| Feline panleukopenia(FPL)is an acute,highly contagious disease infected by feline parvovirus(FPV).The disease is characterized by programmed lymphocyte death and a significant decrease in leukocyte levels,causing significant damage to the companion pet industry,rare animal conservation,and economic animal breeding.Feline calicivirus(FCV)is a common infectious disease caused by Feline calicivirus(FCV).It is highly infectious and transmissible in felines.It can cause Feline upper respiratory tract disease(FURTD)by mixed infection with other pathogens,and it is difficult to differentiate FCV in clinical diagnosis and treatment.FCV mutation evolution speed,high rate of vaccine immune failure,lack of specific therapeutic agents.Therefore,it is of great significance to study the molecular epidemiology,clinical diagnosis and prevention measures of FPV and FCV.This study was conducted to isolate,identify and analyze the genetic evolution of FPV and FCV pathogens in Wuhan,aiming to lay a foundation for subsequent epidemiological investigation,development of therapeutic agents,specific diagnostic technology and vaccine research and development.1)Isolation and identification of FPV and FCVIn this study,fecal swab samples were collected from cats suspected of having FPL based on clinical cases from veterinary hospitals in Wuhan.The pathogen was isolated from 12 of the positive FPV samples by inoculating the clinical samples on feline kidney cells(Crandell Reese Feline Kidney(CRFK))using the pathogen isolation method and amplifying the FPV VP2 gene using the polymerase chain reaction(PCR)technique.The full length of the nucleotide sequence of FPV VP2 gene was amplified by PCR,and the isolated products were sequenced and identified,and two FPV strains were successfully isolated,named FPV-WH-2020.1.6 and FPV-WH-2020.1.9.Eye,nose and mouth swab samples were also collected from cats suspected to be infected with FCV at the veterinary hospitals in Wuhan.Three of the FCV-positive samples were inoculated and passaged on CRFK using the pathogen isolation method,and isolated using reverse transcriptase polymerase chain reaction(RT-PCR).The full-length nucleotide sequence of FCV VP1 gene was amplified by RT-PCR,and the isolated product was sequenced and identified,and an FCV strain was successfully isolated,named FCV-WH-2020.12.10.2)Genetic evolution analysis of FPV VP2 and FCV VP1In this study,the nucleotide sequences of VP2 gene of FPV-WH-2020.1.6 and FPV-WH-2020.1.9 were compared with the domestic and foreign reference strains of FPV included in the NCBI Gen Bank database by MEGA 7.0 software,and the genetic evolution analysis of FPV VP2 gene was performed.The range of genome sequence identity between the isolated strains in this study was high,reaching 99.7%,and the strains were closely related.FPV-WH-2020.1.6 had the highest homology with Thai FPV strain(Gen Bank: JX048608),reaching 99.8%.The range of genome sequence identity among FPV-WH-2020.1.6 and isolates from China was 99.1% ～ 99.5%.FPV-WH-2020.1.9 had the highest homology with the Australian FPV strain(Gen Bank:MN6039760),reaching 99.7%.The range of genome sequence identity among FPV-WH-2020.1.9 and isolates from China was 98.9% ～ 99.6%.The range of genome sequence identity among the two isolates and the vaccine strain(Gen Bank: EU498680,EU498681,M38246)was 98.8% ～ 99.1%,with low homology,two isolates were most closely related to the domestic FPV strain and the Thai FPV strain(Gen Bank: JX048608),and both were distantly related to the vaccine strain,suggesting that the vaccine was less effective in protection.The nucleotide sequences of FCV-WH-2020.12.10 VP1 gene were also compared with the domestic and foreign reference strains of FCV included in the NCBI Gen Bank database using MEGA 7.0 software to analyze the genetic evolution pattern of FCV VP1 gene.The range of genome sequence identity among FCV-WH-2020.12.10 and most isolates from China was 74.4% ～ 88.6%.The range of genome sequence identity between FCV-WH-2020.12.10 and vaccine strains F9(Gen Bank: M86379),2024(Gen Bank:AF479590)and domestic vaccine strain(Gen Bank: D90357)was 76.8%,78.0% and78.4%.The homology with FCV-VSD strain was not high.The range of genome sequence identity among FCV-WH-2020.12.10 and isolates from other countries was about 76% ～ 80%.From the genetic evolutionary tree of FCV VP2 gene,FCV-WH-2020.12.10 belongs to group GIII,it is closely related to the Chinese FCV isolates,while it is more distantly related to vaccine strains such as F9(Gen Bank:M86379)and 2024(Gen Bank: AF479590),indicating that FCV mutates and evolves rapidly.The differences in genome sequence identity and genetic branching between the strains indicate that the vaccine immunity of FCV is poor.The results of genetic evolutionary analysis of FPV VP2 gene and FCV VP1 gene in this study enriched the information about the prevalent strains of FPV and FCV in China,and also showed that the vaccines commonly used in clinical practice cannot provide effective protection against FPV and FCV,so it is urgent to carry out the development of FPV and FCV therapeutic agents,diagnostic technology research and vaccine development. |
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