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Antiviral Research (v.81, #3)

Editorial Board (pp. ii).

Notes to Authors (pp. co3).

No exit: Targeting the budding process to inhibit filovirus replication by Ronald N. Harty (pp. 189-197).

The filoviruses, Ebola and Marburg, cause severe hemorrhagic fever in humans and nonhuman primates, with high mortality rates. Although the filovirus replication pathway is now understood in considerable detail, no antiviral drugs have yet been developed that directly inhibit steps in the replication cycle. One potential target is the filovirus VP40 matrix protein, the key viral protein that drives the budding process, in part by mediating specific virus–host interactions to facilitate the efficient release of virions from the infected cell. This review will summarize current knowledge of key structural and functional domains of VP40 believed to be necessary for efficient budding of virions and virus-like particles. A better understanding of the structure and function of these key regions of VP40 will be crucial, as they may represent novel and rational targets for inhibitors of filovirus egress.

Keywords: Filovirus; Ebola virus; Marburg virus; Viral budding; VP40 matrix protein; Virus-like particles; Antiviral therapy

Using high-throughput genomics to study hepatitis C: What determines the outcome of infection? by Kathie-Anne Walters; Michael G. Katze (pp. 198-208).

High-throughput genomic methods are now being used to study a wide variety of viral diseases, in an effort to understand how host responses to infection can lead either to efficient elimination of the pathogen or the development of severe disease. This article reviews how gene expression studies are addressing important clinical issues related to hepatitis C virus infection, in which some 15–25% of infected individuals are able to clear the virus without treatment, while the remainder progress to chronic liver disease that can lead to cirrhosis and death. Similar methods are also being used in an effort to identify the mechanisms underlying the failure of some hepatitis C patients to respond to interferon-α/ribavirin therapy. By providing a detailed picture of virus–host interactions, high-throughput genomics could potentially lead to the identification of novel cellular targets for the treatment of hepatitis C.

Keywords: Hepatitis C virus; Hepatitis C; Gene expression; Microarray; Viral hepatitis; Genomics; Interferon-α; Antiviral therapy

RNA interference protects horse cells in vitro from infection with Equine Arteritis Virus by Anett Heinrich; Diana Riethmüller; Marleen Gloger; Gerald F. Schusser; Matthias Giese; Sebastian Ulbert (pp. 209-216).

Equine Arteritis Virus (EAV) belongs to the Arteriviridae and causes viral arteritis in horses. In an attempt to develop novel and save therapies against the infection it was tested whether EAV is susceptible to RNA interference (RNAi) in an equine in vitro system. Horse cells were transfected with chemically synthesized small interfering RNA oligonucleotides (siRNAs) and challenged with EAV. Application of these siRNAs led to a significant protection of the cells, and virus titers decreased drastically. siRNAs derived from DNA plasmids expressing small hairpin RNAs (shRNAs) were also effective. The protection was most pronounced with two siRNAs targeting the open reading frame 1 (coding for non-structural proteins), whereas siRNAs targeting sequences for several structural proteins had less or no effect. In addition, it was investigated whether RNAi could be used to treat cells with an already established viral infection. Only application of the siRNAs shortly after viral challenge led to significant survival rates of the cells, whereas transfection at later time points caused much less benefit for the cells. These findings are discussed in a perspective of using RNAi as a therapeutic approach to combat EAV.

Keywords: Equine Arteritis Virus; RNA interference

Pseudorabies virus glycoprotein B can be used to carry foot and mouth disease antigens in DNA vaccination of pigs by Daniel Dory; Michelle Rémond; Véronique Béven; Roland Cariolet; Marija Backovic; Stephan Zientara; André Jestin (pp. 217-225).

To evaluate the feasibility of using pseudorabies virus (PrV) glycoprotein B (gB) as a carrier of foot and mouth disease virus (FMDV) antigens in DNA immunization, FMDV B- and T-cell epitopes were inserted either between the two B-cell epitopes of the N-term subunit of PrV-gB (BT-PrV-gB–N-term construct) or within the B-cell epitope of the C-term subunit of PrV-gB (BT-PrV-gB–C-term construct). Two animal experiments were performed, each with three injections of plasmids 2 weeks apart, followed by a booster inoculation of peptides corresponding to the FMDV epitopes. Control groups of pigs were injected with plasmids encoding either PrV-gB or FMDV-BT, or with empty-pcDNA3. The results of both assays were combined. Significant titers of FMDV neutralizing antibodies were detected after the peptides boost in groups injected with the BT-PrV-gB–C-term construct. Insignificant amounts were detected in groups injected with the BT-PrV-gB–N-term and FMDV-BT constructs. PBMCs from the BT-PrV-gB–N-term groups, isolated after the peptide boost injection, produced IFN-γ and IL-4 mRNAs in vitro when stimulated with FMDV peptides. This was not observed with the other groups. These results imply that PrV-gB can be used to carry FMDV antigens in a DNA vaccine.

Keywords: Foot and mouth disease virus; Pseudorabies virus glycoprotein B; B- and T-cell epitopes; Carrier of antigens; DNA vaccination

Levocetirizine inhibits rhinovirus-induced ICAM-1 and cytokine expression and viral replication in airway epithelial cells by Yong Ju Jang; Jong Hwan Wang; Ji Sun Kim; Hyun Ja Kwon; Nam-Kyung Yeo; Bong-Jae Lee (pp. 226-233).

Levocetirizine inhibits the production of intercellular adhesion molecule (ICAM)-1 and secretion of interleukin (IL)-6 and IL-8, which may have beneficial effects on the pathophysiologic changes related to human rhinovirus (HRV) infection. We investigated the effects of levocetirizine on rhinovirus infection in primary human nasal epithelial cells (HNEC) and A549 cells. Cells were treated with different concentrations of levocetirizine, ranging from 0.5, 5 or 50nM, either starting at the time of infection and continuing thereafter, or beginning 24h before infection and continuing thereafter. Levocetirizine treatment inhibited the HRV-induced increase in ICAM-1 mRNA and protein levels, as well as the HRV-induced expression of IL-6 and IL-8 mRNA and protein levels. Viral titer, as measured by culture in MRC-5 cells, was reduced by levocetirizine. Levocetirizine treatment also reduced the increased nuclear factor-kappa B (NF-κB) expression seen with HRV infection. Levocetirizine inhibited the expression of Toll-like receptor (TLR)3 mRNA and protein levels. These findings indicate that, in HNEC and A549 cells, levocetirizine inhibits HRV replication and HRV-induced upregulation of ICAM-1, IL-6, and IL-8, TLR3 expression and NF-κB activation. The results of this study suggest that levocetirizine may have a possible clinical application in the treatment of airway inflammation caused by HRV infection.

Keywords: Rhinovirus; Levocetirizine; Airway epithelial cells; ICAM-1; Cytokines; TLR3

Recombinant fowlpox virus vector-based vaccine completely protects chickens from H5N1 avian influenza virus by Chuanling Qiao; Yongping Jiang; Guobin Tian; Xiurong Wang; Chengjun Li; Xiaoguang Xin; Hualan Chen; Kangzhen Yu (pp. 234-238).

With the widespread presence of influenza virus H5N1 in poultry and wildlife species, particularly migrating birds, vaccination has become an important control strategy for avian influenza (AI). In this study, the immune efficacy and hemagglutination inhibition (HI) antibody responses induced by a recombinant fowlpox virus (FPV) vector-based rFPV–HA–NA vaccine was evaluated in SPF and commercial chickens. Four-week old SPF chickens vaccinated with one dose of vaccine containing 2×10³ plaque forming units (PFU) of virus were completely protected from H5N1 AI virus 1 week after vaccination, and protective immunity lasted for at least 40 weeks. Two-week old commercial layer chickens were vaccinated with the rFPV–HA–NA vaccine and boosted with the same dose of vaccine following an interval of 18 weeks. The HI antibody titers higher than 4log2 lasted for 52 weeks after the booster immunization. We also examined the efficacy of the rFPV–HA–NA vaccine in SPF chickens administrated by different routes. The results showed that effective application of rFPV–HA–NA vaccine in poultry may be restricted to wing-web puncture, intramuscular or subcutaneous injection. These results demonstrate that the rFPV–HA–NA vaccine is effective in the prevention of infection of H5N1 AI virus.

Keywords: Recombinant fowlpox virus; Vaccine; H5N1; Avian influenza virus

High-throughput screening using pseudotyped lentiviral particles: A strategy for the identification of HIV-1 inhibitors in a cell-based assay by Jean-Michel Garcia; Anhui Gao; Pei-Lan He; Joyce Choi; Wei Tang; Roberto Bruzzone; Olivier Schwartz; Hugo Naya; Fa-Jun Nan; Jia Li; Ralf Altmeyer; Jian-Ping Zuo (pp. 239-247).

Two decades after its discovery the human immunodeficiency virus (HIV) is still spreading worldwide and killing millions. There are 25 drugs formally approved for HIV currently on the market, but side effects as well as the emergence of HIV strains showing single or multiple resistances to current drug-therapy are causes for concern. Furthermore, these drugs target only 4 steps of the viral cycle, hence the urgent need for new drugs and also new targets. In order to tackle this problem, we have devised a cell-based assay using lentiviral particles to look for post-entry inhibitors of HIV-1. We report here the assay development, validation as well as confirmation of the hits using both wild-type and drug-resistant HIV-1 viruses. The screening was performed on an original library, rich in natural compounds and pure molecules from Traditional Chinese Medicine pharmacopoeia, which had never been screened for anti-HIV activity. The identified hits belong to four chemical sub-families that appear to be all non-nucleoside reverse transcriptase inhibitors (NNRTIs). Secondary tests with live viruses showed that there was good agreement with pseudotyped particles, confirming the validity of this approach for high-throughput drug screens. This assay will be a useful tool that can be easily adapted to screen for inhibitors of viral entry.

Keywords: Abbreviations; 3TC; Lamivudine; AZT; Zidovudine; BSL; biosafety level; CPRG; chlorophenol red-β-; d; -galactopyranoside; CPE; cytopathic effect; d4T; Stavudine; ddI; Didanosine; DMSO; dimethyl sulfoxide; FCS; fetal calf serum; GFP; green fluorescent protein; HIV; human immunodeficiency virus; Luc; luciferase; MDR; multi-drug resistant; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NNRTI; non-nucleoside reverse transcriptase inhibitor; NVP; Nevirapine; PDB; Protein Data Bank; SFV; Semliki Forest Virus; TCM; traditional Chinese medicine; VSV-G; Protein G of the Vesicular Stomatitis VirusHIV; Pseudotyped lentiviral particles; High-throughput screening; HTS; Cocktail library; NNRTI

Effects of transferrins and cytokines on nitric oxide production by an avian lymphoblastoid cell line infected with Marek's disease virus by Maria Federica Giardi; Cristina La Torre; Francesco Giansanti; Dario Botti (pp. 248-252).

Marek's disease virus (MDV), the causative agent of Marek's disease (MD), is a herpesvirus that infects poultry causing T lymphomas. Although vaccination may prevent lymphomas formation, it is not known whether it controls viral replication and spreading in the environment. Ovotransferrin (Otrf), a member of the transferrin family, is known to exert in vitro antiviral activity in primary cultures of chicken embryo fibroblasts (CEF). In addition Otrf is produced by CEF and by an avian lymphoblastoid cell line (MDCC-MSB1) following infection/reinfection with MDV. The present work was designed to investigate the effects of reinfection and of Otrf and lactoferrin (Lf) on the production by MDCC-MSB1 of nitric oxide (NO), a molecule naturally exerting an antiviral activity. These effects were also tested with two cytokines (IL-8 and IFN-γ), alone and in association with transferrins. Synergy was found between Otrf and IFN-γ, thus suggesting a possible role in a complementary or alternative strategy against MDV spreading.

Keywords: Marek's disease virus; Ovotransferrin; Lactoferrin; Nitric oxide; Cytokines

Enhanced protective immunity against H5N1 influenza virus challenge by vaccination with DNA expressing a chimeric hemagglutinin in combination with an MHC class I-restricted epitope of nucleoprotein in mice by Pan Tao; Mengcheng Luo; Ruangang Pan; Dawei Ling; Siyu Zhou; Po Tien; Zishu Pan (pp. 253-260).

DNA vaccination is an effective means of eliciting both humoral and cellular immune responses. The hemagglutinin (HA) surface protein of influenza A virus is a major target of protective antibody responses induced by virus infection or by vaccination and is widely considered to be the antigen of choice for an influenza vaccine. Cytotoxic T lymphocyte (CTL) responses directed against the conserved nucleoprotein (NP) are thought to play an important role in clearing virus and promoting survival and recovery from influenza. In this study, we developed a novel DNA vaccine approach using a chimeric plasmid consisting of the HA of H5N1 influenza virus in which an MHC class I-restricted NP-specific CTL epitope (NP147–155) was inserted. Immunogenicity and antiviral efficacy of this vaccine was assessed in mouse models. A similar level of HA expression was achieved in 293T cells transfected with pHA/NP_147–155 compared to that with pHA. Besides eliciting the specific anti-HA antibody responses, vaccination using pHA/NP_147–155 in mice induced NP epitope-specific CD8⁺ T cell responses, which are generally not inducible by vaccination with pHA alone. After H5N1 influenza virus challenge, BALB/c mice vaccinated with pHA/NP_147–155 exhibited reduced inflammation severity and lung viral titers compared to those vaccinated with pHA. Our work may contribute to improvement of HA-based influenza DNA vaccines.

Keywords: H5N1 influenza A virus; Hemagglutinin; CTL epitope; DNA vaccine

Effective ribavirin concentration in mice brain using cyclodextrin as a drug carrier: Evaluation in a measles encephalitis model by H. Jeulin; V. Venard; D. Carapito; C. Finance; F. Kedzierewicz (pp. 261-266).

Ribavirin (RBV) is a water-soluble synthetic nucleoside with broad spectrum antiviral properties, but it is ineffective against major viral encephalitis because of a failure to cross the blood–brain barrier (BBB). The antiviral activity of the complex ribavirin/alpha-cyclodextrin was previously demonstrated to be stronger than free ribavirin, in an in vivo model of measles virus (MV) encephalitis in mice. The role of cyclodextrin (CD) on ribavirin uptake into the brain needs to be defined. Ribavirin specific extraction from brain tissue was developed, based on a solid phase extraction. It was quantified by high performance liquid chromatography at different time points after intraperitoneal injection of single or multiple doses of free ribavirin or of the complex ribavirin/alpha-cyclodextrin. Whatever the tested dose (40 or 100mg/kg), the amount of ribavirin in the brain was significantly higher ( p<0.001) when the drug was injected as a complex with alpha-cyclodextrin, in healthy or measles virus-infected mice.

Keywords: Ribavirin; Alpha-cyclodextrin; Blood–brain barrier; Measles virus; Encephalitis

Design, synthesis, molecular modeling, and anti-HIV-1 integrase activity of a series of photoactivatable diketo acid-containing inhibitors as affinity probes by Mario Sechi; Fabrizio Carta; Luciano Sannia; Roberto Dallocchio; Alessandro Dessì; Rasha I. Al-Safi; Nouri Neamati (pp. 267-276).

The diketo acid (DKA) class of HIV-1 integrase (IN) inhibitors is thought to function by chelating divalent metal ions on the enzyme catalytic site. However, differences in mutations conferring resistance to various DKA inhibitors suggest that multiple binding orientations may exist. In order to facilitate identification of DKA binding sites, a series of photoactivable analogues of two potent DKAs was prepared as novel photoaffinity probes. In cross-linking assays designed to measure disruption of substrate DNA binding, the photoprobes behaved similarly to a reference DKA inhibitor. Molecular modeling studies suggest that such photoprobes interact within the IN active site in a manner similar to that of the parent DKAs. AnaloguesIa-c are novel photoaffinity ligands useful in clarifying the HIV-1 binding interactions of DKA inhibitors.

Keywords: HIV-1 integrase inhibitors; Diketo acids; Photoaffinity labeling; Photoprobes

Inhibitory effect of HMGN2 protein on human hepatitis B virus expression and replication in the HepG2.2.15 cell line by Yun Feng; Fang He; Ping Zhang; Qi Wu; Ning Huang; Hong Tang; Xiangli Kong; Yan Li; Junju Lu; Qianming Chen; Boyao Wang (pp. 277-282).

Natural killer (NK) cells and cytolytic T lymphocytes (CTL) have been implicated as important effectors of antiviral defense. We previously isolated a novel antibacterial polypeptide, which was identified as high mobility group nucleosomal-binding domain 2 (HMGN2), from human mononuclear leukocytes. This study examined the antiviral activity of HMGN2 against human hepatitis virus B. HMGN2 was isolated and purified from the acid soluble proteins of the human THP-1 cell line, and identified by mass spectrum, Western blot and antibacterial assay. The hepatitis B virus (HBV)-transfected HepG2.2.15 cell line was used in the in vitro assay system. In the range of 1–100μg/ml HMGN2, no cytotoxicity for HepG2.2.15 cells was detected by MTT assay. When incubated with HMGN2 at 1–100μg/ml for 72 or 144h, there was a significant reduction in hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) expression, which were detected by ELISA, and a significant reduction in HBV DNA copies, which was determined by the real time quantitative PCR, in the supernatant of HepG2.2.15 cells. Northern and Southern blot analysis also showed that the levels of the HBV 3.5kb and the 2.4/2.1kb mRNA species and HBV replicative intermediate DNA were significantly reduced in the HMGN2-treated HepG2.2.15 cells. These results indicated that HMGN2 protein could markedly inhibit HBV protein expression and replication in vitro.

Keywords: HMGN2; Antiviral activity; Human hepatitis B virus; HepG2.2.15 cell line

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Antiviral Research (v.81, #3)