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1 Centers for Disease Control and Prevention, DVRD/VRZB/Rabies, G33, 1600 Clifton Road NE, Atlanta, GA 30333, USA
2 1899 Olmo Way, Walnut Creek, CA 94598-1446, USA
3 Department of Biology and Center for Disease Ecology, Emory University, Atlanta, GA 30333, USA
4 Viral and Rickettsial Diseases Laboratory, California Department of Health Services, 850 Marina Bay Parkway, Richmond, CA 94804-6403, USA
Correspondence
Richard Franka
rpf5{at}cdc.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are DQ445308–DQ445382.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Currently, most human rabies deaths are typically not related to inadequate biologicals, but rather as a result of a failure to recognize the risk of disease transmission and to seek appropriate prophylaxis following the exposure to an infected animal. A key facet to understanding the epidemiology of rabies involves a better appreciation of viral variants maintained by different mammalian species. High levels of genetic and antigenic diversity of Rabies virus (RABV) have been reported in association with distinct species of mammals in the Americas (Rupprecht et al., 1991; Nadin-Davis et al., 2001; Smith, 2002; Velasco-Villa et al., 2005). Specific genetic and antigenic patterns exist among RABV lineages. Recent studies suggest that viral variants tend to be host-associated and maintained in populations predominantly through intraspecific transmission, as exemplified by viral variants or biotypes found among common North American taxa, such as: domestic dogs (Canis familiaris), grey foxes (Urocyon cinereoargenteus), eastern spotted skunks (Spilogale putorius), striped skunks (Mephitis mephitis), raccoons (Procyon lotor) and bats of several species.
Worldwide, most human rabies cases are caused by rabies viruses associated with dogs. However, in the Americas, RABVs associated with bats are emerging as a disproportionate source for human infection (Rupprecht et al., 1995; Messenger et al., 2002; Belotto et al., 2005). To date, bat species frequently reported rabid in the USA include the big-brown bat (Eptesicus fuscus), the Brazilian (Mexican) free-tailed bat (Tadarida brasiliensis) and in the western region, the California myotis (Myotis californicus). More limited numbers of cases are identified in the hoary bat (Lasiurus cinereus), the red bat (Lasiurus borealis) and the little-brown bat (Myotis lucifugus). Interpretation of surveillance results is complicated by the fact that certain bat species may be difficult to identify and therefore results could be biased because of misidentification. Despite the frequency of reported rabies cases in E. fuscus and T. brasiliensis, 32 of 58 human rabies deaths reported from 1958 to 2000 in the USA were caused by RABV variants harboured by other bat species (16 Pipistrellus subflavus, eight Lasionycteris noctivagans, five T. brasiliensis, two Myotis californicus and one E. fuscus) (Messenger et al., 2002). Two bat species uncommonly submitted to state public health laboratories for diagnosis and rarely found around human dwellings were most frequently associated with human rabies cases: the silver-haired bat (Lasionycteris noctivagans) and the eastern pipistrelle (P. subflavus). The prevalence of Ln/Ps (Lasionycteris noctivagans and P. subflavus) RABV variant in recent human rabies cases is puzzling because Lasionycteris noctivagans and P. subflavus bats are seldom found or reported to be rabid (CDC, 1994). Since both species prefer habitats far from human dwellings, one explanation may be that many bats die of rabies most frequently during summer in forest habitats where they are seldom observed by people. Two other hypotheses have been proposed to explain this phenomenon: (i) increased virulence of Ln/Ps RABV relative to other virus variants, and (ii) the existence of unique host characteristics, such as the so-called ‘small vector’ hypothesis (Morimoto et al., 1996; Messenger et al., 2003). The latter hypothesis describes a failure to recognize or appreciate the significance of a bite when a small bat is involved, because of the limited severity of the lesion produced. Support of the former hypothesis has been suggested by results from experimental data, comparing infectivity of Ln/Ps RABV variant with those from domestic canids (Morimoto et al., 1996; Dietzschold et al., 2000).
In North America, RABV has been detected in almost every bat species (Constantine, 1979). However, due to problems of passive surveillance, only limited data are available for RABV variants associated with less abundant species. Several bat species, such as Pipistrellus hesperus, are more reclusive or restricted in distribution and rarely come into contact with humans or domesticated animals. Relatively few samples obtained from rabid P. hesperus have been available for analysis, even though this species is relatively abundant in desert and grassland habitats of the western USA where they roost in rock crevices (Barbour & Davis, 1969). As the smallest bat in the USA, P. hesperus is an excellent example for consideration to continue study of the ‘small vector’ hypothesis, by using a molecular approach.
In the present study, RABV of P. hesperus was characterized based on the last 264 bp of the 5' end of the nucleoprotein (N) gene coding region. In Arizona and California, 30 samples were obtained from P. hesperus bats during the years 2000–2005, sequenced and compared with nine historical samples (collected during 1981–1997) from P. hesperus from Arizona (Flagstaff), available in GenBank. These sequences were also compared to more than 300 sequences of RABV collected from different mammalian species throughout North America. Additionally, we compared sequences of Ln/Ps RABV from Lasionycteris noctivagans and P. subflavus, to inquire if they have species-specific markers or should be considered as a single RABV variant.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Brains were removed and rabies diagnoses were performed by the direct fluorescent antibody test (Dean et al., 1996). In addition, another 30 samples of P. hesperus, 14 samples of P. subflavus and 22 samples of Lasionycteris noctivagans bats were used for further characterization (Table 1). Total RNA was extracted from infected bat brains with TRIzol reagent (Invitrogen) according to the manufacturer's recommendations. RT-PCR was performed with primer sets designed for the rabies virus nucleoprotein (N): forward primer 113fw (5'-GTAGGATGCTATATGGG-3', position 1013–1029, according to the PV genome); and reverse primer 304 (Trimarchi & Smith, 2002).
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) (all positions are according to the PV genome, GenBank accession no. M13215
[GenBank]
). Because of limited length of sequences available in GenBank and the abundance of RABV sequences from the last 300 nt of the N gene coding region, further comparisons were focused on the variable 264 nt corresponding to bases 1157–1420 and aa 363–450. Use of this fragment allowed us to increase the robustness of the analysis and provided better resolution for the detection of possible spillover events between species. Previous analyses showed that this short fragment from the 5' end of the N gene generates the same branching pattern as the whole N gene sequence (Smith et al., 1992).
A phylogenetic analysis was undertaken using more than 300 RABV sequences (only 133 unique representatives were used for the final analysis and are shown in the Table 1
), originating from different North American mammals, available from GenBank, including nine historical sequences from P. hesperus collected in Arizona (Table 1) and sequences from P. subflavus and Lasionycteris noctivagans available in the CDC archival database or otherwise sequenced for this study. Sequences were edited using BioEdit software (Hall, 1999) and multiple alignments were built using the CLUSTAL X package (Jeanmougin et al., 1998). Duvenhage virus and European bat lyssavirus 2 (EBLV-2) were used as outgroup taxa. A neighbour-joining (NJ) analysis (p-distance model) with 1000 bootstrap replicates was performed using the MEGA computer program, version 2.1. (Kumar et al., 2001). The NJPLOT program from the CLUSTAL X package and the treeexplorer module of MEGA were used to obtain graphical output of phylogenetic estimations. Bootstrap values of more than 70 % were considered as providing support for phylogenetic grouping. Representatives of particular lineages were selected for the final phylogenetic analysis.
In addition, both maximum-likelihood (ML) and Bayesian analyses were performed on the dataset to support or clarify results of NJ analysis. A total of 133 RABV sequences were analysed, including seven sequences from raccoon, skunk and dog RABVs that were used as outgroup taxa. MODELTEST (Posada & Crandall, 1998) analysed 56 models of evolution to determine the most appropriate selection for the dataset. The HKY85 model (Hasegawa et al., 1985) with a gamma distribution (HKY85+G) was selected and subsequently implemented for the ML analysis in PAUP*4.0b10 (Swofford, 2002). Nucleotide frequencies were A=0.34190, C=0.24190, G=0.20470, T=0.21150, the transition-to-transversion ratio=4.3131 and the gamma shape parameter=0.3395.
MrBayes 3.1.1 (Ronquist & Huelsenbeck, 2003) was used for a Bayesian analysis with the general time-reversible model incorporating both invariant sites and a gamma distribution (GTR+I+G) to examine the data with a more complex model than used in the ML analysis. Two simultaneous analyses, each with four Markov chains, were run for 2 000 000 generations and sampled every 100 generations. Trees generated prior to the stabilization of likelihood scores were discarded, (burnin=450). The remaining trees were used to build a 50 % majority rule consensus tree. Posterior probability values were used to assess support at each node (
95=statistical support).
The deduced amino acid sequences from the consensus of particular monophyletic clades were aligned for comparison as well. All new RABV sequences reported in this study were submitted to GenBank (Table 1).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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In the NJ analysis, 79 % (31 samples) of RABV samples originating from P. hesperus bats in California and Arizona (a large part of their geographical range in the USA) formed a monophyletic clade (Ph1) with high bootstrap support, separated from other phylogenetic lineages of RABV (Fig. 2). A high level of nucleotide sequence similarity (98.7 %) within this clade originating from different locations was observed over the period of sample collection (1981–2004). Five samples (13 %) collected from P. hesperus in California in 2002–2005, formed a separate clade (Ph2), which was a sister to a clade consisting of Myotis species and P. hesperus with inconsistent bootstrap support (Fig. 2). Nucleotide sequence similarity within Ph2 clade was 98.8 % and within the Myotis species clade was 95.5 %. Nucleotide sequence similarity between Ph1 and Ph2 was 94.3 %, between Ph2 and the Myotis species clade was 94.3 % and between Ph1 and Myotis species clade was 91.7 %. Only three (8 %) samples collected from P. hesperus belonged to other RABV lineages (Myotis species), suggesting occasional infectious spillover events from Myotis species. Only four of more than 50 sequences (<8 %) of RABV originating from Myotis species were found in the Ph1 clade. The P. hesperus (Ph) variants Ph1 and Ph2 shared a most recent common ancestor with a RABV variant associated with Myotis species (a RABV variant associated with different bat species from the genus Myotis). The separation between the Ph1 and Ph2 clades was based on 11 synonymous nucleotide substitutions within the N gene fragment, G/A1165, C/T1237, C/T1240, G/A1264, C/A1268, T/A1282, T/C1291, T/C1297, G/A1315, A/G1324, A/C1348 and one non-synonymous substitution T/G1205 (aa L/V379).
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, Fig. 1). The cluster of Lasionycteris noctivagans (Ln) included 24 sequences of Lasionycteris noctivagans originating from Ontario, British Columbia, New York, Colorado, Wisconsin, Washington, Idaho, North Carolina and California, collected during the period of 1976–2004, and two sequences from Myotis lucifugus originating from Alberta and British Columbia, collected in 1979 and 1992, respectively (Table 1, Fig. 1). The separation between the Ps and Ln clades was based on three conservative synonymous nucleotide substitutions within the studied N gene fragment, G/A1172, A/T1184 and G/A1283 (according to the PV genome). Spillovers of the Lasiurus borealis RABV variant to P. subflavus, and spillover of the E. fuscus RABV variant to Lasionycteris noctivagans were detected (Fig. 2).
Comparison of amino acid consensus sequences from clades of P. hesperus, P. subflavus, Lasionycteris noctivagans and Myotis species revealed that most of the nucleotide mutations from the last 264 nt of the N coding region in these particular bat variants were synonymous. Moreover, 100 % amino acid similarity was found between the Ln/Ps, Myotis species, Ph2 and LBNA (Lasiurus borealis North America) variants. The amino acid consensus sequence of P. hesperus RABV variant (Ph1) contained a substitution of valine to leucine at position 379 aa (according to PV strain) in comparison with Myotis species, Ln/Ps, Ph2 and LBNA variants (Fig. 3). Interestingly, amino acid consensus sequences of five samples collected from P. hesperus (Ph2), branching separately from the main P. hesperus clade (Ph1) and separately from the Myotis species clade (Fig. 2), while sharing a common ancestor, have a valine at position 379 aa. (Fig. 3).
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) and Bayesian (Fig. 5) analyses resulted in similar topologies. In terms of the overall structure of the trees, two large clades could be identified: one consisting of Ph1, Ph2 and Myotis species (Bayesian posterior probability=94) and another containing Lasionycteris noctivagans, Lasiurus cinereus, Lasiurus borealis, P. subflavus, E. fuscus 3, D. rotundus and T. brasiliensis (Bayesian posterior probability=99). Following the two large clades, a cluster of E. fuscus samples (1 and 2) joined as the basal group of bat RABVs (Bayesian posterior probability=99). Results from the NJ analysis (Fig. 2), however, indicated a different structure. In addition to Ph1, Ph2 and Myotis species found in the first large clade, samples from E. fuscus (1, 2 and 3), D. rotundus and T. brasiliensis were also included here, albeit with low bootstrap support. The second largest clade then consisted of Lasiurus cinereus, Lasiurus borealis, P. subflavus and Lasionycteris noctivagans with bootstrap support at 98.
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). In the Bayesian analysis (Fig. 5), as was also the case in the NJ analysis (Fig. 2), Ph2 was sister to the clade of Myotis species, and that clade was then sister to Ph1. High support was found for the Ph1 clade (bootstrap=93, posterior probability=100) in all analyses.
The placement of taxa within the large clade containing Lasionycteris noctivagans, Lasiurus cinereus, Lasiurus borealis and P. subflavus also differed in the three analyses. According to the NJ tree (Fig. 2), a sister relationship was found between P. subflavus and Lasionycteris noctivagans, but bootstrap support was low. This clade was then sister to a clade comprised of Lasiurus cinereus and Lasiurus borealis, with a bootstrap value of 98 for the association of the four groups. In the ML analysis (Fig. 4), it appeared that the Lasionycteris noctivagans, Lasiurus cinereus/borealis and P. subflavus clades were unresolved in relation to each other. Finally, a clade of Lasiurus cinereus and Lasiurus borealis (posterior probability=100) was sister to Lasionycteris noctivagans, which then grouped with the P. subflavus clade in the Bayesian tree (Fig. 5). Support for the Lasionycteris noctivagans, Lasiurus cinereus, Lasiurus borealis and P. subflavus clade was indicated by a posterior probability value of 100.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Among North American bat RABV variants, a notable division was shown between group (clade) I specimens associated with colonial, non-migratory bats (Myotis species and E. fuscus) and those of group (clade) II harboured mainly by solitary, migratory species (Lasiurus species and Lasionycteris noctivagans). These conclusions, however, will need to be re-evaluated, because many publications have suggested the gregarious behaviour of Lasionycteris noctivagans (Parsons et al., 1986; Campbell et al., 1996; Mattson et al., 1996; Vonhof & Barclay, 1996; Betts, 1998). Certain Myotis species were suggested as reservoirs, an observation often obscured previously by their frequent infection with viral variants from other Chiroptera. An additional group (III) apparently circulates in E. fuscus, while viruses harboured by both Molossidae and Desmodontinae bats of Latin America form a phylogenetically distinct clade (group IV) (Nadin-Davis et al., 2001). Similar branching patterns were presented by Hughes et al. (2005).
In our study RABV variants harboured by North American bats also formed two major clades. However, in contrast with Nadin-Davis et al. (2001), the first clade was formed by the Ph1, Ph2 and Myotis clades (and not by Myotis species and E. fuscus 1 and 2 clades) and a second one by Lasionycteris noctivagans, Lasiurus cinereus, Lasiurus borealis and P. subflavus. Following the two large clades, a cluster of E. fuscus (1 and 2) joined as the basal group of bat RABVs. Interestingly, the overall branching pattern of the trees constructed in our study by using the NJ, ML and Bayesian methods was the same without regards to the method used. The E. fuscus 3 clade represents a group that branches differently if we use different methods. The differences in branching of Myotis species and E. fuscus 1–3 clades between trees constructed in Nadin-Davis et al. (2001), Hughes et al. (2005), and in our study, could be explained by the different methods and evolution models used, and most importantly by different outgroups and datasets used. We used sequences of RABV collected from P. hesperus (Ph), which were not included in the previous publications. These new clades (Ph1 and Ph2) brought novel insights into the relationship between clades, and the different branching of E. fuscus is the result of these changes. However, the position of the E. fuscus 3 clade in our NJ tree was not supported, similarly as it wasn't supported in the NJ tree published by Nadin-Davis et al. (2001).
Our study revealed that 79 % of RABV samples originating from P. hesperus bats from California and Arizona (a large part of their natural geographical range in the USA) formed a monophyletic clade Ph1 with high bootstrap support and high posterior probabilities, separated from other phylogenetic lineages of RABV (Fig. 2, 4 and 5). Genetic consistency over space and time, along with the phylogenetic independence of the Ph1 clade, suggests that this RABV variant has been maintained in the P. hesperus population independently. These findings raise a question of adequacy of current passive surveillance methods and are indicative of the public health risk of the P. hesperus (Ph) RABV variant. Approximately 13 % of P. hesperus samples collected in California (Los Angeles, El Dorado; distance between two sites approximately 550 km) during 2002–2005 formed a separate clade (Ph2). This clade was also supported by a moderate bootstrap value of 78 %. With only five samples (three from the same county from 2003 to 2004) representing this group, it is difficult to interpret conclusively the relationship of this clade to the Myotis species and P. hesperus 1 clades. However, from all three trees (Fig. 2, 4 and 5), it is obvious that both P. hesperus RABV lineages (Ph1 and Ph2) shared a more recent common ancestor with a RABV variant associated with a Myotis species and their clade is separated from the clade formed by Lasionycteris noctivagans, Lasiurus species and P. subflavus. Further analyses are needed to explain the relationship between the Ph1, Ph2 and Myotis species clades. Likely relevant to the differentiation of P. hesperus clades Ph1 and Ph2 through evolution are differences in the geographical and ecological distributions of the P. hesperus host populations that maintain the virus of each clade. The hosts of Ph1 were collected in desert areas east of the mountains (Sierra Nevada and Coastal Mt Transverse Ranges: Tehachapi Mts, San Gabriel Mts and San Bernardino Mts) that separate this P. hesperus population from a population distributed in more humid valley and coastal areas to the west. The pelage of these bats is pale grey in colour, contributing to their early subspecific distinction as P. h. hesperus. Contrastingly, the hosts of Ph2 were collected in more western areas, where the pelage of these bats is more brownish in colour, contributing to their early subspecific distinction as P. h. merriami (Hatfield, 1936; Hall & Dalquest, 1950; Findley & Traut, 1970).
Only two samples collected from P. hesperus belonged to other RABV lineages (Myotis species clade), suggesting relatively rare spillover events from Myotis species. As well, only four of more than 50 sequences of RABV collected from Myotis species were included in the Ph1 clade, which could be explained by spillover events or by misidentification of bat species.
No spillover between Lasionycteris noctivagans, P. subflavus and P. hesperus was detected in the present study. In addition, spillover of the P. hesperus RABV variants into terrestrial mammals has not been detected. In contrast, Messenger et al. (2003) reported a high prevalence of Lasionycteris noctivagans and P. subflavus variants among terrestrial mammals in the same regions where human cases have occurred. They suggested that increased infectivity of these RABV variants is responsible for relatively frequent spillover events. Our analyses showed that P. hesperus harbours RABV variants genetically distinct from the Ln and Ps variants and from other RABV variants. This finding corresponds interestingly to the taxonomic revisions of the relationship between P. subflavus and P. hesperus, recently published by Hoofer & Van Den Bussche (2003) and Hoofer et al. (2006). Two bat species previously classified as members of one genus and now, on the basis of genetic classification, assigned to two different genera harbour two distinct RABV variants. New pathogenesis studies focused on the infectivity of all bat RABV variants, together with improved epidemiological analysis of both bat RABV prevalence and accurate bat species identification, are necessary to evaluate the increased infectivity hypothesis.
Data generated by phylogenetic analyses provided us with basic information about relationships between RABV variants harboured by North American bats. Thoughtful interpretation of these data, coupled with relevant epidemiological findings can lead to insights of new hypotheses that seek to explain the frequency of human rabies cases associated with bat RABV variants. As the smallest North American bat P. hesperus weighs only 2–6 g, compared with the larger P. subflavus (6–10 g) and Lasionycteris noctivagans (9–12 g). P. hesperus becomes aggressive as it develops rabies, and it engages in seemingly unprovoked attacks. This behaviour may be necessary for the Ph RABV variants survival via transmission to other P. hesperus bats, which are strictly solitary and may not be readily approached. Such attacks on larger bats by this diminutive butterfly sized bat could end in its demise through retaliatory bites. Given its occasional tendency to apparently attack people when rabid, no human rabies cases have been linked to the Ph RABV variants. However, such overt exposures may signal the need for post-exposure prophylaxis (Constantine, 1970). Of relevant interest, a significantly greater proportion of smaller bats bit people than larger bats: 39 of 279 rabid bats bit people at rates ranging from 67 % of P. hesperus to only 15 % of E. fuscus (Constantine, 1967). These results might suggest greater aggressiveness of the smaller bats or greater care by people to avoid bites of the larger bats.
The lack of human cases linked to the Ph variants may be related to the biology and ecology of P. hesperus. These bats have few contacts with humans and apparent difficulty inflicting deep wounds by their small teeth. Additionally, specific viral properties such as a higher level of adaptation to a principal host and a relatively lower pathogenicity for other species or limitations in virus excretion in terms of intermittent shedding and viral dose may be operative. The lack of spillover events to terrestrial carnivores and infrequency of spillover to other bat species strongly support the hypothesis that addresses specific viral properties. The thickness of mammalian fur could form a barrier protecting skin from penetration by the small teeth of P. hesperus, but this argument fails to explain the absence of human cases associated with Ph RABV variants. Moreover, terrestrial mammals contact bats primarily by the paws and mouth, where fur coverage is absent (lips, mucosae of mouth cavity). One of the most frequently reported rabid bats in California is Myotis californicus, which is also a small bat species (weight 3.3–5.4 g). However, no human cases caused by this RABV variant have been reported in California, suggesting that human exposures are recognized and treated, or these viruses fail to achieve successful infection. Two human cases caused by Myotis species RABV were reported in 1984 (Pennsylvania, no history of contact with a bat was reported) and in 1995 (Washington, rabid Myotis found in the bedroom, no known animal bite) (Messenger et al., 2002). Interestingly, most of the human rabies cases, which had an unknown history of bite exposure, were caused by different RABV variants, harboured by medium-sized bat species (14 of 16 P. subflavus variant, 6–10 g; five of eight Lasionycteris noctivagans variant, 9–12 g; four of five T. brasiliensis variant, 10–15 g; one of one E. fuscus variant, 11–23 g and two of two Myotis species variant, weight range according to species 3–13 g). Among 15 human rabies cases reported in California since 1958, seven were acquired indigenously and all were characterized as bat RABV variants [four Ln/Ps variant and three T. brasiliensis (Tb) variant] (Messenger et al., 2002).
Thus, given the rather diminutive P. hesperus, small body size alone (small vector hypothesis) may be inadequate to explain the association between human rabies cases and certain RABV isolates from bats. More importantly in this regard seem to be viral properties, such as viral infectivity and relative virulence, in association with particular host characteristics (e.g. receptors, body temperature, immune system response, etc.) coupled with host lifestyle (e.g. more solitary versus colonial bat species).
Hughes et al. (2005) inferred from the estimated substitution rate of the N gene that the initial branching of parental rabies virus in bats gave rise to the current variants associated with infection of T. brasiliensis and D. rotundus. Compartmentalization of RABV into lineages associated with infection of solitary bat species (or small group-forming species, such as P. subflavus and Lasiurus species) and more colonial bat species (E. fuscus, Myotis species) occurred later. Questions remain, as to the degree of sociality and the dynamics of RABV transmission in different bat species. Can we consider bats as solitary only if individuals are solitary all year or also those which form small (5–50 individuals) maternity colonies? There is an obvious difference between large colonies of T. brasiliensis consisting of millions of individuals, seasonal colonies of E. fuscus and Myotis species with hundreds of individuals, and maternity colonies of P. subflavus and Lasionycteris noctivagans with only tens of individuals. The results of Hughes et al. (2005) showed that adaptation of RABV to colonial bat species occurred more quickly than adaptations for the more solitary species, suggesting dependence of this process on host ecology.
In this regard, one interesting result of our analysis is that the Ln and Ps RABV variants have circulated in two different bat species populations (P. subflavus and Lasionycteris noctivagans) probably separately from each other and maintain reasonably high genetic stability over relatively long periods (Ps, 1989–1999; Ln, 1976–2004), especially given the error of replication fidelity in RNA viral genomes. The separation between Ps and Ln clades was based on three conservative nucleotide substitutions within the studied N gene fragment, G/A1172, A/T1184 and G/A1283 (according to the PV genome). Although all three substitutions were synonymous, they suggest that these RABV variants may have circulated independently in these host species. Since bootstrap support for separation of P. subflavus and Lasionycteris noctivagans clades in the NJ tree and similarly posterior probabilities in the Bayesian tree were low, further examination of the RABV variants collected from Lasionycteris noctivagans and P. subflavus will be necessary to understand the evolutionary relationships of these two clades. Taking into account the fact that 16 of 24 human rabies cases in the USA over the last 50 years were associated with P. subflavus, and our recent finding of possible independent circulation of Ln and Ps RABVs in Ln and Ps populations, the finding of Geluso et al. (2005) that P. sublfavus has expanded westward in the USA to New Mexico, South Dakota and Texas in recent years (Fig. 1) should highlight a need for enhanced surveillance.
All nucleotide mutations in the highly variable 264 nt fragment of the N gene from P. hesperus (Ph2), P. subflavus, Lasionycteris noctivagans, Myotis species and LBNA were synonymous with identical amino acid consensus sequences. The only exception was a substitution of the valine for leucine at position 379 aa (according to the PV strain), in Ph1 RABV (Fig. 3). This substitution is, however, structurally conservative. Such findings raise a question of potential constraints of RABV evolution, despite differences in the estimated substitution rate between more solitary or colonial bat species. Comparison of other genomic regions would facilitate a better understanding of differences between these RABV lineages.
Further phylogenetic analysis of additional samples from P. hesperus, P. subflavus and Lasionycteris noctivagans from North America are needed to corroborate the results revealed by our limited dataset. In particular, considering the extent of their distributions and their obvious role in public health in Canada and the USA, greater attention to these bats should occur in Mexico and Central America. Moreover, in vitro and in vivo pathogenesis studies should be conducted for a better understanding of the features of these particular RABV variants.
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ACKNOWLEDGEMENTS |
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Received 6 January 2006;
accepted 20 March 2006.
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