Isolation、Identification And Molecular Characterization Of Complete Genome Analysis Of Rabies Viruses Isolated In China

Rabies is an acute progressive viral disease and invariably fatal once the clinical symptoms are developed. Rabies is widely popular throughout the world. The causative agent is rabies virus (RABV), which infects the central nervous system. RABV has a broad host range and can infect almost all warm-blooded vertebrates. Asia and Africa are the high incidence areas of rabies, and China is also the high incidence region. Since2000, the number of human rabies cases increased consecutively in China, the domestic researches mainly focuse on the molecular epidemiology of nucleoprotein and glycoprotein of RABV, while fewer researches is carried out on the molecular epidemiology analysis from the complete genome perspective which is much better to explain the genetic evolution of RABV.1. Isolation and amplification of rabies virus.The virus isolation and amplification were carried out through injecting the suspension of specimen into1-day-old sulking mice via intracerebral route.9strains of RABV were isolated using this method, which are the basis for sequencing and analyzing the whole genome.2. Sequencing and analyzing of whole genome9street strains of RABVs isolated form different provinces of China were segmented amplified by RT-PCR and RACE. According to sequences published in the Genebank,16pairs of primers for general PCR and three primers for the end sequence are designed by molecular biology software. The amplified productsof these overlap primers covers the entire genome. PCR products are identified by agarose gel electrophoresis, and then sent to the bio-company for sequencing.PCR products were sequenced and all the segmented sequences were assembled to the full-length genome for9strains of RABV by molecular biology software. The full-length genomes of these9strains of RABVs are from11922bp to11924bp, all of which belong to genotype1. All these genomes encode five structural genes, each gene was started with AACA and ended by Poly (A). Genomic GC content is about45%. Based on the full-length genomic sequences of these9RABVs, the genomic structure and molecular evolution of RABVs isolated in China were analyzed.Full genomic sequences of25strains of RABVs isolated in China were selected to conduct homology analysis. The homology analysis results showed that the homologies of nucleotides and the deducted amino acids sequences forN, P, M, G and L gene were88.6%-100%(97.3%-100%)、85.7%-100%(91.6%-100%)、88.7%-100%(94.1%-100%)、82.76.1%-100%(92.0%-100%)、87.5%-99.9%(95.5%-100%), The five structural genes conservative order is N> L> M> G> P.The analysis results show that the major functional sites of the structural proteins were conserved, but some mutations on amino acids were also occurred, such kind of mutations may have an impact in virulence and immunogenicity. The amino acids in the position333of G protein were Arg in these9isolates, which indicated that they were all pathogenic and identified by MIT test. It was found that the L proteins of almost all the Chinese isolates were started with two Met residues, The functions of such kind of mutations in virus evolution and transmission were still unclear and more information may be discovered by further analysis on LP of RABVs isolated from other areas.Whole-genome phylogeny using nucleotide sequences revealed that domestic strains are mostly concentrated in a large branch, Yunnan strains and Thailand strains have a closer relationship, which indicated that the prevention and control of rabies at borderland areas should be reinforced simultaneously when efforts are made to control rabies in our country. Compared with other RABV strains in the world, the genetic relationship between the Chinese RABVs strains was closer, which showed that the RABVs strains were regional distribution in our country.The sequences of these9strains of RABVs enriched the Chinese and international database resources for RABVs, which provided useful information for molecular epidemiological research from the full genome perspective and established the important theoretical basis for effective control of rabies in China.