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Antiviral Research (v.94, #2)
Orthopoxvirus targets for the development of new antiviral agents by Mark N. Prichard; Earl R. Kern (pp. 111-125).
► The replication cycle of vaccinia virus is described in detail noting inhibitors of each stage of replication. ► Potential targets of antiviral drugs for the treatment of orthopoxvirus replication are discussed. ► Progress with effective drugs that are currently in clinical trials, including CMX001 and ST-246 is summarized.Investments in the development of new drugs for orthopoxvirus infections have fostered new avenues of research, provided an improved understanding of orthopoxvirus biology and yielded new therapies that are currently progressing through clinical trials. These broad-based efforts have also resulted in the identification of new inhibitors of orthopoxvirus replication that target many different stages of viral replication cycle. This review will discuss progress in the development of new anti-poxvirus drugs and the identification of new molecular targets that can be exploited for the development of new inhibitors. The prototype of the orthopoxvirus group is vaccinia virus and its replication cycle will be discussed in detail noting specific viral functions and their associated gene products that have the potential to serve as new targets for drug development. Progress that has been achieved in recent years should yield new drugs for the treatment of these infections and might also reveal new approaches for antiviral drug development with other viruses.
Keywords: Orthopoxvirus; Nucleoside analog; CMX001; ST-246; Cidofovir; Drug targets
Expression of an antiviral protein from Lonomia obliqua hemolymph in baculovirus/insect cell system by A.C.V. Carmo; D.N.S. Giovanni; T.P. Corrêa; L.M. Martins; R.C. Stocco; C.A.T. Suazo; R.H.P. Moraes; A.B.G. Veiga; R.Z. Mendonça (pp. 126-130).
► We have demonstrated an antiviral effect in the hemolymph of Lonomia obliqua caterpillars. ► We have observed that this recombinant protein has a potent antiviral action. ► This protein was performed in a baculovirus/Sf-9 system. ► The recombinant protein was able to reduce the picornavirus by at least 4 logs.The control of viral infections, mainly those caused by influenza viruses, is of great interest in Public Health. Several studies have shown the presence of active properties in the hemolymph of arthropods, some of which are of interest for the development of new pharmacological drugs. Recently, we have demonstrated the existence of a potent antiviral property in the hemolymph of Lonomia obliqua caterpillars. The aim of this study was to produce an antiviral protein in a baculovirus/Sf9 cell system. The resulting bacmid contains the sequence coding for the antiviral protein previously described by our group. Total RNA from L. obliqua caterpillars was extracted with Trizol and used in the reverse transcription assay with oligo(d)T primer followed by polymerase chain reactions (RT-PCR) with specific primers for the cDNA coding for the antiviral protein, based on the sequence deposited in the GenBank database. Restriction sites were inserted in the cDNA for ligation in the donor plasmid pFastBac1™. The recombinant plasmid was selected in Escherichia coli DH5α and subsequently used in the transformation of E. coli DH10Bac for the construction of the recombinant bacmid. This bacmid was used for the expression of the antiviral protein in the baculovirus/Sf9 cell system. After identifying the protein by western blot, activity tests were performed, showing that the purified recombinant protein was able to significantly reduce viral replication (about 4 logs). Studies on the optimization of the expression system for the production of this antiviral protein in insect cells are in progress.
Keywords: Antiviral; Insect cells; Baculovirus; Antiviral protein; Hemolymph; Lonomia obliqua
An engineered inhibitor RNA that efficiently interferes with hepatitis C virus translation and replication by Cristina Romero-López; Beatriz Berzal-Herranz; Jordi Gómez; Alfredo Berzal-Herranz (pp. 131-138).
► We analyse the anti-HCV potential of the RNA molecule HH363-24. ► It cleaves the HCV genome and binds to the essential domain IIId of the IRES region. ► HH363-24 blocks the assembly of translationally active ribosomal complexes. ► The inhibitor interferes with viral translation and replication in cell culture.Hepatitis C virus (HCV) translation is mediated by a highly conserved internal ribosome entry site (IRES), mainly located at the 5′untranslatable region (5′UTR) of the viral genome. Viral protein synthesis clearly differs from that used by most cellular mRNAs, rendering the IRES an attractive target for novel antiviral compounds. The engineering of RNA compounds is an effective strategy for targeting conserved functional regions in viral RNA genomes. The present work analyses the anti-HCV potential of HH363-24, an in vitro selected molecule composed of a catalytic RNA cleaving domain with an extension at the 3′ end that acts as aptamer for the viral 5′UTR. The engineered HH363-24 efficiently cleaved the HCV genome and bound to the essential IIId domain of the IRES region. This action interfered with the proper assembly of the translationally active ribosomal particles 48S and 80S, likely leading to effective inhibition of the IRES function in a hepatic cell line. HH363-24 also efficiently reduced HCV RNA levels up to 70% in a subgenomic replicon system. These findings provide new insights into the development of potential therapeutic strategies based on RNA molecules targeting genomic RNA structural domains and highlight the feasibility of generating novel engineered RNAs as potent antiviral agents.
Keywords: Abbreviations; α-IFN; alpha interferon; HCV; hepatitis C virus; IRES; internal ribosome entry site; UTR; untranslatable regionAntiviral tools; Anti-HCV compounds; Aptamer; HCV targeting; RNA-based inhibitors
Antiviral effects of Psidium guajava Linn. (guava) tea on the growth of clinical isolated H1N1 viruses: Its role in viral hemagglutination and neuraminidase inhibition by Nongluk Sriwilaijaroen; Syuichi Fukumoto; Kenji Kumagai; Hiroaki Hiramatsu; Takato Odagiri; Masato Tashiro; Yasuo Suzuki (pp. 139-146).
► Guava tea has anti-2009 pandemic and drug-resistant influenza A viruses in cells. ► Guava tea showed markedly higher anti-influenza activities than did green tea. ► Guava tea has a dual action against both viral HA and NA spike functions.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 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 IC50 of 0.05% and the growth of A/Yamaguchi/20/06 (sensitive strain) and A/Kitakyushu/10/06 (oseltamivir-resistant strain) at similar IC50 values ranging from 0.24% to 0.42% in AX4 cells, being 3.4- to 5.4-fold more potent than green tea (IC50 values: 0.27% for the 2009 pandemic strain and 0.91% to 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 (IC50 of 3.83nM) and A/Yamaguchi/20/06 (IC50 of 11.57nM) but not against that of A/Kitakyushu/10/06 bearing a His274-to-Tyr substitution (IC50 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.
Keywords: Psidium guajava; Linn.; Influenza; Oseltamivir resistance; Pandemic; Neuraminidase; Hemagglutination
EPs® 7630 (Umckaloabo®), an extract from Pelargonium sidoides roots, exerts anti-influenza virus activity in vitro and in vivo by Linda L. Theisen; Claude P. Muller (pp. 147-156).
► EPs® 7630 has a robust effect against multiple different IAV strains in vitro. ► EPs® 7630 inhibits an early step of infection and acts on virus and host cell. ► Prodelphinidins constitute the active antiviral principle. ► Anti-influenza efficacy goes in parallel to the prodelphinidin chain length. ► EPs® 7630 administered by inhalation protects mice against a lethal virus challenge.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 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.
Keywords: Abbreviations; EC; 50; half maximal effective concentration; IAV; influenza A virus; CC; 50; half maximal cytotoxic concentration; MDCK; Madin-Darby canine kidney; MLD; 50; half maximal mouse lethal dose; MOI; multiplicity of infection; TCID; 50; half maximal tissue culture infectious doseInfluenza A virus; Pelargonium sidoides; Polyphenol; Gallocatechin; Antiviral; Prodelphinidin
Viral genome RNA degradation by sequence-selective, nucleic-acid hydrolyzing antibody inhibits the replication of influenza H9N2 virus without significant cytotoxicity to host cells by Aeyung Kim; Ja-Yeong Lee; Sung June Byun; Myung-Hee Kwon; Yong-Sung Kim (pp. 157-167).
Display Omitted► NP-encoding vRNA (NP-vRNA) of H9N2 virus is targeted for degradation by antibody. ► NP25 antibody selectively hydrolyzes a 18-nt ssDNA derived from the NP-vRNA. ► The expression of NP25 does not show any significant cytotoxicity to the host cells. ► NP25 potently inhibits the replication of H9N2 virus in the stable cell lines. ► NP25 selectively hydrolyzes the targeted NP-vRNA in the stable cell lines.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 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 asNP18, 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, asNP18-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 asNP18 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.
Keywords: Influenza A virus; H9N2 virus; Genome targeting; Nucleocapsid protein; Catalytic antibody; Protein engineering
Polyfunctional CD8+ T cells are associated with the vaccination-induced control of a novel recombinant influenza virus expressing an HCV epitope by Amabel C.L. Tan; Emily M.Y. Eriksson; Katherine Kedzierska; Georgia Deliyannis; Sophie A. Valkenburg; Weiguang Zeng; David C. Jackson (pp. 168-178).
► Lipopeptide vaccination elicits polyfunctional CD8+ T cells at multiple organ sites. ► The T cell receptor repertoire of the NS5B-specific response was narrow and “public”. ► Vaccination protects against a recombinant influenza virus. ► Polyfunctional CD8+ T cells are associated with control of the recombinant HCV influenza virus.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 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 NS5B2594–2602 epitope from non-structural protein 5B of hepatitis C virus (PR8-HCVNS5B). We showed that mice inoculated with a lipopeptide containing the NS5B epitope had reduced viral loads following challenge with the PR8-HCVNS5B virus. This reduction was associated with the induction of NS5B2594–2602-specific IFN-γ and TNF-α co-producing CD8+ T cells. The T cell receptor usage in the NS5B2594–2602 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.
Keywords: Abbreviations; HCV; hepatitis C virus; ICS; intracellular cytokine staining; i.n.; intranasal; IAV; influenza A virus; LP; lipopeptideHepatitis C virus; Lipopeptide vaccination; CD8; +; T cells; Liver
Inhibition of Japanese encephalitis virus entry into the cells by the envelope glycoprotein domain III (EDIII) and the loop3 peptide derived from EDIII by 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 (pp. 179-183).
► Recombinant JEV EDIII was expressed and purified. ► The purified EDIII inhibits JEV infection with IC50 at 25μg/ml. ► Loop3 peptide derived from EDIII inhibits JEV infection with IC50 at 10μM. ► Loop3 peptide exerts protective ability on mice experiment.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 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.
Keywords: Japanese encephalitis virus; Envelope protein domain III; Antiviral peptide; Attachment
Long-term monitoring drug resistance by ultra-deep pyrosequencing in a chronic hepatitis B virus (HBV)-infected patient exposed to several unsuccessful therapy schemes by M. Sede; D. Ojeda; L. Cassino; G. Westergaard; M. Vazquez; S. Benetti; F. Fay; H. Tanno; J. Quarleri (pp. 184-187).
► This is 11-year longitudinal analysis of HBV genomics by ultra-deep pyrosequencing. ► Persistent HBV viremia on tenofovir without HBV mutations that reduce the drug-sensitivity. ► The UDPS allowed for low-prevalence HBV variants detection. ► HBV-S gene mutants associated with immune evasion were detected. ► First study describing the dynamics of HBV drug-resistance mutations by UDPS.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 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.
Keywords: HBV; UDPS; Resistance; NRTI
Natural compounds inhibiting the replication of Porcine reproductive and respiratory syndrome virus by Anbu K. Karuppannan; Kan Xing Wu; Jia Qiang; Justin Jang-Hann Chu; Jimmy Kwang (pp. 188-194).
► Natural compounds inhibiting Porcine reproductive and respiratory syndrome virus replicative cycle. ► Ion transport is important for Porcine reproductive and respiratory syndrome virus replicative cycle. ► Sub micro molar concentration inhibitors of Porcine reproductive and respiratory syndrome virus.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 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.
Keywords: PRRSV; Natural product compounds; Viral inhibitor