Objective: To determine the distribution of genotype IV among hepatitis E virus (HEV) infections in Wuhan by sequencing the open reading frame (ORF) 3 gene of HEV clinical isolates.
Methods: Serum samples were collected from 103 individuals who tested positive for the anti-HEV IgM antibody, and total HEV RNA was extracted for targeted gene sequencing analysis. Reverse transcription-nested polymerase chain reaction (PCR) was used to amplify two fragments of the ORF3 gene (5020 to 5392 nt and 5347 to 5956 nt, EF570133). The two PCR products were sequenced and the sequences were stitched with the ContigExpress program and used to determine the HEV genotype.
Results: Both ORF3 gene fragments were amplified in 18 out of the 103 anti-HEV IgM-positive serum samples. These 18 HEV isolates shared 92.5% to 99.4% identity with each other at the nucleotide level. Nucleotide sequence homology analysis of the HEV genotypes I, II, III, and IV indicated the highest homology was with genotype IV; specifically, homology with genotype I was 83.5% to 86.7%, with genotype II was 83.2% to 85.2%, with genotype III was 84.6% to 87.2%, and with genotype IV was 92.0% to 96.5%.
Conclusion: Targeted sequencing of the HEV ORF3 gene facilitated genotyping of clinical isolates. Using this method, it was determined that nearly 20% of HEV clinical isolates from Wuhan belong to genotype IV.