Isolation And Identification Of Feline Herpes Virus HR-1 Strain And Establishment Of Indirect ELISA

Feline herpesvirus-1?FHV-1?is the causative agent of cat infectious rhinotracheitis,which can cause acute and highly contactive upper respiratory diseases in cats.FHV-1 belongs to the herpesvirus family,a member of the alphaherpesvirinae subfamily.It is a double-stranded DNA virus.FHV-1recessive infection and post-infection recovery can be poisoned and detoxified for a long time,becoming a dangerous source of infection.Like other herpes viruses,FHV-1 can be latent in the trigeminal ganglion of cats,and can be reactivated when the cat has low immunity,leading to the onset of the disease,making the diagnosis and prevention of the disease difficult.Therefore,strengthening the isolation and identification of domestic FHV-1 strains and the establishment of diagnostic methods are great significance for the prevention and control of feline infectious rhinotracheitis.In this study,a FHV-1 strain was isolated and purified from the eye and nose swab of a sick cat and identified via a series of experiments.The result showed that the CRFK cells infected by HR-1 can produce typical cytopathic effect.The virus titer can reach 10⁸ TCID₅₀/mL in CRFK cells.Electron microscopy examination showed round virus particles with capsule membrane,and its diameter was about 150 nm.A specific 1125bp fragment for gD gene of FHV-1 was amplified by PCR from the viral genome DNA.The nucleotide sequence analysis of gD gene showed that the isolate shared the similarity 100%with C-27 gD.It showed that the isolated strain belongs to FHV-1,named HR-1.IFA results showed specific fluorescence in monolayer infected CRFK cells.The pathogenicity test in cats showed that HR-1 infection can cause conjunctivitis and rhinotracheitis,pathological sections showed pathological changes of lung epithelial hyperplasia,renal tubular epithelial cells vacuolization and necrosis.Viral nucleic acid were detected in nasal and ocular swabs for several days by RT-PCR and reached the maximum of viral nucleic acid at 4-6 days.It also proved that HR-1 replicates a lot in the trigeminal nerve.The lethal rate of HR-1 to domestic cats was 100%.Secondly,in order to establish the diagnostic method of FHV-1,the highly conserved and immunogenic gD glycoprotein was selected as the research object,and predicted the transmembrane region and antigenic epitope of gD protein by using TMHMM Server v.2.0 and BepiPred 1.0 server online software.The 505 bp-987 bp was identified as the gD epitope enrichment region,which is 483bp long.The solubility and antigenicity of the recombinant gD protein were proved by SDS-PAGE and Western blot,and the recombinant gD glycoprotein was choosed as the coated antigen to establish indirect ELISA diagnostic method.The results showed that the optimal concentration of gD recombinant protein was 2 ng/?L,and the optimal coating time was 2 h in 37°C;The optimum dilution of the serum was 1:400,and the optimum reaction time was 45 min in 37°C;The optimum dilution of the secondary antibody was 1:8000,optimum reaction time was 45 min in 37°C;The best reaction of the substrate was 5 min.Under the optimal conditions,the cut off value was 0.432;There was no cross-reaction with FCV,FPV,FIPV-1 and FIPV-2 positive serum,and the specificity was good.The coefficient of variation within and between groups was less than 10%.The repeatability was good.The positive serum of FHV-1 diluted to 1:3200 still showed positive and the coincidence rate with IFA assay was 92%,indicating good sensitivity.In addition,the established indirect ELISA diagnostic method was used to detect the serum samples from 100 cats,and the positive rate was 77%.In summary,in this study HR-1 strain was isolated and performed its pathogenicity analysis,which provided a reference for the evaluation of vaccine in the later stage.The indirect ELISA diagnostic method established by using gD recombinant protein as the coating is simple,which lays a foundation for prevention and control of feline herpesvirus type-1.