Development of a novel immunoperoxidase monolayer assay for detection of swine Hepatitis E virus antibodies based on stable cell lines expressing the ORF3 protein

Hepatitis E virus (HEV) strains are classified into 4 genotypes by nucleotide sequencing. Genotypes 3 and 4 infect humans and animals via HEV-contaminated food or water. HEV RNA was detected by PCR and antibodies were detected by ELISA. Since human studies showed that HEV IgG antibodies in sera can persist for extended periods, diagnosis of HEV infection in swine or humans is mainly based on serological detection using commercial ELISA kits. However, there is no supplemental method to verify ELISA results. Hence, we developed a novel method used for mutual correction of these common processes. Here, a modified stable HepG2 cell line was transfected with pcDNA3.1-ORF3 to express the swine HEV ORF3 protein. Based on this cell line, a novel immunoperoxidase monolayer assay (IPMA) was developed to detect antibodies against HEV. The results show that this method has good specificity, sensitivity and repeatability. When used to investigate 141 porcine serum samples, the IPMA had a coincidence rate of 92.2% with a commercial ELISA kit. The established IPMA described herein is valuable as a supplemental method to ELISA and can differentiate infections by HEV and other viruses

Hepatitis E virus serosurvey among pet dogs and cats in several developed cities in China.

Infection by Hepatitis E virus (HEV), as a zoonotic disease virus, is well studied in pigs in China, but few studies in pets have been performed. This study was designed to characterize the prevalence of HEV infection among pet dogs and cats in major metropolitan areas of China. We conducted a seroepidemiological survey from 2012 to 2013 in 5 developed cities, Beijing, Shanghai, Canton, Shenzhen and Macao, by enzyme-linked immunosorbent assay (ELISA). The overall HEV seroprevalence in 658 dog and 191 cat serum samples was 21.12% and 6.28%, respectively. The analysis in dogs suggested that there were significant differences among cities, and the positive rate of HEV-specific antibody in all cities ranged from 6.06% (Shenzhen) to 29.34% (Beijing). Older pet cats have a high risk (OR, 10.25) for HEV seropositivity, but no strong relationship was observed between different genders and age groups. Additionally, it was revealed that stray dogs, omnivorous pet dogs and pet cats who share food, such as kitchen residue, with the general population would have a higher risk for HEV seropositivity. The odds ratios for these groups are 2.40, 2.83 and 5.39, respectively, compared with pet dogs and cats fed on commercial food. In this study, we first report that HEV is prevalent in pet dogs and cats in several large cities in China. Swill and kitchen residue may be a potential risk for HEV transmission from human to pets. As the sample size was relatively small in this study and may not be fully representative of China, further investigation is required to confirm the conclusions

The anti-HEV rates of the population in Guangdong province, China, according to age, gender, and pig-exposure status.

<p>a: OR, odds ratio; b: 95%CI, 95%confidence interval; c: X², Chi-Square Test; d: ref, reference; UF, urban female; UM, urban male; FF, swine farm female; FM, swine farm male; **, significant difference; *, different.</p

The prevalence of antibodies against HEV among pigs in 2011–2013, Guangdong Province, China.

<p>a: OR, odds ratio;</p><p>b: 95%CI, 95%confidence interval;</p><p>c: X², Chi-Square Test;</p><p>d: ref, reference.</p

Comparative analysis between HEV ORF3 protein and peptides with deduced amino acids borne by selected phages.

<p>The consensus sequence was mainly identified in the ORF3 C-terminus. Only the sequence from A93 to R114 of ORF3 is presented. “.” represents residues of deduced amino acids differing from the ORF3 C-terminus, “-”indicates no residues. The numbers at the top signify the position of the residue at the ORF3 C-terminus. The first “3” represents position 93, and the last “4” represents position 114.</p

The prevalence of HEV RNA in pig bile samples collected from the Pearl River Delta. ^b

b<p>Pig bile samples have been collected from 9 districts of the Pearl River Delta since 2011.</p

Deduced amino acid sequences of peptides borne by selected phages.

<p>① Isoleucine (I) has similar structure and characteristics as Leucine (L)</p><p>② Isoleucine (I) has similar structure and characteristics as Valine (V). I, L and V all belong to the branched chain amino acid group. The underline indicates the continuous consensus sequence within the C-terminal region of ORF3 protein.</p><p>N/A, not applicable.</p

The information of swine farms and samples.

<p>Bile samples were collected from 22 different swine farms including large-scale farms and family-scale farms in 9 districts of Pearl River Delta. Swine sera were sampled from 12 large-scale swine farm in Guangdong. N, nursery pig; G, growing pig; S, sow; B, boar.</p

Binding analysis of selected phages to ORF3 mAb-1 using ELISA.

<p>Control and selected phage names are shown on the <i>z</i> axis, the OD₄₉₀ values of individual phages and control on the <i>y</i> axis, and logarithm value of the phage concentration on the <i>x</i> axis.</p