| Rabies is a fatal enzootic disease caused by rabies virus (RABV), genus Lyssavirus of the Rhabdoviridae family. RABV infects a wide range of mammalian species and causes fatal ence- phalitis. It is estimated that human death toll due to rabies is approximates 55,00060,000 people each year worldwide and most of them occurred in the developing country (about 55,000), of which approximately 56% occur in Asia and 44% in Africa. In recent years, China has reported the second highest rates of illness and death from human rabies and rabies incidents is on the top of the list of reported infectious diseases in recent years, so rabies is one of the most severe public health problems. The molecular epidemiology of RABV isolates in China is carried out by using antigenic typing and genetic typing in order to better understand the antigenic and genetic diversity of RABV isolates in our country, then provide suggestions for the program of the rabies prevention and control.In order to characterize rabies viruses, brain tissues of rabid animals were subjected to mouse inoculation test (MIT) followed by viral isolation using N2A and BHK-21 cell lines from mouse brain tissue or animal original brain tissue homogenates in Nonclon Delta Tube with coverslip. Once viral growth was confirmed at 96 hrs p.i. by detection of coverslips using fluorescent antibody test (FAT) the infected cells were propagated three times and the viral titers were determined for each passage, and then the glycoprotein gene of cellular adapted viruses were amplified and sequenced. In results, 40 street viruses were isolated in N2A cells from 40 rabid dogs, pigs and cows, while only 37 isolated in BHK-21 cells. The isolated viruses showed a variation of viral titers at passage 3 from 10-1 TCID50/100uL to 10-4.0 TCID50/100uL, indicating different adaptive capacities of the viruses in the cell cultures. And it is also showed that N2A cell line is more sensitive than BHK-21 in isolation of street viruses. The relative stability of virus isolates during propagation in N2A cell is confirmed by the same consensus sequences of original brain tissue and F3, F4, F5, F6, F7, F8 N2A cellular strains of FJ02 strain obtained from the Chongqing City, and the mutation frequency of quasispecies sequences of original brain tissue and F3, F4, F5, F6 N2A cellular strains is 2.54×10-4, 1.27×10-3, 1.34×10-3, 7.97×10-4, 8.25×10-4, respectively, of which showed the cellular adaptivity of virus isolates is associationwith the massive proliferation of genetic variants of virus. The antigenic characterization of RABV isolated from different regions and hosts in our country were carried out by 9 anti-rabies nucleoprotein MAbs, 6 of which reacted with all the 34 virus isolates and the other 3 MAbs only reacted with some of them. Based on the reactivity patterns between virus and MAbs, the analyzed 34 viruses can be classified into 6 antigenic variants (V1-V6), and V1 isolates were widely distributed in China. Phylogenetic analyses indicate that these rabies virus isolates characterized by MAbs can be grouped into four clusters (I-IV), but there was little correlation between genetic typing and antigenic typing.A molecular epidemiological study was performed on 30 China RABV isolates, which were collected from domestic animals diagnosed as rabid between 2004 and 2008. 27 samples were collected from dogs, one from pig and the rest of the two samples from cattle. This study was carried out based on the comparison of nucleotide and amino acid sequences of glycoprotein coding region. The similarity of nucleotide sequences of China RABV isolates was about 82.7%100%. Phylogenetic analysis of these and 24 additional Chinese isolates clearly supported the placement of all Chinese viruses in Lyssavirus genotype 1 and divided all Chinese isolates between eight distinct clusters (I-VIII), cluster VIII viruses originated from the raccoon dog were the Arctic strains that first identified in our country, and the isolates determined by our laboratory belong to the I, II, III, VI cluster, respectively. Six subgroups within the most commonly encountered cluster I were identified according to their geographical origins. A comparison of representative Chinese viruses of different clusters with other isolates retrieved worldwide indicated a close phylogenetic relationship between China cluster I and II viruses and those of Indonesia, all of which formed three clades independently; cluster III formed a unique clade, and cluster IV viruses strain formed a clade together with PM, HEP-Flury, isolates from Mexico; there was high similarity of nucleotide sequence of G gene between cluster V virus strain CHI1-BK and YUG1-GF isolated from Yugoslavia, as well as cluster VI virus DRV and Africa RABV strain; cluster VII viruses were closely association with those of Thailand , Malaysia and Vietnam while cluster VIII viruses formed the Arctic-like strains clade with those of Korea and Siberia, Russia. The identities of nucleotide sequences of glycoprotein coding region between Chinese vaccine strains CTN and cluster I, II, III and VII viruses were higher than that of those between the latter and 3aG, SRV9 and international classical vaccine strains, in contrast, the identities between classical vaccine strains and cluster IV, V, VI and VIII viruses were higher than that of those between the latter and CTN. Thus, our data suggest that RABV isolates currently circulating in China have great genetic diversity, and two or more cluster viruses circulated independently in the same province, such as Hunan, Chongqing, and so on. The close phylogenetic relationship between China RABV and those of other Asia country show that they may have the common orgin. The molecular epidemiological data obtained in our study is useful for the program of rabies prevention and control in China.RABV isolates database that includes the original brain tissue viruses, mouse brain viruses and cellular adaptive strains is initially established in our laboratory through the virus isolation using MIT and RTCIT. This database makes the RABV viruses isolated from China increased and preserved, and these will provide the virus materials for rabies-associated studies; Antigenic variation of Chinese RABV isolates were firstly identified using 9 anti-rabies nucleoprotein MAbs in our study, the results reflect the antigenic diversity of RABV isolates in China; Molecular epidemiology targeting the glycoprotein gene of RABV isolates in China show that rabies viruses currently circulating in our country can be classified into eight distinct clusters, and the VIII cluster viruses is the first reported Arctic-like strains in China. This study is based on the previous studies achievement together with the viruses determined in our lab and other RABV G gene retrieved from the GenBank.The epidemiological data obtained from the present study reflect the current epidemics in China and it has important epidemiological and practical significance in rabies prevention and control for our country.
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