Molecular Epidemiology of Reemergent Rabies in Yunnan Province, Southwestern China

Hai-Lin Zhang1, Yu-Zhen Zhang1, Wei-Hong Yang1, Xiao-Yan Tao, Hao Li, Ji-Chao Ding, Yun Feng, Du-Juan Yang, Juan Zhang, Jiang He, Xin-Xin Shen, Li-Hua Wang, Yun-Zhi Zhang, Miao Song, and Qing Tang

Author affiliations: Yunnan Institute of Endemic Diseases Control and Prevention, Dali, China (H.-L. Zhang, Y.-Z. Zhang, W.-H. Yang, J.-C. Ding, Y. Feng, D.-J. Yang, J. Zhang, J. He, Y.-Z. Zhang); Yunnan Provincial Research Center of Virus and Rickettsia, Dali (H.-L. Zhang, Y.-Z. Zhang, W.-H. Yang, J.-C. Ding, Y. Feng, D.-J. Yang, J. Zhang, J. He, Y.-Z. Zhang); Chinese Center for Disease Control and Prevention, Beijing, China (X.-Y. Tao, H. Li, X.-X. Shen, L.-H. Wang, Q. Tang); Liupanshui Vocational and Technical Collage, Liupanshui, China (M. Song)

Abstract

Yunnan Province in China borders 3 countries (Vietnam, Laos, and Myanmar) in Southeast Asia. In the 1980s, a large-scale rabies epidemic occurred in this province, which subsided by the late 1990s. However, 3 human cases of rabies in 2000 indicated reemergence of the disease in 1 county. In 2012, rabies was detected in 77 counties; 663 persons died of rabies during this new epidemic. Fifty two rabies virus strains obtained during 2008–2012 were identified and analyzed phylogenetically by sequencing the nucleoprotein gene. Of the 4 clades identified, clades YN-A and YN-C were closely related to strains from neighboring provinces, and clade YN-B was closely related to strains from Southeast Asia, but formed a distinct branch. Rabies virus diversity might be attributed to dog movements among counties, provinces, and neighboring countries. These findings suggest that Yunnan Province is a focal point for spread of rabies between Southeast Asia and China.

Thumbnail of Distribution of rabies cases, 2000–2012, and clades of rabies virus isolates, 2008–2012, Yunnan Province, China. Shown are the 16 prefectures in Yunnan Province. Black stars indicate 52 specimens collected in the present study. White stars indicate specimens obtained before the present study. YN-A, YN-B, and YN-C indicate clades identified in different prefectures.

Figure 1. Distribution of rabies cases, 2000–2012, and clades of rabies virus isolates, 2008–2012, Yunnan Province, ChinaShown are the 16 prefectures in Yunnan ProvinceBlack stars indicate 52 specimens collected in the present studyWhite...

Although rabies is distributed globally, it is especially prevalent in developing countries in Asia and Africa. China has the second highest number of deaths caused by rabies, exceeded only by India (1–3). A massive rabies epidemic occurred in China in the 1980s (4–6), which subsided by the mid-1990s. However, rabies has once again become a serious public health concern in China. A sharply increasing dog population and a lack of efficient management and vaccination of dogs, especially in rural areas, has led to a dramatic increase in human rabies cases in many provinces in China (6–9). Yunnan Province in China shows the same temporal pattern of rabies outbreaks as the rest of China; rabies was first reported in this province in 1956 (10,11). However, during the present reemergence of rabies, the threat to public health has intensified because the disease-endemic area has increased (Figure 1).

Yunnan Province, which comprises 16 prefectures and 129 counties, is located in southwestern China. It is adjacent to Guangxi, Guizhou, and Sichunag Provinces, which have the highest incidences of human rabies in China (9). Yunnan Province also has a 4,060 km border with Vietnam, Laos and Myanmar, which are countries to which rabies is endemic (Figure 1). This province has an area of ≈394,000 km2, and mountains account for >84% of the terrain; the highest elevation (6,740 m) is in the northwestern region and the lowest elevation (76.4 m) is in the southeastern region. According to 2010 census data, Yunnan Province has a population of 45.6 million persons; most persons live in the eastern part of the province. In western areas, the difference in altitude between mountain peaks and river valleys can be as much as 3,000 m.

The primary objective of this study was to clarify the epidemiology of rabies in Yunnan Province. During 2008–2012, we obtained brain tissue specimens from patients who had died of rabies and from dogs, and other animals with suspected rabies, and cerebrospinal fluid (CSF) and saliva specimens from surviving patients. Antigens of rabies viruses (RABVs) were tested, and nucleoprotein genes from 52 rabies-positive specimens were sequenced. Results were used to characterize patterns of rabies transmission and to evaluate the factors that may influence spread of rabies. Our secondary objective was to evaluate effects of rabies control and preventive measures adjusted to local conditions.

Human Rabies Case Data

A human case of rabies was defined according to diagnostic criteria (WS281–2008) of the Health Department of the People’s Republic of China. A person with rabies had to have the following features: a history of being bitten or scratched by a dog, cat, or a wild animal, or had a wound that was licked by these animals; and clinical manifestations of itching, pain, numbness, and formication around the healed wound, followed by hyperactivity, hydrophobia, aerophobia, spasms of the pharyngeal muscle, and sympathetic excitability. The prodromal stage of paralytic rabies shows hyperpyrexia, headache, emesis, and pain at the site of the wound. Muscles of patients gradually become paralyzed, and patients with rabies die of cardiorespiratory arrest within a few days.

A case-patient with laboratory-diagnosed rabies had RABV antigen, antibody, or nucleic acid was detected in specimens. In China, rabies is a reportable disease; all human cases are reported to the Chinese Center for Disease Control and Prevention (CDC), and nearly all cases are confirmed by clinical features, rather than laboratory diagnosis. A person who satisfied these criteria was confirmed as having a clinical case of rabies.

Human rabies case data were obtained from an infectious disease database report that was officially compiled by the Yunnan Center for Disease Control and Prevention, and the Chinese CDC. Detailed information was obtained by epidemiologic investigations. These data were sorted and analyzed by using Excel (Microsoft, Redmond, WA, USA) and a descriptive epidemiologic method.

Sample Collection

During 2008–2012, brain tissues from 1 cow and 86 sick dogs suspected of having rabies, including dogs that had bitten humans or animals (during abnormally aggressive incidents), were obtained in Baoshan, Dehong, Honghe, Xishuangbanna, Qujing, Zhaotong, Yuxi, Puer, and Chuxiong Prefectures of Yunnan Province. In addition, brain tissues from 1,069 apparently healthy dogs were collected during depopulation of dogs in 11 villages in which a rabies outbreak suddenly occurred and threatened local inhabitants. Brain tissues from 300 dogs used for meat were also obtained from local restaurants. Human brain tissues were obtained from 3 patients within 24 h of death. In addition, 14 saliva samples and 1 CSF sample were obtained from surviving patients. All specimens were kept in airtight screw-cap tubes, transported in liquid nitrogen, and stored at –70°C until tested.

Detection of RABV Antigen

All brain tissues were analyzed by using a direct immunofluorescence assay (DFA) (12) and fluorescent-labeled monoclonal antibody against RABV nucleoprotein (Rabies DFA Reagent; Chemicon, Temecula, CA, USA). Fluid specimens were also screened for specific gene fragments of the nucleoprotein gene by using a nested PCR.

Reverse Transcription PCR

Viral RNA was extracted by using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and used as template for synthesis of cDNA with Ready-To-Go You-Prime First-Strand Beads (GE Healthcare, Piscataway, NJ, USA). Specific regions of nucleoprotein genes were amplified by using nested PCR.

DNA Sequencing

Complete nucleoprotein gene sequences were obtained by using primers specific for this gene as described (12,13). PCR products were purified by using a QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) and sequenced.

Phylogenetic Analysis

Complete nucleoprotein gene sequences were analyzed by using BioEdit software (http://bioedit.software.informer.com/) and ClustalX version 1.8 (http://www.clustal.org/) software. Nucleotide homologies were analyzed using the MegAlign software version 5 (DNAStar, Madison, WI, USA). Phylogenetic trees were generated by the neighbor-joining algorithm in MEGA version 5 (http://www.megasoftware.net/).

Distribution of Human Cases

Temporal Distribution

On the basis of published reports, during 1956–2012, a total of 1,841 human rabies cases were reported officially in Yunnan Province (annual average incidence 0.115 cases/100,000 population; range 0.00–0.69 cases/100,000 population). However, only 33 cases occurred during 1956–1979. In the 1980s, the number of human cases increased and accounted for 57.9% of the total number since official records were initiated in 1956. The highest numbers of human rabies cases were reported in the late 1980s. From 1990 onwards, the number of reported cases fell from 73 cases in 1991 to 5 cases in 1994; during 1995–1999, human deaths caused by rabies were rare. However, this decreasing trend in the incidence of rabies then reversed; 3 cases were reported in 2000, and the number of cases increased to 130 by 2010. During 2000–2012, a total of 663 human cases were reported.

Regional Distribution

Thumbnail of Distribution of rabies cases in 16 prefectures in Yunnan Province, China, 2000–2012, and data analysis of human and animal specimens. Except for Dali, Lijiang, Nujiang, and Diqing Prefectures, 12 prefectures had reported human cases. The color key is a scale in which each color bar indicates the year and its length indicates the number of human cases in that year. The longest bar indicates 40 human cases. Values indicate number of rabies-positive samples/number of samples submitted

Figure 3. Distribution of rabies cases in 16 prefectures in Yunnan Province, China, 2000–2012, and data analysis of human and animal specimensExcept for Dali, Lijiang, Nujiang, and Diqing Prefectures, 12 prefectures had reported...

During the past 13 years, rabies-endemic areas in Yunnan Province showed an increase in the number of rabies cases. In 2000, a total of 3 human deaths caused by rabies were reported in 1 county in Wenshan Prefecture). By the end of 2012, human cases were detected in 12 prefectures (77 counties). However, no cases have been reported in 4 prefectures in the northwestern Yunnan Province since 2000 (Figures 1–3).

In Yunnan Province, most cases occurred in eastern and central areas (Figures 1, 3). Zhaotong, Qujing, Honghe, and Wenshan were the most affected prefectures in rabies-endemic areas of Yunnan Province, which borders Guizhou, Guangxi, and Sichuang Provinces. Human cases were reported in these 3 provinces (maximum no. cases: 664 in Guizhou Province in 2006; 518 in Gaungxi Province in 2006; and 372 in Sichuang Province in 2007; Center for Public Health Surveillance and Information Service and China CDC). This proximity might have been responsible for introduction of RABV into Yunnan Province. Prefectures with a comparatively low incidence of human rabies are located in southern and southwestern regions of Yunnan Province, which borders Myanmar and Laos.