Establishment Of Detection Method,isolation And Identification And Infectious Clone Construction Of Feline Calicivirus

Feline calicivirus(FCV)belongs to the calicivirus family and the genus Vesicularis virus.It mainly infects felines,can spread across species,and has a worldwide distribution.FCV infection in cats can cause respiratory diseases such as mouth ulcers,chronic stomatitis,and pneumonia.Most caliciviruses are difficult to culture in vitro,but FCV can proliferate in cat kidney cells(F81)at high titers,and the construction of animal models is relatively mature,and it is often used as a model strain for calicivirus research.At present,the existing vaccines against FCV cannot provide complete clinical protection for cats,and the establishment of accurate and rapid FCV detection methods is essential for early diagnosis of the disease.In this study,a Taqman fluorescent quantitative PCR detection method was established,showing high sensitivity and good reproducibility.Using this method,21 positive samples were identified from clinical samples such as nasopharyngeal and fecal swabs of suspected cats in many regions across the country.5 strains of FCV were isolated by cell culture,of which 4samples were infected by FCV alone,named as FCV(BJ19),FCV(SH19),FCV(BJ20),FCV(DL20).One sample was identified as a mixed infection of FCV and Feline herpesvirus-1(FHV-1)via plaque purification,named as FCV(DL19).The results of TCID₅₀ determination shows that the TCID₅₀ of FCV(DL19)strain is significantly higher than that of the other four isolates,and the TCID₅₀ was stable for 20 consecutive passages.In order to further identify the FCV(DL19),indirect immunofluorescence,western blot identification,electron microscopy and other identification tests were carried out,and the animal regression test showed that FCV(DL19)can cause typical clinical symptoms and histopathological changes of feline calicivirus disease.Genetic evolution analysis shows that the homology of 5 strains and 40 domestic and foreign FCV strains was 76.62%?84.88%,amongst them,FCV(DL19)showed highest nucleotide and amino acid homology with two Chinese cheetah-derived FCV,and these 3 trains locate in the same phylogenetic tree branch.FCV(DL20)is closely related to a tiger-derived FCV strain in China.Although FCV(BJ19)is located in a large branch with some domestic isolates,it forms a small branch independently.The comparative analyses with the amino acid sequence of the E region variable region of showed that the FCV(DL19)had five FCV VSD strain specific amino acid mutations.The FCV(DL19)is a FCV VSD strain.In order to further explore the genome structure,functions of FCV(DL19)and its pathogenic mechanism,FCV infectious clone construction is constructed.First,the p Bluescript II SK(+)vector was modified to introduce T7 RNA polymerase promoter,FCV(DL19)5'UTR,3'UTR,poly(A),and the 5'end and 3'end of the genome,the hepatitis delta virus ribozyme,respectively.The rescue system is proved to be effective by verification with the microgenome system.Thereafter,the FCV(DL19)gene component is divided into 2 segments and connected to the modified p Bluescript SK II(+)using a seamless cloning method on the vector,and a synonymous mutation was added at 4505 as a genetic marker to rescue the virus.In this study,the FCV Taqman fluorescent quantitative PCR detection method was successfully established,providing a method for the diagnosis and control of FCV disease.Five strains of FCV were isolated and identified,and the genetic evolution analysis were further performed,providing data for the epidemiological study of FCV.Meanwhile,the microgenome modification and system verification of the vector were completed.Moreover,FCV(DL19)infectious cloning plasmid was constructed.All of these studies lay the foundation for later virus rescue,virus protein function,gene expression regulation,pathogenic mechanism research and vaccine research and development.