Phylogenetics And Pathogenicity Analysis Of Feline Parvovirus In Partial Area Of China In Autumn And Winter Of 2021

Feline parvovirus（FPV）belongs to the genus Parvovirus of the family Parvoviridae,which is a non-enveloped,single-stranded DNA virus.It can naturally infect cats,tigers,lions,leopards,lynx,and other felines,as well as raccoons and other bears,and is a highly contagious acute disease.Cats can develop feline panleukopenia caused by FPV,also known as feline distemper or feline parvoviral disease.Clinical manifestations of the disease are mainly characterized by high fever,persistent vomiting,diarrhea,dehydration,and rapid decrease in white blood cells.Generally,cats under 1 year old are susceptible,with an infection rate of up to 70%,and a death rate of 50%to 60%.The mortality rate of kittens under 5 months old can be as high as80%to 90%,posing a great threat to pet cats and related industries.Vaccination is the main measure to prevent this disease.Understanding the current prevalence and genetic variation of FPV,clarifying its pathogenicity,is of great significance for the development of vaccines and the prevention and control of the disease.In this study,more than 250 clinical samples suspected of FPV infection were collected from pet hospitals in Weifang,Rizhao,Linyi in Shandong,Changsha in Hunan,Shanghai,and some unknown areas from October to December 2021.The virus was cultured using Crandell Reese Feline Kidney（CRFK）cells and cytopathic effects（CPE）were observed.The VP2 gene nucleotide sequence was amplified by Polymerase Chain Reaction（PCR）,and the amplified products were sequenced and identified.Western blot and immunofluorescence techniques were used to identify the VP2 protein.Virus morphology was observed by transmission electron microscopy.A total of 167 FPV strains were successfully isolated.A total of 36 strains of FPV were selected,including 4-6 strains from known regions and 10 strains from unknown regions.Biological characteristics and genetic evolution analysis were conducted.The results showed that the titers of the 36 strains ranged from 10^2.7 to 10^7.2 TCID₅₀/m L,while the hemagglutination titers ranged from1:2² to 1:2¹⁴.The VP2 gene sequences and NS1 gene sequences were compared with domestic and foreign reference strains of FPV in the NCBI Gen Bank database for genetic evolution analysis using MEGA 7.0.The genetic evolution analysis of the VP2gene sequence showed that the 36 isolated strains were clustered into two groups,G1and G2.24 strains（66.7%）were clustered in the G1,while 12 strains（33.3%）were clustered in the G2,including the vaccine strains Purevax and Felocell.The genetic evolution analysis of the NS1 gene sequence showed that 18 strains（50%）were clustered in the G1,while the other 18 strains（50%）were clustered in the G2.Amino acid site comparisons showed that compared with the standard strain（CU-4）,the VP2mutations of the isolated strains were mainly concentrated at 6 amino acid sites（A91S,I101T,I145L,V232I,A300P,V562L）,while the NS1 mutations were mainly concentrated at 12 amino acid sites（N23D,E72K,E73K,S136R,R148H,K201H,H247Q,T248V,H574V,H595Q,V596L and D616N）.Some of these site mutations may lead to changes in protein conformation,alter the ability of the virus to bind host cells,and thereby affect the pathogenicity of the virus.In order to compare the differences in infectivity and pathogenicity between the two groups of viruses,we selected FPV027(10^6.9 TCID₅₀/m L)as the representative strain for G1 and FPV072(10^7.2 TCID₅₀/m L)as the representative strain for G2 to establish a pet cat infection model and determine their 50%infectious doses(ID₅₀).The results showed that the ID₅₀ of FPV072 was 10^3.48 TCID₅₀,while the ID₅₀ of FPV027 was 10^4.2 TCID₅₀,indicating that FPV072 had higher infectivity.In addition,at the same infection dose(10^4.7 TCID₅₀),the weight and white blood cell count of cats in the FPV072 infection group decreased more significantly,the level of diarrhea was higher,and the amount of viral shedding was greater than in the FPV027 infection group.Consistent with this,histopathological results showed that FPV072 infection caused more severe villus necrosis,mucosal erosion,and ulcers.These results suggest that FPV072 is more pathogenic than FPV027.This study both enrich the information on the epidemic strains of FPV in China through isolation and identification,genetic evolution analysis,and pathogenicity research,and provide valuable references for vaccine development and FPV diagnostic technology research.