Molecular Characterization of Cryptically Circulating Rabies Virus from Ferret Badgers, Taiwan - Volume 20, Number 5—May 2014 - Emerging Infectious Disease journal - CDC
Volume 20, Number 5—May 2014
Molecular Characterization of Cryptically Circulating Rabies Virus from Ferret Badgers, Taiwan
Author affiliations: National Taiwan University, Taipei, Taiwan, Republic of China (H.-Y. Chiou, C.-H. Hsieh, C.-R. Jeng, H.-Y. Wang, V.F. Pang); Council of Agriculture, Executive Yuan, Nantou County, Taiwan, Republic of China (F.-T. Chan)
Rabies is possibly one of the oldest zoonotic diseases. It is caused by the rabies virus (RABV), a neurotropic virus in the family Rhabdoviridae, genus Lyssavirus. Except for a small number of countries and regions, particularly islands, RABV is found worldwide. The virus infects nearly all warm-blooded animals and causes severe neurologic signs, which almost invariably lead to death (1). It was estimated that worldwide in 2010, the disease caused >60,000 human deaths, primarily in Africa and Asia (2). Although dogs are considered the principal host of RABV in developing countries, the virus is also dispersed among many species of wild carnivora and chiroptera, especially in those countries of Europe and North America that have well-established vaccination programs (3). Mustelids, including various species of the genera Melogale, Meles, andMellivora of the weasel family Mustelidae, can carry RABV (4–6). In southeastern China, Chinese ferret badgers (CNFB; Melogale moschata moschata) have been associated with human rabies for many years and are considered to be a primary host in this region (7–9).
After what were considered to be the last reported cases of rabies in a human and a nonhuman animal in 1959 and 1961, respectively, Taiwan was rabies free for >50 years until the 2012–2013 outbreak of ferret badger–associated rabies. During May 2012–January 2013, through a government-supported program of routine disease surveillance of free-range dead wild animals that had been killed by vehicles or were receiving treatment for injuries and/or illness at the wildlife first aid station, 3 dead Taiwan ferret badgers (TWFB; M. moschata subaurantiaca) were submitted to the School of Veterinary Medicine, National Taiwan University, for further examination.
Pathologic examination revealed nonsuppurative meningoencephalomyelitis with formation of eosinophilic intracytoplasmic inclusion bodies in all 3 animals; reverse transcription PCR and immunohistochemical staining excluded the possibility of infection with the canine distemper virus. However, the results of fluorescence antibody testing, immunohistochemical staining, and reverse transcription PCR, followed by sequencing for RABV, were positive (H.-Y. Chiou, unpub. data). After the rabies diagnoses for the initial 3 ferret badgers were confirmed, by the end of August of 2013, rabies had been diagnosed by fluorescence antibody testing for an additional 105 dead or ill and euthanized ferret badgers and 1 shrew.
Our objective in this study was to clarify whether the current outbreak of the TWFB–associated rabies is an emerging, a reemerging, or a cryptically circulating disease. We investigated the possible origin of this outbreak and its relations with CNFB-associated rabies in mainland China via genomic organization and characterization and analysis of genetic diversity and phylogeographic origin of RABV-TWFB. In addition, we propose a mechanism that might be contributing to the limited host range of RABV-TWFB.
Materials and Methods
Animals and Specimen Collection
During May 2012–January 2013, three ill TWFB were collected from different regions of central Taiwan (Figure 1). One was in the Xitou nature education area at Lugu Township, Nantou County (R2012–26); one was in Gukeng Township, Yunlin County (R2012–88); and one was in Yuchih Township, Nantou County (R2013–01). These 3 TWFB, respectively, showed the following clinical signs: emaciation, coma, paddling, loss of pain response, reduced body temperature, and a 2-cm skin wound on the chin; extreme weakness and inability to move; and signs of weakness and respiratory signs, including labored breathing and increased breath sounds with hypersalivation and exudation of foamy fluid from the mouth and nose. Initial supportive treatment was provided at the wildlife first aid station, but the ferret badgers died within 1–3 days, and their carcasses were submitted to the School of Veterinary Medicine, National Taiwan University, for routine disease surveillance. Full necropsy was performed, during which half of the left cerebral hemisphere was collected from each animal and stored at −80°C for subsequent nucleic acid extraction. Representative tissue samples were taken from all major organs and fixed in 10% neutral buffered formalin for histopathologic examination.
Sample Preparation and Genome Sequencing
Approximately 25 mg of brain specimen from each animal was homogenized, and 1 mL of TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was added. Total RNA was extracted by using an RNeasy Mini Kit (QIAGEN, Valencia, CA, USA), and cDNA was synthesized by using a Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer’s instructions. To amplify the whole genome, we used 19 pairs of primers (Table 1), including the forward primer for the 5′ end and the reverse primer for the 3′ end designed to be complementary to the respective ends of the genome, as described (10).