Hepatitis G virus

A number of new hepatitis viruses (G, TT, SEN) were discovered late in the past century. We review the data available in the literature and our own findings suggesting that the new hepatitis G virus (HGV), disclosed in the late 1990s, has been rather well studied. Analysis of many studies dealing with HGV mainly suggests the lymphotropicity of this virus. HGV or GBV-C has been ascertained to influence course and prognosis in the HIV-infected patient. Until now, the frequent presence of GBV-C in coinfections, hematological diseases, and biliary pathology gives no grounds to determine it as an "accidental tourist" that is of no significance. The similarity in properties of GBV-C and hepatitis C virus (HCV) offers the possibility of using HGV, and its induced experimental infection, as a model to study hepatitis C and to develop a hepatitis C vaccine.

Hepatitis G virus genomic RNA is pathogenic to Macaca mulatta

AIM: To explore the pathogenicity and infectivity of hepatitis G virus (HGV) by observing replication and expression of the virus, as well as the serological and histological changes of Macaca mulatta infected with HGV genomic RNA or HGV RNA-positive serum.METHODS: Full-length HGV cDNA clone (HGVqz) was constructed and proved to be infectious, from which HGV genomic RNA was transcribed in vitro. Macaca mulatta BY1 was intra-hepatically inoculated with HGV genomic RNA, HGV RNA-positive serum from BY1 was intravenously inoculated into Macaca mulatta BM1, and then BB1 was infected with serum from BM1. Serum and liver tissue were taken regularly, and checked with RT-PCR, in situ hybridization and other immunological, serological,histological assays.RESULTS: Serum HGV RNA was detectable in all the 3Macaca mulattas, serological and histological examinations showed the experimental animals had slightly elevated alanine transaminase (ALT) and developed HGV viremia during the infectious period. The histology, immunohistochemistry, and in situ hybridization in liver tissues of the inoculated animals demonstrated a very mild hepatitis with HGV antigen expression in cytoplasm of hepatocytes.RT-PCR and quantitative PCR results showed that HGV could replicate in liver.CONCLUSION: The genomic RNA from full-length HGV cDNA is infectious to the Macaca mulatta and can cause mild hepatitis. HGV RNA-positive serum, from HGV RNA inoculated Macaca mulatta, is infectious to other Macaca mulattas. Macaca mulatta is susceptible to the inoculated HGV, and therefore can be used as an experimental animal model for the studies of HGV infection and pathogenesis.

Small interfering RNA-mediated inhibition of hepatitis G virus gene expression in human hepatoma cell Huh-7

The RNA interference(RNAi)phenomenon is a recently observed process in which the introduction of a double-stranded,small interfering RNA(siRNA)into a cell causes the specific degradation of a homologous single-stranded RNA.It represents an exciting new technology that could have therapeutic applications for the treatment of viral infections.Since hepatitis G virus(HGV)genome is a positive-sense single-stranded RNA,the replication of HGV does not lead to an integrated DNA genome,suggesting a particularly attractive target for RNAi study that could eliminate viral RNA from infected cells.The eukaryotic expression vector pVAX.EH containing the cDNA sequences of the entire HGV structural genes and hygromycin resistance gene downstream from the encephalomyocarditis virus(ECMV)internal ribosome entry site(IRES)was constructed and transfected into human hepatoma cell Huh-7.The modified cleavage products of the structural proteins of HGV expressed in hygromycin-resistant cell line Huh-7-EH were confirmed by RT-PCR and Western blot methods.Two specific HGV E2 siRNAs(1-E2siRNA,2-E2 siRNA)synthesized with T7 RNA polymerase by transcription in vitro were transfected into the Huh-7-EH cells.With the analyses of Western blot and the formation of hygromycin-resistant colonies,the inhibitions of expression of HGV structural protein by two HGV E2siRNAs were detected and found lasting at least one week.The inhibition of 2-E2 siRNA was stronger and only 1%of the cells treated with 2-E2 siRNA formed hygromycin-resistant colonies.These results support that specific HGV 2-E2 siRNAs mediate the degradation of mRNA spanning from HGV structural gene cDNA to hygromycin resistance gene in a majority of cells.In conclusion,the Huh-7-EH cells expressing HGV structural proteins stably can be used as a cell model for studying the replication of HGV and RNAi and the enlargement of RNAi may exist,in mammalian cells.

嵌合流行毒株G-H环基因重组口蹄疫病毒的拯救及其免疫原性分析

为了研究能有效免疫防控当前流行的O型3个拓扑型口蹄疫病毒(foot-and-mouth disease virus,FMDV)的疫苗候选株,本研究利用FMDV反向遗传操作技术,通过基因替换,构建同时含当前流行的O型三个拓扑型病毒株结构蛋白基因的重组全长克隆,转染表达T7RNA聚合酶的细胞后拯救重组FMDV,分析结构蛋白基因的重构对病毒生物学特性的影响;拯救病毒制备灭活疫苗,免疫猪和牛,用体外中和试验初步研究其作为O型FMD疫苗候选株的潜力。结果表明FMD流行毒株结构蛋白VP1 G-H环基因的替换没有明显影响重组病毒的噬斑表型和复制能力,但不同FMDV G-H环抗原表位对猪诱导机体产生交叉中和抗体水平影响较大,对牛诱导机体产生交叉中和抗体的影响较小,表明FMDV G-H抗原表位是猪免疫优势的表位。本研究为未来FMDV疫苗的设计提供了重要参考。

SEQUENCE ANALYSIS OF THE NS5 REGION OF GBVC/HGV AND DETECTION OF THE VIRUS BY REVERSE TRANSCRIPTASE PCR

GBV C/HGV is a newly identified virus associated with human hepatitis In this study, the nucleotide sequences of the partial NS5 gene of GBV C/HGV derived from sera of 8 Chinese patients were determined The nucleotide homology among the 8 isolates were 92% on average On the basis of sequence analysis, two sets of oligonucleotide primers derived from highly conserved region of GBV C/HGV NS5 gene were designed to establish both sensitive and specific nested PCR for detection of GBV C/HGV RNA 253 Chinese patients were examined for the virus RNA GBV C/HGV RNA positive rates in patients infected with HBV, HCV and patients with chronic non B,non C hepatitis were 18 4%, 19 8% and 8 9% respectively This result suggested that HBV,HCV and GBV C/HGV shared the same transmission risk factors 8 patients with GBV C/HGV and HCV coinfection were retrospectively observed for the response to interferon Coinfection with GBV/HGV did not negatively influence the responsiveness of HCV, and GBV C/HGV was sensitive to interferon to a certain degree

Insertion site of FLAG on foot-and-mouth disease virus VP1 G-H loop affects immunogenicity of FLAG

The G-H loop of the foot-and-mouth disease virus（FMDV） virion contains certain dominant immunogenic epitopes, as well as an arginine-glycine-aspartic acid（RGD） motif that is recognized by cell surface integrin receptors. Previous experiments indicate that it is critical to maintain virus structural integrity when inserting an exogenous epitope into the surface of an FMDV structural protein. However, it remains to be determined how factors such as different insertion positions affect interactions among the virus, cells and host immune system. In this study, one infectious c DNA clone of the swine FMDV Cathay topotype strain O/CHA/90 was constructed. Then, a FLAG marker（DYKDDDDK） was inserted upstream（–4） or downstream（＋10） of the RGD motif to generate tagged viruses vFLAG-O/CHA/90 or vO/CHA/90-FLAG, investigating the possibility of expressing foreign antigen and effect on its immunogenicity. Compared to the parental virus, both tagged viruses exhibited similar plaque phenotypes, suckling mouse pathogenicity and antigenicity. Additionally, the FLAGtag insertion position did not change the use of integrin-mediated cell entry by the tagged viruses. Interestingly, both tagged vaccines protected pigs against challenge with the parental virus O/CHA/90 and induced immune responses against FMDV in BALB/c mice and pigs, but only vaccination with vFLAG-O/CHA/90 generated anti-FLAG antibodies. Our findings demonstrated that two sites（RGD–4 and RGD＋10） tolerated the insertion of an exogenous gene in the swine FMDV O/CHA/90 strain. However, only RGD–4 was a novel and appropriate inserting site which could tolerate exogenous FLAG. The resultant tagged virus is a promising candidate for FMD vaccine which can be differentiating infected from vaccinated animals（DIVA）.

Hepatitis G Viral RNA Co-infection in Plasma and Peripheral Blood Mononuclear Cells in Patients with Hepatitis C

The incidence of the co-infection of hepatitis G virus (HGV) and hepatitis C virus (HCV) and its clinical implication was investigated and the difference in the positive rate of HGV RNA and HCV RNA between plasma and peripheral blood mononuclear cells (PBMCs) observed. By using reverse transcriptase polymerase chain reaction (RT-PCR) assay, HCV-RNA and HGV-RNA in plasma and PBMCs of 72 patients with hepatitis C was detected. It was showed that HGV RNA was positive in plasma of 11 patients, in PBMCs of 15 patients, and simultaneously in both of plasma and PBMCs of 10 patients with the co-infection rate being 22.2 %. Nine patients were both HGV RNA and HCV RNA positive in plasma, 11 patients were both HGV RNA and HCV RNA positive in PBMC, and 6 patients were both HGV RNA and HCV RNA positive in both plasma and PBMC with the positive rate being 12.4 %,15.3 % and 8.3 % respectively. The positive rate of both HGV RNA and HCV RNA in PBMCs was higher than in plasma. It was concluded that the HGV co-infection rate in the patients with hepatitis C was 22.2 %. Simultaneous examination of plasma and PBMC can improve clinically detectable rate.

侵染甘薯的SPCSV、SPVG、SPFMV多重RT-PCR检测方法的建立及应用

为建立可同时检测甘薯褪绿矮化病毒（SPCSV）、甘薯G病毒（SPVG）和甘薯羽状斑驳病毒（SPFMV）多重RT-PCR检测方法,本文根据SPCSV热激蛋白基因（hsp70）及SPVG、SPFMV外壳蛋白基因（CP）基因核苷酸序列的保守区域设计特异性引物,通过引物筛选,优化多重RT-PCR反应条件,建立了能同时检测SPCSV、SPVG和SPFMV 3种病毒的多重RT-PCR检测方法。该体系能有效扩增出大小为304、433、601 bp 3个特异性片段。测序结果表明3种病毒与参考序列的一致性达94%-99%。应用建立的多重RT-PCR检测方法可稳定、准确、灵敏地同时检测单一或复合侵染3种甘薯病毒,为甘薯脱毒和病毒病诊断奠定基础。

新型冠状病毒IgM/IgG胶体金试剂应用情况分析

目的评价新型冠状病毒血清学IgM/IgG胶体金试剂与核酸检测试剂的一致性。方法收集整理2020年3月3日至5月26日中山市出院健康随诊的确诊病例(或无症状感染者)和集中隔离观察期间的密切接触者的检测信息和流行病学信息。结果242人385人次完成血清学检测。出院后第7天、第14天和第3个月开展了血清学检测的确诊病中,IgG阳性率分别为100.0%(12/12)、94.7%(36/38)和77.8%(21/27);10例无症状感染者,出院后14 d,IgG阳性率为80%(8/10);178例核酸检测阴性的密切接触者,IgG阴性率为98.9%(176/178)。除1例确诊病例发生IgG阳性到阴性转化外,其余人群IgG和核酸检测符合率为95.9%(231/241)(Kappa=0.889,P=0.334)。结论新型冠状病毒IgM/IgG胶体金试剂中IgG和核酸检测结果的一致性较好;但新型冠状病毒感染1个月后,随着时间延长,IgG阳性率有降低的趋势。

肝病患者中庚型肝炎病毒感染的状况

目的观察庚型肝炎病毒 (hepatitisGvirus,HGV)在不同肝病中的感染情况 ,讨论其致病性。方法对天津地区 6 0 77例肝病患者采用ELISA法和RT PCR法检测病原 ,对不同类型肝病中HGV感染情况进行分析比较。结果 2 4 7例患者抗 HGV阳性 ,检出率为 4 .0 6 % (2 4 7/ 6 0 77) ,HGVRNA阳性率为 33.91% (38/ 112 )。HGV的感染情况 :单纯感染 4 4例 ,重叠感染 2 0 3例 ,丙型肝炎 13.0 8% (16 / 10 2 ) ,明显高于其它病原型 ;重症肝炎8.10 % (17/ 2 10 )明显高于其它临床型。重症肝炎的发生率 ,抗 HGV阳性组与抗 HGV阴性组分别为 6 .88%(17/ 2 4 7) ,3.31% (193/ 5 830 ) ,病死率分别为 3.2 4 % (8/ 2 4 7) ;1.18% (6 9/ 5 830 )。结论提示HGV具有较强致病性 ,是病毒性肝炎的病原之一 ,感染HGV病情相对较重 。

庚型肝炎病毒RNA NS3区基因分型

目的：探讨HGVNS3区基因变异与基因型的关系。方法：对32株NS3区序列进行酶切位点分析，比较中国株与GBV－C株和已发表的HGVNS3区酶切位点，选择特异性内切酶切点。结果：32株NS3序列酶切位点分析表明存在5种基因型。1组：在4338位无HhaI切点，而在4360位有HhaI切点；2组：4338位及4360位均无HhaI切点；3组：仅在4338位有HhaI切点；4组：4338位及4351位有HhaI及Tsp509I切点；5组：4351位有Tsp509I切点。其核苷酸变异率分别为0．85％、19．0％、20．5％、18．2％、19．6％等。结论：中国株与西非、东非、加拿大、美国及日本株不是同一基因型。

人源抗乙型肝炎病毒基因工程全抗体的研制

目的 研制人源化抗乙型肝炎表面抗原 (HBsAg)的IgG全抗体。方法 采用噬菌体抗体库技术 ,从乙肝疫苗免疫后健康人外周血淋巴细胞中提取抗体基因建立抗体库 ,用纯化的HBsAg筛选Fab抗体 ;再将获得的抗体轻、重链构建到杆状病毒表达载体pAc κ Fc中 ,转染SF9细胞表达IgG全抗体并进行纯化。免疫荧光检测抗体的表达 ,竞争抑制ELISA检测其与抗原的结合活性及特异性。结果 获得了一株抗乙肝表面抗原的Fab抗体 ;在转染后的SF9培养上清中收获到了IgG抗体 ,竞争ELISA证实该抗体针对HBV表面抗原的特异性抗体。结论 本实验得到了一株人源抗HBsAg的IgG抗体 ,并进行了纯化。

SEN病毒部分基因的克隆及序列测定

目的 :了解我国人群中是否存在SEN病毒 (SENV)感染 ,并分析SENV国内分离株的部分基因序列。方法 :在SENV的保守区设计引物 ,建立巢式PCR方法检测血清中SENVDNA ,并对PCR产物进行克隆、测序。结果 :7例非甲～非庚型肝炎 ,且输血传播病毒 (TTV)阴性患者中 ,2例SENV阳性。其中一株的序列与意大利SENV D株 (AX0 2 5730 )相对应位置核苷酸序列同源性为90 %。结论 :本研究证实了我国存在SENV感染 ,建立了检测SENV的PCR方法 ,并对SENV部分基因进行了克隆及测序 。

水中肠道食源性病毒的多重RT-PCR检测法

目的建立能同时检测水环境中肠道病毒(Enterovirus,EVs)、诺如病毒GⅡ(Norovorus,Nov GⅡ)、轮状病毒(Rotavirus,RVs)、腺病毒(Adenovirus,AdVs)的多重RT-PCR检测方法。方法根据GenBank收录的肠道食源性病毒核酸序列,利用软件DNAMAN设计肠道病毒通用引物以及Nov GⅡ、RVs、AdVs的特异性引物;将这4对引物置于同一体系中进行多重RT-PCR,利用各种肠道食源性病毒的核酸模板检测其特异性并通过不同滴度[100～107pfu(copies)/ml]病毒测试其灵敏度;利用加标实验验证该方法的准确性;通过对5份河水或某水厂出水大体积水样(50 L)进行病毒富集与检测,以确定该方法的实用性,同时,采用传统细胞培养法验证其结果。结果该方法对目的病毒均获得了理想的电泳条带,而对其他病毒无非特异扩增;对EVs、AdVs的最低检出滴度均为10 pfu/ml,Nov GⅡ-4为10 copies/ml,对RVs的最低检出滴度为102pfu/ml;加标实验检测结果准确率为100%;对5份河水或某水厂出水大体积水样进行病毒富集与检测,对以上各种病毒均有检出且与细胞培养结果一致。结论该方法不仅特异性强、灵敏度高、简便快捷,而且准确率高,实际应用性强,适用于水中肠道食源性病毒的快速检测。

诺瓦克样病毒实时荧光定量PCR检测方法研究

目的建立贝类中GⅡ型诺瓦克样病毒的实时荧光定量PCR检测方法。方法针对GⅡ型诺瓦克样病毒保守序列，设计出简并引物与探针，建立GⅡ型诺瓦克样病毒实时荧光定量PCR检测方法。并用该方法和常规PCR法对127份贝类标本进行检测。结果研究方法对GⅡ型诺瓦克样病毒检测准确和重复性好，灵敏度为10拷贝。标准曲线的线性范围为10^1～10^5拷贝，相关系数为0．9986，并且对贝类标本的检出率显著高于常规PCR。结论本研究建立了GⅡ型诺瓦克样病毒的实时荧光定量PCR检测方法，可用于贝类中GⅡ型诺瓦克样病毒污染状况及其突发事件的快速定量检测。