Molecular Epidemiology of Rabies, Southern China | CDC EID

Volume 15, Number 8–August 2009
Research
Molecular Epidemiology of Rabies in Southern People's Republic of China
Xiao-Yan Tao, Qing Tang, Hao Li, Zhao-Jun Mo, Hong Zhang, Ding-Ming Wang, Qiang Zhang, Miao Song, Andres Velasco-Villa, Xianfu Wu, Charles E. Rupprecht, and Guo-Dong Liang
Author affiliations: Chinese Center for Disease Control and Prevention, Beijing, People's Republic of China (X.-Y. Tao, Q. Tang, H. Li, Q. Zhang, M. Song, G.-D. Liang); Guangxi Center for Disease Control and Prevention, Nanning, People's Republic of China (Z.-J. Mo); Hunan Center for Disease Control and Prevention, Changsha, People's Republic of China (H. Zhang); Guizhou Center for Disease Control and Prevention, Guiyang, People's Republic of China (D.-M. Wang); and Centers for Disease Control and Prevention, Atlanta, Georgia, USA (A. Velasco-Villa, X. Wu, C.E. Rupprecht)

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Abstract
In recent years, the number of human rabies cases in the People's Republic of China has increased during severe epidemics in 3 southern provinces (Guizhou, Guangxi, and Hunan). To analyze the causes of the high incidence of human rabies in this region, during 2005–2007, we collected 2,887 brain specimens from apparently healthy domestic dogs used for meat consumption in restaurants, 4 specimens from suspected rabid dogs, and 3 from humans with rabies in the 3 provinces. Partial nucleoprotein gene sequences were obtained from rabies-positive specimens. Phylogenetic relationships and distribution of viruses were determined. We infer that the spread of rabies viruses from high-incidence regions, particularly by long-distance movement or transprovincial translocation of dogs caused by human-related activities, may be 1 cause of the recent massive human rabies epidemics in southern China.

Rabies has been endemic to the People's Republic of China for many years; domestic dogs act as the main reservoir (1–3). After efforts to stabilize the dog population, the lowest incidence of human rabies (159 cases) was recorded in 1996. Subsequent annual increases followed: 505 cases were reported in 2000, 1,191 cases in 2002, and 2,651 cases in 2004 (2,4). In 2005, three southern provinces (Guizhou, Guangxi, and Hunan) recorded most of the human rabies cases throughout China (5). During the next 2 years, this region had ≈50% (6,595) of the total number of cases in China and contributed to one of the most serious human rabies epidemics in this country (5,6).

Various studies have been conducted to determine the causes and characteristics of rabies epidemics in China. Descriptive epidemiologic analysis showed that the increase in domestic dog populations and low vaccination coverage have contributed to rabies epidemics in the 3 provinces in southern China (7–9). Other authors have suggested that this trend may be caused by a carrier state in healthy dogs that remains undetected (10). Molecular epidemiologic analysis of dog specimens collected from 5 provinces (Guangxi, Hunan, Guizhou, Jiangsu, and Henan) demonstrated that rabies viruses (RABVs) in China are similar to those reported from previous epidemics (11). However, no studies have investigated the phylogenetic relationships among viruses circulating in different provinces during peaks of rabies incidence.

RABV is the prototype member of the family Rhabdoviridae and the genus Lyssavirus. It encodes 5 structural proteins: nucleoprotein, phosphoprotein, matrix protein, glycoprotein, and RNA-dependent RNA polymerase (12). The nucleoprotein (N) gene has been extensively used for genetic typing and evolutionary studies because of its relatively conserved variation among reservoir-associated variants and geographic lineages (13–16). Since the onset of a human rabies epidemic in China in 2005, we have collected brain specimens from dogs and human patients. Partial N gene sequences were obtained in an attempt to understand the sustained increase in rabies cases in China by analysis of RABVs circulating in the 3 southern provinces with the highest rabies incidence.

Methods
Specimen Collection
Figure 1

Figure 1. Locations of 15 cities selected for specimen collection in Guizhou (GZ), Hunan (HN), and Guangxi (GX) provinces, in southern People's Republic of China, 2005–2007...


Figure 2

Figure 2. Neighbor-joining phylogenetic tree of 60 specimens of rabies virus from the People's Republic of China, 2005–2007...


Figure 3

Figure 3. Neighbor-joining phylogenetic tree of 60 specimens of rabies virus (RABV) from the People's Republic of China, 2005–2007...

From 2005 through 2007, we randomly collected 2,887 brain specimens from apparently healthy domestic dogs used as meat in restaurants of 15 cities in Guizhou, Guangxi, and Hunan provinces (Table 1, Figure 1). In addition, we included 4 brain specimens of suspected rabid dogs that had bitten other animals or humans and 3 brain specimens from patients who had died of rabies.

Detection and Sequencing of RABV
All specimens were examined by using a direct immunofluorescence assay (DFA) with fluorescent-labeled monoclonal antibody against RABV N protein (Rabies DFA Reagent; Chemicon Europe Ltd., Chandlers Ford, UK). For all identified RABV specimens, RNA was extracted with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and used as template for cDNA synthesis with Ready-To-Go You-Prime First-Strand Beads (Amersham Pharmacia Bioscience, Chalfont St. Giles, UK). The 720-nt sequence of the N gene, encoding regions corresponding to nt 704–nt 1423 of the total genetic sequence of the Pasteur strain of RABV, was amplified with primers N644F (5´-AAGATGTGYGCYAAYTGGAG-3´, nt 644–nt 663) and N1537R (5´-GGATTGACRAAGATCTTGCTCAT-3´, nt 1515–nt 1537). Locations of primer sequences were referred to the full genome sequence of the Pasteur strain of RABV (M13215). PCR products were purified by using the QIAquick PCR Purification Kit (QIAGEN Ltd., Crawley, UK) and sequenced with an ABI PRISM 3100 DNA sequencer (Applied Biosystems, Foster City, CA, USA).

Sequence Analysis
Complete alignment of nucleotide sequences was performed by using the ClustalX program, version 1.8 (17). MegAlign software version 5 (DNAStar, Inc., Madison, WI, USA) was used to analyze nucleotide and deduced amino acid sequence homologies. The neighbor-joining method in MEGA3 version 3.1 (18) was used with 1,000 bootstrap replications (19). All taxa used for the comparative analyses were obtained from GenBank (Table 2).

Results
Specimen Detection
Of 2,887 specimens randomly collected from dogs, 66 were positive for RABV (positivity rate = 2.3%) (Table 1). The 7 additional specimens obtained from 4 dogs suspected of having rabies and 3 patients who died of rabies all were positive for RABV.

Sequencing
A 720-bp region of the N gene of the RABV-positive specimens was obtained from 60 specimens (53 sequences were from 66 specimens obtained from healthy dogs and 7 sequences were from the 4 rabid dogs and 3 patients who died of rabies). The 13 other positive specimens were not sequenced. All 60 sequences were submitted to GenBank (accession nos. EF990564–EF990623).

Similarity Analysis
A sequence comparison of 60 N gene fragments showed 87.6%–100% nucleotide similarity. Some specimens from the same province were identical in their entire nucleotide sequence: 6 specimens from Guangxi Province (CGX0602D, CGX0604D, CGX0605D, CGX0513D, CGX0620D, and CGX0622D); 3 specimens from Hunan Province (CHN0505D, CHN0506D, and CHN0507D); and 2 specimens from Guizhou Province (CGZ0506D and CGZ0514D). Identical sequences were also observed in different provinces: 7 specimens from Hunan Province (CHN0614D, CHN0516D, CHN0529D, CHN0609D, CHN0615D, CHN0528D, and CHN0525D) and 2 specimens from Guizhou Province (CGX0612D and CGX0606D), 2 specimens from Guizhou Province (CGZ0512D and CGZ0516D) and 3 specimens from Guangxi Province (CGX0614D, CGX0523D, and CGX0625D), and 4 specimens from Guizhou Province (CGZ0501D, CGZ0505D, CGZ0513D, and CGZ0518D) and 1 specimen from Guangxi Province (CGX0617D). Identity levels of deduced amino acid sequences of the 60 specimens ranged from 95.4% to 99.6%.

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Molecular Epidemiology of Rabies, Southern China | CDC EID