CHAPTER 6
Conclusions and Future Directions
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The goal of this study was to discern which mosquito vectors and vertebrate hosts
most likely contribute to Venezuelan equine encephalitis virus (VEEV) enzootic
maintenance and epizootic emergence in an area of changing ecology. Human
agricultural activities such as raising cattle and food crop production has eliminated much
of the pristine lowland tropical forest that previously occupied coastal Chiapas, Mexico.
As land-use changes the associated fauna may change accordingly and lead to VEEV
emergence at the interface between human occupation and natural virus transmission
foci.
I first conducted a year of systematic trapping of rodents and mosquitoes. It was
anticipated that antibody presence in specific rodents and virus isolations from particular
mosquitoes would help delineate the natural transmission cycle of subtype IE VEEV in
coastal Chiapas. The effects of hurricane Stan, which hit southern Mexico in October
2005, on local ecology cannot be known with certainty. However, the reduced rate of
rodent seropositivity compared to a previously published, pre-Stan study, suggest that
virus circulation foci were disrupted. At the end of the longitudinal study, in October of
2007, rodents were captured for importation and laboratory infection studies. Among
these 72 animals a slightly higher, though statistically insignificant, rate of seropositivity
was encountered that could suggest gradually increasing levels of virus circulation.
In the absence of conclusive seroprevalence data, I chose to evaluate the 5 most
commonly captured rodents for suitability as subtype IE VEEV amplifying hosts. Cotton
rats (Sigmodon hispidus), rice rats (Oryzomys couesi), spiny pocket mice (Liomys salvini)
and pygmy rice rats (Oligoryzomys fulvescens) all survived infection and mounted
sufficient viremia to infect enzootic mosquito vectors. Laboratory conditions were not
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designed to simulate the natural environment of these animals, thus their physiological
response to infection in the laboratory may vary from their physiological response in
nature. Further serosurveys of these animals would be useful in validating the conclusion
from the laboratory study that animals from all four species may participate in natural
subtype IE VEEV transmission cycles.
A fifth rodent, the southern pygmy mouse (Baiomys musculus), developed fatal
disease after experimental infection leading me to question the previously published
theory that sympatry between virus and amplifying host selects for resistance to disease
in the host. Baoimys musculus is active during the day while the other four rodents
evaluated, as well as the putative mosquito vector, are nocturnal. This is the strongest
distinguishing ecological characteristic and it suggests that for true sympatry between
virus and vertebrate host co-incidence in time as well as space is required. In order to test
this hypothesis, diurnal rodents from other sympatric regions, or additional diurnal
rodents from coastal Chiapas ought to be experimentally evaluated.
I also captured, identified and tested for VEEV 34,375 mosquitoes from coastal
Chiapas. Of these, 305 were the enzootic vector Culex taeniopus and 4,038 were the
epizootic vector Aedes taeniorhynchus. Neither of these mosquitoes yielded VEEV
isolates. Because infection rates in inter-epidemic periods can be as low as 0.1%, it is not
surprising that no isolates were made from Culex taeniopus. I hypothesized Aedes
taeniorhynchus to be the primary vector of equine-virulent subtype IE VEEV. However,
no isolates were acquired from these mosquitoes either. The primary finding from the
mosquito census was the discovery of higher numbers of Cx. taeniopus than expected and
a distribution of Cx. taeniopus abundance that correlates with human and bovine
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seroprevalence data from previous studies. This discovery prompted the colonization of
Cx. taeniopus for laboratory infection studies with the new equine-virulent subtype IE
VEEV strains. These strains have previously been evaluated in the epizootic mosquito
vector Ae. taeniorhynchus but, because no colony of Cx. taeniopus existed, they had not
been evaluated in the enzootic mosquito vector.
In order to evaluate whether or not Cx. taeniopus in coastal Chiapas could be an
enzootic vector for subtype IE VEEV as it is known to be in coastal Guatemala, I first
established a colony of this mosquito in the UTMB insectary. Previous experimental
infection studies showed that equine-virulent subtype IE VEEV strains infected epizootic
vector mosquitoes with enhanced efficiency, compared to enzootic subtype IE VEEV
strains. I sought to determine whether this enhanced infectivity in epizootic mosquito
vectors came at a cost to the infectivity efficiency in the enzootic mosquito vectors. By
orally infecting Cx. taeniopus on bloodmeals with a range of titers and testing the
salivary secretions for infectious virus, I found that there is no difference in the rate of
infection or presence of virus in mosquito saliva between enzootic subtype IE VEEV
strains and equine-virulent subtype IE VEEV strains. Additionally, I found that Cx.
taeniopus from Chiapas is refractory to infection by subtype IAB VEEV as was shown
for Guatemalan Cx. taeniopus. Thus, equine virulence is not necessarily a marker for low
infection efficiency in this mosquito.
These findings suggests that the factors that determine infection efficiency for
subtype IE VEEV may differ among mosquito species. Infection studies with reciprocal
chimera VEEV strains showed that the gene encoding the E2 glycoprotein contains
infection determinants for Ae. taeniorhynchus mosquitoes. Experimental infection of Cx.
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taeniopus with these and other chimeric viruses may begin to elucidate the factors that
contribute to the permissive nature this mosquito demonstrates toward enzootic and
equine-virulent subtype IE VEEV.
Based on this work I propose the subtype IE VEEV transmission and emergence
cycles shown in figure 6.1. I believe Cx. taeniopus mosquitoes maintain the virus in
circulation foci near the coast where there remain intact mangrove swamps large enough
to support their populations. Ground-dwelling mammals representing at least 4 species in
this habitat may serve as virus amplifying hosts.
Figure 6.1:
Proposed enzootic maintenance and epizootic emergence cycle for
Venezuelan equine encephalitis virus subtype IE in coastal Chiapas,
Mexico.
The universal feeding preference of these mosquitoes, in conjunction with the proximity
of human settlements and horse stables to these swamps, would allow for occasional
transmission of subtype IE VEEV to a horse or person near the coast. If the horse is
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susceptible it may then mount a viremia sufficient to orally infect Ae. taeniorhynchus
mosquitoes that subsequently bite it. If there are other susceptible horses in the region,
the aggressive biting behavior and long flight range of Ae. taeniorhynchus may quickly
perpetuate an epizootic outbreak.
This model differs from the paradigm in several important aspects. Fist, epizootic
IAB and IC VEEV strains are thought to periodically emerge and then essentially burn
themselves out when they kill or immunize most susceptible hosts. There is no evidence
that they are maintained by enzootic mosquitoes during inter-epizootic periods. In
contrast, equine-virulent subtype IE VEEV strains in Chiapas probably are maintained by
enzootic mosquitoes. Second, epizootic IAB and IC strains are known to cause high
viremia in horses while enzootic subtypes IE and ID do not. Subtype IE VEEV strains
circulating in Chiapas have been shown to cause equine disease in the absence of high
viremia and horse infection studies have been equivocal with respect to the ability of
these strains to induce high viremia. Lastly, traditional epizootic strains are thought to be
best transmitted by epizootic vector species and enzootic strains by enzootic vector
species. In Chiapas, equine-virulent subtype IE VEEV strains can be efficiently
transmitted by both epizootic and enzootic vector species. Thus, the changing ecology of
coastal Chiapas is associated with changing ecology of VEEV. The existence of equinevirulent strains of virus that can be maintained by enzootic vector mosquitoes during
inter-epizootic periods emphasizes the importance of equine vaccination. If Mexican
horses are not regularly vaccinated virulent subtype IE VEEV could spread to the United
States, as it did during the 1971 subtype IAB VEEV outbreak. It also highlights the
variability in natural transmission cycles between virus strains and geographical regions.
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Lastly it demonstrates the need for further studies with the enzootic vector species to
discern how these virus strains are maintained and the genetic factors that determine
infection efficiently in Cx. taeniopus.
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