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.

The H9N2 avian influenza virus circulates worldwide, predominantly in poultry. Its increasing infectivity and adaptation in poultry and mammals have enhanced the possibility of human infection. However, H9N2 human cases are difficult to detect due to their mild clinical symptoms. Serological study is valuable for risk assessment. A total of 15,700 serum samples were collected from occupationally exposed populations in 22 provinces of China and tested using hemagglutination inhibition (HI) and microneutralization (MN) assays. The sera positive rate of A/Guangzhou/333/99 (G9) was significantly higher than that of A/quail/Hong Kong/G1/97 (G1) (p<0.0001). The seroprevalences of H9N2 were significantly higher in live poultry market workers, large-scale poultry farmers and backyard farmers than in poultry slaughtering factory workers and wild bird habitant workers. The seroprevalences of A/ Guangzhou/333/99 (G9) (3.42%) and A/quail/Hong Kong/G1/97 (G1) (1.37%) in Southern China were significantly higher than those in Northern China (p<0.001). The seroprevalence was highest in the elderly, followed by adults and then youths. Our results indicate that sub-clinical human infection with H9N2 avian influenza virus is widely distributed in China. Longer poultry exposure might contribute to the higher seroprevalence in the elderly group. The higher seroprevalence observed in Southern China than in Northern China might be caused by a higher poultry density.

Nonstructural protein 7 (nsp7), which is flanked by nsp6 and nsp8, is one of the most conserved nonstructural proteins of porcine reproductive and respiratory syndrome virus (PRRSV). Nonstructural protein (nsp)-specific antibodies are produced in high titers in response to virus replication, especially against nsp1a, nsp1b, nsp2, and nsp7. However, many regional aspects of nsp7 are still veiled, such as its impact on viral replication and virulence or the immunological mechanism between virus and host. Based on the structure of the predicted nsp7 domain, we have constructed a series of large mutations and deletions. We ultimately demonstrated all mutations (nsp7, nsp7α/nspβ) and the majority of substitutions of nsp7 affected the PRRSV replicative cycle in some ways and were fatal for viral recovery, which indicates that these are significant to structure or function of the nsp7. What's more, the mutant vOKXH-nsp7 (F40A) indeed caused some of the variation compared with the parental virus vOKXH-GD, which shortens the amount of time needed to reach its highest viral titer, and decreases the concentration of the highest viral titer, obstructing viral mRNA and protein synthesis. Consequently, these valuable results possibly provide the first direct evidence that the nsp7 is really a critical protein domain for the RNA synthesis and the translation of viral protein of PRRSV.

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.

Hepatitis E virus (HEV) is responsible for hepatitis E, which represents a global public health problem. HEV genotypes 3 and 4 are reported to be zoonotic, and animals are monitored for HEV infection in the interests of public hygiene and food safety. The development of novel diagnostic methods and vaccines for HEV in humans is thus important topics of research. Opening reading frame (ORF) 2 of HEV includes both linear and conformational epitopes and is regarded as the primary candidate for vaccines and diagnostic tests. We investigated the precise location of the HEV epitopes in the ORF2 protein. We prepared four monoclonal antibodies (mAbs) against genotype 4 ORF2 protein and identified two linear epitopes, G438IVIPHD444 and Y457DNQH461, corresponding to two of these mAbs using phage display biopanning technology. Both these epitopes were speculated to be universal to genotypes 1, 2, 3, 4, and avian HEVs. We also used two 12-mer fragments of ORF2 protein including these two epitopes to develop a peptide-based enzyme-linked immunosorbent assay (ELISA) to detect HEV in serum. This assay demonstrated good specificity but low sensitivity compared with the commercial method, indicating that these two epitopes could serve as potential candidate targets for diagnosis. Overall, these results further our understanding of the epitope distribution of HEV ORF2, and provide important information for the development of peptide-based immunodiagnostic tests to detect HEV in serum.

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Hepatitis E virus (HEV) strains are classified into 4 genotypes by nucleotide sequencing. Genotypes 3 and 4 infect humans and animals via HEVcontaminated 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.