Chenyan Zhao

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.

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.

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.

Hepatitis E Virus Produced from Cell Culture Has a Lipid Envelope

The absence of a productive cell culture system hampered detailed analysis of the structure and protein composition of the hepatitis E virion. In this study, hepatitis E virus from a robust HEV cell culture system and from the feces of infected monkeys at the peak of virus excretion was purified by ultra-centrifugation. The common feature of the two samples after ultracentrifugation was that the ORF2 protein mainly remained in the top fractions. The ORF2 protein from cell culture system was glycosylated, with an apparent molecular weight of 88 kDa, and was not infectious in PLC/PRF/5 cells. The ORF2 protein in this fraction can bind to and protect HEV RNA from digestion by RNase A. The RNA-ORF2 product has a similar sedimentation coefficient to the virus from feces. The viral RNA in the cell culture supernatant was mainly in the fraction of 1.15g/cm3 but that from the feces was mainly in the fraction of 1.21 g/cm3. Both were infectious in PLC/PRF/5 cells. And the fraction in the middle of the gradient (1.06g/cm3) from the cell culture supernatant,but not that from the feces, also has ORF2 protein and HEV RNA but was not infectious in PLC/PRF/5.The infectious RNA-rich fraction from the cell culture contained ORF3 protein and lipid but the corresponding fraction from feces had no lipid and little ORF3 protein. The lipid on the surface of the virus has no effect on its binding to cells but the ORF3 protein interferes with binding. The result suggests that most of the secreted ORF2 protein is not associated with HEV RNA and that hepatitis E virus produced in cell culture differs in structure from the virus found in feces in that it has a lipid envelope.

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 103.5 to 100.5 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.

Relationships among viral diagnostic markers and markers of liver function in acute hepatitis E

BackgroundDiagnosis of acute hepatitis E has been based in many clinics predominantly on detection of anti-HEV (hepatitis E virus) antibody. Now, new assays have been developed to detect other HEV markers. Our aim was to investigate the relationships among HEV diagnostic markers and liver function markers in acute hepatitis E.MethodsSeventy serum samples were collected from non-A, non-B, non-C acute hepatitis patients and tested for HEV markers (HEV antigen and RNA and anti-HEV IgM) and markers of liver function [alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total iron binding capacity (TBA), γ-glutamyl transferase (GGT), total bilirubin (TBIL), and direct bilirubin (DBIL)]. Partial open reading frame (ORF) 2 sequences from HEV RNA-positive samples were cloned and analyzed.ResultsThe concordances between HEV antigen and HEV RNA and between HEV antigen and anti-HEV IgM were 77.1% and 72.9%, respectively, with significant correlations, while that between HEV RNA and anti-HEV IgM was 61.4% with no significant correlation. Eleven of 25 samples negative for anti-HEV IgM were positive for HEV antigen. The ALT, AST, ALP, TBA, GGT, TBIL, and DBIL levels did not differ significantly between the anti-HEV IgM-positive and -negative groups. However, the ALT, AST, ALP, TBA, and GGT levels were significantly higher in the HEV antigen-positive group than in the HEV antigennegative group. All of the HEV isolates cloned belonged to genotype 4.ConclusionsHEV antigen was highly correlated with HEV RNA and elevated ALT, AST, ALP, TBA, and GGT levels. Testing for HEV antigen in combination with anti-HEV IgM is useful for the diagnosis of HEV infection.

Infection dynamics of hepatitis E virus in naturally infected pigs in a Chinese farrow-to-finish farm

To analyze the changes that occur in pigs during hepatitis E virus (HEV) infection, 256 serial serum samples were obtained from 32 pigs from one pig farm at ages 0 (cord blood), 15, 30, 60, 75, 90, 120, and 150 days. All HEV markers were assayed in these samples and showed that total anti-HEV antibodies and IgG formed two peaks. The first peak occurred at 0-60 days and the second after 75 days. No markers of infection, such as HEV RNA, antigen and anti-HEV IgM, were detectable during the first peak. Most newborn piglets (< 24 h of age) were negative for total anti-HEV and IgG. However, colostrum from all of the sows had evidence of these antibodies. Thus, the anti-HEV in the first peak was assumed to be acquired from maternal milk. Some infectious markers were positive at the beginning of second peak. PCR products were cloned and sequenced and the results indicated those sequences belonged to HEV genotype 4. The antibody present during the second peak may be induced by natural infection with HEV. In conclusion, pigs are susceptible to HEV infection and may remain infectious after the first peak of anti-HEV antibody.