Impact of hepatitis C virus heterogeneity on interferon sensitivity:An overview

Hepatitis C virus(HCV)is a major cause of liver disease worldwide.HCV is able to evade host defense mechanisms,including both innate and acquired immune responses,to establish persistent infection,which results in a broad spectrum of pathogenicity,such as lipid and glucose metabolism disorders and hepatocellular carcinoma development.The HCV genome is characterized by a high degree of genetic diversity,which can be associated with viral sensitivity or resistance(reflected by different virological responses)to interferon(IFN)-based therapy.In this regard,it is of importance to note that polymorphisms in certain HCV genomic regions have shown a close correlation with treatment outcome.In particular,among the HCV proteins,the core and nonstructural proteins(NS)5A have been extensively studied for their correlation with responses to IFN-based treatment.This review aims to cover updated information on the impact of major HCV genetic factors,including HCV genotype,mutations in amino acids 70 and91 of the core protein and sequence heterogeneity in the IFN sensitivity-determining region and IFN/ribavirin resistance-determining region of NS5A,on virological responses to IFN-based therapy.

Prevalence and genotype distribution of hepatitis B virus among migrant workers in Lombok Island, Indonesia

Objective:To examine the potential risk of hepatitis B virus(HBV)spread in Indonesia by migrant workers,based on the molecular characteristics of HBV strains.Methods:Sera collected from migrant workers traveling to their destination countries(pre-migrant workers)and those returning to Indonesia(post-migrant workers)were screened for HBsAg by ELISA,followed by HBV DNA detection by PCR and(sub)genotype/subtype determination according to surface region and whole genome sequencing.Results:Of 87 pre-migrant workers,15(17.24%)were HBsAgpositive,whereas 15(12.10%)of 124 post-migrant workers were HBs Ag seropositive.HBV genotype analysis based on the S region showed that HBV-B3/adw2 was predominant(96.15%,25/26)whereas 3.85%(1/26)of isolates were HBV-C3/adrq+.Whole genome sequencing of selected strains and phylogenetic tree analysis identified subgenotype B7 in three samples previously categorized as subgenotype B3 based on S region analysis,supporting a recent argument that subgenotypes B5/B7/B8/B9 could be considered as a quasi-subgenotype of B3.Conclusions:A high prevalence of HBsAg carriers was detected among migrant workers from Lombok Island,with no significant difference in prevalence between before and after returning to Indonesia.All strains were classified into genotypes common in Indonesia,and the results suggested that migrant workers are not a risk factor for HBV transmission into Indonesia.

Antiviral activity of the dichloromethane extracts from Artocarpus heterophyllus leaves against hepatitis C virus

Objective: To determine anti-viral activities of three Artocarpus species: Artocarpus altilis, Artocarpus camansi, and Artocarpus heterophyllus(A. heterophyllus) against Hepatitis C Virus(HCV).Methods: Antiviral activities of the crude extracts were examined by cell culture method using Huh7it-1 cells and HCV genotype 2a strain JFH1. The mode of action for anti-HCV activities was determined by time-of-addition experiments. The effect on HCV RNA replication and HCV accumulation in cells were analyzed by quantitative reverse transcription-PCR and western blotting, respectively.Results: The dichloromethane(DCM) extract of A. heterophyllus exhibited strong antiHCV activity with an inhibitory concentration(IC_(50)) value of(1.5 ± 0.6) mg/mL without obvious toxicity. The DCM extracts from Artocarpus altilis and Artocarpus camansi showed moderate anti-HCV activities with IC_(50) values being(6.5 ± 0.3) mg/mL and(9.7 ± 1.1) mg/mL, respectively. A time-of-addition studies showed that DCM extract from A. heterophyllus inhibited viral entry process though a direct virucidal activity and targeting host cells. HCV RNA replication and HCV protein expression were slightly reduced by the DCM treatment at high concentration.Conclusions: The DCM extract from A. heterophyllus is a good candidate to develop an antiviral agent to prevent HCV grant reinfection following liver transplantation.

Hepatitis C virus NS3/4A with sequence variation at amino-terminus has different serine protease activities and inhibitory activities on IFN-β induction and p53-dependent transcriptional activation

Objective： To construct the point mutation plasmids expressing HCV NS3/4A with different secondary structures at the N-terminus, and to analyze their serine protease activities. Methods： The point mutation plasmid constructs were generated by using the QuickChange site-directed mutagenesis kit with the backbone of M-H05-5 （AI-1）, and were named as subgroup A1-2, A2-1, A2-2, BI-1, B1-2, B2-1, and B2-2 respectively. The transient expression of the constructs was investigated by immunofluorescence assay and Western blot analysis. The difference in in cis and in trans NS3 serine protease activity between each subgroup was determined by Western blot analysis. Luciferase reporter assay was used to observe the inhibitory effects of the constructs on RIG-I induced IFN-β promoter activity and on p53-dependent transcriptional activation. Results： The point mutation plasmid constructs were verified for the correct sequence by DNA sequencing. The immunofluorescence assay revealed 4 subcellular localization patterns of NS3, including dot-like staining, diffuse staining, doughnut-like staining, and rod-shape staining. Western blot analysis indicated that the incomplete cleavage of NS3/4A appeared in subgroups A2-1 and B2-1, indicating that the in cis NS3 serine protease activities of subgroup A2-1 and B2-1 were weaker when compared with the other subgroups. By using NS5A/SBAC as a substrate for NS3/4A serine protease, it was also found that the in trans NS3 serine protease activities of subgroup A2-1 and B2-1 were also weaker compared the other subgroups. Differences in inhibitory effects of HCV NS3 on RIG-I induced IFN-β promoter activity and on p53-dependent transcriptional activation were also observed between subgroup A2-1, B2-1 and the other subgroups. Conclusion： The results suggest that subgroup A2-1 and B2-1 has weaker serine protease activities and weaker inhibitory activities on host cell functions than the other subgroups, which might be explained by the different secondary structure of the 120-aa sequence at N-terminus of NS3.

Molecular epidemiology of hepatitis B virus in Asia

Although safe and effective vaccines against hepatitis B virus(HBV) have been available for three decades, HBV infection remains the leading cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma(HCC) worldwide, especially in Asian countries. HBV has been classified into at least 9 genotypes according to the molecular evolutionary analysis of the genomic DNA sequence and shown to have a distinct geographical distribution. Novel HBV genotypes/subgenotypes have been reported, especially from Southeast Asian countries. The clinical characteristics and therapeutic effectiveness of interferon(IFN) and nucleos(t)ide analogues vary among different HBV genotypes. Mutations at T1653 C in subgenotype C2 from Japan and South Korea, C/A1753 T and C1858 T in subgenotype C1 from Vietnam, and C1638 T and T1753 V in subgenotype B3 from Indonesia were reported to be associated with advanced liver diseases including HCC. Genotype distribution in Japan has been changed by an increasing ratio of subgenotype A2 in chronic hepatitis B. While a large number of epidemiological and clinical studies have been reported from Asian countries, most of the studies were conducted in developed countries such as Taiwan, China, South Korea and Japan. In this review, the most recent publications on the geographical distribution of genetic variants of HBV and related issues such as disease progression and therapy in Asia are updated and summarized.

氨基端不同二级结构的丙型肝炎病毒NS3／4A复合体的丝氨酸酶活性差异研究

目的探讨表达不同氨基端二级结构的丙型肝炎病毒非结构蛋白3／4A（HCVNS3／4A）各亚型丝氨酸酶活性及其对宿主细胞功能抑制作用的差异。方法以pSGS／M。HOS-5／4A为模板（A1-1），构建表达不同氨基端二级结构NS3／4A的点突变质粒，并分别命名为A1-2，A2-1，A2-2，B1-1，B1-2，B2-1，B2-2。Western印迹检测所有构建质粒的表达及各亚型顺式及反式NS3蛋白酶活性的差异。荧光素酶报告试验检测pSGS／M-H05-5／4A各个二级结构亚型对干扰素-β（IFN-β）产生及p53依赖的荧光素酶基因的转录活性的抑制作用以及各亚型之间抑制作用的差异。结果Western印迹显示所有构建质粒均成功表达，而且A2-1和B2-1亚型NS3／4A存在不完全切割现象。表明与其他亚型相比，A2—1和B2—1顺式NS3丝氨酸蛋白酶活性较弱。与其底物NS5A／SBAC共表达后，A2—1和B2-1亚型未切割NSSASB明显多于其他亚型，而切割NS5A则明显少于其他亚型。说明与其他亚型相比，A2—1和B2．1亚型的反式NS3蛋白酶活性亦较弱。荧光素酶试验结果显示，所有亚型M—H05-5／4A均显著抑制IFN-β启动子活性（P〈0．01）和p53依赖的荧光素酶基因的转录活性（P〈0．01），A2-1和B2-1亚型的抑制作用显著弱于其他亚型（P〈0．05）。结论氨基端不同二级结构的HCVNS3／4A复合体具有不同的丝氨酸酶活性和宿主细胞功能抑制作用。