Isolation And Identification Of Bovine Rotavirus And Its Pathogenicity To Suckling Mice In Heilongjiang Area

Heilongjiang is one of the top ten pastoral areas in the country,and it is the"main force"to ensure the safety of the country’s milk supply.However,due to factors such as geographical environment,climate,and pathogens,bovine diarrhea frequently occurs in Heilongjiang,making bovine diarrhea a common disease that endangers the health of cattle.Among them,bovine rotavirus（BRV）is the main cause of calf diarrhea.pathogen.BRV infection can cause severe diarrhea and dehydration in calves,especially calves less than 7 days old are more susceptible,with a disease rate of 80%and a case fatality rate of up to 50%,seriously affecting the healthy development of the cattle industry.Therefore,research on the pathogenicity and prevention of BRV in Heilongjiang is particularly important.However,there are few studies on virus isolation and pathogenicity of BRV in Heilongjiang,resulting in a lack of information on etiology and pathogenicity of BRV in Heilongjiang.In view of this,research on the isolation and identification of BRV in Heilongjiang area,mastering the genotypes of epidemic strains and evaluating their pathogenicity can provide a theoretical basis for the pathogenic prevention and control of BRV in Heilongjiang area.In order to isolate and identify the popular strains of BRV in Heilongjiang area,this study detected the cattle feces samples collected from Heilongjiang area by RT-PCR method and screened the BRV strong positive samples.MA-104 cells were used to isolate the virus from the BRV strong positive fecal samples.The isolated virus strains were identified by methods such as RT-PCR detection,indirect immunofluorescence identification and electron microscope observation;the whole genome sequence of the isolated virus strain was further obtained by using Illumina next-generation sequencing technology,and the sequence analysis of the whole genome of the isolated virus strain was carried out to construct the genetic evolution Tree.The results showed that CPE appeared in MA-104 cells blindly passed to the 5th passage,and CPE appeared stably in the 8th passage.Typical wheel-shaped virus particles were observed in the virus,which conformed to the morphological characteristics of BRV,indicating that a strain of BRV was successfully isolated and named BRV HLJ-H3;the results of whole genome sequencing analysis of the isolated strain showed that its genotype was G6-P[5]-I2-R2-C2-M2-A3-N2-T6-E2-H3 type;through homology analysis,it was found that BRV HLJ-H3 may be composed of bovine,human,sheep,and equine rotaviruses Multiple genetically reassortant strains.In order to further evaluate the pathogenicity of BRV HLJ-H3,7-day-old Kunming suckling mice were challenged with the virus by oral gavage.Through clinical symptom observation,diarrhea score,histopathological analysis and the BRV fluorescence quantitative method established in this study PCR method was used to measure the tissue viral load of suckling mice to evaluate the pathogenicity of the isolated strain to suckling mice.The results showed that all the suckling mice in the BRV HLJ-H3 challenge group had diarrhea in varying degrees,and the suckling mice in the challenge group discharged brown-yellow soft feces and yellow watery stools;the results of diarrhea score showed that the suckling mice in the BRV HLJ-H3 challenge group had The feces score was lower than that of the uninfected control group of suckling mice,and the diarrhea of the suckling mice was the most serious on the third day after the challenge;the results of tissue viral load measurement showed that BRV nucleic acid was detected in the small intestine tissues of the challenge group suckling mice,and the The nucleic acid content was 10^5.14～10^6.09copies/g;the results of histopathological analysis showed that in the BRV HLJ-H3 challenge group,the villous epithelial cells in the small intestinal mucosa of suckling mice were loosely arranged,some cells were necrotic,part of the small intestinal villi fell off,and the number of goblet cells increased.The columnar epithelial cells of the intestinal mucosa were partially exfoliated.The above results indicated that the isolated strain was pathogenic to suckling mice.In summary,this study successfully isolated and identified a G6P[5]-type BRV strain,and clarified the pathogenicity of the isolated strain to suckling mice;enriched the information on the prevalence of BRV genotypes and pathogenicity in Heilongjiang,provide a reference for BRV prevention and control,and provide a research basis for the screening of BRV vaccine candidate strains.