Isolation,Identification Of Feline Panleukopenia Virus And Genetic Evolution Analysis Of VP2 Gene

Feline panleukopenia,also known as feline infectious enteritis and feline plague fever,is an acute highly contagious disease caused by Feline panleukopenia virus?FPV?.FPV infection has a wide host range and causes high mortality,especially for young animals such as cats,minks and raccoons.At present,vaccination is the main measure to prevent the disease.In order to effectively control the occurrence and prevalence of the disease in China's cat population,understanding the genetic variation and prevalence of FPV is of great significance to the research and development of new vaccines and the prevention and treatment of the disease.In order to study the infection characteristics of FPV,34 clinical samples suspected to be infected with FPV were collected from an animal hospital in China from 2018 to 2019.All samples were isolated by F81 cells,and 22 samples produced CPE.One blood stool sample was selected for identification.,After identification by molecular biology,immunofluorescence,electron microscopy and animal regression experiments,a FPV strain named DL-C-3 strain was isolated and identified.Its fifth generation virus titer was 10⁶.08.08 TCID₅₀/0.1 mL.In order to rapidly detect clinical samples,a TaqMan probe real-time fluorescence quantitative PCR?TaqMan q PCR?diagnostic method for detecting FPV was established in this study.The results show that the correlation coefficient?R²?of the standard curve is 0.9997,and the linear correlation is significant.The amplification efficiency is 104.2%,ranging from 90%to 110%.FPV can be specifically detected;The lowest detectable plasmid concentration is 115 copies/?L,which is 10 times that of PCR detection method.The coefficient of variation of Ct value is less than 1%.Because FPV is often mixed with feline hepersvirus 1?FHV-1?clinically,FHV-1 PCR diagnostic method has been established.FHV-1 can be specifically detected;The lowest detectable plasmid concentration is 37.8 copies/?L.The detection results of clinical samples showed that 22 of 34 clinical samples were positive for FPV,and the detection rate of FPV was 64.7%?22/34?.FPV and FHV-1 mixed infection occurred in 1 case?1/34?.In order to analyze the genetic evolution of FPV VP2 gene,this study extracted viral DNA from 22FPV positive samples,cloned and sequenced VP2 gene,and sequenced the VP2 gene sequences of 22positive samples?1755 bp in length?and 226 strains?215 FPV and 11 CPV?VP2 gene reference sequences recorded in Genbank database.Homology analysis results showed that VP2 gene of most samples in this study had high similarity with Asian FPV strains and low similarity with FPV standard strain CU-4 strain.The amino acid types of key sites are consistent with FPV standard strain CU-4.Synonymous mutations were dominant in VP2 gene of 22 positive samples and 9 non-synonymous mutations were found,4 of which were new mutations.The results of selection pressure analysis showed that 30 amino acid sites were found under negative selection pressure.Phylogenetic tree analysis showed that FPV formed 4 gene groups,and VP2 gene of 12 positive samples with Asian isolates aggregated into the third gene group,showing obvious geographi cal origin characteristics in Asia.VP2 gene of 8 positive samples and isolates from all over the world were clustered into the 4th gene group.Only two positive samples have VP2 gene located in the second gene group,which is closely related to the early foreign isolates.The VP2 gene of most of the positive samples in this study has been mutated,which is far from the early isolated strains abroad.Therefore,it is necessary to evaluate the actual efficacy of the current FPV vaccines through cross neutralization experiments using the recently popular FPV strains.Our research results not only provide reference basis for molecular epidemiological investigation of FPV,but also have important guiding significance for research and development of new vaccines in China.