Identification of genotype 4 hepatitis E virus strains from a patient with acute hepatitis E and farm pigs in Bali, Indonesia

A previous study revealed that antibodies to hepatitis E virus (HEV) (anti-HEV) are highly prevalent among healthy individuals and farm pigs in Bali, Indonesia, and suggested that HEV infection may occur via zoonosis among Balinese people. However, there were no reports of acute hepatitis E in Bali. To elucidate whether Balinese HEV strains recovered from infected humans and pigs have significant sequence similarity, serum samples obtained from 57 patients (age, mean +/- standard deviation, 31.1 +/- 11.9 years) with sporadic acute hepatitis and from one hundred and one 2- or 3-month-old farm pigs in Bali were tested for anti-HEV and HEV RNA. Among the 57 patients, 2 (3.5%) had high-titer IgM/IgA class anti-HEV antibodies and one of them had detectable HEV RNA (BaliE03-46). Overall, 58 pigs (57.4%) tested positive for anti-HEV, while 5 pigs (5.0%) had detectable HEV RNA. Based on the 412-nucleotide sequence within open reading frame 2, the BaliE03-46 isolate and the 5 swine HEV isolates recovered from the viremic pigs were phylogenetically classified in genotype 4, but were only 77.3-90.8% identical to the genotype 4 HEV isolates reported thus far in China, India, Japan, Taiwan, and Vietnam. The BaliE03-46 isolate of human origin shared high identities of 97.3-98.3% with 4 of the 5 Balinese swine isolates, but differed by 16.1% from the remaining swine isolate. These results suggest that indigenous HEV strains of genotype 4 with marked heterogeneity are circulating in Bali, Indonesia, and that pigs are reservoirs of HEV for Balinese people who have a habit of ingesting uncooked pigs.     

Prevalence of antibodies to hepatitis E virus among apparently healthy humans and pigs in Bali, Indonesia: Identification of a pig infected with a genotype 4 hepatitis E virus

In Indonesia where hepatitis E virus (HEV) is believed to be highly endemic, only three outbreaks of HEV transmission have been documented to date in restricted areas (West Kalimantan and East Java). A total of 1,115 serum samples collected from apparently healthy individuals in Bali, Lombok, and Surabaya in Indonesia in 1996 where epidemic HEV transmissions have never been reported, were tested for IgG class antibodies to HEV (anti-HEV). In Bali, anti-HEV was detected in 20% (54/276) of the tested population, in remarkable contrast with 4% (17/446) in Lombok and 0.5% (2/393) in Surabaya. On the other hand, antibodies to hepatitis A virus were highly prevalent in all three regions (95% in Bali, 90% in Lombok, and 89% in Surabaya). Although the majority of the population in Indonesia is Moslem, Balinese people are mostly Hindu and have a habit of consuming pork. Therefore, serum samples were obtained from the 99 farm pigs in Bali and tested for anti-HEV and HEV RNA. The sera from 71 pigs (72%) were positive for anti-HEV and a 2-month-old pig had detectable HEV RNA. The swine HEV isolate recovered from the viremic pig was named SB66-Bali. The SB66-Bali isolate was most closely related to the genotype 4 isolates from China, India, Japan, and Taiwan, but shared only 82.6-90.0% identity in the common 241-412 nucleotides within open reading frame 2 (ORF2). These results indicate that a presumably indigenous HEV strain(s) is circulating in Bali, Indonesia and that HEV infection may occur via zoonosis even in developing countries.     

Serological markers of hepatitis B, C, and E viruses and human immunodeficiency virus type-1 infections in pregnant women in Bali, Indonesia

Except for hepatitis B virus (HBV), there have been few data on serological markers of hepatitis viruses such as hepatitis C virus (HCV) and E virus (HEV), and human immunodeficiency virus type-1 (HIV) in Bali, Indonesia. During 5 months from April to August 2003, sera were collected from 2,450 pregnant women at eight jurisdictions in Bali, and they were tested for markers of these viruses. Only one (0.04%) was positive for antibody to HCV, but none for antibody to HIV. Hepatitis B surface antigen (HBsAg) was detected in 46 (1.9%) at a prevalence significantly lower than that in 271 of the 10,526 (2.6%) pregnant women in Bali surveyed 10 years previously (P < 0.045). The prevalence of hepatitis B e antigen in pregnant women with HBsAg decreased, also, from 50% to 28% during the 10 years (P < 0.011). Antibody to HEV (anti-HEV) was examined in 819 pregnant women who had been randomly selected from the 2,450. The overall prevalence of anti-HEV was 18%, and there were substantial regional differences spanning from 5% at Tabanan district to 32% at Gianyar district. Furthermore, the prevalence of anti-HEV differed substantially by their religions. In the Sanglah area of Denpasar City, for instance, anti-HEV was detected in 20 of the 102 (20%) Hindus, significantly more frequently than in only 2 of the 101 (2.0%) Muslims (P < 0.001). Swine that are prohibited to Muslims, therefore, is likely to serve as a reservoir of HEV in Bali. In conclusion, HBV is decreasing, HCV and HIV have not prevailed, as yet, while HEV is endemic probably through zoonotic infection in Bali.     

猪、羊、鸡抗-HEV抗体流行率调查

目的 探讨猪、羊、鸡群中抗-HEV抗体流行率。方法从新疆、广西、广东、北京和河北收集猪、羊、鸡的血清标本498份，应用双抗原夹心酶联免疫法(ELISA)检测抗-HEV抗体。结果 猪抗-HEV阳性率为67．53％(104／154)，其中新疆4～5个月龄猪血清抗-HEV阳性率为100．00％，广西3个月龄以下仔猪抗-HEV阳性率为36．00％(9／25)，6月龄以上猪阳性率为71．67％(86／120)；新疆108份绵羊血清抗-HEV抗体全部阴性；236份鸡血清标本的抗-HEV阳性率仅为1．27％(3／236)。广东罗定、深圳和广西柳州郊区鸡血清标本中各检测出一份抗-HEV阳性，阳性率分别为4．00％(1／25)、1．49％(1／67)、1．49％(1／67)。北京郊区25份和河北52份鸡血清抗-HEV检测全部阴性。结论 猪、鸡群中存在HEV感染，猪抗-HEV抗体流行率最高。猪和鸡在传播人HEV中的作用值得进一步研究。     

Prevalence of Antibodies to Hepatitis E Virus in Veterinarians Working with Swine and in Normal Blood Donors in the United States and Other Countries

Hepatitis E virus (HEV) is endemic in many developing and some industrialized countries. It has been hypothesized that animals may be the source of infection. The recent identification of swine HEV in U.S. pigs and the demonstration of its ability to infect across species have lent credence to this hypothesis. To assess the potential risk of zoonotic HEV infection, we tested a total of 468 veterinarians working with swine (including 389 U.S. swine veterinarians) and 400 normal U.S. blood donors for immunoglobulin G anti-HEV. Recombinant capsid antigens from a U.S. strain of swine HEV and from a human HEV strain (Sar-55) were each used in an enzyme-linked immunosorbent assay. The anti-HEV prevalence assayed with the swine HEV antigen showed 97% concordance with that obtained with the human HEV antigen (kappa = 92%). Among the 295 swine veterinarians tested from the eight U.S. states (Minnesota, Indiana, Nebraska, Iowa, Illinois, Missouri, North Carolina, and Alabama) from which normal blood donor samples were available, 26% were positive with Sar-55 antigen and 23% were positive with swine HEV antigen. In contrast, 18% of the blood donors from the same eight U.S. states were positive with Sar-55 antigen and 17% were positive with swine HEV antigen. Swine veterinarians in the eight states were 1.51 times more likely when tested with swine HEV antigen (95% confidence interval, 1.03 to 2.20) and 1.46 times more likely when tested with Sar-55 antigen (95% confidence interval, 0.99 to 2.17) to be anti-HEV positive than normal blood donors. We did not find a difference in anti-HEV prevalence between veterinarians who reported having had a needle stick or cut and those who had not or between those who spent more time (> or = 80% of the time) and those who spent less time (< or = 20% of the time) working with pigs. Similarly, we did not find a difference in anti-HEV prevalence according to four job categories (academic, practicing, student, and industry veterinarians). There was a difference in anti-HEV prevalence in both swine veterinarians and blood donors among the eight selected states, with subjects from Minnesota six times more likely to be anti-HEV positive than those from Alabama. Age was not a factor in the observed differences from state to state. Anti-HEV prevalence in swine veterinarians and normal blood donors was age specific and paralleled increasing age. The results suggest that swine veterinarians may be at somewhat higher risk of HEV infection than are normal blood donors.     

Clinical and epidemiological implications of swine hepatitis E virus infection

In nonendemic areas, most patients with acute hepatitis E were infected through traveling to endemic areas. However, some patients did not have a history of foreign travel before infection. Furthermore, high seroprevalence rates of antibody to hepatitis E virus (anti-HEV) were found in the general adult population in some countries without any recorded outbreak of hepatitis E. The significance of anti-HEV assay in these subjects remains obscure. To study if swine might be a source of HEV infection, HEV was tested in sera of 235 pigs in Taiwan, and from 5 patients with acute HEV infection who either denied or did not provide any foreign travel history. Three (1.3%) pigs had detectable swine HEV RNA. The swine and human HEV strains from Taiwan formed a monophyletic group, distinct from three previously reported groups: the United States human and swine HEV strains, the Mexico strain, and the largest group composed of the Asian and the African strains. The identity of nucleotide sequences was 84-95% between swine and human HEV strains in Taiwan, and 72-79% between Taiwan strains and those from different areas. The predicted amino acid sequence of a Taiwan swine HEV strain within the peptide 3-2 used in commercial anti-HEV assay showed a high identity (91-94%) with those of other human and swine HEV strains. Swine may be a reservoir of HEV and subclinical swine HEV infection may occur. Cross-reactivity of current anti-HEV assay may account for the high prevalence rate of anti-HEV in the general population in nonendemic areas.     

Design and evaluation of a recombinant multi-epitope-based ELISA for the serological surveillance of HEV infection in northern China

In this study, seven recombinant epitope peptides from within the ORF2 protein of the local genotype 4 swine hepatitis E virus (HEV) DQ strain were designed and analyzed. Then, a new multi-epitope-based ELISA was established. In comparison with a commercial kit, this test exhibited good specificity and sensitivity for anti-HEV genotype 4. Subsequently, this test was applied for analyzing serum samples from either swine herds or human populations in northern China. The overall seroprevalence rate of anti-HEV IgG reached up to 40.4% for swine and 8.1% for humans. A statistical difference was observed for humans in rural and urban areas, with a higher prevalence for people living in rural than urban areas. Moreover, sequencing confirmed that all RNA-positive samples belonged to genotype 4.     

Analysis of the helicase gene of Korean swine hepatitis E virus isolates and trends in viral infection

Hepatitis E virus (HEV) has been considered to be a zoonotic agent and an important public concern worldwide. In this study, a nested RT-PCR was developed to detect the helicase gene of swine HEV (sHEV) from sera of pigs. Using this RT-PCR, 16 out of 821 Korean isolates of sHEV were identified, with 1.9% prevalence. An age-specific prevalence was demonstrated with the highest prevalence in growing pigs (5.4%). Phylogenetic analysis of sHEV Korean isolates identified genotype 3, with 89.4–99.9% nucleotide sequence identity. The viruses were closely related to US and Japanese HEV isolates from swine and humans (89.4–93.1%). The prevalence of anti-HEV IgG in individual pigs and swine herds was 39.5 and 80%, respectively. The seroprevalence rate increased in proportion to the age of the swine. The seroprevalence of HEV was higher than previously reported. These results indicate that sHEV is widespread in Korean swine herds and further raise concerns about possible zoonosis.     

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.     

Identification of the first strain of swine hepatitis E virus in South America and prevalence of anti-HEV antibodies in swine in Argentina

In Argentina, a country considered non-endemic for hepatitis E virus (HEV) infection, serologic evidence of HEV infection has been observed in different human population groups. In other countries, a high degree of genetic relatedness has been observed between human and swine HEV genotype 3 sequences, suggesting zoonosis as one probable route of infection. This is the first identification of swine HEV in South America. HEV RNA was detected and sequenced in the ORF 1 and ORF 2 regions from swine fecal samples from a herd located in Pergamino, in the province of Buenos Aires. These strains all group into genotype 3 and exhibit a close relationship to two novel HEV variants previously identified in Argentina from sporadic acute cases of non-A to -C hepatitis in humans. In addition, using a modified commercial ELISA, the presence of anti-HEV antibodies was surveyed in five provinces across the country and all five showed a prevalence of HEV antibodies, ranging from 4% to 58%. The results suggest that swine could be an important reservoir for virus transmission in Argentina as has been suggested for other non-endemic areas. The Argentine human strains and swine strain described in this article seem to be closely related to a human Austrian strain, suggesting a potential European origin of HEV infection in these cases.     

Prevalence of specific antibody to hepatitis E virus in the general population of the community of Madrid, Spain

Hepatitis E virus (HEV) is an infectious agent causing hepatitis among humans. Although hepatitis E has been reported from many European countries, its incidence in Europe is largely unknown, and the prevalence of the HEV infection is also unknown for most countries of the region. Antibody to HEV (anti-HEV) was tested on 2,305 serum samples from the general population of the Community of Madrid (Spain) collected in the year 2008 among people aged 2-60 years. Total anti-HEV was tested by enzyme-immunoassay (EIA), and reactive samples were retested separately for anti-HEV IgG and IgM by recombinant immunoblot test (RIBT). Fifty samples (2.17%) displayed reactivity for total anti-HEV after EIA testing, and anti-HEV IgG was confirmed by RIBT in 25 (1.08%). The frequency of RIBT-confirmed anti-HEV ranged from 0.97% among the youngest to 3.61% among the oldest, and displayed a statistically significant trend to increasing with age. The rate of RIBT confirmation was also significantly higher among the individuals aged above 20 years old than among those younger of 21 years. HEV infection would be less frequent in the Community of Madrid than in Catalonia or the United Kingdom, and contact with HEV would be very uncommon among children and adolescents of the region. Confirmation of EIA-reactive samples by RIBT reduced the final numbers of anti-HEV testing as much as 50%, and some findings of this study suggest that such testing protocol would reflect better the real prevalence of anti-HEV in settings of low endemicity than the single testing by EIA.     

High hepatitis e virus seroprevalence in forestry workers and in wild boars in france

Hepatitis E virus (HEV) is a fecally and orally transmitted human pathogen of worldwide distribution. In industrial countries, HEV is observed in an increasing number of autochthonous cases and is considered to be an emerging pathogen. A growing body of evidence suggests that HEV is a zoonotic disease, and pig handlers and pig veterinarians have been reported to be high-risk groups for HEV infection. The aims of the present study were to establish the prevalence of anti-HEV in wild boars in France and to identify whether forestry workers are at a higher risk of HEV infection. Three different anti-HEV tests were used to compare their effectiveness in detecting anti-HEV in the general population. The most sensitive test was then used to investigate HEV seroprevalence in 593 forestry workers and 421 wild boars. Anti-HEV was detected in 31% of the forestry workers and 14% of the wild boars. Detection of anti-HEV in humans was correlated with age, geographical location, and occupational activity and in wild boars was correlated with geographical location. HEV infection is frequent in woodcutters in France, and it varies geographically. Further studies are needed to confirm these findings and to elucidate the transmission route and the exact virus reservoirs.     

云南大理猪戊型肝炎病毒分子流行病学调查

目的：了解云南大理农村的猪群中戊型肝炎病毒（ hepatitis E virus, HEV）感染情况,为HEV的防控提供理论依据。方法利用逆转录巢式聚合酶链反应（ RT?nested PCR）技术,对所采集大理41份猪粪便样品进行HEVORF2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进化树构建。结果 RT?nested PCR方法扩增出348 bp目的基因序列,系统进化树显示该病毒属于基因4型h亚型HEV。此次实验共检测41份猪粪便样品,其中23份为阳性,阳性率为56．10％。结论大理猪群存在较高的HEV感染率,存在HEV跨种间感染人的风险,应该加强防控以免HEV爆发流行。     

Molecular and serological survey of hepatitis E virus infection among domestic pigs in Inner Mongolia, China

To evaluate the prevalence and characteristics of swine hepatitis E virus (HEV) infection in Inner Mongolia, China, serum samples obtained from 356 2- to 4-month-old pigs on 14 farms in Inner Mongolia were tested for the presence of anti-HEV antibodies and HEV RNA. Overall, 186 pigs (52%) tested positive for anti-HEV antibodies, while 30 pigs (8%) had detectable HEV RNA levels. The 30 HEV isolates recovered from the viremic pigs were phylogenetically classified into genotype 4 and differed from each other by up to 15.3% in a 412 nt sequence within ORF2. The Inner Mongolian swine HEV strains were most similar to human or swine HEV strains isolated in the other provinces of China but differed by 15.9–18.9% from those in Mongolia (formerly known as Outer Mongolia). These results indicate that farm pigs in Inner Mongolia are frequently infected with markedly divergent genotype 4 HEV strains that may be indigenous to China.     

Differences in capabilities of different enzyme immunoassays to detect anti-hepatitis E virus immunoglobulin G in pigs infected experimentally with hepatitis E virus genotype 3 or 4 and in pigs with unknown exposure

Hepatitis E virus (HEV), a major cause of acute viral hepatitis in humans in many developing countries, is highly prevalent in the pig population worldwide. The objective of this study was to assess the capability of three porcine prototypes of a human enzyme-linked immunosorbent assay (ELISA), an in-house ELISA and a line-immunoassay (LIA) to detect anti-HEV antibodies in pigs infected experimentally with HEV (n = 57), known to be negative for HEV infection (n = 27), or with unknown exposure to HEV infection (field samples, n = 90). All 27 samples from non-infected pigs were negative with all five assays. The earliest detection of anti-HEV antibodies occurred at 14 days post-inoculation (dpi) with four of five assays. From 42 dpi, all samples from infected pigs were detected correctly as anti-HEV positive. Kappa analysis demonstrated substantial agreement among tests (0.62-1.00) at 14 dpi and complete agreement (1.00) at 56 dpi. The overall area under the curve for all quantitative tests as determined by receiver operator characteristic analysis ranged from 0.794 to 0.831 indicating moderate accuracy. The results showed that all five assays can detect anti-HEV IgG antibodies accurately in pigs infected experimentally with HEV. In field samples, a higher prevalence of anti-HEV IgG was found in breeding herds than in growing pigs (100% versus 66.7-93.9%). These serological assays should be very useful in veterinary diagnostic labs for HEV diagnosis in swine.     

Prior infection of pigs with a genotype 3 swine hepatitis E virus (HEV) protects against subsequent challenges with homologous and heterologous genotypes 3 and 4 human HEV

Hepatitis E virus (HEV) is an important human pathogen. At least four recognized and two putative genotypes of mammalian HEV have been reported: genotypes 1 and 2 are restricted to humans whereas genotypes 3 and 4 are zoonotic. The current experimental vaccines are all based on a single strain of HEV, even though multiple genotypes of HEV are co-circulating in some countries and thus an individual may be exposed to more than one genotype. Genotypes 3 and 4 swine HEV is widespread in pigs and known to infect humans. Therefore, it is important to know if prior infection with a genotype 3 swine HEV will confer protective immunity against subsequent exposure to genotypes 3 and 4 human and swine HEV. In this study, specific-pathogen-free pigs were divided into 4 groups of 6 each. Pigs in the three treatment groups were each inoculated with a genotype 3 swine HEV, and 12 weeks later, challenged with the same genotype 3 swine HEV, a genotype 3 human HEV, and a genotype 4 human HEV, respectively. The control group was inoculated and challenged with PBS buffer. Weekly sera from all pigs were tested for HEV RNA and IgG anti-HEV, and weekly fecal samples were also tested for HEV RNA. The pigs inoculated with swine HEV became infected as evidenced by fecal virus shedding and viremia, and the majority of pigs also developed IgG anti-HEV prior to challenge at 12 weeks post-inoculation. After challenge, viremia was not detected and only two pigs challenged with swine HEV had 1-week fecal virus shedding, suggesting that prior infection with a genotype 3 swine HEV prevented pigs from developing viremia and fecal virus shedding after challenges with homologous and heterologous genotypes 3 and 4 HEV. The results from this study have important implications for future development of an effective HEV vaccine.     

北京地区部分人群接触密切的家畜(禽)戊型肝炎病毒感染血清学调查

目的 了解北京地区戊型肝炎(戊肝)病毒感染现状.方法 采用EIA诊断试剂检测人群、猪和鸡血清中戊肝病毒(HEV)抗体.结果 1208份人群血清中有260份HEV-IgG抗体阳性,总阳性率为21.52%,对260份HEV IgG阳性血进行IgM抗体检测,有22份阳性,占8.46%.32份猪血清中有15份阳性,阳性率为46.88%,34份鸡血清HEV抗体全部为阴性.人群不同年龄组HEV抗体阳性率依次为11～20岁为5.60%(14/250),21～30岁为20%(42/210),31～40岁为24.03%(62/258),41～50岁为26.44%(78/295),51～60岁为32.82%(64/195),男女不同性别HEV感染率比较,男性阳性率为29.51%(144/488),女性为21.70%(102/470).结论 北京地区人群和与人接触密切的家畜猪均存在戊肝病毒感染,人群戊肝病毒感染率随年龄增长而升高,尤其是男性戊肝病毒感染率明显高于女性,郊区流动人群戊肝病毒感染率明显高于市内人群,家畜猪感染率是人群的2倍以上.     

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.