Isolation,Identification And Pathogenicity Analysis Of FHV-1 In A Case Of Mixed Feline Respiratory Virus Infection

In recent years,feline upper respiratory tract disease has become a major cause of morbidity and mortality in kittens.Infections are usually caused by two or more pathogens,and common pathogens include feline herpesvirus,feline calicivirus,Chlamydia,Borrelia,Mycoplasma,and eutherian virus.Feline herpesvirus-1（FHV-1）and feline calicivirus（FCV）are the main causes of upper respiratory tract infections in most cats,and FHV-1 is often mixed with FCV,making the isolation of FHV-1 extremely difficult.FHV-1 causes an acute,highly contagious infection of the upper respiratory tract in felines called feline infectious rhinotracheitis,characterized clinically by sneezing,keratoconjunctivitis,lethargy and depression.The incidence of the disease is extremely high,and both adult cats and kittens are susceptible,posing a serious threat to the health of felines.In this study,we collected ocular and nasal swabs from cats with upper respiratory tract infections in a cattery in Harbin,and the initial diagnosis was a mixed infection of FHV-1 and FCV.CRFK cells were used to isolate viruses from the swabs of infected cats.Nucleic acid assay of the first two generations of cell cultures showed coexistence of FHV-1 and FCV,and only FCV was isolated from the third generation,named strain HRB23.FHV-1 was isolated by serum neutralization using the prepared mouse-derived polyclonal antibody of FCV HRB23 strain,which named HRB2019 strain.HRB2019 was identified by morphological observation of virus particles,indirect immunofluorescence assay and gene sequence analysis.And the growth kinetics in vitro of HRB2019 was studied.The results showed that HRB2019 could produce typical cytopathic effect on CRFK cells and viral titer of the fourth generation of virus culture reached 1×10^8.43 TCID₅₀/m L.Electron microscopy examination showed spherical virus particles with vesicle membrane,and its diameter was about 200 nm.IFA results showed that HRB2019could bind to FHV-1 positive serum and appeared specific fluorescence.The g D gene of the isolate was amplified and the nucleic acid sequence analysis showed that the isolate was highly homologous with the domestic and foreign prevalent strains.The one-step growth curve of HRB2019 showed that the virus began to replicate and proliferate from 12 h after inoculation,and the rapid proliferation period was from12 h to 36 h.The viral titers peaked at 48 h to 72 h and began to decline at 84 h.To further clarify the pathogenicity of HRB2019 strain in cats,this study inoculated healthy Chinese field cats with HRB2019 strain with 500μL/cat(1×10^7.43 TCID₅₀/m L)via nasal and eye drops.The results showed that the cats of the infected group showed a significant decrease in body weight and increase in body temperature during the acute infection period,as well as characteristic clinical symptoms such as sneezing,conjunctivitis and respiratory distress,without any mortality.In order to understand the replication dynamics of HRB2019 strain in cats,the FHV-1 SYBR Green I fluorescence quantitative PCR method were established to detect the changes of viral load of HRB2019 strain in various tissue in this study.The results showed that FHV-1 was detected in eye and nasal swabs from day 1 after the challenge,with the highest viral excretion on days 6～7 and continued until day 16.Viraemia was detected from day 2 after the challenge,peaked on day 4,and continued until day 16,and then persisted at low levels.Combined with the detection of virus in eye and nose swabs and blood,it was found that the viral load in cats reached a peak on days 4～7 after virus infection,began to decline on day 8,and continued to excrete virus until the end of the experiment.Tissue viral load measurements showed that FHV-1 was detected in the larynx,trachea,lungs,and genital organs of the infected group of cats 16 days after infection,demonstrating that FHV-1 has similar infection characteristics to human herpesvirus and can be latent in the genital organs.The study showed that feline herpesvirus strain HRB2019 is a virulent strain that is highly pathogenic to cats.In summary,this study successfully isolated a strain of feline herpesvirus from samples infected with a mixture of FCV and FHV-1,named strain HRB2019,and confirmed that the strain was pathogenic to Chinese field cats,laying the foundation for diagnosis,prevention and control of FHV-1 and vaccine research.