These studies suggest that reptiles are susceptible to infection with North American EEEV and that snakes in particular have the potential to serve as a reservoir host for the virus. The garter snake was shown to be a competent host for EEEV, maintaining circulating levels of the virus that would be expected to be infectious for a mosquito for up to 14 DPI. The duration of the viremia in the snakes was also found to be temperature dependent. Circulating virus levels reached lower levels, but were maintained for longer periods, in animals held at lower temperatures. This might be related to the observation that the kinetics of an immune response to a challenge in reptiles is known to be temperature dependent.27
In support of this hypothesis, antibodies to EEEV were not detected in snakes exiting hibernation at 30 dpi when assayed using a plaque reduction neutralization test.25
Recent studies have demonstrated that cotton rats may serve as reservoirs for both North American and South American strains of EEEV.7
Interestingly, the South American strains of the virus seemed to replicate to higher titers in cotton rats than did the North American strain and the South American strains were less pathogenic to cotton rats than the North American strain.7
These data suggested that while both North American and South American strains of the virus were capable of using cotton rats as a reservoir, the South American strain might be better adapted to a small mammal reservoir than North American strain. In this regard, we noted no significant pathology in the reptiles infected with the North American subtype I strain of the virus, suggesting that this strain of the virus may be fairly well adapted to reptile hosts. It would be of interest to determine if other strains of EEEV are equally capable of replicating in reptiles and if so do they induce any significant pathology in infected animals.
Although the viral titers reached in the snakes were lower than those observed in birds, the infectious viremic period was rather prolonged. Garter snakes were able to maintain a potentially infectious viremia for up to 7 dpi. This is longer than the period that avian hosts for the virus usually maintain an infectious EEEV titer, which is in the range of 2–3 days.26
This finding suggests that although snakes might be a less efficient reservoir for EEEV than birds, they could remain infectious to mosquitoes for a longer period of time. This might have been expected, given that the metabolic rate of ectothermic animals and presumably their ability to clear the virus will vary depending upon the temperature of their environment.
The data presented in this study show that EEEV-infected garter snakes can remain viremic during hibernation. This finding is in concordance with previous studies of garter snakes infected with WEEV, another alphavirus related to EEEV, where it was found that WEEV-infected garter snakes remain viremic during hibernation.16
The persistence of EEEV viremia during hibernation lends support to the hypothesis that these animals might serve as overwintering hosts for EEEV.
For snakes to serve as an efficient overwintering host for EEEV, it is necessary that they be fed upon by mosquitoes that can serve as vectors for the virus. The three most common mosquito species that feed frequently upon ectothermic hosts at TNF are Cx. peccator
, Cx. territans
, and Ur. sappharina
EEEV-positive pools from all these species have been collected from the TNF site, indicating that all three species have come into contact with EEEV-infected hosts. Uranotaenia sapphirina
has previously been implicated as a potential bridge vector for EEEV.5
However, the competency of Cx. peccator
and Cx. territans
for EEEV is unknown, and attempts to colonize these species to conduct such vector competency studies have not been successful (Unnasch TR, unpublished). Thus, the role that these mosquito species play in the transmission of EEEV remains to be determined. The role these species play in the dynamics of EEEV transmission will also be determined in part on their feeding preferences. In light of these experiments, which indicate that reptiles may be much more competent hosts of EEEV than the amphibians, Cx. peccator
, which feeds primarily upon reptiles,14
may contribute more to EEEV transmission than Cx territans,
which feeds primarily upon amphibians.14
In addition, Cx. erraticus
has been shown to feed upon reptiles at the TNF site. This is the most common species found at the TNF site and throughout the Southeastern United States21,28–30
and it is believed to represent a major potential vector of EEEV in this region.21,28
It is therefore possible that any or all of these four mosquito species might be responsible for initiating the enzootic transmission cycle through feeding upon EEEV-infected snakes exiting hibernation in the spring.
In conclusion, the data presented above suggest that garter snakes can serve as competent hosts for North American EEEV and that these animals, when infected, can remain viremic through hibernation. This finding, together with the discovery of early season EEEV infections in pools of mosquitoes that feed primarily upon ectothermic hosts provides support to the hypothesis that EEEV may overwinter in seasonal foci in ectothermic vertebrates. These data also reinforce recent studies7
that suggest other animals in addition to birds may play an important role in the dynamics of the EEEV enzootic transmission cycle.