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
). 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
). 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
). 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
). These associations were found by comparing the nucleotide sequences of the last 88 amino acids of the nucleoproteins (3
). Lineages of rabies viruses associated with rabies in carnivore species segregated independently from those associated with bats (1
). 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
). 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
). 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
) 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
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
spp., or Pipistrellus
spp., may suggest that these viruses are spatially structured, perhaps reflecting the ecology of the species involved in disease maintenance (23
). 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
). 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
). In contrast to expectations, given the greater diversity of the Chiroptera
) 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
). In addition, the fact that RVTC has at least three different lineages closely related to RVAB may substantially reduce the differences between phylogroups (1
Recently, some authors have reported a high level of amino acid conservation in monophyletic clades of rabies viruses associated with putative reservoir species (Carnivora
), suggesting that rabies virus diversity within reservoir species is highly constrained and evolving under a model of purifying selection (13
). 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
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
). 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.