Isolation And Identification Of Canine Parvovirus And Inflammatory Effect Of Nonstructural Protein NS1

Canine parvovirus disease is a high-contact acute infectious disease caused by canine parvovirus(CPV)with hemorrhagic enteritis and myocarditis as the main pathological features,accompanied by some clinical symptoms including severe vomiting,blood in the stool,loss of appetite,and depression etc.CPV disease is prevalent all over the world and has become one of the three major infectious diseases in dogs in China,so it has become a hot research topic in canine disease.The current research on the infectious mechanism of the disease is insufficient and there are little studies on the host cell immune responses after an infection of the host cell by CPV.Therefore,the research on the mechanism of CPV infection becomes imminent.In this study,the following experiments were conducted on the study of various cytokines involved in the inflammatory responses after CPV infection:1.The feces of dogs with positive CPV identified by colloidal gold test paper were collected and the whole genome of the virus were identified.The results showed that a CPV was successfully isolated and its whole genomic sequencing were completed.It was identified as New CPV-2b subtype by VP2 amino acid sequence analysis.TCID50 of this isolate virus was 10-4.85/0.1mL;the blood coagulation titer was 1 : 256;the nucleotide homology with 17 domestic reference strains was 96.4%~99.9% and the homology with the Beijing isolate was 99.9%.2.Fluorescence quantitative PCR was used to detect the transcriptional phase of inflammatory factors in C61-infected F81 cells at different time points.The results showed that the expression of IL-2 was significantly increased in the early stage of CPV-infected F81 cells and the level of IL-4 was unchanged,which proved the Th1 type immune response was induced after viral infection.The mRNA levels of IL-6 and TNF-? were significantly changed compared with the levels of the negative group,and the expression levels was significantly up-regulated in the early stage of infection,decreased in the middle stage and then up-regulated in the later stage.The cytokines IL-10 and TGF-? were significantly activated when IL-6 and TNF-? were up-regulated and the levels of them were decreased when IL-6 and TNF-? were down-regulated.This is consistent with the dynamic balance of inflammatory factors in inflammatory response.3.In order to determine the role of CPV non-structural protein NS1 in viral infection,the non-structural protein NS1 gene was cloned and ligated into pEGFP-N1 eukaryotic expression vector by double enzyme digestion to construct pEGFP-N1-NS1.The recombinant plasmid was transfected into 293 T cells by liposome-mediated method,and the transfection efficiency and expression of the recombinant plasmid were detected more quickly and efficiently by using EGFP fluorescent label.4.Recombinant plasmid was transfected into 293 T cells and the transcriptional phase of related inflammatory factors in cells was detected by real-time PCR.The results showed that the level of IL-2 in cells increased significantly and the level of IL-4 did not change significantly in the early stage of transfection,which indicated that NS1 caused a Th1 immune response after transfection;the mRNA levels of IL-6 and TNF-? were significantly changed compared with the blank control and the empty control.The expression level was significantly up-regulated in the early stage and decreased in the middle stage of the infection.In summary,this experiment preliminarily explored the interaction and effect between CPV and host cells in the process of virus infection,which provided theoretical basis for the study of CPV infection mechanism and clinical prevention,and had certain application value.