Evidence of hepatitis E virus infection in specific pathogen-free rabbits in Korea

Rabbits are considered a new natural reservoir of hepatitis E virus (HEV). In this study, HEV infection was verified by the detection of partial genomic sequence of HEV and anti-HEV antibodies in specific pathogen-free (SPF) rabbits. HEV RNA was found in 6.4% serum and 13.5% fecal samples from 126 SPF rabbits. Anti-HEV antibodies were also detected in 4.0% of the SPF rabbits. HEV genetic sequences isolated from the rabbits were clustered into a rabbit HEV clade with other rabbit HEV isolates; they were found to be most closely related with a rabbit HEV sequence previously reported in Korea. Therefore, HEV infection should be diagnosed before conducting experiments involving SPF rabbits.     

急性肝炎恢复期血清检测抗戊型肝炎病毒抗体及RNA的临床诊断意义

目的 在急性非甲-戊型肝炎患者中进一步追踪检测不同时期血清抗戊型肝炎病毒(HEV)IgG，以明确临床诊断。方法 用美国Genelabs公司和北京万泰公司抗HEV诊断试剂检测抗HEV，用PCR方法检测HEVRNA，并进行基因序列分析。结果 95例入院首次血清学诊断为急性非甲-戊型肝炎患者中，在住院后11～25d、25～35d分别测定血清抗HEV,16例血清抗HEV阳性(万泰公司)，急性期血清HEVRNA检测10例HEVRNA阳性，经核苷酸序列分析证明，其中4例为Ⅰ型HEV感染，6例为Ⅳ型HEV感染。GenekLbs诊断试剂检测12例抗HEV阳性，7例HEVRNA阳性，其中4例是HEVⅠ型病毒感染，3例是HEV Ⅳ病毒感染。结论 对非甲-戊型肝炎患者进行急性期HEV RNA检测和恢复期抗HEV检测可以进一步明确病原学诊断，在这部分患者中存在HEV不同基因型感染。可能是HEV感染漏诊的原因之一。     

Investigation of hepatitis E virus infection in swine from Hunan province, China

Nine hundred and four serum samples were collected from pigs from 16 pig farms in China's Hunan province and tested for anti-hepatitis E virus (HEV) antibody and the HEV capsid antigen using EIAs. Of the 904 samples, 617 (68.3%) and 57 (6.3%) were positive for anti-HEV antibody and antigen, respectively. The prevalence of HEV antibodies and detection of antigen varied significantly among pigs from different farms (P < 0.01). The positivity rate for anti-HEV antibody and occurrence of high titer antibody were significantly higher in pigs above, than in those below, 3 months of age, whilst the detection of antigen did not differ significantly between the two groups. Based on these data, 481 serum samples that were positive for HEV antigen or with an S/CO < or =10 for anti-HEV were tested for HEV RNA using real-time RT-PCR; 28 of 481 (5.8%) were positive. The positivity rate of HEV RNA was much higher for the HEV antigen positive sera (40.1%) than the anti-HEV antibody positive (1.4%) or negative (1.1%), antigen negative samples. Sixteen of the 28 samples were positive for HEV RNA using nested RT-PCR and their products were cloned and sequenced. These 16 isolates belonged to HEV genotype 4, including 12 that did not belong to any subtype of HEV genotype 4 reported previously. Thus, the infection rate of HEV in pigs is high and the HEV antigen has a close relationship with HEV RNA. The HEV genotype infecting the pigs was genotype 4 and a novel subtype may exist in Hunan province.     

Serological prevalence, genetic identification, and characterization of the first strains of avian hepatitis E virus from chickens in Korea

Avian hepatitis E virus (avian HEV) is associated with hepatitis-splenomegaly (HS) syndrome or big liver and spleen disease in chickens. At least three genotypes of avian HEV have been identified from chickens worldwide. A total of 297 serum samples collected from chickens in 35 flocks in Korea were tested for avian HEV antibody with an enzyme-linked immunosorbent assay. The results showed that approximately 57?% of chicken flocks and 28?% of chickens from Korea were positive for antibodies to avian HEV. Thirteen pooled fecal samples from chickens were tested for avian HEV RNA by RT-PCR, and three fecal samples were positive. The partial helicase and capsid genes of the Korean avian HEV isolates were determined, and sequence analyses revealed that the Korean avian HEV isolates were clustered together and closely related to the genotype 1 avian HEV from Australia. The complete genomic sequence of a Korean avian HEV strain HH-F9 from a broiler breeder was determined, and shown to be 6,653 nt in length, excluding the poly (A) tail, which is 1 nt shorter than the prototype avian HEV from chicken with HS syndrome in the United States. Compared to the full-length sequences of other 5 known avian HEV strains worldwide, the Korean avian HEV shared approximately 83-97?% nucleotide sequence identity. The finding that Korean avian HEV belongs to genotype 1 avian HEV which was previously identified only from chickens in Australia has significant implication in understanding the global epidemiology of avian HEV.     

Frequent transmission of hepatitis E virus among piglets in farms in Southern France

The present study aimed to assess whether hepatitis E virus (HEV) is present in domestic pigs in Southern France, and to determine the relationship between HEV sequences detected from pigs and from humans. Two hundred fifteen sera, 207 stools, and 107 bile samples were collected from 3‐ or 6‐month‐old pigs from different regions of Southern France. Pig IgG anti‐HEV antibodies testing was performed using a commercial ELISA kit with minor modifications. Pig HEV RNA was tested by real‐time PCR and sequencing assays using “in‐house” protocols. Forty percent of pigs were HEV‐seropositive. Sixty‐five percent of 3‐month‐old pigs and none of 6‐month‐old pigs were HEV RNA‐positive. HEV RNA was significantly more frequently detected from stools than from sera (65% vs. 22%; P < 0.001). Phylogenetic analysis showed that pig HEV sequences belonged to genotype 3 and formed two clusters of genotype 3f and 3e. Nucleotide homology between pig HEV sequences of each cluster was high (>97%), and clusters were correlated with the geographical origin of pigs and with their repartition into pens and buildings in the pig farm. Based on analysis of 331 nucleotides, pig HEV sequences were close genetically to HEV sequences found from humans or pigs in Europe, and one showed complete nucleotide identity with an HEV sequence obtained in France from a human. The present data indicate that 3‐month‐old pigs from Southern France might represent a potential source of HEV transmission to humans, and stress the potential of HEV to cause epizootic infections in population of farm pigs. J. Med. Virol. 81:1750–1759, 2009. © 2009 Wiley‐Liss, Inc.     

Development and validation of a genotype 3 recombinant protein-based immunoassay for hepatitis E virus serology in swine

Hepatitis E virus (HEV) is classified within the family Hepeviridae, genus Hepevirus. HEV genotype 3 (Gt3) infections are endemic in pigs in Western Europe and in North and South America and cause zoonotic infections in humans. Several serological assays to detect HEV antibodies in pigs have been developed, at first mainly based on HEV genotype 1 (Gt1) antigens. To develop a sensitive HEV Gt3 ELISA, a recombinant baculovirus expression product of HEV Gt3 open reading frame-2 was produced and coated onto polystyrene ELISA plates. After incubation of porcine sera, bound HEV antibodies were detected with anti-porcine anti-IgG and anti-IgM conjugates. For primary estimation of sensitivity and specificity of the assay, sets of sera were used from pigs experimentally infected with HEV Gt3. For further validation of the assay and to set the cutoff value, a batch of 1100 pig sera was used. All pig sera were tested using the developed HEV Gt3 assay and two other serologic assays based on HEV Gt1 antigens. Since there is no gold standard available for HEV antibody testing, further validation and a definite setting of the cutoff of the developed HEV Gt3 assay were performed using a statistical approach based on Bayes'' theorem. The developed and validated HEV antibody assay showed effective detection of HEV-specific antibodies. This assay can contribute to an improved detection of HEV antibodies and enable more reliable estimates of the prevalence of HEV Gt3 in swine in different regions.     

Genotypic characterization of symptomatic hepatitis E virus (HEV) infections in Egypt

BackgroundHepatitis E virus (HEV) is a common cause of acute viral hepatitis (AVH) in many developing countries. In Egypt, HEV seroprevalence is among the highest in the world; however, only a very limited number of Egyptian HEV sequences are currently available.ObjectivesThe objectives were to determine the HEV genotype(s) currently circulating in Egypt.Study designAVH patients without serologic evidence of hepatitis A, B, and C viruses were evaluated for possible HEV infection using serologic assays for anti-HEV IgM and anti-HEV IgG and real-time PCR for HEV RNA. Stool suspensions from suspected cases were inoculated into rhesus macaques to confirm the presence of HEV. Sequence analysis was utilized to determine HEV genotype.ResultsOf 287 subjects with AVH enrolled, 58 had serologic evidence of acute HEV infection. Stool samples for two of these patients were repeatedly positive for HEV RNA by real-time PCR. Macaques experimentally inoculated with these human stools also developed viremia. Sequence analysis of open reading frame (ORF) 1 demonstrated that these isolates belonged to HEV genotype 1 and were 3.9–9.5% divergent from other genotype 1 isolates. ORF2 was 5.3–8.7% divergent from previously reported Egyptian isolates.ConclusionsThis study strongly suggests that genotype 1 HEV related to other North African isolates is circulating in acute symptomatic patients in Egypt. Further evaluation of genotypic variability is underway in this highly endemic cohort and is considered an important component of our increased understanding of HEV pathogenesis.     

Novel approach for detection of hepatitis E virus infection in German blood donors

The risk of transfusion-transmitted hepatitis E virus (HEV) infections by contaminated blood products remains unknown. In the present study, we evaluated and compared different nucleic acid amplification technique (NAT) methods for the detection of HEV in blood components. Minipools of a total of 16,125 individual blood donors were screened for the presence of HEV RNA using the highly sensitive RealStar HEV RT-PCR kit, revealing a minimum detection limit of 4.66 IU/ml. Thirteen donors were HEV RNA positive (0.08%), and of these donors, only three already showed reactive IgM antibody titers. The detected HEV strains all belonged to genotype 3 and were most closely related to German HEV strains from wild boars and pigs as well as from human hepatitis E cases. Furthermore, HEV RNA and HEV-specific IgM and IgG titers were determined in 136 blood donors with elevated alanine aminotransferase (ALT) levels and in 200 donors without pathological findings. HEV RNA was not detectable, but 8.08% (elevated ALT) and 0.5% (nonelevated ALT) of donors showed reactive HEV IgM titers. The overall seroprevalence rate of HEV IgG amounted to 5.94% (elevated ALT, 5.88%; nonelevated ALT, 6.0%). The clinical relevance of transfusion-associated hepatitis E infection still requires further investigation. However, in connection with raising concerns regarding blood safety, our NAT method provides a sensitive possibility for HEV testing.     

Frequent detection and characterization of hepatitis E virus variants in wild rats (Rattus rattus) in Indonesia

One hundred sixteen rats (Rattus rattus) captured in Indonesia from 2011 to 2012 were investigated for the prevalence of hepatitis E virus (HEV)-specific antibodies and HEV RNA. Using an ELISA based on HEV genotype 4 with an ad hoc cutoff value of 0.500, 18.1 % of the rats tested positive for anti-HEV IgG. By nested RT-PCR, 14.7 % of the rats had rat HEV RNA, and none were positive for HEV genotype 1-4. A high HEV prevalence among rats was associated with lower sanitary conditions in areas with a high population density. Sixteen of the 17 HEV isolates obtained from infected rats showed >93.0 % nucleotide sequence identity within the 840-nucleotide ORF1-ORF2 sequence and were most closely related to a Vietnamese strain (85.9-87.9 % identity), while the remaining isolate differed from known rat HEV strains by 18.8-23.3 % and may belong to a novel lineage of rat HEV. These results suggest a wide distribution of rat HEV with divergent genomes.     

Prevalence of Antibodies to Hepatitis E Virus in Immigrants: A Seroepidemiological Survey in the District of Foggia (Apulia‐Southern Italy)

Hepatitis E virus (HEV) is the etiologic agent of endemically transmitted viral hepatitis. HEV is endemic in developing countries where it occurs in sporadic and endemic forms, but autochthonous sporadic cases of hepatitis E have been reported in North America and in Europe, including Italy. The aim of the present study was to assess the seroprevalence of antibodies to HEV in immigrants from developing countries to the province of Foggia. The seroprevalence of HEV was determined in a cohort of 412 immigrants (mostly from countries in sub‐Saharan Africa) who had arrived recently in Italy. Serum samples were tested for anti‐HEV by a commercial enzyme immunoassay (EIA) based on recombinant proteins; positive results were confirmed by a Western blot assay (Recomblot HEV). A total of 88 (21.3%) of the 412 serum samples examined were reactive to IgG anti‐HEV. Eighty‐one of these samples (19.7%) were confirmed by Western blot. Anti‐HEV IgM was found in 34/81 subjects (41.9%) of the anti‐HEV IgG positive serum samples. Almost all anti‐HEV positive subjects were asymptomatic clinically, but alanine aminotransferase serum values were elevated in 28/34 (82.3%) patients with IgM anti‐HEV‐positive. The results of this study indicate high circulation of HEV in the immigrant population. The high prevalence of acute hepatitis involved mainly subjects who arrived in Italy during the same period from the same countries (Eritrea, Ethiopia, and Somalia). J. Med. Virol. 85:261–265, 2013. © 2012 Wiley Periodicals, Inc.     

我国急性散发性戊型肝炎病毒部分核苷酸序列分析

用逆转录套式聚合酶链反应（ＲＴ－ｎＰＣＲ）自深圳、长春、杭州等地４１份急性散发性戊型肝炎病人血清中获得２８株ＨＥＶｃＤＮＡ，对其中３株ＨＥＶｃＤＮＡ的ＯＲＦ２基因片段，用荧光法直接测定其核苷酸序列，并与戊型肝炎病毒墨西哥株（Ｍ）、缅甸株（Ｂ）和新疆流行株（ＣＨ１·１）进行了比较，结果该３株散发性ＨＥＶ与Ｍ株的核苷酸序列同源性分别为８０．２％、７９．９％和７９．４％；与Ｂ流行株的同源性为９５．５％、９３．９％和９５．１％；与散发株的同源性为９３．４％、９２．３％和９３．８％；与ＣＨ１·１的同源性为９７．０％、９６．５％和９５．９；表明该３株散发性ＨＥＶ与ＨＥＶ（Ｂ）和ＣＨ１·１的核酸序列同源性较高，可能属同一亚型。     

A nationwide survey of hepatitis E virus infection in the general population of Japan

To investigate nationwide the prevalence of hepatitis E virus (HEV) infection in the general population of Japan, serum samples were collected from 22,027 individuals (9,686 males and 12,341 females; age, mean ± standard deviation: 56.8 ± 16.7 years; range: 20–108 years) who lived in 30 prefectures located in Hokkaido, mainland Honshu, Shikoku, and Kyushu of Japan and underwent health check‐ups during 2002–2007, and were tested for the presence of IgG, IgM, and IgA classes of antibodies to HEV (anti‐HEV) by in‐house ELISA and HEV RNA by nested RT‐PCR. Overall, 1,167 individuals (5.3%) were positive for anti‐HEV IgG, including 753 males (7.8%) and 414 females (3.4%), the difference being statistically significant (P < 0.0001). The prevalence of anti‐HEV IgG generally increased with age and was significantly higher among individuals aged ≥50 years than among those aged <50 years (6.6% vs. 2.7%, P < 0.0001). Although 13 individuals with anti‐HEV IgG also had anti‐HEV IgM and/or anti‐HEV IgA, none of them had detectable HEV RNA. The presence of HEV RNA was further tested in 50 or 49‐sample minipools of sera from the remaining 22,014 individuals, and three individuals without anti‐HEV antibodies tested positive for HEV RNA. The HEV isolates obtained from the three viremic individuals segregated into genotype 3 and were closest to Japan‐indigenous HEV strains. When stratified by geographic region, the prevalence of anti‐HEV IgG as well as the prevalence of HEV RNA or anti‐HEV IgM and/or anti‐HEV IgA was significantly higher in northern Japan than in southern Japan (6.7% vs. 3.2%, P < 0.0001; 0.11% vs. 0.01%, P = 0.0056; respectively). J. Med. Virol. 82:271–281, 2010. © 2009 Wiley‐Liss, Inc.     

散发性戊型肝炎病毒部分核苷酸序列分析

目的为阐明戊型肝炎病毒（ＨＥＶ）毒株的地理分布、流行特征以及研制血清学诊断试剂和基因工程疫苗提供资料。方法选择１例浙江仙居山区散发性ＨＥ患者，从其血清中分离ＨＥＶＲＮＡ，通过逆转录－套式聚合酶链反应法（ＲＴ－ｎＰＣＲ），扩增该散发性ＨＥＶ（Ｚ－３３株）ＯＲＦ２区部分ｃＤＮＡ片段（４９７ｂｐ），然后进行直接序列分析，并与ＨＥＶ各主要代表株序列作比较。结果Ｚ－３３株与新疆流行株ＣＨ１．１的核苷酸及氨基酸序列同源性分别为９６．７％及９８．２％；与缅甸流行株的同源性分别为９４．２％及９９．１％；与缅甸散发株的同源性分别为９５．２％及９９．１％，与墨西哥株的同源性分别为８１．８％及９４．５％。结论浙江仙居散发性ＨＥＶＺ－３３株和新疆流行株ＣＨ１．１一样，与ＨＥＶ缅甸株可能为同一亚型。     

河南地区不同月龄猪戊型肝炎病毒感染情况调查

目的 调查河南地区不同月龄猪戊型肝炎病毒(HEV)感染情况.方法 从河南五个地区共采集623份猪血清,其中3月龄以下血清113份,3月龄以上血清510份,酶联免疫诊断试剂盒分别检测HEV抗原和抗体.对抗原阳性者用套式RT-PCR检测HEV核酸,并对核酸阳性者进行克隆和序列分析.结果 3月以下组和3月以上组的抗体阳性率分别为90.27%和92.55%,差异无统计学;而两组的抗原阳性率分别为15.93%和5.69%,差异有统计学意义(P＜0.001).各地区间的抗原和抗体阳性率存在明显的统计学差异.在47份抗原阳性样本中,检出5例为HEV核酸阳性,序列分析显示4例为HEV4型,1例为HEV 1型.结论 河南地区猪的HEV感染率较高,而且不同地区存在明显差异.