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Antiviral Research (v.89, #3)

Editorial Board (pp. ii).

Notes to Authors (pp. co3).

Human leukocyte antigen class I and class II genes polymorphisms might be associated with interferon α therapy efficiency of chronic hepatitis B by Xilin Zhu; Te Du; Xiaopan Wu; Xinhui Guo; Nifang Niu; Liping Pan; Zhenhui Xin; Li Wang; Zhuo Li; Hui Li; Ying Liu (pp. 189-192).

Certain host genetic polymorphisms in human leukocyte antigen (HLA) genes are reported to be associated with response to interferon α (IFNα) therapy. Two hundred and eighteen IFNα treatment-naïve chronic hepatitis B (CHB) patients were enrolled in the present study. HLA-A, B, C and DQA1, DQB1, DRB1 gene alleles were detected by polymerase chain reaction-sequencing based typing (PCR-SBT) and PCR-sequence specific primer (PCR-SSP), respectively. Frequencies of HLA-DQB1*0303 and DRB1*08 in response group were clearly lower than those in nonresponse group ( P=0.019, OR=1.81, 95%CI=1.07–3.15; P=0.031, OR=2.43, 95%CI=1.02–5.98, respectively). Frequencies of haplotype *1101-*4601-*0102 (HLA-A, B, C) and haplotype *0302-*0303-*09 (HLA-DQA1, DQB1, DRB1) were clearly lower than those in nonresponse group ( P=0.009, OR=4.84, 95%CI=1.29–19.48; P=0.031, OR=1.94, 95%CI=1.01–3.73, respectively). These results suggest that patients with HLA-DQB1*0303 or DRB1*08 alleles, and haplotype *1101-*4601-*0102 (HLA-A, B, C) or haplotype *0302-*0303-*09 (HLA-DQA1, DQB1, DRB1), might be less responsive to IFNα treatment.

Keywords: Chronic hepatitis B; Interferon α therapy; Human leukocyte antigen; Pharmacogenetic study

Rana catesbeiana ribonuclease inhibits Japanese encephalitis virus (JEV) replication and enhances apoptosis of JEV-infected BHK-21 cells by Yu-Hsiu Lee; Chyou-Wei Wei; Jaang-Jiun Wang; Chun-Tang Chiou (pp. 193-198).

Rana catesbeiana ribonuclease (RC-RNase) is a cytotoxic and antitumor RNase isolated from the oocyte yolk granules of the bullfrog R. catesbeiana. Our previous studies have shown that RC-RNase possesses antitumor activity by activating proapoptotic caspases. Here, we demonstrate that RC-RNase also possesses antiviral activity. By using cell viability and caspase activation assays, we show that RC-RNase largely enhances apoptosis of Japanese encephalitis virus (JEV)-infected BHK-21 cells by activating caspase-3, caspase-8, and caspase-9. In addition, immunoblotting experiments revealed that JEV infection enhances the internalization of RC-RNase by cells. In sum, these results indicate that RC-RNase provides a beneficial effect on JEV-infected cells by enhancing apoptosis.

Keywords: JEV; RC-RNase; Caspase; Apoptosis

Preparation of CHO cell-derived rhIFN-ω-Fc with improved pharmacokinetics by Jianmin Li; Bing Li; Jun Zhang; Lihua Hou; Changming Yu; Ling Fu; Xiaohong Song; Ting Yu; Jinglong Zhang; Jun Ren; Chun’e Xu; Wei Chen (pp. 199-203).

Interferon-omega (IFN-ω) may be a useful, promising and alternative antiviral agent, in addition to IFN-α-2a and IFN-α-2b. To improve the pharmacokinetics of IFN-ω for clinical use, the recombinant human IFN-ω-Fc fusion protein (rhIFN-ω-Fc) was expressed in a Chinese hamster ovary cell line (CHO-S), due to the longer serum half-life of rhIFN-ω-Fc compared to the native IFN-ω protein, and purified by affinity chromatography. Physicochemical characterization of the purified fusion protein was performed by SDS–PAGE electrophoresis, dot blot analysis and N-terminal amino acid sequence analysis. The results show that rhIFN-ω-Fc was highly expressed at the predicted size and with the N-terminal amino acid sequence. The antiviral activity was determined by the ability of IFNs to inhibit the cytopathic effects (CPEs) of vesicular stomatitis virus (VSV) on the human amnion WISH cells. The rhIFN-ω-Fc expressed in CHO-S cells has a specific activity of 1.6×10⁷IU/mg compared to rhIFN-ω expressed in yeast, which has a specific activity of 7×10⁷IU/mg. Equimolar concentrations of rhFN-ω and rhIFN-ω-Fc were administered to rabbits for pharmacokinetics comparison. The terminal half-life of rhIFN-ω-Fc was 35 times higher than that of rhIFN-ω. Thus, rhIFN-ω-Fc can be used as a prospective antiviral candidate especially for the treatment of chronic viral disease, such as hepatitis C virus (HCV) infection.

Keywords: rhIFN-ω-Fc; rhIFN-ω; CHO-S; Physicochemical characterization; Pharmacokinetics

Picornavirus non-structural proteins as targets for new anti-virals with broad activity by Heléne Norder; Armando M. De Palma; Barbara Selisko; Lionel Costenaro; Nicolas Papageorgiou; Carme Arnan; Bruno Coutard; Violaine Lantez; Xavier De Lamballerie; Cécile Baronti; Maria Solà; Jinzhi Tan; Johan Neyts; Bruno Canard; Miquel Coll; Alexander E. Gorbalenya; Rolf Hilgenfeld (pp. 204-218).

Picornaviridae is one of the largest viral families and is composed of 14 genera, six of which include human pathogens. The best known picornaviruses are enteroviruses (including polio, PV, and rhinoviruses), foot-and-mouth disease virus (FMDV), and hepatitis A virus (HAV). Although infections often are mild, certain strains may cause pandemic outbreaks accompanied with meningitis and/or paralysis. Vaccines are available for PV, HAV and FMDV. When the oral vaccines are given to immunocompromised individuals, they may be chronically infected, and remain secretors of vaccine-derived variants of virus for years. There is no effective prophylaxis available for these or other picornaviruses. So far, only the 3C protease from viruses in three genera has been fully characterized as an anti-viral target, whereas the mode of action of compounds targeting other non-structural proteins have remained largely unaddressed. Within the EU-supported FP6 project-VIZIER (Comparative Structural Genomics of Viral Enzymes Involved in Replication), the non-structural proteins were studied to identify conserved binding sites for broadly reactive anti-virals. The putative 2C helicase from echovirus-30 was shown to form ring-shaped hexamers typical for DNA-encoded SF3 helicases, and to possess ATPase activity. Hexamer formation of 2C from enterovirus 76 was in vitro shown to be dependent on the 44 N-terminal residues. Crystal structures of three enterovirus 3C proteases were solved and shown to be similar to those of other picornaviruses. A new binding site of VPg to the bottom of the thumb domain of CV-B3 3D polymerase was identified as a potential target. Broad anti-enterovirus compounds against 2C and 3A proteins were also identified, including thiazolobenzimidazoles (active against 2C) and TTP-8307 (targeting 3A). There is a need for more potent inhibitors against PV and other picornaviruses, which are potential silent reservoirs for re-emerging PV-like disease.

Keywords: Picornavirus; Non-structural proteins; Anti-viral compounds; Enterovirus; Polio; Structure

MiR-101 regulates HSV-1 replication by targeting ATP5B by Shu-qi Zheng; Yi-xuan Li; Yan Zhang; Xin Li; Hua Tang (pp. 219-226).

MicroRNAs (miRNAs) are short non-coding RNAs that negatively modulate gene expression at the post-transcriptional level and are known to be involved in the cross-talk between the host and virus. Using a standard plaque assay and real-time PCR method, we found that ectopic expression of miR-101 could significantly suppress herpes simplex virus-1 (HSV-1) replication, and that blocking endogenous miR-101 could increase viral progeny without affecting cell viability. Bioinformatics analysis indicates the 3′ untranslated region (UTR) of mitochondrial ATP synthase subunit beta (ATP5B) has a putative binding site for miR-101. MiR-101 can directly bind to ATP5B 3′UTR and negatively regulate ATP5B expression. Using a RNA interference technique, knockdown of ATP5B significantly inhibited HSV-1 replication, indicating that ATP5B functions as a pro-viral factor. The ectopic expression of ATP5B lacking the 3′UTR could override the suppressive effect of miR-101 on HSV-1 replication. A concordant inverse correlation between miR-101 and ATP5B was observed in HSV-1-infected HeLa cells. Up-regulation of miR-101 expression may play a role in repressing productive HSV-1 replication by targeting ATP5B. Exploring the role of host-encoded miRNA in the regulation of viral infection would enable us to better understand the intricate networks of host–pathogen interactions.

Keywords: Abbreviations; miRNA; microRNA; HSV-1; herpes simplex virus-1; UTR; untranslated region; ATP5B; ATP synthase subunit beta; ASO; antisense oligonucleotide; CPE; cytopathogenic effect; MOI; multiplicity of infection; siRNA; small interference RNA; EGFP; enhanced green fluorescence protein; MTT; (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide); RFP; red fluorescent protein; CT; threshold cycle; ORF; open reading frame; Vhs; virion host shutoffmiR-101; HSV-1; ATP5B; Viral replication

Oxymatrine inhibits hepatitis B infection with an advantage of overcoming drug-resistance by Yu-Ping Wang; Wei Zhao; Rong Xue; Zhen-Xian Zhou; Fei Liu; Yan-Xing Han; Gang Ren; Zong-Gen Peng; Shan Cen; Hong-Shan Chen; Yu-Huan Li; Jian-Dong Jiang (pp. 227-231).

Oxymatrine (OMTR) is an anti-hepatitis drug used in China. Its mechanism of action is unknown. Recently, we found that OMTR inhibits hepatitis B virus (HBV) via down-regulating the expression of heat-stress cognate 70 (Hsc70), a host protein required for HBV DNA replication. Goal of this study was to assess the effect of OMTR on clinical HBV drug-resistance. OMTR monotherapy (oral, 12 months) reduced blood HBV DNA by 96% and HBeAg by 70% in the chronic hepatitis B (CHB) patients resistant to lamivudine ( n=17), equal to its efficacy in the naïve CHB cohort ( n=20). Liver biopsy study showed that OMTR treatment caused a decrease of Hcs70 mRNA in liver cells, parallel with a reduction of intracellular HBV DNA. Combination of lamivudine with OMTR ( n=15) (oral, 12 months) showed an enhanced anti-HBV effect as compared to lamivudine monotherapy ( n=25). The incidence of drug resistance against lamivudine in the combination group was significantly lower than that in the lamivudine group (1/15 vs 7/25; p<0.01). The results were further confirmed in vitro. Treatment of HBV(+) HepH2215 cells with sub-optimal dose of OMTR for 8 months suppressed HBV replication without inducing drug resistance, whereas lamivudine monotherapy caused drug-resistant mutation in 3 months. Combination of OMTR with lamivudine prevented HBV from developing drug resistance.

Keywords: Abbreviations; Hsc70; heat-stress cognate 70; HBV; hepatitis B virus; OMTR; oxymatrine; RT; reverse transcriptase; CHB; chronic hepatitis BOxymatrine; Hsc70; Antiviral therapy; Drug resistance; Hepatitis B virus

Polymorphisms of interferon-inducible genes OAS associated with interferon-α treatment response in chronic HBV infection by Shan Ren; Haibin Yu; Hongwei Zhang; Ying Liu; Yanxiang Huang; Lina Ma; Lai Wei; Hao Wu; Xinyue Chen (pp. 232-237).

To evaluate the role of host single nucleotide polymorphisms (SNPs) of 2′,5′-oligoadenylate synthetase (OAS) in predicting IFN response in patients with HBV infection, OAS gene and four SNPs were examined in 363 patients with chronic HBV infection (including 41 patients with HBsAg seroconversion) and 57 healthy controls. One SNP and three haplotypes were identified after adjustment for age, sex, HBV DNA. The frequency of OAS3T/C heterozygotes is 52.2% in responders (R) and 38.2% in non-responders (NR), with an odds ratio (OR) of 1.511 ( P=0.018). For complete responders (CR) and NR, the OR reached 2.323( P=0.023). Haplotype analyses revealed significant association between three OAS haplotypes and response to IFN-α treatment. Genotype combination and interaction between gene–gene analyses disclosed that there was a positive interaction between OAS2/OAS3 and OAS3/OASL, and the rate of OR was 2.46 (likelihood test, P=0.004) and 4.46 (likelihood test, P=0.004), respectively. Our results suggest that OAS gene variations may play an important role in response to IFN-α and provide a novel strategy for the resolution of HBV infection.

Keywords: Hepatitis B virus (HBV); Interferon-α; HBsAg seroconversion; Single nucleotide polymorphism (SNP); 2′,5′-Oligoadenylate synthetase(OAS); Haplotype

Safety, pharmacokinetics, and antiviral activity of the cyclophilin inhibitor NIM811 alone or in combination with pegylated interferon in HCV-infected patients receiving 14 days of therapy by Eric Lawitz; Eliot Godofsky; Regine Rouzier; Thomas Marbury; Tuan Nguyen; June Ke; MeiMei Huang; Jens Praestgaard; Denise Serra; Thomas G. Evans (pp. 238-245).

Cyclophilin inhibitors have shown activity against a variety of viruses, including HCV. NIM811, a novel, non-immunosuppressive cyclophilin inhibitor was studied in ascending doses in a randomized, double-blind, placebo-controlled 14-day trial in genotype 1 HCV patients. Doses of 10 up to 600mg were given orally once or twice daily as monotherapy (9:3 randomization of NIM811:placebo). 600mg or placebo bid for 14 days was then co-administered with pegylated interferon alpha (PEG-IFN-α) administered on days 1 and 8 to genotype 1 relapsers.NIM811 was well tolerated at all doses. Although lack of antiviral effect was noted in the monotherapy arms, liver transaminase normalization occurred at doses over 75mg. Mild, clinically non-significant elevations of bilirubin, and significant declines in platelet numbers were observed in the 400 and 600mg bid groups. In the combination group, the mean HCV RNA decline was 2.85log, compared to a 0.56log in the PEG-IFN alone arm. The mean ALT (alanine transaminase) declined significantly by day 14 in the combination, but was unchanged in the PEG-IFN alone group. In the combination therapy group, the mean platelets were 203×10⁹/L at baseline and fell to 105×10⁹/L by day 14; for patients treated with PEG-IFN the values were 177×10⁹/L and 139×10⁹/L. There was a significant increase in bilirubin, although this did not reach clinically concerning levels. There were no severe or serious adverse events. The pharmacokinetics in both monotherapy and combination arms were dose linear and not affected by PEG-INF.NIM811 monotherapy resulted in a normalization of liver transaminases in the absence of significant virological response. The combination of NIM811 and pegylated interferon alpha showed significant antiviral activity compared to interferon alone in genotype 1 HCV relapsers. The use of oral cyclophilin inhibitors as part of a combination regime for treatment of hepatitis C, especially to deter resistance, holds promise.

Keywords: Cyclophilin; HCV; NIM811; Pegylated interferon; Antiviral

High-content screening to distinguish between attachment and post-attachment steps of human cytomegalovirus entry into fibroblasts and epithelial cells by Yvonne Ibig-Rehm; Marjo Götte; Daniela Gabriel; David Woodhall; Ashley Shea; Nathan E. Brown; Teresa Compton; Adam L. Feire (pp. 246-256).

Human cytomegalovirus (HCMV) enters cells through a complex pathway involving the interaction of multiple viral glycoproteins and cellular receptors. While HCMV clinical isolates enter a wide range of cell types, entry has historically been studied using a laboratory strain of virus that can only infect fibroblasts. Herein, we have constructed a HCMV reporter strain that contains GFP fused to the abundant tegument protein pp65 to allow for the direct visualization of virus attachment and entry. Furthermore, the UL131 gene of this strain was restored to clinical isolate sequence to expand our studies of entry into physiologically relevant epithelial cell types. Using the HCMV-GFP reporter virus, we developed an image-based assay and screened a library containing 65,000 compounds for the inhibition of virus entry into fibroblasts. In addition to assessing the effect on virus entry, automated image analysis provided information on compound toxicity and whether the compounds acted as attachment or post-attachment inhibitors. To identify therapeutically viable inhibitors capable of blocking entry in multiple cell types, the inhibitors were screened further for their ability to inhibit virus entry into epithelial cells. Compounds were identified that were able to inhibit virus entry into both cell types at either attachment or post-attachment steps.

Keywords: HCMV; Cytomegalovirus; High-content screening; Imaging; Virus entry

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Antiviral Research (v.89, #3)