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J Clin Microbiol. May 2006; 44(5): 1697–1710.
PMCID: PMC1479161
Molecular Diversity of Rabies Viruses Associated with Bats in Mexico and Other Countries of the Americas
Andrés Velasco-Villa,1,2* Lillian A. Orciari,1 Víctor Juárez-Islas,2 Mauricio Gómez-Sierra,2 Irma Padilla-Medina,2 Ana Flisser,3 Valeria Souza,4 Amanda Castillo,4 Richard Franka,1 Maribel Escalante-Mañe,5 Isaias Sauri-González,5 and Charles E. Rupprecht1
Viral and Rickettsial Zoonoses Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd., Mail Stop G33, Atlanta, Georgia 30333,1 Laboratorio de Rabia, Instituto de Diagnóstico y Referencia Epidemiológicos, SSA, 11340 México, D.F., México,2 Departamento de Microbiología y Parasitología, Facultad de Medicina, UNAM, 04510 México, D.F., México,3 Laboratorio de Evolución Molecular y Experimental, Departamento de Ecología Evolutiva, Instituto de Ecología, AP 70-275, UNAM, 04510 México, D.F., México,4 Laboratorio Central Regional de Mérida, Km 4.5 antigua carretera Mérida-Motul, colonia Díaz Ordaz, 97130, Yucatán, México5
*Corresponding author. Mailing address: Rabies Unit, Viral and Rickettsial Zoonoses Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd., NE, Mail Stop G33, Atlanta, GA 30333. Phone: (404) 639-1055. Fax: (404) 639-1564. E-mail: dly3/at/cdc.gov.
Received November 15, 2005; Revised February 15, 2006; Accepted March 10, 2006.
Bat rabies and its transmission to humans and other species in Mexico were investigated. Eighty-nine samples obtained from rabid livestock, cats, dogs, and humans in Mexico were studied by antigenic typing and partial sequence analysis. Samples were further compared with enzootic rabies associated with different species of bats in the Americas. Patterns of nucleotide variation allowed the definition of at least 20 monophyletic clusters associated with 9 or more different bat species. Several lineages associated with distinctive antigenic patterns were found in rabies viruses related to rabies in vampire bats in Mexico. Vampire bat rabies virus lineages associated with antigenic variant 3 are widely spread from Mexico to South America, suggesting these lineages as the most likely ancestors of vampire bat rabies and the ones that have been moved by vampire bat populations throughout the Americas. Rabies viruses related to Lasiurus cinereus, Histiotus montanus, and some other not yet identified species of the genus Lasiurus were found circulating in Mexico. Long-range dissemination patterns of rabies are not necessarily associated with migratory bat species, as in the case of rabies in Desmodus rotundus and Histiotus montanus. Human rabies was associated with vampire bat transmission in most cases, and in one case, rabies transmission from free-tailed bats was inferred. The occurrence of rabies spillover from bats to domestic animals was also demonstrated. Genetic typing of rabies viruses allowed us to distinguish trends of disease dissemination and to address, in a preliminary fashion, aspects of the complex evolution of rabies viruses in different host-reservoir species.
Antigenic and genetic variants of rabies virus are associated with different species of terrestrial carnivores and bats in the Americas (27, 32, 33). However, not all taxa in these two mammalian orders play an equivalent role in maintaining enzootic disease within geographically discrete areas (32, 33). Within each area, a spillover of rabies virus into other mammalian species may occur, especially during epizootics (11).
Approximately 3,600 human rabies cases were reported in Mexico between 1939 and 2003, and more than 90% of these were associated with virus transmission by dogs (2, 28). Efforts to control dog rabies dramatically diminished the number of human rabies cases, from an average of 70 cases per year during the period 1939 to 1989 to a total of 320 cases during the interval 1990 to 2003 (28-30). Introduction of viral antigenic characterization methods for reservoir inference have revealed that more than 30% of the total reported cases of human rabies in the last 7 years have been associated with bats (2, 35). Little is known about the epizootiology of the disease associated with bats in Mexico; active and passive surveillance on bat populations does not exist in the country, and molecular typing of positive specimens obtained from domestic animals is not performed routinely. Thus, the objective of this study was to use a molecular epidemiology approach (7, 19, 25, 32) to (i) infer which bat taxa are involved in the maintenance and transmission to nonreservoir mammals (including humans), (ii) update the geographic distribution of the disease associated with such reservoirs in order to predict potential areas at risk for human infection, (iii) determine geographic trends of disease dissemination due to possible movement of infected bat populations, and (iv) establish possible patterns of interspecies transmission.
(This work was submitted in partial fulfillment of the requirements for the D. Sc. degree for Andres Velasco-Villa at Doctorado en Ciencias Biomedicas, Universidad Nacional Autonoma de Mexico.)
Samples and sequences.
In the present study, 293 rabies virus sequences, including outgroups represented by European bat Lyssavirus 2 and Duvenhage, were analyzed. The analysis included 89 samples obtained from livestock, cats, and humans over a 12-year period (1991 to 2002) from 18 Mexican states (Table (Table1).1). These 89 samples were chosen on the basis of antigenic variant identification to obtain the greatest potential representation of rabies viruses transmitted by bats. The remaining 204 sequences corresponded to historical samples used in context to support more robust comparative associations with putative reservoir species, geographic regions, or time periods. Sequences available in GenBank were edited and organized taking into account statistically supported monophyletic groups associated with rabies in a given bat species, which in turn represent major bat rabies enzootic foci reported in the United States, Canada, and South America (4, 5, 6, 7, 8, 16, 17, 19, 25, 32, 36, 37, 38).
TABLE 1.
TABLE 1.
Mexican samples which encompass major foci of bat rabies, selected on the basis of antigenic characterization with a panel of eight monoclonal antibodies
Antigenic characterization.
The rabies virus N protein was characterized for all Mexican samples, using a panel of eight monoclonal antibodies (MAbs) (7). Use of this panel has identified 11 reactivity patterns linked with different animals involved with rabies virus maintenance and transmission in Latin America (Table (Table22).
TABLE 2.
TABLE 2.
Standard antigenic reactivity profile of MAbs against rabies viruses in Latin Americaa
Partial amplification of the nucleoprotein gene and its sequencing.
Total RNA was extracted from infected brain tissue by using TRIzol (Invitrogen, San Diego, CA [formerly GIBCO-BRL]) according to the manufacturer's instructions. cDNA was produced by reverse transcriptase PCR, using primers 304 and 1066 as described previously (34). Forward- and reverse-sequencing PCRs using both primers mentioned above were accomplished for every purified amplicon. Sequences were obtained using the Applied Biosystems 377 automated DNA sequencer, as described previously (5).
Nucleoprotein region used for construction of phylogeny.
Fragments of 264 bp pertaining to the rabies virus nucleoprotein gene were used to construct phylogenetic trees. This segment encompasses nucleotides 1157 to 1420 and amino acids 363 to 450, according to positions for the fixed laboratory strain SAD B19 (5, 31, 32).
Phylogenetic tree construction.
Multiple alignments were performed by using CLUSTALW (http://www.ebi.ac.uk/clustalw/index.html). The 294 sequences were edited to 264-bp fragments using BioEdit (12). Phylogenetic analyses were conducted using MEGA 2.1 (20). Distance matrix (neighbor-joining) and maximum parsimony algorithms were used to investigate both possible phylogenetic links and spatiotemporal relationships between Mexican sequences and those previously associated with rabies in different bat species in the Americas (Table (Table33 and Fig. Fig.1)1) (4, 5, 6, 7, 8, 16, 25, 38). For both methods, corrected nucleotide substitutions were calculated using Kimura's two-parameter model and the confidence limits were estimated by a bootstrap algorithm applying 1,000 iterations (9). To contrast rabies associated with bats, consensus sequences associated with rabies in carnivores in Mexico and the United States were also used to construct the phylogenies (Fig. (Fig.11 and and2).2). To provide a root to the phylogenetic trees, the Lyssavirus species Duvenhage and European bat Lyssavirus 2 were included as outgroups (Fig. (Fig.11 and and2).2). Nucleotide and amino acid pairwise p-distances were calculated among all lineages (Table (Table3),3), using MEGA 2.1 software (20). A consensus sequence for each lineage was then generated using the program BioEdit (12).
TABLE 3.
TABLE 3.
Nucleotide and protein distance among major clades associated with bat rabies in Mexico and elsewhere in the Americasa
FIG. 1.
FIG. 1.
Distance matrix tree generated by neighbor-joining algorithm. Bootstrap values for each branch were calculated with 1,000 iterations and appear to the left of each node; on the right side, vertical lines indicate arbitrary names for each major clade. (more ...)
FIG. 2.
FIG. 2.
FIG. 2.
(a) Distance matrix tree generated with neighbor-joining algorithm and (b) maximum parsimony tree. Each taxon comprises consensus sequences which represent the major clades shown in Fig. Fig.1.1. Bootstrap values for each branch were calculated (more ...)
To observe any possible phylogenetic relationships among the different lineages associated with rabies in bats, maximum parsimony and distance matrix trees (neighbor joining) were constructed by using a consensus sequence for each monophyletic group defined at external nodes. Sequences that clustered separately were used as independent lineages in the construction of a consensus phylogeny (Fig. (Fig.2).2). Geographic distribution of Mexican bat lineages is shown in Fig. Fig.3,3, and the consensus amino acid sequences from each lineage were compared with each other (Fig. (Fig.44).
FIG. 3.
FIG. 3.
Geographic distribution of major clades associated with bat rabies in Mexico.
FIG. 4.
FIG. 4.
Consensus amino acid sequence of major clades associated with bat rabies in Mexico, compared to viruses in other countries of the Americas. The last 88 amino acids encoded by the rabies virus nucleoprotein gene at the carboxy terminus are shown.
For clarity, lineage was operationally defined as a group of related sequences having an immediate common ancestor (originated in an external node) that was associated with rabies in the same bat species or rabies transmitted by the same bat species. In turn, these sequences share specific patterns of nucleotide variation conserved along extensive geographic areas and over time. Thus, bat species associated with statistically supported and stable rabies virus monophyletic groups were suggested as the most likely reservoirs of the respective genetic variants, as previously assumed for rabies associated with mammalian carnivores and bats (3, 24, 25, 31). Lineages where the bat species could not be identified were referred to as unknown reservoirs.
A clade was defined as a group of lineages that have a common ancestor located at internal nodes. Thus, clades may be integrated or not by lineages associated with the same bat species.
Antigenic diversity.
All rabies-positive samples with a history of association with bat rabies were screened with a panel of eight monoclonal antibodies, obtaining nine known antigenic patterns. These represented all rabies virus variants associated with bat rabies in Latin America and the Caribbean, in the context of the panel of eight MAbs, plus five atypical reaction patterns (ARP) detected in nine samples (Tables (Tables11 and and2).2). Antigenic variant 3 (AgV3) and AgV11 showed an overlap distribution along the eastern coast of Mexico. AgV11 appears to have a continuous distribution over the tropical and subtropical region along the Gulf of Mexico, encompassing also part of the states of Chiapas and Oaxaca within the plains of the Itsmo de Tehauntepec, whereas AgV3 was found in patches from Tamaulipas to Yucatan throughout the region adjacent to the Gulf of Mexico and the Mexican Caribbean. An atypical antigenic variant, sometimes depicting an antigenic pattern identical to southern central United States skunk ARP/AgV8, was found circulating within the subtropical and tropical region of central western Mexico (Fig. (Fig.3).3). Antigenic variants associated with insectivorous bats such as Tadarida brasiliensis and Lasiurus cinereus were found scattered from central to northern Mexico (Tables (Tables11 and and2;2; Fig. Fig.3).3). Five atypical antigenic variants were found in association with bats (the bat species was not recorded in the place where rabies was primary diagnosed), one with humans, two with cows, and one with cats (Tables (Tables11 and and2;2; Fig. Fig.33).
Molecular diversity of rabies virus associated with bats in Mexico and other countries of the Americas.
The pattern of nucleotide variation among the samples analyzed allowed definition of at least 22 monophyletic clusters associated with 9 different bat species. All monophyletic groups related to bat rabies were clearly differentiated from those related to rabies in terrestrial carnivores (Fig. (Fig.11 and and22).
The average pairwise nucleotide identity among samples associated with bat rabies in Mexico ranged from 83.1% to 100%, with an overall average of 93.6%. The pairwise amino acid identity varied from 91.3% to 100%, with an overall average of 97.8%. In contrast, the pairwise values observed for bat rabies in the Americas ranged from 81.5% to 100% with an average of 88.8% for nucleotide identity and from 86% to 100% with an average of 95.6% for amino acid identity (Table (Table3).3). The putative phosphorylation site at serine 389 was conserved in all samples studied, and one to two distinctive amino acid changes were observed within the monophyletic groups (Fig. (Fig.44).
Sequence analysis and distribution of rabies viruses associated with vampire bats in Mexico.
Monophyletic groups Dr1 through Dr7, associated with rabies transmitted by vampire bats, were distributed along the eastern and part of the western tropical and subtropical regions of Mexico (Fig. (Fig.3).3). Most of these sequences were obtained from livestock and humans but were also obtained from a fruit bat and a dog (Table (Table1).1). This is the first report of a spillover event of vampire bat variants into a fruit bat (Artibeus jamaicensis) and into a dog in Mexico. Western clusters of vampire bat rabies Dr6 and Dr7 were clearly differentiated from Eastern lineages Dr1 through Dr5 on the basis of their conserved patterns of nucleotide variation (Fig. (Fig.22 and and4).4). The amino acid alanine and two threonines at positions 377, 433, and 379, respectively, were conserved over time and space in lineages Dr1 to Dr6. Lineage Dr7 presented one extra amino acid change at position 376 (lysine changed to arginine), which has been preserved over at least a 6-year period (Fig. (Fig.4;4; Table Table1).1). Clusters Dr1, Dr2, and Dr3 overlapped temporally and geographically, but Dr3 segregated independently, having a restricted distribution along the eastern coast, similar to that observed for Dr4 and Dr5 (Table (Table1;1; Fig. Fig.3).3). Lineages Dr4 and Dr5 were genetically and antigenically related to Dr3 and DrSA, representing antigenic variant 3, but distinct from Dr1 and Dr2, which were characterized as antigenic variant 11 (Table (Table1).1). In the case of two samples collected in the state of Aguascalientes, 3255agcshr99 and 4817agcsbv00, and one collected in the state of Hidalgo, 4494hgobv02, translocation events were corroborated by means of the genetic analysis. These samples grouped together with rabies specimens collected from a bat and from cows autochthonous to Estado de Mexico and Michoacan. The owners of the cows confirmed that the animals were bought either in Estado de Mexico or Michoacan, whereas the owner of the horse claimed that the animal was used for racing and was constantly moved throughout these same two states to participate in small township competitions.
Comparison of rabies associated with vampire bats in Mexico and South America and molecular relationships to rabies in free-tailed bats.
Over 200 sequences associated with vampire bat rabies in Mexico and South America were analyzed to discern possible disease dissemination patterns and the evolution of rabies virus associated with this host throughout the Americas (Fig. (Fig.11 and and2).2). The overall genetic identity among lineages associated with AgV3 (Dr3 to Dr5) in Mexico was 96.8% (Table (Table3),3), similar to the value obtained (97.1%) for rabies viruses associated with the same antigenic variant circulating in several countries of South America (Table (Table3)3) during the period 1993 to 2005 (4, 5, 6, 8, 16, 17, 25, 33, 37, 38). Values for lineages exclusively circulating in Mexico were 98.2%, 98.7%, and 98.3% for Dr1 to Dr2 (AgV11), Dr6, and Dr7, respectively (Table (Table3).3). The overall p-distance between lineages with AgV3 in Mexico and South America (0.038) was smaller than the one obtained when the Mexican AgV3 lineages were compared with AgV11 lineages (0.054) or with AgV8/ARP (Dr6 and Dr7) lineages (0.06). Strikingly, the genetic distances between South American AgV3 lineages and those lineages exclusive for Mexican Dr1 to Dr2 (AgV11) (0.046) and Dr6 to Dr7 (AgV8/ARP) (0.052) were higher than that observed with intra-Mexican AgV3 lineages (Table (Table33).
The phylogenies suggest that rabies in Desmodus rotundus and rabies in Tadarida brasiliensis occurring in South America appear to have an early common ancestor which in turn gave rise to rabies viruses that are now circulating in vampire bats throughout the Americas and in free-tailed bats in South America (Fig. 2a and b). Amino acid changes conserved over time at positions 379 and 394, a distinctive antigenic profile (AgV4), and its restricted distribution in South America suggest that the lineage of rabies viruses found in Tadarida brasiliensis in South America (TbSA) may have diverged from the common ancestor shared with vampire bat lineages early in the evolution of vampire bat rabies in the Americas (Fig. (Fig.22 and and44 and Tables Tables11 and and3).3). A later (or more recent) common ancestor for vampire bat rabies in the Americas indicates that vampire bat rabies in the eastern coastal area of Mexico, Dr1 to Dr5 (AgV3 and AgV11), is monophyletic with vampire rabies in South America, whereas Dr6 and Dr7 appear to have diverged along with rabies in Tadarida brasiliensis in North America from the same primary source in the subtropical area of western Mexico (Fig. (Fig.11 and and2).2). In addition, rabies viruses (RABV) associated with vampire bat rabies in the Americas (Dr1 to Dr7 and DrSA) and RABV associated with free-tailed bats in North America (TbNA) were barely differentiated by their neutral pattern of nucleotide variation (Fig. (Fig.11 and and2),2), one amino acid substitution (aspartic acid at position 378), and a different antigenic profile (Fig. (Fig.4;4; Table Table1).1). Strikingly, TbNA, along with all clades related to rabies in vampire bats (Dr1 to Dr7 and DrSA), had consensus amino acid substitutions at positions 377, 379, and 433 which are apparently conserved over time. Rabies related to Tadarida brasiliensis in North America seems to have no close early relationship to rabies in the same species in South America (Table (Table1;1; Fig. Fig.4).4). Spillover from this variant into a dog and a human in Mexico is reported for the first time. The TbNA lineage presented four subgroups (with bootstrap values higher than 64), whose taxa were temporally connected with a wide geographic distribution with no apparent spatial structure (Fig. (Fig.11).
Rabies in other insectivorous bats different from Tadarida brasiliensis.
In regard to rabies related to nonhematophagous bats other than Tadarida brasiliensis, all samples segregated into two major clusters. One cluster, which was associated with colonial but nonmigratory bats, was polyphyletic and contained several independent clades related to rabies in Myotis spp. in the United States and Canada (MyNA); a Myotis sp. in Chile (MySA); Eptesicus fuscus, three lineages, in the United States and Canada (Ef1NA and Ef2NA); Pipistrellus hesperus and Antrozous pallidus in the United States; Histiotus montanus in Argentina (HtAg); and a Histiotus sp. in Chile. The other cluster was associated with rabies in solitary bats and contained at least three different clades related to rabies in Lasionycteris noctivagans (silver-haired bat) (LanNA), Lasiurus borealis (red bat) (LbNA), and Lasiurus cinereus (hoary bat) (LcNA) (Fig. (Fig.11 and and2).2). The nomenclature designated for these two major clusters was described earlier (25).
The cluster associated with solitary nonmigratory bats was monophyletic and presented homogeneity with respect to the amino acid sequence, with the exception of samples associated with rabies in hoary bats. This lineage had one amino acid substitution at position 414, conserved from North to South America, at least within the period of collection, 1982 to 2000 (Table (Table1;1; Fig. Fig.4).4). Two Mexican samples, termed LcMx (L. cinereus Mexico), collected in the central region of Mexico clustered within this monophyletic group (Fig. (Fig.11 to to3;3; Table Table3).3). The patterns of nucleotide and antigenic variations, as well as the amino acid and nucleotide identities, suggest that these samples are associated with rabies in L. cinereus (Fig. (Fig.1,1, ,2,2, and and44 and Tables Tables22 and and3).3). Similarly, the sample LMx (Lasiurus Mexico) seems more closely related to rabies in L. cinereus and L. borealis than to rabies in L. noctivagans, given that it presented a common ancestor with the former clade (Fig. (Fig.11 and and2).2). When sample 3870slpbt03 was compared with all monophyletic groups previously defined, no consistent pattern of similarity was found at either the nucleotide or amino acid level (Fig. (Fig.11 and and22).
Two samples collected in the central region of Mexico (UkMx) from one cat and one bat clustered together with viruses associated with Histiotus montanus in Chile and Argentina (Fig. (Fig.1,1, ,2,2, and and3).3). A consensus amino acid change at position 367 (Fig. (Fig.4)4) and a higher identity value with RABV associated with Histiotus montanus (Table (Table3)3) made these Mexican samples very like RABV currently circulating in small big-eared brown bats (Histiotus montanus) in South America.
Human rabies.
Nine of 10 human rabies cases were associated with 6 lineages related to vampire bat rabies, which, in turn, have either different geographic distributions or distinctive genetic and antigenic profiles (Fig. (Fig.1;1; Table Table1).1). The molecular typing of the samples helped to corroborate that the cases were indigenous with respect to the distribution of the enzootic foci, and it facilitated inferring the most likely reservoir. This approach was especially useful in those cases where the history of exposure was not available, which accounted for 44% of the human rabies cases in this study.
Two cases from the state of Nayarit (Table (Table1)1) were linked with a cluster of rabies (Dr6) in vampire bats distributed on the north-central western coast of Mexico (Fig. (Fig.3).3). In one of these two cases, a diagnosis of rabies was going to be neglected because the patient showed slight clinical improvement shortly before death (Table (Table1).1). In both cases a history of exposure was documented but the risk of rabies transmission by bat bites was not recorded.
The first human case associated with an insectivorous bat rabies virus variant in Mexico was detected by the use of sequence data (Fig. (Fig.1).1). In this case, there was no history of exposure, and the antigenic profile did not help to infer the reservoir of the variant (Tables (Tables11 and and2).2). The patient was taken to health care services when marked neurological signs appeared, and she died 2 days later. The sample sequence grouped within the TbNA cluster, which helped to suggest that the most likely source of infection was Tadarida brasiliensis (Fig. (Fig.11 and Tables Tables11 to to33).
Human rabies associated with bat transmission has been constantly reported over the last 6 decades in North America and became acknowledged as an emerging problem in developing countries of the Americas where the dog rabies variant has become controlled or extinct (2, 23). A lack of a history of exposure is a common characteristic of such cases, and therefore the potential source of infection is uncertain (23). The identification of the most likely rabies reservoirs and the geographic distribution of rabid animals play a central role in controlling and preventing the disease in humans (27, 31, 32, 37). Rabies in Desmodus rotundus (vampire bats) represents the major public health threat in Latin America; however, several other bat species might be playing an important role as inconspicuous rabies virus reservoirs in Mexico and in other countries of the Americas (14, 23). The data analyzed here are meant to provide support regarding the actual enzootic circulation of several other rabies virus variants associated with other bat species in Mexico and several other countries in the Americas, stressing the high capability of dispersion of the rabies virus in some of these species of flying mammals, with the inherent consequences for public health.
Rabies viruses obtained from domestic animals and humans distributed throughout Mexico grouped within several statistically supported monophyletic groups associated with rabies in different bat species (considered as rabies reservoirs) in North and South America (4, 16, 25, 27, 31, 38). These associations were found by comparing the nucleotide sequences of the last 88 amino acids of the nucleoproteins (3, 25, 33, 37). Lineages of rabies viruses associated with rabies in carnivore species segregated independently from those associated with bats (1, 13, 33). The tree topologies obtained in the present study were in general agreement with those previously observed for the entire and partial nucleoprotein gene sequences, as well as with those observed for other RABV structural genes (1, 3, 13, 18, 19, 21, 25, 33). These results suggest that the last 88 amino acids of the RABV nucleoprotein may be useful in making accurate inferences on the specific association of rabies virus lineages to certain bat species, to distinguish trends of disease dissemination and to address, in a preliminary fashion, aspects of the complex evolution of RABV in different host-reservoir species.
Close genetic associations, together with temporal and geographic overlap of the Dr1 to Dr7 clusters, suggest that these rabies viruses may be emerging in different vampire bat subpopulations or colonies, as was proposed to explain rabies virus diversity within fox populations in Canada (26). Natural barriers, such as the major mountain chains in Mexico, may play an important role in circumscribing rabies foci, especially among some vampire bat populations (eastern Dr1 to Dr5 versus western Dr6 and Dr7), and thus promote genetic divergence and geographic partitioning (24, 26). Similarly, the high degree of nucleotide identity observed between some lineages may suggest that recent distributions of rabies virus associated with vampire bats are the product of the gradual movement of vampire bat populations, enhanced by the historic expansion of the cattle industry.
Viruses associated with rabies in D. rotundus throughout the Americas seem to share a common ancestor, according to the dendrograms and cladogram presented here. The occurrence of several lineages associated with AgV3 throughout the Americas and the closer genetic distances of lineages from South America to lineages from Mexico (more than that observed for lineages associated with different antigenic variants within Mexico) suggest this vampire bat variant is most likely responsible for vampire bat rabies dissemination and the most likely ancestor for vampire bat rabies in the Americas. The earlier occurrence of lineages associated with AgV3 in the Americas may be supported by the lower diversity observed in lineages associated with AgV11, AgV5, and AgV8/ARP, which in turn also have limited distribution in Mexico and some other countries of the Americas. Thus, dissemination of vampire bat rabies along the eastern coast of the Americas, from the Gulf of Mexico through the Caribbean and Central America to South America, could feasibly have occurred. Although the actual direction of such dissemination seems not to be clear, the relationship between the earliest ancestor of vampire bat rabies and rabies in Tadarida brasiliensis in South America may imply that vampire bat rabies could have occurred earlier in South America. These data are in agreement with previous observations made during the middle of the 20th century that described vampire bat rabies as a migrating epizootic-causing disease both in vampire bats and in cattle (15).
Vampire bat rabies in the Americas shares a relatively recent common ancestry with free-tailed bat rabies in North America, as suggested previously (14). The latter might be evidence of a cross-species adaptation event of rabies virus from vampire bat origins, as previously suggested (31). These two clades also presented higher amino acid identity with each other than when individually compared with all other clades. Vampire bats may feed upon free-tailed bats, as observed in captivity, which represents a good opportunity for disease transmission (10). In addition, interspecies transmission events of rabies from vampire to fruit bats (Artibeus lituratus) have been reported rather frequently in Brazil (17). In the present study, a fruit bat, A. jamaicensis, was found infected with a genetic variant associated with D. rotundus. Other species of bats, including some that may migrate to the United States, may be fed upon by vampire bats that share the same roosts, especially during inclement weather, a time when bats may be confined (10).
According to amino acid and nucleotide identities, TbSA remain more closely related to vampire bat and TbNA rabies viruses than to those of colonial nonmigratory and solitary bats. Moreover, the observation that TbSA do not share a recent common ancestor with TbNA suggests that the enzootics they are associated with may have had different origins or at least that they are not products of same recent dissemination events, contrary to what was observed in rabies viruses associated with D. rotundus and Lasiurus cinereus from North and South America, which presented a clear recent common ancestor throughout the Americas. Samples obtained from free-tailed bats collected in both Mexico and the United States grouped together in a monophyletic cluster. However, despite the fact that rabies cases in this species have occurred with a scatter distribution, they tend to form subgroups, suggesting that rabies in this species might be related to migration routes and that rabies may be occurring in different subspecies or in independent populations of Tadarida brasiliensis mexicana bats.
According to the phylogenetic data, rabies in solitary bats may be a relatively recent event and one that subsequently may have undergone spillover events with further cross-species divergence. However, it is not clear from the data available which species (L. noctivagans, L. cinereus, or L. borealis) was first in maintaining the enzootic disease or which direction was the actual direction of these hypothetical spillover events. Among viruses associated with L. cinereus, the high degree of nucleotide and amino acid homogeneity, as well as the phylogenetic data, suggests that disease dissemination might be occurring throughout the Americas. This observation suggests long-range migratory patterns for this species, with the inherent ability to move rabies virus with it. Nonetheless, the time frame in which these geographic dissemination events may be taking place is uncertain. Given that, the high degree of nucleotide and amino acid conservation observed within RABV associated with hoary bats in the Americas may be due to a high degree of purifying selection that is taking place within this reservoir-host.
Public awareness regarding the risk of Lasiurus cinereus transmitting rabies in Mexico should be stressed by health care workers, given that the natural distribution of the species encompasses almost the whole country, with the exception of the Yucatan peninsula (22), and bats carrying this RABV variant were found circulating in Mexico. Another two samples collected in northern Mexico (Coahuila and Baja California Sur) were found to cluster within the clade associated with solitary bats in the Americas. Although these two samples segregated in an independent lineage within such clade, the genetic distance indicates these viruses are more closely related to viruses currently harbored by species of the Lasiurus genus. The two bat species (Lasionycteris noctivagans and Pipistrellus subflavus) related to the higher number of human rabies cases in the United States (23, 31) were not found to be implicated in rabies cases in Mexico so far. However, rabies associated with these two bat species should be taken into account in future studies, since these species are naturally distributed within Mexico, maintaining the risk of latent rabies transmission (22, 31).
Rabies virus clades associated with colonial but nonmigratory bats were highly heterogeneous, with conserved amino acid differences. A tendency of having subgroups within lineages and in some instances being nonmonophyletic (unlike RABV in D. rotundus or L. cinereus rabies), particularly for those associated with E. fuscus, Myotis spp., or Pipistrellus spp., may suggest that these viruses are spatially structured, perhaps reflecting the ecology of the species involved in disease maintenance (23, 25, 31). In these clades, rabies may have had several independent origins.
Two rabies viruses collected in the central region of Mexico within a span of at least 5 years were related to RABV associated with Histiotus montanus in Chile, Argentina, and Brazil on the basis of their conserved patterns of nucleotide and amino acid variation (4, 38). The natural distribution of this bat species has not been reported for Mexico and North America, making this an extraordinary finding. Very little is known about the biology of this bat species; the description and marginal distribution of some species of this genus date from the middle of the 19th century to the beginning of the 20th century (http://www.funet.fi/pub/sci/bio/life/mammalia/chiroptera/vespertilionidae/histiotus/). Perhaps there is a possibility that this species is migratory like Lasiurus cinereus, and thus it may have a wider distribution over the Americas; alternatively, perhaps there is another bat species, not yet identified, which is carrying such viruses from one continent to the other.
The diversity of rabies viruses associated with bats (RVAB) in the Americas was similar to that reported for rabies associated with terrestrial carnivores (RVTC) in North America on the basis of partial and complete nucleoprotein sequences (21, 36). In contrast to expectations, given the greater diversity of the Chiroptera (25, 31) over the Carnivora species, RVAB did not exceed RVTC. These results may reflect that rabies virus has achieved specific adaptation in relatively few Chiroptera species (or that such adaptation processes have taken place relatively recently from RABV spillovers coming from older bat rabies reservoirs), as has been observed for Carnivora species (1, 13). In addition, the fact that RVTC has at least three different lineages closely related to RVAB may substantially reduce the differences between phylogroups (1, 13).
Recently, some authors have reported a high level of amino acid conservation in monophyletic clades of rabies viruses associated with putative reservoir species (Carnivora and Chiroptera), suggesting that rabies virus diversity within reservoir species is highly constrained and evolving under a model of purifying selection (13, 18). Within this study, the same observation was obtained for clades of rabies viruses associated with vampire bats in Mexico and South America, TbNA, solitary bats, Histiotus montanus, Myotis spp., etc. However, some amino acid residues were conserved over time in specific association with some bat species (i.e., amino acids A 377 and T 379 in D. rotundus rabies viruses, S 414 in L. cinereus rabies viruses, and D 378 in TbNA rabies viruses) as previously reported for rabies in terrestrial carnivores (36). Positive selection on rabies viruses along their plethora of putative reservoirs has not been proven; however, the specific pattern of amino acid conservation within some lineages suggests that similar analyses should be reconducted using more carefully selected sets of sequences. Alternatively, current surveillance methods are highly biased and may not be an adequate reflection of natural tendencies.
Clearly, improved methods for disease surveillance in wildlife, coupled with phylogeography and the ecology of the putative reservoir species, are required to better understand rabies epizootiology, evolution, and viral diversity. Such investigations may contribute to focusing rabies control measures on those species most likely to be involved in disease maintenance and transmission on a regional basis (11, 23, 31). More accurate actions in this respect may help to prevent unwarranted destruction of beneficial bat species. Educational programs about the risk of rabies transmission by different bat species may be promoted to prevent human infection and fatalities in high-risk areas.
 
. . . .
Acknowledgments
We thank our colleagues working at the National Network of Public Health Rabies Laboratories as well as the epidemiologists and personnel of the rabies control program in Mexico, who provided all samples.
The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the funding agency.
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