Standardization Of Hepatitis E Virus Genotyping

The hepatitis E virus (HEV), a positive-sense, single-stranded RNA virus, is responsible for the majority of cases of hepatitis E, an acute viral hepatitis which is epidemic and sporadic in developing countries and rare in industrialized nations. It is important to define the genotypes of HEV for its diagnosis and vaccine design. At present, no consensus exists on genotype classification of HEV. Due to the tedious methods of full length sequencing, the detected HEV strains are genetically characterized in laboratories on the basis of partial sequences. However, there is a high degree of variability in the regions of the HEV genome targeted for analysis, which cause difficulty for comparison of experimental results. Furthermore, different nucleotide divergence levels and genetic distances are used for the criterion defining HEV genotypes by different investigators. In addition, the designation for genotypes and subgenotypes of HEV has not come to a consensus.In present study, we aim at standardizing the HEV genotyping based on the RT-PCR amplicons of different regions of HEV genome. It consists of following two parts:Comparison and analysis of genotyping of HEV based on full length and RT-PCR amplicons of different genomic regions Four sets of degenerate primers (MJ-A~MJ-D) were designed based on conserved regions by alignments of 37 HEV strains with complete or nearly complete genome. Other 20 reported sets of universal primers and corresponding amplicons were also evaluated. Nucleotide identity and genetic distance calculated from 37 HEV strains based on 24 genomic regions and full-length genome were compared and analyzed by statistical methods to determine a genomic region which has no significant difference with the complete genome. Phylogenetic analysis results are compared based on the full length and the selected region to further prove the statistical results.According to the statistical analysis of nucleotide identity and phylogenetic analysis, one HEV RNA polymerase region based on MJ-C primers yields evolutionary relationships similar to those produced from the full-length genome inter-genotypes and intra-genotype. With genetic distance data, a slight different region based on another set of primers (GV-A) had no significant difference with that of full-length genome only inter-genotypes, but not intra-genotype.According to the data of full length of HEV genome, the cretirion for defining genotype and subgenotype was established: for genotype, percent of nucleotide divergence>24%, genetic distance>0.30; for subgenotype, percent of nucleotide divergence>10%, genetic distance>0.11. The percent of nucleotide identity and genetic distance and phylogenetic analysis based on MJ-C amplified region had good correlation with that of full-length genome.Detection of HEV by RT-PCR using the MJ-C primersHEV stains with known genotypes and sera of 38 patients with a clinical diagnosis of hepatitis E were determined by RT-nested PCR using the MJ-C primers to evaluate their efficiency.For HEV detection, the MJ-C primers work sufficiently well to amplify all genotypes HEV. 13 of 38 isolates were positive for HEV RNA. All the positive isolates belonged to genotype IV, and can be further divided into 4 subgenotypes.In conclusion, a HEV RNA polymerase region approved to be the consencus region for HEV genotyping by statistical and phylogenetic analysis. The RT-PCR primers (MJ-C) for this region are conserved enough for HEV detection of all genotypes. A standard for HEV genotyping is established in this study: the nucleotides divergence of different genotypes of HEV>24%, genetic distance of that >0.30; between subgenotypes, the nucleotides divergence>10%, while genetic distance >0.11. Furthermore, a nomenclature for HEV genotypes is also set up: the Roman numeral and the English letter could be used as the HEV genotype and subgenotype designation respectively in the strains isolation time order.