Qigai HE1,2,3,4*, Zhonghua LI1,3, Xioazhen GUO1,3, Fangzhou CHEN1,3,4
1Key State Laboratory of Agricultural Microbiology, College of Veterinary Medicine; 2International Research Center for Animal Disease, Ministry of Science and Technology of PR China; 3The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan, Hubei province, PR China 4Key Laboratory of Preventive Veterinary Medicine in Hubei Province, PR China
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Abstract:
Since 2010, the variant porcine epidemic diarrhea virus (PEDV) has been the etiological agent responsible for the outbreak of porcine epidemic diarrhea (PED) worldwide. In this study, a variant PEDV strain YN1 was isolated, serially propagated on the Vero cells and was characterized for 200 passages. To better elucidate the molecular basis of Vero cell adaptation of variant PEDV strains, we sequenced, compared, and analyzed the full-genome sequences of parental YN1 and passages 15, 30, 60, 90, 144, and 200. The results showed that the variations increased with the viral passage. The nucleotides sequences of non-structural protein (NSP)2, NSP4-7, NSP10, NSP12 and NSP13 genes did not change during the Vero cell adaptation process. After comparison of the variation characteristic of classical, variant virulent/attenuated strains, it was found that attenuation of PEDV virus was associated with 9-26 amino acid (aa) changes in open reading frames (ORF) 1a/b and S protein, early termination in ORF3, 1–3 aa changes in E, M and N protein and some nucleotide sequences’ synonymous mutations. The aa deletion at about 144 aa of S protein could be the attenuation marker for the PEDV. The pig study showed that the early termination in ORF3 was more important for virus cell adaptation than virus attenuation. Based on the sequence differences between virulent, attenuated as well as CV777 strain, a multiplex RT-PCR is developed for etiological investigation.
To better understand the pathogenesis mechanism and the virus-host interaction during infection with both PEDV YN13 and YN144 strains, a comparative proteomic analysis was carried out to investigate the proteomic changes produced in the primary target organ, using isobaric tags for relative and absolute quantitation (iTRAQ) labeling, followed by liquid chromatography tandem-mass spectrometry (LC-MS/MS). A total of 269 and 301 differently expressed proteins (DEPs) were identified in the jejunum tissues of the piglets inoculated with YN13 and YN144, respectively. Bioinformatics analysis revealed that these proteins were involved in stress responses, signal transduction, and the immune system. All of these involved interferon-stimulated genes (ISGs) which were up-regulated in jejunums by both of the PEDV-infected groups. Based on the comparative analysis, we proposed that different changes induced by YN13 and YN144 in heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), eukaryotic initiation factor 4G1 (eIF4G1), and some members in the heat shock protein (HSP) family, may be responsible for differences in their pathogenicity.
Keywords: PEDV, attenuated vaccine, RT-PCR, proteomics, pathogenicity
Acknowledgement: The research was funded by State Key Research and Development Project (2016YFD0500702), National Science Foundation of China (31272572), China Agriculture Research System (CARS-35) and International cooperation project of Wuhan Science and Technology Department.