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by: Dr. Sister Schiller

Pharmacology BSC 597

Dr. Sister Schiller

GPA 3.58

Frank Miskevich

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Frank Miskevich
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This 8 page Class Notes was uploaded by Dr. Sister Schiller on Friday October 30, 2015. The Class Notes belongs to BSC 597 at Texas A&M University - Commerce taught by Frank Miskevich in Fall. Since its upload, it has received 30 views. For similar materials see /class/232396/bsc-597-texas-a-m-university-commerce in Biology at Texas A&M University - Commerce.


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Date Created: 10/30/15
DDT 39Volume 10 NumberS Mt Yossef Kliger E 72 Pinthas Rosen Tel Aviv 59512 Israel ermailkigerompugenoil REVIEWS From genome to antivirals SARS as a test tube Yossef Kliger ErezYLevanon and Doron Gerber The severe acute respiratory syndrome SARS epidemic brought into the spotlight the need for rapid development of effective antiviral drugs against newly emerging viruses Researchers have leveraged the 20year battle against AIDS into a variety of possible treatments for SARS Most prominently based solely on viral genome information silencers of viral genes viralenzyme blockers and viralentry inhibitors were suggested as potential therapeutic agents for SARS In particular inhibitors of viral entry comprising therapeutic peptides were based on the recently launched antiHIV drug enfuvirtide This could represent one of the most direct routes from genome sequencing to the discovery of antiviral drugs Severe acute respiratory syndrome SARS is a pul monary infection that has been identified in multiple outbreaks around the world emerging initially in Guangdong Province China in November 2002 The number of reported cases increased exponentially and reached 8422 which resulted in 916 deaths by August 2003 WHO website The syndrome is caused by a previously unknown virus 7 SARSassociated coronavirus SARSGOV 13 The global SARS out break has been contained mainly owing to strict patient isolation and aggressive containment of infected regions but the virus itself has potential to reappear This concern is supported by studies reporting a cyclic pattern for other humaninfective coronaviruses that attack mainly in the winter sometimes skipping years for example the related human coronavirus HCoV OC43 which breaks out every two to four years 4 To date there are 36 antiviral drugs half of which were developed in the past 15 years to treat a single virus HIVI Development of these antivi ral drugs gained from the advances in molecular and structural biology coupled with advances in medic inal chemistry and in the industrialization of the drug discovery process The SARS epidemic has l3597644604Srsee ontmatter 2005 ElsevlerLtd All rlgbts reserved PH 385976446014 emphasized the need to develop drugs against emerging viral infections quickly and demonstrates the usage of genomic technologies in antiviral research In the past two decades biology has become an informationdriven science as a result of the emergence of genomic technologies and the expan sion of the Internet that allows analysis of genomic databases at every researcher s desktop These ad vanced genomic technologies led to rapid sequencing of SARSGOV 56 and for the first time in history the sequencing of a genome of an infective agent preceded the understanding of its basic biology and etiology Armed with this genomic information research groups around the world suggested multi disciplinary approaches to attain antiSARS drugs In general physicians tried to relieve the symptoms mainly by modulating parts of the immune system while vaccinologists began the long process of developing a vaccine against the virus Molecular and structuralbiologists suggested ways to interfere with the viral life cycle and these are the focus of this review Figure 1 The strategy starts from virus identification goes through genome sequencing and hopefully ends with an antiviral drug Drug development remains a challenge but the acceleration 345 0332073 wwwdrugdisoverytod ayom E 8 gt x g 0 u 2 n u D x n II 2 E i U a c m E III n o lt I11 5 lt I o E lt REVIEWS DDTVoume10 Number 5 March 2005 Capping Inhibitors Polymerase Inhibitos Helicase Inhibitors 4 4 Protein Maturation g 1 Proteinase Inhibitors History lessons HIV and AIDS In the early 1980s a sudden increase in lifethreatening opportunistic infections and Kaposi s sarcoma considered rare until then was observed among homo sexuals and injecting drug users These infections were attributed to an acquired immunodeficiency syndrome AIDS About two years of extensive studies passed until the human immunodeficiency virus HIV was identified as the etiological agent 9 10 An additional two years passed before completing the sequencing of its genome 11 The huge efforts invested in developing the first drug to treat AIDS patients bore fruit in record speed when the FDA approved azidothymidine AZT in 1987 AZT a nucleoside analog inhibim the viral reverse transcriptase 7 an enzyme that is essential for HIV replication Unfortunately isolates from these patienm showed decreased sensitivity after six months of AZT administration The find ing that some of these isolates also ex hibited crossresistance to other nucleo side analogs 12 raised further concern AZT and other nucleoside reverse tran f scriptase inhibitors were still the sole treat ment available for almost a decade The next important milestones were the devel opment of inhibitors against the protease another essential viral enzyme and non nucleoside reverse transcriptase inhibitors first approved in 1995 and 1997 respec tively While the nucleoside reverse tran scriptase inhibitors were discovered by cell a J FIGURE1 The SARSCOV 39 39 39 39 39 39 cellular receptors Outside the cell blocking the interaction of SARSrCoV with the cellular receptor w prevent the virus from attaching to host cellstCorreceptor antagonists will prevent the initiation of the next step 2 Membrane fusion ofthe virion with the host Fusion inhibitors will block merging ofthe viral mbrane with the host cell membra net3 Viral RNA processingtWithin the cetra nscription and multiplication oftheviral RNA can be blocked by polymerase and helicase inhibitorstTranslation oftheviral proteins might be inhibited by blocking mRNA cappingt 4 Protein maturationtViraI proteinswill not mature in the presence ofprotease inhibitors rendering them useless in drug discovery offered by genome technologies will hope fully enable significant timetable cuts in achieving antiviral medicine Other more classical antiviral strate gies used to treat SARS patients like the use of interferon will not be discussed here The interested reader is referred to 78 This review includes a retrospective summary of the development of antiHIV drugs followed by an appraisal of antiSARS strategies and their applicability for rapid development of antivirals against SARSCoV if it does resurface and against the next probably inevitable viral threat 346 wwwdrugdismverytodaytmm 1Virus binding to culture screening directed towards a spe cific class of chemical agent the nonnu cleoside reverse transcriptase inhibitors were discovered by HTS of large com pound libraries The discovery of HIV pro tease inhibitors represents one of the best examples of the application of protein structural knowledge to rational drug de sign Since then AIDS patients have been treated with a cocktail of inhibitors against HIV reverse transcriptase and protease However despite the unprecedented successes in the therapy of HIV infection HIV continues to spread causing more than 14000 new infections every day 95 of these in the developing world WHO website A major problem with current AIDS treatment is the high frequency of HIV mutations resulting in drug resistance Thus efforts are being made to develop agents addressing yet untargeted steps in the HIV life cycle Viralinduced fusionbetween viral and host cell membranes was acknowledged as a DDTVoume 10 Number 5 March 2005 REVIEWS useful drug target with the approval of the HIV fusion inhibitor enfuvirtide Fuzeon in 2003 13 Unlike other HIV dnigs that are small molecules developed against viral enzymes enfuvirtide is a peptide that corresponds to a specific segment of the viral envelope protein Importantly this segment can be directly pinpointed by computational sequence analysis 1415 This strategy seems promising in developing antiviral therapeutic peptides to other viruses that possess type 1 viral fusion proteins eg measles virus and respiratory syncytial vinrs RSV which share some structural motifs with HIV Little is known about viralinduced membrane fusion of other viruses that do not share these motifs Entry inhibitors Vinises can be divided into two groups based on the com position of their outer surface a nonenveloped vinises are enclosed by a protein shell called a capsid b enveloped viruses are surrounded by a membrane stolen from their last host In order to infect host cells that is to inject their genetic material into the cell enveloped viruses need to overcome both viral and cellular mem brane barriers Viral entry of many enveloped viruses including SARSCoV involves two major steps First the virion binds to receptors localized on the surface of its host cell and second the viral membrane fuses with the host cell membrane SARSCoV spike glycoprotein respon sible for these two steps is translated as a large polypep tide that is subsequently cleaved to produce two func tional subunits S1 and S2 S1 is the peripheral protein which binds to cellular receptors whereas S2 is a type I transmembrane protein that catalyzes the membrane fusion reaction Both steps are crucial for viral infection and there fore were suggested as targets for antivirals Blocking the interaction between SARSCoV and its cellular receptors Since the identification of CD4 as the cellular receptor for HIV in 1984 1617 several therapeutic agents aiming to inhibit the binding of HIV to CD4 were suggested Unfortunately these efforts have yet to bear fruit Major difficulties that slow down the development of inhibitors for the binding of CD4 to gp120 include i the gp120 binding site for CD4 consists largely of a recessed pocket ii antibodies that bind to CD4 antigen are likely to block virus attachment but can be immunosuppressive because they will lead to depletion of CD4 cells Currently both a recombinant CD4IgG2 fusion protein PRO542 and a smallmolecule EMS488043 aiming to prevent HIV from attaching to CD4 are in clinical trials These efforts reflect the motivation of inhibiting the first step in the viral life cycle that is the binding of the virus to its host cell This approach was strengthened when CXCR4 and CCR5 were identified as additional essential cellular receptors for HIV 1819 and with the discovery that CCRS deficient people are resistant to infection by HIV 20 Similar to HIV binding of the viral spike glycoprotein to some receptors on host cells is the first step in SARS CoV infection Blocking the interaction between these receptors and the virus could prevent infection thus inspiring the search for SARSCoV cellular receptors Recently human angiotensin converting enzymerelated carboxypeptidase ACE2 a type I integral membrane metalloprotease was identified as a receptor for SARSCoV 21 A soluble form of ACE2 and an antibody recognizing SARSCoV S1 efficiently neutralized SARSCoV in Vitm supporting the speculation that the ACE2binding site of the spike glycoprotein is an attractive target for vaccine and drug development 2122 This is further supported by an ACE2 inhibitor which also inhibits SARSCoV infection in Vina 23 Notably a 193amino acid fragment of SARSCoV S1 which efficiently bound ACE2 blocked spike glycoproteinmediated infection with an ICSO of less than 10 nM 24 More recently a human lung cDNA library was screened to identify receptors for SARSCoV revealing that human CD209L can also mediate infection by SARSCoV although it is a much less efficient receptor than ACE2 Interestingly CD209L is expressed in human lung in type II alveolar cells which are an important target for SARSCoV infection 25 It is still not known whether interactions between ACE2 and CD209L play a role in SARSCoV infection and pathogenesis Fusion inhibitors HIV entry involves the binding of the viral envelope glycoproteins comprising gp120 and gp41 which are the homologous of SARSCoV S1 and S2 respectively to CD4 on the host cell plasma membrane This induces confor mational changes enabling the Nterminal heptad repeat region NHR of gp41 to be exposed At this stage enfu virtide binds to the NHR of gp41 hence blocking further conformational changes required for membrane fusion Enfuvirtide is a synthetic peptide inhibitor corresponding to a segment of gp41 known as the Cterminal heptad repeat C HR Following the CD4induced conformational change of gp41 plasma membrane CCRS or CXCR4 molecules are recruited to the binding site and bind to the CD47envelope complexes This triggers a highly stable interaction between the CHR and the NHR regions of gp41 which drives the membrane fusion reaction to completion Thus enfuvirtide can no longer inhibit the fusion process 26 Slower engagement of the coreceptor with the CD47envelope complexes results in a stronger inhibition by CHRderived peptides 2728 Furthermore reduction in CCRS binding efficiency resulted in slower fusion kinetics and increased sensitivity to enfuvirtide 2829 Further support for this model is provided by the finding that CCR5 and CXCR4 antagonists showed strong antiHIV synergy with enfuvirtide against CCRSdependent and CXCR4dependent HIV isolates respectively 3031 In addition PRO542 acts in concert with enfuvirtide in virusecell and cellecell fusion assay by triggering formation wwwxlrugdisoverytoday0m 347 J 2 3539 E a vac g g g A 39lt a g 39lt REVIEWS 348 DDTVoume 10 Number 5 March 2005 a b C E d R Q Y Q S R L L L l I D A S L s K E N D R V L S A R s V E L Q N N T A s Q L V K N A L T s T Q l SARSCoV R 11511185 SARSCoV K NHR 9131000 E E E S N I T E V D Q A D Q S N L K T Q L K N G G s L I N N Q V Q D N A L Q s V L G s F I T L L I I E L v R v D Q T L T Q V A A L Drug DIscuvery Today FIGURE 2 Similarity between the fusion proteins of HIV1 and SARSCoV Schematic illustration ofaHIV71 gp41 and b the equivalent 52 protein from the SARSrCoVIA Leucinelsoleucine heptad repeat adjacent to the Nrterminus of both proteins appears in redIThe CVHR is in greenICysteine residues purple con ning a loop structure are located between thetwo heptad repeatslAn aromatic residuesrrich motifis marked blueand the 39 g isin orange Ap p 394 quot to the CVHR M h inhibitorofHIVrl entryintothe ceappearsin yellowlThe helical wheel is a top view ofa single strand ofa coiled coil In the wheel projection ofthe NVHR c and CVHR cl ofSARSrCoV 52 protein each ofthe seven positions arg corresponds to the location ofan amino acid residuethat makes up the coiled coillThe arrows betweenthe seven positions indicate the relative locations ofadjacent 39 39 min 394 L The helical h 39 39 39 a potential antiviral drug can be discovered based solely on sequence information of gp41 fusion intermediates enabling enquirtide to act on free HIV1 Virions 32 There are no peptide fusion inhibitors for influenza Virus It is noteworthy that in uenza Virus uses a different mechanism to enter its host cells it is first endocytosed into the cell followed by a pHdependent fusion between the Viral and the endosome membranes Strikingly it takes only few milliseconds from the time the pH drops in the endosomes until the fusion process is completed 33736 In contrast the time scale of HIV infection is about 20 minutes allowing ample time for binding of entry inhibitors 262737 SARSCoV entry kinetics resembles that of HIV At 5 minutes after exposure the SARSCoV lined the plasma membrane of Vero cells 38 Fusion and entry of the Viral load into the cytoplasm was observed mainly between 15 and 20 minutes 38 The timescale similarity between HIV and SARS CoV fusion process as opposed to the fast membrane fusion of in uenza Virus indicates that entry inhibitors could be successful with SARS CoV Despite the lack of sequence wwwdrugd ismverytadaylcom DDTVolume 10 Number 5 March 2005 REVIEWS homology and the difference in length between SARSCoV 52 and HIV gp41 homologous regions of the NHR and C HR in SARSCoV 52 were identified immediately after the SARSCoV genome sequence was published Figure 2 Thus a similar strategy might be applied to inhibit the entry of SARSCoV 39 httpwwwvirologynetArticles sarssZmodelhtml Indeed preliminary reports revealed antiSARS activity for peptides corresponding to the C HR of SARSCoV 52 protein 40742 and indicated a mode of action similar to that of enfuvirtide 43746 The kinetic similarity of SARSCoV and HIV entries sug gests a synergism between SARSCoV spike glycoprotein inhibitors and agents that block some of its receptors The role of different cellular receptors in SARSCoV entry should be characterized to discover the receptors that trigger conformational changes and transform the spike protein into the stable fusogenic form Antagonists for these receptors could synergize with fusion inhibitors The synergy between SARS fusion inhibitors and ACEZ or CD209L antagonists has not yet been investigated The first step is to determine optimal fusion inhibitors lntriguingly whereas polar residues disrupt the heptad repeat in the C HR of HIV1 gp41 the CHR of SARSCoV 52 has a perfect leucineisoleucine heptad repeat Figure 2d This could explain why the exact sequence bound aries of the C HRderived peptides are crucial for efficient inhibition 41424447 as aggregation of the peptides in solution could abolish antiviral activity Interestingly two reports demonstrate that NHRderived peptides are also active 4041 while others found that only C HR derived peptides have antiSARS activity 4247 It is note worthy that the reason for the poor inhibitory activities of NHRderived peptides in other viruses is contributed to their tendency to aggregate in solution suggesting that similar to the C HRderived peptides the exact sequence boundaries of the NHRderived peptides are important The main advantage of fusion inhibitors is their immediate discovery as they are simply the corresponding fragments of a known protein However their drawbacks as therapeutic peptides are lack of oral bioavailability and high production costs Auspiciously SARS is a respiratory syndrome thus peptidic fusion inhibitors could be given by inhalation This approach was applied successfully in RSVinfected mice 48 SARSCoV enzymes as targets for antivirals To serve as dnig targets viral proteins should fulfill two criteria i they should be essential for the viral life cycle and ii they should exhibit low similarity to host proteins SARSCoV genome analysis was performed to predict its proteome 49 and three viral enzymes were suggested as targem for drug discovery the helicase the RNAdependent RNA polymerase and the main protease These enzymes are crucial for replication transcription translation and post translational polyprotein processing Box 1 Assay devel opment based on these three SARSCoV target enzymes BOX 1 SARSCoV enzymes as targets for antiviral agents Currently inhibitors target three enzymescrucial for SARSCoV life cycle Helicase Proteinfold recognition methods followed by a biochemical study suggested a dual use of SARSCoV helicase in both RNA synthesis and cap formation suggesting new avenues to treat the virus 6566 Screening ofa compound library by plaque reduction assay resulted in a helicase inhibitor at the low microM range In vitro assay confirmed SARSCoV helicase as a validated drug target 67 RNAdependent RNA polymerase Molecular modeling revealed that SARSCoV RNAdependent RNA polymerase does not contain a hydrophobic pocket for nonnucleoside inhibitorsThis is in contrast with the nonnucleoside inhibitors activity against HIV1 reverse transcriptase 68 Of the many nucleoside analogues screened SARSCoV most selective nucleoside analogue inhibitor is BetaDN4 hydroxycytidine albeit at low efficacy EC9O of6 microM by virus yield reduction assay 69 Ribavirina broadspectrum nucleoside analogue efficacious in the treatment of several viral infections was used in various countries against SARS CoVWhile ribavirin was promising in vitro 70 recent reports revealed that ribavirin did not appearto confer any benefit for patients with SARS 7172 Main protease Sequence similarity was found between the substratebinding sites of SARSCoV main protease and the main protease of related viruses Remarkablythe SARSCoV main protease cleaved the porcine coronavirus transmissible gastroenteritis coronavirus TGEV main protease substrate 73This rationalizes screening of known protease inhibitors for antiSARS activityThis approach was reinforced by the crystallization ofthe SARSCoV main protease together with a TGEV inhibitor 74 The findings revealed that homology modeling is often inadequate for the prediction ofthe mutual orientation ofdomains in multidomain proteins HowevertheTGEV based homology model also shows that a reasonable model ofa substratebinding site can serve to develop useful ideas for inhibitor design that can inspire medicinal chemists to start a synthesis program long before the 3D structure ofthe target enzyme is experimentally determined reviewed in 75 In parallel HTS of compound libraries identified inhibitors ofthe SARSCoV main protease in the low M range 677677There are a few studies demonstrating that inhibitors ofthe HIV protease can also inhibit SARSCoV albeit with much lower efficiency 5278 was initiated 5052 thus paving the way for high throughput in vitro screening approaches to identify candidate inhibitors in compound libraries Other approaches The traditional and in many cases the most costefficient way of dealing with viruses has been through vaccines The logic of vaccine development against SARSCoV emerges from the combination of several findings i reinfection with SARSCoV causes only mild illness ii SARS is fatal mainly to old people who have difficulty in producing good humoral and cellular immune responses and iii the case fatality ratio of SARS ranges from 050 depending on the age group affected with an overall wwwxlrugdiscoverytodayxom 349 REVIEWS DDTVolume 10 Number 5 March 2005 estimate of case fatality of 14715 WHO website Thus most infected individuals recover from SARS Furthermore the success of a vaccine against other mammalinfective coronaviruses is encouraging 5354 Modern antiviral vaccine development depends heavily on the viral genome The availability of the human genome together with the recent sequencing of the SARSCoV genome largely increases the probability of success of vaccine development The fulllength spike glycoprotein of SARS CoV expressed by vaccinia virus induces binding and neutralizing antibody and protectively immunizes mice against a subsequent infection with SARSCoV 55 In addition DNA vaccine encoding the spike glycoprotein of the SARSCoV induces Tcell and neutralizing antibody responses as well as protective immunity in a mouse model 5 6 The discovery of RNAi raises many hopes regarding antiviral strategies and carries the promise of a shortcut in the drug discovery process Usually target discovery is followed by exhaustive HTS andor structurebased screening of many thousands of compounds in the hope that some of them will efficiently bind to the target Theoretically with smallinterfering RNA siRNA as a drug the course from target to drug is much shorter Encouraging results in mice were obtained using an RNAi based therapy against hepatitis B virus HBV transfection with plasmids expressing short hairpin RNAs shRNAs homologous to HBV mRNAs effectively inhibited repli cation initiation in cultured cells and mice liver showing that such an approach could be useful in the treatment of viral diseases 5 7 Currently there are attempts to use siRNA as antiSARS drugs but they are still in preliminary in vitra stages 5860 The application of this relatively Discove App roval Identi cation ofinfective agent nI Sequencing viral genome Crystal structure of rst g viral protein Entry inhibitors invitra il ry of cellular receptor of rst antiviral drug I 0 5 10 15 Time from epidemiological recognition years Drug Discuvery Tuday FIGURE3 r 5kg 39 nine Iclmc h Fffarriua 39 39 39 quot39 39 39t hquot 39 quoti 39 4 g 39 ceieiaieuthe understanding ofviral diseases It is anticipated that these research achievements will also lead to faster drug discovew and development 350 wwwdrugd ismverytodayxnm new technology to therapeutics faces several safety and technical issues including delivery of the RNA molecule into the virusinfected cells and the activation of interferon system 6162 The challenges ahead SARSCoV reminds us that viral infections are a global threat It is vital that the scientific community acquire the ability to develop antiviral therapy promptly We can be encouraged by the remarkable speed with which the global community acted in a coordinated research effort to investigate SARSCoV Immediately after the last nucleotide of the SARS CoV genome was verified the sequence was distributed through the internet to the worldwide scientific community Among the genomic based approaches that followed inhibitors of the viral induced membrane fusion seem the most promising The mutation rate of SARSCoV is much slower than that of HIV1 and is among the lowest of RNA viruses 6364 However viral resistance will be an obstacle The solution could lie in the use of a dnig cocktail combining antiviral drugs with different modes of action eg pro tease and polymerase inhibitors to lower the chances for drugresistant vinises to arise In addition drug cocktails are beneficial when the optimal dose of a drug given as a monotherapy is toxic 7 then combining drugs with distinct modes of action in suboptimal doses might alleviate toxicity issues Moreover the recent advance ment in the understanding of HIV entry into its host cell revealed an opportunity for synergism based on the molecular mechanism of viral entry Drugs that inhibit the interaction between CCR5 and the CD47envelope complexes enhance the efficiency of HIV fusion inhibitors by elongating the exposure time of their target site The timescale similarity of the SARSCoV fusion process to that of HIV is encouraging Hopefully future identification and characterization of SARSCoV receptors will open a way for an efficient antiviral strategy by synergistically com bining viral entry inhibitors Within a few months scientism have managed to lever age the technological advances of the past 20 years of antiAIDS research into an unprecedented antiviral cam paign against SARS Figure 3 SARS served as a test tube for novel approaches developed following the AIDS epi demic The current SARS epidemic was finally contained yet quick development of antivirals is still high priority Today we are closer than ever to achieving therapeutic solutions for a viral epidemic shortly after viral outbreak Acknowledgements We thank L Rychlewski A Wool E Eisenberg N Rabbie A Toporik M Olshansky and SG Peisajovich for critical reading of the manuscript We are also grateful to artist R Lieber for capturing the central idea of the article with her illustration DDTVoume 10 Number 5 March 2005 References 1 Ksiazek TG et al 2003 A novel coronavirus associated with severe acute respiratory syndrome N 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67 Kao RY et al 2004 Identification of noVel smallmolecule inhibitors of seVere acute 68 Xu X et al 2003 Molecular model of SARS coronaVirus polymerase implications for biochemical functions and drug design Nucleic Acids Res 31 711777130 69 Barnard DL et al 2004 Inhibition of seVere A a A coronaVirus SARSCoV by calpain inhibitors and betaDN4hydroxycytidine Antivir Chem hemather 15 15722 70 Chen F et al 2004 In vitro susceptibility of 10 clinical isolates of SARS coronaVirus to selected antiViral compounds I Clin Virol 31 69775 71 Leong HN et al 2004 InVestigational use of ribaVirin in the treatment of seVere acute DDTVolume 10 Number 5 March 2005 respiratory syndrome Singapore 2003 Trop Med Int Health 9 9237927 72 Wang WK et al 2004 Temporal relationship of Viral load ribaVirin interleukin IL6 IL8 and clinical progression in patients with seVere acute respiratory syndrome Clin Infect Dis 39 107171075 73 Anand K et al 2003 CoronaVirus main proteinase 3CLpro structure basis for design of 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