Da Shi

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.

Erratum to: Immunogenicity and antigenic relationships among spike proteins of porcine epidemic diarrhea virus subtypes G1 and G2.

Porcine epidemic diarrhea virus (PEDV) is a coronavirus that infects cells lining the small intestine of swine, resulting in vomiting, diarrhea, and dehydration. The amino acid sequence of the spike (S) protein, which is the principal target recognized by host immune cells, has multiple mutations that distinguish the two PEDV genotypes, G1 and G2. To determine whether these mutations lead to changes in antigenicity, as suggested by the failure of PEDV vaccines in China, we first optimized the codons of typical S genes of the CV777 vaccine strain (G1 subtype) and LNCT2 strain (G2 subtype) and expressed the recombinant full-length sequence of the S protein in a eukaryotic expression system. The IgG antibody levels of serum from mice immunized with purified S protein were markedly high. Antigenicity was compared by detection of polyclonal antibodies (PAbs) against the virus and S protein using an enzyme-linked immunosorbent assay (ELISA), an indirect immunofluorescence assay (IFA), and a serum cross-neutralization (SN) assay. Reactivity with the PAbs revealed significant cross-reactivity between the two PEDV subtypes, although there was a twofold difference in the antigenic responses based on PAb titers in the ELISA and IFA. Consistent with the variation in the S gene sequences, the SN titer suggested differences in the neutralization activity of the S protein between the two subtypes, which could explain the antigenic variation between the PEDV subtypes G1 and G2.

Analysis of protein expression changes of the Vero E6 cells infected with classic PEDV strain CV777 by using quantitative proteomic technique.

Abstract Recent outbreaks of porcine epidemic diarrhea virus (PEDV) have caused widespread concern. The identification of proteins associated with PEDV infection might provide insight into PEDV pathogenesis and facilitate the development of novel antiviral strategies. We analyzed the differential protein profile of PEDV-infected Vero E6 cells using mass spectrometry and an isobaric tag for relative and absolute quantification. A total of 126 proteins were identified that were differentially expressed between the PEDV-infected and mock-infected groups (P <0.05, quantitative ratio ≥1.2), among which the expression of 58 proteins was up-regulated and that of 68 proteins was down-regulated in the PEDV-infected Vero E6 cells, involving in integrin β2/β3, cystatin-C. The Gene Ontology analysis indicated that the molecular function of the differentially expressed proteins (DEPs) was primarily related to binding and catalytic activity, and that the biological functions in which the DEPs are involved included metabolism, organismal systems, cellular processes, genetic information processing, environmental information processing, and diseases. Among the disease-related functions, certain anti-viral pathways and proteins, such as the RIG-I-like receptor, Rap1, autophagy, mitogen-activated protein kinase, PI3K-Akt and Jak-STAT signaling pathways, and integrin β2/β3 and cystatin-C proteins, represented potential factors in PEDV infection. Our findings provide valuable insight into PEDV-Vero E6 cell interactions.

EF1A interacting with nucleocapsid protein of transmissible gastroenteritis coronavirus and plays a role in virus replication.

Abstract Transmissible gastroenteritis coronavirus (TGEV) is an enteropathogenic coronavirus that causes diarrhea in pigs, which is correlated with high morbidity and mortality in suckling piglets. Using the method of GST pull-down with the nucleocapsid (N), N protein was found to interact with swine testes (ST) cells elongation factor 1-alpha (EF1A), an essential component of the translational machinery with an important role in cells. In vitro and in virus-infected cells interaction was then confirmed by co-precipitation. Knockdown of EF1A impairs N protein proliferation and TGEV replication in host cell. It was demonstrated that EF1A plays a role in TGEV replication. The present study thus provides a protein-related information that should be useful for underlying mechanism of coronavirus replication.

Molecular characterization of a porcine kobuvirus variant strain in China

In this study, we report the complete nucleotide sequence of porcine kobuvirus CH/HZ/2011 (CH/HZ) isolated from infected piglets in China. Dendrograms indicated that CH/HZ is closely related to other porcine kobuviruses. The complete genome sequence of CH/HZ has an adenine insertion at position 124, an 18-nt deletion in the 5′UTR, and a 90-nt deletion in the 2B coding region. A neighbor-joining tree based on the VP1 gene indicated that 31 porcine kobuvirus strains are divided into four clusters (Thai-Japanese group, Hungarian group, Chinese group I and Chinese group II). The strain CH/HZ was included in Chinese group II.

Molecular Characterizations of Subcellular Localization Signals in the Nucleocapsid Protein of Porcine Epidemic Diarrhea Virus

The nucleolus is a dynamic subnuclear structure, which is crucial to the normal operation of the eukaryotic cell. The porcine epidemic diarrhea virus (PEDV), coronavirus nucleocapsid (N) protein, plays important roles in the process of virus replication and cellular infection. Virus infection and transfection showed that N protein was predominately localized in the cytoplasm, but also found in the nucleolus in Vero E6 cells. Furthermore, by utilizing fusion proteins with green fluorescent protein (GFP), deletion mutations or site-directed mutagenesis of PEDV N protein, coupled with live cell imaging and confocal microscopy, it was revealed that, a region spanning amino acids (aa), 71–90 in region 1 of the N protein was sufficient for nucleolar localization and R87 and R89 were critical for its function. We also identified two nuclear export signals (NES, aa221–236, and 325–364), however, only the nuclear export signal (aa325–364) was found to be functional in the context of the full-length N protein. Finally, the activity of this nuclear export signal (NES) was inhibited by the antibiotic Lepomycin B, suggesting that N is exported by a chromosome region maintenance 1-related export pathway.

Generation of a mouse scFv library specific for porcine aminopeptidase N using the T7 phage display system.

Abstract Porcine aminopeptidase N (pAPN) is a common cellular receptor for swine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV). To investigate single-chain fragment variable (scFv) repertoire against pAPN, the genes encoding the immunoglobulin light chain variable region (VL) and heavy chain variable region (VH) were amplified by reverse transcript polymerase chain reaction (RT-PCR) using a series of degenerate primers from the spleen of BABL/c mice immunized with native pAPN. The VL and VH amplicons were combined randomly by a 12 amino acid flexible linker by splicing by overlap extension PCR (SOE-PCR), which produced the scFv gene repertoire. After ligation of the scFv gene repertoire into the T7Select10-3b vector, a mouse scFv phage library specific for pAPN was produced through in vitro packaging. The primary scFv library against pAPN contained 2.0×107 recombinant phage clones, and the titer of the amplified library was 3.6×109 pfu/mL. BstNI restriction analysis and DNA sequencing revealed that 28 phage clones from the primary pAPN scFv library showed excellent diversity. The effectiveness of the scFv library against pAPN was verified further by phage ELISA using the recombinant protein of the pAPN C subunit as coating antigen. The construction and evaluation of a murine scFv library against the common receptor pAPN of porcine coronaviruses TGEV and PEDV using the T7 phage display system are described.