Susceptibility of Ae. aegypti (Diptera: Culicidae) to infection with epidemic (subtype IC) and enzootic (subtypes ID, IIIC, IIID) Venezuelan equine encephalitis complex alphaviruses.

To test the hypothesis that enzootic and epidemic Venezuelan equine encephalitis (VEE) complex alphaviruses can infect and be transmitted by Ae. aegypti, we conducted a series of experimental infection studies. One set of experiments tested the susceptibility of geographic strains of Ae. aegypti from Peru and Texas (U.S.A.) for epidemic (subtype IC) and enzootic (subtype ID) strains from Colombia/Venezuela, whereas the second set of experiments tested the susceptibility of Ae. aegypti from Iquitos, Peru, to enzootic VEE complex strains (subtypes ID, IIIC, and IIID) isolated in the same region, at different infectious doses. Experimental infections using artificial bloodmeals suggested that Ae. aegypti mosquitoes, particularly the strain from Iquitos, Peru, is moderately to highly susceptible to all of these VEE complex alphaviruses. The occurrence of enzootic VEE complex viruses circulating endemically in Iquitos suggests the possibility of a dengue-like transmission cycle among humans in tropical cities.

Vector competence of eastern and western forms of Psorophora columbiae (Diptera: Culicidae) mosquitoes for enzootic and epizootic Venezuelan equine encephalitis virus.

Venezuelan equine encephalitis virus (VEEV) continues to circulate enzootically in Mexico with the potential to re-emerge and cause disease in equines and humans in North America. We infected two geographically distinct mosquito populations of eastern Psorophora columbiae form columbiae (Chiapas, Mexico and Texas, United States) and one mosquito population of western Psorophora columbiae form toltecum (California, United States) with epizootic and enzootic IE VEEV and epizootic IAB VEEV. We detected no differences between epizootic and enzootic IE viruses in their ability to infect any of the mosquito populations analyzed, which suggested that neither species selects for epizootic IE viruses. Psorophora columbiae f. columbiae (Texas) were significantly less susceptible to infection by epizootic IE than Ps. columbiae f. columbiae (Mexico). Psorophora columbiae f. toltecum populations were more susceptible than Ps. columbiae f. columbiae populations to epizootic IE and IAB viruses.

Analysis of Venezuelan equine encephalitis virus capsid protein function in the inhibition of cellular transcription.

The encephalitogenic New World alphaviruses, including Venezuelan (VEEV), eastern (EEEV), and western equine encephalitis viruses, constitute a continuing public health threat in the United States. They circulate in Central, South, and North America and have the ability to cause fatal disease in humans and in horses and other domestic animals. We recently demonstrated that these viruses have developed the ability to interfere with cellular transcription and use it as a means of downregulating a cellular antiviral response. The results of the present study suggest that the N-terminal, approximately 35-amino-acid-long peptide of VEEV and EEEV capsid proteins plays the most critical role in the downregulation of cellular transcription and development of a cytopathic effect. The identified VEEV-specific peptide C(VEE)33-68 includes two domains with distinct functions: the alpha-helix domain, helix I, which is critically involved in supporting the balance between the presence of the protein in the cytoplasm and nucleus, and the downstream peptide, which might contain a functional nuclear localization signal(s). The integrity of both domains not only determines the intracellular distribution of the VEEV capsid but is also essential for direct capsid protein functioning in the inhibition of transcription. Our results suggest that the VEEV capsid protein interacts with the nuclear pore complex, and this interaction correlates with the protein's ability to cause transcriptional shutoff and, ultimately, cell death. The replacement of the N-terminal fragment of the VEEV capsid by its Sindbis virus-specific counterpart in the VEEV TC-83 genome does not affect virus replication in vitro but reduces cytopathogenicity and results in attenuation in vivo. These findings can be used in designing a new generation of live, attenuated, recombinant vaccines against the New World alphaviruses.

A novel, rapid assay for detection and differentiation of serotype-specific antibodies to Venezuelan equine encephalitis complex alphaviruses.

An epitope-blocking enzyme-linked immunosorbent assay was developed for the rapid differentiation of serologic responses to enzootic variety IE and ID versus epizootic variety IAB and IC strains of Venezuelan equine encephalitis (VEE) virus. Two monoclonal antibodies that differentially recognize epizootic versus enzootic VEE virus epitopes were used to measure the serotype-specific blocking abilities of antibodies in sera of naturally infected humans, equines, and bovines, as well as in experimentally infected equines. The assay is simple, species-independent, rapid, and sensitive, and will improve surveillance for VEE emergence. It could also be used to determine the epidemic potential of a VEE virus following an intentional introduction for bioterrorism.

Venezuelan equine encephalitis virus, southern Mexico.

Equine epizootics of Venezuelan equine encephalitis (VEE) occurred in the southern Mexican states of Chiapas in 1993 and Oaxaca in 1996. To assess the impact of continuing circulation of VEE virus (VEEV) on human and animal populations, serologic and viral isolation studies were conducted in 2000 to 2001 in Chiapas State. Human serosurveys and risk analyses indicated that long-term endemic transmission of VEEV occurred among villages with seroprevalence levels of 18% to 75% and that medical personnel had a high risk for VEEV exposure. Seroprevalence in wild animals suggested cotton rats as possible reservoir hosts in the region. Virus isolations from sentinel animals and genetic characterizations of these strains indicated continuing circulation of a subtype IE genotype, which was isolated from equines during the recent VEE outbreaks. These data indicate long-term enzootic and endemic VEEV circulation in the region and continued risk for disease in equines and humans.

Natural enzootic vectors of Venezuelan equine encephalitis virus, Magdalena Valley, Colombia.

To characterize the transmission cycle of enzootic Venezuelan equine encephalitis virus (VEEV) strains believed to represent an epizootic progenitor, we identified natural vectors in a sylvatic focus in the middle Magdalena Valley of Colombia. Hamster-baited traps were placed into an active forest focus, and mosquitoes collected from each trap in which a hamster became infected were sorted by species and assayed for virus. In 18 cases, a single, initial, high-titered mosquito pool representing the vector species was identified. These vectors included Culex (Melanoconion) vomerifer (11 transmission events), Cx. (Mel.) pedroi (5 transmissions) and Cx. (Mel.) adamesi (2 transmissions). These results extend the number of proven enzootic VEEV vectors to 7, all of which are members of the Spissipes section of the subgenus Melanoconion. Our findings contrast with previous studies, which have indicated that a single species usually serves as the principal enzootic VEEV vector at a given location.

Glycosaminoglycan binding properties of natural venezuelan equine encephalitis virus isolates.

Equine-virulent, epidemic/epizootic strains of Venezuelan equine encephalitis (VEE) virus (VEEV) arise via mutation of progenitor enzootic strains that replicate poorly in equines. Sequencing studies have implicated positively charged amino acids on the surface of the E2 envelope glycoprotein in the acquisition of equine virulence and viremia potential, suggesting that changes in binding to cell surface glycosaminoglycans (GAGs) may mediate VEE emergence. Therefore, we evaluated the binding of natural enzootic and epizootic VEEV isolates to Chinese hamster ovary (CHO) cells expressing normal, high levels of GAGs as well as to mutant CHO cells lacking GAG expression. Binding to GAGs was not consistently associated with the epizootic phenotype, and cell culture passages resulted in increased GAG binding. The low levels of GAG binding exhibited by some low-passage, equine-virulent subtype IC VEEV strains indicate that the positive-charge E2 mutations implicated in VEE subtype IC emergence are not artifacts of laboratory passage and suggest that GAG binding does not play a major role in mediating VEE emergence. The increased GAG binding exhibited by VEEV strain CPA201 from the 1993 Mexican epizootic, when compared to that of closely related enzootic subtype IE strains, was shown to result from a Glu-to-Lys mutation at position 117 of the E2 envelope glycoprotein.

Contrasting sylvatic foci of Venezuelan equine encephalitis virus in northern South America.

The ecology of Venezuelan equine encephalitis (VEE) virus transmission was compared at three enzootic foci: two forest sites in the Catatumbo region of western Venezuela that have yielded small numbers of virus isolates since the 1970s, and another focus in the middle Magdalena Valley of Colombia that has consistently yielded many VEE virus isolates. Our results demonstrated dramatic differences in VEE virus isolation rates from sentinel hamsters, as well as differences in mosquito species composition and captured mammals with antibodies to VEE virus, between the Colombian and Venezuelan study sites. The higher isolation rate of enzootic VEE virus in the Colombian site was associated with a more abundant fauna of spiny rats (Proechimys spp.), known reservoir hosts of enzootic VEE virus. Mosquito collections demonstrated that the Colombian forest had a higher mosquito diversity and species evenness than either of the Venezuelan forests. The Colombian focus was especially richer in its Culex (Melanoconion) spp. fauna, a subgenus that includes all proven enzootic vectors for VEE virus. Our results suggest that the greater abundance, diversity, and stability of enzootic vector populations, combined with the greater density of rodent reservoir hosts, explains the higher levels of VEE virus circulation in the Colombian focus compared with the Venezuelan forests.