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.     

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.     

四川地区部分人群和与人接触密切的家畜禽戊型肝炎病毒感染的调查及基因分型的研究

目的 了解四川地区人群和与人接触密切的家畜猪和鸡戊型肝炎(戊肝)病毒感染的分布及病毒基因分析.方法 采用万泰生物药业的HEV EIA诊断试剂检测人群、猪和鸡血清中戊肝病毒(HEV)IgG抗体.采用逆转录套式PCR方法(RT-nPCR)检测猪胆汁和血清中戊肝病毒核酸,并对阳性产物进行纯化和部分核苷酸序列分析.结果 学龄前儿童血清672份中有41份阳性,阳性率6.1%,成人血清661份中有280例阳性,阳性率42.36%,猪血清36份中有32份阳性,阳性率为88.89%,59份鸡血清HEV抗体均阴性.对15份HEV IgM抗体阳性的患者血清和54份猪胆汁进行RT-nPCR,从1份患者血清和3份猪胆汁扩增到戊肝病毒核酸,4株核苷酸序列之间同源性92.1%～98.6%,与GenBank HEV参考株ORF2相对应序列比较,核苷酸序列与Ch-T21(我国散发性急性肝炎人分离株HEV4 B亚型)之间同源性最高占90.1%～96.9%.结论 四川地区部分人群和与人接触密切的家畜猪均存在HEV感染,尤其是家畜猪感染率高达90%左右,从人和猪分离到的病毒株核苷酸同源性很高,均属于戊肝病毒基因4型.     

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.     

Analysis of the full-length genome of hepatitis E virus isolates obtained from farm pigs in Mongolia

Although no outbreaks of hepatitis E have been reported in Mongolia, a significant proportion of the general population had antibodies to hepatitis E virus (HEV). To investigate whether pigs are possible reservoirs of HEV in Mongolia, serum samples obtained from 243 2- or 3-month-old pigs on four swine farms surrounding Ulaanbaatar, the capital city of Mongolia, were tested for the presence of anti-HEV antibodies and HEV RNA. Overall, 223 pigs (91.8%) tested positive for anti-HEV, while 89 pigs (36.6%) had detectable HEV RNA. The 89 HEV isolates obtained from the viremic pigs were 78.7-100% identical to each other, and 80.9-85.9% similar to the prototype genotype 3 HEV isolate (US1) in the 412-nucleotide (nt) sequence within open reading frame 2. They were classified into two novel phylogenetic groups within genotype 3, differing by 16.4-21.3%. The swMN06-A1288 and swMN06-C1056 isolates, representing each of the two clusters within genotype 3, had a genomic length of nucleotides (nt) 7,222 nt and 7,223 nt, respectively, excluding the poly(A) tail, and shared only 81.6% over the entire genome. Upon comparison with the 25-reported genotype 3 HEV isolates over the entire genome, swMN06-A1288 had identities of merely up to 84.9%, while swMN06-C1056 of only up to 85.9%. Phylogenetic analysis confirmed the remote relatedness of the Mongolian swine isolates to the genotype 3 HEV isolates reported thus far. These results indicate that farm pigs in Mongolia are frequently infected with presumably indigenous HEV strains of genotype 3 and could be a source of HEV infections in humans in Mongolia.     

Seroprevalence and molecular detection of hepatitis E virus in Yunnan Province, China

In this study, the prevalence and characteristics of hepatitis E virus (HEV) in pigs and the general population in the Yunnan province, China, were evaluated. Nine hundred sixty sera, 95 liver and 60 feces samples were randomly collected from pig farms and abattoirs, in addition 173 human sera were sampled in the provincial capital city for a serological survey and an RT-nPCR assay. The screening results showed that among 621 samples collected from five pig farms, the HEV-specific IgG positive rate ranged from 73.2% to 83.5%, and the overall seroprevalence was 78.9% (490/621). A further analysis revealed that the seroprevalence increased with age. The positive rate of human serum samples was 39.9% (69/173). HEV RNA was detected in five swine feces, six swine liver and one anti-HEV-IgM-positive human serum sample by RT-nPCR. Sequence and alignment of the 348-nt PCR-amplified products of 12 HEV strains identified nine distinct nucleotide sequences. Phylogenetic and molecular evolutionary analysis revealed that these nine sequences shared 84.2% to 100.0% nucleotide sequence identity with each other, with all isolates belonging to genotype 4 HEV and clustering with other Chinese swine and human HEV sequences determined earlier. This study results suggest that the prevalence of genotype 4 HEV is serious, both in pig herds and in the human population, and authorities should pay more attention to the prevalence of HEV in southwest China.     

Swine HEV infection in south India and phylogenetic analysis (1985-1999)

Hepatitis E is endemic in India. It was recently noted that although all the Indian human hepatitis E virus (HEV) isolates (1976-2001) were placed in genotype I, the swine HEV recovered from western India (2000) belonged to genotype IV. This was in contrast to reports from the United States and Taiwan wherein both human and swine HEV belonged to the same genotype, i.e., genotypes III and IV, respectively. In order to validate these findings further, we retrospectively examined serum samples collected from pigs from southern India. Sequential serum samples from 45 (1985-1987) and 12 (1999) pigs from Karnataka state, south India, were screened for the presence of HEV RNA (nested PCR) and IgG-anti-HEV (ELISA). PCR products (Open Reading Frame-2 region) were sequenced and subjected to phylogenetic analysis. In this study, 42/45 (1985-1987) and 12/12 (1999) pigs showed seroconversion to IgG anti-HEV antibodies, with a mean age at seroconversion of 4.8 +/- 1.6 months. Four samples collected in 1999 and two samples collected during 1985 were HEV RNA positive. All swine HEV sequences clustered with genotype IV, demonstrating that swine HEV was prevalent among south Indian pigs for at least for 16 years and, similar to western India, belonged to genotype IV. Thus, genotype I and IV HEV continue to circulate in humans and pigs, respectively, from India. Whether swine HEV infects humans remains to be determined.     

烟台沿海地区人源和猪源戊型肝炎病毒基因型别的研究

目的调查烟台市沿海地区人源与猪源戊型肝炎病毒（HEV）基因型别的相关性。方法应用逆转录一巢式聚合酶联反应（RT—nPcR）方法对当地急性散发戊型肝炎患者、正常人群中抗HEV-IgM阳性者和当地养猪场的猪进行HEVRNA检测,并对HEVRNA阳性标本进行克隆测序和序列分析。结果16例急性散发戊型肝炎患者中有7例粪便标本HEVRNA阳性;51份IgM阳性正常人群血清标本中有1份HEVRNA阳性;34份猪胆汁标本中有1份HEVRNA阳性。序列分析发现该地区HEV人株与猪株在ORF2部分区域的核苷酸序列同源性为87％～98．1％。7株患者的戊肝病毒基因型和1株猪的戊肝病毒基因型均为Ⅳ型,基因序列同源性在87％～98．1％之间;其中有6例患者和猪的基因序列同源性在93．9％～98．1％之间,为Ⅳ型a亚型;1例患者和猪的基因序列同源性为87％,为Ⅳ型d亚型。正常人群的1例戊肝病毒基因型为Ⅰ型d亚型。该地区人与猪HEV的ORF2的部分基因片段与HEVⅠ～Ⅳ型的代表株进行比较,核苷酸序列同源性分别是82．5％～100％,81．7％～92．9％,81．4％～93．9％,84．9％～100％。结论该地区人群中流行的HEV存在2个基因型3个亚型,主要以基因Ⅳa型为主,与猪群中流行的HEV基因Ⅳa型同源性较高;HEVI型在人群中散在存在。     

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.     

Analysis of complete genome sequences of swine hepatitis E virus and possible risk factors for transmission of HEV to humans in Korea

The hepatitis E virus (HEV) is an emerging zoonotic agent, for which pigs are the most important reservoir. Complete genome sequences of two swine HEV strains, designated swKOR‐1 and swKOR‐2, were determined via RT‐PCR and RACE‐PCR. The strains contained genomes composed of 7,222‐ and 7,221‐bp excluding the poly(A) tails, respectively. The swKOR‐1 and swKOR‐2 strains were classified into subtype 3a of genotype 3 via phylogenetic analysis. These strains formed a distinctive cluster in the phylogenetic tree with human and swine HEVs isolated in the USA and human HEVs isolated in Japan. Anti‐HEV antibodies were identified via ELISA in 8 of 99 (8.1%) cats, whereas, among 115 cattle and 213 dogs, no HEV‐specific antibodies were detected. The conserved RNA‐dependent RNA polymerase (RdRp) gene of HEV could be detected via RT‐PCR in 8.7% of raw oysters collected from coastal regions in Korea. The HEV RNAs detected in oysters were identified as belonging to subtype 3a. The HEV RNAs in oysters most closely resembled that of the swKOR‐2 strain. They also showed a close genetic relationship with the swKOR‐1 strain and the swine and human HEVs isolated in the USA. This is the first report describing the detection in oysters of HEV that may have originated from genotype 3 swine HEV in Korea. Pigs and cats infected with HEV, as well as oysters contaminated with HEV, are potential risk factors for HEV transmission to humans. J. Med. Virol. 82:583–591, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

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.     

Full‐length sequence of genotype 3 hepatitis E virus derived from a pig in Thailand

Hepatitis E virus (HEV) infection in pigs was investigated in two principal swine farming areas in Thailand. Anti‐HEV antibodies and HEV RNA in sera were examined in 258 pigs reared on five commercial farms from age 1 to 6.5 months and sows. Overall, 167 of 258 (64.7%) pigs were positive for anti‐HEV IgG, while 20 of 258 (7.75%) had detectable HEV RNA. Sequence analysis of 20 HEV isolates obtained from viremic pigs revealed that they were 92.3–100% identical to each other and had 82.2–88.2% nucleotide similarity to other reported genotype 3 isolates in 415 nucleotide sequences within ORF2 region. Further characterization by sequencing the complete genome of the Thai swine HEV isolate (named Thai‐swHEV07) and phylogenetic analysis showed that Thai‐swHEV07 segregated into a cluster consisting of swine isolates from Japan, Mongolia, and Kyrgyzstan within the HEV genotype 3. The Thai‐swHEV07 had a genomic length of 7,229 nt excluding the polyadenylated region at 3′ terminus of the genome. Comparison of Thai‐swHEV07 and 27 reported strains of genotype 3 revealed 80.4–85.9% nucleotide identity, with the highest identity of 85.9% to the novel swHEV strain from Mongolia. These findings suggest that genotype 3 HEV isolates are markedly heterogeneous. J. Med. Virol. 81:657–664, 2009 © 2009 Wiley‐Liss, Inc.     

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.     

Use of a swine bioassay and a RT-PCR assay to assess the risk of transmission of swine hepatitis E virus in pigs

The objective of this study was to assess the risk of transmission of swine hepatitis E virus (swine HEV) to na?ve pigs by inoculation with tissues or feces collected from pigs infected experimentally with swine HEV. Seventy-five, 3-week-old pigs were assigned randomly to 24 groups of 3-4 pigs and inoculated with homogenates of tissues (liver, heart, pancreas, or skeletal muscle) or a suspension of feces from swine HEV-infected pigs collected at 3, 7, 14, 20, 27, or 55 days post inoculation (DPI). Each inoculum was prepared as a 10% suspension (w/v) in PBS buffer and tested by a semi-quantitative RT-PCR for swine HEV RNA and by the swine bioassay. The inoculation route was intravenous for liver, heart and pancreas, and via stomach tube for skeletal muscle and fecal suspension. The liver homogenate inocula and feces collected at 3-7 and 14-20 DPI were positive for swine HEV RNA by RT-PCR. The pigs inoculated with liver homogenates collected at 3-7 and 14-20 DPI developed anti-HEV antibodies and swine HEV RNA was detected in their sera. Pigs inoculated with heart, pancreas, skeletal muscle homogenates or fecal suspensions failed to develop anti-HEV antibodies. These findings suggest that there is a potential risk of transmission of swine HEV via liver tissue from infected pigs in the early stages (3-20 DPI) of infection and the in vitro RT-PCR assay correlates well with the swine bioassay.     

Prevalence of hepatitis E virus antibodies in Canadian swine herds and identification of a novel variant of swine hepatitis E virus.

Swine hepatitis E virus is a newly identified potentially zoonotic virus from pigs of particular concern for possible direct transmission to a human xenotransplant recipient by organ transplantation. In the present study, prevalence of serum antibodies to hepatitis E virus was examined in Canadian swine herds. A total of 998 serum samples collected from 6-month-old healthy slaughter hogs were examined by enzyme immunoassay and Western blot analysis for antibodies to the recombinant open reading frame 3 (ORF3) protein of hepatitis E virus expressed in Escherichia coli. These samples represented more than 80 different swine production units from five major swine-producing provinces across Canada. From this study, 594 samples (59.4%) were found to be positive for hepatitis E virus antibody. The seroprevalence was higher in Quebec (88.8%) and Ontario (80.1%) than in Alberta and Saskatchewan (38.3%). By PCR using a pair of oligonucleotide primers deduced from the ORF2 sequence of human hepatitis E virus, a specific hepatitis E virus sequence was recovered from feces of pigs. The nucleotide sequence identity between the U.S. swine hepatitis E virus and the Canadian isolate (SK3) was only 85.8%, suggesting that genotypic variations may exist in swine hepatitis E virus in North America. Among 165 serum samples collected from humans in Saskatchewan, 2.4% were found to be positive for antibodies to the hepatitis E virus ORF3 protein. Our data indicate that hepatitis E virus is highly prevalent in commercial swine populations in Canada and support the suggestion that the swine hepatitis E virus may be an important zoonotic agent for humans.