Estimation On Virulence Of And Vaccination Protection Against Different Street Rabies Viruses

Rabies is a zoonotic disease caused by Rabies virus(RABV).RABV is a typical neurophilic virus,which invades the central nervous system,causes obvious neurological symptoms and leads to the death of the host.In recent years,the diversity of RABV strains in China has increased significantly,with the emergence of new evolutionary clones,such as the Arctic related group,the steppe type of the world group and the Indian subcontinent group.There are some differences in the biological characteristics of different strains.At present,there are many studies on the virulence and biological characteristics of the Asian rabies with a long epidemic time,but there are no studies on the biological characteristics such as the virulence of the newly emerged strains,and the immune protection effect of the existing rabies vaccine against the newly emerged strains is not clear.In this study,the gene sequence characteristics,infection and pathogenicity of the new evolutionary strains,and whether the commercial rabies vaccine for human has cross immunity protection ability to rabies representative strains were studied.The main research contents are as follows.1.Sequencing and analysis of the whole genome of rabies virus street strainThe Illumina Novaseq 6000 Hiseq X Ten platform was used to sequence 50existing strains in the laboratory.Each strain was sequenced with 6G sequencing data,and the whole genome sequence with the length of 11875-12084 bp was obtained by stitching with DNAstar software.By phylogenetic analysis,the 50 virulent strains were divided into 3 groups,including 23 strains from Asian group,24 strains from world group and 3 strains from Arctic related group.The genomic characteristics of the above strains were identified.Among the five structural genes,there were amino acid mutations in some epitopes of antigen,major epitopes of lymphocyte,glycine sequence and hypervariable region of individual strains,but there were no mutations in virulence determining sites.The nucleotide substitution rates of the five structural genes of RABV were N,L,M,G and P,respectively.The RABV TMRCA mean is estimated at 1160-1362.The quasispecies of 50 RABV strains were sequenced,among which 12 strains had quasispecies mutations,including 6 strains of Asian group,4 strains of world group steppe type,and 2 strains of Arctic associated group.The mutation sites were mainly concentrated in L gene.2.Analysis of infection characteristics of street rabies virus from different hostsIn order to evaluate the virulence of street virus from different evolutionary groups in mice,three street virus strains GDMMD16,NMALSF01 and Nei Meng927A from different evolutionary groups and different host sources were selected to evaluate the virulence in mice.The standard challenge strain CVS-11 was used as a reference.Each strain attacked 12 young mice with 10 LD₅₀.The mice infected by three street virus strains all showed the clinical symptoms of weight loss,hair fold,hind limb extension and paralysis after the onset of rabies,while some mice in GDMMD16 group showed manic clinical symptoms of abnormal mental excitement,while those in NMALSF01and Nei Meng927A groups showed the same clinical symptoms as CVS-11.In the open field test,all the four groups showed the same clinical symptoms.The results showed that the symptoms of anxiety and decreased motor ability were most obvious in NMALSF01 group;in the rotating rod test,the motor ability decreased in all four groups,and the most obvious in NMALSF01 group;the histopathological observation of the brain tissue and spinal cord of the dead mice showed that there were different degrees of neuronal necrosis and lymphocyte infiltration in the brain tissue and spinal cord.CVS-11 group showed neuronal necrosis and lymphocyte infiltration in the brain and spinal cord while GDMMD16 group and NMALSF01 group showed no lesions in the hippocampus,and no pathological changes in cerebellum in Nei Meng927A group.No RABV was detected in the heart,liver,spleen,lung and kidney of 3 dead mice in each group.3.Analysis of the immune protection of rabies vaccine against street virusIn this paper,we prepared the cytotoxin of street rabies virus GDMMD16,NMALSF01 and Nei Meng927A from different evolutionary groups and different host sources,and carried out the modified rabies fluorescent antibody virus neutralization test(mfavn)on 23 human serum,22 dog serum,1 fox serum and 1 raccoon dog serum immunized with rabies vaccine,respectively.The results showed that the fox serum had no protective effect on GDMMD16 street virus The neutralization titers of the rest sera against GDMMD16,NMALSF01 and Nei Neng927A Street strains were higher than the standard recommended by WHO(0.5 IU/m L),which indicated that some commercial rabies vaccines for human and animal could protect the three street strains.