Seroprevalence of Hepatitis E Virus Infection, Rural Southern People’s Republic of China

HEV infection is thought to have been endemic in southern China for >60 years; swine are now the main source of human infection

Recombination analysis reveals a double recombination event in hepatitis E virus

Recombination of Hepatitis E Virus (HEV) has rarely been reported. In the present study, phylogenetic and recombination analyses were performed on 134 complete HEV genomes. Three potentially significant recombination events, including both intra-genotype and one inter-genotype, were identified by recombination detection analysis. Recombination events I and II occurred intra-genotype and inter-genotype, respectively, among three isolates, including the lineage represented by CHN-XJ-SW13 (GU119961, swine isolate), E067-SIJ05C (AB369690, human isolate), and JJT-Kan (AB091394, human isolate), and lead to the recombinant swine isolate swCH31 (DQ450072). Recombination event III occurred between the lineage represented by the NA1 (M73218) and K52-87 (L25595), which resulted in the recombinant Xingjiang-1 (D11092). Our analyses proved that that recombination could occur between human and swine HEV strains, double recombination events existed in HEV, and recombination event could happen within ORF2 region of HEV. These results will provide valuable hints for future research on HEV diversity

Characterization of hepatitis E virus in southern Africa

M.Sc. (Med.), Faculty of Health Sciences, University of the Witwatersrand, 2004Endemic circulation of hepatitis E virus (HEV) in Namibia was suspected from serological data during an outbreak of non-A non-B hepatitis in Rundu, in 1995. The source of the outbreak was surmised to be the water supply, which had been compromised approximately six months earlier. This study examines nucleotide and amino acid sequence data obtained from the open reading frame 2 (ORF2) region towards the carboxy terminal end (3’-end) of the HEV genome extracted from the stool of Namibian patients infected during this outbreak. The overall aim is to establish a polymerase chain reaction (PCR) for molecular diagnosis of HEV, to isolate HEV from specimens collected from acute viral hepatitis outbreaks such as this one in Namibia, to characterize, at the genomic level, the strain of the virus involved, to compare the strain to those involved in other outbreaks of HEV in southern Africa and to relate this information to available data from other parts of the world. A nested reverse transcriptase-polymerase chain reaction (RT-PCR) was developed for molecular diagnosis of HEV and four representative HEV isolates from this outbreak have been successfully amplified, sequenced and analysed over a 451 base pair (bp) region of a subgenomic fragment from the 3’-end of the genome in ORF2. Phylogenetic analysis showed the four Namibian HEV isolates clustered with a Mexican isolate in genotype II and shared a 85.8-86.3 percent (%) nucleotide identity with the 1987 Mexican isolate but were only 77.6-79.6 % similar to other African isolates. HEV isolated from the same region of Namibia in 1983 was reported to cluster into genotype I and this analysis clearly indicates the difference between the strains involved in the two outbreaks. Virus from sporadic cases of HEV isolated in 1997/8 in Nigeria were also found to be from genotype II. This is the first study performed in South Africa to isolate, amplify by PCR and sequence the HEV. It is also the first to characterize HEV as the causative agent of the hepatitis outbreak that occurred in Namibia in 1995. As it reports the presence of a second unique HEV strain in southern Africa, we conclude that HEV genotypes may be more widely distributed than previously thought

Prevalence of Hepatitis E Virus in Swine Fed on Kitchen Residue

The aim of this study was to investigate the prevalence of swine hepatitis E virus (HEV) in pigs fed different feedstuffs (kitchen residue or mixed feeds) and genetic identification of HEV isolated in Hebei province, China. Serum and fecal samples were collected from adult swine. Anti-HEV antibody was evaluated by double sandwich antigen enzyme immunoassay. HEV RNA was extracted from fecal samples and amplified by nested RT-PCR. The reaction products were sequenced, and the sequence analyzed. Virus-like particles were distinguishable by negative staining in the electron microscope. Histopathological observation and immunohistochemical localization were used in the animal models. Overall, the anti-HEV positive percentage of serum samples from pigs fed on kitchen residue was 87.10% (27/31), and 53.06% (130/245) from pigs fed on complete feed. The HEV RNA positivity rate of fecal samples from pigs fed on kitchen residue was 61.54% (8/13), but zero for pigs fed on complete feed. Sequence analysis of these eight samples and comparison with the published sequence showed that there were eight groups that belonged to genotype 4 d and the nucleotide identity was 95.6–99.3%. swHE11 is most closely related to strain CCC220, and the other seven HEV isolates were most closely related to strains swGX40, SwCH189 and V0008ORF3, which are isolates from human and pigs. Histopathological observation showed that there was liver damage in the experimental group, and immunohistochemistry indicated that the HEV antigens were strongly positive at 7 days after infection. The results demonstrated that the prevalence of HEV in pigs fed on kitchen residue was higher than in those fed on complete feed (P<0.05)

Analysis of a strain of hepatitis E virus associated with acute liver failure

Hepatitis E virus (HEV) infection often results in a relatively mild, self-limiting hepatitis, although acute liver failure occurs in some patients, particularly women in the later stages of pregnancy. It is uncertain whether the outcome of infection is predominantly influenced by host or viral factors, or whether an interplay of both is important. At the time of project inception, most HEV sequences in the nucleotide databases were from virus associated with uncomplicated cases of hepatitis E, and many were derived from virus passaged in monkeys. We set out to determine the nucleotide sequence of an HEV strain associated with acute liver failure in a male, using liver tissue as the source of viral RNA. The consensus nucleotide sequence of this HEV strain was determined from standard length amplicon and full length genomic HEV PCR products. Comparison of the consensus sequence with published HEV sequences showed this strain of HEV to conform to the previously described genomic structure. The 5' non-coding region of this strain contained a unique nucleotide change at position 11. There were no obvious features that could account for the increased pathogenicity. The closest matches in the database were with genotype 1 HEV sequences. Expression in-vitro of full length HEV genomes and individual ORF 2 and 3 generated proteins of the size expected from sequence data and published results. Transient expression in COS-7 cells failed to show the presence of HEV specific proteins by western blot or radioimmunoprecipitation or clear evidence of HEV RNA by northern blot. Northern blotting of total liver RNA did not indicate the presence of previously described subgenomic RNAs. The outcome of infection with HEV is likely to be the result of the host immune response rather than altered viral pathogenicity, however further analysis of HEV strains associated with acute liver failure is required

Expression in animal cells and characterization of the hepatitis E virus structural proteins

Hepatitis E virus (HEV) is a major human pathogen in much of the developing world. It is a positive-strand RNA virus with a 7.5-kb polyadenylated genome consisting of three open reading frames (ORFs). In the absence of an in vitro culture system, the replication and expression strategy of HEV and the nature of its encoded polypeptides are not well understood. We have expressed the two ORFs constituting the structural portion of the HEV genome in COS-1 cells by using simian virus 40-based expression vectors and in vitro by using a coupled transcription-translation system. We show here that the major capsid protein, encoded by ORF2, is an 88-kDa glycoprotein which is expressed intracellularly as well as on the cell surface and has the potential to form noncovalent homodimers. It is synthesized as a precursor (ppORF2) which is processed through signal sequence cleavage into the mature protein (pORF2), which is then glycosylated (gpORF2). The minor protein, pORF3, encoded by ORF3 is a 13.5-kDa nonglycosylated protein expressed intracellularly and does not show any major processing. pORF3 interacts with a cellular protein of about 18 kDa which we call 3IP, the pORF3-interacting protein. The significance of these findings are discussed in light of an existing model of HEV genome replication and expression

Molecular characterization and prevalence of hepatitis E virus in Swedish wild animals

Observation of chronic hepatitis E virus (HEV) in immunosuppressed patients, and unexplained high hepatitis E virus (HEV) prevalence in the human population raises public health concern. The aim of this thesis is to molecular characterize and investigate the prevalence of HEV in Swedish wild life and their association with HEV transmission to humans. A novel virus was detected in a sample from a Swedish moose (Alces alces). The genome was highly divergent with sequence identity of 30-60% to other HEVs. Genome sequence and phylogenetic analysis showed closest relationship with HEV genotypes1-7 (gt1-7). In addition, three open reading frames (ORFs) was also detected, and all these observed properties suggested the virus as a member of the Hepeviridae family. Markers for ongoing (HEV RNA) and/or past HEV infection (anti-HEV) was demonstrated in 67 (29%) of 231 Swedish moose samples collected from various Swedish provinces. Thus, moose are frequently infected with HEV. Its closest similarity with the HEV gt1-7 group, which includes strains that also infects humans, may indicate a potential for zoonotic transmission of this HEV. A survey detected HEV markers in the wild life, which included samples from wild boars (Sus scrofa) and different deer species, fallow deer (Darna darna), red deer (Cervus elaphus), roe deer (Capreolus capreolus) and moose (Alces alces). These markers were ongoing and/or past infections, and were found in 53 (22%) out of 245 animal samples. The viral nucleic acid sequences strains were sequenced and compared with autochthonous Swedish human HEV cases, of whom three were found infected with strains similar to wild boar strains. These results indicate that Swedish wild animals are often infected with HEV and may be an important source of HEV transmission to humans who come into contact with wild animals or eat game meat. The introduction of a single amplicon PCR of near complete HEV genomes enabled the identification of possible virulence marker, and the detection of possible recombination events between Swedish swine and wild boar, and that there may have been zoonotic transmission of HEV strains between Spain and France