Hepatitis E virus infection in central China reveals no evidence of cross-species transmission between human and swine in this area

Abstract

Hepatitis E virus (HEV) is a zoonotic pathogen of which several species of animal were reported as reservoirs. Swine stands out as the major reservoir for HEV infection in humans, as suggested by the close genetic relationship of swine and human virus. Since 2000, Genotype 4 HEV has become the dominant cause of hepatitis E disease in China. Recent reports showed that genotype 4 HEV is freely transmitted between humans and swine in eastern and southern China. However, the infection status of HEV in human and swine populations in central China is still unclear. This study was conducted in a rural area of central China, where there are many commercial swine farms. A total of 1476 serum and 554 fecal specimens were collected from the general human and swine populations in this area, respectively. The seroepidemiological study was conducted by enzyme-linked immunosorbent assay. Conserved genomic sequences of open reading frame 2 were detected using reverse transcription-PCR. The results indicated that the overall viral burden of the general human subjects was 0.95% (14/1476), while 7.0% (39/554) of the swine excreted HEV in stool. The positive rate of anti-HEV IgG and IgM in the serum samples was 7.9% (117/1476) and 1.6% (24/1476), respectively. Phylogenetic analysis based on the 150 nt partial sequence of the capsid protein gene showed that the 53 swine and human HEV isolates in the current study all belonged to genotype 4, clustering into three major groups. However, the HEV isolates prevalent in the human and swine populations were classified into known distinct subgenotypes, which suggested that no cross-species transmission between swine and humans had taken place in this area. This result was confirmed by cloning and phylogenetic analysis of the complete capsid protein gene sequence of three representative HEV strains in the three major groups. The cross reactivity between anti-HEV IgG from human sera and the two representative strains from swine in central China was confirmed by Dot-blot assay. In conclusion, although all the HEV strains prevalent in central China belonged to genotype 4, there is no evidence of cross-species transmission between human and swine in this area.

Figure 1. The phylogenetic tree of human (black squares) and swine (white squares) hepatitis E virus (HEV) isolates in central China, 2007–2008.

The phylogenetic tree was produced with a 150-nt ORF2 sequence alignments of 42 isolates in the present study and other 16 representative reference sequences. The scale bar indicates a genetic distance of 0.02% nucleotide substitution per position. Values for various branches are percentages of the tree obtained from 1000 resamplings of the data. Genotype 1, 2 and 3 strains are included as outgroup. The three black triangles show the three representative strains whose complete sequences of ORF2 were determined in the present study.

Figure 2. The phylogenetic tree constructed by alignment of the complete 2025-nt nucleotide sequence of ORF2 using Mega 4 software.

Virus strains included the three isolates in this study and the referenced genotype 4 isolates. Percent bootstrap support is indicated at each node. GenBank accession no., source and country of origin are indicated. Genotype 1, 2 and 3 strains are included as outgroup. The isolates identified in this study were marked with black triangles.

Figure 3. The results of Dot-blot assay.

(A) The Dot-blot assay using the 9 serum samples which were positive for IgM, IgG and HEV RNA; (B) The Dot-blot assay using the 20 randomly selected serum samples which were positive for anti-HEV IgG but not for IgM and HEV RNA. Line NC(fecal) mean presents the reactivity between HEV-negative fecal suspension and the serum samples; Column NC(serum) presents the reactivity between HEV antibody negative serum sample with the fecal suspension.