Hongyan Shi

Identification of two novel B cell epitopes on porcine epidemic diarrhea virus spike protein

Abstract S1D (residues 636–789) is a neutralizing epitope region on the spike protein (S) of porcine epidemic diarrhea virus (PEDV). To accurately identify epitopes on S1D, the S1-phage library containing the gene encoding the S1D region of PEDV S protein was micropanned by six specific monoclonal antibodies (McAbs) against the S1D region. These micropanned epitope regions (MER) were focused on 696–779 amino acids of the S protein. To further map epitopes of the MER, seven overlapping mini-fragments covering MER nucleotides were separately synthesized and expressed in Escherichia coli BL21 with a GST tag. These mini-GST fusion proteins were scanned by ELISA and Western blotting with the six McAbs, and the result showed that S1D5 (residues 744–759) and S1D6 (residues 756–771) are two linear epitopes of the PEDV S protein. The antisera of the epitopes S1D5 and S1D6 could react with the native S protein of PEDV. Furthermore, Pepscan of the two linear epitopes demonstrated that SS2 (748YSNIGVCK755) and SS6 (764LQDGQVKI771) are two core epitopes on S1D5 and S1D6, respectively, located on the S protein of PEDV.

Molecular epidemiology of porcine epidemic diarrhea virus in China

Since early 2006, porcine epidemic diarrhea virus (PEDV) has been reemerging in immunized swine herds. Open reading frame 3 (ORF3) is the only accessory gene in the PEDV genome. The entire ORF3 genes of 12 PEDV field strains and one vaccine strain were sequenced. The ORF3 genes of Chinese PEDV field strains (excluding CH/GSJIII/07) contain a single 672- or 675-nucleotide (nt) ORF, which encodes a 223- or 224-aa-long peptide. However, the CV777 vaccine strain and CH/GSJIII/07 contain a 276-nt ORF because of a 49-nt deletion at nt 245–293. The Chinese PEDV field strains and PEDV reference strains are divided into three groups based on the phylogenetic relationship of their ORF3 genes. Chinese PEDV field strains (excluding CH/GSJIII/07) have a close phylogenetic relationship to Korean strains and are genetically different from the PEDV vaccine strains. However, CH/GSJIII/07 has a close phylogenetic relationship to two vaccine strains, suggesting that it might have evolved from a live vaccine strain. Chinese PEDV field strains (excluding CH/GSJIII/07) can be differentiated from PEDV vaccine strains by a nested RT-PCR method.

The papain-like protease of porcine epidemic diarrhea virus negatively regulates type I interferon pathway by acting as a viral deubiquitinase

Porcine epidemic diarrhea virus (PEDV) is the cause of an economically important swine disease. Previous studies suggested that PEDV does not elicit a robust IFN response, but the mechanism(s) used to evade or block this innate immune response was not known. In this study, we found that PEDV infection blocked synthetic dsRNA-induced IFN-β production by interfering with the activation of interferon regulatory factor 3 (IRF3). We identified PEDV replicase encoded papain-like protease 2 (PLP2) as an IFN antagonist that depends on catalytic activity for its function. We show that levels of ubiquitinated proteins are reduced during PEDV infection and that PEDV PLP2 has deubiquitinase (DUB) activity that recognizes and processes both K-48 and K-63 linked polyubiquitin chains. Furthermore, we found that PEDV PLP2 strongly inhibits RIG-I- and STING-activated IFN expression and that PEDV PLP2 can be co-immunoprecipitated with and deubiquitinates RIG-I and STING, the key components of the signalling pathway for IFN expression. These results show that PEDV infection suppresses production of IFN-β and provides evidence indicating that the PEDV papain-like protease 2 acts as a viral DUB to interfere with the RIG-I- and STING-mediated signalling pathway.

PEDV ORF3 encodes an ion channel protein and regulates virus production

Several studies suggest that the open reading frame 3 (ORF3) gene of porcine epidemic diarrhea virus (PEDV) is related to viral infectivity and pathogenicity, but its function remains unknown. Here, we propose a structure model of the ORF3 protein consisting of four TM domains and forming a tetrameric assembly. ORF3 protein can be detected in PEDV‐infected cells and it functions as an ion channel in both Xenopus laevis oocytes and yeast. Mutation analysis showed that Tyr170 in TM4 is important for potassium channel activity. Furthermore, viral production is reduced in infected Vero cells when ORF3 gene is silenced by siRNA. Interestingly, the ORF3 gene from an attenuated PEDV encodes a truncated protein with 49 nucleotide deletions, which lacks the ion channel activity.

Molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in China.

Six porcine epidemic diarrhea viruses (PEDVs) were isolated from the fecal samples of piglets infected with PEDV in 2006 in China. The membrane (M) protein genes of six PEDV isolates were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR), then cloned, sequenced, and compared with each other as well as those ten PEDV reference strains. The M protein genes of six Chinese PEDV isolates consisted of 692 nucleotides containing a single open reading frame (ORF) of 681 nucleotides, which encoded a 226aa-long peptide. The conserved intergenic motif (ATAAAC), as previously recognized in Br1/87, was found in the 5 nucleotides upstream of the initiator ATG of M protein genes of six Chinese PEDV isolates. The hexamer motif was also found in CV777, JMe2, LZC, and QH. The M protein of six isolates had three main transmembrane domains (aa20–38, aa43–65, aa75–97). The M protein of one isolate, CH/IMT/06, had one potential glycosylation site, but those of the other five isolates had two. The glycosylation sequence Asn-Phe-Thr was highly conserved in the M proteins of six PEDV isolates. The six PEDV isolates showed nucleotide sequence homology between 98.8 and 100% and deduced amino acid sequence homology between 98.2 and 100% with each other. The nucleotide and amino acid identity of M protein genes between the six PEDV isolates and ten reference PEDV strains varied from 97.2 to 99.4% and 96.9 to 100%, respectively. On the basis of the phylogenetic relationship of M protein genes, six Chinese PEDV isolates composed of a separate cluster including one Chinese strain JS-2004-02, however, not including the Chinese strain LJB/03. These results demonstrated that there was a new genotype of PEDV prevailing in China.

Detection and molecular diversity of spike gene of porcine epidemic diarrhea virus in China.

Since late 2010, porcine epidemic diarrhea virus (PEDV) has rapidly disseminated all over the China and caused considerable morbidity and high mortality (up to 100%) in neonatal piglets. 79.66% (141 of 177) pig farms in 29 provinces (excluding Tibet and Hainan, China) and 72.27% (417 of 577) samples were positive for PEDV confirmed by reverse transcription-polymerase chain reaction (RT-PCR). The full-length S genes of representative field strains were sequenced. 33 field strains share 93.5%–99.9% homologies with each other at the nucleotide sequence level and 92.3%–99.8% homologies with each other at the amino acids sequence level. Most field strains have nucleotide deletion and insertion regions, and show lower homologies (93.5%–94.2%) with Chinese classical strain CH/S, however higher homologies (97.1%–99.3%) with recent strain CHGD-1. The phylogenetic analysis showed there are classical strains and variants prevailing in pig herd in China. PEDV has a high detection rate in pig herds in China. Sequence analysis indicated the S genes of recent field strains have heterogeneity and the variants are predominant.

Complete Genome Sequence of a Chinese Virulent Porcine Epidemic Diarrhea Virus Strain

ABSTRACT CH/S is a virulent porcine epidemic diarrhea virus (PEDV) strain and is used as the virulent strain to evaluate the protection rates of vaccines against PEDV infection in China. Here, we report the complete genome sequence of strain CH/S, which may aid in understanding the molecular characteristics of this strain.

Complete Genome Sequence of a Porcine Epidemic Diarrhea Virus Variant

ABSTRACT In 2011, outbreaks of viral diarrhea were observed on most swine-breeding farms in most of the provinces of China. The disease is characterized by vomiting, severe diarrhea, and a high mortality rate (82.3%) in newborn piglets. The clinical appearance was similar to that of porcine epidemic diarrhea virus (PEDV) infection. PEDVs were detected in samples (feces or small intestines) from most farms. In order to investigate whether there is a PEDV variant circulating in China, we sequenced and analyzed the complete genome of the recently identified field strain, CH/FJND-3/2011. The sequence data indicate that this PEDV variant prevails in China.

Identification of a conserved linear B-cell epitope in the M protein of porcine epidemic diarrhea virus

BackgroundThe major structural protein of coronaviruses, the membrane (M) protein, can elicit the formation of protective antibodies, but little information is available about the M protein of porcine epidemic diarrhea virus (PEDV). Identification of epitopes on the PEDV M protein will be helpful in the elucidation of the antigenic properties of this protein.ResultsOne hybridoma cell line secreting anti-M protein monoclonal antibody (McAb) was generated and designated 4D4. To map the epitopes on the PEDV M protein, a total of 17 partially overlapping fragments covering the C-terminus of M protein were expressed as fusion proteins with a 6×His tag or a GST tag. A linear motif, 193TGWAFYVR200, was identified by enzyme-linked immunosorbent assay (ELISA) and western blot (WB) analysis using McAb 4D4. The motif 195WAFYVR200 was the minimal requirement for reactivity, as demonstrated by removing amino acids individually from both ends of the motif 193TGWAFYVR200. The result of WB analysis showed that the 4D4-defined epitope could be recognized by PEDV-positive serum, but not transmissible gastroenteritis virus (TGEV)-positive serum. Furthermore, this epitope was highly conserved among different PEDV strains, as shown by alignment and comparison of sequences.ConclusionA McAb, 4D4, directed against the M protein of PEDV, was obtained, and the 4D4-defined minimal epitope sequence was 195WAFYVR200. The McAb could serve as a candidate for development of a McAb-based antigen capture ELISA for detection of PEDV. The epitope identified provides a basis for the development of epitope-based differential diagnostic techniques and may be useful in the design of epitope-based vaccines.

Molecular characterization of a rare G9P[23] porcine rotavirus isolate from China

The fifth most important G genotype, G9 rotavirus, is recognized as an emerging genotype that is spreading around the world. Sequence analysis was completed of a rare group A rotavirus, strain G9P[23], that was designated rotavirus A pig/China/NMTL/2008/G9P[23] and abbreviated as NMTL. It was isolated from a piglet with diarrhea in China. Nucleotide sequence analysis revealed that the VP7 gene clustered within the G9 lineage VId. The VP4 gene clustered within the rare P[23] genotype. NMTL is the first porcine G9 stain reported in China. Thus, to further characterize the evolutionary diversity of the NMTL strain, all gene segments were used to draw a phylogenetic tree. Based on the new classification system of rotaviruses, the NMTL sequence revealed a G9–P[23]–I5–R1–C1–M1–A8–N1–T1–E1–H1 genotype with close similarity to human Wa-like and porcine strains. The results showed that (i) NSP2 and NSP4 genes of NMTL exhibited higher genetic relatedness to human group A rotaviruses than to porcine strains, (ii) the VP2 and VP4 genes clustered with porcine and porcine-like human strains, and (iii) VP1 genes clustered apart from the Wa-like human and porcine clusters. In view of rotavirus evolution, this report provides additional evidence to support the notion that the human and porcine rotavirus genomes might be related.