Screening and identification of Hantaan virus Vero cell adaptation strains
Objective To study the adaptability of Hantaan virus(HTNV)PS-6 strain in Vero cells after passing through the brain of suckling mice,and to screen out a high titer HTNV strain adaptable to Vero cells.Methods HTNV PS-6 strain was cultured continuously in the brain of suckling mice,and then continued to pass on Vero cells for 10 times.The cyto-pathic effect(CPE)was observed for each generation of the virus,and the virus titer was determined by plaque method.After the virus titer increased on Vero cells,the monoclonal virus was screened by plaque cloning and purification and the titer of monoclonal virus was measured.After infecting Vero cells at MOIs of 0.01,0.05,and 0.1,the growth curve of the virus was drawn.The LD50 of the virus was calculated by Reed-Muench method,the specificity of the virus was initially iden-tified by Western blot,and the size and structure of the virus before and after screening were observed by transmission elec-tron microscopy.The nucleotide sequences of the whole genome and amino acid sequences were compared with those of HTNV PS-6 strain.The strain adaptive to Vero cells,V-PS-6,was injected intraperitoneally into 10 female BALB/c mice,which were dissected when dying.The tissues of heart,liver,spleen,kidney,brain,lung,small intestine and muscle were taken and analyzed by immunohistochemistry.Results The passage strain in mouse brain had good adaptability to Vero cells,with an initial virus titer of 1.3 lgPFU/mL,and the virus titer gradually increased with the increase of passage times and stabilized at 7.5-7.9 lgPFU/mL.The plaque boundary of the adapted strain V-PS-6 was clear,round and visible to naked eyes,the size was like a needle tip,the LD50 was 10-7.8,and the specific protein band was found at the relative molec-ular mass of about 50 000,consistent with that of the pre-adapted virus strain HTNV PS-6.The diameter of V-PS-6 strain was 80-210 nm,with no significant difference in structure between V-PS-6 strain and HTNV PS-6 strain under electron micro-scope,and the homology of nucleotide sequences and amino acid sequences was 99.39％and 98.85％,respectively.The V-PS-6 strain was obviously distributed in kidney,liver,small intestine,muscle of hind leg and heart of mice.Conclusion A HTNV strain well adaptive to Vero cells with high titer was successfully screened and had good genetic stability,laying a foundation for the research and development of HTNV Vero cell vaccine and the study of related mechanisms.
NETL
NSTL
万方数据
