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Antiviral Research (v.95, #2)

Prelim II: Editorial Board (pp. ii).

Inside Back Cover: Notes to Authors (pp. ibc).

No influence of antiretroviral therapy on the mutation rate of the HCV NS5B polymerase in HIV/HCV-coinfected patients by Federico Alejandro Di Lello; Juan Macias; Zulema Plaza; Silvia García-Rey; Vicente Soriano; Celia Cifuentes; Maria del Mar González; Manuel Parra-Sánchez; Pablo Labarga; Eva Recio; Eva Poveda; Juan Antonio Pineda (pp. 67-71).

► We assess the impact of HIV coinfection on mutation rate and dN/dS of HCV-NS5B. ► We assess the impact of antiretroviral treatment on mutation rate and dN/dS of HCV. ► Mutation rate of NS5B in HIV/HCV-coinfected patients is similar to HCV-monoinfected. ► The use of antiretroviral treatment does not affect the mutation rate of HCV-NS5B. ► The use of antiretroviral treatment does not affect the dN/dS of NS5B of HCV.To assess the impact of antiretroviral treatment (ART), including nucleoside analogues retrotranscriptase inhibitors (NRTIs), on the mutation rate of hepatitis C virus (HCV) NS5B polymerase and on the ratio of substitution at synonymous and nonsynonymous sites (dN/dS) this polymerase in HIV/HCV-coinfected patients.Sixty-one patients on defined ART were included in this study. The NS5B polymerase of HCV was sequenced at baseline and after at least two years of ART. The mutation rate and the dN/dS were calculated at both times.The NS5B gene from forty-nine (80.3%) patients including; 19 HCV-1a (38.8%), 13 HCV-1b (26.5%), 8 HCV-3a (16.3%) and 9 HCV-4d (18.4%), could be sequenced. Thirty-two (65.3%) patients received non-nucleoside analogues and 41 (83.7%) received protease inhibitor. The mean estimated substitution rates at baseline and at the end of follow-up were from 1.38 to 3.5×10⁻³substitution/site/year (s/s/y) and from 1.39 to 3.18×10⁻³s/s/y, respectively, varying according to HCV genotype. All HCV genotypes at baseline and the end time point had values of dN/dS <1. At the end of follow-up, most of sites experienced negative selection and positive selection occurred only in a few sites.The mutation rate of NS5B in HIV/HCV-coinfected patients is within the range previously reported in studies in HCV-monoinfected patients. Additionally, the use of ART, including NRTIs, in these patients does not affect neither mutation rate nor the dN/dS of the HCV NS5B protein, suggesting that its use would not generate new resistance mutants to the polymerase inhibitors of HCV.

Keywords: Drug resistance; BEAST; Hepatitis C variability

Longitudinal analysis of the 5′UTR, E2-PePHD and NS5A-PKRBD genomic regions of hepatitis C virus genotype 1a in association with the response to peginterferon and ribavirin therapy in HIV-coinfected patients by Federico Bolcic; Natalia Laufer; Carolina Torres; Lucila Cassino; Rita Reynoso; Jorge Quarleri (pp. 72-81).

► Longitudinal analysis of IFN non-responsiveness on HIV-HCV and HCV infection. ► The number amino acid mutation at NS5A-ISDR was higher when HIV coexists. ► IFN-based therapy did not affect diversity of HCV-1a 5′UTR, E2-PePHD, NS5A-PKRBD.The rate of non-response to pegylated interferon plus ribavirin (peg-IFN+RBV) in HCV/HIV coinfected patients is higher than in HCV-monoinfected patients. In this sense, the contribution of HCV genetic variability is unknown. The 5′ untranslated (5′UTR), the nonstructural 5A (NS5A) and the second envelope (E2) HCV genomic regions have been implicated to peg-IFN therapy response. The proteins appear to block interferon (IFN)-induced RNA-dependent protein kinase (PKR) and the 5′UTR may influence the viral lymphotropism.We examined comparatively the pretreatment HCV variability between HIV coinfected and HCV monoinfected patients as well as assessed longitudinally the impact of peg-IFN+RBV on HCV variability when HIV is co-present. For this purpose, 15 HIV coinfected and 20 HCV monoinfected patients were compared. They were peg-IFN+RBV non-responders and infected with HCV 1a.Irrespectively of the HIV-coexistence, at baseline the amino acid variation in the NS5A-related domains was significantly higher than in the E2-PePHD ( p<0.001). The number of amino acid variations (mean±SD) at the NS5A-ISDR domain was higher among HCV/HIV patients than HCV-monoinfected ones (1.80±0.77 vs. 0.95±1.05; p=0.009) but such difference was slightly lower when comparing NS5A-PKRBD sequences (2.47±1.13 vs. 1.60±1.57; p=0.06). No differences were found at the E2-PePHD (0±0 vs. 0.2±0.4). At intra-HIV coinfected patient level, only minor (HCV genetic analysis) or no (HCV substitution rate and quasispecies heterogeneity) changes were observed during therapy (basal, 24h, 4weeks, and 12weeks).Among HCV-1a/HIV coinfected and HCV-monoinfected peg-IFN+RBV non-responder patients, the HCV variability at the 5′UTR, E2-PePHD and NS5A-PKRBD/ISDR domains was mostly comparable exhibiting a low number of variations. Four well-defined amino acid substitutions in NS5A-ISDR domain appeared most frequently when HIV coexists. The interferon-based therapy did not exert any effect in the variation, selection or diversity in the above mentioned HCV regions that could influence clinical responsiveness to IFN therapy.

Keywords: HCV; Genotype 1a; HIV; Pegylated interferon; Ribavirin

Schmallenberg virus: A new Shamonda/Sathuperi-like virus on the rise in Europe by Mutien-Marie Garigliany; Calixte Bayrou; Déborah Kleijnen; Dominique Cassart; Sandra Jolly; Annick Linden; Daniel Desmecht (pp. 82-87).

► In the summer-fall of 2011, a nonspecific febrile syndrome emerged in livestock in Europe. ► In the winter, an outbreak of arthrogryposis–porencephaly emerged in newborn kids, lambs and calves. ► Both syndromes are associated with a new bunyavirus, named Schmallenberg virus.In the summer-fall of 2011, a nonspecific febrile syndrome characterized by hyperthermia, drop in milk production and watery diarrhea was reported in adult dairy cows from a series of farms located in North-West Europe. Further, in November 2011, an enzootic outbreak of abortion, stillbirth and birth at term of lambs, kids and calves with neurologic signs and/or head, spine or limb malformations emerged throughout several European countries. Both syndromes were associated with the presence in the blood (adults) or in the central nervous system (newborns) of the genome of a new Shamonda-Sathuperi reassortant orthobunyavirus provisionally named Schmallenberg virus after the place where the first positive samples were collected. The clinical, pathological, virological and epidemiological facts that were made publicly available during the first 6months after the emergence are presented here. Current knowledge of the epidemiology of the phylogenetically closest relatives of the newcomer (Shamonda, Sathuperi, Aino and Akabane viruses) is not exhaustive enough to predict whether the current outbreak of Schmallenberg virus is the prelude to endemicity or to a 2years long outbreak before the infection burns out when serologically naïve animals are no longer available. In the future, cyclic epizootic reemergences are a possibility too, either synchronized with a global decrease of herd immunity or due to antigenic variants escaping the immunity acquired against their predecessors. The latter hypothesis seems unlikely because of the wide array of biologic constraints acting on the genome of viruses whose life cycle requires transmission by a vector, which represses genetic drift. The remarkable stability of the Shamonda virus genome over the last forty years is reassuring in this regard.

Keywords: Orthobunyavirus; Emergence; Arthrogryposis; Hydranencephaly; Schmallenberg

Effects on maribavir susceptibility of cytomegalovirus UL97 kinase ATP binding region mutations detected after drug exposure in vitro and in vivo by Sunwen Chou; Morgan Hakki; Stephen Villano (pp. 88-92).

► CMV UL97 kinase ATP-binding p-loop is newly implicated in maribavir resistance. ► UL97 variants found in maribavir clinical trials conferred no maribavir resistance. ► UL27 and UL97 mutations combined to increase the level of maribavir resistance.Resistance to the experimental human cytomegalovirus (CMV) UL97 kinase inhibitor maribavir has been mapped to UL97 mutations at codons 353, 397, 409 and 411, in the kinase ATP-binding region, and to mutations in the UL27 gene. We studied the maribavir susceptibility phenotypes of additional UL97 mutations observed in vitro and in clinical trials, and the effect of simultaneous mutation in both UL97 and UL27. In vitro selection under maribavir identified a new locus of UL97 mutation within the conserved kinase p-loop (L337M), which conferred low grade maribavir resistance (3.5-fold increased EC50) without ganciclovir cross-resistance. During maribavir Phase III CMV prevention clinical trials, three previously unknown UL97 sequence variants were detected in plasma samples after 27–98days of drug exposure (I324V, S334G and S386L). These variants did not confer any drug resistance despite proximity to mutations that confer maribavir resistance. The UL27 resistance mutation R233S, when added to strains containing UL97 mutations L337M or V353A, doubled their maribavir EC50s. These results expand the range of UL97 maribavir-resistance mutations into another part of the kinase ATP-binding region, but offer no genotypic evidence that development of drug resistance affected the outcomes of Phase III maribavir clinical trials after drug exposure of up to 14weeks. There is a potential for increased maribavir resistance in UL27–UL97 double mutants.

Keywords: Cytomegalovirus; Maribavir; UL97; Kinase; UL27; Antiviral drug resistance

Understanding the molecular mechanism of sequence dependent Tenofovir removal by HIV-1 reverse transcriptase: Differences in primer binding site versus polypurine tract by Pinar Iyidogan; Karen S. Anderson (pp. 93-103).

► HIV-1 RT shows distinct sequence specificity difference during Tenofovir removal. ► The sequence dependent helical structure of DNA modulates removal efficiency. ► We show further mechanistic insight on the synergistic effects of Atripla components.Tenofovir (TFV) is a nucleotide reverse transcriptase inhibitor (NtRTI) that is often administered as first-line therapy against human immunodeficiency virus type-1 (HIV-1) infection and acts as a chain terminator when incorporated into viral DNA. However, HIV-1 reverse transcriptase (RT) excises TFV in the presence of either ATP or pyrophosphate, which is an important drug resistance mechanism that would interfere with the effective treatment. Previous studies have shown conflicting results on excision efficiencies for TFV-terminated primer-templates derived from either primer binding site (PBS) or polypurine tract (PPT) sequences. To provide mechanistic insight into the variation in TFV removal from both sequences that are vital for the HIV-1 life cycle, we compared the efficiencies of removal reaction in response to sequence dependence via utilizing blocked PBS and PPT primer-templates. We found an enhanced TFV excision with PPT sequence over PBS sequence through ATP-mediated removal and a subsequent incorporation of ATP into the unblocked primers. Furthermore, the rate of pyrophosphorolytic excision of TFV from PPT sequence was 21-fold higher than that for the PBS sequence. However, the addition of efavirenz, nonnucleoside reverse transcriptase inhibitor (NNRTI), to the removal reaction effectively inhibits the TFV excision from both primers by forming a stable complex that would leave TFV inaccessible for excision. These results illuminate the degree of primer-template sequence contribution on TFV removal as well as increase our understanding of the molecular mechanism for the beneficial effects of widely used combinations of antiretroviral regimens in the context of synergistic antiviral activity and drug resistance.

Keywords: Abbreviations; HIV-1; human immunodeficiency virus type 1; WT RT; wild type reverse transcriptase; N[t]RTI; nucleoside [nucleotide] reverse transcriptase inhibitor; TFV; tenofovir; NNRTI; nonnucleoside reverse transcriptase inhibitor; EFV; efavirenz; PPT; polypurine tract; PBS; primer binding site; dNTP; deoxynucleoside triphosphate; ATP; adenosine triphosphate; PP; _i; inorganic pyrophosphateHuman immunodeficiency virus type 1; Reverse transcriptase; Antiretroviral agents; Nucleoside reverse transcriptase inhibitor; Tenofovir

Dobrava-Belgrade virus: Phylogeny, epidemiology, disease by Anna Papa (pp. 104-117).

► Dobrava-Belgrade virus (DOBV) is the principal cause of hemorrhagic fever with renal syndrome in southeastern Europe. ► The epidemiology of DOBV and phylogenetic relationships among DOBV strains are described. ► Different DOBV genotypes present different levels of pathogenicity for humans. ► Approaches used for drug development and vaccine design are summarized.Dobrava-Belgrade virus (DOBV) is an Old World hantavirus that causes hemorrhagic fever with renal syndrome in humans. With a case fatality rate up to 12%, DOBV infection is the most life-threatening hantavirus disease in Europe. The virus was initially identified in the Balkans, but the discovery of new endemic foci have expanded its recognized geographic range. The recent description of novel genetic variants with different degrees of pathogenicity have complicated its taxonomic analysis. The original rodent host of DOBV is Apodemus flavicollis, however additional Apodemus species, such Apodemus agrarius and Apodemus ponticus, have been found to serve as hosts of the various DOBV genotypes. The complex evolution and genetic diversity of the virus are still under investigation. The present review aims to provide an update on the phylogeny of DOBV and the epidemiology of infection in rodents and humans; to describe the clinical characteristics of the disease; to present current knowledge about laboratory diagnosis, treatment and prevention; discuss the current state of the art in antiviral drug and vaccine development.

Keywords: Dobrava-Belgrade virus; Hantavirus; Bunyavirus; Hemorrhagic fever with renal syndrome; Zoonosis

Identification of substituted [3, 2-a] pyrimidines as selective antiviral agents: Molecular modeling study by Kilaru Ravendra Babu; Valasani Koteswara Rao; Yellapu Nanda Kumar; Kishore Polireddy; Kadiam Venkata Subbaiah; Matcha Bhaskar; Valluru Lokanatha; Chamarthi Naga Raju (pp. 118-127).

► Biological activity of thiazolo [3, 2-a] pyrimidine derivatives was studied. ► The molecular descriptors of4b, 5b and6b explained their drug like properties.► 4b, 5b and6b showed best docking scores in silico with HN viral envelop protein.► 4b, 5b and6b showed antiviral activity against NDV virus infected chicks in vivo. ► Both in silico, in vivo assays ensures4b, 5b and6b as potential antiviral drugs.A series of novel substituted dihydropyrimidine and 5H-thiazolo [3, 2-a] pyrimidine derivatives were designed and synthesized as a potential target to discover drugs fighting against the viral diseases. The main objective of the present work is to carry out the QSAR studies for all the series of the compounds starting from4a to6j to find out their molecular descriptors and predict the biological properties. All of them are showing the best QSAR descriptors, hence chosen for the prediction of anti-viral activity against Newcastle disease virus (NDV). Initially their inhibitory activity was predicted by molecular docking of these compounds against haemaglutinin–neuraminidase (HN) protein using molecular operating environment (MOE) software. Based on the best affinity and highest docking scores4b, 5b and6b were assayed in vivo on NDV infected chicks and it was found that there is significant improvement in the survival of the chicks with the treatment ( P<0.05).4b and6b showed better curative effect than5b at the dose concentration of 40mg/kg body weight of chicks. The results from molecular docking study and biological assays can be inferred to consider these molecules as potential antiviral drugs.

Keywords: Antiviral inhibitors; Newcastle disease virus; Dihydropyrimidine; Haemaglutinin–neuraminidase; Molecular modeling

Prevention of human enterovirus 71 infection by kappa carrageenan by Ya-Huang Chiu; Yi-Lin Chan; Li-Wen Tsai; Tsung-Lin Li; Chang-Jer Wu (pp. 128-134).

► Pre-treatment of carrageenan can inhibit EV 71 replication. ► Carrageenan inhibits EV 71 infection by blocking virus from entering into cells. ► Carrageenan can associate with EV 71 to form a complex. ► Carrageenan inhibits EV 71 induced cytopathic effects and apoptosis in Vero cells.Enterovirus 71 (EV 71), the newest member of Enteroviridae, is notorious for its etiological role in epidemics of the hand-foot-and-mouth disease, particularly in association with fatal neurological complications in young children. Searching for new and more effective agents against EV 71 infections has never relented as corresponding vaccines or antiviral drugs remain unavailable. Sulfated polysaccharides from seaweed are known to possess a broad range of biological activities across anti-virus, anti-tumor, immunomodulation, anti-coagulation, etc. In this study, we report kappa carrageenan also has a strong and effective anti-EV 71 activity able to reduce plaque formation, prevent viral replication before or during viral adsorption, as well as inhibit EV 71-induced apoptosis. In virus binding assay, kappa carrageenan was shown able to bind EV 71 firmly, forming carrageenan-viruses complexes, whereby the virus-receptor interaction is likely disrupted. Added together, kappa carrageenan may be an ideal candidate worthwhile to develop into anti-EV 71 agents.

Keywords: Enterovirus 71; Sulphated polysaccharide; Kappa carrageenan; Antiviral agent; Adsorption inhibitor

Recent progress in henipavirus research: Molecular biology, genetic diversity, animal models by Barry Rockx; Richard Winegar; Alexander N. Freiberg (pp. 135-149).

► Henipaviruses are emerging zoonotic paramyxoviruses and potential bioterrorism agents. ► Nipah and Hendra viruses cause fatal encephalitis and/or respiratory disease. ► Fruit bats are the natural reservoir and recent studies suggest that the henipaviruses originate from Africa. ► Data on mutation rates and genetic diversity are summarized and discussed.Nipah and Hendra virus are members of a newly identified genus of emerging paramyxoviruses, the henipaviruses. Both viruses have the ability to cause severe pulmonary infection and severe acute encephalitis. Following their discovery in the 1990s, outbreaks caused by these zoonotic paramyxoviruses have been associated with high public health and especially economic threat potential. Currently, only geographic groupings in Asia and Australia have been described for the henipaviruses. However, while few viral isolates are available and more detailed characterization is necessary, there has been recent evidence that divergent henipaviruses might be present on the African continent. This review endeavours to capture recent advances in the field of henipavirus research, with a focus on genome structure and replication mechanisms, reservoir hosts, genetic diversity, pathogenesis and animal models.

Keywords: Henipavirus; Nipah virus; Hendra virus; Animal models

Down-regulation of viral replication by lentiviral-mediated expression of short-hairpin RNAs against vesicular stomatitis virus ribonuclear complex genes by Lisbeth Ramirez-Carvajal; Charles R. Long (pp. 150-158).

► Transgenic cells stably expressed shRNAs that diminished VSV target RNA. ► Diminished VSV target mRNA does not always reduce virus production. ► Silencing of ribonuclear complex genes resulted in variable VSV inhibition. ► Only shRNAs targeting the N-gene reduced virus yield after VSV infection. ► RNAi-based antiviral effects can be elicited from stably integrated transgenes.Vesicular stomatitis virus (VSV) causes great economic impact to livestock industry and is a prototype for studying non-segmented negative-stranded RNA (NSNR) viruses. In this study, we evaluated the antiviral potential of unique short-hairpin RNA (shRNA) targeting genes that form the ribonuclear protein (RNP) complex of VSV serotype Indiana (VSIV). We used lentiviral vectors to construct cell lines that stably expressed one of seven shRNAs targeting the RNP genes of VSIV, namely nucleocapsid (N), phosphoprotein (P), or polymerase (L). We reported two N-shRNA sequences targeting the 5′ or 3′ end of N that significantly reduced N, P, and L viral transcripts ( p<0.001), reduced viral protein expression, and reduced the viral particles shed in Vero cells ( p <0.01). When we analyzed the sequence diversity in the target region of this N-shRNA from two field isolates, we detected a single base substitution outside the seed region. We also reported five other shRNA sequences targeting components of the viral RNA that significantly reduce N, P, and L viral transcripts ( p<0.001) but failed to efficiently impair viral replication. The differences in the efficiency of the shRNAs tested were not due to mismatches within the target region in the genome of VSIV. Although partial silencing of viral transcripts by single shRNAs impaired but did not block VSIV replication, the combination of the shRNAs identified here into a multiple shRNA vector may result in inhibition of viral replication. These data contribute to ongoing development of RNAi-based technologies to combat viral diseases.

Keywords: VSV; shRNA; Lentivirus; Nucleocapsid; RNAi; Ribonuclear complex

SKI-1/S1P inhibition: A promising surrogate to statins to block Hepatitis C virus replication by Matthieu Blanchet; Nabil G. Seidah; Patrick Labonté (pp. 159-166).

► Inhibition of the lipogenic pathways regulator SKI-1/S1P limits HCV propagation. ► HCV antiviral effect of SKI-1/S1P inhibitor is superior to the tested statins. ► SKI-1/S1P inhibition impairs HCV genome replication and assembly. ► SKI-1/S1P inhibition alters infectivity but not secretion of HCV particles.Hepatitis C virus (HCV) is often associated with steatosis, cirrhosis and hepatocellular carcinoma (HCC). Statins (HMG-CoAR inhibitors) have been shown to exert an antiviral effect in vitro, principally on replicon harboring cells, but the effect of their use alone in vivo remains controversial. In clinical trials, when used in combination with the standards of care (SOC), they led to an increased proportion of sustained virological responder (SVR). Here we investigated the implication of SKI-1/S1P, a master lipogenic pathways regulator upstream of HMG-CoAR, on different steps of HCV life cycle. We compared the HCV antiviral effect of the most potent SKI-1/S1P small molecule inhibitor (PF-429242) with a set of two statins on different steps of the viral life cycle, and showed that SKI-1/S1P inhibitor blocked HCVcc (strain JFH-1) RNA replication (EC₅₀= 5.8μM) more efficiently than statins. Moreover, we showed that PF-429242 could reduce lipid droplets accumulation in Huh7 cells. Interestingly, PF-429242 dramatically reduced infectious particles production (EC₉₀= 4.8μM). Such inhibition could not be achieved with statins. SKI-1/S1P activity is thus essential for viral production and its inhibition should be considered for antiviral drug development.

Keywords: Abbreviations; HCV; hepatitis C virus; HCC; hepatocellular carcinoma; SOC; standards of care; SKI-1/S1P; subtilisin/kexin-isoenzyme-1/site-1 protease; SREBP; sterol regulatory-element binding protein; LD; lipid droplet; VLDL; very low density lipoprotein; PEG-IFN-α; pegylated interferon-αHCV; SKI-1/S1P; HMG-CoAR; FASn; Statins; Lipids metabolism

The European Virus Archive: A new resource for virology research by E.A. Gould; X. de Lamballerie; B. Coutard; A.R. Fooks; M. Outlaw; C. Drosten; S. Guenther; B. Klempa; D. Pinschewer; T. Avsic-Zupanc; C. Sabeta; A. Lukashev; M. Eropkin; A. Koslov; V. Zverev; D. Lvov; A. Zhebrun; G. Shipulin; M. Niedrig; G. Gao Fu; G. Dong Liang; G. Ippolito; E. Koray; J.L. Romette (pp. 167-171).

► EVA is a globally available virus collection serving academia, public health and industry. ► EVA is a unique, networked, quality-controlled non-profit virus archive that benefits science. ► EVA provides wide-ranging access to virus collections held in laboratories worldwide. ► Laboratories in developing countries contribute to the pool of quality-controlled reagents.The European Virus Archive (EVA) was conceived as a direct response to the need for a coordinated and readily accessible collection of viruses that could be made available to academia, public health organisations and industry, initially within Europe, but ultimately throughout the world. Although scientists worldwide have accumulated virus collections since the early twentieth century, the quality of the collections and the viruses collected may vary according to the personal interests and agenda of the scientists. Moreover, when laboratories are re-organised or closed, collections are no longer maintained and gradually cease to exist. The tragedy of 9/11 and other disruptive activities have also meant that some previously available biological reagents are no longer openly exchanged between countries. In 2008, funding under the FP7–EU infrastructure programme enabled the initiation of the EVA. Within three years, it has developed from a consortium of nine European laboratories to encompass associated partners in Africa, Russia, China, Turkey, Germany and Italy. There is every reason to believe that EVA will continue to expand and ultimately exist as a globally networked, quality-controlled non-profit archive for the benefit of science. Organizations or individuals who would like to be considered as contributors are invited to contact the EVA coordinator, Jean–Louis Romette, at jean-louis.romette@univmed.fr.

Keywords: Virus; Archive; Access; Reagents; Kits; Diagnosis; Storage; Freeze-drying

Prophylactic and therapeutic combination effects of rimantadine and oseltamivir against influenza virus A (H3N2) infection in mice by Lora Simeonova; Galina Gegova; Angel S. Galabov (pp. 172-181).

► We studied the combination rimantadine+oseltamivir on influenza infection in mice. ► Treatment was applied prophylactically or therapeutically against H3N2 virus type. ► Survival of animals, lung viral titers and pneumonia parameters were evaluated. ► Combination of oseltamivir+rimantadine is protective against influenza virus A (H3N2).The combined effect of rimantadine and oseltamivir in a prophylactic context (therapy beginning 4h pre-virus infection) and therapeutic context (therapy started at 24h post-viral inoculation) course on influenza H3N2 virus infection in mice was studied. In the prophylactic course 5 and 10mg/kg/day rimantadine with 0.2 and 0.4mg/kg/day (25:1 dose ratio) oseltamivir showed a protection index (PI) of 79.6% and 75%, respectively and a mean survival time (MST) of 13.1 and 12.9days. The individual effects of the same doses ranged from 0% to 33.3% PI and 8.2 to 10.3days MST, respectively. Lung virus titers were decreased 630-fold in the combination-treated groups as compared to monotherapy and placebo groups. The reduction of surface lung pathology in combination-treated groups demonstrated a protective effect for the combination of both antivirals. In the therapeutic course 5 and 10mg/kg rimantadine combined with 0.2 and 0.4mg/kg oseltamivir showed no beneficial effect. At higher dosage (0.8, 1.6, 3.2mg/kg oseltamivir and 20, 40, 80mg/kg rimantadine) preserving the 25:1 ratio, the resultant PI ranged from 57.6% to 80.5% and the MST was 12.8–13.4days. Used alone at the same doses the compounds’ protection varied between 10.7% and 71.8% PI, MST 9.8–12.8days (8.7days in PBS control). Compared to vehicle and individual treatment, a decrease in infectious viral titers of up to 1000-fold and other viral pneumonia parameters were also recorded. The therapeutic effect of the drugs’ optimal effective doses combinations was characterized as synergistic. Survival of animals was 81.2–100% and MST was extended by 5–7days compared to placebos. Monotherapy protection was from 9.1% to a maximum of 56.5%, MST being prolonged only by 1.3–4.2days compared to 7.5days in the PBS control group. Lung viral titers were decreased 1445-fold for the most efficacious combination groups and a significant reduction in lung parameters was observed. These data emphasize that prophylactic and therapeutic courses using a combination of oseltamivir and rimantadine have a significant protective effect in mice experimentally infected with drug-sensitive influenza virus A (H3N2).

Keywords: Influenza; Rimantadine; Oseltamivir; Combination therapy; Mice

Discovery of substituted N-phenylbenzenesulphonamides as a novel class of non-nucleoside hepatitis C virus polymerase inhibitors by Marina M. May; Dirk Brohm; Axel Harrenga; Tobias Marquardt; Bernd Riedl; Joerg Kreuter; Holger Zimmermann; Helga Ruebsamen-Schaeff; Andreas Urban (pp. 182-191).

► SPBSs could be identified as novel non-nucleoside inhibitors of the HCV RNA polymerase. ► SPBSs reversibly bind to the allosteric thumb site II of NS5B. ► SPBSs showed both overlapping and nonoverlapping cross-resistance to known NNI-2 inhibitors. ► SPBSs efficiently inhibit genotype 1a and 1b NS5Bs, but failed to inhibit non-genotype 1 NS5Bs. ► SPBSs showed antiviral activity in the HCV replicon system with an EC₅₀ about 1μM.The RNA-dependent RNA polymerase NS5B of the hepatitis C virus (HCV) has emerged as one of the key targets for antiviral drug discovery. Here we describe a novel non-nucleoside inhibitor (NNI) chemotype identified by screening: The substituted N-phenylbenzenesulphonamides (SPBS) which showed reversible inhibition of NS5B from HCV genotype 1b with IC₅₀ values up to 40nM. Based on the decreased inhibitory activity against a recombinant NS5B protein carrying the mutation L419M or M423T we assumed that the SPBS inhibitors bind to the thumb site II which has already been described as the allosteric binding site for the NNI carboxy thiophene. The postulated binding site was consequently confirmed by solving two co-crystal structures of NS5B in complex with SPBS analogues at 2.3 and 2.2Å resolutions. The inhibitors are hydrogen-bonded to the main chain Ser476 and Tyr477 and to the side chain of Arg501. In addition, the inhibitors displayed van der Waals interactions with several residues of the hydrophobic binding pocket Leu419, Ile482, Leu497, Met423 and Trp528. Notably, the two SPBS analogues reported here revealed significant differences in addressing the NH-group of the main chain Tyr477 by hydrogen-bonds, water-mediated or directly, which provoked a shift of the carboxyphenyl group of the inhibitors towards the His475 position for the water-mediated binding mode. Interestingly, the differences observed in the binding mode led to a different cross resistance profile at positions M423 and I482. Using a panel of 38 individual NS5B proteins derived from different HCV genotypes, we could demonstrate inhibitory activity of the SPBS against polymerases from HCV genotypes 1a and 1b whereas the inhibitor class failed to inhibit any of the non-genotype 1 polymerases efficiently. Furthermore we demonstrated initial antiviral activity for SPBS against the subgenomic replicons of HCV genotypes 1a and 1b, respectively, and no considerable cytotoxic potential against a panel of ten different cell types.

Keywords: Hepatitis C virus; RNA polymerase; NS5B; Non-nucleoside polymerase inhibitors; Resistance; Genotype coverage

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