Establishment Of Rabbit Splenic Fibroblast Type Cell Line RS-17 And Its Infection Tests Of The RHDV SCH04 Strain

Rabbit hemorrhagic disease is a acute,highly contagious and highly pathogenicity disease that caused by rabbit hemorrhagic disease virus;with the mainly pathological characteristics of the respiratory systems hemorrhagic,parenchymal organs congested and edematous,livers necrotic,congested and hemorrhagic,this disease threatens the development of the rabbit industry seriously.RHDV is a single-positive-stranded RNA virus that belongs to the family Caliciviridae,with the genome is about 7.5kb.At present,there are no stable cells for RHDV cultured in vitro,which affects the researches of the viral cell infection mechanism and prevention and control.In order to explore the mechanism of RHDV infection,the primary cells of the rabbit were cultured by enzyme digestion method and then the cell line was established,and RHDV infection test was carried out based on the cell lines and the specific steps as follows:1.In the process of cultivating rabbit primary cells,a fibroblast type cell was isolated from spleen and named RS-17.The subculture conditions was optimized and cellular marker proteins were detected by immunofluorescence,and the growth curve,karyotype and tumorigenicity of the RS-17 were detected for researching the growth characteristics and biological characteristics of cells.The results showed that the RS-17 were fibroblast cells and had the normal karyotype,normal growth characteristic,no tumorigenicity.The cell has been passaged stably to the 95 generations.2.The RHDV SCH04 strain gene sequences were divided into seven sections for RT-PCR amplification according to the viral nucleic acid,following the homologies and genetic evolution were analyzed by comparing with 32 strains viral complete genomes,VP60 gene sequences,and ORF2 encoding gene sequences in GenBank.The fragment length of the SCH04 strain complete genome is 7439 bp,with other 32 reference strains complete genomes homologies are 78.3%~99.8%,and over 79.1% of VP60 gene sequences comparison and 84.2%~99.2% of ORF2 encoding gene sequences.Phylogenetic trees showed the strain belong to the antigenic variation RHDV a(GI.1a)group.The complete genome determination and genetic evolution analysis of the SCH04 strain enrich the epidemiological data of RHDV,and provided reliable background information ofRHDV for the infection test.3.The RHDV SCH04 infection tests were included the rabbit primary cells infection,RS-17 infection and SCH04 RNA transfection cells RS-17.After infection,the CPE of the cells was observed and the cells were collected for the quantitative analysis by the TaqMan-based fluorescent quantitative PCR method.Results showed that the primary cells had the slight CPE appearance after infection the virus,and the viral nucleic acid content detection positive in the cells by the fluorescence quantitative method;RS-17 cells appeared CPE at the first time of infection,but with the continuous blindness passaged,the CPE of cells were weakened.The quantitative detection of viral nucleic acid content decreased with the increased of the numbers of passaged,and the viral nucleic acid content could not be detected within fourth times after subculturing.After transfection the SCH04 strain RNA into the cells RS-17,the cells began to shrink,and over time,the more obvious the CPE appearance.The viral nucleic acid content of cells was positive by fluorescence quantitative method.