南京地区戊型肝炎病毒基因型鉴定和变异分析

为了解南京地区戊型肝炎病毒(Hepatitis E virus,HEV)基因型的分布以及变异状况,本研究采用逆转录套式聚合酶反应(RT-nPCR)的方法检测南京地区40份急性散发性戊型肝炎患者的粪便标本,对PCR产物进行测序,利用生物信息学软件比较核苷酸的同源性、遗传距离,进行基因分型和变异分析.40份粪便标本中检测到14份阳性HEV、RNA,检出率为35%,基因分型均为HEV Ⅳ型,且分属2个不同的亚型;利用生物信息学软件对国内Ⅰ型和Ⅳ型毒株加以比较,发现HEV Ⅳ型毒株比Ⅰ型变异程度高,不同年份的HEV Ⅳ型毒株变异更大,有新的亚型出现,且变异有随时间推移而增大的趋势.     

新疆猪粪便戊型肝炎病毒RNA的检测及序列分析

从戊型肝炎病毒(Hepatitis Evirus,HEV)IgG检测阳性的新疆某猪场采集70份猪粪便,利用逆转录套式聚合酶链方法(RT-nPCR),检测HEV RNA,其中13份为阳性,阳性率18．57％。将PCR扩增产物克隆到pMD18-T载体上,构建成重组质粒并测序,结果表明,13株猪源HEV分离株在HEV ORF2 348bp核苷酸序列的同源性为97．1％～100％,为同一基因型;与HEVⅠ、Ⅱ、Ⅲ、Ⅳ的同源性分别为74．1％-77．6％,71．6％-74．1％,73.3％～78．2％和82．8％-91.4％,与ⅣA亚型的同源性同源性最高达89．4％-91．4％。以该核苷酸片段绘制的基因进化树显示13株猪源HEV与HEV Ⅳ T1株在同一分支上,属基因Ⅳ型;与国内其他猪源HEV分离株该片段核苷酸序列的同源性为82．6％-91．3％,提示中国猪源HEV的基因型比较一致,同属HEV Ⅳ型。     

检测猪粪便中基因4型戊型肝炎病毒的实时荧光定量RT-PCR方法的建立

针对基因4型戊型肝炎病毒(Hepatitis E virus,HEV)基因组ORF3设计特异性引物和探针,建立了一套TaqMan实时荧光定量RT-PCR方法。评价了该方法的准确性和灵敏性,同时与常规RT-PCR和巢式RT-PCR方法进行了对比分析。结果表明,生成的标准曲线显示各浓度范围内具有良好的线性关系,相关系数(r2)为0.994,斜率为-3.312,PCR效率为100%。组内和组间重复性试验相同稀释度标准质粒Ct值之间差异均不显著,并且能够准确的从HEV阳性猪粪便样品中检测到基因4型HEV RNA,具有较好准确性。可检测到戊型肝炎病毒数量为(1.7×101)copies,比普通RT-PCR的灵敏度高100倍,比Nested RT-PCR高10倍。     

新疆猪粪便戊型肝炎病毒RNA的检测及序列分析

从戊型肝炎病毒(Hepatitis E virus,HEV)IgG检测阳性的新疆某猪场采集70份猪粪便,利用逆转录套式聚合酶链方法(RT-nPCR),检测HEV RNA,其中13份为阳性,阳性率18.57%.将PCR扩增产物克隆到pMD18-T载体上,构建成重组质粒并测序,结果表明,13株猪源HEV分离株在HEV ORF2 348bp核苷酸序列的同源性为97.1%～100%,为同一基因型;与HEV Ⅰ、Ⅱ、Ⅲ、Ⅳ的同源性分别为74.1%～77.6%,71.6%～74.1%,73.3%～78.2%和82.8%～91.4%,与ⅣA亚型的同源性同源性最高达89.4%～91.4%.以该核苷酸片段绘制的基因进化树显示13株猪源HEV与HEVⅣT1株在同一分支上,属基因Ⅳ型;与国内其他猪源HEV分离株该片段核苷酸序列的同源性为82.6%～91.3%,提示中国猪源HEV的基因型比较一致,同属HEVⅣ型.     

福建省2000～2003年戊型肝炎病毒分离株的分子流行病学研究

为研究福建省2000～2003年急性散发性戊型肝炎病毒（HEV）分离株的分子流行病学特征,采用RT—PCR方法扩增HEV ORF2的基因片段,经纯化、克隆、测序后,对其序列用Neighbor-Joining法和Bootstrap法进行基因进化分析。从158例急性散发性戊型肝炎的血清标本中扩增出72份HEV RNA,经基因进化树分析,这72株HEV均属于HEV基因Ⅳ型,且被明显地分为A、B、C、D共4个组群。A群的同源性最高,群内同源性为93．3％～100％;D群的同源性最低,群内同源性在86．6％～100％之间。A群和B群流行株所占比例没有差异;C群流行株所占比例由2002年的7．1％逐渐增加到2003年的26．3％（x^2=10．553,P=0．014）;D群则由85．7％逐渐减少到44．7％（x^2=8．136,P=0．043）。引起福建地区急性散发性戊型肝炎流行的HEV均属于HEV基因Ⅳ型,基因型内存在不同的组群,其中D群的变异较大。     

不同基因型戊型肝炎病毒存在多种类型抗原表位

以戊型肝炎病毒(HEV)ORF2重组蛋白p166Us为免疫原制备单克隆抗体(McAbs),采用间接ELISA和免疫印迹法,检测McAbs与不同基因型和亚型HEV重组蛋白p166Bur(Ⅰa型)、p166Pak(Ⅰb型)、p166Mor(Ⅰc型)、p166Mex(Ⅱ型)、p166Us(Ⅲ型)、p166Nz(猪HEV,Ⅲ型)和p166Chn(Ⅳ型)的反应性,采用抗原或抗体竞争ELISA分析p166蛋白与天然HEV颗粒之间抗原表位的关系。结果获得4D3、2E3、11E11、12H5、3A3和1F16株稳定分泌McAbs的杂交瘤细胞株。4D3分泌的McAb与7种p166均发生反应,其与免疫原p166Us的结合可被Ⅰ、Ⅱ、Ⅲ或Ⅳ型天然HEV颗粒或病人血清竞争抑制。2E3、11E11和12H5分泌的McAbs只与p166Us、p166Nz和p166Chn发生反应,它们与p166Us的结合仅能被Ⅲ和IV型病毒或血清所抑制。3A3分泌的McAb只与p166Us及p166Nz结合,1F1分泌的McAb只与p166Us结合,两者均能被Ⅲ型美国株竞争抑制,而Ⅰ、Ⅱ、Ⅳ型不能抑制它们与p166Us的结合。由此可见,不同基因型和亚型HEV ORF2编码蛋白p166上存在多种类型抗原表位,其中包括Ⅰ、Ⅱ、Ⅲ、Ⅳ基因型共同的,Ⅲ、Ⅳ基因型共有的和第Ⅲ基因型特异的等,这些表位与天然HEV颗粒上的抗原表位具有相同的免疫学特征。     

戊型肝炎病毒通用性PCR引物的设计及其基因分型的研究

针对戊型肝炎病毒(HEV)基因分型尚无统一标准的现状,本研究通过分析GenBank中现有的82株HEV基因组全序列,设计一组HEV通用性PCR引物(HEVuPrimer)用于扩增不同基因型HEV可测序长片段,分别基于全基因序列、HEVuPrimer扩增序列和较常用的MXJ引物扩增序列对82株HEV进行基因分型,再以HEVuPrimer扩增1～4型HEV参考株和HEVIg M抗体阳性的临床标本,进行HEV基因分型的研究。研究结果表明HE-VuPrimer扩增区段与全基因序列对82株HEV的基因分型结果完全一致;HEVuPrimer与HEV序列的匹配程度明显高于MXJ引物,且HEVuPrimer区段的基因分型结果较MXJ区段的更为准确。HEVuPrimer可同时扩增出不同基因型HEV基因片段。124份临床标本中,60份测出特异性HEV RNA,阳性率为48.4%,基因分型结果均为4型HEV,但分属于4个不同的基因亚型,核苷酸同源性为80.0%～99.9%,其中有6例近期分离的HEV毒株形成一新的基因亚型。因此,基于HEVuPrimer扩增区段的HEV基因分型方法具有较高的可靠性和可信度,为建立统一、可行的HEV基...     

广西14市猪戊型肝炎的分子流行病学分析

为调查广西猪戊型肝炎(HE)的感染情况,本研究应用套式RT-PCR方法对采自广西14市43个猪场的508份3月龄左右猪只粪样及市售182份猪肝扩增猪戊型肝炎病毒(HEV)ORF2基因特异序列,并进行核苷酸序列同源性分析及遗传进化分析。结果表明,在508份猪粪样中194份可以检测到HEV RNA,阳性率为38.2%;182份猪肝样本中,33份可检测到HEV RNA,阳性率为18.1%;43个猪场中检出HEV RNA的有35个,猪场阳性率为81.4%。所获取的39株广西猪HEV ORF2基因特异序列同源性为89.9%～100%,提示在所调查的广西猪场中已较普遍存在HEV感染,所获取的39株广西HEV阳性样品均属于基因Ⅳ型。本研究结果佐证了感染猪及市售带毒猪肝脏是HEV重要的储存库和传染源,应该重视其公共卫生学意义,预防粪-口途径或者食源性HEV感染。     

戊型肝炎病毒IV型衣壳蛋白缺失突变体原核表达与性质

采用PCR技术扩增基因IV型HEV(Hepatitis E Virus,HEV)开放阅读框2(Open Reading Frame 2,ORF2)的缺失突变体(aa384-606),亚克隆到表达载体后,转化到大肠杆菌中进行诱导表达,表达蛋白命名为rP24。SDS-PAGE和免疫印迹实验表明,rP24获得了高效表达,且和单克隆抗体15B2具有强的反应活性。rP24经过包涵体洗涤、溶解复性、离子交换层析和分子筛层析纯化后,免疫印迹实验表明,纯化rP24能与抗HEV ORF2中和单克隆抗体8C11以及HE(Hepatitis E,HE)阳性血清发生很强的免疫反应性,说明rP24上具有构象型中和表位,模拟了HEV衣壳蛋白的空间结构。动态光散色测量结果表明,rP24的平均水化半径为7.48 nm;纯化rP24免疫动物实验表明,rP24具有强的抗原性,小鼠阳转周期短,抗体持续时间长;纯化rP24作为包被抗原检测HE阳性血清和阴性血清,结果显示rP24对HE阳性血清和阴性血清检出率与北京万泰公司的抗HEV-IgG检测试剂盒的检出率一致。这些实验结果说明,具有较好免疫反应性和免疫原性的rP24获得了高效表达,该蛋白模拟了天然病毒衣壳蛋白的中和表位,为进一步研究基因I型和基因IV型HEV感染不同宿主细胞差异的分子机制奠定了基础。     

新疆羊粪便戊型肝炎病毒RNA的检测与序列分析

【目的】为了了解新疆地区羊群中是否存在戊型肝炎的感染,我们从戊型肝炎病毒(Hepatitis E virus,HEV)IgG检测阳性的新疆某羊场采集54份1-3岁的羊粪便,【方法】利用逆转录套式聚合酶链方法(RT-nPCR),检测HEVRNA,其中6份为阳性,阳性率11.11%。【结果】将PCR扩增产物克隆,测序并进行序列分析,结果表明,6株羊源HEV检测株在HEV ORF2 189bp 99.38%-100%,为同一基因型;与HEVⅠ、Ⅱ、Ⅲ和Ⅳ型的同源性分别为78.67%-85.33%、81.33%-82.67%、78.67%-84.00%和84.67%-95.36%,与Ⅳ型最高同源性达94.04%-95.36%。以该189bp片段绘制进化树,发现与新疆牛源分离株、中国大陆猪源分离株(FJ610232)和中国大陆人源分离株(AJ272108)在同一分枝上,同属基因Ⅳ型;【结论】提示新疆羊群中可能存在HEV感染,并且羊可能是人类HEV传染源中除猪之外的新宿主。     

南京地区戊型肝炎病毒基因型鉴定和变异分析

为了解南京地区戊型肝炎病毒(Hepatitis E virus,HEV)基因型的分布以及变异状况,本研究采用逆转录套式聚合酶反应(RT-nPCR)的方法检测南京地区40份急性散发性戊型肝炎患者的粪便标本,对PCR产物进行测序,利用生物信息学软件比较核苷酸的同源性、遗传距离,进行基因分型和变异分析。40份粪便标本中检测到14份阳性HEV、RNA,检出率为35%,基因分型均为HEV IV型,且分属2个不同的亚型;利用生物信息学软件对国内I型和IV型毒株加以比较,发现HEV IV型毒株比I型变异程度高,不同年份的HEV IV型毒株变异更大,有新的亚型出现,且变异有随时间推移而增大的趋势。     

戊型肝炎病毒荧光定量RT-PCR快速检测技术的建立和初步应用

利用DNAMAN软件对GenBank登录的戊型肝炎病毒四个主要基因型代表株的序列进行分析, 选择其高度保守的ORF2区域设计合成引物和探针, 并用包含有扩增区域的核苷酸片段进行体外转录制备标准品cRNA。在对荧光定量RT-PCR的反应条件优化的基础上, 建立了适用于戊型肝炎病毒主要基因型检测的荧光定量RT-PCR检测技术。该检测技术可以有效检测I型和IV型戊型肝炎阳性病料, 而对猪的其它几种疫病阳性病料则为阴性结果, 证实本技术的特异性强、可靠性好。对阳性标准品的检测结果表明, 所建立的TaqMan荧光定量RT-PCR灵敏度可达2.0×101拷贝/反应, 相比于巢式RT-PCR方法, 其灵敏度高10~100倍以上。在对54份临床样品的检测中, 进一步证实了该方法快速、灵敏且重复性好, 可满足戊型肝炎病毒早期快速诊断的需要。     

戊型肝炎病毒荧光定量RT-PCR快速检测技术的建立和初步应用

利用DNAMAN软件对GenBank登录的戊型肝炎病毒四个主要基因型代表株的序列进行分析, 选择其高度保守的ORF2区域设计合成引物和探针, 并用包含有扩增区域的核苷酸片段进行体外转录制备标准品cRNA。在对荧光定量RT-PCR的反应条件优化的基础上, 建立了适用于戊型肝炎病毒主要基因型检测的荧光定量RT-PCR检测技术。该检测技术可以有效检测I型和IV型戊型肝炎阳性病料, 而对猪的其它几种疫病阳性病料则为阴性结果, 证实本技术的特异性强、可靠性好。对阳性标准品的检测结果表明, 所建立的TaqMan荧光定量RT-PCR灵敏度可达2.0×101拷贝/反应, 相比于巢式RT-PCR方法, 其灵敏度高10~100倍以上。在对54份临床样品的检测中, 进一步证实了该方法快速、灵敏且重复性好, 可满足戊型肝炎病毒早期快速诊断的需要。     

上海部分地区戊型肝炎病毒（HEV）基因型的分析

为了解戊型肝炎病毒 (HEV)在上海部分地区流行的基因型 ,采用RT nPCR的方法检验 35例急性散发性戊型肝炎患者中HEVRNA ,并对阳性产物进行克隆测序 ,然后对其基因型进行分析。结果显示在 35例急性散发性戊型肝炎患者中PCR阳性为 9例 ,测序证实 8例为HEV的基因序列 ;其中 1例为HEV 1型 ,7例为HEV 4型。提示在上海部分地区的急性散发性戊型肝炎中以HEV 4型感染为主     

First isolation of hepatitis E virus genotype 4 in Europe through swine surveillance in the Netherlands and Belgium

Hepatitis E virus (HEV) genotypes 3 and 4 are a cause of human hepatitis and swine are considered the main reservoir. To study the HEV prevalence and characterize circulating HEV strains, fecal samples from swine in the Netherlands and Belgium were tested by RT-PCR. HEV prevalence in swine was 7-15%. The Dutch strains were characterized as genotype 3, subgroups 3a, 3c and 3f, closely related to sequences found in humans and swine earlier. The HEV strains found in Belgium belonged to genotypes 3f and 4b. The HEV genotype 4 strain was the first ever reported in swine in Europe and an experimental infection in pigs was performed to isolate the virus. The genotype 4 strain readily infected piglets and caused fever and virus shedding. Since HEV4 infections have been reported to run a more severe clinical course in humans this observation may have public health implications.     

Detection of the virus of hepatitis E isolates and elucidation of the possibility of their replication in vitro

Blood serum samples collected from patients with acute hepatitis symptoms admitted to Infectious Disease Hospitals of Novosibirsk, Barnaul, and Irkutsk were studied. The serum samples were tested for the IgM and IgG antibodies to HEV using ELISA. Seropositive samples were tested using RT-PCR for HEV RNA. Two HEV strains were isolated, and thus HEV infection was identified for West Siberia. One of this strains is classified as HEV genotype I; the other, as genotype III. Cell culturing of these strains in green monkey kidney (4647) cells showed an ability of HEV genotype I strain to cause persistent infection.     

Experimental Infection of Rabbits with Rabbit and Genotypes 1 and 4 Hepatitis E Viruses

Background

A recent study provided evidence that farmed rabbits in China harbor a novel hepatitis E virus (HEV) genotype. Although the rabbit HEV isolate had 77–79% nucleotide identity to the mammalian HEV genotypes 1 to 4, their genomic organization is very similar. Since rabbits are used widely experimentally, including as models of infection, we investigated whether they constitute an appropriate animal model for human HEV infection.

Methods

Forty-two SPF rabbits were divided randomly into eleven groups and inoculated with six different isolates of rabbit HEV, two different doses of a second-passage rabbit HEV, and with genotype 1 and 4 HEV. Sera and feces were collected weekly after inoculation. HEV antigen, RNA, antibody and alanine aminotransferase in sera and HEV RNA in feces were detected. The liver samples were collected during necropsy subject to histopathological examination.

Findings

Rabbits inoculated with rabbit HEV became infected with HEV, with viremia, fecal virus shedding and high serum levels of viral antigens, and developed hepatitis, with elevation of the liver enzyme, ALT. The severity of disease corresponded to the infectious dose (genome equivalents), with the most severe hepatic disease caused by strain GDC54-18. However, only two of nine rabbits infected with HEV genotype 4, and none infected with genotype 1, developed hepatitis although six of nine rabbits inoculated with the genotype 1 HEV and in all rabbits inoculated with the genotype 4 HEV seroconverted to be positive for anti-HEV IgG antibody by 14 weeks post-inoculation.

Conclusions

These data indicate that rabbits are an appropriate model for rabbit HEV infection but are not likely to be useful for the study of human HEV. The rabbit HEV infection of rabbits may provide an appropriate parallel animal model to study HEV pathogenesis.     

合成肽抗原在戊型肝炎病毒感染诊断中的应用

An ELISA for the detection of anti HEV using synthetic peptide antigens was developed. The synthetic antigens were encoded by OFR2 and OFR3 genes of HEV. The purpose of this study was to determine the applicability of the synthetic antigens in the serodiagnosis of hepatitis E. The anti HEV detection using synthetic antigens was carried out in 47 healthy subjects and 89 patients with acute or chronic viral hepatitis. The results showed that the positive rate of anti HEV IgG in healthy subjects was 4.2%(2/47), and no IgM antibody to HEV was found. The positive rates of IgG and IgM antibodies to HEV in the hepatitis patients were 8.9% and 10% respectively. In addition, we compared the detecting efficacy of the synthetic antigens with that of the market reagent in 57 serum samples, the total coincident rate was 87.7% (50/57). All of the results accorded with the literatures reported. This study suggests that the ELISA based on the synthetic peptide antigens was specific, sensitive and convenient in diagnosis of HEV infection, it can be widely used in both clinical and epidemiological reseaches.