Prevalence of hepatitis E virus antibodies in Canadian swine herds and identification of a novel variant of swine hepatitis E virus

Abstract

Swine hepatitis E virus is a newly identified potentially zoonotic virus from pigs of particular concern for possible direct transmission to a human xenotransplant recipient by organ transplantation. In the present study, prevalence of serum antibodies to hepatitis E virus was examined in Canadian swine herds. A total of 998 serum samples collected from 6-month-old healthy slaughter hogs were examined by enzyme immunoassay and Western blot analysis for antibodies to the recombinant open reading frame 3 (ORF3) protein of hepatitis E virus expressed in Escherichia coli. These samples represented more than 80 different swine production units from five major swine-producing provinces across Canada. From this study, 594 samples (59.4%) were found to be positive for hepatitis E virus antibody. The seroprevalence was higher in Quebec (88.8%) and Ontario (80.1%) than in Alberta and Saskatchewan (38.3%). By PCR using a pair of oligonucleotide primers deduced from the ORF2 sequence of human hepatitis E virus, a specific hepatitis E virus sequence was recovered from feces of pigs. The nucleotide sequence identity between the U.S. swine hepatitis E virus and the Canadian isolate (SK3) was only 85.8%, suggesting that genotypic variations may exist in swine hepatitis E virus in North America. Among 165 serum samples collected from humans in Saskatchewan, 2.4% were found to be positive for antibodies to the hepatitis E virus ORF3 protein. Our data indicate that hepatitis E virus is highly prevalent in commercial swine populations in Canada and support the suggestion that the swine hepatitis E virus may be an important zoonotic agent for humans.

FIG. 1

Expression of the HEV ORF3 protein in E. coli. The entire ORF3 coding sequence was expressed as a fusion protein with the GST gene. JM109 cells containing pGEX-HEVF3 were grown to log phase, and the protein expression was induced by adding IPTG to the bacterial culture at a final concentration of 2 mM. Cells were harvested and lysed with a mixture of detergents. The cell lysate was then subjected to partial purification of the recombinant protein as insoluble aggregates. The partially purified aggregate preparation was resolved by SDS-PAGE on a 12% polyacrylamide gel and visualized by Coomassie blue staining. Lanes: 1, molecular mass standard; 2, uninduced pGEX-HEVF3; 3, IPTG-induced pGEX-HEVF3. The arrowhead indicates the HEV recombinant ORF3 protein expressed as a fusion protein upon IPTG induction.

FIG. 2

Immunoreaction of the recombinant ORF2 protein of human HEV with swine sera. One microgram of the HEVF3-GST fusion protein was loaded into a wide preparative well and electrophoresed by SDS-PAGE. The gel was transblotted to nitrocellulose membrane, and the membrane was cut into 10 longitudinal strips so as to contain 0.1 μg of the antigen per strip. The individual strips were subjected to Western blot immunoassay with various dilutions of swine serum known to be positive or negative for HEV by ELISA. Lanes: M, molecular mass standards; 1 through 5, pig serum positive for human HEV diluted 1:200, 1:400, 1:800, 1:1,600, and 1:3,200, respectively; 6 through 10, pig serum negative for human HEV diluted 1:200, 1:400, 1:800, 1:1,600, and 1:3,200, respectively; 11, rabbit anti-GST antibody diluted 1:200.

FIG. 3

Amplification by PCR of HEV sequence from pig. (A) Total RNA was extracted from swine feces and the first-strand cDNA was synthesized using a primer pair deduced from the sequence of the swine HEV U.S. isolate. The first-strand cDNA was amplified by two consecutive rounds of nested PCR, and the amplified product was electrophoresed on a 2% agarose gel. Lanes: M, 100-bp ladder molecular mass standard; 1 through 6, feces collected from six different barns; 7, swine HEV (U.S. isolate) as a positive control. (B) Nucleotide sequence of HEV recovered from a Saskatchewan pig in Canada and the comparison with U.S. swine HEV. Dots represent identical nucleotides.

FIG. 4

Phylogenetic trees of human and swine HEVs based on the nucleotide sequences representing a portion of the ORF2 gene. Strains of HEV are indicated, and their GenBank accession numbers are presented in parentheses. Swine forms of HEV are underlined. The bar indicates a 10-nucleotide difference every 100 nucleotides.