| 1. Hepatitis E virus (HEV) , the causative agent of human hepatitis E (HE), is an important public health problem in many countries. The discoveries of swine HEV from pigs in many countries, suggest that HE may be a zoonosis. In order to investigate the serological epidemicology of swine hepatitis E virus (HEV) infection in Xinjiang, an indirect ELISA has been developed for the detection of antibody to HEV in pig serum samples. Four recombinant proteins from different genotype and sub genotype of HEV ORF2 (452~617aa), including Burma strain from subgenotype I a (p166Bur), Pakistan strain from subgenotype I b (p166Pak), Mexican strain from genotype II (p166Mex), and US strain from genotype III (p166Us) , were used as coating antigen. HRP-SPA was replace enzyme labeled secondary antibody. Seventy five pig sera were tested for anti-HEV IgG The result showed that the positivity rate and liter of sera detected with p166Us (77.3%) were higher than those detected with p166Pak (57.3%\ p166Mex (36%) and p166Bur (26.7%) . Therefore, p166Us protein(about 30ng/well) was selected for the subsequent sero-provalence studies.2. The purpose of the present study is to determine the prevalence of swine hepatitis E vims (HEV) infection in Xinjiang. 813 swine serum samples collected from 1 to 12 months of age at 9 swine farms in Xinjiang region were tested by ELISA for the presence of IgG antibodies against HEV. The recombinant protein pUS166 containing region 452-617aa of the ORF2 of HEV US strain was used as coating antigen. The result showed that anti -HEV IgG were detected in 205 of 405 pigs (50.62%) in one group and 238 of 408 pigs (58.33%) in another group , and that the seropositivity rate was not related to geographic district and breeds, but differed remarkably by age, being 40% among the 1- to 3-month-old piglets, but 77.33% among ones over 3-month-old. It suggested that swine HEV was widespread in different geographic regions of Xinjiang.3. To investigate hepatitis E vims (HEV) infection in swine and wild life and the extent of genetic variation among Chinese animal HEV strains, seventy pig feces from the first farm, sixty pig feces from the second farm in Xinjiang, respectively, twenty four wild life feces from HongShan zoo in Nanjing and ten fox feces from Anhui were tested for the presence of HEV RNA by reverse transcription nested polymerase chain reaction (RT-nPCR). Thirteen of 70 (18.57%) pigs were positive in the first farm and 42 of 70 (70%) in the second farm for HEV RNA. In contrast, all of the feces from 24 wild life and 10 fox feces were negative for HEV RNA. The nucleotide sequence of 348bp region within open reading frame 2(ORF2) of the 20 swine HEV isolates was determined. The DNAstar softwares were used for nucleotide sequences analysis. Twenty swine HEV isolates shared 89.7%~100% nucleotide sequence identities and belong to the same genotype, and shared 75.9%~80.2%, 76.3%~78.0%, 76.2%~81.7%, and 84.0%~91.1% homogeneity to human HEV genotypes I, II .HI and IV. Phylogenetic analyses further revealed that these swine HEV isolates were closely related to HEV IV T1 strain isolated from patients with acute hepatitis in China, but swine HEV isolates from two farm were clustered together respectively in the phylogenetic tree. Meanwhile these swine HEV isolates had 83.7%~92% homogeneity to other Chinese swine HEV and were grouped into genotype IV. Our findings further support the hypothesis that swine severed as reservoirs for HEV infection.4. To analyze the full length sequence of swCH25, isolated from a pig fecal sample in Xinjiang in 2001, eight overlapping fragments of swCH25 genomes were amplified with reverse transcription nested polymerase chain reaction (RT~nPCR) and the 5'and 3' ends were amplified with RACE. The PCR products were cloned into pMD18~T vector and sequenced. The result showed that its genome consisted of 7243nt excluding the poly(A) tail ,and contained three ORFs(ORFsl~3) that encoded proteins of 1707,674 and 114aa. Sequence analysis revealed that the overall nucleotide sequence id... |
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