家兔自然感染戊型肝炎病毒的动态观察

目的 了解自然条件下家兔感染戊型肝炎病毒(HEV)的方式和感染标志物的动态变化。方法 对养兔场28 d龄家兔断乳、分笼饲养,每周1次采集粪便检测HEV RNA,粪便HEV RNA转为阳性的家兔,每周采集静脉血,所有家兔每满1月龄时采集静脉血,进行血清HEV RNA、抗HEV-Ab及转氨酶检测,动态观察各种指标的变化直至家兔满5个月龄出栏。结果 共对47只家兔追踪观察,7周至5个月龄的10只家兔(21.3%,10/47)粪便中检测到HEV RNA,排毒时间在3~10周以上;其中7只家兔(14.9%,7/47)形成了病毒血症,病毒血症的出现晚于粪便排毒1~3周,持续时间为2~5周;至出栏时共有11只家兔(23.4%,11/47)转化为抗HEV-Ab阳性。基因序列分析显示HEV分离株均为兔HEV。结论 家兔从断乳到出栏的整个生长过程均对HEV均易感。家兔粪便排出病毒的时间比病毒血症持续时间长,但有家兔出栏时还处于病毒血症时期,因而家兔粪便及肉类制品可能具有传播兔HEV的危险性。     

Lack of evidence of hepatitis E virus infection among renal transplant recipients in a disease‐endemic area

Persistent hepatitis E virus (HEV) infection has been reported among solid‐organ transplant recipients in nonendemic areas. Such chronic infections have all been related to genotype 3 HEV, which is prevalent in these areas. Whether persistent infection occurs with genotype 1 HEV, prevalent in areas where the infection is hyperendemic, is unclear. We therefore tested sera from renal transplant recipients receiving immunosuppressive agents in India, where genotype 1 HEV infection is endemic, for alanine aminotransferase levels, and presence of IgM and IgG anti‐HEV antibodies and HEV RNA. Of the 205 subjects studied [aged 16–65 (median, 38) years, 182 male], 46 (22.4%) had abnormal ALT levels (>40 IU/mL). IgG anti‐HEV was detected in 52 (20.5%) and IgM anti‐HEV was detected in 14 (6.8%) subjects, including four who had IgG anti‐HEV; antibody positivity had no relation with serum ALT or serum creatinine. All the sera tested were negative for HEV RNA. These findings suggest that chronic infection with genotype 1 HEV is infrequent.     

A Putative Novel Hepatitis E Virus Genotype 3 Subtype Identified in Rabbit,Germany 2016

Hepatitis E is an emerging viral disease that is the leading cause of viral hepatitis in the world. The vast majority of hepatitis E cases in developed countries are caused by zoonotic genotypes 3 and 4 of hepatitis E virus (HEV) for which pig and wild boar and to lesser extent rabbits are the main reservoir. According to recent reports rabbits are a source of human HEV infection and highlight the risk of zoonotic foodborne transmission. Here we report the molecular analysis of a novel HEV strain identified in a rabbit during a countrywide surveillance of rabbits and hares in Germany, 2016. The analysis of the complete genome reveals characteristics of a putative novel recombinant subtype of the species Orthohepevirus A within the clade of genotype 3 but not closely related to any known subtypes. Importantly, the genome of this strain possesses a nucleotide exchange in the overlapping region of open reading frames ORF2/ORF3 interfering with a broadly applied diagnostic real-time RT-PCR. In conclusion, a new type of HEV strain was identified in a German rabbit with atypical and novel sequence characteristics.     

Characterization of hepatitis E virus natural infection in farmed rabbits

Rabbit hepatitis E virus (HEV3‐ra) is widely distributed in rabbits worldwide and several recent reports found that HEV3‐ra can infect humans. Therefore, people exposed to rabbits are at high risk of HEV infection. This study was conducted to investigate the characteristics and outcomes of HEV3‐ra natural infection in rabbits. Seventy farmed rabbits (3‐month‐old) were surveyed in a farm in Beijing, China. Rabbits tested positive for HEV RNA were followed weekly for testing of HEV RNA, antigen, antibody and alanine aminotransferase (ALT) level. Liver and kidney tissue was collected for histopathology. Complete genome sequencing of the isolated HEV3‐ra strain was performed (CHN‐BJ‐r4, GenBank: MT364355). The infectivity of CHN‐BJ‐r4 was tested in ten naïve rabbits by intravenous injection or gavage. Anti‐HEV antibody and HEV RNA were tested positive in 7.14% (5/70) and 11.4% (8/70) of rabbits, respectively. Eight naturally infected rabbits were followed, and 37.5% (3/8) of the observed rabbits were found to have fecal shedding of HEV ranging from 3‐22 weeks with high viral load (10⁵‐10⁷ copies/g). Two out of eight rabbits showed temporary viremia. Naturally infected rabbits presented elevated ALT level, seroconversion, and liver histopathology. Complete genome of HEV3‐ra isolated in this study shared 84.61%‐94.36% nucleotide identity with known HEV3‐ra complete genomes. The isolated HEV3‐ra strain was infectious and could infect other rabbits through intravenous and fecal–oral route. Naturally infected rabbits showed up to 22‐week fecal virus shedding with high viral load. These features increased the risk of rabbit‐to‐rabbit and rabbit‐to‐human transmission.     

Identification of HEV in symptom‐free migrants and environmental samples in Italy

Hepatitis E virus (HEV) is considered an emerging pathogen in industrialized countries. The occurrence of HEV genotypes in samples of faeces from asymptomatic migrants arriving on the coasts of South Italy and environmental samples was investigated. Analyses of sequences were used to compare human and environmental genotypes. A total of 40 stool specimens, 12 samples of untreated urban sewage, 12 samples of treated urban sewage and 12 samples of surface water were analysed. Viruses were concentrated from water samples by the tangential flow ultrafiltration technique. The presence of HEV RNA was detected by nested RT‐PCR. Viral isolates were sequenced and phylogenetically characterized. Two (5%) of the 40 faecal samples tested were found to be positive for HEV RNA (G1 and G3 genotypes). The virus was detected in 25% (3/12) of the untreated sewage samples and 25% (3/12) of the surface water samples: all isolates belonged to G3 genotype. None of the treated sewage samples were found to be HEV RNA positive. The virus was detected in the faeces of two asymptomatic subjects, suggesting a potential role for symptom‐free HEV carriers as a human reservoir. G3 HEV strains were detected in the untreated sewage, as observed in similar studies conducted in other European countries but differing from another study conducted in Italy recently. Moreover, our results show the first case of HEV isolated from fresh surface waters.     

Cross‐genotype‐specific T‐cell responses in acute hepatitis E virus (HEV) infection

Hepatitis E is an inflammatory liver disease caused by infection with the hepatitis E virus (HEV). In tropical regions, HEV is highly endemic and predominantly mediated by HEV genotypes 1 and 2 with >3 million symptomatic cases per year and around 70 000 deaths. In Europe and America, the zoonotic HEV genotypes 3 and 4 have been reported with continues increasing new infections per year. So far, little is known about T‐cell responses during acute HEV genotype 3 infection. Therefore, we did a comprehensive study investigating HEV‐specific T‐cell responses using genotypes 3‐ and 1‐specific overlapping peptides. Additional cytokines and chemokines were measured in the plasma. In four patients, longitudinal studies were performed. Broad functional HEV‐specific CD4⁺ and CD8⁺ T‐cell responses were detectable in patients acutely infected with HEV genotype 3. Elevated of pro‐ and anti‐inflammatory cytokine levels during acute HEV infection correlated with ALT levels. Memory HEV‐specific T‐cell responses were detectable up to >1.5 years upon infection. Importantly, cross‐genotype HEV‐specific T‐cell responses (between genotypes 1 and 3) were measurable in all investigated patients. In conclusion, we could show for the first time HEV‐specific T‐cell responses during and after acute HEV genotype 3 infection. Our data of cross‐genotype HEV‐specific T‐cell responses might suggest a potential role in cross‐genotype‐specific protection between HEV genotypes 1 and 3.     

烟台市2015-2018年人感染戊型肝炎病毒基因型分析北大核心CSCD

目的分析2015-2018年烟台市人感染戊型肝炎病毒(HEV)基因型别变化,探讨分子流行病特征。方法选取烟台市2015-2018年国家法定传染病报告系统上报的戊型肝炎病例为研究对象,采集血清标本检测HEV-IgM抗体,对血清HEV-IgM阳性病例的血清和粪便标本提取HEV核酸,采用逆转录PCR方法对HEV开放式阅读框架2区域内的核苷酸片段(644 bp)进行扩增,测序后进行系统进化分析。结果共采集360份血清样本,198份(55%)样本HEV-IgM阳性。HEV-IgM阳性的血清标本,HEV核酸检出率27.78%(55/198);粪便标本HEV核酸检测阳性率39.58%(19/48)。总阳性病例数72例,男性48例,女性24例。本次检测的72例HEV病毒株基因型全部是Ⅳ型,以4d亚型为主,占77.78%(56株);其次是4b亚型,占19.44%(14株);4a亚型占2.78%(2株)。72株HEV核苷酸序列的遗传距离为0.002~0.303。56株4d亚型HEV序列与山东地区猪源(KF176351)、羊源(KU904267)、牛源(KU904278)HEV序列的遗传距离为0.021~0.160、0.010~0.178、0.008~0.176。结论基因Ⅳ型HEV在2015-2018年烟台地区占主导趋势,4d亚型为主要流行株。本地人感染HEV可能与进食动物制品或密切接触动物相关。     

Evaluation of an antigen‐capture EIA for the diagnosis of hepatitis E virus infection

An enzyme immunoassay (EIA) has been developed for hepatitis E virus (HEV) antigen (HEV‐Ag) detection and marketed in China. This study aimed to evaluate the sensitivity of the assay and assess the value of HEV‐Ag detection in the diagnosis of HEV infection in comparison with HEV RNA detection. Using serial dilutions of a genotype 4 HEV strain, significant correlation was found between the EIA (S/CO) and HEV RNA (IU/mL) concentration in the range 10^3.5 to 10^0.5 IU/mL HEV RNA, the Pearson correlation coefficient r approached 0.97. The EIA detection limit was 54.6 IU/mL, compared to 24 IU/mL for HEV RNA using real‐time RT‐PCR. In clinical samples from hepatitis E patients, the HEV‐Ag and HEV RNA positivity rates were 55.6% (65/117) and 60.7% (71/117) in sera and 76.7% (56/73) and 84.9% (62/73) in stools, and the concordance of these two markers was 77.8% in sera and 80.8% in stools. In serum samples, the HEV‐Ag positivity rate and the concordance between HEV‐Ag and HEV RNA were inversely proportional to the presence of anti‐HEV antibody. The presence of anti‐HEV IgG could reduce the S/CO of the HEV‐Ag EIA. These results reveal a significant correlation between the detection of HEV‐Ag and HEV RNA. The sensitivity of the HEV‐Ag EIA was lower than real‐time RT‐PCR but could be higher than conventional nested RT‐PCR. Therefore, the detection of HEV‐Ag in serum and faeces is valuable for the diagnosis and prognosis of HEV infection in developing regions where real‐time RT‐PCR is not available.     

云南某农村猪戊型肝炎病毒分子流行病学调查

目的 了解云南农村散养猪群HEV感染情况,为HEV防控提供理论依据。方法 利用反转录套式聚合酶链反应（RT-nested PCR）技术,对所采集云南猪群78份粪便样品进行HEV ORF2基因部分片段扩增,经琼脂糖凝胶电泳后对目的条带进行回收纯化及克隆测序。序列利用DNAStar和MEGA4.0软件进行同源性比较和系统进化树构建。结果 RT-nested PCR方法扩增出了350bp目的基因序列,该序列与GenBank中HEV各型的同源性在77.4~97.4%之间,与基因4型同源性最高（97.4%）,与3型同源性最低（77.4%）。系统进化树显示测定的序列与基因4型聚为一支,表明该序列属于基因4型。本次实验共检测了78份猪粪便样品,其中8份为阳性,阳性率为10.26%。结论 云南农村人群存在感染HEV的风险,应该加以防控以免HEV暴发流行。     

与人类关系密切的7种动物对戊型肝炎病毒易感性的初步研究

目的　用双抗原夹心法ELISA (DS -ELISA)和逆转录巢式PCR法 (RT -nPCR)研究与人类生活关系密切的猪、犬、山羊、鸡、鸭、鸽子和兔等 7种动物对戊型肝炎病毒 (HEV)的易感性。方法　用多基因型HEV重组蛋白建立检测HEV抗体的DS -ELISA ,并用于 5 91份猪、警犬、家犬、宠物犬、山羊、鸡、鸭、鸽子和兔等多种动物血清标本的HEV抗体检测 ;用HEV多基因型通用性引物RT -nPCR扩增 14 3份犬血清标本HEVRNA。结果　在猪、家犬和宠物犬血清标本中检测到HEV抗体 ,阳性率分别为 4 3 93%、15 19%和 4 6 5 % ,但在警犬、山羊、鸡、鸭、鸽子和兔血清标本中未检测到HEV抗体 ;14 3份犬血清标本用RT -nPCR未扩增出HEVRNA。结论　用多基因型HEV重组蛋白建立的双抗原夹心法ELISA适用于多种动物血清标本HEV抗体的检测 ;猪和犬对HEV易感 ,在HEV传播中可能起重要作用。     

Immunogenicity and Antigenicity of Rabbit Hepatitis E Virus-Like Particles Produced by Recombinant Baculoviruses

Rabbit hepatitis E virus (HEV) is a novel HEV belonging to genotype 3 (HEV-3) in the Orthohepevirus A species of the genus Hepevirus, family Hepeviridae. Rabbit HEV was originally isolated from rabbits and found to cause zoonotic infection. Although rabbit HEV can be successfully grown in culture with several cell lines, including the human carcinoma cell line PLC/PRF/5, it is difficult to obtain the large amounts of viral antigen required for diagnosis and vaccine development. In this study, we expressed N-terminal 13 and 111 aa-truncated rabbit HEV ORF2 proteins using recombinant baculoviruses and obtained two types of virus-like particles (VLPs), RnVLPs and RsVLPs with ~35 and 24 nm diameter, respectively. Anti-rabbit HEV IgG antibodies were induced in high titer by immunizing rabbits with RnVLPs or RsVLPs. The antibody secretion in the serum persisted more than three years. RsVLPs showed stronger antigenic cross-reactivity against HEV-1, HEV-3 and HEV-4 than rat HEV. Moreover, anti-RsVLPs antibodies neutralized not only the cognate virus but also HEV-1, HEV-3 and HEV-4 ex vivo, indicating that rabbit HEV had the same serotype as human HEVs. In contrast, the antibody did not block rat HEV infection, demonstrating that rat HEV belonged to a different serotype. Animal experiments indicated that immunization with either RnVLPs or RsVLPs completely protected the rabbits from challenge by rabbit HEV, suggesting that the VLPs are candidates for rabbit HEV vaccine development.     

Current status of hepatitis e virus infection in Korea

Hepatitis E virus (HEV) is an emerging pathogen associated with acute viral hepatitis, and HEV is becoming increasingly recognized. Approximately 2% of acute viral hepatitis is caused by HEV, and 18 cases of hepatitis E have been reported in Korea. Of these cases, only two have involved a history of travel from India, which suggests that they were imported cases. The remaining reported cases include a sporadic case of acute hepatitis E with genotype 4 HEV isolates and identification of the full genome sequence, as well as another case of genotype 4 HEV hepatitis that developed after ingestion of the raw bile juice of a wild bear living on a mountain in southern Korea. Moreover, genotype 3 HEV, which shows close genetic homology with swine HEV in Korea, has been detected in collected human serum samples. Therefore, genotypes 3 and 4 HEV are currently circulating in the Korean community and may be related to zoonotic transmission and food-borne infection. The reported anti-HEV seroprevalence of 17% to 27% in the Korean population suggests that HEV infection has been autochthonously circulating, thereby resulting in subclinical infection in Korea. Given the discrepancies among anti-HEV assays, the diagnosis of hepatitis E should be made with caution using adequate antibody assays, and HEV RNA should be preferably detected from the stool. Further virological characterization and epidemiological study of the virus are warranted.     

A nationwide survey for prevalence of hepatitis E virus antibody in qualified blood donors in Japan

Background and Objectives In previous studies, we reported the transmission of hepatitis E virus (HEV) by transfusion, and the frequent detection of HEV markers in Japanese blood donors with elevated ALT levels. For the current study, we carried out a nationwide survey of the prevalence of IgG anti‐HEV in qualified blood donors throughout Japan. Materials and Methods The 12 600 samples from qualified blood donors were collected from seven blood centres (1800 per centre) representing nearly all regions of Japan. Samples were from age‐ and sex‐matched blood donors who tested negative for all the current blood screening tests. The samples were screened using the in‐house IgG anti‐HEV ELISA. Sequentially, the positive samples were tested by the commercial IgG anti‐HEV ELISA. Results Of 12 600 samples, 431 (3·4%) were regarded as positive for IgG anti‐HEV. The prevalence of IgG anti‐HEV was higher in eastern Japan (5·6%) than in western Japan (1·8%) (P < 0·001), and was also age‐dependent and higher in men (3·9%) than in women (2·9%) (P = 0·002). Conclusion The spread of the domestic infection of HEV was observed in qualified blood donors in Japan. A higher prevalence of IgG anti‐HEV was observed in male donors, older donors and in donors residing in eastern Japan. Further studies are necessary to clarify the potential risk of transfusion–transmission of HEV in Japan.     

武汉地区散发性急性戊型肝炎流行特点和病毒基因型分析

目的：探讨武汉地区散发性急性戊型肝炎流行特点和病毒基因型。方法：采用逆转录-巢式聚合酶链反应（RT-nPCR）检测162份急性戊型肝炎患者血清HEV RNA,并对PCR产物进行测序及同源进化分析。结果：武汉地区急性戊型肝炎患者男女比例为4.40∶1,30～59岁年龄段占71.60%,162份急性戊型肝炎患者血清有41份HEVRNA阳性,阳性率为25.31%,同源进化分析表明41株HEV毒株均属于基因Ⅳ型,各毒株间的核苷酸同源性为84.7%～100%,基因Ⅳ型又可进一步分为5种亚型,即Ⅳ-A、Ⅳ-B、Ⅳ-C、Ⅳ-D和Ⅳ-E,各亚型内部的核苷酸同源性分别为91.3%～100%、98.0%、92.0%～100%、98.0%～100%、100%,其中Ⅳ-A与上海株、西安株和广西株的核苷酸同源性分别为92.0%～97.3%、94.7%～99.3%和91.3%～96.0%,Ⅳ-D与北京株的核苷酸同源性为95.3%～96.7%。结论：武汉地区急性戊型肝炎以男性多见,好发于30～59岁年龄段,且均由基因Ⅳ型HEV引起,基因Ⅳ型又可进一步分为Ⅳ-A、Ⅳ-B、Ⅳ-C、Ⅳ-D和Ⅳ-E 5种亚型,其中Ⅳ-A与上海株、西安株和广西株亲缘关系较近,Ⅳ-D与北京株亲缘关系较近。     

High prevalence of hepatitis A and E viruses in environmental and clinical samples from West Argentina

Environmental surveillance of water sources is important to monitoring viral hepatitis transmission in clinical settings. This study investigated the circulation of hepatitis A (HAV) and E (HEV) viruses in sewage and clinical samples from Argentina. Between 2016 and 2017, 80 raw sewage samples and 86 clinical samples (stool and serum) from suspected cases of hepatitis A and hepatitis E were obtained. HAV and HEV were tested by both real-time and nested PCR. Positive samples were sequenced for genotype determination and phylogenetic analysis. Overall, HAV was recovered in 39% of sewage samples and 61.1% of clinical samples. HEV was detected in 22.5% of sewage samples and 15.9% of clinical samples. HAV was found more frequently in sewage during the winter and in clinical samples in spring; HEV was more prevalent in sewage during summer and in clinical samples in autumn. All HAV isolates belonged to genotype IA and HEV isolates belonged to genotype 3, the most prevalent genotypes in South America. High prevalence of HAV and HEV in environmental and clinical samples in Mendoza, Argentina was observed. These findings reinforce the importance of environmental surveillance and implementation of health strategies to control the spread of HAV and HEV in developing countries.     

Temporal profile of HEV RNA concentration in blood and stool from patients with acute uncomplicated hepatitis E in a region with genotype 1 predominance

Acute hepatitis E virus (HEV) is associated with viremia and faecal excretion of the virus. The information on duration and temporal pattern of viremia and faecal shedding in HEV infection is important, but is not available. Serial serum and stool specimens were collected from patients with acute hepatitis E (typical clinical picture, serum alanine aminotransferase levels > 5‐folds the upper limit of normal and presence of IgM anti‐HEV), beginning from within 7 days of the onset of symptoms. HEV RNA concentrations were measured in sera and 10% stool suspensions, using a real‐time Taqman‐based nucleic acid amplification assay. Seventeen patients (median age 25 [range 19‐61] years; all men) were enrolled within a median of 5 (range 3‐8) days of the onset of the first symptom and provided 113 serum specimens and 71 stool specimens. The median (range) highest levels of serum bilirubin, alanine aminotransferase and aspartate aminotransferase in the patients were 10.3 (5.9‐43.4) mg/dL, 1817 (442‐4642) IU/L and 1016 (88‐4561) IU/L, respectively. All the 17 patients had demonstrable viremia, and 12 of the 13 patients who were tested had faecal excretion at one or more time points. The HEV RNA titres were the highest in the early phase of disease and declined rapidly with time, becoming nondetectable in the serum by day 20 and in the stool by day 21. In most of the patients with acute uncomplicated acute hepatitis E, the degree of viremia and faecal shedding decline quickly after the onset of clinical illness and rapidly disappear in parallel with each other.     

Experimental infection of pregnant rabbits with hepatitis E virus demonstrating high mortality and vertical transmission

A high mortality rate of approximately 20% in pregnant women with hepatitis E has been reported in previous studies. However, other studies showed no difference between pregnant and nonpregnant women in the severity of hepatitis E. To determine the effects of HEV infection on pregnancy, we successfully established HEV infection in six pregnant rabbits (PR) and six nonpregnant rabbits (NPR) with a rabbit HEV isolate, taking three PR and one NPR without HEV infection as controls. Tests for HEV RNA by RT‐PCR, anti‐HEV antibodies by ELISA and HEV antigen via immunohistochemistry and histopathology were carried out. Two of six infected PR miscarried and three of the remaining four PR died which may be attributed to severe liver necrosis caused by HEV infection. Moreover, vertical transmission was found to be associated with the replication of HEV in placenta, indicated by the presence of HEV RNA and antigen in placenta from the infected PR. Our findings strongly suggest that HEV infection could lead to adverse outcomes in pregnancy and vertical transmission, suggesting the necessity for pregnant women at risk of HEV infection to be vaccinated.