Identification of epitopes on nonstructural protein 7 of porcine reproductive and respiratory syndrome virus recognized by monoclonal antibodies using phage-display technology.

Nonstructural protein 7 (nsp7) of porcine reproductive and respiratory syndrome virus (PRRSV) is considered to be a suitable reagent for the development of serological diagnostic assays. It can be expressed as a soluble recombinant protein in Escherichia coli, and its antibody response may continue up to 202 days post-infection. Furthermore, the region encoded by nsp7 is highly homologous among various strains within the genotype, and the results of nsp7-based enzyme-linked immunosorbent assay (ELISA) showed high agreement with previous Idexx ELISA results. All these evidences suggest the existence of important epitopes on nsp7, though the characteristics of these epitopes remain unclear. In the present study, we prepared three monoclonal antibodies against nsp7 protein and used them to screen the epitope-distribution characteristics of PRRSV nsp7 protein by phage-display technology. We identified a linear epitope NAWGDEDRLN at amino acids 153-162 type II PRRSV nsp7ß subunit. This newly defined epitope showed excellent reactivity with PRSSV-positive serum samples. These results further our understanding of the antigenic structure of nsp7 protein, and provide efficient reagents for PRRSV serological tests.

A Proline-Rich Domain in the Genotype 4 Hepatitis E Virus ORF3 C-Terminus Is Crucial for Downstream V105DLP108 Immunoactivity.

The hepatitis E virus (HEV) is responsible for serious viral hepatitis worldwide. Animals are considered a reservoir of HEV, particularly pigs. While HEV infection in pigs and dogs is always asymptomatic, the virus causes high death rates in patients with pre-existing chronic liver disease and pregnant women in developing countries. HEV open reading frame 2 (ORF2) has been used as a diagnostic target to detect specific antibodies against HEV in serum samples. Recent research has additionally supported the potential utility of the ORF3 protein as a target in serum anti-HEV detection. However, the epitope distribution of ORF3 protein remains ambiguous. In the current study, we showed that continuous amino acid motif, VDLP, at the C-terminus of genotype 4 HEV ORF3 is a core sequence of the ORF3 protein epitope. Moreover, cooperative interaction with upstream elements is essential for its immunoactivity. Three proline residues (P99, P102 and P103) in the upstream proline-rich domain exerted significant effects on the immunocompetence of VDLP. ELISA results revealed that SAPPLPPVVDLP and SAPPLPPVVDLPQLGL peptides containing the identified VDLP epitope display weaker reactions with anti-HEV serum than the commercial ELISA kit. Our collective findings provide valuable information on the epitope distribution characteristics of HEV ORF3 and improve our understanding of the influence of the proline-rich domain on the immunoactivity of downstream amino acids in the C-terminal region.

Serological evidence of avian influenza virus and canine influenza virus infections among stray cats in live poultry markets, China.

From January 2010 to January 2012, we collected sera samples from 700 stray cats living in close proximity to poultry farms or poultry markets in 4 provinces in China. A number of cats had evidence of avian and canine influenza virus infection: avian H9N2 [24 by HI ≥1:20 and 16 by microneutralization (MN) assay ≥1:80]; avian H5N1 (9 by HI ≥1:20 and 3 by MN assay ≥1:80) and canine H3N2 (32 by HI ≥1:20 and 18 by MN ≥1:80). Bivariate analyses revealed that cats sampled near live poultry markets and cats with influenza-like-illness were at increased risk of having elevated antibody titers by HI against avian H9N2, avian H5N1, or canine H3N2 viruses. Hence, cats may play a very important role in the ecology of novel influenza viruses and periodic epidemiological surveillance for novel influenza infections among stray cats could serve as an early warning system for human threats.

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 prevalence of hepatitis E virus infections among swine, swine farmers and the general population in Guangdong Province, China.

Hepatitis E virus (HEV) infection is widespread in China, but few studies have been carried out in Guangdong Province. This study aimed to characterize the prevalence of HEV infections among swine, swine farmers and the general population in Guangdong Province. We conducted an epidemiological study that included swine, swine farmers and health examination attendees in Guangdong from 2011 to 2013. The overall seroprevalence of anti-HEV antibodies in swine was 64.7%. The results revealed that growing pigs, sows and boars (OR ranges from 3.5 to 21.5) have a higher risk than nursery pigs. HEV RNA in swine bile showed that HEV is epidemic in swine in the Pearl River Delta, with the highest prevalence of 22.73% in Foshan. Some genomes of HEV strains from each district were sequenced. Phylogenetic analysis of partial open reading frame 2 (ORF2) shows that they belong to genotype IV and are most closely related to isolates from China. In total, 307 participants were enrolled in the study, including 114 swine farmers and 193 attendees from hospitals. IgG anti-HEV was detected in 48.25% of swine farmers and in 38.34% of the general population. Seroprevalence rates were almost stratified by age, with a higher positive rate for males compared to females across all age groups. Women on swine farms appeared to have a lower risk of infection compared to the general population, revealing that the risk factors for HEV infection are not unique. The results suggested that there were other risk factors for HEV infection. HEV infection is prevalent in Guangdong, but due to the small sample sizes, more investigations are needed to assess the potential impact of HEV infection, and many additional risk factors should be considered.