Youchun Wang

A novel genotype of hepatitis E virus prevalent among farmed rabbits in China.

In total, 335 serum samples were collected from rabbits from two farms in Gansu province, China, and tested for anti‐hepatitis E virus (HEV) antibody using EIA and for HEV RNA using nested RT‐ PCR with ORF2 primers. The overall prevalence of anti‐HEV antibody and HEV RNA was 57.0% (191/335) and 7.5% (25/335), respectively. The positivity rate of HEV RNA in the anti‐HEV antibody negative group (7.6% (11/144)) did not differ significantly from that in the positive group (7.3% (14/191)). The concordance between HEV RNA and anti‐HEV antibody was 43.3% with no significant correlation (P < 0.05). All 25 amplicons from the ORF2 region were cloned and sequenced. On the basis of nucleotide sequence comparison, they had 84–99% identity to each other and 73–77%, 70–76%, 75–82%, 71–77%, and 53–65% with the corresponding regions of genotypes 1, 2, 3, 4, and avian HEV, respectively. Samples that were positive with the ORF2 primers were amplified using ORF1 region primers; 17 were positive and shared 71–78%, 73–76%, 74–82%, 72–78%, and 39–58% identity with the corresponding regions of genotypes 1, 2, 3, 4, and avian HEV, respectively, at the nucleotide level. Two representative full‐length sequences were determined. These two sequences shared 85% identity with each other and had 74%, 73%, 78–79%, 74–75%, and 46–47% identity to full‐length genotypes 1, 2, 3, 4, and avian HEV, respectively. Thus, the sequences isolated from the rabbits represent a novel genotype of HEV. This study provides novel information about HEV genotypes infecting rabbits as well as evidence of a new mammalian genotype of HEV. J. Med. Virol. 81:1371–1379, 2009.

A divergent genotype of hepatitis E virus in Chinese patients with acute hepatitis

Recent studies have reported and provided nucleotide sequence data from divergent isolates of hepatitis E virus (HEV), including isolates from North America and Africa. Sera were investigated from 29 Chinese patients with a diagnosis of acute hepatitis and who were negative for hepatitis viruses A-E by serology (HEV was excluded by testing for IgG antibody only). To determine whether some patients were infected with HEV but had yet to seroconvert to antibody positivity, RT-PCR was carried out with primers designed within conserved sequences of the HEV open reading frame (ORF) 1 and ORF2 regions. Fifteen patients were found to harbour sequences related to HEV. Analysis of the HEV products revealed that nucleotide sequences from nine of the sera closely matched Burmese-like HEV sequences (more than 92% nucleotide identity across ORF1 and 88% in ORF2). The remaining six HEV isolates were similar to each other but divergent from all other known HEV sequences (74 to 83% nucleotide identity in ORF1 or ORF2). Phylogenetic analysis suggests that the six divergent isolates represent a fourth genotype of HEV, distinct from the previously described Burmese, Mexican and United States variants (genotypes 1, 2 and 3). This novel variant, referred to here as the Chinese genotype (genotype 4), may be responsible for a significant proportion of cases of acute hepatitis in China, as seen by the fact that 40% of the HEV-infected patients in this study were genotype 4 positive.

Detection of Sporadic Cases of Hepatitis E Virus (HEV) Infection in China Using Immunoassays Based on Recombinant Open Reading Frame 2 and 3 Polypeptides from HEV Genotype 4

ABSTRACT We reported previously on the complete sequence of hepatitis E virus (HEV) genotype 4, isolated from patients with sporadic cases of acute HEV infection in China. At least eight HEV genotypes have now been described worldwide, and further isolates await classification. Current immunoassays for the detection of anti-HEV antibodies are based on polypeptides from genotypes 1 and 2 only and may be inadequate for the reliable detection of other genotypes. Because genotypes 1 and 4 predominate in China, we wished to investigate the antigenic reactivities of HEV genotype 4 proteins. Four overlapping regions of open reading frame 2 (ORF2) (FB5, amino acids [aa] 1 to 130; E4, aa 67 to 308; F2-2, aa 288 to 461; E5, aa 414 to 672) and the entire ORF3 product were expressed in Escherichia coli as fusion proteins. Enzyme immunoassays based on each of the five purified polypeptides were evaluated with sera from patients with sporadic cases of acute HEV infection. Individual immunoassays derived from HEV genotype 4 detected more cases of acute hepatitis E than a commercial assay. Some serum samples, which were positive for anti-HEV immunoglobulin G only by assays based on HEV genotype 4, were positive for HEV RNA by reverse transcription-PCR. Polypeptide FB5, from the N terminus of ORF2, had the greatest immunoreactivity with sera from patients with acute hepatitis E. These data indicate that the N terminus of ORF2 may provide epitopes which are highly reactive with acute-phase sera and that assays based on genotypes 1 and 2 alone may be inadequate for the detection of HEV infection in China, where sporadic cases of HEV infection are caused predominantly by HEV genotypes 4 and 1.

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.

The serological prevalence and genetic diversity of hepatitis E virus in farmed rabbits in China

We identified and characterized a novel virus, designated rabbit hepatitis E virus (HEV), in rex rabbits farmed in China. Rabbit HEV is genetically related to but distinct from other known mammalian HEVs and avian HEV and may represent a novel genotype. To evaluate the spread and genetic variation of rabbit HEV, a total of 1094 serum samples were collected from various breeds of rabbits across ten counties in China. All sera were screened for the presence of anti-HEV antibody, HEV antigen and viral RNA. A total of 169 samples (15.4%), from nine of the ten counties, were found to be positive for HEV antibody. The seroprevalence was highest in Wuhan, Hunan Province (53.4%, 55/103). Samples positive for HEV antigen were detected in seven counties and the overall prevalence was 3.7% (41/1094). HEV RNA was detected in 22 samples and all but one of these samples was found to be positive for HEV antigen. Sequence analysis of the 304 bp amplicons within open reading frame 2 showed that all HEV isolates in this study clustered with known rabbit HEV strains, in a branch separate from genotypes 1 to 4. The rabbit HEV strains were genetically heterogeneous and divided into divergent groups. Strains from the same geographic region tended to cluster together. These results indicate that rabbit HEVs with considerable genetic diversity are prevalent in farmed rabbits in China. The potential zoonotic risk of rabbit HEV needs to be investigated and evaluated further.

Detection and assessment of infectivity of hepatitis E virus in urine

BACKGROUND & AIMS Hepatitis E virus (HEV) is known to be excreted in the stool but there has been no report of its presence in urine. This study investigated the presence of HEV RNA and antigen (HEV-Ag) in urine and its possible transmission. METHODS Serum and urine samples from patients with chronic or acute HEV infection and HEV infected monkeys were tested for viral and biochemical markers. Liver and kidney biopsies from the infected monkeys were analyzed by histopathology and immunohistochemistry. The infectivity of HEV from urine was assessed by inoculation into monkeys. RESULTS HEV RNA and HEV-Ag were detected persistently in the urine of a patient with chronic HEV infection. Subsequently, HEV RNA was detected in the urine of three of the eight (37.5%) acute patients, all of whom had detectable HEV-Ag in their urine. HEV RNA and HEV-Ag were also detectable in the urine of HEV infected monkeys. The ratio of HEV-Ag to RNA in the urine of the infected monkeys was significantly higher than in their sera and feces. The parameters of routine urinalysis remained within the normal ranges in the hepatitis E patients and infected monkeys, however, pathological changes and HEV-Ag were observed in the kidneys of the infected monkeys. Furthermore, one of two monkeys became infected with HEV after inoculation with urine from another infected monkey. CONCLUSIONS HEV infection may result in kidney injury and the urine may pose a risk of transmission. HEV-Ag detection in urine may be valuable for diagnosis of ongoing HEV infection.

Seroepidemiology and genetic characterization of hepatitis E virus in the northeast of China.

The aim of this study was to analyze the prevalence of infection and genotype of hepatitis E virus (HEV) in people and animals in the northeast of China (Heilongjiang, Jilin and Liaoning provinces). This seroepidemiological study was conducted using enzyme immunoassays and human sera positive for HEV antigen or anti-HEV IgM, and animal sera positive for HEV antigen or with an S/CO <or=10 for anti-HEV were tested for HEV RNA using real-time RT-PCR and nested RT-PCR. In humans, the overall prevalence of anti-HEV IgG was 31.6% (311/985), 28.6% (147/514) and 21.1% (841/3994) in individuals frequent, infrequent, and very rare contact with swine, respectively. The overall prevalence of anti-HEV was 81.6% (1737/2127) in pigs above 3 months of age, 66.4% (1644/2473) in pigs below 3 months of age, 18.7% (301/1612) in cattle and 12.4% (162/1302) in sheep. 1211 samples were tested for HEV RNA using real-time RT-PCR and 71 were positive. 30 of the 71 samples also were positive for HEV RNA using nested RT-PCR. These 30 isolates shared 81.2-100% sequence identity with each other at the nucleotide level and belonged to HEV genotype 4, regardless whether from human or animals. The results indicate that HEV infection is widely spread in the northeast of China. The prevalence of anti-HEV in individuals with frequent contact with pigs was significantly higher than those without and the HEV sequences isolated from such individuals were related more closely to isolates from pigs. These support strongly the hypothesis of a zoonotic origin of hepatitis E.

Persistent Hepatitis E Virus Genotype 4 Infection in a Child With Acute Lymphoblastic Leukemia

Introduction: In general, the hepatitis E virus (HEV) causes acute, self-limiting hepatitis. Prolonged and chronic infections caused by HEV genotype 3 have been found in some immunosuppressed patients in developed countries. Case Presentation: Here we report a Chinese boy with acute lymphoblastic leukemia, who developed hepatitis E during a period of intensive chemotherapy. Twenty months after the initial infection, HEV viremia was reappeared in the patient, with detectable anti-HEV IgM and IgG and modestly elevated serum transaminases. Sequence analysis of the viral RNAs revealed the reactivation of the HEV genotype 4d strain, indicating viral persistence in the patient. Conclusions: To our knowledge, this is the first chronic case confirmed by the prolonged presence of HEV RNA in china. It is also the first reported persistent hepatitis E infection caused by HEV genotype 4.

Serological Prevalence of Hepatitis E Virus in Domestic Animals and Diversity of Genotype 4 Hepatitis E Virus in China

Pigs have been confirmed to be reservoirs of some genotypes of hepatitis E virus (HEV), and other nonhuman species are also likely infected with the virus. To assess the prevalence of HEV infection in domestic animals in China, 3579 serum samples, including 1967 swine, 700 goat, and 912 cattle sera, were collected from 26 provinces across the country and tested for HEV antibodies and antigen using enzyme immunoassays. The results showed that 82.2% of the swine samples, but only 10.4% and 28.2% of cattle and goat sera, were anti-HEV positive respectively. The prevalence of anti-HEV antibody in animals varied from province to province, ranging from 10.9% to 100% in pigs, 0% to 48% in goats, and 0% to 92.9% in cattle. About 1.9% of pigs, 1.6% of goats, and 0.8% of cattle tested in the study were positive for HEV antigen. Some samples, including all HEV antigen-positive samples, were tested for HEV-specific RNA using reverse transcription polymerase chain reaction. Fifteen swine samples, but none from the goats or cattle, were found to be HEV RNA positive. Sequence and phylogenetic analyses classified all the swine HEV isolates into HEV genotype 4, which was further divided into four subgroups. This study demonstrated that HEV infection is widespread in domestic animals, particularly pigs, in China. The HEV genotype infecting pigs in China was genotype 4. However, the isolates displayed considerable genetic diversity.

Cross-protection of hepatitis E virus genotypes 1 and 4 in rhesus macaques

The purpose of this study was to determine cross‐protection between HEV genotypes 1 and 4, which are prevalent in China. Fecal suspensions of genotypes 1 and 4 from patients, as well as genotype 4 from swine, were inoculated intravenously into rhesus macaques. Each inoculum contained 5 × 104 genome equivalents of HEV. After infection, serum and fecal samples were collected serially and the levels of alanine aminotransferase (ALT) and anti‐HEV IgG and IgM in sera, and HEV RNA in fecal samples, were measured. Liver biopsies were carried out. All the infected monkeys (12/12) developed anti‐HEV IgG and exhibited fecal shedding of virus. IgM was detected in 11 of 12, and ALT elevation occurred about 2–6 weeks post‐inoculation in 10 of 12, infected monkeys. Hepatic histopathology was consistent with acute viral hepatitis and the ORF2 antigen of HEV was detected in the granular cytoplasm of hepatocytes by immunohistochemistry. After recovery from their initial HEV infection, the monkeys were challenged with a heterologous genotype or heterologous source of HEV and monitored for hepatitis and fecal shedding. Previous infection with HEV completely or partially protected against subsequent challenge with a heterologous virus, because 7 of 11 monkeys did not develop HEV infection or shed virus in the feces, and none of them developed hepatitis or exhibited ALT elevation or liver biopsy findings of hepatitis. In conclusion, previous HEV infection may give rise to cross‐genotype and cross‐host‐species protection. J. Med. Virol. 80:824–832, 2008.