Studies On Complete Genome Sequence Analysis Of Viruses And Pathology Of Cattle Rabies

Rabies is a zoonosis involving the central nervous system (CNS) which is caused by rabies virus (RABV), whose fatality rate is almost100%. There were cows discovered with symptoms similar to rabies occured at some dairy farms and grazing areas in Inner Mongolia region during the period from the end of2010to the beginning of2013. Combining with these cows’ symptom, studies of molecular biology characteristics and pathogenicity of bovine rabies virus in the area have been carried out to provide the basis for the prevention of rabies.In this study Ⅰ observed the incidence, epidemiological investigation and clinical symptoms of17cases (12cases of cows and5cases of dogs) from24dairy farms and1grazing area, and performed autopsy on the onset and dead cows for histopathological observation, immunofluorescence staining and nucleic acid detection. I successfully isolated three strains of virus from neonatal mice which had intracranial inoculation of three brain tissue of typical cases. According to the rabies virus gene sequences from GenBank, I designed16pairs of primers for PCR and3primers for terminal sequencing. I applied RACE and RT-PCR for the segmented amplification of whole genome sequence and finally obtained the whole genome sequence of three strains of RABV. Then I performed sequence alignment and evolutionary analysis. ICR mice of different ages (1-day-old,4-day-old,10-day-old,2-week-old,3-week-old and4-week-old) were selected for intramuscular and intracranial injection respectively with three different strains of RABV(CNM1101C, CNM1103C and CTN) so that I could explore the virulence and distribution characteristics of different RABV strains in mice brain.10-day-old mice infected with3RABV strains through intracerebral inoculation. were selected as research object. Their brain tissues were sampled when those mice were killed at the agonal stage after onset. Histopathological staining, immunofluorescence, transmission electron microscopy (TEM), in situ hybridization and other experimental techniques were utilized to research the histopathological changes, ultrastructure pathological changes, cell apoptosis, inflammatory cells’ characteristic, nerve cells neurite (MAP-2) lesions. This research also carried out the correlation of virus antigen with nerve cells neurite lesions, and with positive cells expressing three kinds of inflammatory factors IL-1β mRNA, RANTES mRNA and TIMP-1mRNA. Results showed that:(1) RABV nucleic acid positive rate of seventeen cases was100%; the main histopathological changes in Rabies cows were visible intracytoplasmic eosinophilic inclusions in neurons, non suppurative encephalitis, granulomatous-like lesions, metamorphic changes.(2) Three strains were isolated by inoculating sucking mice. Three strains of virus were named CNM1101C, CNM1103C and CNM1104D respectively. The full-length genome were11924nt,11917nt and11924nt respectively, and GenBank accession numbers were KC193267, KC252633and KC252634respectively. All had the typical structure characteristics of rabies virus genome, the genomics (G+C)%content averaged about44.56%, all belonged to genotype I.(3) The homology analysis of every encoding protein, gene in the three isolated virus strains and60virus strains from GenBank indicated that the homology of N, P, M, G and L genes with encoding protein were of88.7%～100%(97.2%-100%),85.4%～100%(91.3%～100%),88.3%～100%(93.4%～100%),85.8%～100%(91.6%～100%) and87.3%～99.9%(95.2%～100%) respectively. The conservatism of five proteins were P<G<M<L<N in turn.(4) For the three strains of virus determined in this study, the main functional sites of each structural protein were conservative. But there also were amino acid mutations at some sites, which might have an impact on the virus virulence and immunogenicity. In addition, CNM1103C strain had close relationship with the isolates from Mongolian wolves and Russian red foxes, while CNM1101C and CNM1104D strains had close relationship with the epidemic strains in mainland.(5) Different ages and infection routes of onset and mortality results caused by three virus strains showed that:the virulence of three strains were CNM1101C>CNM1103C> CTN in turn. Most part of the dying mice’s brain was infected by the inoculated three strains of virus, and the dominant areas were cerebellum and brain-stem. But there was slight difference in distribution among different strains.(6) For the neonatal mice inoculated with RABV CNM1101C. CNM1103C and CTN of different virulence, the pathological changes of brain tissue were different. The positive cells with virus antigen were neurons, astrocytes. oligodendrocytes and microglia. (7) Both virulent and attenuated rabies virus infection could cause higher expression of TIMP-1mRNA, RANTES mRNA and IL-1β mRNA of neonatal rat brain tissue. Expression of three kinds of inflammatory factor caused by attenuated virus is higher than virulent virus. Expression quantity of the three kinds of inflammatory factor and their focus expression position in the brain caused by attenuated virus varied in different strains.Research has shown that the examined cases of eleven cows, one cattle and five canines were all rabies. Three strains of virus were successfully isolated in this study, which had typical structure characteristics of RABV genome, and belonged to genotype I; the main functional sites of each structural protein were conservative, but also with amino acid mutations at some sites. CNM1103C strain had close relationship with the isolates from Mongolian wolves and Russian red foxes, while CNM1101C and CNM1104D strains had close relationship with the epidemic strains in mainland. For the neonatal mice inoculated with RABV of different virulence, the pathological changes of brain tissue were different. The positive cells with virus antigen were neurons, astrocytes, oligodendrocytes and microglia.