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Antiviral Research (v.89, #1)

Editorial Board (pp. ii).

Notes to Authors (pp. co3).

Cellular IMPDH enzyme activity is a potential target for the inhibition of Chikungunya virus replication and virus induced apoptosis in cultured mammalian cells by M. Khan; R. Dhanwani; I.K. Patro; P.V.L. Rao; M.M. Parida (pp. 1-8).

Inosine monophosphate dehydrogenase (IMPDH) catalyzes an essential step in the de novo biosynthesis of guanine nucleotide, namely, the conversion of IMP to XMP. The depletion of the intracellular GTP and dGTP pools is the major event occurring in the cells exposed to the inhibitors such as mycophenolic acid. The present study was undertaken with an objective to assess the antiviral potential of mycophenolic acid (MPA) against Chikungunya virus via inhibition of IMPDH enzyme in Vero cells. The inhibitory potential of MPA on CHIKV replication was assessed by virus inhibition assay (cytopathic effect, immunofluorescence), virus yield reduction assay and cell viability assay. Inhibition of virus induced apoptosis was analyzed by Hoechst staining, DNA fragmentation, immunoblotting of Caspase-3, PARP and Bcl-2. Percentage apoptotic cell population was determined by flow cytometry. Total genome infectivity was determined by analyzing the ratio of total infectious viral particles to the genome copy number. Non-toxic concentration of MPA (10μM) reduced ≥99.9% CHIKV titre in Vero cells. MPA via depletion of substrate for polymerase (GTP), inhibited CHIKV induced apoptosis. By limiting the rate of de novo synthesis of guanosine nucleotide, MPA could apparently block the formation of the CHIKV progeny. The antiviral activity of MPA against Chikungunya virus is mediated through depletion of GTP pool via inhibition of IMPDH as demonstrated by Immunoblotting and different microscopic analysis.

Keywords: Mycophenolic acid; Chikungunya; Virus inhibition; Apoptosis

Oligomeric proanthocyanidins from Rumex acetosa L. inhibit the attachment of herpes simplex virus type-1 by Kirsten Gescher; Andreas Hensel; Wali Hafezi; Andrea Derksen; Joachim Kühn (pp. 9-18).

The polyphenole-enriched acetone–water extract R2 from the aerial parts of Rumex acetosa L. containing high amounts of oligomeric and polymeric proanthocyanidins and flavonoids was tested for antiviral activity. R2 exhibited strong antiviral activity against herpes simplex virus type-1 (HSV-1) while the replication of adenovirus 3 was not affected. By plaque reduction test and MTT assay on Vero cells, the HSV-1-specific inhibitory concentration (IC₅₀) and cytotoxic concentration (CC₅₀) were determined. R2 exibited an IC₅₀ of 0.8μg/mL and a selectivity index (SI) (ratio of IC₅₀ to CC₅₀) of approximately 100 when added to the virus inoculum for 1h at 37°C prior to infection. The antiviral activity was due to the presence of flavan-3-ols and oligomeric proanthocyanidins in the extract. Structure–activity analyses indicated that flavan-3-ols and proanthocyanidins with galloylation at position O-3 are highly potent compounds (SI>40), while ungalloylated compounds did not exhibit antiviral effects (SI<1).R2 and a major proanthocyanidin from R2, epicatechin-3-O-gallate-(4β→8)-epicatechin-3-O-gallate abolished virus entry into the host cell by blocking attachment to the cell surface. When added after attachment at a concentration of ≥12.5μg/mL, R2 inhibited also penetration of HSV-1 into the host cell. R2 and epicatechin-3-O-gallate-(4β→8)-epicatechin-3-O-gallate were shown to directly interact with viral particles leading to the oligomerisation of envelope proteins as demonstrated for the essential viral glycoprotein gD.Using raft cultures with three-dimensional organotypic human skin equivalents it was shown that treatment of cultures with R2 after infection with HSV-1 resulted in a reduced viral spread.

Keywords: Rumex acetosa; Proanthocyanidins; Epicatechin-3-O-gallate; Galloylation; Herpes simplex virus type-1; Attachment; Penetration; Organotypic skin model; Antiviral

Sensitivity of seven HIV subtyping tools differs among subtypes/recombinants in the Spanish cohort of naïve HIV-infected patients (CoRIS) by Gonzalo Yebra; Miguel de Mulder; Leticia Martín; Santiago Pérez-Cachafeiro; Carmen Rodríguez; Pablo Labarga; Federico García; Cristina Tural; Àngels Jaén; Gemma Navarro; Á. Holguín (pp. 19-25).

HIV-1 group M is classified into 9 subtypes and recombinants (CRFs/URFs). Variants other than subtype B (non-B) cause 90% of infections worldwide. HIV is often subtyped using automated tools instead of the gold-standard phylogenetic analysis. We evaluated the reliability of subtyping tools vs. phylogeny in a panel of HIV-1 pol sequences from the cohort of naïve patients of the HIV/AIDS Spanish Research Network (CoRIS).HIV-1 subtyping was performed using seven automated subtyping tools (Stanford, Geno2pheno, Rega, NCBI, EuResist, STAR, TherapyEdge) in HIV-1 pol sequences from 670 CoRIS patients previously subtyped by phylogeny (587 subtype B/83 non-B). Sensitivity with respect to phylogeny was assessed.Most tools correctly classified subtype B, although up to 15% of non-B sequences were wrongly identified as B depending on the tool. For subtype B and CRF02_AG identification, Stanford/NCBI and Geno2pheno/Rega presented the highest/lowest sensitivities, respectively. EuResist and Geno2pheno correctly classified all 13 non-B “pure”subtypes at pol. The efficacy of all subtyping tools dropped clearly when identifying recombinants different from CRF02_AG. Only NCBI05, Rega and STAR identified URF, but with very low sensitivities. NCBI classified the highest number of subtypes B as non-B, and overestimated recombinants, especially when including references of 2009.Automated tools are useful for subtype B identification, although they present serious limitations in classifying variants uncommon in developed regions, especially recombinants. Their sensitivity depends on the prevalence of non-B variants in the population, and decreases drastically when the frequency of recombinants increases. Furthermore, HIV-1 variant distribution differs according to the tool used.

Keywords: HIV-1 non-B subtypes; Subtyping tools; Phylogenetic analysis; Recombinants

Combination of α-glucosidase inhibitor and ribavirin for the treatment of dengue virus infection in vitro and in vivo by Jinhong Chang; Wouter Schul; Terry D. Butters; Andy Yip; Boping Liu; Anne Goh; Suresh B. Lakshminarayana; Dominic Alonzi; Gabriele Reinkensmeier; Xiaoben Pan; Xiaowang Qu; Jessica M. Weidner; Lijuan Wang; Wenquan Yu; Nigel Borune; Mark A. Kinch; Jamie E. Rayahin; Robert Moriarty; Xiaodong Xu; Pei-Yong Shi; Ju-Tao Guo; Timothy M. Block (pp. 26-34).

Cellular α-glucosidases I and II are enzymes that sequentially trim the three terminal glucoses in the N-linked oligosaccharides of viral envelope glycoproteins. This process is essential for the proper folding of viral glycoproteins and subsequent assembly of many enveloped viruses, including dengue virus (DENV). Imino sugars are substrate mimics of α-glucosidases I and II. In this report, we show that two oxygenated alkyl imino sugar derivatives, CM-9-78 and CM-10-18, are potent inhibitors of both α-glucosidases I and II in vitro and in treated animals, and efficiently inhibit DENV infection of cultured human cells. Pharmacokinetic studies reveal that both compounds are well tolerated at doses up to 100mg/kg in rats and have favorable pharmacokinetic properties and bioavailability in mice. Moreover, we showed that oral administration of either CM-9-78 or CM-10-18 reduces the peak viremia of DENV in mice. Interestingly, while treatment of DENV infected mice with ribavirin alone did not reduce the viremia, combination therapy of ribavirin with sub-effective dose of CM-10-18 demonstrated a significantly enhanced antiviral activity, as indicated by a profound reduction of the viremia. Our findings thus suggest that combination therapy of two broad-spectrum antiviral agents may provide a practically useful approach for the treatment of DENV infection.

Keywords: Abbreviations; DENV; dengue virus; ER; endoplasmic reticulum; DNJ; deoxynojirimycin; BVDV; bovine viral diarrhea virus; FOS; free oligosaccharide; IC; ₅₀; 50% inhibitory concentration; EC; ₅₀; 50% effective concentration; CC; ₅₀; 50% cytotoxicity concentration; CFI; cell-based flavivirus immunodetection; PK; pharmacokineticα-Glucosidase; Imino sugar; Dengue virus; Ribavirin; Combination

Anti-hepatitis C virus activity of Acacia confusa extract via suppressing cyclooxygenase-2 by Jin-Ching Lee; Wei-Chun Chen; Shou-Fang Wu; Chin-kai Tseng; Ching-Yi Chiou; Fang-Rong Chang; Shih-hsien Hsu; Yang-Chang Wu (pp. 35-42).

Chronic hepatitis C virus (HCV) infection continues to be an important cause of morbidity and mortality by chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC) throughout the world. It is of tremendous importance to discover more effective and safer agents to improve the clinical treatment on HCV carriers. Here we report that the n-butanol–methanol extract obtained from Acacia confusa plant, referred as ACSB-M4, exhibited the inhibition of HCV RNA replication in the HCV replicon assay system, with an EC₅₀ value and CC₅₀/EC₅₀ selective index (SI) of 5±0.3μg/ml and >100, respectively . Besides, ACSB-M4 showed antiviral synergy in combination with IFN-α and as HCV protease inhibitor (Telaprevir; VX-950) and polymerase inhibitor (2′- C-methylcytidine; NM-107) by a multiple linear logistic model and isobologram analysis. A complementary approach involving the overexpression of COX-2 protein in ACSB-M4-treated HCV replicon cells was used to evaluate the antiviral action at the molecular level. ACSB-M4 significantly suppressed COX-2 expression in HCV replicon cells. Viral replication was gradually restored if COX-2 was added simultaneously with ACSB-M4, suggesting that the anti-HCV activity of ACSB-M4 was associated with down-regulation of COX-2, which was correlated with the suppression of nuclear factor-kappaB (NF-κB) activation. ACSB-M4 may serve as a potential protective agent for use in the management of patients with chronic HCV infection.

Keywords: Acacia confusa; Hepatitis C virus; Cyclooxygenase-2; Nuclear factor-kappaB

Long-term inhibition of HIV-1 replication with RNA interference against cellular co-factors by Julia J.M. Eekels; Dirk Geerts; Rienk E. Jeeninga; Ben Berkhout (pp. 43-53).

In this study we tested whether HIV-1 replication could be inhibited by stable RNAi-mediated knockdown of cellular co-factors. Cell lines capable of expressing shRNAs against 30 candidate co-factors implicated at different steps of the viral replication cycle were generated and analyzed for effects on cell viability and inhibition of HIV-1 replication. For half of these candidate co-factors we obtained knockdown cell lines that are less susceptible to virus replication. For three co-factors (ALIX, ATG16 and TRBP) the cell lines were resistant to HIV-1 replication for up to 2 months. With these cells we could test the hypothesis that HIV-1 is not able to escape from RNAi-mediated suppression of cellular co-factors, which was indeed not detected.

Keywords: HIV-1; Cellular co-factors; RNAi; shRNA; Gene silencing

Measuring antiviral activity of benzimidazole molecules that alter IRES RNA structure with an infectious hepatitis C virus chimera expressing Renilla luciferase by Shuanghu Liu; Cassie A. Nelson; Li Xiao; Ling Lu; Punit P. Seth; Darrell R. Davis; Curt H. Hagedorn (pp. 54-63).

Major progress has been made in developing infectious HCV cell culture systems and these systems have been useful in identifying novel HCV antivirals. However, more rapid and sensitive assays using infectious cell based HCV systems would facilitate the development of additional antivirals, including small molecules directed at unique targets such as the HCV RNA internal ribosomal entry site (IRES). We have found that the V3 region (28 aa) of NS5A of HCV JFH1 can be deleted from the genome with only modest effects on the titer of infectious virus produced in cell culture. Moreover, the V3 region can be replaced with the Renilla reniformis luciferase (Rluc) gene resulting in an infectious virus that stably expresses an NS5A–Rluc fusion protein. Infected cells cultured in 96-well plates provided a robust luciferase signal that accurately reflected the production of infectious virus. This infectious HCV reporter system was used to test the activity of three benzimidazole compounds that bind the HCV RNA IRES. Compounds in this chemical class of small molecules bind and alter the IRES RNA structure at low to sub-micromolar concentrations and interfere with viral replication. The current study shows that these compounds inhibit HCV replication in an infectious HCV cell culture system, defines their IC₅₀ in this system, and provides a platform for the rapid testing of next generation inhibitors.

Keywords: Abbreviations; Rluc; Renilla reniformis; luciferase; RLU; relative light units; IFA; immunofluorescence assayHepatitis C; HCV; IRES; Benzimidazole; Renilla; luciferase; NS5A

Inhibition of poxvirus spreading by the anti-tumor drug Gefitinib (Iressa™) by Stefan Langhammer; Robert Koban; Constanze Yue; Heinz Ellerbrok (pp. 64-70).

The threat of smallpox virus as a bioterrorist weapon is raising international concerns again since the anthrax attacks in the USA in 2001. The medical readiness of treating patients suffering from such infections is a prerequisite of an effective civil defense system. Currently the only therapeutic option for the treatment of poxvirus infections relies on the virostatic nulceosid analog cidofovir, although severe side effects and drug resistant strains have been described. A growing understanding of poxvirus pathogenesis raises the possibility to explore other appropriate targets involved in the viral replication cycle. Poxvirus encoded growth factors such as the Vaccinia Growth Factor (VGF) stimulate host cells via the Epidermal Growth Factor Receptor (EGFR) and thereby facilitate viral spreading. In this study we could visualize for the first time the paracrine priming of uninfected cells for subsequent infection by orthopoxviruses directly linked to EGFR phosphorylation. Since EGFR is a well known target for anti-tumor therapy small molecules for inhibition of its tyrosine kinase (TK) activity are readily available and clinically evaluated. In this study we analyzed three different EGFR receptor tyrosine kinase inhibitors for inhibition of orthopoxvirus infection in epithelial cells. The inhibitor shown to be most effective was Gefitinib (Iressa™) which is already approved as a drug for anti-tumor medication in the USA and in Europe. Thus Gefitnib may provide a new therapeutic option for single or combination therapy of acute poxvirus infections, acting on a cellular target and thus reducing the risk of viral resistance to treatment.

Keywords: Bioterrorism; Antiviral therapy; Smallpox; Orthopoxvirus; Receptor; Tyrosine kinase inhibitors

Viral entry inhibitors block dengue antibody-dependent enhancement in vitro by Cindo O. Nicholson; Joshua M. Costin; Dawne K. Rowe; Li Lin; Ekachai Jenwitheesuk; Ram Samudrala; Sharon Isern; Scott F. Michael (pp. 71-74).

Severe dengue virus (DENV) disease symptoms, including dengue hemorrhagic fever and dengue shock syndrome, have been correlated with the presence of pre-existing antibodies that enhance rather than neutralize infections in Fc receptor bearing cells. These antibodies can originate from previous infection with a different serotype of dengue, or from waning antibody titers that occur in infants and young children as they are weaned from breast milk that contains protective dengue-specific antibodies. Despite the apparent importance of this antibody dependent enhancement (ADE) effect, there has been no description of any specific inhibitors of this process. We explored DENV entry inhibitors as a potential strategy to block ADE. Two different peptide entry inhibitors were tested for the ability to block antibody-mediated DENV-2 infection of human, FcRII bearing K562 cells in vitro. Both peptides were able to inhibit ADE, showing that entry inhibitors are possible candidates for the development of specific treatment for severe DENV infection.

Keywords: Abbreviations; DENV; dengue virus; DMEM; Dulbecco's modified Eagle's medium; DMSO; dimethylsulfoxide; FBS; fetal bovine serum; FFU; focus forming unit; PBS; phosphate buffered saline; qRT-PCR; quantitative reverse transcriptase polymerase chain reactionDengue; Antibody; Enhancement; Peptide; Entry; Inhibitor

Single-dose intranasal administration with mDEF201 (adenovirus vectored mouse interferon-alpha) confers protection from mortality in a lethal SARS-CoV BALB/c mouse model by Yohichi Kumaki; Jane Ennis; Ramtin Rahbar; Jeffrey D. Turner; Miles K. Wandersee; Aaron J. Smith; Kevin W. Bailey; Zachary G. Vest; Jason R. Madsen; Joseph K.-K. Li; Dale L. Barnard (pp. 75-82).

Interferons (IFNs) are a first line of defense against viral infection. Herein we describe the use of an adenovirus vectored mouse IFN alpha gene (mDEF201) as a prophylactic and treatment countermeasure in a SARS-CoV-infected BALB/c mouse model. Complete survival protection was observed in mice given a single dose of mDEF201 administered intranasally 1, 3, 5, 7, or 14 days prior to lethal SARS-CoV challenge ( p<0.001), and body weights of these treated mice were unaffected by the challenge. In addition, low doses of mDEF201 protected lungs in a dose dependent manner as measured by a reduction in gross pathology. Intranasal treatment with mDEF201 ranging from 10⁶ to 10⁸PFU significantly protected mice against a lethal SARS-CoV infection in a dose dependent manner up to 12h post infection ( p<0.001). The data suggest that mDEF201 is a new class of antiviral agent further development as treatment for SARS-CoV infections.

Keywords: Interferon; mDEF201; SARS; Treatment; Prophylaxis

Activity of Melaleuca alternifolia (tea tree) oil on Influenza virus A/PR/8: Study on the mechanism of action by A. Garozzo; R. Timpanaro; A. Stivala; G. Bisignano; A. Castro (pp. 83-88).

Our previous study demonstrated that Melaleuca alternifolia (tea tree) oil (TTO) had an interesting antiviral activity against Influenza A in MDCK cells. In fact, when we tested TTO and some of its components, we found that TTO had an inhibitory effect on influenza virus replication at doses below the cytotoxic dose; terpinen-4-ol, terpinolene, and alfa-terpineol were the main active components.The aim of this study was to investigate the mechanism of action of TTO and its active components against Influenza A/PR/8 virus subtype H1N1 in MDCK cells.None of the test compounds showed virucidal activity nor any protective action for the MDCK cells. Thus, the effect of TTO and its active components on different steps of the replicative cycle of influenza virus was studied by adding the test compounds at various times after infection. These experiments revealed that viral replication was significantly inhibited if TTO was added within 2h of infection, indicating an interference with an early step of the viral replicative cycle of influenza virus.The influence of the compound on the virus adsorption step, studied by the infective center assay, indicated that TTO did not interfere with cellular attachment of the virus.TTO did not inhibit influenza virus neuraminidase activity, as shown by the experiment measuring the amount of 4-methylumbelliferone, cleaved by the influenza virus neuraminidase from the fluorogenic substrate 2′- O-(4-methylumbelliferyl)- N-acetylneuraminic acid.The effect of TTO on acidification of cellular lysosomes was studied by vital staining with acridine orange using bafilomycin A1 as positive control. The treatment of cells with 0.01% (v/v) of TTO at 37°C for 4h before staining inhibited the acridine orange accumulation in acid cytoplasmic vesicles, indicating that TTO could inhibit viral uncoating by an interference with acidification of intralysosomal compartment.

Keywords: Melaleuca alternifolia; Essential oil; Antiviral activity; Influenza virus

Lung surfactant DPPG phospholipid inhibits vaccinia virus infection by Julien Perino; David Crouzier; Danièle Spehner; Jean-Claude Debouzy; Daniel Garin; Jean-Marc Crance; Anne-Laure Favier (pp. 89-97).

Vaccinia virus (VACV) was used as a surrogate of Variola virus (genus Orthopoxvirus), the causative agent of smallpox, to study orthopoxvirus infection via the respiratory airway. Lung surfactant, a physiological barrier to infection encountered by the virus, is predominantly composed of phospholipids whose role during orthopoxvirus infection has not been investigated. An attenuated Lister strain, derived from the traditional smallpox vaccine and the Western Reserve (WR) strain, lethal for mice infected by the respiratory route, were examined for their ability to bind various surfactant phospholipids. Dipalmitoyl phosphatidylglycerol (DPPG) was found to interact with both VACV strains. DPPG incorporated in small unilamellar vesicle (SUV-DPPG) inhibited VACV cell infection, unlike other phospholipids tested. Both pre-incubation of virus with SUV-DPPG and pretreatment of the cell with SUV-DPPG inhibited cell infection. This specific DPPG effect was shown to be concentration and time dependent and to prevent the first step of the viral cycle, i.e. virus cell attachment. Cryo-electron microscopy highlighted the interaction between the virus and SUV-DPPG. In the presence of the phospholipid, virus particles displayed a hedgehog-like appearance due to the attachment of lipid vesicles. Mice infected intranasally with VACV-WR pre-incubated with SUV-DPPG survived a lethal infection. These data suggest that DPPG in lung surfactant could reduce the amount of orthopoxvirus particles able to infect pneumocytes at the beginning of a respiratory poxvirus infection. The knowledge acquired during this study of virus–DPPG interactions may be used to develop novel chemotherapeutic strategies for smallpox.

Keywords: Abbreviations; DPPG; dipalmitoyl-phosphatidylglycerol; DPPC; dipalmitoyl-phosphatidylcholine; PC; phosphatidylcholine; VACV; vaccinia virus; VARV; Variola virus; SUV; small unilamellar vesicleOrthopoxvirus; Phospholipids; Dipalmitoyl-phosphatidylglycerol; Pulmonary surfactant; Lung; Mice

Pentagalloylglucose downregulates cofilin1 and inhibits HSV-1 infection by Ying Pei; Yang-Fei Xiang; Jia-Nan Chen; Chun-Hua Lu; Jing Hao; Qian Du; Chi-choi lai; Chang Qu; Shen Li; Huai-Qiang Ju; Zhe Ren; Qiu-Ying Liu; Sheng Xiong; Chui-Wen Qian; Fan-Li Zeng; Pei-Zhuo Zhang; Chong-Ren Yang; Ying-Jun Zhang; Jun Xu; Kaio Kitazato; Yi-Fei Wang (pp. 98-108).

To investigate the anti-herpesvirus mechanism of pentagalloylglucose (PGG), we compared the proteomic changes between herpes simplex virus type 1 (HSV-1) infected MRC-5 cells with or without PGG-treatment, and between non-infected MRC-5 cells with or without PGG-treatment by 2-DE and MS-based analysis. Differentially expressed cellular proteins were mainly involved with actin cytoskeleton regulation. Significantly, PGG can down-regulate cofilin1, a key regulator of actin cytoskeleton dynamics. PGG can inhibit HSV-1-induced rearrangements of actin cytoskeleton which is important for infectivity. Furthermore, cofilin1 knockdown by siRNA also inhibited the HSV-1-induced actin-skeleton rearrangements. Both PGG-treatment and cofilin1 knockdown can reduce HSV-1 DNA, mRNA, protein synthesis and virus yields. Altogether, the results suggested that down-regulating cofilin1 plays a role in PGG inhibiting HSV-1 infection. PGG may be a promising anti-herpesvirus agent for drug development.

Keywords: Herpes simplex virus type 1; Pentagalloylglucose; Two-dimensional gel electrophoresis; Actin cytoskeleton; Cofilin1

Artificial recombinant cell-penetrating peptides interfere with envelopment of hepatitis B virus nucleocapsid and viral production by Xiao-Ben Pan; Lai Wei; Jin-Chao Han; Hui Ma; Kai Deng; Xu Cong (pp. 109-114).

Hepatitis B virus (HBV) is a major human infectious pathogen, with over 300 million chronically infected patients worldwide. Current therapeutics for chronic HBV infection have shown only limited success. The plasma membrane represents an impermeable barrier for development of most macromolecular antiviral agents. To develop new anti-HBV macromolecules that can cross the membrane barrier, we designed a series of artificial recombinant peptides including cell penetrating sequence oligoarginine R7 and several nucleocapsid binding subunits (NBS). The anti-HBV function of these peptides was evaluated in a HBV DNA replicative cell line HepG2.2.15. Our results showed that the synthetic recombinant cell penetrating peptides retained the activity of cell penetrating in the living cells. HBV DNA in culture medium markedly decreased in cells treated with cell penetrating peptides bearing NBS for three days. Intracellular HBcAg and HBV DNA replicative intermediates increased by 2–3 fold. In conclusion, the synthetic recombinant cell penetrating peptides bearing NBS can efficiently enter into the cells; block nucleocapsid assembly and inhibit HBV release. Cell penetrating subunit presents a high efficiency tool to deliver synthetic antiviral peptides into cells.

Keywords: Abbreviations; HBV; hepatitis B virus; cccDNA; covalently closed circular DNA; pgRNA; pregenomic RNA; HPLC; high-performance liquid chromatography; NBS; nucleocapsid binding subunits; HBsAg; hepatitis B surface antigen; R7; RRRRRRR; FQPCR; fluorescence quantitative PCRHepatitis B virus; Nucleocapsid; Cell penetrating peptide; Assembly; Antivirus

Genotypic and phenotypic resistance of pandemic A/H1N1 influenza viruses circulating in Germany by Susanne C. Duwe; Marianne Wedde; Patricia Birkner; Brunhilde Schweiger (pp. 115-118).

In response to the rapid global spread of an antigenically novel A/H1N1 influenza virus in 2009, the World Heath Organization (WHO) recommended surveillance and monitoring for antiviral resistance of influenza viruses. We designed and evaluated pyrosequencing (PSQ)-based genotypic assays for high-throughput analysis of the susceptibility of pandemic A/H1N1 influenza viruses to neuraminidase (NA) inhibitors. A total of 1570 samples circulating in Germany between April 2009 and April 2010 were tested for determination of molecular markers of resistance to the NA inhibitors oseltamivir and zanamivir, and 635 of them were evaluated by phenotypic fluorescence-based assay with MUNANA substrate. Eight (0.5%) viruses were resistant to oseltamivir due to the H274Y NA substitution (N2 numbering). Six of these oseltamivir-resistant cases were treatment-related; four of them were selected in immunocompromised patients, two in patients suffered from chronic diseases. The two remaining oseltamivir-resistant viruses seem to have evolved in the absence of drug treatment and were isolated from immunocompetent healthy patients. All tested A/H1N1 pandemic viruses were sensitive to zanamivir. In addition, analysis of 1011 pandemic A/H1N1 virus samples by a PSQ-based assay according to the WHO protocol revealed the presence of mutation S31N in the M2 protein that conferred resistance to M2 ion channel inhibitors. Our data demonstrate a low incidence of oseltamivir-resistant pandemic A/H1N1 influenza variants isolated under drug selection pressure as well as community-acquired or naturally evolving viruses.

Keywords: Oseltamivir resistance; H274Y; Pandemic A/H1N1 virus; Pyrosequencing; Antiviral drug

Immunization with the transmembrane protein of a retrovirus, feline leukemia virus: Absence of antigenemia following challenge by Stefan Langhammer; Janine Hübner; Oswald Jarrett; Reinhard Kurth; Joachim Denner (pp. 119-123).

A major challenge in the development of vaccines against retroviruses is the induction of neutralizing antibodies since they prevent infection of the cells where the virus may persist. The transmembrane envelope (TM) protein contains highly conserved domains and seems to be a suitable target. To study whether vaccinating with a TM protein of a retrovirus could protect from infection in vivo, cats were immunized with the TM protein p15E of feline leukemia virus (FeLV) and subsequently challenged. For the first time we show that immunization with a retroviral TM protein prevented antigenemia. The level of neutralizing antibodies after the boost immunization correlated with the outcome of FeLV infection.

Keywords: Neutralizing antibodies; Antiretroviral vaccine

CpG oligodeoxynucleotides protect against the 2009 H1N1 pandemic influenza virus infection in a murine model by Tao Jiang; Hui Zhao; Xiao-Feng Li; Yong-Qiang Deng; Juan Liu; Li-Juan Xu; Jian-Feng Han; Rui-Yuan Cao; E-De Qin; Cheng-Feng Qin (pp. 124-126).

The 2009 H1N1 influenza virus pandemic poses a global public health threat, and there is a critical need for antiviral drugs for pandemic control. CpG oligodeoxynucleotides have strong immunostimulatory properties and are expected to be used as prophylactic agents to protect against microbial infections. The present study evaluated the efficacy of synthetic CpG oligodeoxynucleotide (ODN) 1826 against pandemic H1N1 virus infection in a murine model. A single injection of 15μg ODN 1826 intraperitoneally prior to virus challenge inhibits virus replication in lungs, reduces lung lesions and prevents mortality in mice, indicating CpG ODNs as a possible strategy for future influenza pandemics control.

Keywords: CpG oligodeoxynucleotide; H1N1 pandemic; Influenza A virus

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Antiviral Research (v.89, #1)