Wang, Youchun; (2000) Studies of a divergent genotype of hepatitis E virus from Chinese patients. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Sera were investigated from 50 Chinese patients with a diagnosis of acute hepatitis and who were negative for hepatitis viruses A-E by serology (hepatitis E virus (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 using primers designed within conserved regions of HEV. The results showed that fifteen patients were found to harbour sequences related to HEV. The nucleotide sequences from nine of these sera closely matched the Burmese-like HEV (genotype 1), known to be endemic in China. The remaining six HEV sequences were similar to each other but were divergent from all known HEV genotypes. These new variants were designated as genotype 4 HEV. The complete genomic sequence of a representative isolate (HEV-T1) of genotype 4 HEV was determined. The results showed that the HEV-T1 genome comprises 7232 nt, excluding the 3' poly(A) tail. It has three open reading frames located in the same positions as other known HEV sequences. However, insertion of a single nucleotide (U) was detected at position nt 5159. This insertion may affect ORF2 and ORF3 translation. HEV-T1 was 74.5–75.8%, 74.5% and 75.3–76.3% identical to genotypes 1, 2 and 3, respectively, at the nucleotide level. This result confirmed our previous conclusion that these Chinese isolates belong to novel genotype, genotype 4, In order to study the immune reactivity of the HEV structural protein (ORF2 protein), the ORF2 protein with the N-terminal 124 amino acids deleted was expressed in a baculovirus system. This protein showed strong reactivity to HEV antibody in a Western-Blot. Because most nucleotide substitutions occur in the third base of the codon and do not affect the amino acid sequence. So the current antibody assays may be suitable for the detection of IgG responses to genotype 4 infections but this will require confirmation using validated sera.

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