Abstract: Ocular and nasal swabs were collected from cats with upper respiratory tract infections in a cattery in Harbin, and the initial diagnosis was a mixed infection of feline herpesvirus-1 (FHV-1) and feline calicivirus (FCV) by PCR. 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 that FHV-1 and FCV were both positive, and only FCV was isolated from the third generation. FHV-1 was isolated by serum neutralization using the prepared mouse-derived polyclonal antibody of the isolated FCV, which named HRB2019 strain. HRB2019 was identified by morphological observation of viral particles, indirect immunofluorescence assay (IFA) 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 1×10^8.43TCID₅₀·mL^-1. Electron microscopy examination showed spherical virus particles with vesicle membrane, and its diameter was about 200 nm. IFA results showed that HRB2019 could bind to FHV-1 positive serum and appeared specific fluorescence. The gD 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 the virus showed that the virus began to replicate and proliferate from 12 h after inoculation, and the rapid proliferation period was from 12 to 36 h. The viral titers peaked at 48 to 72 h and began to decline at 84 h. In this study, FHV-1 was successfully isolated for the first time from samples infected with a mixture of FCV and FHV-1, which provided important data for epidemiological investigation and pathogenetic study of FHV-1 and provided methodological basis for isolation of growth-vulnerable strains in mixed infection.

Key words: feline herpesvirus-1, feline calicivirus, isolation, identification