The Studies On Biological Characteristics And Reverse Genetics Of Rabies Virus BDo6 Strain

Rabies is an important and widespread zoonotic disease in the world. Especially in Asia and African countries, the disease is a significant public health problem. Every year about 55,000 people died from rabies World-wide, 56% of these deaths occurred in Asia and 44% in Africa. In developing countries, the human rabies is mainly caused by rabid dog bitting. The programs aimed at population reduction of at risk animal supported by the government has been proved limited, for those stray and house dogs, especially in rural area, this is more laborious and not cost-effective. And there aren't corresponding programs or policies for wild animals in China. However, the dog rabies id still endemic in many rural areas of China.Face the difficulty of rabies control in our country, we have a molecular epidemiology investigation of popular rabies street strains in China to find the genetic relationship among them, and chosed a classical isolate named BD06, designed the experiments to study the physical and chemical Properties,biological characteristics and constructed a reverse genetics system based on this street strain.All of the results may be a good understand of the pathogenic molecular mechanism and for the new vaccine development of rabies virus.The main results of this study are as following:1. The molecular epidemiology investigation of rabies virus isolates in ChinaWe collected the rabies suspected samples from seven provinces for the molecular epidemiology , the RT-PCR and mouse inoculation test two methods were used for the rabies identify, we isolated 18 street strains and had a phylogenitic analysis basd on the complete ORF of N and G gene, the phylogenitic analysis result showed: the 18 isolates can be divided three clusters(Ⅱ,Ⅲ,Ⅳ) , the results based on N and G gene were the same.The isolates isolated from HeBei province showed a close relationship , and isolates from other provinces showed difference diverse.BD06 was a classical isolate among them, the phylogentic analysis showed it belong to the Lyssavirus genotype 1, it was closed with other popular isolates.The similarity of N gene nucleotide sequences of BD06 and other Chinese isolates was about 86.3%-99.7% , and the similarity of G gene was 96.2%-99.6%.The nucleotide sequences similarity of N and G gene were higher than the CVS between Chinese isolates.And the G gene of BD06 was conservative, no mutates were found of some important sites which response for the antigen and pathogenicity.2. The biological characteristics studies on BD06 isolateWe study the physical and chemical properties, the pathogenicity to mouse and dog, the adaptability cells choice, the growth kinetics in cells and mouse brain and the genetic stability of BD06.The results showed:BD06 was sensitive to temperature, organic solvent, ultraviolet ray, weak acid,strong base.100℃10mins, 56℃2mins, ultraviolet ray exposure 2mins, PH5 and PH 10, in 75% ethanol 2mins, in chloroform 5mins, 30% acetone and 0.05% formaldehyde 6h, 10% formaldehyde 5mins can inactivated virus.BD06 was not sensitive to the trypsin. This isolate can killed mouse, inoculation 105LD50 with the dogs, 80% of the dogs can be dead, and the incubation period was about 9-14 days, the course of disease was about 2 days. The highest cell culture titer was 107.5TCID50/ml. BD06 was passaged to 80 times, this isolate showed a high genetic stability, only 1 mutate was found in N gene and 4 mutates in G gene. The mutations on 365 and 496 nucleotide of G gene were all found both in BHK-21 and DK cell lines. The growth kinetics in cells and mouse brain results showed that the virus antigen can be found at 24h after cultured in cells and reached the peak at 96h by the DFA test; the antigen can be found at 156h after inoculation in mouse brain by the immunohistochemistry test.3. The reverse genetics system of BD06 isolateIn order to obtain the full genome of BD06, a rabies steet strain, we designed 12 pairs specificity primers, then get all of the sequences information about BD06, based on these result,we constcuted five plasmids named pcDNA3.1-BD, pcDNA3.1-N, pcDNA3.1-P, pcDNA3.1-G, pcDNA3.1-L, all of these plasmids transfected BHK-21 cell, RT-PCR and DFA results showed we successfully get the recombinant virus,named BD06-L .