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Research
Emerging Pattern of Rabies
Deaths and Increased Viral Infectivity
Sharon L. Messenger,* Jean S. Smith,* Lillian A. Orciari,* Pamela
A. Yager,* and Charles E. Rupprecht*
*Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Suggested citation for this article: Messenger
SL, Smith JS, Orciari LA, Yager PA, Rupprecht CE. Emerging Pattern of
Rabies Deaths and Increased Viral Infectivity. Emerg Infect Dis [serial
online] 2003 Feb [date cited]. Available from: URL: http://www.cdc.gov/ncidod/EID/vol9no2/02-0083.htm
Most human rabies
deaths in the United States can be attributed to unrecognized exposures
to rabies viruses associated with bats, particularly those associated
with two infrequently encountered bat species (Lasionycteris
noctivagans and Pipistrellus subflavus). These
human rabies cases tend to cluster in the southeastern and northwestern
United States. In these regions, most rabies deaths associated with
bats in nonhuman terrestrial mammals are also associated with virus
variants specific to these two bat species rather than more common bat
species; outside of these regions, more common bat rabies viruses contribute
to most transmissions. The preponderance of rabies deaths connected
with the two uncommon L. noctivagans and P. subflavus
bat rabies viruses is best explained by their evolution of increased
viral infectivity.
Bites by rabid dogs are the source of 35,000–50,000 human rabies deaths
each year globally (1), yet most human rabies deaths
in the United States are attributed to unrecognized exposures to rabid
bats. Particular attention has focused upon two relatively rare bat species
(Lasionycteris noctivagans and Pipistrellus subflavus) because
rabies variants associated with these species account for approximately
70% of human cases and 75% of cryptic rabies deaths (2–6).
Molecular typing (i.e., phylogenetic analysis of DNA data) has shown
that rabies viruses associated with insectivorous bats (L. noctivagans
and P. subflavus variants in particular) are the culprits in what
otherwise would have been unsolved cryptic human rabies deaths. However,
phylogenetic analyses of human rabies cases have not provided insights
into why an unexpectedly large proportion of human rabies deaths involve
the uncommon L. noctivagans and P. subflavus variants. Passive
surveillance systems used by state public health departments confirm that
human encounters with Eastern Pipistrelle bats (P. subflavus) and
Silver-haired Bats (L. noctivagans) are rare. Neither species exceeded
5% of all bats submitted for rabies testing in the southeastern United
States, and Silver-haired Bats account for <12% of all bats submitted
in the Northwest (7). Moreover, the prevalence of rabid
individual bats within each species is also low and similar to the estimated
prevalence in other, more common, bat species (8,9).
While these surveillance studies are known to produce biased estimates
of the true prevalence of rabies in natural bat populations, they should
accurately reflect the prevalence of rabies in bat species encountered
by the public. In addition, we have determined that although other bat
species occasionally are infected with L. noctivagans and P.
subflavus variants, the frequency of such spillover is low (6,10).
A survey of rabid “house bats” (i.e., Eptesicus fuscus and
Myotis lucifugus) in the United States revealed that only
2 of 117 E. fuscus and 4 of 15 M. lucifugus
were infected with L. noctivagans and P. subflavus variants
(6). The sample size of M. lucifugus is
notably small because this species is rarely found rabid, despite submissions
of thousands of individual bats each year (11). Thus,
all available data suggest that L. noctivagans and P. subflavus
variants are rare.
Given that L. noctivagans and P. subflavus variants appear
to be infrequently encountered, two explanations have been proposed to
explain their prevalence among cryptic human rabies deaths associated
with bats. The small vector hypothesis suggests a failure to recognize
that a bite has occurred when a small bat is involved (7,12).
Absence of a bite history may result from inaccurate documentation when
a patient could not be questioned directly or was not lucid. In 18 of
34 cases with no bite history, however, documented contact with a potentially
rabid animal could have contributed to an unrecognized bite (i.e., physical
contact with bats in 11 cases, bats in residence in 4 cases, and contact
with a known sick domestic animal in 3 cases). Among these contacts, 11
involved L. noctivagans and P. subflavus variants. In addition,
bites by smaller bat species, such as Eastern Pipistrelles, may be more
likely to go unnoticed than bites by larger bats, which may explain why
more cases are associated with this species. Wounds from the teeth of
small bat species are not easily seen without careful examination (13,14),
possibly leading to the impression that a bite has not occurred.
A second hypothesis was suggested by results from experimental data comparing
rabies virus isolates from Silver-haired Bats with those from domestic
dogs and a coyote (15,16). These data showed that, although
both viruses replicated equally well in neuroblastoma cells, rabies viruses
from Silver-haired Bats replicated to higher titers in fibroblast and
epithelial cells, particularly at low temperatures 34°C. Such growth characteristics
might facilitate successful infection after a superficial bite. Thus,
superficial contact may occur frequently between bats and terrestrial
mammals (including humans), but may be unlikely to result in a productive
infection unless L. noctivagans and P. subflavus variants
are involved (the increased infectivity hypothesis). Although these experimental
data suggest that L. noctivagans and P. subflavus variants
have evolved increased infectivity, comparison between viruses associated
with Silver-haired Bats and dogs (including one coyote) is not sufficient
to pinpoint whether increased infectivity evolved in the common ancestor
of L. noctivagans and P. subflavus variants and, thereby,
is relevant to the noted prevalence of these variants among human rabies
deaths.
We proposed a novel test of the increased infectivity hypothesis using
a comparative phylogenetic approach in which we characterized transmission
patterns from bats to nonhuman terrestrial mammals. Transmission of bat
rabies to terrestrial mammals can be viewed as a natural experiment that
removes the confounding effects of vector size and, therefore, can be
used as a control in understanding transmission patterns between bats
and humans. That is, such comparisons take advantage of a fundamental
difference between rabies exposures in humans and rabies exposures in
other terrestrial mammals (nonhuman terrestrial mammals cannot initiate
postexposure prophylaxis even if they are aware of a bite). Given that
size of the vector species is not a factor in terrestrial mammal deaths,
we can control for the effect of bat vector size in our comparison. Therefore,
a disproportionate number of L. noctivagans and P. subflavus
cases among terrestrial mammals would be consistent with increased infectivity
of the L. noctivagans and P. subflavus variants. If these
variants are not overrepresented in terrestrial mammal rabies cases, we
can reject the increased infectivity hypothesis, leaving the small vector
hypothesis as the most plausible alternative.
Methods
Sequence Data
Sequences were obtained from reverse transcriptase polymerase chain reaction
(RT-PCR)–amplified portions of the nucleoprotein (positions 1,157–1,477)
regions based on the reference strain M13215 (17). The
taxa include 32 rabies virus samples obtained between 1958 and 2000 from
frozen (n=27) and formalin-fixed (n=5) U.S. human brain tissue specimens,
17 of 41 U.S. bat species (n=54), and 98 nonhuman terrestrial mammals
(24 cats, 3 dogs, 5 cattle, 9 horses, 1 sheep, 1 llama, 42 foxes [grey,
red, and kit], 1 raccoon, 1 ringtail cat, and 11 striped skunks), determined
to have been infected by a bat rabies virus by using monoclonal antibody
screening. Reservoir species from the eight U.S. terrestrial rabies-endemic
regions (n=24) were included as outgroups. Nucleotide sequences included
in this study will be deposited in GenBank but are also available as an
aligned matrix (click here to view).
Phylogenetic Analyses
Phylogenetic analyses used PAUP* 4.0b2 (18), employing
the neighbor-joining search algorithm (minimum evolution) using maximum
likelihood to estimate Ti:Tv ratio and nucleotide base frequencies (settings
correspond to Hasegawa-Kishino-Yano [HKY] 1985 model of nucleotide sequence
evolution). We assessed tree support using the nonparametric bootstrap
method (1,000 replicates). Phylogenetic analyses presented here included
only frozen human brain samples (n=27). Additional analyses (not shown)
that included incomplete taxa (formalin-fixed human case samples) did
not alter tree topology but decreased nonparametric bootstrap proportions
for those nodes including formalin-fixed taxa.
Results
and Discussion
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Figure
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Click to view
enlarged image
Figure 1. Phylogenetic tree of
bat-associated rabies cases. Taxa represent 203 rabies virus variants
from 27 human rabies cases...
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Figure
2
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Click
to view enlarged image
Figure 2. Geographic distribution
of (A) human (including 5 formalin-fixed samples not shown in Figure
1) and (B) terrestrial mammal cases identified by rabies virus variant
isolated... |
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Phylogenetic analysis (Figure 1) links 20 of 27
bat-associated human cases (24/32 cases, including formalin-fixed samples
not shown in Figure 1) with L. noctivagans
and P. subflavus variants. In contrast to the patterns seen in
humans (Figures 1 and 2A),
a wide variety of bat rabies variants are implicated in the terrestrial
mammal cases across a broad geographic area and largely correspond to
the most common bat rabies virus variants in that geographic region (Figures
1 and 2B). Two notable exceptions
to this general geographic pattern occur in the northwestern and the southeastern
United States (Figure 2B). In these two regions,
L. noctivagans and P. subflavus variants account for a substantially
larger percentage of transmission events to terrestrial mammals than expected,
given the rarity of the host bat species and L. noctivagans and
P. subflavus variants in those geographic areas (Figure
2C) (because spillover of these variants to other bat species is rare,
we used the prevalence of rabid Silver-haired Bats and Eastern Pipistrelles
as a surrogate for the prevalence of L. noctivagans and P. subflavus
variants). These two geographic regions also match very closely the geographic
distribution of human rabies deaths in the United States associated with
these variants (Figure 2A). In the northwest region,
delimited by the human cases associated with Silver-haired Bats (clade
1), the L. noctivagans rabies virus variant accounted for 57% of
bat-associated cases in terrestrial mammals and 80% of bat-associated
cases in humans, despite the fact that rabies-positive Silver-haired Bats
account for only 5% of all bats submitted for testing. Similarly, in the
southeast region, delimited by human cases associated with Eastern Pipistrelles
(clade 2), where neither Eastern Pipistrelles nor Silver-haired Bats exceed
an average of 2% of all rabies-positive bats submitted, the P. subflavus
variant accounted for 63% of bat-associated cases in terrestrial mammals
and 89% of bat-associated cases in humans. The high prevalence of L.
noctivagans and P. subflavus variants among terrestrial mammals
in the same regions where human cases have occurred suggests that a similar
mechanism, such as increased infectivity of these rabies virus variants,
is responsible for both epidemiologic patterns.
Our study included 13 terrestrial mammal species, both domesticated and
wild, suggesting that exposure to bat species likely occurred in a variety
of habitats, including more remote forest habitats. Our data set was comprised
mostly of foxes and cats (two species likely to capture and feed upon
bats), but we had no reason to suspect that their behavior would create
a disproportionate opportunity for infection by L. noctivagans
and P. subflavus variants compared with other bat variants.
While our data do not invalidate the small vector hypothesis, the terrestrial
mammal data show that even when vector size does not play a role in determining
whether bat bites are recognized, L. noctivagans and P. subflavus
variants are still the most prevalent rabies virus variants among bat-associated
terrestrial mammal deaths in the northwestern and southeastern United
States. For human rabies cases, small vector size may still play a role
in the probability of detecting a bat bite, while the increased infectivity
of L. noctivagans and P. subflavus variants enhances the
likelihood of a successful infection following a superficial bite. Additional
experimental data (e.g., site-directed mutagenesis) will be necessary
to show definitively whether L. noctivagans and P. subflavus
variants have evolved genetic changes associated with increased infectivity.
Nonetheless, comparisons of phylogenetic patterns between independent
datasets in which one data set can be used to control for one or more
potentially relevant parameters offers a valid method of hypothesis testing.
Additionally, through phylogenetic analyses such as these, we can target
those taxa and genomic regions that warrant further investigation.
Acknowledgments
We thank the staff members at the U.S. and foreign state
health departments for their submissions of invaluable samples included
in our analyses. We also thank J. Bull, F. Burbrink, J. Childs, M. Hellberg,
B. Mahy, J. McGuire, M. Noor, J. O’Connor, D. Pollock, and the scientific
staff in Viral and Rickettsial Zoonoses Branch, Division of Viral and
Rickettsial Diseases, National Center for Infectious Diseases, Centers
for Disease Control and Prevention for their fruitful discussions and
helpful suggestions.
S.L.M. was supported by the American Society for Microbiology–National
Center for Infectious Diseases postdoctoral research associates program.
Dr. Messenger has
worked on bioterrorism preparedness programs at Louisiana State University
and the Centers for Disease Control and Prevention. Her work includes
training, development of rapid diagnostics laboratory procedures, and
rapid diagnostic testing of clinical and environmental samples. She
continues to employ the tools of molecular genetic and phylogenetic
analyses to investigate the evolution and ecology of infectious disease
pathogens and how that knowledge can be applied to improved detection,
surveillance, and forensic technologies.
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