As human populations continue to expand into rural environments, the incidence of emerging and re-emerging zoonotic pathogens will continue to climb. This has already been observed with other arboviral viruses, such as chikungunya, dengue, yellow fever, and Japanese encephalitis viruses 
. Similar trends have also been observed with enzootic strains of VEEV that have caused endemic disease as well as outbreaks in Peru, Central America, and Mexico 
. Historically, studies of VEEV emergence have focused on epidemic strains within subtypes IAB and IC; however, enzootic ID and IE strains can also cause a large burden of endemic disease, which can often be misdiagnosed as dengue fever 
. Recent studies have also shown that the primary mosquito vector of enzootic IE, C. taeniopus
, can be an efficient vector of newly emerged epizootic IE strains in Mexico 
. Considering the growing risk of enzootic VEEV strains in causing human disease, it is important to understand the determinants and dynamics for viral infection of the primary enzootic vector, which we hypothesize to be different from what is known about the epizootic virus-vector interaction.
We first examined the genetic determinants of infection and dissemination utilizing chimeric viruses to analyze the molecular determinants for VEEV specificity to the enzootic mosquito vector, C. taeniopus. We used two viruses with distinct phenotypes for these chimeras, to help identify the major genome regions that contribute to specific infection of the enzootic vector. However, because these viruses have such a wide genetic divergence (10.2% at the amino acid level), extrapolation of this method to clarify the roles of each gene during enzootic mosquito infection could be prone to bias from incompatibilities between open reading frames within each chimera (). Therefore, to ensure that chimerization did not result in general attenuation of virus replication, the parental and chimeric strains were evaluated using in vitro replication curves on Vero and C6/36 cell monolayers; no replication deficiencies were observed. It was noted that the replication in mosquito cells was different from what was observed in the in vivo mosquito model in that the parental IAB virus, which showed no deficiencies in vitro, was unable to infect the in vivo model. Similarly, the differences observed between the IE parental and the four chimeras in the in vivo model were not demonstrated in the in vitro model. These results emphasize the importance of using an in vivo mosquito model to detect differences in viral replication, which may not be detected in a mosquito cell line.
Identity percentage between the IAB TrD and 68U201 genomes.
We orally exposed C. taeniopus mosquitoes to varying doses of two parental strains, subtype IAB TrD and subtype IE 68U201, as well as four chimeric strains, IAB/IE/IAB, IE/IAB/IE, IAB/IE, and IE/IAB, and evaluated the role of the nonstructural and structural protein genes and the 3′ UTR as determinants of infection and dissemination in C. taeniopus. We hypothesized that, unlike the epizootic virus strains and their vectors, the genetic determinants for enzootic infection include multiple genes and they are not limited to a single region in the structural portion of the genome. Our data supported this hypothesis, as all four chimeras were able to infect and disseminate in C. taeniopus, albeit at rates lower than the wild-type IE parental strain. We included multiple replicates within each viral group to compensate for variations within the mosquito colony. If the E2 or the structural regions were the primary determinants of infection and dissemination, those chimeras with IE-derived structural regions would have infected mosquitoes at a higher rate than those chimeras with IAB-derived structural regions, and this was not observed.
We anticipated that the chimeras with mismatched 3′UTR regions would show diminished infection and dissemination rates based on previous alphavirus studies examining the effects of mismatched cis
-acting elements 
; however, our IE/IAB chimera showed a significantly higher rate of infection than that of the other three chimeras. This suggests that the 3′ UTR plays some role in infection of the enzootic vector. A closer examination of the effect of the 3′ UTR on infection showed that the chimera with IAB structural genes and IAB 3′ UTR (IE/IAB) had the highest infection rate, while the chimeras with a mixed structural-3′ UTR makeup (IE/IAB/IE or IAB/IE/IAB) had intermediate infection abilities, and the chimera with IE in the structural and the 3′ UTR (IAB/IE) actually had the lowest rate of infection. This suggests that 3′ UTR acts in concert with other portions of the genome, although it is unclear which specific regions are important for this cooperative effect. These potential interactions should be further explored with 3′UTR-specific chimeras, such as a IAB virus backbone with a IE derived 3′UTR and a IE backbone with a IAB-derived 3′ UTR. While the role of these regions was not mirrored by in vitro
mosquito infections, our C6/36 data were based on cells from A. albopictus
, which in laboratory experiments has been shown to be equally susceptible to epizootic IC and enzootic ID VEEV strains 
. Previous studies examining chimeras between Ross River virus (RRV) and Sindbis virus (SINV), two genetically distant alphaviruses, have shown that mismatched 3′ UTR regions can result in depressed RNA synthesis in vitro
, although the effects on replication in vivo
have not been examined 
. However, studies of chimeras between more closely related alphaviruses, such as o'nyong-nyong (ONNV) and chikungunya (CHIKV) viruses, indicate that chimerization does not have a deleterious affect on the infection of the CHIKV mosquito vector, A. aegypti
. There was also no indication that mismatched 3′ UTRs altered infection rates 
There were no statistical differences in the infection rates between chimeras IAB/IE, IAB/IE/IAB, and IE/IAB/IE, indicating that both the structural and nonstructural protein regions of the enzootic virus play a role in vector infection, as none of the chimeras displayed infection rates as high as the parental IE strain. However, we observed a trend in which the two chimeras with IE-derived nonstructural protein genes showed higher rates of infection at higher doses. Specifically, the chimeras with IE-derived nonstructural protein genes reached 100% infection at the highest doses, while the chimeras with IAB-derived nonstructural protein gene regions never reached 100% infection even at the highest doses. The diminished infection of all chimeras implies that there are multiple determinants of infection that reside in different genome regions and may act synergistically. Our results show that the determinants for infection of the enzootic vector do not reside solely in the structural protein genes, specifically not only in the E2 glycoprotein of the genome, which supports our hypothesis that infection determinants for VEEV in the enzootic mosquito vector relies on both structural or nonstructural protein regions of the genome. Interestingly, our findings suggest that in the enzootic model, the nonstructural elements are stronger determinants of vector infection.
We also hypothesized that the characteristics of initial midgut infection of the enzootic mosquito vector would be inherently different than those used by the epizootic virus in A. taeniorhynchus
. To test this hypothesis, we exposed the enzootic vector, C. taeniopus
, to replicon particles generated from a subtype IE enzootic strain. Examination of the initial sites of infection in the midgut indicated multiple locations in the abdominal portion with no predilection for either the anterior or the posterior region; we detected no infection of the cardial epithelium at the midgut/foregut junction. Similar to what was observed in A. taeniorhynchus
, a clear response was observed between the oral dose and the number of infected midgut cells, although the ID50
for C. taeniopus
was lower and the maximum number of infected cells was higher than the 100 susceptible A. taeniorhynchus
cells previously estimated 
. The greater number of infected cells (>1700) in C. taeniopus
following high oral doses indicates that a larger number of its midgut epithelial cells is susceptible to VEEV IE infection compared to A. taeniorhynchus
and VEEV IC. This observation, in conjunction with our observation of no co-infected midgut cells in the mixed replicon experiments, supports the hypothesis that the population size of enzootic VEEV virions during initial infection of the midgut is not severely restricted by a limited number of susceptible C. taeniopus
epithelial cells. The average population of cells infected by the 68UGFP replicon at the highest dose did not differ from the average number of cells infected by the 68UCFP replicon. This suggests that there is no effect of co-exposure on individual particle infection rates. Utilizing the same methods, previous studies in the epizootic VEEV/mosquito model found an average of 26 midgut cells co-infected with two replicons, which was greater than what we observed in the enzootic model. This indicates that the initial infection of the enzootic vector differs from that of the epizootic VEEV strain. Using the Poisson distribution and given the epizootic data (a model with a small population of susceptible cells), we determined the probability of observing less than a single co-infected cell out of our five C. taeniopus
midgut replicates to be 5.1×10−12
, indicating an extremely low likelihood that there is a subpopulation of midgut epithelial cells with enhanced susceptibility.
Our studies illustrate the contrast in the virus-vector interactions between the enzootic and epizootic VEEV cycles. Not only do these interactions persist in different ecological cycles and infect different species of mosquitoes, but they also behave differently within their respective vectors. This difference may be explained by the dissimilar selective pressures that are exerted on each subtype during transmission. For example, epizootic viruses produce a very high level of viremia in equids, which facilitates VEEV transmission by epizootic vectors even if only a small population of their midgut cells are initially infected. However, the highly susceptible enzootic vector, which appears to have a greater number of susceptible midget cells that can be initially infected even after small oral doses, can transmit efficiently among populations of rodents that develop only moderate viremia titers 
As the growing impact of enzootic VEEV on human health is becoming more apparent, especially after the recent emergence of epizootic-like IE strains, understanding how these viruses interact with vectors is critical to estimating their threat to human health and for refining public health prevention strategies as well as developing vaccines. For instance, the design strategy of a vaccine that is protective against epizootic and enzootic strains that are currently causing human disease must also consider mosquito vectors that could potentially acquire and transmit should a vaccinee become viremic. If the epizootic vector only has a few susceptible midgut cells and is examined for competence for a given vaccine strain, it may appear to be incompetent. However, the same vaccine may be able to establish an infection in the enzootic vector. Considering that the determinants for infection appear to differ between the two vector types, vaccine strains that are derived from epizootic VEEV and depend on the elimination of mosquito infection may not necessarily reflect how infectious these vaccine candidates would be for enzootic vectors. As enzootic habitats are encroached upon and enzootic cycles gain close proximity to epizootic habitats, it is essential to consider the contribution of enzootic vectors to viral emergence and the potential introduction of vaccine strains into natural cycles.