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Antiviral Research (v.84, #1)

Editorial Board (pp. ii).

Notes to Authors (pp. co3).

Vaccinia virus vaccines: Past, present and future by Bertram L. Jacobs; Jeffrey O. Langland; Karen V. Kibler; Karen L. Denzler; Stacy D. White; Susan A. Holechek; Shukmei Wong; Trung Huynh; Carole R. Baskin (pp. 1-13).

Vaccinia virus (VACV) has been used more extensively for human immunization than any other vaccine. For almost two centuries, VACV was employed to provide cross-protection against variola virus, the causative agent of smallpox, until the disease was eradicated in the late 1970s. Since that time, continued research on VACV has produced a number of modified vaccines with improved safety profiles. Attenuation has been achieved through several strategies, including sequential passage in an alternative host, deletion of specific genes or genetic engineering of viral genes encoding immunomodulatory proteins. Some highly attenuated third- and fourth-generation VACV vaccines are now being considered for stockpiling against a possible re-introduction of smallpox through bioterrorism. Researchers have also taken advantage of the ability of the VACV genome to accommodate additional genetic material to produce novel vaccines against a wide variety of infectious agents, including a recombinant VACV encoding the rabies virus glycoprotein that is administered orally to wild animals. This review provides an in-depth examination of these successive generations of VACV vaccines, focusing on how the understanding of poxviral replication and viral gene function permits the deliberate modification of VACV immunogenicity and virulence.

Keywords: Poxvirus; Orthopoxvirus; Smallpox; Biodefense; Vaccinia virus; Vaccine vector; Vaccination complications; Vaccinia E3L

Novel HCV replication mouse model using human hepatocellular carcinoma xenografts by Carl Guévin; Alain Lamarre; Patrick Labonté (pp. 14-22).

In the absence of an immunocompetent mouse model for HCV replication, we developed a convenient xenograft mouse model that produces infectious viral particles. For this purpose, HCV-permissive tumors were generated in SCID/beige mice using a tumorigenic population of the human hepatocarcinoma-derived Huh7 cell line. Following infection, HCV RNA increased in the mouse sera and the human tumor by up to 10⁵GE/ml and 10⁷GE/μg of RNA, respectively. Immunohistochemistry analysis revealed that active viral replication had taken place within the tumor. Moreover, virus recovered from infected mice sera was readily infectious in cell culture. Finally, we showed that interferon-α and the protease inhibitor BILN-2061 inhibited the cell culture HCVcc strain JFH1 replication in vivo. In conclusion, we developed a simple and inexpensive mouse model that allows the production of infectious HCV particles in vivo. Such a model will be an extremely valuable tool for the characterization of promising drug candidates.

Keywords: HCV; Mouse model; Antiviral; JFH1

A novel combined vaccine candidate containing epitopes of HCV NS3, core and E1 proteins induces multi-specific immune responses in BALB/c mice by Ruihong Zeng; Guangxue Li; Shigan Ling; Heqiu Zhang; Zhiyan Yao; Bingshui Xiu; Feng He; Rui Huang; Lin Wei (pp. 23-30).

Hepatitis C virus (HCV) has emerged as the major pathogen of liver disease worldwide. The mechanisms of HCV infection and interaction with a host are poorly understood. What exactly is required for efficient control of HCV infection is largely unknown. Standard treatment combining interferon-α (IFN-α) and ribavirine is effective in about 50% of the treated patients, however associated with significant toxicity and cost. Therefore, the development of new drugs or vaccines is urgently needed. An efficient vaccine against HCV infection requires induction of broad cellular and humoral immune responses against several viral proteins. We have engineered the combined vaccine candidate mT+mE1, an inclusion of multiple epitopes from HCV NS3, core (C) and E1 proteins. mT contains multiple conserved CD4⁺ and CD8⁺ T cell epitopes from HCV NS3 and C proteins. mE1 is based on eight dominant neutralizing epitopes of E1 protein from six HCV genotypes. In current study, we showed that immunization with mT+mE1 induced high titers of IgG, IgG1 and IgG2a antibodies to mE1, and high level of NS3- or C-specific CTLs. Furthermore, mT+mE1 elicited a Th1-biased immune response with secretion of high amounts of IFN-γ, compared with mT alone. Prophylactic as well as therapeutic administration of mT+mE1 in BALB/c mice led to protecting mice against SP2/0 tumor cells expressing HCV NS3 protein. These results suggested that mT+mE1 elicited strong humoral immune responses and multiple specific cellular immune responses. The vaccine candidate is now being tested in pre-clinical trials.

Keywords: Hepatitis C virus; Combined vaccine; mT; +; mE1; Humoral immune response; Cellular immune response

Inhibition of Junín virus replication by small interfering RNAs by María C. Artuso; Paula C. Ellenberg; Luis A. Scolaro; Elsa B. Damonte; Cybele C. García (pp. 31-37).

Junín virus (JUNV), the etiological agent of the Argentine hemorrhagic fever, has a single-stranded RNA genome with ambisense expression which encodes for five proteins. In previous works we have demonstrated that the Z arenavirus matrix protein represents an attractive target for antiviral therapy. With the aim of studying a new alternative therapeutic mechanism, four Z-specific siRNAs (Z1- to Z4-siRNAs) were tested showing variable efficacy. The most effective inhibitor was Z2-siRNA targeted at the region encompassed by nt 179–197 of Z gene. The efficacy of this Z2-siRNA against JUNV was also demonstrated in virus-infected cells, by testing infectious virus plaque formation (92.8% JUNV yield reduction), viral RNA level or antigen expression, as well as in cells transfected with Z-specific reporter plasmids (91% reduction in expression of Z-EGFP fusion protein). Furthermore, the lack of effect of this Z-siRNA on the expression of other JUNV proteins, such as N and GPC, confirmed the specificity of action exerted by Z2-siRNA on Z transcript. Thus, the present study represents the first report of virus inhibition mediated by RNA interference for a New World arenavirus.

Keywords: Junín virus; Arenavirus; Matrix protein; RNA interference; siRNAs

The candidate sulfonated microbicide, PRO 2000, has potential multiple mechanisms of action against HIV-1 by Dana Huskens; Kurt Vermeire; Albert T. Profy; Dominique Schols (pp. 38-47).

PRO 2000 is a polyanionic compound under development as a topical antimicrobial gel for the potential prevention of HIV-1 transmission. It has been shown that PRO 2000 binds to HIV-1 gp120 and interferes with virus attachment to and/or fusion with CD4⁺ T cells.Here, we demonstrate that PRO 2000 interacts not only with viral gp120 but also with CD4 and CXCR4 receptors on the cell surface. Minor or no effects were noticed on DC-SIGN and on CCR5. PRO 2000 dose-dependently inhibited the interaction of CXCL12 with the CXCR4 receptor as demonstrated with CXCL12^AF647-labeled binding, CXCL12-induced calcium signaling, CXCR4 internalization and chemotaxic assays. These CXCR4 antagonistic properties of PRO 2000 are a potential additional mechanism of action that could explain the observation that PRO 2000 is described to be more active against X4 viruses than R5 viruses.The cellular activation potential and inflammatory properties of PRO 2000 were also examined in PBMCs. PRO 2000 had minor effects on the expression level of several cellular activation markers and enhanced the production of a small number of cytokines/chemokines, as determined by the Bio-Plex human cytokine 27-plex assay system. PRO 2000 showed less mitogenic and stimulatory activity than cyanovirin-N, but careful monitoring for potential side-effects is still advised.

Keywords: PRO 2000; HIV-1; Microbicide; CXCR4; CD4; Cytokines

Identification of allosteric inhibitors blocking the hepatitis C virus polymerase NS5B in the RNA synthesis initiation step by Stéphane Betzi; Cécilia Eydoux; Cécile Bussetta; Marilyne Blemont; Pieter Leyssen; Claire Debarnot; Mohamed Ben-Rahou; Jacques Haiech; Marcel Hibert; Françoise Gueritte; David S. Grierson; Jean-Louis Romette; Jean-Claude Guillemot; Johan Neyts; Karine Alvarez; Xavier Morelli; Hélène Dutartre; Bruno Canard (pp. 48-59).

Hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B constitutes a target of choice for the development of anti-HCV drugs. Although many small molecules have been identified as allosteric inhibitors of NS5B, very few are active in clinical applications. We have screened 17,000 compounds in an enzymatic assay involving the purified NS5B in order to increase the therapeutic arsenal. We hoped to shed some light on the precise mechanism of RNA synthesis. We succeeded in isolating a series of 21 original inhibitors of the RNA synthesis by NS5B. Four of these non-nucleoside inhibitors (NNIs) could be mapped to the known binding site called ‘B’ as judged by the decrease in their inhibition potency when assayed with a ‘B’ site mutant, M423T NS5B. Incidentally, our in silico model pointed to Y477 as a key residue for inhibitor binding. In vitro, Y477F mutant loses its sensitivity to the newly discovered inhibitors but is unable to extend primers during the elongation phase. Our results demonstrate that elements of the ‘B’ site are involved in the conformational changes required in the switch between the different RNA synthesis steps and that compounds targeting this site could lock the enzyme in its initiation phase.

Keywords: Abbreviations; HCV; hepatitis C virus; CMV; cytomegalovirus; HSV; herpes simplex virus; HBV; hepatitis B virus; dsRNA; double-stranded ribonucleic acid; ssRNA; single-stranded ribonucleic acid; RdRp; RNA-dependent RNA polymerase; WT; wild-type; GTP; guanosine triphosphate; NI(s); nucleoside inhibitor(s); NNI(s); non-nucleoside inhibitor(s); NS5B; non-structural protein 5B; P; _x; products made of; x; nucleotides (where; x; is a number of nucleotides); neg; negative; pos; positive; DMSO; dimethyl sulfoxide; %; percentage; SBDD; structure-based drug design; PDB; Protein Data Bank; PDB ID; Protein Data Bank Identifier; LGA; Lamarckian genetic algorithm; RMSD; root-mean-square deviation; AU; arbitrary unit; IC; ₅₀; 50% inhibitory concentration; LIMS; laboratory information management system; SAR; structure–activity relationship; DTT; dithiothreitol; Mn; manganese; Mg; magnesiumPolymerase; Hepatitis C virus; Inhibitor screening; Mechanism of inhibition; Antiviral

Expression of exogenous IFN-α by bypassing the translation block protects cells against FMDV infection by Yi Xiong; Meijuan Lin; Bing Yuan; Tingting Yuan; Congyi Zheng (pp. 60-66).

Foot-and-mouth disease virus (FMDV) is the most contagious pathogen of cloven-hoofed animals. Previous studies have demonstrated that type I interferons [alpha/beta interferons (IFN-α/βs)] can suppress FMDV replication and spread. Conversely, FMDV can also inhibit IFN-α expression in infected cells by blocking cap-dependent translation. To overcome the blockade on IFN-α mRNA translation during FMDV infection, we generated an IRES-IFN construct that carries FMDV's internal ribosome entry site (IRES) cDNA sequence between the promoter and porcine IFN-α gene. ELISA assays indicated that expression of IFN-α regulated by wild-type IRES increased to 125% of pre-infection level after infection for 24h, but the expression of IFN-α regulated by nonfunctional IRES mutants were only ∼50% of pre-infection level. Correspondingly, the former could suppress the replication of FMDV to 20% of the latter and protect cells against FMDV for a longer time. Therefore, these findings provide a new strategy to anti-FMDV therapy.

Keywords: FMDV; IRES; Alpha interferon (IFN-α)

A sugar binding protein cyanovirin-N blocks herpes simplex virus type-1 entry and cell fusion by Vaibhav Tiwari; Shripaad Y. Shukla; Deepak Shukla (pp. 67-75).

Herpes simplex virus type-1 (HSV-1) causes significant health problems from periodic skin and corneal lesions to encephalitis. It is also considered a cofactor in the development of age-related secondary glaucoma. Inhibition of HSV-1 at the stage of viral entry generates a unique opportunity for preventative and/or therapeutic intervention. Here we provide evidence that a sugar binding antiviral protein, cyanovirin-N (CV-N), can act as a potent inhibitor of HSV-1 entry into natural target cells. Inhibition of entry was independent of HSV-1 gD receptor usage and it was observed in transformed as well as primary cell cultures. Evidence presented herein suggests that CV-N can not only block virus entry to cells but also, it is capable of significantly inhibiting membrane fusion mediated by HSV glycoproteins. While CV-N treated virions were significantly deficient in entering into cells, HSV-1 glycoproteins-expressing cells pretreated with CV-N demonstrated reduced cell-to-cell fusion and polykaryocytes formation. The observation that CV-N can block both entry as well as membrane fusion suggests a stronger potential for this compound in antiviral therapy against HSV-1.

Keywords: Cyanovirin-N; Microbicide; HSV-1; Viral entry

Inhibition of rabies virus replication by multiple artificial microRNAs by Nipan Israsena; Pornpan Supavonwong; Nitipol Ratanasetyuth; Pakamatz Khawplod; Thiravat Hemachudha (pp. 76-83).

The RNA interference (RNAi) technology has been recognized as a promising antiviral therapy for a few years. One of the potential limitations for applying this technology against wild type rabies virus is its high rate of genetic variation. Recently, an RNAi vector system that incorporated modified dsRNA within microRNA structure [or artificial miRNAs(amiRNAs)] has been described. This allowed expression of multiple amiRNAs of single or multiple targets from a single construct. In this study, we evaluated a benefit of using amiRNA vector against different rabies strains. We found that applying single targeting amiRNA against challenged rabies virus standard (CVS) rabies nucleocapsid (N) mRNA resulted in more than 90% reduction of viral genome in Neuro2A cells up to 72h after infection. Multiple amiRNAs aiming at single or multiple NmRNA target(s) yielded comparable inhibitory results as with a single amiRNA against perfectly matched target. Although the level of each mature miRNA generated from multiple amiRNA construct was slightly reduced as assessed by stem-loop RT and real-time PCR techniques, its effectiveness remained unchanged even when an ineffective or scrambled amiRNA was also included in the transcript. Against highly pathogenic wild type virus, single amiRNA construct activity was reduced when mismatching with target sequence occurred at critical site whereas multiple targeting amiRNA construct remained highly effective. Our results suggest the benefit of using multiple targeting amiRNAs when confronting with wild type rabies virus, the sequence of which is not completely known.

Keywords: miRNA; RNAi; Rabies; Viral replication

Antiviral activity of the hemolymph of Lonomia obliqua (Lepidoptera: Saturniidae) by Katia N. Greco; Rita Maria Z. Mendonça; Roberto H.P. Moraes; Dalva. A.P. Mancini; Ronaldo Z. Mendonça (pp. 84-90).

Potent antiviral activity against measles, influenza and polio viruses was observed in the hemolymph of Lonomia obliqua. The antiviral protein responsible for this activity was isolated, purified by gel filtration chromatography using a gel filtration column system (Superdex 75) and further fractionated using a Resource-Q ion exchange column system. Experiments with the purified protein led to a 157-fold reduction (from 3.3±1.25×10⁷ to 2.1±1.5×10⁵ TCID₅₀mL⁻¹) in measles virus production and a 61-fold reduction (from 2.8±1.08×10⁹ to 4.58±1.42×10⁷mL⁻¹) in polio virus production. Heating and freezing seem to have no influence over its antiviral activity. Also, the protein does not display virucidal activity and does not act on receptors on the cell membrane. The observations suggest an intracellular mechanism of action and that the protein may act as a constitutive agent that affects the innate antiviral immune response.

Keywords: Antiviral; Lonomia obliqua; Purification; Hemolymph; Measles; Polio; Influenza

Emergence of oseltamivir-resistant influenza A H1N1 virus during the 2007–2008 winter season in Luxembourg: Clinical characteristics and epidemiology by Joël Mossong; Matthias Opp; Nancy Gerloff; Patrick Hau; Jacques Kremer; Angie Lackenby; Vicki Gregory; Jos Even; Pierrette Huberty-Krau; Claude P. Muller; François Schneider (pp. 91-94).

Human cases of oseltamivir-resistant influenza A H1N1 virus emerging in 2007–2008 in Luxembourg were not associated with treatment, prophylaxis or stockpiling of oseltamivir. Following initial local seeding, oseltamivir-resistant strains spread synchronously to sensitive strains causing a similar epidemiology and clinical symptoms.

Keywords: Oseltamivir; Influenza; H1N1; Epidemiology; Resistance

Inhibitory activity of oxyresveratrol on wild-type and drug-resistant varicella-zoster virus replication in vitro by Pattaraporn Sasivimolphan; Vimolmas Lipipun; Kittisak Likhitwitayawuid; Masaya Takemoto; Pornpen Pramyothin; Masao Hattori; Kimiyasu Shiraki (pp. 95-97).

The anti-herpes simplex virus (HSV) compound, oxyresveratrol, purified from a Thai traditional medicinal plant of Artocarpus lakoocha, was evaluated for its anti-varicella-zoster virus (VZV) activity. This compound exhibited IC₅₀ values (50%-inhibitory concentrations for virus plaque formation) of 12.82, 12.80, 12.99 and 12.82μg/ml against wild type, thymidine kinase-deficient and two types of DNA polymerase mutants with acyclovir-resistance, respectively. Thus oxyresveratrol showed a broad spectrum of anti-VZV activity with a mechanism of action different from that of acyclovir.

Keywords: Oxyresveratrol; Varicella-zoster virus; Drug-resistant virus; Acyclovir; Foscarnet

Corrigendum to “Recombinant fowlpox virus vector-based vaccine completely protects chickens from H5N1 avian influenza virus” [Antiviral Res. 81 (3) (2009) 234–238] by Chuanling Qiao; Yongping Jiang; Guobin Tian; XiurongWang; Chengjun Li; Xiaoguang Xin; Hualan Chen; Kangzhen Yu (pp. 98-99).

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Antiviral Research (v.84, #1)