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Antiviral Research (v.96, #2)

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

Prelim II: Editorial Board (pp. ii).

Design and synthesis of pinanamine derivatives as anti-influenza A M2 ion channel inhibitors by Xin Zhao; Yanling Jie; Matthew R. Rosenberg; Junting Wan; Shaogao Zeng; Wei Cui; Yiping Xiao; Zhiyuan Li; Zhengchao Tu; Marco G. Casarotto; Wenhui Hu (pp. 91-99).

Display Omitted► A series of pinanamine derivatives were design and synthesized for M2 inhibitors. ► Several imidazole and guanazole substituted compounds were found to inhibit WT A/M2 as potent as amantadine. ► Compound12 was identified to be a novel inhibitor against both A/M2-WT and A/M2-S31N mutant channels.The adamantanes are a class of anti-influenza drugs that inhibit the M2 ion channel of the influenza A virus. However recently, the clinical effectiveness of these drugs has been called into question due to the emergence of adamantane-insensitive A/M2 mutants. Although we previously reported (1R,2R,3R,5S)-3-pinanamine3 as a novel inhibitor of the wild type influenza A virus M2 protein (WT A/M2), limited inhibition was found for adamantane-resistant M2 mutants. In this study, we explored whether newly synthesized pinanamine derivatives were capable of inhibiting WT A/M2 and selected adamantane-resistant M2 mutants. Several imidazole and guanazole derivatives of pinanamine were found to inhibit WT A/M2 to a comparable degree as amantadine and one of these compounds12 exhibits weak inhibition of A/M2-S31N mutant and it is marginally more effective in inhibiting S31NM2 than amantadine. This study provides a new insight into the structural nature of drugs required to inhibit WT A/M2 and its mutants.

Keywords: M2 ion channel; Influenza A virus; Pinanamine derivatives

Comparison of rescue strategies in lamivudine-resistant patients with chronic hepatitis B by Pan Zhao; Chunya Wang; LiLi Huang; Dongping Xu; Tanshi Li (pp. 100-104).

► We simultaneously compare the efficacy among four rescue strategies for lamivudine-resistant chronic hepatitis B. ► Combination of adefovir and entecavir is effective in lamivudine-resistant patients. ► Switching to adefovir monotherapy is widely used in real-world clinical practice in China, in spite of its low efficacy. ► Switching to entecavir monotherapy is not optimal in lamivudine-resistant patients due to the high incidence of resistance.Lamivudine (LAM) resistance now poses a major problem in the management of patients with chronic hepatitis B virus (HBV) infection. We retrospectively collected clinical data on chronic HBV-infected patients who had developed LAM resistance under de novo LAM monotherapy and subsequently took nucleos(t)ide analogs as rescue strategy in our hospital. From initiation of rescue therapies to January 2012, incidence of antiviral drug resistance was 23.67%, 18%, 6.94% and 0% ( P=0.007) in the group of switching to adefovir dipivoxil (ADV) monotherapy, switching to entecavir (ETV) monotherapy, adding on ADV and switching to combination of ADV and ETV. At month 12, the median levels of serum HBV DNA were respectively 9300IU/mL, 4648IU/mL, 2054IU/mL and 100IU/mL ( P<0.001), and the cumulative rates of serum ALT normalization were respectively 75%, 84%, 93% and 100% ( P=0.003). Additionally, the strategy of switching to ADV monotherapy induced more single rtA181T mutations. In conclusion, switching to ADV monotherapy has been widely used in real-world clinical practice in China, however, due to the high incidence of drug resistance, switching to neither ADV nor ETV monotherapy is optimal when LAM resistance occurs; combination of ADV and ETV is most effective, whereas the strategy of adding on ADV is rational for most of LAM-resistant Chinese patients with chronic hepatitis B.

Keywords: Abbreviations; HBV; hepatitis B virus; LAM; lamivudine; ADV; adefovir dipivoxil; ETV; entecavir; CHB; chronic hepatitis B; HBeAg; hepatitis B e antigen; ALT; alanine aminotransferaseChronic hepatitis B; Lamivudine; Resistance; Adefovir dipivoxil; Entecavir

Significance of amino acid substitutions in the thymidine kinase gene of herpes simplex virus type 1 for resistance by Andreas Sauerbrei; Kristin Liermann; Kathrin Bohn; Andreas Henke; Roland Zell; Simon Gronowitz; Peter Wutzler (pp. 105-107).

► Report of methods to clarify significance of amino acid changes in HSV-1 TK gene. ► Recombinant proteins were synthesized after cloning and site-directed mutagenesis. ► A novel test for measuring phophorylation activity of HSV-1 TK was evaluated. ► Significance of seven amino acid substitutions for acyclovir resistance was clarified.The analysis of the viral thymidine kinase (TK) genotype is of rising significance for testing resistance of herpes simplex virus (HSV) to antivirals especially acyclovir. However, numerous of the described amino acid (aa) substitutions are diagnostically less conclusive because of the pronounced natural polymorphism of this gene. In this study, several aa substitutions in the TK sequence of HSV-1 with unclear significance for resistance were analyzed by expression of recombinant TK proteins and determination of enzymatic activity on the basis of an enzyme linked immunosorbent assay using bromodeoxyuridine (BrdU) as TK substrate. The recombinant TK wild-type protein resulted in high TK activity and TK mutant with stop of translation showed negative results. The recombinant TK proteins containing the aa substitutions R41H or V348I had high phosphorylation activities suggesting most likely natural gene polymorphisms. By contrast, the aa changes Y53H, L139V, R163H, L298A and L315S were accompanied by negative or weakly positive TK activities indicating resistance association. In conclusion, the combination of methods described here represents a useful tool to evaluate the significance of aa substitutions for resistance of clinical HSV-1 strains.

Keywords: HSV-1; Resistance; Thymidine kinase; Site-directed mutagenesis

Hepatitis B virus basal core promoter mutations A1762T/G1764A are associated with genotype C and a low serum HBsAg level in chronically-infected HBeAg-positive Chinese patients by Chun-Hui Yan; Cheng-Yu Zhao; Hai Ding; Ya-Qin Peng; Peng-Yuan Jin; Ling Yan; Hui Zhuang; Tong Li (pp. 108-114).

► A1762T/G1764A and G1896A mutations of HBV were analyzed among 192 chronically-infected HBeAg-positive Chinese patients. ► Mutations A1762T/G1764A rather than G1896A were more prevalent in genotype C than in genotype B. ► HBV subpopulations accounted for the most of the mutations. ► A1762T/G1764A were associated with a low HBsAg level.The present study was aimed to obtain baseline information of basal core promoter A1762T/G1764A and precore G1896A mutations of hepatitis B virus (HBV) in 192 HBeAg-positive chronically-infected Chinese patients, who were potential candidates for antiviral treatment. The detection of these mutations (including minor mutant subpopulations) was achieved by direct sequencing, whose sensitivity for minor mutant subpopulations identification was confirmed by clone sequencing. Patients enrolled were infected with either genotype B (46.35%) or C (53.65%) HBV identified by routine tests in our laboratory. The A1762T/G1764A or G1896A mutations were detected in 125specimens (125/192, 65.10%), in which 77 (77/125, 61.60%) existed as subpopulations. The A1762T/G1764A mutations were found to be more prevalent in genotype C than that in genotype B HBV [62.14% (64/103) vs. 20.22% (18/89), P<0.0001]. There is no statistically significant link between G1896A and genotypes. The emergence of A1762T/G1764A mutations was also found to be associated with an older age, an elevated ALT/AST level, and a lower HBsAg level in serum [wild-type vs. mutant: 4.57 (3.46–5.42) vs. 3.93 (2.51–5.36), P<0.0001]. In conclusion, HBV basal core promoter mutations A1762T/G1764A are associated with genotype C and a low serum HBsAg level in chronically-infected HBeAg-positive Chinese patients.

Keywords: Abbreviations; HBV; hepatitis B virus; BCP; basal core promoter; preC; precore; nt; nucleotide; G; guanine; A; adenine; T; thymine; HBeAg; hepatitis B e antigen; HCC; hepatocellular carcinoma; NA; nucleos(t)ide analogues; HBsAg; hepatitis B surface antigen; ALT; alanine aminotransferase; AST; aspartate aminotransferase; CMIA; chemiluminescent microparticle immunoassay; nPCR; nested polymerase chain reaction; bp; base pairs; W; A/T; R; A/GHepatitis B virus; A1762T/G1764A; G1896A; HBeAg-positive; Mutation

Structural biology of dengue virus enzymes: Towards rational design of therapeutics by Christian G. Noble; Pei-Yong Shi (pp. 115-126).

► Review of the latest structural data for the dengue MTase, protease and polymerase. ► First MTase structure with a flavivirus-specific compound. ► First crystal structure of the ‘active’ conformation of the dengue protease. ► First dengue polymerase structure bound to a small molecule.Development of anti-dengue therapy represents an urgent un-met medical need. Towards antiviral therapy, recent advances in crystal structures of DENV enzymes have led to the possibility of structure-based rational design of inhibitors for anti-dengue therapy. These include (i) the structure of the ‘active’ form of the DENV protease in complex with a peptide substrate; (ii) the structure of DENV methyltransferase bound to an inhibitor that selectively suppresses viral methyltransferase, but not human methyltransferases; (iii) the structure of DENV RNA-dependent RNA polymerase in complex with a small-molecule compound. This review summarizes the structural biology of these three key enzymes (protease, methyltransferase, and polymerase) that are essential for DENV replication. The new structural information has provided new avenues for development of anti-dengue therapy.

Keywords: Flavivirus; Dengue virus; Enzyme; Crystal structure; Structure-based design

Efficacy of an antiviral compound to inhibit replication of multiple pestivirus species by Benjamin W. Newcomer; M. Shonda Marley; Julia F. Ridpath; John D. Neill; David W. Boykin; Arvind Kumar; M. Daniel Givens (pp. 127-129).

► Antiviral testing of a novel compound was performed using five diverse pestiviruses. ► All viruses tested were inhibited by antiviral concentrations ⩾0.20μM. ► Cytotoxicity was not observed at antiviral concentrations less than 50μM. ► The compound represents a potential therapeutic agent for diverse pestiviral infections.Pestiviruses are economically important pathogens of livestock. An aromatic cationic compound (DB772) has previously been shown to inhibit bovine viral diarrhea virus (BVDV) type 1 in vitro at concentrations lacking cytotoxic side effects. The aim of this study was to determine the scope of antiviral activity of DB772 among diverse pestiviruses. Isolates of BVDV 2, border disease virus (BDV), HoBi virus, pronghorn virus and Bungowannah virus were tested for in vitro susceptibility to DB772 by incubating infected cells in medium containing 0, 0.006, 0.01, 0.02, 0.05, 0.1, 0.2, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5 or 25μM DB772. The samples were assayed for the presence of virus by virus isolation and titration (BDV and BVDV 2) or PCR (HoBi, pronghorn and Bungowannah viruses). Cytotoxicity of the compound was assayed for each cell type. Complete inhibition of BVDV 2, BDV, and Pronghorn virus was detected when DB772 was included in the culture media at concentrations of 0.20μM and higher. In two of three tests, a concentration of 0.05μM DB772 was sufficient to completely inhibit HoBi virus replication. Bungowannah virus was completely inhibited at a concentration of 0.01μM DB772. Thus, DB772 effectively inhibits all pestiviruses studied at concentrations >0.20μM. As cytotoxicity is not evident at these concentrations, this antiviral compound potentially represents an effective preventative or therapeutic for diverse pestiviruses.

Keywords: Pestivirus; Antiviral; Bovine viral diarrhea virus; Border disease virus

Oseltamivir–zanamivir bitherapy compared to oseltamivir monotherapy in the treatment of pandemic 2009 influenza A(H1N1) virus infections by Vanessa Escuret; Catherine Cornu; Florent Boutitie; Vincent Enouf; Anne Mosnier; Maude Bouscambert-Duchamp; Ségolène Gaillard; Xavier Duval; Thierry Blanchon; Catherine Leport; François Gueyffier; Sylvie Van Der Werf; Bruno Lina (pp. 130-137).

► We compared oseltamivir plus zanamivir vs oseltamivir alone in a clinical trial. ► Clinical trial conducted in community adults infected with A(H1N1)pdm09 virus. ► We could not detect any significant difference between treatment groups. ► Mean viral load decreased at around 1 log₁₀cgeq/μl per day. ► We did not detect any oseltamivir-resistant influenza viruses.The emergence of oseltamivir resistance in 2007 highlighted the need for alternative strategies against influenza. To limit the putative emergence of resistant viruses this clinical trial aimed to evaluate the antiviral efficacy and tolerability of oseltamivir–zanamivir (O+Z) bitherapy compared to oseltamivir monotherapy (O). This clinical trial was designed in 2008–2009 and was conducted during the A(H1N1) influenza virus pandemic in 2009–2010. The A(H1N1)pdm09 viruses were reported to be sensitive to oseltamivir and zanamivir but resistant to amantadine.During the pandemic phase in France, adults with influenza-like illness for less than 42h and who tested positive to influenza A were randomised into treatment groups: (O+Z) or (O). Patients had a nasal wash at day 0, before the beginning of treatment and daily at days 1 to 4. They also had a nasal swab at days 5 and 7 to check for the negativation of viral excretion. Virological response was assessed using the GAPDH adjusted M gene quantification.Analysis was possible for 24 patients, 12 in the (O+Z) arm and 12 in the (O) arm. The mean viral load decreased at around 1 log₁₀cgeq/μl per day regardless of allocated treatment group. We could not detect any significant difference between treatment groups in the duration needed to alleviate symptoms. All treatments were well tolerated. No oseltamivir-resistant H275Y NA mutated virus has been detected in patients of both treatment groups.The sample size of our study is too limited to be fully informative and we could not detect whether combination therapy (O+Z) improves or reduces the effectiveness of oseltamivir in the treatment of influenza A(H1N1)pdm09 virus infection in community patients. Additional studies are needed to improve the antiviral treatment of patients infected with influenza virus.

Keywords: Oseltamivir; Zanamivir; Combination therapy; Pandemic; Influenza A(H1N1)pdm09 virus

Poly (4-styrenesulfonic acid-co-maleic acid) is an entry inhibitor against both HIV-1 and HSV infections – Potential as a dual functional microbicide by Min Qiu; Yu Chen; Siwei Song; Hongyong Song; Ying Chu; Zhongping Yuan; Lin Cheng; Datong Zheng; Zhiwei Chen; Zhiwei Wu (pp. 138-147).

► Poly (4-styrenesulfonic acid-co-maleic acid) (PSM) is found as an antiviral agents against HIV-1 and HSV-2. ► PSM inhibits viral infection at entry step. ► PSM shows low toxicity to human genital epithelial cells, and did not interrupt vaginal microflora. ► PSM also shows potential anti-HSV-2 genital infection in vivo. ► PSM is a potential tropical microbicide to prevent HIV-1 and HSV-2.Genital herpes is one of the most prevalent sexually transmitted diseases (STD) caused by herpes simplex viruses type 1 and 2 (HSV-1 and -2). HSV is considered as a major risk factor in human immunodeficiency virus type-1 (HIV-1) infection and rapid progression to acquired immunodeficiency syndrome (AIDS). Here, we reported the finding of a polymer of styrenesulfonic acid and maleic acid (PSM) which exhibited antiviral activity with low cytotoxicity. PSM exhibited in vitro inhibitory activity against HIV-1 pseudovirus and HSV-1 and -2. In vivo efficacy of PSM against HSV-2 (G) was also investigated. We found that both 1% and 5% PSM gels protected mice from HSV-2 vaginal infection and disease progression significantly. Mechanistic analysis demonstrated that PSM was likely an entry inhibitor that disrupted viral attachment to the target cells. In particular, PSM disrupted gp120 binding to CD4 by interacting with the gp120 V3-loop and the CD4-binding site. The in vitro cytotoxicity studies showed that PSM did not stimulate NF-κB activation and up-regulation of proinflammatory cytokine IL-1β and IL-8 in vaginal epithelial cells. In addition, PSM also showed low adverse effect on the growth of vaginal Lactobacillus strains. PSM is, therefore, a novel viral entry inhibitor and a potential microbicide candidate against both HIV-1 and HSV.

Keywords: Poly (4-styrenesulfonic acid-co-maleic acid) (PSM); Human immunodeficiency virus type 1 (HIV-1); Herpes simplex virus; Antiviral activity; Microbicide; Entry inhibitor

Selection pressure on the hepatitis B virus pre-S/S and P open reading frames in Tongan subjects with a chronic hepatitis B virus infection by William G.H. Abbott; Peter Tsai; Howard A. Ross; Malakai ‘Ofanoa; Alexander J. Trevarton; John Hornell; Stephen R. Munn; Edward J. Gane (pp. 148-157).

► We need a therapeutic vaccine to control viral replication in chronic hepatitis B. ► The best antigens to include in the vaccine have not been identified. ► CD8+ T cell responses to HBV core antigens control replication in inactive healthy carriers. ► We could not detect CD8+ T cell responses to pre-S/S and P ORF antigens in inactive healthy carriers. ► pre-S/S and P ORF antigens may not be needed in a therapeutic vaccine for chronic hepatitis B.Identification of the full repertoire of hepatitis B virus (HBV) peptides that are presented to CD8+ T cells by common HLA class I alleles will be useful for designing immunotherapies for chronic hepatitis B. One hundred and seventy five cloned sequences containing the pre-S/S and P open reading frames (ORF) of the HBV were obtained from serum HBV-DNA of HBeAg-positive ( n=4) and HBeAg-negative (inactive healthy carriers (IHC), n=16) Tongan subjects with an inactive chronic HBV infection. In addition, 34 and 32 sequences were obtained 5.2±1.4 (mean±SD) years apart from eight subjects. PAML was used to identify codons in the pre-S/S and P ORFs that were under positive selection pressure ( ω>1). The number of non-synonymous substitutions in these codons was compared in IHC who were homozygous for either HLA-B∗4001 ( n=9) or HLA-B*5602 ( n=7), and who were either positive ( n=6) or negative ( n=10) for HLA-A*02. 34 codons in the pre-S/S and 11 codons in the P ORFs were under positive selection pressure. There was a higher number of non-synonymous substitutions in these codons in HBeAg-negative versus HBeAg-positive subjects in the P ( p=0.02) but not the pre-S/S ( p=0.64) ORF. There was no association between any HLA class I allele and non-synonymous substitutions in these codons. There was no increase in positive selection pressure on the pre-S/S and P ORFs with time. In conclusion, we could not find HLA class I-restricted selection pressure on any pre-S/S or P ORF amino acid; raising the possibility that peptide-based immunotherapies for chronic hepatitis B may not require peptides from these ORFs.

Keywords: Abbreviations; CHB; chronic hepatitis B; IHC; inactive healthy carrier; ORF; open reading frameHepatitis B virus; Phylogenetics; HLA class I; Polymerase gene; Envelope gene; HBeAg

WFDC1/ps20: A host factor that influences the neutrophil response to murine hepatitis virus (MHV) 1 infection by Erin Rogers; Ben X. Wang; Zhu Cui; David R. Rowley; Steven J. Ressler; Annapurna Vyakarnam; Eleanor N. Fish (pp. 158-168).

► Host factor, WFDC1/ps20 influences the innate immune response. ► WFDC1/ps20 confers protection against MHV-1 infection. ► WFDC1/ps20 regulates a neutrophil response.The whey acidic protein family member, WFDC1/ps20 is a permissivity factor in HIV infection. Herein we describe a contrasting role for ps20 in limiting MHV-1 infection. Intranasal MHV-1 infection produces a respiratory infection in mice. Using ps20 knockout mice we provide evidence that intranasal MHV-1 infection results in increased lung viral titers in ps20^−/− compared to ps20^+/+ mice. Accompanying MHV-1 infection we observe an increase in the number of neutrophils infiltrating the BAL and an increase in the percentage of neutrophils in the lung draining lymph nodes of ps20^−/− compared with ps20^+/+ mice. Gene expression levels for the neutrophil chemoattractants CXCL1 and CXCL2 are elevated in the lungs of ps20^−/− mice post-MHV-1 infection. Characterization of the immune cell profile in naïve ps20^−/− mice revealed an increase in circulating neutrophils compared to ps20^+/+ mice. No notable differences in other immune cell profiles were observed between the ps20^+/+ and ps20^−/− mice. Accordingly, we examined MHV-1 infection of neutrophils and provide evidence that neutrophils isolated from ps20^−/− mice are more susceptible to MHV-1 infection than neutrophils isolated from ps20^+/+ mice. These data suggest roles for ps20 in regulating expression of neutrophil-specific chemotactic factors, thereby potentially modulating neutrophil migration, and in modulating neutrophil susceptibility to MHV-1 infection.

Keywords: ps20; WFDC1; MHV-1; Neutrophils; Chemokines

The antiviral activity and mechanism of action of ( S)-[3-hydroxy-2-(phosphonomethoxy)propyl] (HPMP) nucleosides by Wendy C. Magee; David H. Evans (pp. 169-180).

► HPMP nucleosides are analogs of deoxynucleoside monophosphates. ► The HPMP nucleosides inhibit a wide range of DNA viruses. ► The HPMP nucleosides have a complex mechanism of action against viral polymerases. ► The development of resistance to the HPMP nucleosides has been described.One class of compounds that has shown promise as antiviral agents are the ( S)-[3-hydroxy-2-(phosphonomethoxy)propyl] (HPMP) nucleosides, members of the broader class of acyclic nucleoside phosphonates. These HPMP nucleosides are nucleotide analogs and have been shown to be effective inhibitors of a wide range of DNA viruses. Prodrugs of these compounds, which achieve higher levels of the active metabolites within the cell, have an expanded activity spectrum that also includes RNA viruses and retroviruses. Because they are analogs of natural nucleotide substrates, HPMP nucleosides are predicted to target polymerases (DNA polymerases, RNA polymerases and reverse transcriptases), resulting in the inhibition of viral genome replication. Previous work using the replicative enzymes of different viruses including human cytomegalovirus (HCMV) and vaccinia virus DNA polymerases and human immunodeficiency virus type 1 (HIV-1) reverse transcriptase has shown that the activated forms of these compounds are substrates for viral polymerases and that incorporation of these compounds into either the primer strand or the template strand inhibits, but does not necessarily terminate, further nucleic acid synthesis. The activity of these compounds against other viruses that do not encode their own polymerases, like polyoma viruses and papilloma viruses, suggests that host cell DNA polymerases are also targeted. This complex mechanism of action and broad activity spectrum has implications for the development of resistance and host cell genome replication, and suggests these compounds may be effective against other viruses such as influenza virus, respiratory syncytial virus and Dengue virus. This class of nucleotide analogs also points to a potential avenue for the development of newer antivirals.

Keywords: HPMP nucleoside; Nucleotide analog; DNA polymerase; Reverse transcriptase

Eight flavonoids and their potential as inhibitors of human cytomegalovirus replication by Sébastien Cotin; Claude-Alain Calliste; Marie-Christine Mazeron; Sébastien Hantz; Jean-Luc Duroux; William D. Rawlinson; Marie-Cécile Ploy; Sophie Alain (pp. 181-186).

► We report in vitro activity of four flavonoids amongst eight, as new inhibitors of HCMV, without increased toxicity. ► We analyze their level of action in the viral cycle. ► We analyze their activity in combination.The drugs currently available for treatment of severe human cytomegalovirus (HCMV) infections suffer from many drawbacks, particularly toxicity, and potential teratogenicity contraindicating their use in target populations such as pregnant women. The emergence of drug-resistant strains is still a problem for disease management, particularly in immunosuppressed populations where antivirals are used for extended periods of time. The flavonoid family of drugs contains promising candidates as they have low toxicity and inhibit different targets to currently available antivirals. We report here that, unlike their chalcon homologs, four flavonoids (baicalein, quercetin, quercetagetin and naringenin) inhibit various stages of HCMV replication, the most active anti-HCMV compound being baicalein and the less active and less selective being quercetagetin. These drugs could provide potential inhibitors of virus replication alone or in combination, without increased toxicity.

Keywords: Cytomegalovirus; Flavonoids; Antiviral; Toxicity

A small molecule screen in yeast identifies inhibitors targeting protein–protein interactions within the vaccinia virus replication complex by Olivier Flusin; Laurent Saccucci; Céline Contesto-Richefeu; Amel Hamdi; Carine Bardou; Thomas Poyot; André Peinnequin; Jean-Marc Crance; Pierre Colas; Frédéric Iseni (pp. 187-195).

► We report the use of a forward duplex yeast two-hybrid assay to screen molecules. ► Two molecules interfere with protein interactions within the VACV replication complex. ► Molecules inhibit VACV in vitro and display a similar antiviral activity than CDV. ► Molecules inhibit CPXV but not HSV-1, thus demonstrating a good specificity. ► We showed that, as expected, the two molecules interfere with VACV DNA synthesis.Genetic and biochemical data have identified at least four viral proteins essential for vaccinia virus (VACV) DNA synthesis: the DNA polymerase E9, its processivity factor (the heterodimer A20/D4) and the primase/helicase D5. These proteins are part of the VACV replication complex in which A20 is a central subunit interacting with E9, D4 and D5. We hypothesised that molecules able to modulate protein–protein interactions within the replication complex may represent a new class of compounds with anti-orthopoxvirus activities. In this study, we adapted a forward duplex yeast two-hybrid assay to screen more than 27,000 molecules in order to identify inhibitors of A20/D4 and/or A20/D5 interactions. We identified two molecules that specifically inhibited both interactions in yeast. Interestingly, we observed that these compounds displayed a similar antiviral activity to cidofovir (CDV) against VACV in cell culture. We further showed that these molecules were able to inhibit the replication of another orthopoxvirus (i.e. cowpox virus), but not the herpes simplex virus type 1 (HSV-1), an unrelated DNA virus. We also demonstrated that the antiviral activity of both compounds correlated with an inhibition of VACV DNA synthesis. Hence, these molecules may represent a starting point for the development of new anti-orthopoxvirus drugs.

Keywords: Vaccinia virus; Small molecules; Yeast two-hybrid; Protein interaction inhibitors; Anti-orthopoxvirus drugs

Resveratrol inhibits Epstein Barr Virus lytic cycle in Burkitt’s lymphoma cells by affecting multiple molecular targets by Alessandra De Leo; Giuseppe Arena; Egidio Lacanna; Giorgio Oliviero; Francesca Colavita; Elena Mattia (pp. 196-202).

► Resveratrol inhibits EBV lytic cycle initiation and particles release. ► The effects on EBV replication occur at the post-transcriptional level. ► We report that the polyphenol down regulates protein synthesis, ROS levels and NF–kB and AP-1 activities.Resveratrol (RV), a polyphenolic natural product present in many plants and fruits, exhibits anti-inflammatory, cardio-protective and anti-proliferative properties. Moreover, RV affects a wide variety of viruses including members of the Herpesviridae family, retroviruses, influenza A virus and polyomavirus by altering cellular pathways that affect viral replication itself. Epstein Barr Virus (EBV), the causative agent of infectious mononucleosis, is associated with different proliferative diseases in which it establishes a latent and/or a lytic infection. In this study, we examined the antiviral activity of RV against the EBV replicative cycle and investigated the molecular targets possibly involved. In a cellular context that allows in vitro EBV activation and lytic cycle progression through mechanisms closely resembling those that in vivo initiate and enable productive infection, we found that RV inhibited EBV lytic genes expression and the production of viral particles in a dose-dependent manner. We demonstrated that RV inhibited protein synthesis, decreased reactive oxygen species (ROS) levels, and suppressed the EBV-induced activation of the redox-sensitive transcription factors NF–kB and AP-1.Further insights into the signaling pathways and molecular targets modulated by RV may provide the basis for exploiting the antiviral activity of this natural product on EBV replication.

Keywords: Abbreviations; RV; resveratrol; EBV; Epstein Barr Virus; IE; immediate early; E; early; NF–kB; nuclear factor kappa-light-chain-enhancer of activated B cells; AP-1; activator protein 1Epstein Barr Virus; Resveratrol; Lytic infection; ROS; Protein synthesis; Transcription factors

The maximum potential market for dengue drugs V 1.0 by Geoffrey Dow; Eric Mora (pp. 203-212).

► Global economic burden of dengue was US $1.7billion in 2006. ► Maximum potential market for dengue drugs is US $338million per annum. ► Potential sales of a dengue drug exceed drug R+D costs. ► Dengue vaccines will not substantially impact potential sales of a dengue drug. ► Drug pricing based on shared division of economic costs is an alternative to tiered pricing.Drugs offer a complementary approach to vaccines for preventing the progression of symptoms and onset of the severe manifestations of dengue. Despite the rapid maturation of the research and development infrastructure for dengue drugs and the increasing frequency of dengue inhibitors reported in the scientific literature, the potential size of the market for dengue drugs has not been articulated. In the present work, extrapolating from publicly available information, we explored the economic burden attributable to dengue, the impact of dengue vaccines on clinical case loads, a possible alternative to tiered pricing for products for neglected diseases, and defined the maximum potential market for a dengue drug. Our projections suggest that in 2006, the annual global burden of dengue was US $1.7billion. Our proposed alternative to existing tiered pricing structures is that during a temporary period of market exclusivity, individual countries would pay 50% of the per-case equivalent of economic costs saved through the use of a dengue drug. This would yield prices per case of US $13–$239 depending on drug effectiveness and cost of medical and indirect costs and lost productivity in different countries. Assuming that such a pricing scheme was embraced, the maximum potential market for a dengue drug or drugs that on average reduced 40% of economic costs might be as high as US $338million annually. Our simulations suggest that dengue vaccines will begin to reduce the clinical case load of dengue in 2022, but that the number of cases will not decrease below 2006 levels and the proportion vaccinated will remain well below that required for the onset of herd immunity during the period of market exclusivity after the licensure of the first wave of dengue drugs.

Keywords: Dengue; Market; Economic burden; Tiered pricing; Dengue drug; Dengue vaccine

Enhanced inhibition of foot-and-mouth disease virus by combinations of porcine interferon-α and antiviral agents by Su-Mi Kim; Jong-Hyeon Park; Kwang-Nyeong Lee; Se-Kyung Kim; Young-Joon Ko; Hyang-Sim Lee; In-Soo Cho (pp. 213-220).

► We measured EC₅₀ and CC₅₀ of the five well-known or potential antiviral agents. ► The antiviral effect against of 6-Azauridine was demonstrated. ► We tested the combination effects of the pairs of antiviral agents. ► Enhanced antiviral effect of Ad-porcine IFN-α with Ribavirin was demonstrated. ► Enhanced antiviral effect of Ad-porcine IFN-α and Ad-siRNA was demonstrated.Foot-and-mouth disease (FMD) is an economically significant animal disease because of the speed of its transmission. The current FMD vaccine provides no protection until 7days after the vaccination, which reduces its effectiveness in the case of an outbreak. Therefore, to find an alternative method of applying antiviral agents for rapid and enhanced inhibition of the FMD virus (FMDV), we compared the antiviral effects of promising antiviral agents and attempted to apply them in combination. First, we measured and compared the 50% effective concentration (EC₅₀) to the mean inhibition effects of FMDV, and the 50% cytotoxic concentration (CC₅₀) to the mean cytotoxicity of antiviral agents such as ribavirin, guanidine-hydrochloride (guanidine-HCl), 6-azauridine, and recombinant adenovirus expressing three small interference RNAs (Ad-siRNA) or porcine interferon-α (Ad-porcine IFN-α) in swine kidney cells (IBRS-2). The selectivity indices of ribavirin (35.2) and 6-azauridine (34.6) were higher than that of guanidine-HCl (26.9). The selectivity indices of Ad-siRNA or Ad-porcine IFN-α were 7×10³ or 7×10⁴ based on the adenoviral titer. Next, we tested the combined effects of the FMDV inhibition agents. Enhanced inhibition effects were observed in the IBRS-2 cells and in suckling mice from the combination of Ad-porcine IFN-α and Ad-siRNA or ribavirin. The combined application of these recombinant adenoviruses and ribavirin may enhance their inhibitory effect on FMDV and overcome FMDV resistance against antiviral agents.

Keywords: Abbreviations; CPE; cytopathic effect; CC50; cytotoxic concentration required to reduce cell viability by 50%; EC50; effective concentration required to reduce virus-induced cytopathogenicity by 50%; FMD; foot-and-mouth disease; FMDV; foot-and-mouth disease virus; Guanidine-HCl; Guanidine hydrochloride; IFN; interferon; IP; intraperitoneal; SD; standard deviation; siRNA; small interference RNA; shRNA; short hairpin RNA; RNAi; RNA interference; RT-PCR; reverse transcriptase polymerase chain reaction; TCID; ₅₀; 50% tissue culture infective dose; DPC; days post challenge; P.I.; post infection; LD; ₅₀; 50% lethal doseFoot-and-mouth disease virus; Antiviral agent; Interferon; Small interfernce RNA; Ribavirin; Combination

Safety and efficacy of tenofovir/IQP-0528 combination gels – A dual compartment microbicide for HIV-1 prevention by Charlene S. Dezzutti; Cory Shetler; Alamelu Mahalingam; Shweta R. Ugaonkar; Garry Gwozdz; Karen W. Buckheit; Robert W. Buckheit Jr (pp. 221-225).

► Tenofovir/IQP-0528 microbicide combination gels were tested for safety and efficacy. ► Polarized ectocervical and colorectal tissues were used to evaluate these gels. ► Combination gels were safe toward mucosal tissue and blocked HIV infection. ► These gels provide the foundation of a dual compartment, combination product microbicide.Tenofovir (TFV) is a nucleotide reverse transcriptase inhibitor and IQP-0528 is a non-nucleoside reverse transcriptase inhibitor that also blocks virus entry. TFV and IQP-0528 alone have shown antiviral activity as microbicide gels. Because combination therapy will likely be more potent than mono-therapy, these drugs have been chosen to make a combination microbicide gel containing 2.5% TFV/1% IQP-0528. Safety and efficacy testing was done to evaluate five prototype combination gels. The gels retained TZM-bl cell and ectocervical and colorectal tissue viability. Further, the epithelium of the ectocervical and colorectal tissue remained intact after a 24h exposure. The ED₅₀ calculated from the formulations for IQP-0528 was ∼32nM and for TFV was ∼59nM and their inhibitory activity was not affected by semen. The ED₅₀ of TFV in the combination gels was ∼100-fold lower than when calculated for the drug substance alone reflecting the activity of the more potent IQP-0528. When ectocervical and colorectal tissue were treated with the combination gels, HIV-1 p24 release was reduced by ⩾1log₁₀ and ⩾2log₁₀, respectively. Immunohistochemistry for the ectocervical tissues treated with combination gels showed no HIV-1 infected cells at study end. With the increased realization of receptive anal intercourse among heterosexual couples often in conjunction with vaginal intercourse, having a safe and effective microbicide for both mucosal sites is critical. The safety and efficacy profiles of the gels were similar for ectocervical and colorectal tissues suggesting these gels have the potential for dual compartment use.

Keywords: HIV prevention; Combination microbicide; Rectal microbicide; Pyrimidinedione; Topical gel

In vitro profiling of the vaginal permeation potential of anti-HIV microbicides and the influence of formulation excipients by Carolien Grammen; Patrick Augustijns; Joachim Brouwers (pp. 226-233).

► An in vitro strategy to predict the tissue permeation potential of microbicides is described. ► This strategy is based on solubility and permeability profiling of microbicides. ► Tenofovir exhibits the best permeation compared to darunavir, saquinavir and dapivirine. ► Dapivirine permeation can be enhanced by including excipients in HEC-based gels. ► The proposed biopharmaceutical profiling may reduce microbicide failure in vivo.In the search for an effective anti-HIV microbicidal gel, limited drug penetration into the vaginal submucosa is a possible reason for failed protection against HIV transmission. To address this issue in early development, we here describe a simple in vitro strategy to predict the tissue permeation potential of vaginally applied drugs, based on solubility, permeability and flux assessment. We demonstrated this approach for four model microbicides (tenofovir, darunavir, saquinavir mesylate and dapivirine) and additionally examined the influence of formulation excipients on the permeation potential. When formulated in an aqueous-based HEC gel, high flux values across an HEC-1A cell layer were reached by tenofovir, as a result of its high aqueous solubility. In contrast, saquinavir and dapivirine fluxes remained low due to poor permeability and solubility, respectively. These low fluxes suggest limited in vivo tissue penetration, possibly leading to lack of efficacy. Dapivirine fluxes, however, could be enhanced up to 30-fold, by including formulation excipients such as polyethylene glycol 1000 (20%) or cyclodextrins (5%) in the HEC gels. Alternative formulations, i.e. emulsions or silicone elastomer gels, were less effective in flux enhancement compared to cyclodextrin-HEC gels. In conclusion, implementing the proposed solubility and permeability profiling in early microbicide development may contribute to the successful selection of promising microbicide candidates and appropriate formulations.

Keywords: HIV microbicide; Vaginal gel; Solubility; Permeability

A novel minicircle vector based system for inhibting the replication and gene expression of Enterovirus 71 and Coxsackievirus A16 by Zhuo Yang; Guodong Li; Yingqiu Zhang; Xiaoman Liu; Po Tien (pp. 234-244).

► A novel minicircle derived siRNAs were designed to inhibit EV 71 and CA 16 infections. ► siRNAs interfered with viral expression and replication in cells and mice model. ► The minicircle system can be further developed into an effective treatment against HFMD.Enterovirus 71 (EV 71) and Coxsackievirus A16 (CA 16) are two major causative agents of hand, foot and mouth disease (HFMD). They have been associated with severe neurological and cardiological complications worldwide, and have caused significant mortalities during large-scale outbreaks in China. Currently, there are no effective treatments against EV 71 and CA 16 infections. We now describe the development of a novel minicircle vector based RNA interference (RNAi) system as a therapeutic approach to inhibiting EV 71 and CA 16 replication. Small interfering RNA (siRNA) molecules targeting the conserved regions of the 3C^pro and 3D^pol function gene of the EV 71 and CA 16 China strains were designed based on their nucleotide sequences available in GenBank. This RNAi system was found to effectively block the replication and gene expression of these viruses in rhabdomyosarcoma (RD) cells and virus-infected mice model. The inhibitory effects were confirmed by a corresponding decrease in viral RNA, viral protein, and progeny virus production. In addition, no significant adverse off-target silencing or cytotoxic effects were observed. These results demonstrated the potential and feasibility of this novel minicircle vector based RNAi system for antiviral therapy against EV 71 and CA 16 infection.

Keywords: Enterovirus 71; Coxsackievirus A16; Minicircle vector; RNA interference; Antiviral therapy

Fluorescent primuline derivatives inhibit hepatitis C virus NS3-catalyzed RNA unwinding, peptide hydrolysis and viral replicase formation by Jean Ndjomou; Rajesh Kolli; Sourav Mukherjee; William R. Shadrick; Alicia M. Hanson; Noreena L. Sweeney; Diana Bartczak; Kelin Li; Kevin J. Frankowski; Frank J. Schoenen; David N. Frick (pp. 245-255).

Display Omitted► Characterized NS3 RNA helicase inhibitors synthesized from the dye primuline. ► Discovered that a subset of primuline derivatives also inhibit the NS3 protease. ► Showed that some primuline derivatives are also potent inhibitors of Dengue virus NS3. ► Found a primuline derivative active against the HCV subgenomic replicon.The hepatitis C virus (HCV) multifunctional nonstructural protein 3 (NS3) is a protease that cleaves viral and host proteins and a helicase that separates DNA and RNA structures in reactions fueled by ATP hydrolysis. Li et al. (2012) recently synthesized a series of new NS3 helicase inhibitors from the benzothiazole dimer component of the fluorescent yellow dye primuline. This study further characterizes a subset of these primuline derivatives with respect to their specificity, mechanism of action, and effect on cells harboring HCV subgenomic replicons. All compounds inhibited DNA and RNA unwinding catalyzed by NS3 from different HCV genotypes, but only some inhibited the NS3 protease function, and few had any effect on HCV NS3 catalyzed ATP hydrolysis. A different subset contained potent inhibitors of RNA stimulated ATP hydrolysis catalyzed by the related NS3 protein from Dengue virus. In assays monitoring intrinsic protein fluorescence in the absence of nucleic acids, the compounds cooperatively bound NS3 with K_ds that reflect their potency in assays. The fluorescent properties of the primuline derivatives both in vitro and in cells are also described. The primuline derivative that was the most active against subgenomic replicons in cells caused a 14-fold drop in HCV RNA levels (IC₅₀=5±2μM). In cells, the most effective primuline derivative did not inhibit the cellular activity of NS3 protease but disrupted HCV replicase structures.

Keywords: Motor protein; ATPase; RNA-binding proteins; Protease; Direct acting antiviral; Benzothiazole

Are novel combination therapies needed for chronic hepatitis B? by Fabien Zoulim (pp. 256-259).

► HBV infection is characterized by the persistence of a viral minichromosome (cccDNA). ► Viral suppression is achieved in the majority of patients with current treatments. ► cccDNA eradication is currently not possible with antiviral therapy. ► The new treatment endpoints are HBsAg clearance and/or immune control of infection. ► Novel viral and host targets are required to develop true combination therapy.The treatment of chronic hepatitis B remains limited to monotherapy with pegInterferon-alpha or one of 5 different nucleoside analogues (NUC). While viral suppression can be achieved in approximately 95% of patients with new-generation NUCs, the rate of HBeAg seroconversion ranges from only 20% with NUCs to 30% with pegInterferon-alpha. HBsAg loss is achieved in only 10% of patients with both classes of drugs after a follow-up of 5years. Attempts to improve the response by administering two different NUCs or a combination of NUC and pegInterferon-alpha have been unsuccessful. This situation has led researchers to investigate a number of steps in the HBV replication cycle as potential targets for new antiviral drugs. Novel targets and compounds could readily be evaluated in in vitro and in vivo models of HBV infection. The addition of one or more new drugs to the current regimen should offer the prospect of markedly improving the response to therapy, reducing the future burden of drug resistance, cirrhosis and hepatocellular carcinoma.

Keywords: Hepatitis B virus; Antivirals; Drug resistance; Viral persistence

Interference with ERK and STAT signaling pathways and inhibition of hepatitis C virus replication by ribavirin by Lan-Juan Zhao; Wen Wang; Yuan Liu; Hao Ren; Zhong-Tian Qi (pp. 260-268).

► Ribavirin reduced phosphorylation of ERK and STAT1 but increased STAT3 phosphorylation. ► IFN-α regulated ERK and STAT3 synergistically with ribavirin but up-regulated expression and phosphorylation of STAT1. ► ERK and STAT pathways were down-regulated by ribavirin following HCVcc infection. ► Ribavirin decreased HCV RNA and protein expression levels.Ribavirin in combination with interferon (IFN)-α is the approved treatment for hepatitis C virus (HCV) infection. Interference of ribavirin with signaling events is involved in its biological activities. However, little is known of signaling pathways induced by ribavirin following HCV infection. In human hepatoma cells, effects of ribavirin on ERK and signal transducers and activators of transcription (STAT) pathways, HCV replication, and antiviral gene expression were evaluated before and after cell culture-derived HCV infection. Ribavirin reduced phosphorylation of Raf, MEK, ERK, Tyk2, and STAT1, but selectively increased STAT3 phosphorylation. IFN-α synergistically regulated ERK and STAT3 phosphorylation with ribavirin, and up-regulated expression and phosphorylation of STAT1. Ribavirin dose-dependently decreased HCV RNA replication and HCV protein expression, with slight induction of IFN regulatory factor 9 and IFN-stimulated gene 15. Ribavirin and IFN-α exerted a synergetic inhibitory effect on HCV. ERK and STAT pathways were down-regulated by ribavirin following HCV infection. These results suggest that ribavirin may mediate anti-HCV activity through interference with ERK and STAT pathways.

Keywords: Ribavirin; Hepatitis C virus; ERK; STAT

Corrigendum to “Inhibition of rotavirus infection in cultured cells by N-acetyl-cysteine, PPARγ agonists and NSAIDs” [Antiviral Research 96(1) (2012) 1–12] by Carlos A. Guerrero; Andrea Murillo; Orlando Acosta (pp. 269-269).

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Antiviral Research (v.96, #2)