Isolation and characterization of two new hepatitis E virus genotype 1 strains from two mini-outbreaks in Lahore, Pakistan

Abstract

Background: Pakistan is a highly endemic area for hepatitis E virus (HEV) infection. The aim of the current study was to isolate and characterize strains of HEV in two mini outbreaks.

Results: RNA was extracted and reverse transcribed to cDNA. Nested PCR was done for the detection of HEV RNA. The positive bands were eluted, cloned in TA vector and sequenced in both directions using genetic Analyzer (Applied Biosystem Inc, USA). Phylogenetic analysis was done using MEGA4 software. We isolated two new HEV genotype-1 strains from Lahore, Pakistan, based on cloning and sequencing of ORF2 region.

Conclusions: Our study suggest that both the human HEV strains were closely related to the Sar-55 but different from the Abb-2B and 87-Pakistan-B HEV isolates sharing 88-91% sequence identity to Pakistani isolate Sar-55. These results indicated that Sar-55 is the main endemic HEV strain in various areas of the country.

Figure 1

Phylogenetic analysis based on the ORF2 sequence of both the isolates in current study and other HEV genotypes isolated from different regions of the world, using the neighbor-joining method and evaluated using the interior branch test method with Mega 4 software. Percent bootstrap support is indicated at each node. The scale bar represents nucleotide substitutions per base. GenBank accession numbers of sequences used for phylogenetic analysis are given in parenthesis.

Figure 2

Phylogenetic analysis based on the complete ORF2 sequence of the isolate in this study and other HEV genotypes 1-4, using the neighbor-joining method and evaluated using the interior branch test method with Mega 4 software. Percent bootstrap support is indicated at each node. The scale bar represents nucleotide substitutions per base. GenBank accession numbers of sequences used for phylogenetic analysis were: HEV type 1 (M80581), type 2 (M74506), type 3 (AB089824) and type 4 (AB097812).