Epidemiologic Survey Of Bovine Rotavirus And Construction Of Rotavirus Reassortant And Immunogenicity Evaluation Of Bivalent Attenuated Virus Strains

Group A rotaviruses are one of the major causes of nonbacterial diarrhoea in young animals. Bovine rotaviruses compose a genus rotavirus within the family Reoviridae. Mature virus particles contain an 11-segmented double-stranded RNA genome, which encodes six structural and five nonstructural proteins. The calves with age of less than 1 month are susceptible to rotavirus, and rotaviruses cause disease that varies in severity, including asymptomatic infection, mild and self-limiting diarrhea, or severe diarrhea with excessive fluid loss and severe electrolyte imbalance.In order to determine the prevalence and the main serotypes of bovine rotavirus infections in China, we collected 115 stool samples of the calves suffered from diarrhea from Inner Mongolia, Heilongjiang, Xinjiang, Beijing and Anhui for detection of rotavirus using RNA PAGE electrophoresis and RT-PCR. Results showed that 12 stool samples were detected to be rotavirus positive, and the average positive ratio is 10.4% (12/115) with range of rotavirus positive ratio from 0 to 100% in different regions. A subset of the positive samples was examined for their G-type specificities by RT-PCR , indicating that 1 sample from Xinjiang and 4 samples from Inner Mongolia were determined as G10 serotype, and that 2 samples from another cattle farm from Xinjiang and 2 samples from Heilongjiang were determined as G6 serotype. A G10P[11] bovine rotavirus, designated as the HM26 strain, was isolated from rotavirus positive feces of the calves suffered from diarrhea in the MA104 cell line. This is the first report for G10 bovine rotavirus to be isolated in China. The HM26 was molecularly characterized to show that the VP4, VP7 gene segments and the VP6, NSP1, NSP4 gene segments derived from different bovine rotavirus stains, suggesting that the isolate HM26 was a reassortant within different bovine rotavirus strains. Meanwhile, 2 bovine reovirus stains and 2 bovine enterovirus strains were isolated, and what part was they play in diarrhea remains unknown.Rotavirus as a major cause of neonatal bovine diarrhea was found to be responsible for significant scours cases in dairy calves around worldwide, and rotavirus serotypes G6 and G10 predominate in cattle, and the approaches of rotavirus reassortant had been successfully employed to develop various rotavirus candidate vaccines. In this study, based on the approach which had been succeeded in human rotavirus vaccines, we constructed a bivalent vaccine candidates which could provide antigenic coverage for serotypes G6 and G10. We constructed and characterized a reassortant rotavirus stain, which bears a single bovine rotavirus VP7 gene encoding G serotype 6 specificity while the remaining 10 genes are derived from the ovine rotavirus LLR-85. The reassortant rotavirus strain, named as R191, and its parental virus strain LLR-85 were combined as bivalent vaccine candidates to orally inoculate neonatal calves. Seventeen colostrum-deprived newborn calves without antibodies to rotavirus were divided into four groups to orally inoculate candidate vaccines and placebo within 24 h after their birth. Calves were orally inoculated with the reassortant R191 (group 1), the parental rotavirus LLR-85 (group 2), or R191 and LLR-85 (group 3). A fourth group was orally inoculated with placebo. Serum specimens from the neonatal calves inoculated with the vaccine candidates and placebo were analyzed to determine the immune response of IgA, IgG and neutralizing antibodies. Results showed that seroconversion to positivity for IgG and IgA antibodies occurred at postinoculation day (PID) 10 in all of the calves in vaccinated groups. The highest titers of serum IgG (range 1:800 to 1:6400) and IgA (range 1:800 to 1:3200) antibodies were obtained at PID 21 for all calves in the vaccinated groups. Moderate levels (range 1:16 to 1:256) of neutralizing antibodies to rotavirus NCDV and LLR-85 (with G6 and G10 serotype specificity) were detected at PID 21 for all calves in the vaccinated groups. Meanwhile, virus shedding was detected after inoculation, showing that it was limited only to the strain LLR-85 of the bivalent vaccine candidates with a very low shedding rate (2.6%, 2/77). The results suggested that the rotavirus strain R191 and LLR-85 are promising bivalent vaccine candidates for prevention of bovine rotavirus infection.