Jianfei Chen

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

n Abstractn n 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.n n

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.

Infectious bronchitis virus: S1 gene characteristics of vaccines used in China and efficacy of vaccination against heterologous strains from China

The entire S1 protein genes of eight infectious bronchitis (IB) vaccine strains used in China were compared with those of the IB virus isolates present in the field in China. The nucleotide and amino acid similarities between the eight IB vaccine strains and the field strain, tl/CH/LDT3/03, which was isolated from a teal (Anas sp.), were not more than 81.1% and 79.2%, respectively. Phylogenetic analysis based on the S1 genes showed that the vaccines and field strains belonged to different clusters and showed larger evolutionary distances, and indicated that they were of different genotypes. Four out of the eight vaccines, in addition to the Massachusetts type vaccine H120, were used for protection tests against challenge by the IB virus isolate tl/CH/LDT3/03. This revealed that each of the five IB vaccines induced poor protection against the teal isolate, as assessed by respiratory protection, clinical signs and mortality, indicating the necessity of developing vaccines from local strains for IB control in China. Les gènes complets codant la protéine S1 de huit souches de vaccins de la bronchite infectieuse (IB) utilisés en Chine ont été comparés à ceux des souches de virus de la bronchite infectieuse (IBV) isolées sur le terrain en Chine. Les similarités respectives des nucléotides et des acides aminés entre les huit souches vaccinales de lIB et la souche du terrain tl/CH/LDT3/03, isolée dune sarcelle (Anas sp.), nont pas été supérieures à 81,1% et 79,2%. Lanalyse phylogénétique basée sur les gènes S1 a montré que les souches vaccinales et la souche du terrain appartenaient à des groupes différents, présentaient des distances dévolution importantes, et a indiqué quelles correspondaient à des génotypes différents. Quatre des huit vaccins, en plus du vaccin H120 de type Massachusetts, ont été utilisés dans des tests de protection vis-à-vis de la souche dépreuve tl/CH/LDT3/03. Les résultats ont montré que, vis-à-vis de la souche isolée de la sarcelle, les cinq vaccins de lIB ont induit une protection faible, mesurée sur la base de la protection respiratoire, les symptômes et la mortalité, indiquant la nécessité de développer des vaccins à partir de souches locales pour le contrôle de lIB en Chine. Die vollständigen S1-Protein-Gene von acht in China gebräuchlichen infektiöse Bronchitis (IB)-Impfstämmen wurden mit den von in China vorkommenden Feldisolaten des Virus der infektiösen Bronchitis (IBV) verglichen. Die Nukleotid- und Aminosäurenübereinstimmungen zwischen den acht IB-Impfstämmen und einem Feldstamm, tl/CH/LDT3/03, der aus einer Krickente (Anas sp.) isoliert worden war, betrug nicht mehr als 81,1 % bzw. 79,2 %. Die auf den S1-Genen basierende phylogenetische Analyse zeigte, dass die Impf- und Feldstämme zu verschiedenen Clustern gehörten, größere evolutionäre Distanzen von einander hatten und verschiedene Genotypen darstellten. Zusätzlich zu der Massachusetts-Typ-Vakzine H120, wurden vier der acht Vakzinestämme für Protektionstests gegen eine Belastungsinfektion mit dem IBV-Isolat tl/CH/LDT3/03 eingesetzt. Durch die Ermittlung der Schutzwirkung auf den Respirationstrakt, der klinischen Symptome und der Mortalitätsrate wurde festgestellt, dass die fünf IB-Vakzinen nur einen geringen Schutz gegen das Krickentenisolat induzierten, was die Notwendigkeit der Impfstoffentwicklung aus lokalen Stämmen für die IB-Bekämpfung in China deutlich macht. Se compararon los genes completos de la proteína S1 de 8 cepas vacunales de bronquitis infecciosa (IB) utilizadas en China con los de aislamientos del virus de bronquitis infecciosa (IBV) presentes en el campo en China. Las similitudes nucleotídicas y aminoacídicas entre las ocho cepas vacunales de IB y la cepa de campo tl/CH/LDT3/03, que se aisló de una cerceta (Anas sp.), no superaron el 81.1% y el 79.2%, respectivamente. El análisis filogenético basado en los genes S1 mostró que las cepas vacunales y de campo pertenecían a grupos distintos y mostraban distancias evolutivas mayores, e indicó que pertenecían a genotipos distintos. Cuatro de las ocho vacunas, además de la vacuna del tipo Massachussets H120, se utilizaron en pruebas de protección frente al desafío con el aislamiento de IBV tl/CH/LDT3/03. Ésta reveló que cada una de las cinco vacunas inducía una protección pobre frente al aislamiento de cerceta, evaluada mediante protección respiratoria, signos clínicos y mortalidad, lo cual indicó la necesidad de desarrollar vacunas a partir de cepas locales para el control de la IB en China.

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.

S1 gene sequence heterogeneity of a pathogenic infectious bronchitis virus strain and its embryo-passaged, attenuated derivatives

Infectious bronchitis virus CK/CH/LDL/97I was attenuated by serial passage in chicken embryos. Virus of passage 115 was attenuated as determined by clinical response to inoculation to 15-day-old specific pathogen free chickens. The vaccination-challenge test showed that the attenuated passage 115 virus could afford protection against the homologous pathogenic virus, passage 5, by the clinical response. Based on the sequence analysis and comparison of the S1 gene, both the pathogenic and attenuated CK/CH/LDL/97I viruses were populations that each included at least two subpopulations. Also, from analysis of the amino acid and nucleotide sequences of S1 gene, we speculate that recombination between the minor and dominant subpopulations plus accumulation of mutations in the S1 region of pathogenic passage 5 might lead to the formation of the embryo-passaged, attenuated passage 115.