Isolation And Identification Of Bovine Rotavirus HQ09Strain

Bovine rotavirus (BRV) belongs to the reovirus Branch (Reoviridae), Rotavirus, is the causative agent of calf viral diarrhea. In2009in Qiqihar City, Heilongjiang Province, we obtained a calf diarrheal stool, and isolated an BRV designated HQ09via MA104cell continuous passage, at the15th passages, the measured was104.384TCID50/100μl; virus particle morphology observed by the electron microscope showed that the diameter size of the virus about70nm, displaying a typical wheel-like virus structure. The nucleic acid type of experiment and the lipid solvent sensitivity experiment confirmed that the viruses are enveloped RNA viruses; at50℃water bath for30min, the infectivity of of the virus significantly decreased; the virus reacted with bovine rotavirus positive serum, neutralizing antibody titer was1:145. The identified BRV was named HQ09.BRV HQ09strain VP5, VP6, VP7and VP8genes were amplified by RT-PCR. Sequencing results showed that HQ09VP6gene is1194bp, encoding a length of398amino acids of the protein. Phylogenetic tree analysis revealed that HQ09, NCDV, B223isolated in North America located at the same branch, and their nucleotide homology was99.6%and87.8%, respectively. Topology of phylogentic tree indicated that BRV HQ09belongs to group A rotavirus. HQ09VP4gene is2331bp, encoding a length of777amino acids, and the phylogenetic tree analysis showed that HQ09, P [1] type of standard strains NCDV, North American strains of O Agent, the Japanese strains A5, China Shandong isolates CHLY located in the same branch. Sequence homology was99.9%,97.2%,80.9%,99.6%, respectively, showing that HQ09is P [1] type. The HQ09VP7gene is981bp, encoding a length of327amino acids. Phylogenetic analysis showed that HQ09, G6standard strain NCDV of WC3, Argentina, North American vaccine strains, strains1730, China’s Shandong isolates CHLY located in the same branch. Sequencee homology among them was98.9%,97.4%,98.7%, and99.6%, respectively, indicating that the BRV HQ09is G6type.Totally,0.2mL of BRV HQ09was used to inoculate mice orally subsequently, infected mice developed diarrhea; diarrhea incubation period ranged from1to2days; diarrhea was more obvious at3days to4; at5days to6, diarrhea disappeared. Other clinical symptoms included fur shiny, lethargy, aggressive, etc.; pathological examinations indicated that the intestinal wall became meager and lumen was filled with yellow thin mucoid or watery contents; small intestine histopathological changes were significant; the midgut gland and epithelial cells of the intestinal villi showed, hyperplasia, swelling, mucoid degeneration, necrosis and shedding the top of the small intestine villi damage. The lamina propria showed mild cell infiltration and glandular atrophy destruction.These results indicated that the mouse can be used as BRV infection model.