Rabies is one of the most important and widespread zoonotic diseases. The disease is caused by the rabies virus in the genus lyssavirus, family Rhabdoviridae. Rabies virus possesses a single-stranded, negativesense RNA genome consisting of five genes in the order 3' N-P-M-G-L 5', which encode nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase (L) respectively (19, 22). The virus is transmitted usually through a bite of rabid animals. Exposure to the virus among animals and human will result in an almost invariably fatal encephalomyelitis (8). Although an effective vaccine is available, worldwide human deaths caused by canine rabies were estimated at 55, 000 people each year with 56% of these deaths estimated to occur in Asia and 44% in Africa (21).
Rabies remains a serious public health problem in China as in many developing countries. During the past ten years, human rabies cases have been increasing gradually, from 159 in 1996 to 2, 548 in 2005. Furthermore, the third rabies epidemic apparently has not reached its peak (26), and the number in 2006 increased by almost 30% when compared with that in 2005. Moreover, human rabies suddenly occurred in some areas in 2004, where no human rabies cases had been reported over the ten years, and the incidence rate reached up to 10.73 per 100, 000 habitants (25, 28). In addition, the percentage of the cases with an incubation period of ≤20 days apparently increased in the endemic areas (27, 28). Although rabies has been prevalent in China, knowledge of molecular epidemiology and the genetic characteristics of the virus is scant (18, 26), and there are particularly few virus isolations from patients. In this study, we confirmed two patients as positive for rabies, and characterized the rabies virus derived from one patient.
The first patient was a young boy who lived in Wenzhou, Zhejiang province. He was bitten by a dog, and did not receive post exposure prophylaxis in 2004. The second one was a peasant who lived in Xinning, Hunan province. He was also bitten by a stray dog in 2004, and did not receive post exposure prophylaxis. According to the clinical signs, both patients were suspected as being positive for rabies in their local hospital.
Saliva and blood sample were collected from two patients by the local physicians in The Affiliated Hospital of Wenzhou Medical College and Xinning Center of Disease Control and Prevention respectively. Saliva samples were supplemented with 0.75% bovine serum albumin (BSA), penicillin (500U/mL) and streptomycin (2mg/mL), frozen immediately at -70℃, and transported to the laboratory for further analysis. One-day-old suckling mice were inoculated with the saliva by the intracerebral route and were observed for 30 days.
The recombinant nucleoprotein of rabies virus was separated by SDS-PAGE. A stacking gel containing 5% polyacrylamide and a resolution gel containing 12% polyacrylamide were run in a vertical slab gel apparatus (Bio-Rad). Western blot was performed by using a semi-dry transfer cell (Bio-Rad). The blotted nitrocellulose sheet was quenched by incubation in blocking solution containing 5% non-fat dry milk. After washing twice in TBST (10 min per washing), the sera from the patients were added, and incubated at 37℃ for 2 h. After washing in TBST again, the strips were incubated at 37℃ for 1 h with affinity -purified peroxidase-conjugated goat polyclonal antibodies specific for human IgG (1:2, 000) (Sigma Chemical Co. St. Louis, MO, US), and developed with 4-chloro-1-napthol (4CN) (Sigma Chemical Co. St. Louis, MO, US). The strips were dried and photographed.
Total RNA was extracted from saliva or virus-infected mouse brain with Trizol reagent according to the manufacturer's instructions (Invitrogen, Beijing). RNA was also extracted from the normal saliva or the uninfected mouse brains for use as negative controls. RNA was used to amplify N and G gene sequences by RT-PCR. cDNA from the N gene was obtained with primer RHN1 (12). The initial amplification of N gene was performed with primers RHN1 and RHN3 (12), the nested PCR with primers AraN-O1 (1) and 304 (10). The conditions of PCR reaction were as follows: incubated at 94 for 5 min, 94℃ for 1 min, 37℃ for 1 min, 72℃ for 3 min (with a 2s auto extension) for 30 cycles and a final extension at 72℃ for 10 min.
In order to amplify the G gene sequences, primer P3 (15) was used for cDNA synthesis of the G gene. Nested PCR for amplification of the G gene was performed with two pairs of primers P3 and P4 (15) as outer primers, and PIMC (17) and XCYW1 (5' TC (T/C) TGAATCTACCCTGTTGC 3') as inner primers, which was designed by the authors. The condition of PCR reactions was identical to those used for amplifying N gene sequences.
The final PCR products were purified by using the Agarose Gel DNA Purification Kit (TaKaRa Biotechnology Co., Ltd, Dalian) according to the manufacturer's instructions. Purified DNA fragments were cloned into pMD18-T vector provided by TaKaRa. The ligated products were transformed into JM109 competent cells (Promega, Beijing). DNA sequencing was performed with the ABI-PRISM Dye Termination Sequencing kit and an ABI 373-A genetic analyzer.
Sequences were edited and aligned with the DNAStar software package (version5.01). The nucleotide and amino acid identities were calculated with the same software. The PHYLIP program package (3.65) was used to construct the phylogenetic trees by using the maximum likehood (ML) method with 1, 000 replicates. The rabies virus sequences used in the comparisons were obtained from GenBank. The N and G gene sequences used in this study are listed in Table 1.
Table 1. Sequences of rabies virus strains used in this study