the compounds of the invention is against viruses such as those belonging to the Lentrviras family, and the Coronovirus family family of viruses. For example, the compounds of the invention exhibit antiviral activity against viruses such as Human hnmunodeficiency Virus (HIV), Severe Acute Respiratory Syndrome virus (SARS), Mouse Hepatitis virus 0, and Hepatitis C virus (HCV). According to a fourth aspect of the present invention, there is provided a pharmaceutical composition ojmprismg an antiviral compound according to any one of the first, second or third aspects, and optionally one or more pharmaceutical acceptable earners or derivatives, wherein said compound is capable of reducing, retarding or otherwise mhibitirtg viral growth and/or replication. Preferably, the antiviral activity of the compounds of the invention is against viruses such as those belonging to the Lentivirus family, and the Coronovirus family of viruses. For example, the compounds of the invention exhibit antiviral activity against viruses such as Human Irnmunodefidency Virus (HIV), Severe. Acute Respiratory Syndrome virus (SARS), Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV), Hepatitis C virus (HCV) and Equine Arteritis Virus (EAV). Other Coronaviruses which can be inhibited or their infections treated by the compounds of the invention are those listed in Table 1. The compositions of the invention may further comprise one or more known antiviral compounds or molecules. According to a fifth aspect, there is provided a method for reducing, retarding or . otherwise inhibiting growth and/or replication of a virus comprising contacting a cell infected with said virus or exposed to said virus with a compound according to any one of the first, second or third aspects. Preferably, the virus is from the Lentivirus family, or the Coronavirus family. More preferably, the virus is Human Immunodeficiency Virus (HIV), Severe Respiratory Syndrome virus (SARS), Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV), Porciitie Respiratory Coronavirus (PRCV), Moiuse Hepatitis'virus (MHV), Hepatitis C virus (BCV), or Equine Arteritis virus (EAV). Most preferably, the virus is HTV-1, HIV-2, the SARS virus. Coronavirase 229E, Coronaviros OC43, PRCV, BCV, HCV, or EAV. Other Coronaviruses wliich can be inhibited or their infections treated by the compounds of the invention are those listed in Table 1. According to a sixth aspect, there is provided a method for preventing the infection of a cell exposed to a virus comprising contacting said cell with a compound according to any one of the first, second or third aspects. Preferably, the virus is from the Lentivirus family, or the Coronavirus family. More preferably, the virus is Human Immunodeficiency Virus (HIV), Severe Respiratory Syndrome virus ( SARS), Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV), Mouse HepatiuYvinis (MHV), Hepatitis C virus (HCV), or Equine Arteritis V:rus (EAV). Most preferably, the virus is HIV-1, HIV-2, the SARS virus, Coronaviruset 229E, Coronavirus OC43, PRCV, BCV, HCV, EAV. Other Coronaviruses which can be inhibited or their infections treated by the compounds of the invention arc those listed in Table 1. According to a seventh aspect of the invention, there is provided a method for the therapeutic or prophylactic treatment of a subject infected with or exposed to a virus, comprising the admirasttation of a compound according to any one of the first, second or third aspects, to a subject in need of said treatment Preferably, infection with a virus or exposure to a virus occurs with viruses belonging to the Lentivirus family, or the Coronovirus family. More preferably, infection or exposure occurs with HIV, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis vims (MHV), Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV), Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Most peferably, infection or exposure occurs with HIV-1, HIV-2, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Hepatitis C virus (HCV), or Equine Arteriti 5 Virus (EAV). Other Coronaviruses which can be inhibited or their infections treated by the compounds of the invention are those listed in Table 1. The subject of the viral inhibition is generally a mammal such as but not limited to human, primate, livestock animal (e.g. sheep, cow, horse, donkey, pig), companion animal (e.g. dog, cat), laboratory test animal (e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animal (e.g. fox, deer). Preferably, the subject is a . primate, or horse. Most preferably, the subject is a human. According to a eighth asp set, mere is provided a method of down regulating a membrane ion channel functional activity in a cell infected with a virus, comprising contacting said cell with a compound according to any one of the first, second or third aspects. The membrane ion channel maybe endogenous to the cell or exogenous to the celL Preferably, the membrane ion channel of which functional activity is down - regulated is that which Lentiviruses,- and Coronaviruses utilise for mediating viral- replication and include, for example, me HIV membrane ion channel Vpu, the HCV membrane ion channel P7, the Coronavirus E protein membrane ion channel, and the SARS E protein membrane ion channel. Preferably, infection with a virus or exposure to a virus occurs with viruses belonging to the Lentivirus family, or the Coronovirus family. More preferably, infection or exposure occurs with HIV, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV), Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Most preferably, infection or exposure occurs with IHV-1, HIV-2, SARS, Human Coronav irus 229E, Human Coronavirus OC43, Hepatitis C virus (HCV), or Equine Arteritia Virus (EAV). According to an. ninth aspecr of the present invention, there is provided a method of reducing, retarding or otherwise inhibiting growth and/or replication of a virus that has infected a cell, said method comprising contacting said infected cell with a compound according to any one of the first, second or third aspects, wherein said compound down regulates functional activity of a membrane ion channel derived from said virus and expressed in said infected cell. Preferably, infection occurs with a virus belonging to the Lentivirns family, or the Coronoviras family. More preferably, infection or exposure occurs with HIV, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MHV); Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV), Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Most preferably, infection or exposure occurs with HIV-1, HIV-2, SARS, Human Coronavirus 229E, Human . Coronavirus OC43, Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Other Coronaviruses which can be inhibited or their infections treated by the compounds of the invention are those listed in Table 1. Preferably, the membrane ion channel of which functional activity is down regulated is that which Lentiviruses, and Coronaviruses utilise) for mediating viral •replication and include, for example, the HIV membrane ion channel Vpu, the HCV membrane ion channel P7, and he Cotonavirns E protein membrane ion channel. According to an tenth aspect, the present invention provides a method of reducing, retarding or otherwise inhibitmglgrowth and/or replication of a virus that has infected a cell in a mammal, said method comprising administering to said mammal a compound according to any one of the first, second or third aspects, or a pharmaceutical composition according to the fourth aspect, wherein, said compound or said composition down regulates functional activity of a membrane ion channel expressed in said infected celL Preferably, infection occurs with a virus belonging to the Lentivirus family, or the Coronovirus family. More preferably, infection or exposure occurs with HIV, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV), Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Most preferably, infection or exposure occurs with HIV-1, HIV-2, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Other Coronaviruses which can be inhibited or their infections treated by the compounds of the invention are those listed in Table 1. Preferably, the membrane ion channel of which functional activity is down regulated is that which LentiviruseS, and Coronaviruses utilise for mediating viral replication and include, for example, the HIV membrane ion channel Vpu, the HCV membrane ion channel P7, and the Coronavirus E protein membrane ion channel. The subject of the viral i nhibition is generally a mammal such as but not limited to human, primate, livestock animal (e.g. sheep, cow, horse, donkey, pig), companion animal (e.g. dog, cat), laboratory test animal (e.g. mouse., rabbit, rat, guinea pig, hamster), captive wild animal (e.g. fox, deer). Preferably, the subject is a primate, or horse. Most preferably, the subject is a human. According to a eleventh aspect, the present invention provides a method for the therapeutic or prophylactic treatment of a subject infected with or exposed to a virus. . comprising administering to said subject a compound according to any one of the first, second or third aspects, or a pharmaceutical composition according to the fourth aspect, wherein said compound or said composition down-regulates functional activity of a membrane ion channel derived from said virus. Preferably, infection occurs with a virus belonging to the Lendvirus family, or the Coronovirus family of viruses. More preferably, infection or exposure occurs with HIV, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV), Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Most preferably, infection or exposure occurs with HIV-1, HIV-2, SARS", Human Coronavirus 229E, Human Coronavirus OC43, Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Other Cpronaviruses which can be inhibited or their infections treated by the compounds of the invention are those listed in Table 1. Preferably, the membrane ion channel of which functional activity is down regulated is that which Lentiviruses, and Coronaviruses utilise for mediating viral replication and include, for example, the HIV membrane ion channel Vpu, the HCV membrane ion channel P7, and the Coronavirus E protein membrane ion channel,. The subject of the viral inhibition is generally a mammal such as but not limited to human, primate, livestock animal (e.g. sheep, cow, horse, donkey, pig), companion animal (e.g. dog, cat), laboratory test animal (e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animal (e.g. fox, deer). Preferably, the subject is a primate, or horse. Most preferably, the subject is a human. According to a thirteenth aspect, the present invention provides a pharmaceutical composition comprising a compound according to the twelfth aspect, and optionally one or more pharmaceutical acceptable carriers or derivatives.. Preferably, the pharmaceutical composition may farther comprise one or more known antiviral compounds or molecules. Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to", BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS Figure 1 is a schematic representation of plasmids used for expression of Vpu in E. coli. A. The amino acid sequence ( 1) encoded by the vpu open reading frame (ORF) generated by PCR from an HIV-1 strain HXB2 cDNA clone. The vpu ORF was cloned in-frame at tie 31 end of the GST gene in p2GEX to generate p2GEXVpu (B). It was subsequently cloned into pPL451 to produce the plasmid pPL + Vpu (C). Figure 2 is a photographic representation of the expression and purification of Vpu in E. coli. A. Western blotting after SDS-PAGE was used to detect expressed Vpu in E, coli extracts. Lanes 1-4 contain samples, at various stages of purity, of Vpu expressed from p2GEXVpu: lane 1, GST-Vpu fusion protein isolated by glutathione-agarose affinity chromatography; lane 2, Vpu liberated from the fusion protein by treatment with thrombin; lane 3, Vpu purified by HPLC anion exchange chromatography; Iane 4, Vpu after passage through, the immunoaffinity column. Lanes 5 and 6, membrane vesicles prepared from 42'C incluced cells containing pPL+Vpu or pPL451, respectively. B. Silver stained SDS-PAGE gel: Iane 1, Vpu purified by HPLC anion exchange chromatography; Iane 2, Vpu after passage through the immunoaffinity column. Figure 3 is a graphical representation of ion channel activity observed after exposure of lipid bilayers to aliquots containing purified Vpu. In A and B, the CIS chamber contained 500mM NaCl and the TRANS chamber contained 50mM NaCl; both solutions were buffered at pH 6.0 with 10 mM MES. B shows a current versus voltage curve generated from data similar to that shown in A. Figure 4 is a photographic representation of bacterial cross-feeding assays. For all plates, the Met, Pro- auxotrophic strain was used to seed a soft agar overlay. Plates A . and B contain minimal drop -out medium minus proline; in plate C the medium was minus methionine. To control for viability of the cells in the background lawn, the discs labelled P and M contained added proline or methionine, respectively. The discs labelled C and V were inocu lated with Met4, Pro+ B. coli cells containing the plasmids pPL451 or pPL+Vpu, respectively.' Plates were incubated at 37oC (A and C) or 30°C (B) for two days and photographed above a black background with peripheral illumination from, a fluorescent light located below the plate. The images were recorded on a Novaline video gel documentation system. Light halos around the discs labelled P or M on all plates .and around the disc labelled V on plate A indicate growth of the background lawn strain Figure 5 is a graphical representation of the. screening of drugs for potential Vpu channel blockers. The photograph shows a section of a minimal medium-lacking adenine - agarose plate onto, which a lawn of XL-I-blue E. coli cells containing the Vpu expression plasmid pPLVpu has been seeded. Numbers 6-11 are located at the sites of application of various drugs being tested, which were applied in 3^1 drops and allowed to soak into the agarose. The plate was then incubated at 37°C for 4Shr prior to being photographed. The background grey shade corresponds to areas of no bacterial growth. The bright circular area around " 10" represents bacterial cell growth as a result of application of adenine at that location (positive control). The smaller halo of bacterial growth around "9" is due to the application of 5-CN.N- hexamethyiene)-amiloride at that location. Figure 6. SARS E protein ion channel activity observed in NaCl solutions after exposure of lipidbikyer to 3-10µg of E protein. A. The closed state is shown as solid line, openings are derivations irom the line. Scale bar is 300ms and 5pA. The CIS chamber contained 50mM NaCl in 5mM HEPES buffer pH 7.2, the TRANS chamber contained 500mM NaCl in 5mM HEPES buffer pH 7.2. The CIS chamber was . earthed and the TRANS chamber was held at various potentials between -100 to +2 00mV. B. Largest single op suing events of a single channel. Figure 7. SARS E protein ion channel activity observed in NaCl solutions after exposure of lipid bilayer to 3-10µg of E protein. A. The closed state is shown as solid line, openings are derivations irom the line. Scale bar is 300ms and 5pA. The CIS chamber contained 50mM NaCl in 5mM HEPES buffer pH 7.2, the TRANS chamber contained 500mM NaCl in 5mM HEPES bufferpH 7.2. The CIS chamber was earthed and the TRANS chamber was held at various potentials between -100 to +r00mV, B. Largest single opening events of a single channel. Figure 8. Cinnamoylguanidine (Bit036) inhibits SARS E protein ion channel activity in NaCl solution. A, Representative currents at holding potential of-40mV. Scale bar is 300mS and 5pA. E protein ion channel activity and E protein channel activity after the addition of 100µM Bit036. B. All points histogram at holding potential of- 40mV. E protein ion channel activity before and after the addition of 100µM Bit036. C, Average current (pA), before formation of E protein ion channel, E protein ion channel activity and after addition of 100µM Bit036. Figure 9 229E E protein Ion channel activity in lipid bilayers in KCl solutions. Figure 10: Part A shows raw currents generated by the 229E-E protein ion channel in a planar lipid bilayer. The top trace shows current activity prior to drag addition and the lower trace shows the effect of addition of lOO^M cinnamoylguanidine on channel activity. Part B is a graphical representation of the average current flowing across the bilayer (in arbitrary units), before and after addition of cinnamoylguanidine. Figure 11: MHV E protein Ion channel activity in lipid bilayers NaCl solutions. Figure 12: Part A shows raw currents generated by the MHV-E protein ion channel in a planar lipid bilayer. The top lace shows current activity prior to drug addition and the lower trace shows the effect of addition of 100µM cinnamoylguanidine on channel activity. Part B is a graphical representation of the average current flowing across the bilayer (in arbitrary units), before and after addition of cirmamoylguanidine. DETAILED DESCRIPTION OF THE INVENTION The present invention is based, in part, on the surprising determination that certain compounds that fell under the classification of substituted acylguanidines have antiviral activity against viruses from a range of different virus families. Without intending to be bound by any particular theory or mechanism of action, the negative impact of the compounds of the present invention on viral replication may be mediated by the inhibition or otherwise down-regulation of a membrane ion channel relied upon by the virus for replication. This membrane ion channel may be a viral membrane ion channel (exogeaous to the host cell) or a host cell ion channel induced as a result of viral infection (endogenous to the host cell). As an example, the compounds of the present invention may inhibit Vpu or p7 function and thereby inhibit the continuation of the respective HIV or HCV life cycle. The SARS virus encodes an E protein which is shown for the first time, by the present inventors, to act as an ion channel As similar E proteins are present in other coronaviruses, the compounds, compositions and methods of the present invention would have utility in the inhibition and/or treatment of infections by other coronaviruses. While the present inversion is concerned with novel antiviral compounds falling under the classification of substituted acylguanidines, it does not include in its scope the use of compounds 5-(N,N-hexamethylene)armiloride and 5-(N,N-dimethyl)- amiloride for retarding, reducing or otherwise inhibiting viral growth and/or functional activity of HIV. It will be understood by those skilled in the art that the compounds of the invention may be administered ia the form of a composition or formulation comprising pharmaceutically acceptable carriers and excipients. The pharmaceutical compositions of the invention may further comprise one or more known antiviral compounds or molecules. Preferably, the known antiviral compounds are selected from the group consisting of Vidarabine, Acyclovir, Ganciclovir, Valganciclovir, Valacyclovir, Cidofovir, Famciclovir, Ribavirin, Amantadine, Rimantadine, Interferon, Oseltamivir, Palrvizumab, Rimantadine, Zanarnivir, nucleoside-analo greverse transcriptase inhibitors (NRTI) such as Zidovudine, Didanosine, Zaloitabine, Stavudine, Lamivudme and Abacavir, non- nucleoside reverse transcriptase inhibitora (NNRTI) such as NeYiiapine, Delavirdine and Efavirenz, protease inhibitors such as Saquinavir, Ritonavir, Indinavir, Nelfinavir, Amprenavir, and other known antiviral compounds and preparations. Known antiviral compounds or molecules may in some cases act synergistically with the antiviral compounds of the invention. The present observations and findings now permit the use of agents such as certain substituted acylguani dines, as anti-viral agents for the therapy and prophylaxis of viral conditions caused by different viruses. The methods and compositions of the present invention may be par ticulariy effective against viruses which rely on ion channel formation for their replication, however it will be understood that this is not the only mechanism relied on by viruses tor replication and that the compounds and methods of the present invention are not limited to agents which exert their action by retarding or inhibiting the function of ion channels. Reference to."membranetion channel" should be understood as a reference to a structure which transports ions across a membrane. The present invention extends to ion channels which may function by means such as passive, osmotic, active or exchange transport. The ion channel may be formed by intracellular or extracellular , means, For example, the.ion chamel maybe an ion channel which is naturally formed by a cell to facilitate its normal functioning, Alternatively, the ion channel may be formed by extracellular means. Extracellular means would include, for example, the formation of ion channels due to introduced chemicals, drugs or other agents such as ionophores or due to the functional activity of viral proteins encoded by a virus which has entered a cell. The ion channels which ire the subject of certain embodiments of the present invention facilitate the transport of ions across membranes. Said membrane maybe any membrane and is not Iimi:ed to the outer cell wall plasma membrane. Accordingly, "membrane" as used herein encompasses the membrane surrounding any cellular organelle, such as the Golgi apparatus and endoplasmic reticulum, the outer cell membrane, the membrane surrounding any foreign antigen which is located within the cell (for example, a viral envelope) or the membrane of a foreign organism which is located extracellularly. The membrane is typically, but not necessarily, composed of a fluid lipid bilayer. The subject ion channel may be of any structure. For example, the Vpu ion chat cnel is formed by Vpu which is an integral membrane protein encoded by HIV-1 which associates with, for example, the Golgi and endoplasmic reticulum membr anes of infected cells. Reference hereinafter to "Vpu ion channels" is a reference tc all related ion channels for example P7 HCV and M2 of influenza and the like. Reference to "HIV", "SARS" Coranavirus" or "HCV" should he understood as a reference to any HIV, SARS, Coronavirus or HCV virus strain and including homologues and mutants. Reference to the "functional activity of an ion channel should he understood as a reference to any one or mon: of the functions which an ion channel performs or is involved in. For example, the Vpu protein encoded ion. channel, in addition to facilitating the transportation of Na+, K+ CI and PO43', also plays a role in the ' degradation of the CD4 molecule in the endoplasmic reticulum. Without wishing to be hound by a particular theory, the Vpu protein encoded ion channel is also thought to play a role in mediating the HIV life cycle. The present invention is not limited to treating HIV infection via the mechanism of inhibiting the HIV life cycle and, in particular, HIV replication. Rather, the present invention should be understood to encompass anylmrehlaiisrrl bywhicStfie compounds of the present invention exert their anti-viral activity and may include inhibition of HIV viability or junctional activity. This also applies to HCV, Coranaviruses, and to other viruses. Reference to the "functional activity" of a virus should be understood as a reference to any one or more of the functions which a virus performs or is involved in. Reference to the " viral replication" should, he understood to include any one or more stages or aspects of the viral life cycle, such as inhibiting the assembly or release of virions. Ion channel mediation of viral replication may be by direct or indirect means. Said ion channel mediation is by direct means if the ion channel interacts directly with the virion at any one or more of its life cycle stages. Said ion channel mediation is indirect if it interacts with a molecule other than those of the virion, which other molecule either directly or indirectly modulates any one or more aspects or stagey of the viral l)fe cycle. Aixordingly, the method of the present invention encompasses the mediation of viral replication via the induction of a cascade of steps which lead tc the mediation of any one or more aspects or stages of the viral life cycle. Reference to "down-rsgulatizig' ion channel functional activity, should be understood as a reference to the partial or complete inhibition of any one or more aspects of said activity by both direct and indirect mechanisms. For example, a suitable agent may interact directly -with an ion channel to prevent replication of a virus or, alternatively, may act indirectly to prevent said replication by, for example, interacting with a molecule other than an ion channel. A further alternative is that said other molecule interact! with and inhibits the activity of the ion channel. Screening for molecules that have antiviral activity can be achieved by the range of methodologies described herein. . Reference to a "cell" infected with a virus should be understood as a reference to any cell, prokaryotic or eukaryotic, which has been infected with a virus. This includes, for example, immatal or primary cell lines, bacterial cultures and cells in situ. In a suitable screening system for antiviral compounds, the preferred infected cells would be macrophages/monocytes or hepatocytes/lymphoid cells infected with - either HIV or HCV respectively. Without limiting the present invention to any one theory or mode of action, the compounds of the present invention are thought to inhibit viral replication or virion release from cells by causing ion channels, namely VPU of HIV, the E protein of SARS and other Coronaviruses, or P7 of HCV to become blocked. The present invention encompasses antiviral compounds that are substituted acylgnanidines. The present invention also includes the use of compounds 5-(N,N- hexamethylene)amilorid9 and 6-(N,N-dimethyl)-amiloride in the control of viral replication and/or growth other than HIV. The subject of the viral inhibition is generally a mammal such as but not limited to human, primate, livettock animal (e.g. sheep, cow, horse, donkey, pig), companion animal (e.g. dog, cas:), laboratory test animal (e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animal (e.g. fox, deer). Preferably, the subject is a human or primate. Most preferably, the subject is a human. The method of the present invention is useful in the treatment and prophylaxis of viral infection such as, for example, but not limited to HIV infection, HCV infection and other viral infections. For example, the antiviral activity may be effected in subjects known to be infected with HIV in order to prevent replication of HIV thereby preventing the onset of AIDS. Alternatively, the method of the present invention maybe used to reduce serum viral load or to alleviate viral infection symptoms. Similarly, antivir tf treatment may be effected in subjects known to be infected with, for example, H 2V, in order to prevent replication of HCV, thereby preventing the further hepatot yte involvement and the ultimate degeneration of liver tissue. The method of the present invention may be particularly useful either in the early stages of viral infection to prevent the establishment of a viral reservoir in affected cells or as a prophylactic treatment to be applied immediately prior to or for a period after exposure to a possible source of virus. Reference herein to "therapeutic" and "prophylactic" is to be considered in their broadest contexts. The teim "therapeutic" does not necessarily imply that a mammal is treated until total recovery. Similarly, "prophylactic" does not necessarily mean that the subject will not eventually contract a disease condition. Accordingly, therapy and prophylaxis include amelibration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. The term "prophylaxis" may be considered as reducing the seventy of onset of a particular condition. Therapy may also reduce the severity of an existing condition or the frequency of acute attacks. In accordance with the methods of the present invention, more than one compound or composition may be co-administered with one or more other compounds, such as known anti -viral compounds or molecules. By "co- administered" is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes. By "sequential" administration is meant a time difference of from seconds, minutes, hours or days between the administration of tha two or more separate compounds. The subject antiviral compounds may be administered in any order. Routes of administration include but are not limited to intravenously, intraperitionealy, subcutaneously, intracranialy, intradermally, intramuscularly, intraocularly, intrathecaly, intracsrebrally, intranasally, transmucosally, by infusion, orally, tectally, via iv drip, patch and implant Intravenous routes are particularly preferred. Compositions suitable for injectable use include sterile aqueous solntions (where water soluble) and sterile powders for the extemporaneous preparation of - sterile injectable solutions. The carrier can be a solvent or dispersion medium containing, for example, wansr, ethanol, poryol (for example, glycerol; propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The preventioa of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, calorobutanol, phenol, sorbic acid, thinnerotial and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositioms can be brought about by the useinlhe compositions of agents delaying absorption, for example,, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated abovu, as required, followed by, for example, filter sterilization or sterilization by other appropriate means. Dispersions are also contemplated and these may be prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients fron those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, a preferred method of preparation includes vacuum frying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution. When the active ingredients are suitably protected, they maybe orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be. enclosed in hard or soft shell gelatin capsule, or it may be compressed' into tablets. For oral therapeu ;ic administration, the active compound may be incorporated with excipients iind used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparation 5 should contain at least 0.01 % by weight, more preferably 0.1% by weight, even more preferably 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently "be between about 1 to about 99%, more preferably about 2 to about 90 %, even more preferably about 5 to about 80% of the weight of the unit The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 ng and 2000 mg of autive compound. The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter, A binder such as gum, acacia, com starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as com starch, potato starch, atginic acid and he like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil c f wintergreen, or cherry flavouring. "When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various timer materials may be present as coatings of to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A Syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry ox orange flavour. Any material used in preparing any dosage unit form should be pharmaceuticaliy pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations. The present invention ttlso extends to forms suitable for topical application such as creams, lotions and gel 3. In such forms, the anti-clotting peptides may need to be modified to permit penetrati on of the surface barrier. Procedures for the preparation of dosage unit forms and topical preparations are readily available to those skilled in the art from texts such as Pharmaceutical Handbook A Martinddle Companion Volume Ed. Amley Wade Nineteenth Edition The Pharmaceutical Press London, CRC Handbook of Chemistiy and Physics Ed Robert C. WeastPhD. CRCPress Inc.; Goodman and Gilman's; Tlie Pharmacological basis ofTherapeutics. Ninth Ed McGraw Hill; Remington; and The Science and Practice of Pharmacy. Nineteenth Ed. Ed. Alfonso R. Gennaro Mack Publishing Co. Easton Pennsylvania. PhannaceuticaHy acctsptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. . It is especially advantageous to formulate parenteral compositions in dosage' unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated/to produce the desired therapeutic effect in association with therequired pharmaceutical carier. The specification for the novel dosage unit forms' of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved and (b) the limitations inherent in the art of compounding. Effective amounts contemplated by the present invention will vary depending on the severity of the pain and the health and age of the recipient. la general terms, effective amounts may vary from 0.01 ng/kg body weight to about 100 mg/kg body weight. Alternative amounts include for about 0.1 ng/kg body weight about 100 . mg/kg body weight or from 1.0 ng/kg body weight to about 80 mg/kg body weight. The subject of the viral inhibition is generally a mammal such as but not limited to human, primate, livestock animal (e.g, sheep, cow, horse, donkey, pig), companion animal (e.g. dog, cat), laboratory test animal (e.g. mouse, rabbit, rat, guinea pig, hamster), captive will animal (e,g. fox, deer). Preferably, the subject is a human or primate. Most preferab ly, the subject is a human. The methods of the present invention, is useful in the treatment and prophylaxis of viral infection such as, for example, but not limited to HIV infection, HCV infection and other viral infections. For example, the antiviral activity may be. effected in subjects Imown to be infected with HIV in order to prevent replication of HIV thereby preventing the onset of AIDS. Alternatively, the methods of the present invention maybe used to recitce serum viral load or to alleviate viral infection symptoms. Similarly, antiviral treatment may be effected in subjects known to be infected with, for example, ECV, in order to prevent replication of HCV, thereby preventing the furiher.hepato syte involvement and the ultimate degeneration of liver tissue. The methods of the present invention may be particularly useful either in the early stages of viral infection to prevent the establishment of a viral reservoir in affected cells or as aprophylastic treatment to be applied immediately prior to or for a period after exposure to a possible source of virus. The present invention will now be described in more detail with reference to specific but non-limiting examples describing studies of viral membrane ion channels and screening for antiviral activity. Some examples involve the use of the SARS virus. It will be clear from the description herein that other lehtiviruses, and coronaviruses and other compounds may be used effectively in the context of the present invention. It is to be understood, however, that the detailed description is included solely for the purpose of exemplifying the present invention. It should not be understood in any way as a resbiction on the broad description of the invention, as set out above. Example 1. Synthesis of the Compounds of the Invention. The compounds of the present invention may be made from the corresponding acid chlorides or methyl esters as: shown in Scheme 1. Both of these methods are well described in the literature.. To a solution of trans-cinnamic acid (1.50 g, 10.12 mmol) in dry benzene (30mL) containing a drop of N,J V^imemylformamide was added oxalyl chloride (5.14 g, 40.5 ramol) causing the solution to effervesce. After refluxing for 2 h, the solution was evaporated to drynsss under reduced pressure. The resulting solid was dissolved in dry tetrahydrofuran (20mL) and added slowly to a solution of guanidine hydrochloride in 2M aqueous sodium hydroxide (25mL). The reaction was stirred at room temperature for 1h then extracted with ethyl acetate (3x50mL). The combined extracts were dried over magnesium sulfate and evaporated to give an orange oil. The crude product was purified by column chromatography. Elution with 10% to 20% methanol in dichloromethane gave Cinamoylguanidine as a cream solid (0.829 g, 43%). To a solution of methyl 3-amino-5,6-dichloro-2-pyrazinecarboxylate (0.444 g, 2.0 mmol) in tetrahydrofuran (5 mL) / water (10 mL) / toluene (20 mL) was added phenyl boronic acid (0.536 g, 4.4 mmol),. sodium carbonate (0.699 g, 6.6 mmol) and tetrakis(triphenylphosphine)- palladium(0) (0.116g, 0.10 mmol). The reaction was evacuated and purged with nitrogen several times before being refluxed for 6 h. The organic layer was separated and the aqueous layer extracted with toluene (3 x 20 mL). The combined organic extracs were dried over magnesium sulfate, filtered and evaporated under reduced pressure to give methyl 3-amino-6-chloro-5- phenyl-2-pyrazinecarboxylate as a yellow solid (0.43 g, 82%). To a solution of sodiutl (0.040 g, 1.74 mmol) dissolved in methanol (5 mL) was added guanidine hydmchloridfi (0.258 g, 2.70 mmol) and the mixture refluxed for 30 min after which it was filtered. To the filtrate was added methyl 3-amin0'6- cb1oro-5-pnenyl-2-pyrazinecarboxylate (6.264g, 1.0 mmol) in N,N- dimediyiformamide (5 mL) and the solution heated at 75oC for 12 h. The solvent was removed under reduced pressure and the residue chromatographed on silica gel eluting with 1% triethylamine 5% methanol / dichloromethane. The resulting solid was suspended in chloroform, filtered and dried under high vacuum to give N- Amidino-3-amino-5-phenyl-6-chloro-2-pyrazinecarboxamide as a yellow solid (0.04 g, 14%). Example 4. To a solution of methyl 5-armno-5,6-dichloro-2-pyrazmecarboxylate(1.11 g, 5.0 mmol) in tetrahydrofuran (50 mL) was added hexaroemyleneimine (1.49 g, 15.0 mmol) and the reaction was refluxed for 1 h. The reaction was allowed to cool and the solid hexamethyleneimine hydrochlorideremovedby filtration. The filtrate was To a solution of methyl 3-aniino-6-ohloro-5-hexamethyleneimino-2- pyrazinecarboxylate (0.350g, 1.23 mmol) in dimethylsutfoxide (5 mL) was added phenyl boronic acid (0.166 g, 135 mmol), potassium carbonate (0.511 g, 3.70 mmol) and[1,1'-bis(diphenylphosphino)feirbcene]dichloropalladium(II)-dichloromethane complex (0.041 g, 0.05 mmol). The reaction was heated at 90oC for 16 h before being poured into water (50mL) and extracted with ethyl acetate (3 x 50mL). The combined extracts were dried over magnesium sulfate, filtered and evaporated to give a brown oil which was purified by chromatography on silica gel. Elution with dicbloromethane followed by 10% ethyl acetate/dichloromethane gave methyl 3- amino-5-kexamethyleneimino-6-phenyl-2-pyrazmecarboxylate as a yellow solid (0.309 g, 77%). To a solution of sodium (0.090 g, 6.17 mmol) dissolved in methanol (8 mL) was added guanidine hydrochloride (0.598 g, 6.26 mmol) and the mixture was refiuxed for 30 min after which it was filtered. To the filtrate was added methyl 3- ammo-5-hexamefhyleneimmo-6-phenyl-2-pyrazniccarboxylate (G.310 g, 0.95 mmol) in tetrahydrofuran (10 mL) and the solution refluxed for 72 h. The solvent was removed under reduced pressw e and the residue chromatographed on silica gel. Elution with 5% memanol/dihoromethame gave N-amidino-3-amino-5- hexamethylmeimino-6-phenyl-2-pyrazinecarboxamide as a yellow solid (0.116 g, 35%). Example 5. Viral Studies Construction of recombinattt plasmids containing open reading frames encoding varions virus proteins, Complimentary DNA (cDNA) fragments for the various viral proteins listed in Table 2 were obtained eitlier by PCR amplification from a parental virus genome clone, or by direct chemical synthesis of the polynucleotide sequence. For example, the open reading frame encoding Vpu (Fig la) was amplified by PCR from a cDNA clone of an Nde I fragment of the HIV-1 genome (isolate HXB2, McFarlane Burnet Centre, Melbourne, Australia) as follows: Native Pfu DNA polymerase (Stratagene; 0.035 U//D) was chosen to catalyse the PCS. reaction to minimise possible ?CR introduced errors by virtue of the enzyme's proofreading activity. The 5\ sense, primer AGTAGGATCCATGCAACCTATACC (2) introduces a BamHl site (underlined) for cloning in-framo with the 3' end of the GST gene in p2GEX (41). This primer also-repairs the start oodon (bold T replaces a Q of the vpu gene which is a threonine codonin the HXB2 isolate. The 3', antisense, primer TCTGGAATTLTACAGATCA1' CAAC ( 3) introduces an EcoRl site (underlined) to the other end of the PCR product to iacilitate cloning. After 30 cycles of94°C for 45 sec, 55°C for 1 milL and 72°C for 1 min in 0.5 ml thin-walled eppendorf tubes in a Perkin-Elmer thermocycler, the 26Sbp fragment was purified, digested with BamHl and EcoRl and ligated to p2GEX prepared by digestion with the same two enzymes. The resultant recombinant plasmid is illustrated in Fig lb. The entireVpu open reading frame and the BamH1 and EcoR1 ligation sites were sequenced by cycle sequencing, usng the Applied Biosystems dye-terminator kit, to confirm the DNA sequence. Other cDNAs were synthesised for us using state of the art methods by GenScript Corporation (New Jersey, USA). Codon sequences were optimised for expression in bacterial, insect or mammalian cells, as appropriate. Restriction endonuclease enzyme recognition sites .were incorporated at the 5* and 3' cods of the synthetic cDNAs to facilitate cloning into plasmid expression vectors, pcDNA3.1,pFastBac and pPL451 for expression of the encoded virus proteins in. mamma lian, insect or bacterid cells, respectively. Standard techniques ofmolecular biology were used in cloning experiments. For example, to prepare the Vpu open reading frame for insertion into the pPL451 expression plasmid, p2GEXVpu was first digested with BamHI and the 5' base overhang was filled in the Klenow DNA polymerase in the presence of dNTPs. The , Vpu-encodtng fragment was then liberated by digestion with EcoRL purified from an agarose gel and ligated into pPL451 which had been digested with Hpal and EcoRl. Western blots subsequently confirmed that the pPLVpu construct (Fig 1c) expressed Vpu after induction of cultures at 42°C to inactivate the cIS57 repressor of the PR and PL promoters. Example 6. Purification of Recombinant Vpu from E, Coli Cultures of E. coli strain XLI-blue cells containing p2GEXVpu were grown at 30oC with vigorous aeration in LB medium supplemented with glucose (6g/L) and ampicillion (50mg/L) to a density of approximately 250 Klett units, at which time IPTG was added to a final concentration of 0.01roM and growth was continued for a further 4hr. The final culture densiy was approximately 280 Klett units. Since early experiments revealed that the majority of expressed- GST-Vpu fusion protein was associated with both the cell debris and 30 membrane fractions, the method of Varadhachary and Moloney (Varadhachary and Maloney, 1990) was adopted to isolate osmotically disrupted cell ghosts (combining both cell debris and membrane fractions) for the. initial purification steps. Cells were harvested, washed, weighed and resuspended to 10ml/g wet weight in MTPBS containing DTT (ImM) and MgCl2 (10mM). Lysozyme (0.3 mg/ml; chicken egg white; Sigma) was added and incubated on ice for 30 min with gentle agitation followed by 5 min at 37°C. The osmotically sensitised eells were pelleted itt 12,000g and resuspended to the original volume in water to burst the cells. The suspension was then made up to 1xMTPBS/DTT using a 10x buffer stock and the ghosts: were isolated by cenoifugation and resuspended in MTPBS/DTT to which was then sequentially added glycerol (to 20 % wt/vol) and CHAPS (to 2 % wt/vol) to give a final volume of one quarter the original volume. This mixture was stirred on ice for 1hr and then centrifuged at 400,000g for 1hr to remove insoluble material. The GST-Vpu fusion protein was purified from the detergent extract by affinity chromatography on a glutathione agarose resin (Sigma). The resin was thoroughly washed in 50mM Tris pH 7.5 containing glycerol (5 %), DTT (ImM), and CHAPS (0,5 %) (Buffer A) andthen the Vpu portion of the fusion protein was liberated and eluted from the resin-bound GST by treatment of a 50% (v/v) suspension bf the beads with human fhrombm(100U/ml; 37°C for 1hr). PMSF . (0.5mM) was added to the eluant to eliminate any remaining thrombin activity. This Vpu fraction was further purified on a column of MA.7Q anion exchange resin attached to a BioRad HPLC and eluted with a linear NaCl gradient (0-2M) in buffer A. The Vpu was purified to homogeneity - as determined on silver stained gels - on an immunoaffinity column as follows: HPLC fractions containing Vpu were desalted on a NAP 25 column (Pharmacia) into buffer A and then mixed with the antibody- agarose beads for Ihr at room tei operature. The beads were washed thoroughly and Vpu was eluted by increasing tha salt concentration to 2M. Proteia was quantitated using the BioRad dye binding assay- Example 7. Expression and Pgrificatiott of Vpu in E,Coli The piasmid p2GEXVpu (Fig. 1) was constructed to create an in-frame gene fusion between the GST and Vpu open-reading frames. This system enabled IPTG-inducible expression of the Vpu polypeptide fused to the C-terrninus of GST and allowed purification of the fusion protein by affinity chromatography on glutathione agarose.. Optimal levels of GST-Vpu expression were obtained by growing the cultures at 30°C to a cell density of approximately 250-300 Klett units and' inducing with low levels of 1PTG (0.01mM). To purify the GST-Vpu, a combined cellular fraction containing the cell debris and plasma membrane was prepared by lysozyme treatment of the induced cells followed by a low-speed centrifugation. Approximately 50% of the GST-Vpu protein could bs solubilised from this fraction using the zwitterionic detergent CHAPS. Affinity chiomatography using glutathione-agarose beads was used to enrich the fusion proteia andthrombin was used to cleave the fusion protein at the high affinity thrombin she between the fusion partners, liberating Vpn (Fig. 2A). In fractions eluted from the anion exchange column Vpu was the major protein visible on silver stained gels (Fig. 2B, lane 1). Finally, Vpu was purified to apparent homogeneity on an immunoaffnity column (Fig. 2B, lane 2). The N-terminal amino acid'sequence of the protein band (excised from SDS-PAGE gels) corresponding to the immunodetected protein confirmed its identity as Vpu. Example 8. Reconstitution of Vpu in Phospholipid Vesicles. Proteoliposornes containing Vpu were prepared by the detergent dilution method (New, 1990). A mixture of lipids (PE:PC:PS; 53:2; Img total lipid) dissolved in chloroform was dried under a stceam of nitrogen gas and resuspendedin 0.1 ml of potassium-phosphate buffer (50mM pH 7.4) containing DTT (ImM). A 25ul aliquot containing purified Vpu was adcted, followed by octylglucoside to a final concentration of 1.25 % (wt/vol). This mixture was subject to three rounds of freezing in liquid nitrogen, thawing and sonication in a bath type sonicator (20-30 sec) and was then rapidly diluted into 200 volumes of the potassium phosphate buffer. Proteoliposornes were collected by centrifugation at 400,000g for lbr and resuspended in approximately 150ui of phosphate buffer. Example 9. Assaying Vpn Ion Channel Activity Purified Vpu was tested ibr its ability to induce channel activity in planar lipid bilayers using standard techniques as described elsewhere (Miller, 1986; and Piller et al, 1996). The solutions in the C3S and TRANS chambers were separated by a Delrin™ plastic wall containing a small circular hole of approximately 100µm diameter across wnicn a lipid bilayer was painted so as to form a high resistance electrical seal. Bilayers were painted from a mixture (8:2) of pahnitoyl-oleoly- phosphatidyl-emanolaniine and pahnitoyl-oleolyphosphatidyl-choline (Avanti Polar Lipids, Alabaster, Alabama) in n-decane. The solutions in the two chambers contained MES buffer (10mM, pH 6.0) to which various NaCl or KCl concentrations were added. Currents were recorded with an Axopatch™ 200 amplifier. The electrical potential between the two chambers could be manipulated between +/-200mV (TRANS relative to grounded CIS). Aliquots containing Vpu were added to the CIS chamber either as a detergent iolution or after incorporation of the protein into ! phospholipid vesicles; The chamber was stirred until currents were observed. Example 10. Vpu Forms Ion Channels in Lipid Bilayers. To assay for ion:channt:l formation by Vpu, reconstitution into planar lipid bilayers was performed. When samples (containing between 7 and 70ng of protein) of purified recombinant Vpu were added to the 1ml of buffer in the CIS chamber of the bilayer apparatus, current fluctuations were detected after periods of stirring that varied from 2 to 30 min (Fig. 3). This time taken to observe channel activity " approximately correlated with the amount of protein added to the chamber. No channels were detected when control buffer aliquots or control lipid vesicles were added to the CIS chamber. la those control experiments the chambers could be stirred for more than an hour without abearance of channel activity. Example 11. Properties of The Vpu Channels. Channel activity was obs srved in over 40 individual experiments with Vpu . samples prepared from five independent purifications. In different experiments, the amplitude of the currents varied over a large range and, again, seemed to approximately correlate with the amount of protein added. The smallest and largest channels measured had conductances of 14 pS and 280 pS, respectively. The channels were consistently smaller when lipid vesicles containing Vpu were prepared and fused to the bilayer rather than when purified protein in detergent solution.was added. This may be because the former method included treatment with high concentrations of detergent and a dilution step that may have favoured the breakdown of large aggregates into monomers. Hie relationship between current amplitude and voltage was linear and the reversal potential in solutions containing atea-fold gradient of NaCl (500mM CIS; 50mM TRANS) was +3GmV (Fig. 3B). A similar reversal potential was obtained when solutions contained K.(2 instead of NaCl. In 5 experiments with either NaCl or KCI in the solutions on either side of the membrane, the average reversal potential was 31.0 +/-1.2mV (+/-SEM). This is more negative than expected for a channel selectively permeable for the cations alone. Using ion activities in the Goldman- Hodgkin-Katz equation give s a Pjfe/Pd ratio of about 5-5 indicating that the channels are also permeable to chloride ions. An attempt was made to reduce the anion current by substituting phosphate for chloride ions. When- a Na-phosphate gradient (150mM Na+ & 100mM phosphate CIS; 15mM Na+ & 10mM phosphate TRANS, pH 6.8) was used instead of the NaCl gradient, the reversal potential was 37.1 +/- 0.2 (+/-SEM, n-2) again indicating a cation/anion permeability ratio of about 5. (For calculations involving the phosphate solutions, the summed activities of the mono and bivalent - anions were used and it was assumed that the two species were equally permeable). - The current-voltage curve now exhibited rectification that was not seenin the NaCl solutions. It can be concluded that the channels formed by Vpu are equally permeably to Na4" and K* and are also parmgable* though to a lesser' extent} to chloride as well as phosphate ions. Example 12. Bacterial Bio -Assay for Screening Potential Ion Channel-Blocking Drags. This bio-assay is basud on the observation that expression of Vpu in & colt results in an active Vpu charnel located in the plasmalemmathat dissipates the transmembrane sodium gradient. As a consequence of this Vpu channel activity, metabolites whose accumulation within the cells is mediated by a sodium dependent co-transporter (for example proline or adenine) leak out of the cell faster than they canbe synthesised so that the metabolites' intracellular levels become limiting for growth of the cell. Thereby, an E. coli cell expressing Vpu is unable to grow hi minimal drop-out media lading adenine or proline. However, in the presence of a drug that blocks the Vpu channel, the cell is once again able to re-establish its transmembrane sodium gradient - due to the action of other ion pumps in the membrane - and the leakage of metabolites is prevented enabling growth. Experiments to demonstrate that Vpu can form sodium channels in the plasma membrane of R coH were performed as follows. To express unfiised Vpu in E. coli, the vpu open-reading frame was cloned into the plasmid pPL451 to create the recombinant plasmid pPL-Vpu (Fig. lb). la this vector me strong PL and PR lambda promoters are used to drive expression of Vpn under control of the temperature sensitive c1857 represser, such that when grown at 30oC expression is tightly repressed and can be induced by raising the temperature to between 37oC and 42oC. On agar plates, cells containing pPL-Vpu grew when incubated at 30°C and 37°C but not at 42oC, while control strains grew well at 42°C Liquid cultures of cells containing pPL-Vpu were grown at 30°C to OD600,=0.84 then moved to grow at 42°C for two hours (the final cell density was OD600,=0.75). The plasma membrane fraction was; prepared and western blotting, using an antibody that specifically binds to the C-teraunus of Vpu, detected a single band at approximately 16KDa, indicating that Vpu was expressed and assodated wth the membranes (Fig. 2A,.lane 5). Example 13. Cross-Feeding Experiments Reveal That Proline Leaks Out of Cells Expressing VPU. Uptake of proline by E. noli is well characterised and active transport of the amino acid into the cells is knovm to use the sodium gradient as the energy source (Yamato et al, 1994). To detect ivhether proline leakage occurs, the following cross- feeing assay was used: A lawn of an E. coli strain auxotrophic for proline and methionine (Met Pro), was seecsd and poured as a soft agar overlay on rmnimai drop-out media plates lacking proline but ccrafaining methionine. Sterile porous filter discs were inoculated with a Met+ Pro+ strain (XL-1 blue) containing either the pPL451 control plasmid or pPL-Vpu and placed onto the soft agar. The plates were then incubated at 37°C or 30°C fixr two days. After man time a halo growth of the Met" Pro" strain was clearly visible surrounding the disc inoculated with the cells containing pPL-Vpu incubated at 37°C (Fig. 4A). This growth can only be due to the leakage of proline from the Vpu-expressing cells on the disc. No such leakage was apparent from the control strain at 37°C nor around either strain on plates grown at 30oC (Fig. 4B). la contrast to proline transport, the K coU methionine permease is known to belong to the ABC transports family (Rosen, 1987) and hence be energised by ATP. Identical crossfeeding experiments to those described above were set us except that the Met" Pro" strain was spreed on minimal drop-put plates lacking methionine but containing proline. No growth of this strain was evident around any of the discs (Fig. 4C), indicating that methiohnie was not leaking out of the XL-1 blue cells even when Vpu was being expressed. Example 14, E.COLI Cells Expressing Vpa Require Adenine in the External Medium for Growth. It was observed that, due to an uncharacterised mutation in the adenine synthesis pathway, growth of.E coli cells of the XLI-blue strain expressing Vpu at 37°C was dependant on the prtjsence of adenine in the medium. This allowed the development of an even simplisr bioassay for Vpu ion-channel activity than the proline cross-feeding assay described above: A lawn of XLI-blue cells containing the pPL-VpuplasSiid is seeded on to an agarose plate lacking adenine in the medium, small ah'quots of drugs to be tested for inhibition of the Vpu channel are spotted onto the agarose in discrete locations and the plates are incubated at 37°C for a suitable period of time (12-36 hours). Halos of growth around a particular drug application site indicate that the drug has inhibited expression of the Vpu ion channel activity that prevents growth in the absence of the drug. (Figure 5). Example 15 Assay of Compounds in Planar Lipid Bilavers for Vpu Channel Blocking. Activity Comopuhds were characterized for their ability to block Vpu ion channel activity reconstituted into planar lipid bilayers. Vpu N-terminal peptide (residues 1- 32) dissolved in trifluoroethanol was added to the CIS chamber of the bilayer apparatus and the solutions was stirred until ion currents were observed, indicating incorporation of one or more Vpu ion channels into the bilayer. After recording the channel activity for a few minutes, drugs were added to the solutions in the CIS and TRANS chambers - with stirring - to a final concentration of 100µM. Channel activity was then recorded for at east a further three minutes and the effect of drug addition on ion current was determind by comparing the channel activity before and after drag addition. For each experiment drag effect was classified into four categories: "Stong block", if current wasinhibrted approximately 90-100%; "weak block", approx. 50-90% inhibition; "partial block", . Experiments were disregarded if currents larger than ±50pA were generated after . addition of Vpu N-peptide because in such cases it is possible that non-native peptide aggregates contribute to bilaysr breakdown. Snch aggregates, by virtue of their disorganized structure may not be specifically blocked by the drags at the concentrations tested. Table 3 summarises the results of the bilayer experiments. A novel outcome of these experiments was the strong blocking of Vpu channels observed with Phenamil. Phenamil has a phenyl group derivative at the guanidine group of amiloride. Amiloride itself is not a blocker of Vpu, whereas addition of the hexamethylene group at the 5- position of the syrazine ring created a structure (HMA) that blocks the channel at concentrations as low as 25µM; Thesenewresults with Phenamil, however, now show that a bulky hydrophobic derivative at the opposite end of the molecule can also turn amiloride into an effective Vpu channel blocker. Interestingly, benzamil, with a very similar structure was much less effective at blocking the Vpu channel. of BIV isolates and allowed to absorb for 2hr before complete aspiration of the medium, washing once with virus-free medium and resuspension in fresh medium. The cells were exposed to various concentration of compound either 24 hr prior to infection or after infection. Subsequent HIV replication, at various times after infection! was compared in cells exposed to drugs and in cells not exposed to drugs (controls). The progression and extent of viral replication was assayed using either an HEV DNA PCR method (Fear et al, 1998) or an ELISA method to quantitate p24 in culture supematants (Kelly et id, 1998). Table 5 provides examples of results obtained using this assay and test antiviral compounds. Example 18 SARS Coronavirus. BARS E protein, forms an ion channel Peptide Synthesis A peptide corresponding to the fufl-length SARS-CoV (isolate Tor2 and Urbani) E protein (^SFVSBETGTLIVNSVIXFIAFVVFLLVTLAILTALRLCA YCasnVNVSLVEPTVYVYSRVKNLNSSEGVPDLLV) and a second peptide jomprising the first 40 amino acids of the full length E protein which correspond to the transmembrane domain (MYSFVSEKrGTLIVNSVLLFLAFWF LLVTIAJLTALRLC) were synthesized manually using FMOC chemistry and solid phase peptide synthesis The synthesis was done at the Biomolecular Resource Facility (John Curtin School of Medical Research, ANU, Australia) using a SymphonyR Peptide Synthesiser from Protein Technologies Inc. (Tucson, AZ, USA) according to the manufacturers instructions. Example 19. Peptide purification Mass spectral analysis of the synthetic peptide revealed that the preparation contained significant amounts of material with lower m/z ratio than expected, for the full-length product The majorit y of these are presumably truncated peptides generated during the peptide synthesis process. To enrich the full-length E protein, the following procedure was used, which relies on differential solubility of the smaller molecules and full-length peptide. The crude preparation was suspended at 12 nag/ml in 70% CH3CN, 0.1%TFA and vortexed for 10 minutes. This suspension was centrifuged at l'0,000g for 10 mir-utes at 20aC. The supernatant was discarded and the insoluble fractions was extracted with 70% CH3CN, 0.1% TFA, as above, two . more times. The insoluble material containing the E protein was dried using Speedvac an the weight of the final product was used to calculate the yield. The purified peptide was analysed by Bruker Omaiflex MALDKTOR mass spectrometry in HABA matrix at 2.5mg/ml in rr ethanol at a 1:1 ratio and spectra were obtained in the positive linear mode. A clearpeat at m/z ratio- of 8,360.1 was seen as expected for the calculated molecular weight of full-length E protein and 4422.3 for the N-terminal E protein. Example 20. Planar Lipid Bilavers The SARS virus E protein was resuspended at 1mg/ml in 2,2,2-trifluoroethanol The SARS virus E protein's ability to form ion channels was tested on a Warner (Warner instruments, Inc. 1125 Djxwel [ Avenue, Hamden, CT 06514) bilayer rig as follows; A lipid mix of 3:1:1, l-Palmitoyl -2-oleolyl phosphatidyl Eftaaolamnie: l-Pahmtoyl-2- oleolyl phosphatidyl Serine: l-PaInritoyl-2-oleolyi phosphatidyl choline in CHCl3 was dried under N2 gas and resuspended to 50mg/ml in n-decane. Bilayers were painted across a circular hole of approximately 100µm diameter in a Delrin™ cup separating aqueous solution in he CIS and TRANS chambers. The CIS chamber contained a solution of 500mM Nad or KCL in a 5mM HEPES /buffer pH 7.2, the TRANS chamber contained a solution of 50mM NaCl or KCl, in a 5mM HEPES buffer pH 72. Silver electrodes coated in chloride with 2% agarose bridges are placed in the CIS and TRANS chamber solutions. The SARS E protein full-length or N- tenninal peptides (3-10µg) were: added to the CIS chamber, which was stirred until channel activity was detected, -The CIS chamber was earthed and the TRANS chamber was held at various holding potentials ranging between +100 to -lGOmV. Currents were recorded using a SVamer model BD-525D amplifier, filtered at 1kHz, sampling at 5 kHz and digitally recorded on the hard disk of a PC using software developed in house. Drugs to be tested for their ability to inhibit SARS E protein ion channel activity were made up at 50mM in a solution of 50% DMSO: 50% methanol For experiments testing the ability of compounds to inhibit E protein ion channel activity 100 uM to 400 ixM of compound was added to the CIS chamber while stirring for 30 seconds. Bilayer currents were t worded before channel activity, during channel activity and after the addition of the drag. Among the compounds tested was cinnataoylguaniditte (Bit036), a compound which was shown in earlier experiments to be antiviral and to inhibit ion channel proteins from other viruses. Example 20.1. Polyaerylamide gel electrophoresis Purified E protein was dis solved to 1 mg/ml, 5 mg/ml and 10 mg/ml in, 6 M Urea, 10% Glycerol, 5% SDS, 503 mMDTT, 0.002% Bromophenol Blue, 62.5 mM Tris HC1 (pH 83). Peptides in solutions were heated at 100°C for 20 minutes before 30 p,L samples were run on stacking gel 4-20% (Gradipore). SeeBlue® pre-stained standard (Eavitrogen) was used for molecular weight markers. Example 20.2 Resnlts . To test if the SARS P, protein forms ion channels the purified synthetic peptide was reconstituted into planar lipid bilayers (21). Typically, 3µg of SARS fell-length E protein was added to the CIS chamber, while stirring. This CIS chamber contained 500 mMNaCl and the TRANS chamber contained 50 mM NaCl. In 60 experiments, ion currents due to SARS E protein ion.charmel activity were observed after about 5-15 minutes of s tirring. Activity was detected more rapidly and reliably with a holding potential of approximately-100mV across the bilayer. Currents recorded at-100mV, (A) and at-60mV (B) in one of these experiments are shown in Figure 6. In that experiment tine reversal potential was about +48mV and the channel conductances ware calculated to be 52pS arid 26pS, respectively. This indicates that the current-voltege,(IV) relatipnship is not linear In ten other experiments, where no protein was added to the CIS chamber, no ion channel activity was detected, even after recording for over I hour. Figure 7a shows typical current traces recorded oyer a range of potentials in NaCl solutions. In that experiment the direction of current flow reversed at +48mV (Fig 7b). The IV curve shows that at the lower voltages the average current flow across the bilayer is small but at higher potentials there is an increase in average current across the bilayer, resulting in a non-linear IV relationship. In seven independent experiments, the average feversal potential was +48.3 ± 2,3 mV (mean ± 1SEM), indicating that the channels were about 37 times more permeable to Na+ ion than to CI" ions. The reversal potential is close to the Na+ equilibrium potential (+53m,V), therefore the channel is selective for Na+ ions. For these 7 experiments the channel conductance varied between 95-164 pS; the average conductance was 130 ± 13 pS. SARS E protein ion channel in sh'ghtly less selectivity for K+ ions than Na+ ions. Figure 8b shows recording of cur rents in KCl solutions at a range of potentials. In this experiment the currents reversed, at +31 mV. In seven similar experiments E protein ion channel average reversal potential was +34.5 ± 2.5 mV. Therefore the SARS E protein ion channel is about 7.2 times more permeable to K+ ions than CT ions. In seven experiments, the channel conductance varied ranging between 24-166 pS, the average conductances was 83.4 + 26 pS. Similar results were obtaintsd with a second synthetic peptide, which corresponded to the first forty N-eiminal ammo acids of the SARS E protein "N-terminal peptide" (21). The average reversal potential in NaCl solution in four experiments was +46.3' + 2.5 mV, indicating that the: ion channel formed by N-terminal peptide is about 25 times more permeable to Na+ ion than to Cl-ions. The SARS E protein N-terminal peptide was sufficient for the formation of ion channels with properties like those of the full length SARS E protein. Therefore, the selectivity filter for the SARS E protein is most likely contained within the first forty amino acids of the N-terniinaL SARS E protein N-terminal pqptide also formed ion channels in KG solution that were similarly" selective for EH- ions compared to the full-length E protein. In five independent experiments the average channel reversal potential was +39.5 + 3.6 mV, • therefore the channel is about 11 times more permeable to K+ ions than CI" ions. - SDS-PAGE of the purified mil-length E potein peptide showed bands correspending to the full-length Eprotein (Data not shown). Larger bands of varying size up to about 20 kDa were detected, suggesting that SARS E protein may form homo- oligomers. Example 21. SARS E protein ion channel is blocked by cmnamovlguanidme and other compounds E protein ion channel activity in NaCl solutions was significantly reduced (p> 0.01, n=6 experiments) by addition of 100 to 200 µM cinnamoylguahidine to the CIS chamber. The average current across the bilayer was reduced to baseline by 100µM cmnarnoylguanidine. In experiments when E protein ion channels had higher conductance, 100 to 200 uM cimamoulguanidme reduced the average current across the bilayer about 4 fold;. Similarly, in four other experiments, 100 to 200 uM cinnamoylguanidine blocked channels formed by full-length E protein in KCI solutions. la two additional experiments, the SARS E protein N-terminal peptide was blocked by 100 to 200 µM cinn.unoylguani:dine, demonstrating that the cumamoylguam'drnedrug-binding site is located within the first forty amino acids of the E protein N-tenninal dansin. Other compounds tested in bilayers for their effect on the SAR5 B protein are shown in below in Table 6. Example 21.1 Results and Pisccssion. We have shwon that SARS E protine can form ion channels in lipid bilayer membranes. The ion currents reversed at positive potentials, which demonstrates that E protein ion channels are selective for monovalent cations over monovalent anions. " E protein Ion channels were about 37 times more selective for Na+ ions over Cl-ions and about 13. times more selective for K+ ions over CI- ions. In over 60 experiments theNa+ conductance of the E piotein ion channel varied from as km as 26 PS to as high as 154 pS. SBS-PAGE showed that the E protein forms homo-oligomers, and we surmised that the larger conductances were probably due to aggregation of the E protein peptide leading to larger Ion channels or the synchronous opening of many ion channels. Single channel currents were observed in several experiments and from these the channel conductance wsis calculated to be voltage dependent.. The first 40 amino acids cf the N-terminal which contains the hydrophobic domain of the'SARS virus E protein is sufficient for the formation of ion channels on planar lipid bilayers. The N-termiaal E protein ion channel has the same selectivity and conductance as the full-lengrr.. E protein ion channel. The SARS virus fall length E protein ion channel activity and N-terminal domain E protein ion channel acrMty on planar lipid bilayers in NaCl and K.C1 solutions was inhibited by addition of between 100µM. to 200µM cinnamoylguanidime to the CIS chamber. Inhibition or partial, inhibition of the E protein ion channel activity by cinnamoylguamdine has been observed in seven independent experiments in NaCl solution and four independent experiments in KCI solution. AH known coronaviruses encode an E protein with a hydrophobic N-terrm'mis transmembrane domain therefore all coronaviruses E proteins could form ion channels on planar lipid bilayers. This indicates that the E protein could be a suitable target for antiviral drags and potentially stop the spread of coronavirus from infected host cells. Drugs that block the E protein ion channel could be effective antiviral therapy for the treatment of several significant human and veterinary coronavirus diseases including SARS and the common cold. Example 22. Bacterial Bio-Assay for Screening Potential SARS-CoV E protein Ion Channel-Blocking Drugs, SARS-CoVE protein Ion Channel inhibits Bacterial Cell growth. A-bio-assay of SARS-GbV-Bpmteinfunctioninbacterial ceils was developed." A synthetic cDNA fragment encoding SARS-CoV E protein was cloned into the expression plasmid pPL451, creating a vector in which E protein expression is . temperature inducible, as described in Example 4. Inhibition of 1he growth of E.co cells expressing E protein at 37°C was observed as an indicator of p7 ion channel function dissipating the normal Na+ gradient maintained by the bacterial cells. Example 33. Compound Screening using the Bacterial Bio-Assay for SARS coronavirus E protein. The halos of growth around the site of application of particular drugs - as described in example 14-were scored as decribed in example 15. Table 7 lists the scores for inhibition of SARS-CoV E protein in the bacterial bio- assay+ Example 24. SAKS Antiviral Assay for testing compounds against replication of SARS coronavirns (SABS-CoV) Compoimds were tested against SARS-CoV (Hong Kong strain) using virus plaque purified three times in Vera cells. .Stock virus was generated by infecting Vero cells at MOI = 1x TCID50 per 100 cells. Example 24.1 Screening for anii-virsl activity using the virns microtitre assay Monolayers of Vero cells grown in 25cm2 flasks were infected at a multiplicity of 1:50 and treated raimediately post infection with compounds at two concentrations, 10µM and 2µM. A control infected monolayer remained untreated. Samples of culture media were taken at 48 hours post infection. Two aliquots from each of the samples (titrations 1 and 2) were serially log diluted and 12 replicates of log dilutions -4 to -7 added to cells in microtitre plates. Pour days later, wells in the microtitre plates were scored tor cytopathic effect (CPE) and the titration values calculated based on the number of CPE positive wells at the 4 dilutions. Control titres were 4.8 and 5.9 TCDD50 x 106 (average 5.35 x 106) Example 25; Effect of compounds in SARS CoV antiviral assavt Three selected compouads were tested for activity against SARS-CoV according to the method descri ted in example 21, For tf ans-3-(l- napfliyl)acryloylguanidine and cannamoylguanidine a decrease in virus titre of approximately 80% was observed-at a.concentration of-10µM and a reduction of approximately 50% was seen tc persist at 2uM rrans-3-(l-napmyl)acryIoylguantdine. Table 8 provides Virus titration data presented as % of a control (SARS CoV grown for 48 hours in the absence of compounds). Example 26. Human 229E Coronavirus Synthesis and Parifilcation of a Peptide Correspondnig to the 229E-E Protein A peptide corresponding to the full-length 229E-E protein (sequence: MFLKLVDDHALVVNVILWCVLIVILLVCITIIKLCFCHMFTNFCNRTVYGPI . KNVYHiyQSYMHIDPFPKRVIDF; accession number NP_073554) was synthesized manually using FMOC chemistry and solid phase peptide synthesis. The synthesis was done at the Biomoleculir Resource Facility (John Curtin School of Medical Research, ANU, Australia) using a SymphonyR Peptide Synthesiser from Protein Technologies Inc..(Wobum, MS, USA) according to the manufacturers instructions to give C-terminal amides, the coupling was done with HBTU and. hydroxybenzotriazole in N-memylpyrrolidone, Each of the synthesis cycles used double coupling and a 4-fold excess of the amino acids. Temporary -N Fmoc- proteciing groups were removed using 20% piperidinc in DMF. The crude synthetic peptide was purified using the ProteoPhis™ kit (Qbiogene inc. CA), fallowing manufactures instructions. Briefly, the peptides were diluted in loading buffer (60mM Tris-Ha pH 8.3,6M urea, 5% SDS, 10% glycerol, 0.2% Bromophenol blue, ±100 mM p-mercaptoethanol) and run on 4-20% gradient polyacrylamide gels (Gradipons, NSW, Australia) in tris-glycine electrophoresis buffer (25 mM Tris, 250 mM glycine, 0.1% SDS). The peptides were stained with gel code blue (Promega, NSW) auci the bands corresponding to the full-length peptide were excised out of the gel. The gel-slice was transferred to ifce ProteoPLUS™ tube and filled with tris-glycine electrophoresis buffer. The tubes: were emerged in tris-glycine electrophoresis buffer and subjected to 100 volts for ap proximately 1 hour. The polarity of the electric current was reversed for 1 minute to increase the amount of protein recovered. The peptides were harvested and cent ifuged at 13, 000 rpm for 1 minute. The purified peptides were dried in a Speedvac. and the weight of the final product was used to calculate the yield. Example 27.229E-E protein forms ion channels in planar lipid bflayers. Lipid bilayer studitss were performed as described elsewhere (Sunstrom, 1996/ Miller, 1986). A-lipid mixture of palmitoyl-^Ieoyl-phosphatidylethanolaQiirie, palmitoyk)leoyl-phosphafodylserine and palmitoylKjleoyl-phosplratidylcholine (5:3:2) (Avaafi Polar Lipids, Alabaster, Alabama) was used. The \ipid mixture was painted onto an aperture of 150-200 urn m the wall of a. I ml delrin cup. The aperture separates two chambers, cis and trans, both containing salt solutions at different concentrations. The cis chamber was connected to ground and the trans chamber to the input of an Axopateh 20!) amplifier. Normally the cis chamber contained either " 500 mM NaCI or 500mMKCl and the trans 50 mMNaCl or 50mMKCL The bilayer formation was monitored electrically by the amplitude of the currentpulse generated by a current ramp. The potentials were measured in the trans chamber with respect to the cis. The synthetic peptide was added to the cis chamber and stirred until channel activity was seen. The currents were filtered at 1000 Hz, digitized at 5000 Hz and stored on magnetic disk. The'229E E synthetic peptide was dissolved in 2,2,2-triflnorethanol (TFE) at 0.05mg/ml to 1 mg/inl. 10 µl of this was added to the cis chamber (1ml aqueous volume) of the bilayer apparatus, which was stirred via a magnetic "flea". Ionic currents, indicating channel activity in the bilayer, were typically detected within 15- 30 ruin. After channels were detected the holding potential across the bilayer was varied between -100mV and+100mV to characterise the size and polarity of current flow and enable the reversal potential to be determined. In 15 experiments when the cis chamber contained 500mM NaCI solution and the trans chamber contained 50 mM NaCI solution, the average reversal potential of the channel activity was calculated to be 22 ±7 (SEM) mV. In 13 experiments where the cis chamber contained 500mM KC1 solution and the trans chamber contained 50 mM KC1 solution, the average reversal potential of the channel activity was calculated to be 38 ±4 (SEM) mV. These results indicate that the 229EB protein - forms cationselective ion charmds that are slightly more selective for K+ than for Na+ ions. Figure 9 shows examples of mw current data for the 229E B ion channel at various • holding potentials (cis relative to trans) in asymmetrical KC1 solutions (500/50 mM). The graph is a representative plot of average bilayer current (pA; y-axis) versus holding potential (mV; x-axis). Example 28. Chemical coatuonnds mhifrit the ion channel activity of the 229E E protein synthetic peptide. To test compounds for their ability to block or otherwise inhibit the ion channel formed by 229E E proitem, small aliquqts of solutions containing the compounds were added to the aqueous solutions bathing planar lipids in which the peptide channel activity had been reconstituted and the effect of the compound addition ori the ionic currents was recorded and measured. Compound stock solutions were typically prepared at 500 mM in DMSO. This solution was fttrther diluted to 50 mM, or lower concentration in 50% DMSO/50% -methanol and-2 yd of the appropriatelydttuted compound was added to the cis and/or trans chambers to yield the desired final concentration- In the example shown in Figure 10, addition of 100µm cirmamoylgaanidroe to the cis chamber greatly reduced current flow through the 229E E ion channel. Example. 29. Bacterial Bio-Assay for Screening Potential 229E-CW E protein Ion ChanneHBIocldng Drugs. 229E-CoV E-protein Ion Channel inhibits Bacterial Cell growth. A bio-assay of 229E-CoV E-protein function in bacterial cells was developed. A synthetic cDNA fragment encoding 229E-GoV E-protein was cloned into the expression plasmid pPL451, creating a vector in which E protein expression is temperature inducible, as described in Example 4. Inhibition of the growth of Exoli cells expressing E protein at 37°C was observed as an indicator of p7 ion channel function dissipating the normal Na+ gradient maintained by the bacterial ceils. Example 30 Compound Screening using the Bacterial Bio-Assay for 279E-CoV E-protein. The halos of growth around the site of application of particular drugs - as described in example 14-wore scored as decribed in example 15. Table 9 list the scores for inhibition of 229E-CW E-protein in the bacterial bio-assay. Example 31: Antiviral Assay jar testing compounds against replication of human coronaviriis 229E (229E). To determine the antiviral activity of compounds against human coronaviriis 229E repUcation (ATCC VR.-740), an assay measuring reduction in the number of plaques formed in monolayer of 229E infected MRC-5 cells (human lung fibroblasts ATCC CCL-171) was deveiopedr First, a virus working stock was prepared by amplification in MRC-5 cells. This was then used to infect confluent monolayers of MRC-5 cells grown in 6-well tissue culture plates by exposure to the virus at an MOI of approx. 0.01 pin/cell for 1 hour at 35°C in 5%CO2- The infective inoculum was removed and replaced with fresh medium (DMEM supplemented with 10% fetal calf serum) containing various test concentrations of compounds or the appropriate level of solvent used for'the compounds (control). Plates were subsequently incubated at 35°C (in 5% CQ2) for 3 -5 days post infection, after which time culture supernatant was removed and the cells were stained with 0.1% crystal violet solution in 20% ethanol for 10 minutes. Plaques were counted in all wells and the percentage reduction in plaque number compared to solvent control was calculated. Measurements were performed in duplicate tq quadruplicate wells. Example 32 Human OC43 Coronaviras QC43 Antiviral Assay for testing compounds against replication of human coronavirns OC43. To determine the antiviral activity of compounds against human coronaviras OC43 replication (ATCC VR-759), an ELISA assay was developed measuring the release of the viral N-protein into culture supernatants from monolayers of OC43- infected MRC-5 cells (human lung fibroblasts ;ATCC CCL-171); First, a virus working stock was prepared by amplification in MRC-5 cells. This was then used to infect confluent monolayers of MRC-5 cells grown in 6-well tissue culture plates by exposure to the virus at an MOI of approx. 0.01 pfu/cell for 1 hour at 35°C in 5%CO2. The infective inoculutn was removed and replaced with fresh medium (DMEM supplemented with 10% fetal calf serum) containing Various test concentrations of compounds or the appropriate level of solvent used for the compounds (control). Plates were subsequently incu bated at 35°C (in 5% CO2) for 5 days post infection, after which time cnlrure supernatant was harvested and celular debris remdved by centrifugation at 5000 x g for 10 minutes. For N-atitigen.detection, 100µl samples of clarified culture supernatant were added to duplicate wells of a 96-well Maxi-Sorb plate; 100µl of RIP A buffer was added per well with mixing and the plate was covered and incubated at 4°C overnight to enable protein binding to the plastic wells. The next day, the coating soli lion was discarded, wells were washed thoroughly with PBST, and blocking of unbcc spied protein binding sites was performed by incubation in 1% BSA in PBS for 1.5 harass. The antibody recognising OC43 N-protein was used at 1/800 dilution in PBS (1hr at 37°C) and the secondary antibody (goat-anti- mouse alkaline phosphatase) was used for the colour development reaction. Optical density of the wells was read at 405 nm and the effect of compounds deteimined by comparison of the level of signal in presence of compound to level of signal from the solvent control. Example 33; Effect of compounds in OC43 antiviral assay Compounds were screened for activity against OC43 replication according to the method described in example 22. Results are shown in Table 11. Example 34. Mouse Hepatitis Vinis (MHV). Synthesis and Purification oi a Peptide Corresponding to the MHV-A59 E Protein. A peptide corresponding to the fhll-length MHV-A59 E protein (sequence: MFNLFLTDTVWYVGQIIFIiAVCLMVTIIVVAFLASIKLCIQLCGLCNTL VLSPSIYLYDRSKQLYKYYNEEMRLPLLEVDDI; accession number NP_068673) was synthesized manually using EMOC chernistry and solid phase peptide synthesis The synthesis was done at the Biomolecular Resource Facility (John Curtin School of Medical Research, ANU, Australia) using a SymphonyR Peptide Synthesiser from Protein Technologies Inc.(Wbburn, MS, USA) according to the manufacturers ' - instructions to give C-terminal amides, the coupling was done with HBTU and hydroxybenzofriazolc htN-metb.ylpyxrolidone. Each of the synthesis cycles used double coupling and a 4-Md excess of the amino acids. Temporary -N Fmoc- protecting groups were remova I using 20% piperidine in DMF. The crude synthetic peptide was purified using the ProteoPlus™ kit (Qbiogene inc. CA), following manufactures i astructions. Briefly, the peptides were diluted in loading buffer (60mM Tris-HClpH 8.3,6M urea, 5% SDS, 10% glycerol, 0.2% Bromophenol blue, +100 tnM p-mercaptoethariol) and ran on 4-20% gradient polyacrylamide gels (Gradiporo, NSW, Australia) in tris-glyeine electrophoresis buffer (25 mM Tris, 250 mM glycine, 0.1 % SDS). The peptides were stained with gel code blue (Praraega, NSW) and the bands corresponding to the full-length peptide were excised out of the gel. The gel sEce was transferred to. the ProteoPLUS™ tube and filled with tris- glycine electrophoresis buffer, The tubes were emerged in tris-glyciue electrophoresis buffer and subjected to 100 volts for approximately 1 hour. The polarity of the electric current was reversed for 1 minute to increase the amount of protein . recovered. The peptides were harvested and centrifuged at 13, 000rpm for 1 minute. The purified peptides were dried in a Speedvac and the weight of the final product was used to calculate the yield Example 35; MHV-E protein forms ion channels in planar lipid bilayers. lipid, bflayer studies were performed as described elsewhere (Sunstrom, 1996; Miller, 1986). A lipid mixture of palinitoyl-oleoyl-phosphatidylethanolainiae, pahnitoyl-oleoyl-phosphatidylserine andpalmitoyl-oleoyl-phosphatidylcholine (5:3:2) (Avanti Polar Lipids, Alabaster, Alabama) was used. The lipid mixture was painted onto an aperture of 150-200µm in the wall of a 1 ml delrin cup. The aperture separates two chambers, cis and xans, both containing salt solutions at different concentrations. The cis chamber was connected to ground and the trans chamber to the input of an Axopatch 200 amplifier. Normally the cis chamber contained either 500 mM NaCl or 500mM KC1 aid the trans 50 mM NaCl or 50mM KCL The bilayer formation was monitored electrically by the amplitude of the current pulse generated by a current ramp. The potentials were measured in the trans chamber wira respect to the cis. The synthetic peptide was; added to the cis chamber and stirred until channel activity was seen. The currents ware filtered at 1000 Hz, digitized at 5000 Hz and stored on magnetic disk. The MHV E synthetic peptide was dissolved in 2,2,2-trifluorethanol (TFE) at 0.05mg/ml to 1 mg/ml. 10 µl of this was added to the cis chamber (1ml aqueous volume) of the bilayer apparatus, which was stirred via a magnetic "flea", Ionic currents, indicatmg channel activity in the bikyer, were typically detected within 15- 5 30 mm. After channels were detected the holding potential across the Mayer was varied between -100mV and f 100mV to characterise the stee and polarity of current flow and enable the reversal potential to be determined. In 14 experiments whtare the cis chamber contained 500mM NaCl solution and the trans chamber contained 50 mMNaCl solution, the average reversal potential of the channel activity was calculated to be 49 ±1 (SEM) mV. In 11 experiments where the cis chamber contained 500mM KC1 solution and the trans chamber contained 50 mM KG solution, the average reversal potential of the channel activity was calculated to be 13 ±6 (SEM) mV. These results indicate that the MHV E protein forms cation selective ion chatinels that are more selective for Na+ than for K. ions. Figure 11 shows examples of raw current data for the MHV E ion channel at various holding potentials (cis relative to trans) in asymmetrical NaCl solutions (500/50 mM). The graph is a representative plot of average bilayer current (pA; y- . axis) versus holding potential (MV; x-axis). Example 3S. Chettrical compounds inhibit the ion channel activity of the MHV E protein synthetic peptide. To test compounds for tlieir ability to block or otherwise inhibit the ion channel formed by MHV E protein, small aliquots of solutions containing the compounds were added to the aqueous solutions balhing planar lipids in which the peptide channel activity had been reconstituted and the effect of the compound addition on the ionic currents wsis recorded and measured. Compound stock solutions were typically prepared at 500 mM in DMSO. This solution was further diluted to 50 mM, or lower concentration in 50% DMSO/50% methanol and 2 {il of the appropriately diluted, compound was added to the cis and/or trans chambers to yield the desired final concentration. In the example stow a in Figure 12 below, addition of 100µm cirraamoylguairidine to the cis chamber greatly reduced current fiow through the MHV E ion channel. Example 37.Bacterial Channel-Blocking Drugs. MHVE-protein Ion Channel inhibits Bacterial Cell growth. A bio-assay of MHV E-proteia function in bacterial cells was developed. A synthetic cDNA fragment encoding MEV E-proteia was cloned into the expression plasmid pPL451, creating a vector in which E protein expression is temperature inducible, as described in Example 4. Inhibition of the growth of E.coli cells expressing E protein at 37°C was observed as an indicator of p? ion channel function dissipating the normal Na+ gradient maintained by the bacterial cells. . example 38. Compound Screening nsttta tfte Bacterial mo-Assay tor MJAY JK protein. The halos of growth around the site of application of particular drugs - as described in example 14 -were scored as decribed in example 15. Table 12 lists the scores for mhi bition of MTiV E protein in the bacterial bio-assay. Example 39, MHV Antiviral Assay for testing compopnds against replication of naonse hepatitis vims (MHV). To determine the antiviral activity of compounds against MHV" replication (strain MHV-A59: ATCC VR- 764), an assay measuring reduction in the number of plaques formed in monolayers of MHV infected L929 cells (ATCC CCL-a) was developed: First, a virus workiag stock was prepared by amplification in NCTC clone 1469 cells (ATCC CCL-9.1). This was then used to infect confluent monolayers of L929 cells grown in 6-well tissue culture platesby exposure to the virus at an MOI of 0.01 pfu/cell or 1 pfu/cell for 30 minutes at 37°C in 5%CO2. The infective inoculum was removed and replaced with fresh medium (DMEM supplemented with 10% horse s erum) containing various test concentrations of compounds or the appropriate Itrvel of solvent used for the compounds (control). Plates were subsequently incubated at 37°C (in 5% CO2) for 16 - 24 hours post infection, after which time culture supernatant was removed and the cells were stained with 0.1% crystal violet solution in 20% ethanol for 10 minutes. Plaques were counted in all wells and this percentage reduction in plaque number compared to solvent control was calculated. Measurements were performed in duplicate to quadruplicate wells. Example 40. Effect of compounds in MHV antiviral assay. Table 13 provides the results obtained from this study. Example 41. Porcine Respiratory Corottavirns (PRCV) Antiviral Assay for testing compounds against replication of porcine respiratory coronavirns (PRCV). coronavirus replication (ATCC VR-2384).,, an assay measuring reduction in the number of plaques formed in monolayers of PRCV infected ST cells (procine fetal testis cell line, ATCC CRL-174S) was developed: Confluent ST cells in 6 well plates were infected with a quat srnary passage of porcine respiratory virus (PRCV) strain AR310 at three dilutions 10'1,50'1 and 10"2 in PBS'to provide a range of plaques numbers to count. 100µl of diluted virus was added per well in a volume of lml of media. Plates were.incu bated for one hour on a rocking platform at room temp erature to allow virus to ad ;orb to cells. The viral supernatant was removed and 2mlAvell of overlay containing ] % Seaplaque agarose in lx MEM, 5% FCS was added to each well. Compounds to be tested were added to the overlay mixture by diluting the compounds from frozen stock to a concentration so that the same volume of compound/solvent would be added to the overlay for each concentration of compound. The volume of compound/solvent never exceeded 0.07% of the volume of the overlay. The solvent used to dissolve compounds was DMSO and methanol mixed in equal proportions. Compounds were tested for anti-plaque forming activity at four concentrations, 0. 1uM, 1uM, 10uM and 20uM. Either duplicates or quadruplicates were performed a: each concentration. Controls were performed where the same volume of sol vent was added to the overlay. The overlay was allowed to set at room temp for 20 mins. The plates were then incubated at 37°C for 2 days. The monolayers were then fixed and stained overnight at room temperature by adding Iml/well of 0.5% methylene blue, 4% formaldehyde. Overlay agarose and stain was then rinsed off to visualize stained and fixed monolayer Example 42: Effeet of eompqnnds in PRCV antiviral assay Compounds were screened for activity against PRCV replication according to the memod described in example 29. Table 14 provides EC50 values for some tested compounds. Example 43. Bovine Coronavirus. Antiviral Assay for testing compounds against replication of bovine coronavirns To determine the antiviral activity of compounds against bovine coronavirus replication (ATCC VR-874), an assay measuring reduction in the number of plaques formed in monolayers of BCV hdected MDBK cells (bovine kidney cell line ;ATCC CCL-22) was developed: Confluent MDBK cells in 6 well plates were infected with a secondary passage of BCV.wita serially diluted virus diluted to 10~3,5"5 and 10"4 in PBS to provide a range of plaques numbers to count lOOul of diluted virus was added per well in a volume of 1ml of media. Plates were incubated for one hr to allow virus to adsorb to cells. The viral supernatant was removed and 2ml/well of overlay containing 1% Seaplacue agarose in 1xMEM, 5% FCS was added to each well. Compounds to be tested were added to the oveday'mixture by diluting the compounds from a 0.5M frozen stock to a concentration so that the same volume of compound/solvent would be added to the overlay for each concentration of compound, The volume of compound/solvent never exceeded 0.07% of the volume of the overlay. The solvent ussd to dissolve compounds was DMSO and methanol mixed in equal proportions. Compounds were tested for .anti-plaque forming activity at four concentrations, 0.1uM, 1uM, 10uM and 20uM.Quadruplicates were performed at each concentratio % Controls were performed where the same volume of solvent was added to the overlay. The overlay was allowed to set at room temp for 20 mins. The plates were then incubated at 37°C for 7 days. The monolayers were then fixed and stained by adding 1rmVwell of 0.5% methylene blue, 4% fonnaldehyde Examole 44: Effect of comnoands in BCV antiviral assay Compounds were screened for activity against BCV replication according to the method described in example 31. Table 15 provides EC50 values for some tested compounds. Example 45 Hepatitis C Virus Ion channel activity of Hepgtffis C virus P7 Protein Testing of a Synthetic P7 Peptide for channel activity in artificial lipid bilavers A peptide mtmicMng the protein P7 encoded, by the hepatitis C virus (HCV) was synthesised having the following amino acid sequence: AIENLVILNAASLAGTHGLVSFLVFFCFAWYLKGRWVPGAVYAFGMWPLL LLLLALPQRAYA Lipid bilayer studies were performed as described elsewhere (Miller, 1986). A lipid mixture of palmitoyl-olEoyl-phospha1idylethanolaxmine, palmitoyl-oleoyl- phosphatidylserine and palmitoyl-oleoyl-phosphatidylcholiae (5:3:2) (Avanti Polar Lipids, Alabaster, Alabama). was used. The lipid mixture was painted onto an aperture of 150-200 um in the wall of a i ml dehin cup. The aperture separates two chambers, cis and trans, both ccmtaining stdt solutions at different concentrations. The cis chamber was connected to ground, and the trans chamber to the input of an Axopatch 200 amplifier. Normally the cis chamber contained 500 mM KC1 and the trans 50 mM KCl. The bilayer formation was monitored electrically by the amplitude of the current pulse generated by a cur rent ramp. The potentials were measured in the trans chamber with respect to the cis. The protein was added to the pis chamber and stirred until channel activity was seen. The currents were filtered at 1000 His,, digitized at 2000 Hz and stored on magnetic disk. The .P7 peptide was dissolved in 2,2,2- Mfluorethanol (TEE) at 10mg/ml. 10 ul of this was added to the cis chamber of the bilayer which was stirred. Cham iel activity was seen within 15-20 rain. When the P7 peptide was added to the cis chamber and stirred, channel activity was recorded. The potential in the trans chamber was -80 mV and the currents are downwards. The currents reversed at +50 mV close to the potassium equihbrium potential in these solutions indica ing that the channels were cation-selective. The amplitude of the open-channel peuk is 1.7 pA corresponding to a channel conductance of about 14 pS. In most experiments, "single channels" had a much larger size, presumably because of aggregation of the P7 peptide. The currents reversed at about +40 mV in this experiment. In some experiments the solution in the cischamberwas 150mMKCl and 15tnMKClin the trans chamber. The P7 peptide again produced currents that reversed. Similar results were obtained when both chambers contained NaCl. Currents recorded in an experiment when the d$ chamber contained 500 mM Nad and the trans chamber 50 mMNaCL Again the currents reversed between +40 and+60 mV, close to the Na+ equilibrium potential indicating that channels were much more permeable to Na+ than to K+. The channels formed by tie P7 peptide were blocked by 5-(N,N- hexamethylene) amiloride (HMA), Addition of the P7 peptide; produced channel activity.- Addition of 2 ul of 50 uM HMA to the cis chamber followed by stirring resulted in disappearance of the chanel activity. Block of channel activity produced by the P7 peptide with 100 µM HMA was recorded in 4 experiments. In 2 experiments, sodium channels (500/50) were blocked by 500 µM HMA When 10 mM CaCl2 was added to the cis chamber (K solutions) the reversal potential of the currents produced by P7 peptide shifted to more negative potentials indicating a decrease in the PK/PCI ratio. When the cis chamber contiined 500 mM CaCl2 and the trans chamber 50 mM CaCfe, both positive and negative currents were seen at potentials around +20 mV and it was not possible to determine a reversal potential. Example 46. Recombinant Expression of HCV p7 protein. Two cDNA fragments, eath encoding the same polypeptide corresponding to the amino acid sequence of the HCV-1 ap7 protein, were "synthesised commercially by GeneScript. The two cDNAs differed in nucleotide sequence such that in one cDNA ("cDp7.coli") the codons were optimised for expression of the p7 protein in. E.coli while in the other cDNA ("Dp7.ttiatn)" codons were biased for expression in mammalian cell lines. cDp7 coli was cloned into the plasmid pPL451 as a BamHl/EcoRI fragment for expression in E.coli. cDp7.mam was cloned into vectors (for example, pcDNA3.1 vaccinia virus, pfastBac-1) for expression of p7 in mammalian cell lines. Example 47. Role of p7 in enhancement of Gag VLP Budding. The budding of virus-like particles (VLP) from cultured HeLa cells results from the expression of retroviial Gag proteins in the cells and co-expression of small viralion. channels, such as M2, Vpu and 6K, with the Gag protein enhances budding. Ihteresttrigly, the viral ion channels can enhance budding of heterologous virus particles. Therefore, to assess budding enhancement by p7 it wag co-expressed with the HEV-1 Gag protein in HeLg, cells, and VLP release into the culture medium was measured by Gag ELISA. To achieve mis, the plasmids pcDNAp7 (pc DNA3.1 = pcDp7.mam as described in Example 20, p7 expressed under control of the T7~ - - promoter) andpcDNAGag (HIV-1 Gag protein expressed under control of the T7 promoter) were cotransfected into HeLa cells infected with the vaccinia virus strain vTF7.3 (expresses T7 SNA polymerase) and culture supernatants were collected for ELISA assay alter 16 hours inctbation. Example 48, Assay of the ability of compounds to inhibit p7 ion channel functional activity. The two methods of detecting p7 ion channel functional activity, described in Examples 33-35, were employed to assay the ability of compounds to inhibit the p7 channel. In the case of Example 33, compounds were tested for their ability to inhibit p7 channel activity in planar lipid bilayers. In the case of Example 35 compounds were tested for their ability to reduce the number of VLPs released from cells expressing both p7 and HIV-1 Gsg. - Example 49. Bacterial Bio-Assav for Screening Potential HCV p7 protein Ion Channel- Blocking Drugs. HCV n7 Ion Channel inhibits Bacterial Cell growth. A bio-assay of p7 function in bacterial cells was developed. The p7-ehcbding • synthetic cDNA fragment cDp7.coli was cloned into the expression plasmid pPL451, creating the vector pPLp7, in which p7 expression is temperature inducible, as described in Example 4. Inhibition of the growth of E.coli cells expressing p7 at 37oC was observed as an indicator of p7 ion channel function dissipating the normal Na+ gradient maintained by the bacterial cells. Example 50 Compound Sqieenhig using the Bacterial Bio-Assay for HCV p7 protein. The halos of growth around the site of application of particular drugs—as described in example 14=werescored'isdecribed in example' 15 Table 16 lists the scores for in [ubition of HCV p7 protein in the bacterial bio-assay. Table 16 Example 51: Equine Arteritis Virus (EAY) Antiviral Assay for testing contpoands against replication of equine arteritis virasfEAV). To determine the antiviral activity of compounds against EAV replication (strain Bueyrus; ATCC VR-796), an assay measuring reduction in the number of plaques formed in monolayers oi'EAV infected BHK-21 cells (ATCC CCL-10) was developed: A virus stock was amplified in RK-13 cells (ATCC CCL-37) and this was then used to infect confluent monolayers of BHK-21 cells grown in 6-well tissue . culture plates by exposure to the virus at an MOI of 5X10-3 pfu/cell for 1 hour at 37°,C 5% CO2. The infective inoculum was removed and nd the cells were overlayed with a 1% sea plaque overlay (Cambrex Bio Science) .in MEM containing 10% FCS containing and 10,5 or 1 pM of compounds to be tested or the appropriate level of solvent used for the compounds (uontrol). Plates were subsequently incubated at 37oC (in 5% CO2) for 3 days post infection, after which time culture supernatant was . removed and the cells were stained with 0.1% crystal violet solution in 20% ethanol for 10 minutes. Plaques were counted in all wells and the percentage reduction in plaque number compared to solvent control was calculated. Measurements were performed in duplicate to quadruplicate wells. Example 52: Effect of compounds in EAV antiviral assay Compounds were screened for activity against EAV replication according to the method described in example 35. Results expressed as IC50 values are shown in Table 17. Example 53 Dengue FIavivirus Peptide Synthesis of Dengue virus M Protein The C- terminal 40 amino acids of the M protein of the Dengue virus type 1 strain Singapore S275/90 (Fu et al 1992) (AISHPGFrVTAIFRLAHAIGTSITQKGnFILLMLVTPSMA) was synthesised using the Fmoc method,. The synthesis was done on a Symphony Peptide Synthesiser form Protein Technologies Inc (Tucson, Arizona) as used to give C-terminal amides, the coupling was done with HBTU t nd hydroxybenzotriazole in N-methylpyrrolidone. Each of the synthesis cycle used double coupling and a 4-fold excess of the amino acids. Temporary cc~N Fmoc-p.ro :ecting groups were removed using 20% piperidme in DMF. Example 54. Incorporation of Dengue M virus protein into lipid hilayers. Lipid bilayer studies were performed as described elsewhere (Sunstrom, 1996; Miller, 1986), A lipid mixture of palmitoyl-oleoyl-phosphatidylethanolamirie, palrnitoyl-oleoyl-phosphatiaylserifle and palmitoyl-oleoyl-phosphatidylcholin. (5:3:2) (Avanti Polar Lipids, Alabaster, Alabama) was used The lipid mixture was painted onto an aperture of 150-200 µm in the wall of a 1 ml delrin cup. The aperture separates two chambers, cis and trans, both containing salt solutions at different concentrations. The cis chamber was connected to ground and the trans chamber to the input of an Axopatch 200 amplifier. Normally the cis chamber contained S00 mM KCl and the trans 50 mM KCl. The bilayer formation was monitored electrically by the amplitude of the current pulse generated by a current ramp. The potentials were measured in the trans chamber with respect to the cis. The protein was added to the cis chamber and stirred until channel activity was seen. The currents were filtered at 1000 Hz, digitized at 5000 Hz and stored on magnetic disk.. The dengue virus M protein C- teaminal peptide (DMVC) was dissolved in 2,2,2- trifluorefnanol (TFE) at 0.05me/ml to 1 mg/ral, 10 pi of this was added to the cis chamber of the bilayer which was starred Channel activity was seen within 15-30 min. Example 55; Hexamethvlene amiloride (HMA) to inhibits ion channel activity the dengue virus M protein C-termmal peptide. Solutions of 50 mM HMA. were prepared by first making a 500 mM solution in DMSO. This solution was further diluted to 50 mM HMA using 0.1 M HC1.2 µl of the 50 mM HMA was added to the cis chamber after channel activity was seen. The cis chamber contained 1 ml of solution making the final concentration of HMA 100 µM. Example 56, Antiviral Assay for testing compounds against Effects of Dengne flavivirns against cytoproliferation. Compounds were tested at 10,5, 2.5, L25 and 0.625 uM for activity against Dengue 1 strain Hawaii using a cyloproliferation assay which measures the effect of dengue virus infection on proliferation of LLC-MK2, rhesus macaque monkey kidney ceils. The LLC-MK2 cells were infected with a predetermined amount of virus, titrated such that cell proliferation ii infected cultures would be significantly reduced compared to uninfected controls. The infected cells were then plated at 1.5x103 cells per well in a 96 well plate. Negative controls (no vims, no experimental compound). positive controls (virus, no experimental compound), and cytotoxicity controls (experimental compound, nc virus) were run with each drug assay. The cultures wei allowed to grow for 7 days and then Alamar Blue, a fluorescent dye that measures th metabolism of the cultures (red/ox), was added to each culture and the fluorescence value for each culture was raesasured. The negative control without experimental compound or virus was fixed at 100%. The positive controls and the cultures with compound were scored by calculating their average fluorescence as a percentage of the negative control. At least six replicate wells were measured for each experiments condition. Example 57 Effect of compounds in Dengue antiviral assay: A correlative study was performed to measure correlation between the activity scores assigned to compounds tessted m the Vpu bacterial assay and the ability of these compounds to inhibit HIV -1 in the anti-viral assay. Example 58.2. Methodology „.„,The p24-antigen, data for.twelve compounds.representing.various substituted.acyl guanidinss was compared with the activity scores obtained for those compounds in the Vpu bacterial assay. The data from each assay was initially rank ordered for effectiveness. The rank order for the Vpu bacterial assay was determined from all activity scores, the highest score indicating the greatest effectiveness. The rank order for the anti-HIV-1 assay was dettsrmined based on the overall average value of p24 antigen measured in culture supenatants at all of the drug concentrations tested, with the lowest score indicating the grsatest effectiveness. The two rank orders generated were then compared statistically by generating the Spearman's Rank correlation coefficient. Example 58.3. Resnlts and Conclusion The Spearman's correlation coefficient was 0.785 which, by comparison with a statistical table of critical values (for n=12), indicates that the two rank orders are significantly positively correlated (P In addition, a different con rparison of the Vpu Bacterial assay rank order with a yes/no score for whether the anti -viral data indicated a p24 reduction of at least one order of magnitude, aligned the 'y JS' group of compounds with the top 6 compounds by the bacterial assay (Table 19b).. These results are indicative that a positive correlation easts between bacterial assays and the antiviral assajs as peiformed-according to the present invention. The bacterial assay may therefore be a useful tool in screening for compounds that exhibit anti-viral activity. Table 19a. Comparison of Rank order of efficacy of 12 substituted acyl- guanidines in tb.e Vpu bacterial assay and anti-HIV assay. Example 58.4. Correlation Between Percent inhibition of MHV plaque formation and MHV-E bacterial bio-assay score. A positive correlation was seen between the activity scores assigned to .. • compounds when tested in the Mouse Hepatitis Virus E-protein bacterial bio-assay and the percent inhibition exhibited by these compounds in the Mouse Hepatitis Vims plaque assay. Example 58.5. Method; MHV plaque reduction activity data for 96 compounds screened were sorted from greatest to least percent plaque reduction and rank orders, were, assigned to the list of compounds, This was performed for the data generated by exposure to both lO^M and 1µM concentrations of the compounds, giving rise to two rank order lists. Similarly, a rank order list was generated for the MHVE bacterial bioassay scores for the same 96 compounds. Where or e or more compounds had the same score, the rank values for that group were averaged. Spearman's statistical test for [as described in "Mathematical Statistics with Applications" (2nd edn): Mendenhall W., Scheaffer, RL..& Wackerly, DD. Duxbury Press, Boston Massachusatts - 3981] was used to compare rank orders. Briefly, this involved calculating the Sum of squares (SS) of the differences between rank values for . each compound, and then generating the Spearman's Rank Correlation coefficient (Rs) according to the formula: Rs =] -(6.SS/n(nZTl)), where n is the number of compounds ranked (96 in this case). Rs is then compared to a Table of critical values to determine statistical significance (P values) Example 58.6. Summary of Results: This table summarises the Rs and P values generated as a result of the indicated pairwise comparisons between mnk orders. Example 58.7. Conclusions: The rank order comparison of 96 compounds assayed in the bacterial bio- assay and the antiviral assay show that MHVE bacterial assay rank order for the compounds tested is significantly positively correlated with, the rank orders generated by the MHV plaque redaction assay. The significant correlation between trie assays is highly indicative that either assay may be utilised to identify compounds that may be useful. The bacterial assay nay thereby be ai useful tool in screening for compounds that exhibitanti-vhal-activityr Example 58.8. Correlation Between Percent inhibition of 229E plaque formation and 229E-E bacterial bio-assay score. A positive correlation was seen between the activity scores assigned to compounds when tested in the Eirraan Coronavirus 229E E-protein bacterial bio- assay and the percent inhibition exbibited by these compounds in the Human Coronavirus 229B plaque assay. Example 58.9. Method: 229E plaque reduction activity data for 97 compounds screened against 2.5 µM compound concentration were sorted from greatest to least percent plaque reduction and rank orders were assigned to the list of compounds. Similarly, a rank order list was generated for the 229E E bacterial bioassay scores for the same 97 compounds. Where one or more compounds had the same score, therank values for that group were averaged. Spearman's statistical test for [as cescribed in. 'Mathematical Statistics with Applications" (2ni edn): Mendenhall, W., Scheaifer, RL.,& Wackerly, Dp. Duxbury Press, Boston Massachusetts -1981] was used to compare rank orders. Briefly, this involves calculating the Sum of squares (SS) of file differences between rank values for each compound, and then generating the Spearman's Rank Correlation coefficient (Rs) according to the formula: Rs = 1-(6.SS/u(n2-).)), where n is the number of compounds ranked (97 in this case). Rs is then compared to a Table of critical values to determine statistical significance (P values). Example 58.9.1. Summary of Results and Conclusions This table summarises the Rs and P values generated as a result of the indicated pairwise comparisons between rank orders. The results above indicate that the 229E bacterial assay rank order for the compounds tested is significan fly positively correlated with the rank orders generated by the 229B plaque reduction assay. This result combined with that shown in Examples 49.1 and 49.4, provides strong evidence that either assay may be utilised to identify compounds that may be useful. The bacterial assay may thereby be a useful tool in screening for compounds that exhibit anti-viral activity. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this sperificatioti, individually or collectively, and any and all combinations of any two or more of said steps or features. BIBLIOGRAPHY Barry, M., Mubahy, F. and Back, D - J., Br J Clin Pharmacol, 45:221 (1998) Desks, S.G., West J Med, 168:133 (1998) MUes, S.A,, J Acquir Bnmtrae Defic Syndr Hum Retroviral, 16 Suppl 1: S36 (1997) Miles, S.A., J. Acquir immune Defic Syndr Hum Retroyirol, 16 Suppl 1: SI (1998) Moyle, G.J., Gazzard,B.G., Cooper; D.A.and Gatell, J.,Drugs, 55:383 (1998) Rachlis, A.R and Zarowny, E.P., Cmaj, 158:496 (1998) Vena, S., Fragola, V. and Pafcaisano, L„ J Biol Regul Homeost Agents, 11:60 (1997) Vofoerding, P.A. and Deeks, H.G., Jama, 279:1343 (1998) Vplbeiding, P.A., Hosp Pract (Off Ed), 33:81-4, S7-90,95-6 passim. 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IRL Press, Oxford (1990) Grice, AX., Kerr, LD. and Sansom, M.S., FEBSLett, 405(3):299-304 (1997) Moore, P.B., Zhong, Q., Husslein, T. and Kleio, MX., FEBS Lett, 431(2):143-148 (199$) Schubert, U., Bour, S., Ferrennontiel, A.V., Montal, M\, Maldarelli, F. and Strebel, K.Joumal of Virology. 70(2):8Q Sunstrom NA, Premkumar LS, Piemkumar A, Ewart G, Cox GB, Gage PW {1996), Ion channels formed by NB, an iifluenza B virus protein. J Membr Biol. 1996 Mar,150(2):127-32 Willbald, D., Hotftaatm, S. . and Rosch, P., Eur J Biochem, 245(3):581-8 (1997) Wray, V.,Kinder, R., Federau, T., Henkein, P.. Bechinger, B. and Schubert, U., Biochemistry, 3S(16):5272-82 (1999) We Claim:- and wherein X = hydrogen, hydroxy, nitro, halo. C1-6alkyl, C1-6alkyloxy. C3-6cycloalky I, halo-substituted C1-6alkyl, halo-substituted C1-6alkyloxy, phenyl, C1-6alkeneyl, C3-6cycloalkeneyl, C1-6alkeneoxy, or benzo; Ra, Rb , Re. Rd, Re Rt, Rb, Rk, RL , Rm Rn, Ro, Rp independently = hydrogen, amino, halo, C1-5alkyl, C1-5alkyloxy, hydroxy, aryl. substituted aryl, substituted amino, mono or dialkyl-substituted amino, cycloalkyl-substi uted amino, aryl-substituted amino. cannot be hydrogen, benzyl or substituted benzyl or BODIPY-FI; when R1 is C6H5CH=CH, R2 is hydrogen and R3 is phenyl, R4 cannot be phenyl: when R1 is phenyl. R2 is hydrogen, and R3 is benzoyl, R4 cannot be benzoyl; when R1 is phenyl. R2 is substituted benzyl, R3 is hydrogen and R4 is hydrogen. Rn, Ro and RP cannot all be hydrogen; when Ri is phenyl R:, is hydrogen and R4 is hydrogen. R2 cannot be benzyl or phenyl; when Ri is phenyl, R2 is hydrogen, R3 cannot be phenyl together with R4 as benzoyl; and when Ri is pheny1, R2 is hydrogen, R3 and R4 cannot both be benzyl. 2. A pharmaceutical composition comprising an antiviral compound as claimed in claim 1 and optionally one or more pharmaceutical acceptable carriers or derivatives. 3. The pharmaceutical composition as claimed in claim 2, comprising one or more known anti /iral compounds or molecules. A compound as claimei in claim 1, wherein said compound is an antiviral for Lentivirus including Human Immunodeficiency Virus or Human Immunodeficiency Virus-1. selected from the group consisting of: (3-Chlorocinnamoyl)guanidine, (3-Bromocinnamoyl)guanidine, (2-Chlorocinnamoyl)guanidine, (2-Bromocinnamoyl)guanidine, 3-(trifluoromethyl)cinamoylguanidine, 5-bromo-2-fluorocinnamoylguanidine, 3-methylcinnamoylg ianidine, 2-methylcinnamoyluanidine, 2,3-dimethylcinnamc ylguanidine, Cinnamoylguanidine 6-methoxy-2-naphthoylguanidine, trans-3-(l-napthyl)acryloylguanidine, 3,4-dichlorocinnamo ylguanidine, 2,6-dichlorocinnamoylguanidine, 4-phenylbenzoylguaitidine, 2-ethylcinnamoylguenidine, (4-Chlorocinnamoyr guanidine, 2-napthoylguanidine 2,5-dimethylcinnamoylguanidine, 3-isopropylcinnamo} lguanidine hydrochloride, (5-Phenyl-penta-2,4-dienoyl)guanidine, 3-phenylcinnamoylgianidine, (4-Bromocinnamoyl guanidine, 5-(3'-bromophenyl)penta-2,4-dienoylguanidine, 3-(cyclohex-l-en-l-} l)cinnamoylguanidine, 3-(trifluoromethoxy)cinnamoylguanidine, 2-(trifluoromethyl)cinnamoylguanidine, N,N'-bis(3phenylpropanoyl)-N' -phenylguanidine. 2-ethoxycinnamoylguanidine, N-(3-phenylpropano/l)-N'-pheriylguanidine, 4-(trifluoromethyl)cinnamoylguanidine, (4-Methoxycinnamoyl)guanidine, 2-t-butylcinnamoylgianidine, 4-methylcinnamoylg uanidine, 2-fluorocinnamoylguanidine, 2-phenylcinnamoylg uanidine, N-(6-Hydroxy-2-naf thoyl)-N'-phenylguanidine, 3-t-butylcinnamoylguanidine. 3,4-difluorocinnamc ylguanidine, 5-(N,N-hexamethykne)amiloride, 3-fluorocinnamoylguanidine, 5-bromo-2-methoxy;innamoylguanidine, 3-ethoxycinnamoylg uanidine. 3,4-(methylenedicxy)cinnamoylguanidine, (2-Methoxycinnarnoyl)guanidine, 2'4 DichloroBenazamil HC1 2,3,5,6,-tetramethylcinnamoyIguanidine, 3-(2-napthyl)acry oylguanidine, 2-( 1 -napthyl)acetoylguanidine, 2,3-difluorocinnanoy lguanidine, (3-Methoxycinna noyl)guanidine. 4-isopropylcinnatnoylguanidine, 2,4,6-trimethylcinnamoylguanidine, N-(cinnamoyl)-N'phenylguanidine, 2-(cyclohex-1 -en -1yl)cinnamoylguanidine, 2-(2-napthyl)acetoylguanidine, (4-Hydroxycinnamoyl)guanidine, 4-phenylcinnamc ylguanidine, 4-fluorocinnamo /lguanidine, N,N'-bis-(cinnarroyl)-N"-phenylguanidine, (2-Furanacryloyl lguanidine, Phenamil methanesulfonate salt, Benzamil hydrochloride. (3-Nitrocinnarnoyl)guanidine, Benzyoylguanid ne, (4-Phenox.ybenz oyl)guanidine, 3-(trans-hept-1 -<. n-1 lguanidine> 5-(N-Methyl-N- sobutyl)amiloride, 2-cyclohexylciniamoy lguanidine, 4-ethoxycinnam oylguanidine, 2,4-dichlorocinr amolyguanidine, 5-(N-EthyI-N-is:>propyl)amiloride, N-amidino-3-amino-5-hexamethyleneimino-6-phenyl- 2-pyrazinecarbcxamide. (a-Methylcinnainoyl)guanidine, cinnamoylguanidine hydrochloride, [(4-Chlorophenoxy-acetyl]guanidine, N-amidino-3-ariino-5-phenyl-6-chloro-2- pyrazinecarbox.imide, 5-(4-fluoropheryl)amiloride, (trans-2-Phenyl;yclopropanecarbonyl)guanidine, (2-Nitrocinnam oylguanidine, trans-3-Furanac ryoylguanidine, 1 -napthoylguan idine. 5-tert-butylami lo-amiloride, 3-methoxy -HMA, (3-phenylpropanoyl)guanidine, 4-t-butylcinnamoy lguanidine, 5-(N,N-Dimetryl)amiloride hydrochloride, N,N'-Bis(3-phenylpropanoyl)guanidine. N-Benzoyl-N'- ;innamoylguanidine and 1 -bromo-2-naptho\ lguanidine. A compound as claimed in claim 4, wherein said compound is selected from the group consisting of 4-phenylbenzoylguanidine, (3- bromocinnamoyl)guar idine, 3-(trifluoromethyl)cinnamoylguanidine. 5-(N,N- hexamethylene)amiloide, and (5-Phenyl-penta-2,4-dienoyl)guanidine. A compound as claimed in claim 1, wherein said compound is an antiviral for Coronavirus including Severe Acute Respiratory Syndrome Virus (SARS), selected from the group consisting of 2,3-difluorocinnamoylg uanidine. 3.4-dichlorocinnamoylguanidine, 4-t-butylcinnamoyiguanidine, 3-(2-napthyl)acryloylguanidine, (3-Chlorocinnamoyl)gu anidine. 3-(cyclohex-l-en-l-yl)cinnamoylguanidine, 2,5-dimethylcinnamoy guanidine, trans-3-(1 -napthyl)acry loylguanidine, 4-isopropylcinnamoyhj uanidine. (3-Bromocinnamoyl)guanidine, 6-methoxy-2-naphthoy Iguanidine, 5-(N-Methyl-N-isobutyl)amiloride, 3-phenylcinnamoylguE nidine, (2-Chlorocinnamoyl)g aanidine, 2'4 DichloroBenazam 1 HC1 4-phenylcinnamoylgu 4-(trifluoromethyl)cinnamoy Iguanidine, 3-(trifluoromethoxy)cinnamoylguanidine. 3-(trifluoromethyl)ciniamoy Iguanidine, 2-ethoxycinnamoylgu;tnidine, cinnamoylguanidine hydrochloride, 4-ethoxycinnamoylguinidine, (2-Bromocinnamoyl)g uanidine. 2,6-dichlorocinnamoy Iguanidine, 3,4,5-trimethoxycinnamovlguanidine, 5-tert-butylamino-amiloride, 3-t-butylcinnamoy Igu anidine, 5-bromo-2-fluorocinnamoylguanidine, (4-Chlorocinnamoyl)guanidine, 2-t-butylcinnamoyigu anidine, 2-cyclohexylcinnamoylguanidine, 6-Iodoamiloride. 3-(trans-hept-1 -en-1-yl)cinnamoylguanidine, (4-Bromocinnamoyl) guanidine, (4-Hydroxycinnamoyl)guanidine, N-(3-phenylpropano)l)-N'-pheny Iguanidine, (3-Nitrocinnamoyl)guanidine, 3-fluorocinnamoylguanic ine, 2-( 1-napthyl)acetoylguar idine, 2-ethylcinnamoylguanidine, 5-(N,N-Dirnethyl)amilonde hydrochloride, 2-napthoylguanidine, 5-(4-fluorophenyl)amiloride. 2-(trifluoromethyl)cinnanoylguanidine, N-(6-Hydroxy-2-napthoyl)-N'-phenylguanidine, (trans-2-Phenylcyclopropanecarbonyl)guanidine, N,N'-bis(3phenylpropan3yl)-N"-phenylguanidine„ 1 -napthoylguanidine, Benzamil hydrochloride, 3-methoxy -HMA, 4-methylcinnamoylguar idine, 4-fluorocinnamoylguan dine, 3,4-(methylenedioxy)ci nnamoylguanidine, 5-(N,N-hexamethylene)amiloride, N-(cinnamoyl)-N'phen) Iguanidine, 5-(N-EthyI-N-isopropyl)amiloride, 3-methylcinnamoylguaiiidine, 2-methylcinnamoylgua lidine, 2,3,5,6,-tetramethylcinnamoylguanidine, trans-3-Furanacryoylgianidine, (4-Methoxycinnamoyl)guanidine, (2-Furanacryloyl)guanidine, (3-phenylpropanoyl)guanidine, 2-(2-napthyl)acetoylguanidine, Cinnamoylguanidine, (2-Methoxycinnamoylguanidine. [3-(3-Pyridyl)acryloyl]guanidine, 4-phenylbenzoylguani-iine. 2,4-dichlorocinnamol) guanidine, (3-Methoxycinnamoyl )guanidine, 2-fluorocinnamoylguanidine, (4-Phenoxybenzoyl)guanidine, (a-Methylcinnamoyl)§.uanidine. 5-(3'-bromophenyI)peita-2,4-dienoy Iguanidine, (5-Phenyl-penta-2.4-dienoyl)guanidine. (Quinoline-2-carbonyi)guanidine, (Phenylacetyl)guanidine, N,N'-Bis(amidino)napthalene-2,6-dicarboxarnide, 6-bromo-2-napthoylgianidine, 1 -bromo-2-napthoylguanidine, 2-chloro-6-fluorocinramoylguanidine, [(4-Chlorophenoxy-acetyl]guanidine, Phenamil methanesul fonate salt, N-Benzoyl-N'-cinnarioylguanidine and N-(2-napthoy!)-N'-preny)guanidine. A compound as claimed in claim 6, wherein said compound is selected from the group consisting of cinnamoylguanidine, trans-3-(l- napthyl)acryloylguanidine, and 6-methoxy-2-naphthoylguanidine. A compound as claimed in claim 1, wherein said compound is an antiviral for Coronavirus including Human Coronavirus 229E , selected from the group consisting of 4-isopropyicinnamcylguanidine, 3,4-dichlorocinnamoylguanidine, 3-(trifluoromethoxy)cinnamoy[guanidine, 4-t-butylcinnamoylguanidine, 3-isopropylcinnamoylguanidine hydrochloride, 3-t-butylcinnamoylguanidine. 2-t-butylcinnamoylg uanidine. trans-3-(1-napthyl)acryloylguanidine, 5-bromo-2-methoxycinnamoylguanidine, 2,3-difluorocinnamcylguanidine, 3-(2-napthyl)acryloylguanidine, 2-phenylcinnamoylg uanidine. 3-phenylcinnamoylg uanidine, 3-(cyclohex-1 -en-1 -yl)cinnamoylguanidine, 4-phenylbenzoylguaiidine, 3-(trifluoromethyl)c nnamoylguanidine, (4-Phenoxybenzoyl) guanidine. 4-(trifluoromethyl)cinnamoylguanidine, 2-(cycIohex-l-en-lyl)cinnamoylguanidine. (4-Bromocinnamoyl iguanidine. 5-(N,N-hexamethylene)amiloride, 1-napthoylguanidine. 5-(4-fluorophenyl)aniloride, (5-Phenyl-penta-2,4-dienoyl)guanidine, (3-Bromocinnamoyl guanidine, 2,5-dimethylcinnamoylguanidine, 2-(trifluoromethyl)cinnamoylguanidine, 6-methoxy-2-naphthoylguanidine, (4-Chlorocinnamoyl) guanidine, (3-Methoxycinnamoyl)guanidine, 5-bromo-2-f[uorocinnamoylguanidine. 5-(N.N-Dimethyl)amiloride hydrochloride. Cinnamoylguanidine (2-Methoxycinnamoyl)guanidine, (a-Methy lcinnamoyl)guanidine. 4-phenylcinnamoy Iguanidine, 2,6-dichlorocinnamoylguanidine, (2-Bromocinnamoyl) guanidine. 2,4,6-trimethylcir namoylguanidine, (trans-2-Phenylcyclopropanecarbonyl)guanidine, (3-Chlorocinnamoyl)guanidine, 2-( 1-napthyl)acetoylguanidine, 2-ethylcinnamoyl guanidine, 2-cyclohexylcinnamoylguanidine, (4-Hydroxycinnamoyl)guanidine, 2-ethoxycinnamoylguanidine, 3-methylcinnamoylguanidine, 2-methylcinnamoylguanidine, 3-fluorocinnamoy lguanidine, cinnamoylguanidine hydrochloride, 2,3-dimethylcinnamoylguanidine, 2-fluorocinnamoylguanidine. 4-fluorocinnamoy lguanidine. 3,4-difluorocinnainoylguanidine, 5-tert-butylamino-amiloride, 2-napthoylguanidine, N,N'-Bis(amidino napthalene-2,6-dicarboxamide, N,N'-Bis(3-pheny]propanoyl)guanidine. 4-methylcinnamoy lguanidine, 5-(3'-bromopheny )penta-2,4-dienoylguanidine, 2,3,5,6,-tetramethylcinnamoylguanidine, 3-ethoxycinnamo) lguanidine, N,N'-bis(3phenylpropanoyl)-N"-phenylguanidine, (4-Methoxycinnanioyl)guanidine, (2-Chlorocinnamoyl)gunidin, (3-Nitrocinnamoyl )guanidine, 4-ethoxycinnamoyluanidine., 3,4,5-trimethoxycinnamoylguanidine. 2-(2-napthyl)acetoyguanidine, N-(3-phenylpropar oyl)-N'-phenylguanidine, 5-(2'-bromophenyl )penta-2,4- dienoylguanidine, (4-Bromocinnamoyluanidine, (2-Nitrocinnamoyl)lguanidine. (3-Chlorocinnamoyl)guanidine, (4-Methoxycinnarroyl)guanidine, 4-(trifluoromethyl)cinnamoylguanidine, [(E)-3-(4-Dimethylaminophenyl)-2- methylacryloyl]guanidine, N-Benzoyl-N'-cinnamoylguanidine, 4-phenylbenzoylguanidine, trans-3-Furanacryoy lguanidine, N-amidino-3-amino-5-phenyl-6-chloro-2- Pyrazinecarboxamide, N-(cinnamoyl)-N'p leny lguanidine, Cinnamoylguanidint, 3-methoxy-amiloridi:, (3-phenylpropanoyl)guanidine, 3-methoxy -HMA, Benzyoylguanidine, N-amidino-3,5-dianr ino-6-phynyl-2- Pyrazinecarboxamic e. (Quinoline-2-carboryl)guanidine, [3-(3-Pyridyl)acrylcyl]guanidine, N-Cinnamoyl-N',N'-dimethylguanidine, N-(2-napthoyt)-N'-phenylguanidine and (Phenylacetyl)guan dine. A compound as claimed in claim 8, wherein said compound is selected from the group consisting of 2-t-butylcinnamoylguanidine, 4-isopropylcinnainoylguanidine, 3,4-dichlorocinnf.moylguanidine, 3-(trifluoromethcxy)cinnamoylguanidine, 2,6-dichlorocinnamoylguanidine, 2-(cyclohex-l-en-lyl)cinnamoylguanidine, 2-cyclohexylcinr amoylguanidine, 5-bromo-2-methoxycinnamoylguanidine, 2-phenylcinnamoylguanidine, 4-t-butylcinnamoylguanidine, 3-phenylcinnamoylguanidine, (3-Bromocinnamoyl)guanidine, 5-(N,N-hexametliylene)amiloride, trans-3-( 1 -napth /l)acryloylguanidine. 3-(2-napthyl)acryloylguanidine, 2,4-dichlorocinramolyguanidine, 3-(trifluoromethyl)cinnamoylguanidine, 5-bromo-2-fluoiocinnamoylguanidine, 4-methylcinnarr oylguanidine, (4-Chlorocinnainoyl)guanidine, 3-fluorocinnamoylguanidine, 3-(cyclohex-l-en-l-yl)cinnamoylguanidine. (a-Methylcinnanoyl)guanidine, 2,3,5,6,-tetramethylcinnamoylguanidine, 2-fluorocinnam oylguanidine, (3-Nitrocinnamoyl)guanidine, 2,5-dimethylcinnamoylguanidine, 3-t-butylcinnamoylguanidine, (3-Methoxycimamoyl)guanidine. 3-methylcinnamoylguanicline, 3-isopropylcinnamoylguanidine hydrochloride, (2-Bromocinmmoyl)guariidine, 3-ethoxycinnainoylguanidine, (5-Phenyl-penla-2,4-dienoyl)guanidine, (2-Chlorocinnamoyl)guanidine, 4-ethoxycinnamovlguanidine, 4-fIuorocinnamoj lguanidine, 3,4-difluorocinna Tioy lguanidine, N-(3-phenylprop£ noyl)-N'- Phenylguanidine, 2,4,6-trimethylcinnamoylguanidine. 2-methylcinnamc ylguanidine, (trans-2-Phenylcyclopropanecarbonyl)- guanidine, (4-Phenoxybenzoyl)guanidine, (2-Methoxycinnt moyl)guan idine, Cinnamoylguani iine. 3.4-(methylened oxy)cinnamoylguanidine, N,N'-Bis(amidino)napthalene-2,6- Dicarboxamide, 2,3-dimethylcinnamoylguanidine, 5-(3'-bromopheryl)penta-2,4-dienoylguanidine, N,N'-Bis(3-pheriylpropanoyl)guanidine, 2,3-difluorocinnamoylguaridine, 1 -napthoy lguanidine, 6-methoxy-2-naphthoylguanidine, 5-(N,N-Dimethyl)amiloride hydrochloride, 2-ethoxycinnamoy lguanidine, 2-napthoylguan dine, 3,4,5-trimethox/cinnamoylguanidine, 2-(trifIuoromettyl)cinnamoylguanidine, cinnamoylguan dine hydrochloride, (4-Hydroxycinr amoyl)guanidine. 5-(4-fluoropher yl)amiloride, 2-(l-napthyl)acetoy lguanidine, (2-Furanacryloyl)guanidine, N-Cinnamoyl-T4',N'-dimethylguanidine, 2-(2-napthyl)ac etoylguanidine and N,N'-bis(3pherylpropanoyl)-N"- Phenylguanidine. A compound as claimed in claim 1, wherein said compound is an antiviral for Coronavirus including human Coronavirus OC43 ,selected from the group consisting of 3-methyicinnamoy lguanidine, trans-3-(1-napthyl )acryloylguanidine, (3-Bromocinnamc yl)guanidine, (2-Chlorocinnamc yl)guanidine. 3,4-dichlorocinnamoylguanidine. 3-(trifluoromethy )cinnamoylguanidine, (trans-2-Phenylcyclopropanecarbonyl)guanidine, 4-isopropylcinnarnoylguanidine, Cinnamoylguanidine, 6-methoxy-2-naphthoylguanidine, 2,4-dichlorocinnamo lyguanidine, (4-Chlorocinnamoyl guanidine, 5-(N,N-hexamethylene)amiloride, (4-Bromocinnamoyl lguanidine, 2,6-dichlorocinnamcylguanidine, 5-bromo-2-methoxycinnamoylguanidine, (5-Phenyl-penta-2,4-dienoyl)guanidine, 3-(trifluoromethoxy)cinnamoylguanidine and 2-t-butylcinnamoylguanidine. 11. A compound as claimed in claim 1, wherein said compound is an antiviral for Coronavirus including porcine respiratory Coronavirus (PRCV),selected from the group cons sting of 5-(N.N-hexamethylene)amiloride. 6-methoxy-2-naphthoy lguanidine, Cinnamoylguanidine, N-(3-phenylpropanoyl)-N'-phenylguanidine, 3-methylcinnamoylguanidine, (3-Bromocinnamoyl)guariidine, (trans-2-Phenylcyclopropanecarbonyl)guanidine, trans-3-( 1 -napl hyl)acryloylguanidine and 2-(2-napthyl)acetoylguanidine. 12. A compound as clai ned in claim I, wherein said compound is an antiviral , for Coronavirus including bovine Coronvirus (BCV),selected from the group consisting of (3-Bromocinnamoyl)guanidine, 3-(trifluoromethyl)cinnamoylguanidine, 6-methoxy-2-naphthoylguanidine, 5-(N.N-hexamethylene)amiloride, trans-3-( 1 -napth /l)acryloylguanidine, Cinnamoylguan dine, (5-Phenyl-penta-2,4-dienoyl)guanidine, 2-(2-napthyl)acctoylguanidine, (trans-2-Phenykyclopropanecarbonyl)guanidine, N-(3-phenylpropanoyl)-N'-phenylguanidine and 4-phenylbenzoy guanidine. 13. A compound as clained in claim 1, wherein said compound is an antiviral for Coronavirus includirg any one of the known coronavirus isolates listed in Table 1,selected fron the group consisting of 4-isopropylcinnamoylguanidine, 3,4-dichlorocinnamcylguanidine, 3-(trifluoromethoxy)cinnamoylguanidine, 4-t-butylcinnamoylg aanidine, 3-isopropylcinnamo;lguanidine hydrochloride, A compound as claimed in claim 1, wherein said compound is an antiviral for Hepatitis C virus , selected from the group consisting of 2,3-dimethylcinnamoy guanidine, 2,4,6-trimethylcinnamoylguanidine, 5-bromo-2-fluorocinnamoylguanidine, (4-Bromocinnamo\i)guanidine, 2,5-dimethylcinnamoy guanidine, 3-(trifluoromethyl)cinramoy lguanidine, 4-(trifluoromethyl)cinramoylguanidine, 6-methoxy-2-naphthov lguanidine, (2-Chlorocinnamoyl)guanidine, (4-Chlorocinnamoyl)guanidine, (2-Bromocinnamoyl)guanidine. 2,6-dichlorocinnamoylguanidine, (3-Bromocinnamoyl)g aanidine, (3-Chlorocinnamoyl)g lanidine, 2-(trifIuoromethyl)cinnamoy lguanidine, (4-Phenoxybenzoyl)guanidine, 3,4-dichlorocinnamoyl guanidine, 4-isopropylcinnamoyl|;uanidine, trans-3-( 1 -napthy l)acr 'loylguanidine, 4-t-butylcinnamoylgu&nidine, 2-t-butylcinnamoy lguanidine, 2-ethylcinnamoy lguanidine, 4-methylcinnamoy lguanidine, 5-bromo-2-methoxycinnamoylguanidine, 3-(trifluoromethoxy)cinnamoylguanidine, 2-cyclohexylcinnamoy lguanidine, 1 -napthoylguanidine, 3-t-butylcinnamoy lguanidine, 4-pheny Ibenzoylguani dine, (5-Phenyl-penta-2,4-d enoyl)guanidine, N-(cinnamoyl)-N'pherylguanidine, 3-isopropylcinnamoyl guanidine hydrochloride, Benzamil hydrochloride, N-(3-phenylpropanoyl )-N'-pheny lguanidine, N,N'-bis(3phenylpropinoyl)-N"-phenylguanidtne, 3-(2-napthyl)acryloylguanidine, 5-(N-Methyl-N-isobu1yl)amiloride, 2'4 DichloroBenazamil HCl, 5-tert-butylamino-amiloride, 5-(N-Ethyl-N-isoprop/l)amiloride, (4-Methoxycinnamoyl)guanidine, 4-fluorocinnamoylguanidine, (3-Nitrocinnamoyl)guanidine, 4-ethoxycinnamoylguanidine, (4-Hydroxycinnamoyl)guanidine, (trans-2-Phenylcyclopropanecarbonyl)guanidine, 3-ethoxycinnamoylguanidine, 2,3,5,6,-tetramethylcinnamoylguanidine, 4-phenylcinnamoylguanidine, trans-3-Furanacryoy guanidine, N-(6-Hydroxy-2-napthoyl)-N'-phenylguanidine, (2-Furanacryloyl)guanidine, 3-(cyclohex-l-en-1-yl)cinnamoylguanidine, cinnamoylguanidine hydrochloride, 5-(N,N-hexamethylene)amiloride, 2,3-difluorocinnamoylguanidine, 2-( 1 -napthyl)acetoylguanidine, (a-Methylcinnamoyl guanidine. (2-Nitrocinnamoyl)guanidine, 6-lodoamiloride, 3,4-(methylenedioxy)cinnamoy lguanidine, 2-ethoxycinnamoylg janidine, Cinnamoylguanidine, 2-phenylcinnamoylguanidine, 2-(cyclohex-1-en-ly)cinnamoy lguanidine, 2-napthoylguanidine 3-phenylcinnamoylguanidine, 5-(N,N-Dimethyl)arr iloride hydrochloride, 5-(4-fluorophenyl)amiloride. (3-Methoxycinnamoyl)guanidine, 2-fluorocinnamoy lguanidine. 5-(3'-bromophenyl)penta-2,4-dienoylguanidine, [(4-Chlorophenoxy-acetyl]guanidine, (3-phenylpropanoyl)guanidine, 2-chloro-6-fluorocinnamoylguanidine, 3-fluorocinnamoylguanidine. 2-methylcinnamoylguanidine, (2-Methoxycinnamoyl)guanidine, 1 -bromo-2-napthoylg uanidine. 3,4,5-trimethoxycinnamoylguanidine, 3-methylcinnamoylg janidine, 3-(trans-hept-l-en-1-yl)cinnamoylguanidine, Phenamil methanesu fonate salt, 2,4-dichlorocinnamolyguanidine, (4-Nitrocinnamoyl)guanidine, 3,4-difluorocinnamoylguanidine and [(E)-3-(4-Dimethylarninophenyl)-2- methylacryloyl]guan dine. A compound as claimed in claim 1, wherein said compound is an antiviral for Equine Arteritis virus selected from the group consisting of 5-CN,N-hexarnethylene)arniloride, (3-Bromocinnamoyl)guanidine, trans-3-(l-napthyl)acryloylguanidlne, 2-t-butylcinnamoylguanidine and 2-(cyclohex-1 -en -1yl)cinnamoylguanidine. A compound as claimed in any one of claims 4 to 15, wherein said compound is provided as a pharmaceutical composition as claimed in claim 2 or claim 3. An antiviral compound selected from the group consisting of N-(3,5-Diamino-6-chloro-pyrazine-2-carbonyl)-N'-phenyl-guanidine, 5-(N-methyl-N-guanidinocarbonyl-methyl)amiloride, 5-(N-Metriyl-N-isobutyl)amiloride, 5-(N-Ethyl-N- sopropyl)amiloride, 5-(N,"N-Dimethyl)amiloride hydrochloride, 5-(N,N-hexamethylene)amiloride, 5-(N,N-Diethyl)amiloride hydrochloride, 3-hydroxy-5-hexamethyleneimino-amiloride, 5-(4-fluoropht nyl)amiloride, 5-tert-butylam ino-amiloride, N-amidino-3-amino-5-phenyl-6-chloro-2-pyrazinecarboxamide, 3-methoxy-5-N,N-Hexamethylene)-amiloride, 3-methoxy-amiloride, hexamethyleneimino-6-phenyl-2-pyrazinecarboximide, N-amidino-3,5-diamino-6-phenyl-2-pyrazinecarboxamide, 1 -napthoylguanidine, 2-napthoylgumidine, N-(2-napthoyl)-N'-phenylguanidine, N,N'-bis(2-napthoyl)guanidine. N,N'-bis(1-napthoyl)guanidine. N,N'-bis(2-m pthoyl)-N"-phenylguanidine, 6-methoxy-2 naphthoylguanidine. 3-quinolinoylguanic ine, cinnamoylguanidine, 4-phenylbenzoylguanidine, N-(cinnamoyl)-N'phenylguanidine, (3-phenylpropanoyl )guanidine, N,N'-bis-(cinnamoyl)-N"-phenylguanidine, N-(3-phenylpropanoyl)-N'-phenylguanidine. N,"N'-bis(3phenylpropanoyl)-M"-phenylguanidine, trans-3-furanacryo) lguanidine, N-(6-Hydroxy-2-napthoyl)-N'-phenylguanidine, (4-Phenoxybenzoyl)guanidine, N,N'-Bis(amidino)napthalene-2,6-dicarboxamide, N"-Cinnamoyl-N,N'-diphenylguanidine, (Pheny lacetyl)guan idine, N,N'-Bis(3-phenylpropanoyl)guanidine, benzyoylguanidine (4-Chlorophenoxy-acetyl]guanidine, N-benzoyl-N'-cinn;imoylguanidine, [(E)-3-(4-DimethylaminopherLyl)-2-methylacryloyl]guanidine, (4-Chlorocinnamo; 'l)guanidine, (4-Bromocinnamoyl)guanidine, (4-Methoxycinnarroyl)guanicline, (5-Phenyl-penta-2,4-dienoyl)guanidine, (3-Bromocinnamoyl)guanidine, (3-Methoxycinnanioyl)guanidine, (3-Chlorocinnamoyl)guanidine, (2-Chlorocinnamoyl)guanidine, (2-Bromocinnamo yi)guanidine, (2-Methoxycinnamoyl)guanidine, (trans-2-Phenylcyc lopropaneearbonyl)guanidine, [3-(3-Pyridyl)acry oyl]guanidine, (4-Hydroxycinnamoyl)guanidine, (Quinoline-2-carbonyl)guanidine, or pharmaceutical y acceptable salts thereof. 18. A pharmaceutical c omposition comprising a compound as claimed in claim 17, and optionally one or more pharmaceutical acceptable carriers or derivatives. 19. The pharmaceutical composition as claimed in claim 18, comprising one or more known antiviral compounds. 20. The compound as claimed in claim 1, wherein said compound is an antiviral for Flavrirus including Dengue virus selected from the group consisting of cinnamoylguanidine, (2-chlorocinnamoyl)guanidine or trans - 3-( 1 -napthyl)acryloylguanidine. Dated this 20th day of January. 2006 The invention relates to compounds having antiviral activity and methods utilizing the compounds to treat viral infections.

Documents:

159-KOLNP-2006-(30-03-2012)-CORRESPONDENCE.pdf

159-KOLNP-2006-(30-03-2012)-FORM-27.pdf

159-KOLNP-2006-FORM 27.pdf

159-kolnp-2006-granted-abstract.pdf

159-kolnp-2006-granted-claims.pdf

159-kolnp-2006-granted-correspondence.pdf

159-kolnp-2006-granted-description (complete).pdf

159-kolnp-2006-granted-drawings.pdf

159-kolnp-2006-granted-examination report.pdf

159-kolnp-2006-granted-form 1.pdf

159-kolnp-2006-granted-form 18.pdf

159-kolnp-2006-granted-form 2.pdf

159-kolnp-2006-granted-form 3.pdf

159-kolnp-2006-granted-form 5.pdf

159-kolnp-2006-granted-pa.pdf

159-kolnp-2006-granted-reply to examination report.pdf

159-kolnp-2006-granted-specification.pdf