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• IFN-lambda inhibits HIV-1 integration and post-transcriptional events in vitro, but there is only limited in vivo repression of viral production.

  Authors: Ren-Rong Tian, Hong-Xiong Guo, Ji-Fu Wei, Chuan-Kun Yang, Shao-Heng He, Jian-Hua Wang

  Antiviral research.

  The lambda interferons (IL-28a, 28b, and IL-29) inhibit the replication of many viruses, but their role in the inhibition of HIV-1 infection remains unclear. During this study, we monitored IL-29The lambda interferons (IL-28a, 28b, and IL-29) inhibit the replication of many viruses, but their role in the inhibition of HIV-1 infection remains unclear. During this study, we monitored IL-29 production in HIV-1 infected individuals and analyzed the in vitro and in vivo inhibition of HIV-1 production. Prior treatment with IL-28a or IL-29 induced an antiviral state in cultured primary T-cells, which suppressed HIV-1 integration and post-transcriptional events. The antiviral factors MxA, OAS, and PKR were up-regulated. In HIV-1 infected patients, IL-29 level was increased along with the depletion of CD4(+) T-cells in peripheral blood, while the elevated IL-29 did not show a significantly negative correlation with viral load. Further analysis of HIV-1 infected individuals showed that IL-29 was positively correlated with IFN-β and anti-inflammatory cytokine IL-10, and was negatively correlated with IFN-γ, which might suggest that IFN-lambda participates in modulating antiviral immune responses during HIV-1 infection in vivo. Together, although IFN-lambda impeded HIV-1 infection of T-cells in vitro, IFN-lambda showed only limited in vivo repression of viral production. The modulation of IFN-lambda on inflammatory factors might be worthy for further concentrating on for better understanding the host immune response during HIV-1 infection.

• Inhibition of herpes simplex virus infection by oligomeric stilbenoids through ROS generation.

  Authors: Xiaoqing Chen, Haishi Qiao, Taixiang Liu, Ziying Yang, Lanfang Xu, Yunxia Xu, Hui Ming Ge, Ren-Xiang Tan, Erguang Li

  Antiviral research.

  Stilbenoids including resveratrol contain the basic structural unit of 1,2-diphenylethylene. Naturally occurring stilbenoids have broad structural features due to oligomerization and modificationsStilbenoids including resveratrol contain the basic structural unit of 1,2-diphenylethylene. Naturally occurring stilbenoids have broad structural features due to oligomerization and modifications and some have demonstrated potent biological activities. In an effort to identify bioactive stilbenoids, we screened a group of dimeric and oligomeric stilbenoids against HSV-1 and HSV-2 infection. Several trimeric and tetrameric derivatives showed anti-herpetic activity at single digit micromolar concentrations. HSV-1 and HSV-2 replication requires for NF-κB and MAPK activation. The compounds showed no inhibitory activity against NF-κB and Erk/MAPK activation, instead those compounds promoted rapid and transient release of reactive oxygen species (ROS). Addition of N-acetylcysteine (NAC), a scavenger of ROS, reversed the inhibitory effect of those compounds against HSV replication. In addition to the identification of resveratrol derivatives with potent anti-HSV activity, our results uncover a mechanism of polyphenol-mediated anti-HSV response, linking anti-herpetic activity of oligomeric stilbenoids to innate immunity.

• In vitro antiviral activity of dehydroepiandrosterone, seventeen synthetic analogs and ERK modulators against herpes simplex virus type 1.

  Authors: Nicolás I Torres, Viviana Castilla, Andrea C Bruttomesso, Javier Eiras, Lydia R Galagovsky, Mónica B Wachsman

  Antiviral research.

  In the present study the in-vitro antiviral activity of dehydroepiandrosterone (DHEA) and seventeen synthetic derivatives against herpes simplex type 1 (HSV-1) was determined. DHEA, epiandrosteroneIn the present study the in-vitro antiviral activity of dehydroepiandrosterone (DHEA) and seventeen synthetic derivatives against herpes simplex type 1 (HSV-1) was determined. DHEA, epiandrosterone (EA), two synthetic DHEA analogs and three synthetic EA analogs showed a selective inhibitory effect on HSV in vitro multiplication. DHEA and E2, a synthetic derivative of EA, were not found to be virucidal to cell-free HSV-1 and did not impair virus adsorption or penetration. We determined that treatment with both compounds decreased viral protein synthesis. Moreover, inhibitory effect of DHEA and E2 on extracellular viral titer was stronger than the inhibition found on total viral infectivity, suggesting that the antiherpetic activity of these compounds may also be in part due to an inhibition in virus formation and release. Since DHEA is a known Raf/MEK/ERK signaling pathway activator, we studied the role of this pathway on HSV-1 infection. ERK1/2 phosphorylation was stimulated in HSV-1 infected cultures. UO126, a Raf/MEK/ERK signaling pathway inhibitor, impaired viral multiplication, while anisomycin, an activator of this pathway, enhanced it. Treatment with DHEA 6 h before infection enhanced HSV-1 multiplication. On the contrary, pre-treatment with E2, which does not modulate Raf/MEK/ERK signaling pathway, did not produce an increase of viral replication. Taking together these results, the antiviral activity of DHEA seems to occur via a mechanism independent of its ability to modulate ERK phosphorylation.

• A Cell Free Protein Fragment Complementation Assay for Monitoring the Core Interaction of the Human Cytomegalovirus Nuclear Egress Complex.

  Authors: Margit Schnee, Felicia Wagner, Ulrich H Koszinowski, Zsolt Ruzsics

  Antiviral research.

  Certain viral protein-protein interactions provide attractive targets for antiviral drug development. Recently, we described a β-lactamase based protein fragment complementation assay (PCA) to studyCertain viral protein-protein interactions provide attractive targets for antiviral drug development. Recently, we described a β-lactamase based protein fragment complementation assay (PCA) to study the core interaction of the nuclear egress complex (NEC) of different herpesviruses in cells. Now, to have a cell free assay for inhibitor screens, we expressed split β-lactamase tagged interaction domains of the viral pUL50 and pUL53 proteins representing the NEC of human cytomegalovirus (HCMV) in bacteria. After validation and basic characterization of this NEC-PCA, we tested peptide inhibitors of the pUL50-pUL53 complex. We show that peptides resembling sequences of the first conserved region of pUL53 can inhibit the NEC-PCA. This, on one hand, indicated that the core interaction in the HCMV NEC is mediated by a linear motif. On the other hand it proved that this new pUL50-pUL53 interaction assay allows a simple cell free test for small molecular inhibitors.

• Discovery of Substituted N-Phenylbenzenesulphonamides as a Novel Class of Non-Nucleoside Hepatitis C Virus Polymerase Inhibitors.

  Authors: Marina M May, Dirk Brohm, Axel Harrenga, Tobias Marquardt, Bernd Riedl, Joerg Kreuter, Holger Zimmermann, Helga Ruebsamen-Schaeff, Andreas Urban

  Antiviral research.

  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)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 IC(50) values up to 40 nM. 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 Å and 2.2 Å 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.

• Brain Injury Caused by HIV Protease Inhibitors: role of Lipodystrophy and Insulin Resistance.

  Authors: Sunita Gupta, Alecia G Knight, Boriss Y Losso, Donald K Ingram, Jeffrey N Keller, Annadora J Bruce-Keller

  Antiviral research.

  HIV-associated neurocognitive disorders (HAND) remain prevalent even with widespread use of combination antiretroviral therapy (ART), suggesting a potential role for co-morbidities in neurologicHIV-associated neurocognitive disorders (HAND) remain prevalent even with widespread use of combination antiretroviral therapy (ART), suggesting a potential role for co-morbidities in neurologic decline. Indeed, it is well established that ART drugs, particularly HIV protease inhibitors, can induce hyperlipidemia, lipodystrophy, and insulin resistance; all of which are associated with neurologic impairment. This study was designed to determine how metabolic dysfunction might contribute to cognitive impairment and to reveal specific metabolic co-morbidities that could be targeted to preserve brain function. Adult male C57BL/6 mice were thus treated with clinically relevant doses of lopinavir/ritonavir for 4 weeks, and subjected to thorough metabolic, neurobehavioral, and biochemical analyses. Data show that lopinavir/ritonavir resulted in manifestations of lipodystrophy, insulin resistance, and hyperlipidemia. Evaluation of neurologic function revealed cognitive impairment and increased learned helplessness, but not motor impairment following treatment with lopinavir/ritonavir. Further analyses revealed a significant linear relationship between cognitive performance and specific markers of lipodystrophy and insulin resistance. Finally, analysis of brain injury indicated that lopinavir/ritonavir treatment resulted in cerebrovascular injury associated with decreased synaptic markers and increased inflammation, and that the cerebral cortex was more vulnerable than the cerebellum or hippocampus. Collectively, these data reveal an intimate link between metabolic co-morbidities and cognitive impairment, and suggest that remediation of selective aspects of metabolic syndrome could potentially reduce the prevalence or severity HIV-associated neurocognitive disorders.

• In-vivo Selection of the Mutation F121Y in a Patient Failing Raltegravir Containing Salvage Regimen.

  Authors: Jaqueline de Souza Cavalcanti, André Minhoto Lança, João Leandro de Paula Ferreira, Margareth da Eira, Daniel Soares de Souza Dantas, Luís Fernando de Macedo Brígido

  Antiviral research.

  Raltegravir is an Integrase inhibitor (INI) licensed for clinical use and other INI are in advanced stage of development. Different resistance mutations in HIV Integrase from patients using theseRaltegravir is an Integrase inhibitor (INI) licensed for clinical use and other INI are in advanced stage of development. Different resistance mutations in HIV Integrase from patients using these antiretroviral drugs have been described and G148H/R/K, N155H and less frequently Y143C/H/R are considered major resistant mutations to raltegravir. Both Stanford Database and Geno2Pheno list F121Y as conferring intermediate resistance "in vitro" both to raltegravir and elvitegravir. We report for the first time the "in vivo" selection F121Y and evolution to Y143R in a 31 years old male clade B HIV-1 infected patient failing a raltegravir-containing salvage regimen. Plasma samples nine months prior to raltegravir (RAL-Naïve) and at weeks 32, 40 and 88 after RAL-containing regimen were analyzed. INIs and pol susceptibility was evaluated at Stanford and Geno2Pheno from sequences obtained with RT-PCR. After a Viral load at week 12 below 50 copies/mL, viremia raised at week 20 to 4.5log10. The emergence of F121Y was observed at week 32 and 40, alongside with L74I, T97A, Q137H and V151I. At week 88 F121Y was no longer detected, L74I and T97A were maintained and Y143R emerged. F121Y might be an alternative pathway to Y143R. Changing of RAL- ontaining regimen upon the identification of F121Y might avoid the evolution of raltegravir resistance.

• Correlation between hepatitis B virus protein and microRNA processor Drosha in cells expressing HBV.

  Authors: Min Ren, Dongdong Qin, Kai Li, Jialin Qu, Liying Wang, Zengchan Wang, Ailong Huang, Hua Tang

  Antiviral research.

  Drosha regulates the biogenesis of microRNAs (miRNAs) and plays an essential role in the regulation of gene expression. Infection with hepatitis B virus (HBV) causes chronic hepatitis and liverDrosha regulates the biogenesis of microRNAs (miRNAs) and plays an essential role in the regulation of gene expression. Infection with hepatitis B virus (HBV) causes chronic hepatitis and liver cirrhosis. It is also a major risk factor for hepatocellular carcinoma. Emerging evidence suggests that HBV alters miRNA expression profiles, but the mechanisms underlying this process have not yet been fully elucidated. We therefore examined how HBV affected the production of miRNAs. We found that Drosha mRNA and protein expression were downregulated in cells expressing the HBV genome. This was associated with a reduction in the activity of the Drosha gene promoter. Gene silencing of HBx by RNA interference significantly restored the expression of Drosha. In conclusion, our data show that HBV could downregulate Drosha expression by inhibiting promoter activity, and the transcription factors SP1 and AP-2α may be involved in this process. This provides a new understanding of the mechanism of HBV-induced miRNAs dysregulation.

• Profile of stress and toxicity gene expression in human hepatic cells treated with Efavirenz.

  Authors: Leysa J Gomez-Sucerquia, Ana Blas-Garcia, Miguel Marti-Cabrera, Juan V Esplugues, Nadezda Apostolova

  Antiviral research.

  Hepatic toxicity and metabolic disorders are major adverse effects elicited during the pharmacological treatment of the human immunodeficiency virus (HIV) infection. Efavirenz (EFV), the most widelyHepatic toxicity and metabolic disorders are major adverse effects elicited during the pharmacological treatment of the human immunodeficiency virus (HIV) infection. Efavirenz (EFV), the most widely used non-nucleoside reverse transcriptase inhibitor (NNRTI), has been associated with these events, with recent studies implicating it in stress responses involving mitochondrial dysfunction and oxidative stress in human hepatic cells. To expand these findings, we analyzed the influence of EFV on the expression profile of selected stress and toxicity genes in these cells. Significant up-regulation was observed with Cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1), which indicated metabolic stress. Several genes directly related to oxidative stress and damage exhibited increased expression, including Methalothionein 2A (MT2A), Heat shock 70kDa protein 6 (HSPA6), Growth differentiation factor 15 (GDF15) and DNA-damage-inducible transcript 3 (DDIT3). In addition, Early growth response 1 (EGR1) was enhanced, whereas mRNA levels of the inflammatory genes Chemokine (C-X-C motif) ligand 10 (CXCL10) and Serpin peptidase inhibitor (nexin, plasminogen activator inhibitor type 1), member 1 (SERPINE1) decreased and increased, respectively. This profile of gene expression supports previous data demonstrating altered mitochondrial function and presence of oxidative stress/damage in EFV-treated hepatic cells, and may be of relevance in the search for molecular targets with therapeutic potential to be employed in the prevention, diagnosis and treatment of the hepatic toxicity associated with HIV therapy.

• Serotype specificity of recombinant fusion protein containing domain III and capsid protein of dengue virus 2.

  Authors: Alienys Izquierdo, Iris Valdés, Lázaro Gil, Lisset Hermida, Sheila Gutiérrez, Angélica García, Lidice Bernardo, Alekis Pavón, Gerardo Guillén, María G Guzmán

  Antiviral research.

  Recombinant fusion protein containing domain III of the dengue envelope protein fused to capsid protein from dengue 2 virus was immunogenic and conferred protection in mice against lethal challengeRecombinant fusion protein containing domain III of the dengue envelope protein fused to capsid protein from dengue 2 virus was immunogenic and conferred protection in mice against lethal challenge in previously report. Here, the antigenic specificity of this recombinant protein using anti-dengue antibodies from mice and humans and the cross-reactive humoral and cellular response induced in immunized mice were evaluated. The homologous anti-dengue antibodies showed a higher reactivity to the recombinant protein compared to the wide cross-reactivity observed for viral antigen as determined by ELISA. The IgG anti-dengue and functional antibodies, induced by the recombinant proteins in mice, were highly serotype specific by ELISA, hemaglutination inhibition and plaque reduction neutralizing tests. Accordingly, the cellular immune response determined by the IFNγ and TNFα secretion, was serotype specific. The specificity of serotype associated to this recombinant protein in addition to its high antigenicity, immunogenicity and protecting capacity suggest its advantage as a possible functional and safe vaccine candidate against dengue in a future tetravalent formulation.

• Alphaviruses: Population genetics and determinants of emergence.

  Authors: Scott C Weaver, Richard Winegar, Ian D Manger, Naomi L Forrester

  Antiviral research.

  Alphaviruses are responsible for several medically important emerging diseases and are also significant veterinary pathogens. Due to the aerosol infectivity of some alphaviruses and their ability toAlphaviruses are responsible for several medically important emerging diseases and are also significant veterinary pathogens. Due to the aerosol infectivity of some alphaviruses and their ability to cause severe, sometimes fatal neurologic diseases, they are also of biodefense importance. This review discusses the ecology, epidemiology and molecular virology of the alphaviruses, then focuses on three of the most important members of the genus: Venezuelan and eastern equine encephalitis and chikungunya viruses, with emphasis on their genetics and emergence mechanisms, and how current knowledge as well as gaps influence our ability to detect and determine the source of both natural outbreaks and potential use for bioterrorism. This article is one of a series in Antiviral Research on the genetic diversity of emerging viruses.

• In vitro anti-influenza virus and anti-inflammatory activities of theaflavin derivatives.

  Authors: Mian Zu, Fan Yang, Weiling Zhou, Ailin Liu, Guanhua Du, Lishu Zheng

  Antiviral research.

  The theaflavins fraction (TF80%, with a purity of 80%) and three theaflavin (TF) derivatives from black tea have been found to exhibit potent inhibitory effects against influenza virus in vitro. TheyThe theaflavins fraction (TF80%, with a purity of 80%) and three theaflavin (TF) derivatives from black tea have been found to exhibit potent inhibitory effects against influenza virus in vitro. They were evaluated with a neuraminidase (NA) activity assay, a hemagglutination (HA) inhibition assay, a real-time quantitative PCR (qPCR) assay for gene expression of hemagglutinin (HA) and a cytopathic effect (CPE) reduction assay. The experimental results showed that they all exerted significant inhibitory effects on the NA of three different subtypes of influenza virus strains [A/PR/8/34(H1N1), A/Sydney/5/97(H3N2) and B/Jiangsu/10/2003] with 50% inhibitory concentration (IC(50)) values ranging from 9.27 to 36.55μg/mL, and they also displayed an inhibitory effect on HA; these inhibitory effects might constitute two major mechanisms of their antiviral activity. Time-of-addition studies demonstrated that TF derivatives might have a direct effect on viral particle infectivity, which was consistent with the inhibitory effect on HA. Subsequently, the inhibitory effect of TF derivatives on the replication of the viral HA gene as assayed by qPCR and on the nuclear localization of the influenza virus vRNP further demonstrated that they may primarily act during the early stage of infection. Interestingly, besides the activity against functional viral proteins, TF derivatives also decreased the expression level of the inflammatory cytokine IL-6 during viral infection, expression of which may result in serious tissue injury and apoptosis. Our results indicated that TF derivatives are potential compounds with anti-influenza viral replication and anti-inflammatory properties. These findings will provide important information for new drug design and development for the treatment of influenza virus infection.

• Inhibition of adenovirus multiplication by short interfering RNAs directly or indirectly targeting the viral DNA replication machinery.

  Authors: Doris Kneidinger, Mirza Ibrišimović, Thomas Lion, Reinhard Klein

  Antiviral research.

  Human adenoviruses are a common threat to immunocompromised patients, e.g., HIV-positive individuals or solid-organ and, in particular, allogeneic stem cell transplant recipients. Antiviral drugsHuman adenoviruses are a common threat to immunocompromised patients, e.g., HIV-positive individuals or solid-organ and, in particular, allogeneic stem cell transplant recipients. Antiviral drugs have a limited effect on adenoviruses, and existing treatment modalities often fail to prevent fatal outcome. Silencing of viral genes by short interfering RNAs (siRNAs) holds a great promise in the treatment of viral infections. The aim of the present study was to identify adenoviral candidate targets for RNA interference-mediated inhibition of adenoviral replication. We investigated the impact of silencing of a set of early, middle, and late viral genes on the replication of adenovirus 5 in vitro. Adenovirus replication was inhibited by siRNAs directed against the adenoviral E1A, DNA polymerase, preterminal protein (pTP), IVa2, hexon, and protease genes. Silencing of early and middle genes was more effective in inhibiting adenovirus multiplication than was silencing of late genes. A siRNA directed against the viral DNA polymerase mRNA decreased viral genome copy numbers and infectious virus progeny by several orders of magnitude. Since silencing of any of the early genes directly or indirectly affected viral DNA synthesis, our data suggest that reducing viral genome copy numbers is a more promising strategy for the treatment of adenoviral infections than is reducing the numbers of proteins necessary for capsid generation. Thus, adenoviral DNA replication was identified as a key target for RNAi-mediated inhibition of adenovirus multiplication. In addition, the E1A transcripts emerged as a second important target, because its knockdown markedly improved the viability of cells at late stages of infection.

• Design of modified U1i molecules against HIV-1 RNA.

  Authors: Stefanie A Knoepfel, Amaya Abad, Xabi Abad, Puri Fortes, Ben Berkhout

  Antiviral research.

  Several gene therapeutic approaches have been proposed to add to current antiretroviral therapy against HIV-1. U1 interference (U1i) is a promising new gene therapy tool that targets mRNAs withSeveral gene therapeutic approaches have been proposed to add to current antiretroviral therapy against HIV-1. U1 interference (U1i) is a promising new gene therapy tool that targets mRNAs with modified U1 snRNAs. For efficient inhibition, the 3'-terminal exon of pre-mRNAs must be recognized by the modified U1 snRNA. Subsequent interaction between the U1-associated 70K protein and poly(A) polymerase leads to inhibition of polyadenylation and consequently degradation of the pre-mRNA. We designed 14 new U1i inhibitors against HIV-1 mRNA regions that are 100% complementary to at least 70% of HIV-1 sequences listed in the HIV database. All U1i inhibitors were tested transiently in HIV-1 production assays as well as luciferase reporter experiments and three candidates were examined further in stably lentivirus-transduced T cell lines. We identified U1i-J that targets the region encoding the NF-κB binding sites as the most effective inhibitor that substantially reduced viral protein expression. The potency of J is determined in part by the presence of a duplicated target within the HIV-1 mRNA. The stably transduced SupT1 T cells were challenged with HIV-1 but no antiviral effect was detected. U1i inhibitors can be potent suppressors of HIV-1 production in transient assays but further optimization of this antiviral approach is needed.

• Polyfunctional CD8(+) T cells are associated with the vaccination-induced control of a novel recombinant influenza virus expressing an HCV epitope.

  Authors: Amabel C L Tan, Emily M Y Eriksson, Katherine Kedzierska, Georgia Deliyannis, Sophie A Valkenburg, Weiguang Zeng, David C Jackson

  Antiviral research.

  In hepatitis C virus (HCV) infection, CD8(+) T cell responses have been shown to be important in viral clearance. Examining the efficacy of CD8(+) T cell vaccines against HCV has been limited by theIn hepatitis C virus (HCV) infection, CD8(+) T cell responses have been shown to be important in viral clearance. Examining the efficacy of CD8(+) T cell vaccines against HCV has been limited by the lack of an HCV infectious model in mice, and the differences between MHC restriction in humans and mice. Using HLA-A2 transgenic HHD mice, we demonstrate that intranasally delivered Pam2Cys-based lipopeptides containing HLA-A2-restricted HCV epitopes can induce polyfunctional CD8(+) T cell responses in several organs including the liver. To examine the activity of these responses in an infectious context, we developed a recombinant influenza virus that expresses the NS5B(2594-2602) epitope from non-structural protein 5B of hepatitis C virus (PR8-HCV(NS5B)). We showed that mice inoculated with a lipopeptide containing the NS5B epitope had reduced viral loads following challenge with the PR8-HCV(NS5B) virus. This reduction was associated with the induction of NS5B(2594-2602)-specific IFN-γ and TNF-α co-producing CD8(+) T cells. The T cell receptor usage in the NS5B(2594-2602) response was found to exhibit a Vβ8.1/8.2 bias that was characterized by a narrow repertoire and a common CDR3β motif. This work has identified CD8(+) T cell functions induced by lipopeptides that are associated with viral control and demonstrate the potential of lipopeptide-based vaccines as candidates for treatment of HCV infection.

• Natural compounds inhibiting the replication of Porcine reproductive and respiratory syndrome virus.

  Authors: Anbu K Karuppannan, Kan Xing Wu, Jia Qiang, Justin Jang-Hann Chu, Jimmy Kwang

  Antiviral research.

  Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogenic virus in the swine production. Current vaccines against PRRSV do not induce sterile immunity andPorcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogenic virus in the swine production. Current vaccines against PRRSV do not induce sterile immunity and the virus evolves at a rapid rate with frequent appearances of new strains. In this study, we screened a library of 502 highly purified natural product compounds to identify specific inhibitors of PRRSV replication cycle. Our observations showed that many of the inhibitory compounds identified have activity on the cellular ion transport mechanisms. We identified for the first time, four compounds which inhibit the PRRSV replication cycle at micro molar concentration or less, namely, 12-deoxyphorbol 13-phenylacetate 20-acetate, ouabain, bufalin and valinomycin. Further, we have identified 15 other compounds which can inhibit the PRRSV replication at the concentration of 8μM. This study provides a basis for further development of pharmacological agents to inhibit PRRSV replication.

• Viral genome RNA degradation by sequence-selective, nucleic-acid hydrolyzing antibody inhibits the replication of influenza H9N2 virus without significant cytotoxicity to host cells.

  Authors: Aeyung Kim, Ja-Yeong Lee, Sung June Byun, Myung-Hee Kwon, Yong-Sung Kim

  Antiviral research.

  Influenza A virus infection is a great threat to avian species and humans. Targeting viral proteins by antibody has a limited success due to the antigen drift and shift. Here we present a novelInfluenza A virus infection is a great threat to avian species and humans. Targeting viral proteins by antibody has a limited success due to the antigen drift and shift. Here we present a novel antibody-based antiviral strategy of targeting viral genomic RNA (vRNA) for degradation rather than neutralizing viral proteins. Based on the template of a sequence-nonspecific nucleic acid-hydrolyzing, single domain antibody of the light chain variable domain, 3D8 VL, we generated a synthetic library on the yeast surface by randomizing putative nucleic acid interacting residues. To target nucleocapsid protein (NP)-encoding viral genomic RNA (NP-vRNA) of H9N2 influenza virus, the library was screened against a 18-nucleotide single stranded nucleic acid substrate, dubbed asNP(18), the sequence of which is unique to the NP-vRNA. We isolated a 3D8 VL variant, NP25, that had ∼15-fold higher affinity (∼54nM) and ∼3-fold greater selective hydrolyzing activity for the target substrate than for off targets. In contrast to 3D8 VL WT, asNP(18)-selective NP25 constitutively expressed in the cytosol of human lung carcinoma A549 cells does not exhibit any significant cytotoxicity and selectively degrades a reporter mRNA carrying the target asNP(18) sequence in the stable cell lines. NP25 more potently inhibits the replication of H9N2 influenza virus than 3D8 VL WT in the stable cell lines. NP25 more selectively reduces the amount of the targeted NP-vRNA than 3D8 VL WT from the early stage of virus infection in the stable cell lines, without noticeable harmful effects on the endogenous mRNA, suggesting that NP25 indeed more specifically recognizes to hydrolyze the target NP-vRNA of H9N2 virus than off-targets. Our results provide a new strategy of targeting viral genomic RNA for degradation by antibody for the prevention of influenza virus infection in humans and animals.

• EPs® 7630 (Umckaloabo®), an extract from Pelargonium sidoides roots, exerts anti-influenza virus activity in vitro and in vivo.

  Authors: Linda L Theisen, Claude P Muller

  Antiviral research.

  A prodelphinidin-rich extract from Pelargonium sidoides DC, EPs® 7630 (Umckaloabo®), which is licensed to treat respiratory tract infections such as acute bronchitis, was investigated for itsA prodelphinidin-rich extract from Pelargonium sidoides DC, EPs® 7630 (Umckaloabo®), which is licensed to treat respiratory tract infections such as acute bronchitis, was investigated for its antiviral effects. EPs® 7630 showed dose-dependent anti-influenza activity at non-toxic concentrations against pandemic H1N1, oseltamivir-sensitive and -resistant seasonal H1N1, seasonal H3N2 and the laboratory H1N1 strain A/Puerto Rico/8/34, while it had no antiviral activity against adenovirus or measles virus. The extract inhibited an early step of influenza infection and impaired viral hemagglutination as well as neuraminidase activity. However, EPs® 7630 did not exhibit a direct virucidal effect, as virus preincubation (unlike cell preincubation) with the extract did not influence infectivity. Importantly, EPs® 7630 showed no propensity to resistance development in vitro. Analysis of EPs® 7630 constituents revealed that prodelphinidins represent the active principle. Chain length influenced antiviral activity, as monomers and dimers were less effective than oligo- and polymers. Importantly, gallocatechin and its stereoisomer epigallocatechin exert antiviral activity also in their monomeric form. In addition, EPs® 7630 administered by inhalation significantly improved survival, body weight and body temperature of influenza-infected mice, without obvious toxicity, demonstrating the benefit of EPs® 7630 in treatment of influenza.

• Quercetin inhibits rhinovirus replication in vitro and in vivo.

  Authors: Shyamala Ganesan, Andrea N Faris, Adam T Comstock, Qiong Wang, Suparna Nanua, Marc B Hershenson, Uma S Sajjan

  Antiviral research.

  Rhinovirus (RV), which is responsible for the majority of common colds, also causes exacerbations in patients with asthma and chronic obstructive pulmonary disease. So far, there are no drugsRhinovirus (RV), which is responsible for the majority of common colds, also causes exacerbations in patients with asthma and chronic obstructive pulmonary disease. So far, there are no drugs available for treatment of rhinovirus infection. We examined the effect of quercetin, a plant flavanol on RV infection in vitro and in vivo. Pretreatment of airway epithelial cells with quercetin decreased Akt phosphosphorylation, viral endocytosis and IL-8 responses. Addition of quercetin 6h after RV infection (after viral endocytosis) reduced viral load, IL-8 and IFN responses in airway epithelial cells. This was associated with decreased levels of negative and positive strand viral RNA, and RV capsid protein, abrogation of RV-induced eIF4GI cleavage and increased phosphorylation of eIF2α. In mice infected with RV, quercetin treatment decreased viral replication as well as expression of chemokines and cytokines. Quercetin treatment also attenuated RV-induced airway cholinergic hyperresponsiveness. Together, our results suggest that quercetin inhibits RV endocytosis and replication in airway epithelial cells at multiple stages of the RV life cycle. Quercetin also decreases expression of pro-inflammatory cytokines and improves lung function in RV-infected mice. Based on these observations, further studies examining the potential benefits of quercetin in the prevention and treatment of RV infection are warranted.

• Long-term monitoring drug resistance by ultra-deep pyrosequencing in a chronic hepatitis B virus (HBV)-infected patient exposed to several unsuccessful therapy schemes.

  Authors: M Sede, D Ojeda, L Cassino, G Westergaard, M Vazquez, S Benetti, F Fay, H Tanno, J Quarleri

  Antiviral research.

  The aim of this study was to analyze the spectrum and dynamics of low-prevalent HBV mutations in the reverse transcriptase (rt) and S antigen by ultra-deep pyrosequencing (UDPS). Samples wereThe aim of this study was to analyze the spectrum and dynamics of low-prevalent HBV mutations in the reverse transcriptase (rt) and S antigen by ultra-deep pyrosequencing (UDPS). Samples were obtained from a chronically infected patient who was followed throughout a thirteen-year period. This technology enabled simultaneous analysis of 4084 clonally amplified fragments from the patient allowing detecting low prevalent (<1%) mutations during the follow-up. At baseline, HBV sequences were predominately wild-type. Under sequential HBV monotherapies including lamivudine, adefovir and entecavir, a high frequency of rtM204I mutation was detected initially as unique and then coexisting with rtM204V. Both mutations were statistically associated with rtA200V and rtV207I, respectively. Once the entecavir and tenofovir combined therapy was started, polymerase and consequently envelope gene mutations appeared at several positions at a higher frequency than before, including the entecavir resistance-associated mutation rtT184L.

• Antiviral effects of Psidium guajava Linn. (guava) tea on the growth of clinical isolated H1N1 viruses: Its role in viral hemagglutination and neuraminidase inhibition.

  Authors: Nongluk Sriwilaijaroen, Syuichi Fukumoto, Kenji Kumagai, Hiroaki Hiramatsu, Takato Odagiri, Masato Tashiro, Yasuo Suzuki

  Antiviral research.

  Rapid evolution of influenza RNA virus has resulted in limitation of vaccine effectiveness, increased emergence of drug-resistant viruses and occurrence of pandemics. A new effective antiviral isRapid evolution of influenza RNA virus has resulted in limitation of vaccine effectiveness, increased emergence of drug-resistant viruses and occurrence of pandemics. A new effective antiviral is therefore needed for control of the highly mutative influenza virus. Teas prepared by the infusion method were tested for their anti-influenza activity against clinical influenza A (H1N1) isolates by a 19-h influenza growth inhibition assay with ST6Gal I-expressing MDCK cells (AX4 cells) using fluorogenic quantification and chromogenic visualization. Guava tea markedly inhibited the growth of A/Narita/1/2009 (amantadine-resistant pandemic 2009 strain) at an IC(50) of 0.05% and the growth of A/Yamaguchi/20/06 (sensitive strain) and A/Kitakyushu/10/06 (oseltamivir-resistant strain) at similar IC(50) values ranging from 0.24% to 0.42% in AX4 cells, being 3.4- to 5.4-fold more potent than green tea (IC(50) values: 0.27% for the 2009 pandemic strain and 0.91-1.44% for the seasonal strains). In contrast to both teas, oseltamivir carboxylate (OC) demonstrated high potency against the growth of A/Narita/1/09 (IC(50) of 3.83nM) and A/Yamaguchi/20/06 (IC(50) of 11.57nM) but not against that of A/Kitakyushu/10/06 bearing a His274-to-Tyr substitution (IC(50) of 15.97μM). Immunofluorescence analysis under a confocal microscope indicated that both teas inhibited the most susceptible A/Narita/1/2009 virus at the initial stage of virus infection. This is consistent with results of direct inhibition assays showing that both teas inhibited viral hemagglutination at concentrations comparable to their growth inhibition concentrations but inhibited sialidase activity at about 8-times higher concentrations. Guava tea shows promise to be efficacious for control of epidemic and pandemic influenza viruses including oseltamivir-resistant strains, and its broad target blockage makes it less likely to lead to emergence of viral resistance.

• Polymorphisms in ADAR1 gene affect response to interferon alpha based therapy for chronic hepatitis B in Han Chinese.

  Authors: Xiaopan Wu, Zhenhui Xin, Xilin Zhu, Liping Pan, Zhuo Li, Hui Li, Ying Liu

  Antiviral research.

  Host genetic polymorphisms in interferon pathway genes are reported to be associated with response to interferon therapy. Five hundred and forty-eight α interferon treatment-naïve chronic hepatitis BHost genetic polymorphisms in interferon pathway genes are reported to be associated with response to interferon therapy. Five hundred and forty-eight α interferon treatment-naïve chronic hepatitis B patients were enrolled in the retrospective nested case-control study. All patients received α interferon based treatment and were examined for therapy efficacy. We genotyped 115 polymorphisms from 16 interferon pathway genes using the MassArray system. We identified rs4845384 in ADAR1 gene is strongly associated with the outcome of interferon therapy allele dose-depended (P=0.0005), with decreased odds ratios of 0.69 and 0.27 for GA and AA genotypes, respectively (95% confidence interval, 0.47-0.99 for GA; 0.11-0.64 for AA). Our study suggested that rs4845384 in ADAR1 associates with treatment-induced clearance of chronic hepatitis B.

• Combinations of favipiravir and peramivir for the treatment of pandemic influenza A/California/04/2009 (H1N1) virus infections in mice.

  Authors: E Bart Tarbet, Masako Maekawa, Yousuke Furuta, Y S Babu, John D Morrey, Donald F Smee

  Antiviral research. 94(1):103-110.

  Favipiravir, an influenza virus RNA polymerase inhibitor, and peramivir, an influenza virus neuraminidase inhibitor, were evaluated alone and in combination against pandemic influenzaFavipiravir, an influenza virus RNA polymerase inhibitor, and peramivir, an influenza virus neuraminidase inhibitor, were evaluated alone and in combination against pandemic influenza A/California/04/2009 (H1N1) virus infections in mice. Infected mice were treated twice daily for 5d starting 4h after virus challenge. Favipiravir was 40%, 70%, and 100% protective at 20, 40, and 100mg/kg/d. Peramivir was 30% protective at 0.5mg/kg/d, but ineffective at lower doses when used as monotherapy. Combinations of favipiravir and peramivir increased the numbers of survivors by 10-50% when the 0.025, 0.05, and 0.1mg/kg/d doses of peramivir were combined with 20mg/kg/d favipiravir and when all doses of peramivir were combined with 40mg/kg/d favipiravir. Three-dimensional analysis of drug interactions using the MacSynergy method indicates strong synergy for these drug combinations. In addition, an increase in lifespan for groups of mice treated with drug combinations, compared to the most effective monotherapy group, was observed for the 0.025, 0.05, and 0.1mg/kg/d doses of peramivir combined with favipiravir at the 20mg dose level. Therefore, the 20mg/kg/d dose of favipiravir was selected for further combination studies. Increased survival was exhibited when this dose was combined with peramivir doses of 0.1, 0.25 and 0.5mg/kg/d (1mg/kg/d of peramivir alone was 100% protective in this experiment). Improved body weight relative to either compound alone was evident using 0.25, 0.5, and 1mg/kg/d of peramivir. Significant reductions in lung hemorrhage score and lung weight were evident on day 6 post-infection. In addition, virus titers were reduced significantly on day 4 post-infection by combination therapy containing favipiravir combined with peramivir at 0.25 and 0.5mg/kg/d. These data demonstrate that combinations of favipiravir and peramivir perform better than suboptimal doses of each compound alone for the treatment of influenza virus infections in mice.

• Inhibition of Japanese encephalitis virus entry into the cells by the envelope glycoprotein domain III (EDIII) and the loop3 peptide derived from EDIII.

  Authors: Chen Li, Li-Ying Zhang, Ming-Xia Sun, Peng-Peng Li, Li Huang, Jian-Chao Wei, Yi-Lin Yao, Hassan Isahg, Pu-Yan Chen, Xiang Mao

  Antiviral research.

  Japanese encephalitis virus (JEV) infection is a major cause of acute viral encephalitis both in humans and animals. The domain III of virus envelope protein (EDIII) plays important roles inJapanese encephalitis virus (JEV) infection is a major cause of acute viral encephalitis both in humans and animals. The domain III of virus envelope protein (EDIII) plays important roles in interacting with host cell receptors to facilitate virus entry. In this study, recombinant JEV EDIII was expressed and purified. The protein showed the ability to inhibit JEV infection in BHK-21 cells with 50% inhibition at a concentration of 25μg/ml. Based on NMR structure of JEV EDIII, we chose several loop peptides that were reported to be related to receptor binding to test their possible inhibitory activities on virus infection. Our in vitro experiments demonstrated that one of the loop peptides (loop3) can prevent JEV infection with 50% inhibition at concentration of 10μM by interfering in virus attachment to the cells. Our in vivo experiments on mice showed the loop3 was the most protective peptide when administered before virus challenge. Therefore, the loop3 peptide may be served as basis for the development of novel antiviral agents against Japanese encephalitis virus or other flaviviruses infection.

• Amino acid derivatives of the (-) enantiomer of gossypol are effective fusion inhibitors of human immunodeficiency virus type 1.

  Authors: Tai An, Wenjie Ouyang, Wei Pan, Deyin Guo, Jurong Li, Longlong Li, Gang Chen, Jian Yang, Shuwen Wu, Po Tien

  Antiviral research.

  T20 and maraviroc are the only two currently available entry inhibitors that have shown efficacy in treating HIV-1-infected individuals who have failed to respond to first-line antiretroviral drugs.T20 and maraviroc are the only two currently available entry inhibitors that have shown efficacy in treating HIV-1-infected individuals who have failed to respond to first-line antiretroviral drugs. Gossypol is a polyphenolic aldehyde extracted from cotton plants. By modifying the (-) enantiomer of gossypol with a series of small molecules, we have found that neutral amino acids with aliphatic group derivatives of (-) gossypol show the strongest inhibitory activity and the lowest cytotoxicity in vitro among all the derivatives tested. Additionally, the selectivity index of the (-) gossypol-neutral amino acid conjugates is increased 100-fold when compared with (-) gossypol alone. It is widely accepted that gossypol and gossypol derivatives inhibit HIV-1 replication by targeting reverse transcriptase. However, from the results of our time-of-addition assay, HIV-1-mediated cell fusion assay and VSV-G pseudotyped virus assay, we demonstrate that the alanine-(-) gossypol derivative ((-)G-Ala) is an effective HIV-1 entry inhibitor. Further mechanistic analysis revealed that (-)G-Ala neither blocks gp120-CD4 binding nor interacts with the HIV-1 co-receptor CXCR4. Results from sandwich ELISA, native-PAGE and circular dichroism (CD) show that (-)G-Ala inhibits the cell fusion-activated gp41 core domain. Moreover, (-)G-Ala binds to the HIV-5-Helix protein and blocking D-peptide (PIE7) binding to the hydrophobic pocket on the surface of the gp41 internal trimeric coiled-coil domain. The contraceptive properties of (-) gossypol and amino acid derivatives of (-) gossypol are also discussed. Collectively, our results indicate that (-)G-Ala may bind to the gp41 hydrophobic pocket and block the formation of the cell fusion-activated gp41 core to inhibit HIV-1-mediated membrane fusion and subsequent viral entry.

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