Molecular epizootiology of rabies associated with terrestrial carnivores in Mexico
Introduction
Rabies has been recorded since the first human civilizations, with the dog as the main transmitter (Wilkinson, 2002). In developed countries, traditional descriptive approaches have been applied to infer patterns of disease transmission to humans (Krebs et al., 2003). This approach has been reinforced by the process of rabies virus typing at the antigenic and genetic level (Bourhy et al., 1993, Rupprecht et al., 1987, Smith et al., 1992). The detection and analysis of subtle differences within the rabies virus proteins and genes have permitted the identification of viral variants specifically maintained by different animals, such as dogs, foxes, raccoons, skunks, and bats. The combined use of those latter approaches, together with an efficient surveillance system for disease detection in animal populations, have allowed detailed descriptions of the distributions of major rabies foci, as well as the likely hosts responsible for maintenance (Bourhy et al., 1999, Nadin-Davis et al., 1999, Smith et al., 1995). Data provided by such molecular approaches have permitted insights to virus-reservoir relationships, patterns of transmission and dissemination, as well as viral evolution (Badrane and Tordo, 2001; Bourhy et al., 1993, Bourhy et al., 1999, Holmes et al., 2002, Nadin-Davis et al., 1999, Smith et al., 1995). In contrast, developing countries are faced with less than ideal surveillance in animal populations. The reduced resources available are prioritized for diseases with overwhelming human morbidity and mortality. Often, sample availability is limited and wholly dependent upon human rabies occurrence or the perception of major outbreaks. During the past decade, different analytical approaches have been applied to Mexican rabies virus samples. These approaches have provided insights regarding rabies virus diversity and the likely reservoirs responsible for transmission and maintenance (De Mattos et al., 1999, Loza-Rubio et al., 1999, Velasco-Villa et al., 2002). However, previous studies have been limited by sample sizes or the approach applied, to address the deeper descriptive epizootiology of rabies in Mexico. In the present work, we incorporate 138 sequences comprising a 30-year period. The objective of this study was the molecular analysis of rabies viruses associated with terrestrial carnivores in Mexico, describing updated disease distributions, suggested trends of interspecies transmission, and predicted patterns of dissemination, by using 88 amino acids of the nucleoprotein C terminus.
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Antigenic characterization
The rabies virus N protein was characterized with a panel of eight monoclonal antibodies, previously used to infer rabies virus reservoir species associations in Latin America and the Caribbean (Diaz et al., 1994). This reduced panel was able to identify 11 reactivity patterns associated with different animals involved with rabies virus maintenance and transmission in Mexico and South America (Table 1).
Samples and sequences
In the present study, 138 sequences were analyzed, including the outgroup represented by the
Results
Analysis of the 136 sequences indicated the occurrence of 142 conserved sites, 178 variable sites, and 160 parsimonious informative sites. The nucleotide identity in the 136 sequences ranged from 78.7 to 100% with an average of 90.3%, whereas the amino acid identity ranged from 89 to 100% with an average of 97.2%. Similarly, consensus sequences varied from 79.3 to 97.7% with an average of 89.5% for nucleotide identity, and from 90.8 to 100%, with an average of 97% for amino acid identity (Table
Discussion
Overall, a relatively high degree of variation was found within the last 264 nucleotides encoding for 88 amino acids at the C terminus of the rabies virus nucleoprotein. Most regions contributing to putative B and Th cell epitopes were retained, as was the putative phosphorylation site, at Ser 389 (Dietzschold et al., 1987, Ertl et al., 1991). Some amino acid positions conserved along time were found in association with specific lineages, suggesting that traces of positive selection may also be
Acknowledgments
We thank our colleagues working at the National Network of Public Health Rabies Laboratories in Mexico and in the US as well as the epidemiologists and personnel of the rabies control program in Mexico, who provided samples and epidemiological information about them; Jose Luis Marrufo Olivares and Isaias Sauri from the Laboratorio Central Regional of Merida, Yucatan for providing information about Yucatan samples; and to Caroline J. Henderson, Jennifer M. Snaman and Leslie Real who kindly
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