Isolation Of Canine Parvovirus And Feline Panleukopenia Virus In Guangzhou And Establishment Of TaqMan Probe Fluorescence Quantitative Method For Canine Parvovirus And Feline Panleukopenia Virus

Feline panleukopenia virus(FPV)is a viral disease known since the beginning of the 20 th century that occurs in cats,causing severe leukopenia,gastroenteritis and nervous signs.Canine parvovirus type 2(CPV-2)causes acute haemorrhagic enteritis in dogs since the late 1970 s.In 1980 s,two subtypes(CPV-2a and CPV-2b)emerged,eventually replaced the original CPV-2,and a new variant,CPV-2c was reported in Italy in 2000.At present,FPV,CPV and their various subtypes are widely distributed worldwide,with high pathogenicity and high mortality,which poses a serious threat to the population of dogs and cats.Mutations of the capsid protein VP2 of canine and cat parvovirus has been proved to affect the evolution of different antigen variants.Sequence analysis and subsequent characterization of viral VP2 gene play a very important role in the study of viral phylogeny.In the past 2016~2018,feces and swabs from dogs and cats with symptoms of disease were collected from various animal hospitals in Guangzhou,and all samples were subjected for virus isolation in F81 cell line.8 viurs could be isolated include 6 CPV and 2 FPV.The sequence of VP2 gene was amplified and sequenced,and the corresponding phylogenetic tree was analyzed to estimate phylogenetic relationship of field virus with the reference strains.The results showed that 6 strains of canine parvovirus were all on the branch of Southeast Asia.Two of them were New-CPV-2a(297 and 440 amino acid residues were alanine),and four strains were CPV-2c(426 amino acid residues were glutamate).Two isolates of New-CPV-2a showed that they were from different ancestors,while CPV-2c isolates together constituted a branch,and phylogenetic analysis showed that all CPV-2c isolates were close to the CPV-SD/14/12 isolates in Shandong,China.In the past 2016~2018,the prevalence of CPV strains in Guangzhou is mainly New-CPV-2a and CPV-2c.The analysis of FPV phylogenetic tree showed that two isolates were both in the same branch of China,indicating that the isolated strains were endemic in geographical location in China,while the isolated strain,FPV-LM,which was isolated in 2017 has 3 mutations that did not occur on VP2 capsid protein residues at 16,67 and 75 sites.Whether these mutation sites are stable in other strains need extensive sequencing and analysis.On the basis of virus isolates,a fluorescence quantitative method by TaqMan fluorescent probe for CPV-2 and FPV is established.It is proved that the method has high sensitivity and can detect 82.3 copies/?L virus DNA,which is more sensitive than that of conventional PCR.It shows the excellent applicability of the diagnosis of CPV and FPV by real-time fluorescence quantitative PCR method.It provides reliable means for clinical diagnosis and treatment of canine and cat parvovirus diseases.