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Antiviral Research (v.76, #1)

Notes to Authors (pp. co3).

Editorial Board (pp. ii).

CYSTUS052, a polyphenol-rich plant extract, exerts anti-influenza virus activity in mice by Karoline Droebner; Christina Ehrhardt; Anne Poetter; Stephan Ludwig; Oliver Planz (pp. 1-10).

Influenza, a respiratory disease caused by influenza viruses, is still a worldwide threat with a high potential to cause a pandemic. Beside vaccination, only two classes of drugs are available for antiviral treatment against the pathogen. Here we show that CYSTUS052, a plant extract from a special variety of Cistus incanus that is rich in polymeric polyphenols, exhibits antiviral activity against a highly pathogenic avian influenza A virus (H7N7) in cell culture and in a mouse infection model. In vitro and in vivo treatment was performed with an aerosol formulation, because the bioavailability of high molecular weight polyphenols is poor. In MDCK cells, a 90% reduction of plaque numbers on cells pre-incubated with the plant extract was achieved. For in vivo experiments we used a novel monitoring system for influenza A virus-infected mice that allows measurement of body temperature and gross motor-activity of the animals. Mice treated with CYSTUS052 did not develop disease, showed neither differences in their body temperature nor differences in their gross motor-activity and exhibited no histological alterations of the bronchiolus epithelial cells.

Keywords: Influenza A virus; Cystus incanus; Polyphenols

Enhanced protective efficacy and reduced viral load of foot-and-mouth disease DNA vaccine with co-stimulatory molecules as the molecular adjuvants by Chong Xiao; Huali Jin; Yanxin Hu; Youmin Kang; Junpeng Wang; Xiaogang Du; Yu Yang; Ruiping She; Bin Wang (pp. 11-20).

To improve efficacy of DNA vaccination, various approaches have been developed, including the use of plasmid expressing co-stimulatory molecules as molecular adjuvants. In this study, we investigated whether co-inoculation of a construct expressing either 4-1BBL or OX40L as the molecular adjuvant with FMDV DNA vaccine, pcD-VP1, can increase immune responses and protective efficacies. Compared to the group immunized with pcD-VP1 alone, the co-inoculation of either molecular adjuvant induced a higher ratio of IgG2a/IgG1, higher levels of expression of IFN-γ in CD4⁺ and CD8⁺ T cells and antigen-specific CTL responses, and more importantly provided an enhanced protection against the live FMDV challenge in animals. Concurrently, 4-1BBL as the molecular adjuvant dramatically reduced the viral loads of FMDV in vivo after the challenge. Together, the results demonstrate that co-stimulatory molecules 4-1BBL and OX40L can enhance the antigen-specific cell-mediated responses elicited by VP1 DNA vaccine and provide an enhanced protective efficacy with the reduced viral loads.

Keywords: Abbreviations; RT-PCR; reverse transcriptase PCR; GAPDH; glyseraldehyde-3-phosphate dehydrogenase; MTS; 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PMS; phenazine methosulfate; CFSE; carboxyfluorescein succinimidyl ester; TCID50; 50% of tissue culture infectious doseFMDV DNA vaccine; Protection; Viral load; Co-stimulatory molecules; Neutralizing antibody; in vivo CTL; Cell-mediated immune responses; 4-1BBL; OX40L

Antiviral activity of carbohydrate-binding agents against Nidovirales in cell culture by F.J.U.M. van der Meer; C.A.M. de Haan; N.M.P. Schuurman; B.J. Haijema; W.J. Peumans; E.J.M. Van Damme; P.L. Delputte; J. Balzarini; H.F. Egberink (pp. 21-29).

Coronaviruses are important human and animal pathogens, the relevance of which increased due to the emergence of new human coronaviruses like SARS-CoV, HKU1 and NL63. Together with toroviruses, arteriviruses, and roniviruses the coronaviruses belong to the order Nidovirales. So far antivirals are hardly available to combat infections with viruses of this order. Therefore, various antiviral strategies to counter nidoviral infections are under evaluation. Lectins, which bind to N-linked oligosaccharide elements of enveloped viruses, can be considered as a conceptionally new class of virus inhibitors. These agents were recently evaluated for their antiviral activity towards a variety of enveloped viruses and were shown in most cases to inhibit virus infection at low concentrations. However, limited knowledge is available for their efficacy towards nidoviruses. In this article the application of the plant lectins Hippeastrum hybrid agglutinin (HHA), Galanthus nivalis agglutinin (GNA), Cymbidium sp. agglutinin (CA) and Urtica dioica agglutinin (UDA) as well as non-plant derived pradimicin-A (PRM-A) and cyanovirin-N (CV-N) as potential antiviral agents was evaluated. Three antiviral tests were compared based on different evaluation principles: cell viability (MTT-based colorimetric assay), number of infected cells (immunoperoxidase assay) and amount of viral protein expression (luciferase-based assay). The presence of carbohydrate-binding agents strongly inhibited coronaviruses (transmissible gastroenteritis virus, infectious bronchitis virus, feline coronaviruses serotypes I and II, mouse hepatitis virus), arteriviruses (equine arteritis virus and porcine respiratory and reproductive syndrome virus) and torovirus (equine Berne virus). Remarkably, serotype II feline coronaviruses and arteriviruses were not inhibited by PRM-A, in contrast to the other viruses tested.

Keywords: Nidovirales; Glycosylation; In vitro assay; MTT; Immunoperoxidase; Luciferase

Treatment of hepatitis B virus-infected cells with α-glucosidase inhibitors results in production of virions with altered molecular composition and infectivity by Catalin Lazar; David Durantel; Alina Macovei; Nicole Zitzmann; Fabien Zoulim; Raymond A. Dwek; Norica Branza-Nichita (pp. 30-37).

Trimming of the N-glycans attached to the envelope proteins of hepatitis B virus (HBV) is required in different steps of the viral life cycle. Inhibition of the host enzymes α-glucosidases, involved in the endoplasmic reticulum (ER)-associated processing of the N-linked glycans, results in misfolding of the HBV envelope proteins, prevention of HBV secretion and accumulation of viral DNA within infected cells. However, the impact of these effects on HBV morphogenesis and infectivity of the viral particles that are still released from cells with inhibited α-glucosidase has not been addressed so far. Using N-butyldeoxynojirimycin (NB-DNJ), a competitive inhibitor of the ER α-glucosidases, we analyzed the role of these enzymes on HBV assembly and infectivity of the virions released from HepG2.2.2.15 cells. HBV secreted from drug-treated cells contained an envelope with altered composition of the disulfide-linked oligomers and no detectable middle (M) protein. These molecular changes had a significant effect on HBV infectivity, reducing it to 20% compared to controls, for the highest concentrations of NB-DNJ used. Our data show for the first time that an active α-glucosidase activity is crucial for production of infectious HBV and provide new insights into the controversial role of the M protein in this process.

Keywords: HBV; Infectivity: HepaRG cells; Alpha-glucosidase inhibition; NB-DNJ

A polyphenol rich plant extract, CYSTUS052, exerts anti influenza virus activity in cell culture without toxic side effects or the tendency to induce viral resistance by Christina Ehrhardt; Eike R. Hrincius; Virginia Korte; Igor Mazur; Karoline Droebner; Anne Poetter; Stephan Dreschers; Mirko Schmolke; Oliver Planz; Stephan Ludwig (pp. 38-47).

Infections with influenza A viruses still pose a major threat to humans and several animal species. The occurrence of highly pathogenic avian influenza viruses of the H5N1 subtype capable to infect and kill humans highlights the urgent need for new and efficient countermeasures against this viral disease. Here we demonstrate that a polyphenol rich extract (CYSTUS052) from the Mediterranean plant Cistus incanus exerts a potent anti-influenza virus activity in A549 or MDCK cell cultures infected with prototype avian and human influenza strains of different subtypes. CYSTUS052 treatment resulted in a reduction of progeny virus titers of up to two logs. At the effective dose of 50μg/ml the extract did not exhibit apparent harming effects on cell viability, metabolism or proliferation, which is consistent with the fact that these plant extracts are already used in traditional medicine in southern Europe for centuries without any reported complications. Viruses did not develop resistance to CYSTUS052 when compared to amantadine that resulted in the generation of resistant variants after only a few passages. On a molecular basis the protective effect of CYSTUS052 appears to be mainly due to binding of the polymeric polyphenol components of the extract to the virus surface, thereby inhibiting binding of the hemagglutinin to cellular receptors. Thus, a local application of CYSTUS052 at the viral entry routes may be a promising approach that may help to protect from influenza virus infections.

Keywords: Influenza A virus; CYSTUS052; Polyphenols; Plant extract; Virus entry

Inhibition of hepatitis C virus p7 membrane channels in a liposome-based assay system by Corine StGelais; Tobias J. Tuthill; Dean S. Clarke; David J. Rowlands; Mark Harris; Stephen Griffin (pp. 48-58).

Chemotherapy for patients chronically infected with hepatitis C virus (HCV) is ineffective in over 50% of cases, generating a high demand for new drug targets. The p7 protein of HCV displays membrane channel activity in vitro and is essential for replication in vivo though its precise role in the virus life cycle is unknown . p7 channel activity can be specifically inhibited by several classes of compounds, making this protein an attractive candidate for drug development, though techniques used to date in characterising this protein are unsuited to compound library screening. Here we describe an assay for the channel forming ability of p7 based on the release of a fluorescent indicator from liposomes. We show that recombinant p7 from genotype 1b HCV causes a dose-dependent release of dye when mixed with liposomes and that this property is enhanced at acidic pH. We demonstrate that this activity is due to the formation of a size-selective pore rather than non-specific disruption of liposomes and that activity can be blocked by amantadine and several other compounds, validating it as a measure of p7 channel function. This system provides the first convenient in vitro assay for exploiting p7 as a therapeutic target.

Keywords: Hepatitis C virus; p7; Amantadine; Ion channel; Viroporin; Liposome permeability

Immunogenicity of plasmids encoding P12A and 3C of FMDV and swine IL-18 by Ma Mingxiao; Jin Ningyi; Liu Hui Juan; Zheng Min; Shen Guoshun; Zhu Guangze; Lu Huijun; Huo Xiaowei; Jin Minglan; Li Xu; Ma Haili; Ji Yue; Yin Gefen; Jin Kuoshi (pp. 59-67).

In this paper, two recombinant plasmids (pVIR-P12AIL18-3C and pVIR-P12A-3C) containing foot and mouth disease virus (FMDV) capsid polypeptide, 3C coding regions of O/NY00 and using/or not swine IL18 as a genetic adjuvant were constructed, and evaluated for their ability to induce humoral and cellular responses in mice and swine. In addition, the ability to protect swine against homologous virus challenge was examined. Mice and swine were given booster vaccination twice and once, respectively, and swine were challenged 10 days after the booster vaccination. Control groups were inoculated with pVAX1 or phosphate-buffered saline (PBS). All animals vaccinated with pVIR-P12AIL18-3C and pVIR-P12A-3C developed specific anti-FMDV ELISA antibody and neutralizing antibody and T lymphocyte proliferation and CTL cytotoxic activity was observed. In addition, we found that pVIR-P12AIL18-3C possessed stronger immunogenicity than pVIR-P12A-3C. The pVIR-P12AIL18-3C and pVIR-P12A-3C provided full protection in 3/4 and 2/4 swine from challenge with FMDV O/NY00, respectively. The results demonstrate the potential viability of a DNA vaccine in the control and prevention of FMDV infections.

Keywords: Foot and mouth disease virus; P12A; IL-18; DNA vaccine

Effects of apricitabine and other nucleoside reverse transcriptase inhibitors on replication of mitochondrial DNA in HepG2 cells by Michel P. de Baar; Esther R. de Rooij; Karlijn G.M. Smolders; Harm B. van Schijndel; Eveline C. Timmermans; Richard Bethell (pp. 68-74).

Several nucleoside reverse transcriptase inhibitors are associated with mitochondrial toxicity resulting from inhibition of DNA polymerase-γ. This study compared the effects on mitochondrial DNA of apricitabine (previously referred to as AVX754 or SPD754), a novel cytidine analogue under development for the treatment of human immunodeficiency virus (HIV)-1 infection, and other reverse transcriptase inhibitors. Human HepG2 hepatoblastoma were cultured for up to 16 days with test compounds at concentrations of 0.3–300μM. Mitochondrial DNA replication was assessed by means of a duplex nucleic acid sequence-based amplification technique, which measures the ratio of the number of mitochondrial DNA copies to the number of genomic DNA copies. Apricitabine and tenofovir had no effect on the mitochondrial DNA content. In contrast, alovudine, zalcitabine, didanosine and stavudine markedly reduced mitochondrial DNA content, whereas abacavir, emtricitabine, lamivudine and zidovudine produced slight increases in mitochondrial DNA, which may reflect an adaptive cellular response to mitochondrial dysfunction. These results suggest that apricitabine shows a favorable mitochondrial toxicity profile, which is important for long-term clinical use. Further studies are warranted to define the clinical implications of these findings.

Keywords: Apricitabine; Mitochondrial DNA; NRTI

Humoral and cellular immune responses to airway immunization of mice with human papillomavirus type 16 virus-like particles and mucosal adjuvants by Véronique Revaz; Rinaldo Zurbriggen; Christian Moser; John T. Schiller; Françoise Ponci; Martine Bobst; Denise Nardelli-Haefliger (pp. 75-85).

Cervical cancer results from cervical infection by human papillomaviruses (HPV), especially HPV16. Intramuscular administrations of HPV16 virus-like particle (VLP) vaccines have been shown to induce strong neutralizing antibody responses and protect women against genital HPV16 infection and associated lesions. However, an alternative route of administration that avoids parenteral injection might facilitate vaccine implementation, particularly in developing countries which account for the majority of the worldwide cases of cervical cancer. In addition, inducing mucosal immunity could partially overcome the substantial variation in HPV16 antibodies at the cervix seen in ovulating women. Aerosol vaccination with HPV16 VLPs was previously shown to be immunogenic in mice and in women. Here, we examine whether exposure to other respiratory viral antigens may interfere with the HPV16 VLP-specific humoral response and whether two known mucosal adjuvants, CpG oligodeoxynucleotides and a natural non-toxic Escherichia coli heat-labile enterotoxin (HLT), can enhance the immunogenicity of airway immunization (nasal or aerosol-like) of mice with HPV16 VLPs. Our data show that HLT can significantly improve anti-VLP humoral responses in serum and mucosal secretions, as well as VLP-specific proliferative responses and IFN-γ production by CD8 T cells, and that recent exposure to influenza surface antigens can diminish mucosal, but not systemic, antibody responses to the VLPs.

Keywords: Human papillomavirus type 16; Virus-like particles; HLT; CpG ODN; Mucosal immunization; Antibody and cell-mediated immune responses

Activity of compounds from Chinese herbal medicine Rhodiola kirilowii (Regel) Maxim against HCV NS3 serine protease by Guoying Zuo; Zhengquan Li; Lirong Chen; Xiaojie Xu (pp. 86-92).

Treatment of the chronic hepatitis C virus (HCV) infection is an unmet medical need, and the HCV NS3 serine protease (NS3-SP) has been used as an attractive target of antiviral screening against HCV. To find naturally chemical entities as lead compounds from which novel anti-HCV agents could be developed, bioassay-guided fractionation and isolation were performed on a crude ethanol extract from rhizomes of the Chinese medicinal herb Rhodiola kirilowii (Regel) Maxim using column chromatography (CC) techniques and in vitro inhibitory activity against HCV NS3-SP. The partition of the extract between water and different organic solvents led to the isolation and identification of 12 compounds in the ethyl acetate part which proved to be the most active. These compounds were tested for in vitro activity against HCV NS3-SP, among which four (−)-Epicatechin derivatives: 3,3′-Digalloylproprodelphinidin B2 (Rhodisin,1); 3,3′-Digalloylprocyanidin B2 (2); (−)-Epigallocatechin-3-O-gallate (EGCG,3); and (−)-Epicatechin-3-O-gallate (4, ECG) represented the most potent ones with IC₅₀ of 0.77, 0.91, 8.51, and 18.55μM, respectively. Salidroside, the commonly known compounds, together with the other compounds showed no activity up to 100.0μM. Methylation and acylation of the hydroxyl groups of1–4 caused a decrease of activity. Cell viability and secreted alkaline phosphatase (SEAP) activity assays with1–4 revealed little if any toxicity. These nonpeptide inhibitors of HCV NS3-SP might serve as potential candidate anti-HCV agents.

Keywords: Antiviral; HCV NS3-SP; (−)-Epicatechin derivative; Rhodiola kirilowii; (Regel) Maxim

Identification and characterization of mutations conferring resistance to an HCV RNA-dependent RNA polymerase inhibitor in vitro by Liangjun Lu; Tatyana Dekhtyar; Sherie Masse; Ron Pithawalla; Preethi Krishnan; Wenping He; Teresa Ng; Gennadiy Koev; Kent Stewart; Dan Larson; Todd Bosse; Rolf Wagner; Tami Pilot-Matias; Hongmei Mo; Akhteruzumman Molla (pp. 93-97).

Compound A-837093, a non-nucleoside HCV RNA-dependent RNA polymerase inhibitor, displayed nanomolar potencies against HCV genotypes 1a and 1b replicons. It also exhibited an excellent metabolic profile and achieved high plasma and liver concentrations in animals. In order to characterize the development of resistance to this anti-HCV agent, HCV subgenomic 1b strain N replicon cells were cultured in the presence of A-837093 with G418. Mutations S368A, Y448H, G554D, Y555C, and D559G in the NS5B polymerase gene were identified that led to substantial decreases in the susceptibilities of 1b genotype replicons to the inhibitor A-837093. However, the resistant mutants remained susceptible to HCV protease inhibitor BILN-2061 and alpha interferon as well as to a different class of non-nucleoside HCV polymerase inhibitor. In addition, each single resistant mutation identified significantly reduced the replication capacity of mutant compared to wild-type replicon. These findings provide a strategic guide for the future development of non-nucleoside inhibitors of HCV NS5B polymerase.

Keywords: Antiviral; HCV; Polymerase inhibitor; Resistance

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Antiviral Research (v.76, #1)