Isolation, Identification And The Full Genome Sequencing Of A Street Rabies Virus BDRV

Rabies is a drastic infection disease caused by rabies viruses and rabies relative viruses. The mortality rate is 100% once they infect it.Rabies is spreaded all over the world, and almost all the homeothermal animals can infect rabies virus. It is infected mostly by wild animals in Europe, and infected by cattle in Asia and Africa. Homo sapien is abiogenesis host, but according to the examination lately, 99% of anthropo-rabies were causing by canine. Rabies virus can be divided into seven serotypes now, and several rabies virus have not been classified. The number of rabies infections increases these years, and it suggests the other epidemic peak coming. Studies on isolation, identification, characterization of BDRV, sequencing and analyzing of the complete genome were carried out. This study is assisting to the study of molecular epidemiology of the street virus isolated from the nature and might offer an effective way to pervent rabies infection.Virus is isolated and identified by electron microscopic technology, Mouse Inoculation Test (MIT), Fluorescent Antibody Test (FAT), Cell-culture Isolation Techniques (CIT) and RT-PCR from a diseased dog brain tissue in Hebei province. The result of the above methods is positive.The isolated rabies virus is passaged in mouse and cellular level. Incubation period has been decreased from 14d to 6d while have an inoculation in mouse, and the pathogenetic mouse appeared the typical clinical symptom of rabies. The LD₅₀ of BDRV are 10^5.75/30μl and 10^3.30/50μl by intracellular inoculation and intramuscular injection. After passaged in BHK-21 and N2a cell line, the virus BDRV adapted tissue culture and the titre (TCID50) was 103/100μl approximatelly. But freeze-thawing could reduce the infectivity significantly. The virus was freezed in the supernatant of cell culture for 60 days, innoculated BHK-21 cells and followed by high passaged, the titre increased to 106.25/100μl.In this study, the cDNA fragments are amplified by RT-PCR, cloned into pMD18-T vector, and sequenced. Thirteen pairs of primers are used in RT-PCR which cover all the genome of the isolated rabies virus. These primers are designed by molecular biology according to the complete genome of rabies virus published previously. The products of RT-PCR are purified and cloned into pMD18-T vector, and then transferred, indentification. The target vector is indentified and sent to be sequenced.The result of the complete genome sequence of BDRV is gained by linking all the fragments using biological tools. The length of complete genome is 11 932bp. The isolated rabies virus are made up of five genes, the start codon is"ATG", and the stop codon is"TAA"or"TAG"in each gene coding region, and each position of their transcriptional signal is precise. Completed with other rabies viruses of serotype 1, the length of the structural gene is invariant, which shows that there is no inserted or missed gene in the gene coding region of the isolated rabies virus.Analyzing the five structural genes in the isolated rabies virus using comparing nucleotide method based up on informational theory and data. The homology of each structural gene among rabies viruses is analyzed, and five phylogenic trees are made respectively. The result of analysis show that there is several type of mutating including replacing, inserting and missing nucleotide in non-coding regions, but some sequences are highly conserved in this regions, which is considered to be associated with transcription of gene. It is found that there are mutated amino acids in most antigen sites of the isolated rabies virus by comparing functional sites of N gene and G gene, which can affect the virulence and immunity of virus. It is shown thatComparing the five structural genes of the isolated virus with other rabies virus in which are included in GenBank shows that the highest homology of N, P, M, G, L genes. The highest homology of this genes are N gene of Hunan_Xx35(98%), P gene of CNX8601(89%), M gene of CNX8601(91%), G gene of JSL29(99%), L gene of Nishigahara(88%). It is shown that G gene has the highest speed during the evolution and has unique inquilinous body in order to escape immune capture from host by analyzing phyligenic trees of five genes in the whole genome. N gene is the most conservative gene and be treated as the basis of genotyping. The evolution of L gene is torpidity and can be used to analyzing the relationship of remote relatives virus. It is disproportion in the evolution of P and M gene.As one part of the virus genome, the main and secondary genealogy have take their parts in the evolution, so every gene should be considered when analyzing the evolution of rabies virus.