Isoaltion And Identification Of Canine Parvovirus SH15 And Studies On Apoptosis Induced By Canine Parvovirus In MDCK Cells

Canine parvovirus?CPV?belongs to the genus Parvovirus within the family Parvoviridae.CPV is an important pathogen in domestic dogs.Dogs infected with CPV caused severe diarrhea,accompanied by loss of appetite,vomiting,fever,leukopenia and so on.It is seriously damage to the canine health and influence the economic development.Because the virus is easy to mutate and appear in the clinical immune failure,so the virus of different isolates of the control of canine parvovirus disease is of great significance.In order to better understand epidemiology of canine parvorious in Shanghai,a canine parvovirus isolated from CPV infected dead dogs was named CPV-SH15.It could produced a HA titer of 2⁹,the virus titer of TCID₅₀ was 10^5.2/mL.The coding region complete gene sequence was 4869 bp in length.And analyzed VP2 gene,the strain belongs to new CPV-2a type.To explore the proliferation dynamics of CPV in MDCK cells,a SYBR-Green fluorescent quantitative real-time PCR technology was established.The results showed that it was specific and sensitive for the detection of CPV.This method could detect 1.29×10²copies/?L viral load at least and the coefficient of variation?CV?was less than 1%,but the other viruses could not be detected.Virus began to proliferate when the cells infected with CPV,and viral load achieved the highest value during 60 to72 hours and then decreased gradually.To investigate the mechanism of apoptosis induced by CPV infection in MDCK cells.MDCK cells infected with CPV were detected by Annexin V-FITC/PI Apoptosis Detection Kit.It was found that CPV could induce MDCK cells apoptosis.The mRNA expression levels of apoptosis factors such as caspase-8,caspase-9 and caspase-12 were detected by real-time fluorescent quantitative PCR at the established time points.The results showed that the activation of caspase-8 and caspase-12,indicated that there are death receptor pathway and endoplasmic reticulum pathway in cell apoptosis.The activation of caspase-9 and the increase of Bax/Bcl2ratio indicated that mitochondrial pathway pathway is also involved.The activation of p53 may regulated the up-regulation and down-regulation of Bax and Bcl-2,respectively,and then mediated cell apoptosis.In conclusion,a canine parvovirus CPV-SH15 was isolated and identified in this study.The whole genome was obtained and genotyped.Canine parvovirus were detected by real-time fluorescent quantitative PCR.And then explored the mechanisms of CPV-induced apoptosis in MDCK cells.All of these provided the basic theoretical support for the further study of CPV-SH15.