Serological evidence of flaviviruses and alphaviruses in livestock and wildlife in Trinidad.

Seroprevalence rates of selected arboviruses in animal populations in Trinidad were determined using serum samples collected between 2006 and 2009 from horses (n=506), cattle (n=163), sheep (n=198), goats (n=82), pigs (n=184), birds (n=140), rodents (n=116), and other vertebrates (n=23). The sera were screened for antibodies to West Nile virus (WNV), St. Louis encephalitis virus (SLEV), Ilheus virus (ILHV), Bussuquara virus (BSQV), Venezuelan equine encephalitis virus (VEEV), eastern equine encephalitis virus (EEEV), and western equine encephalitis virus (WEEV), using hemagglutination inhibition assay (HIA) and epitope-blocking enzyme-linked immunosorbent assays (ELISA). Antibodies to SLEV were detected in a total of 49 (9.7%) horses, 8 (4.9%) cattle, 1 (1.2%) goat, 2 (1.4%) wild birds, and 3 (2.2%) wild rodents by both methods. In contrast, antibodies to EEEV, VEEV, and WNV were detected only in horses, at rates of 4.3%, 0.8%, and 17.2%, respectively, by ELISA, and IgM capture ELISA was WNV-positive in 3 (0.6%) of these sera. Among locally bred unvaccinated horses that had never left Trinidad, seroprevalence rates against WNV were 12.1% and 17.2% by ELISA and HIA, respectively. The presence of WNV- and SLEV-specific antibodies in a representative sample of horse sera that were both ELISA- and HIA-seropositive was confirmed by plaque reduction neutralization testing (PRNT). Antibodies to ILHV and BSQV were not detected in any of the serum samples tested (i.e., sera from horses, other livestock, and wild birds in the case of ILHV, and wild mammals in the case of BSQV). The data indicate the presence of WNV in Trinidad, and continuing low-level circulation of SLEV, EEEV, and VEEV.

Eilat virus, a unique alphavirus with host range restricted to insects by RNA replication.

Most alphaviruses and many other arboviruses are mosquito-borne and exhibit a broad host range, infecting many different vertebrates including birds, rodents, equids, humans, and nonhuman primates. Consequently, they can be propagated in most vertebrate and insect cell cultures. This ability of arboviruses to infect arthropods and vertebrates is usually essential for their maintenance in nature. However, several flaviviruses have recently been described that infect mosquitoes but not vertebrates, although the mechanism of their host restriction has not been determined. Here we describe a unique alphavirus, Eilat virus (EILV), isolated from a pool of Anopheles coustani mosquitoes from the Negev desert of Israel. Phylogenetic analyses placed EILV as a sister to the Western equine encephalitis antigenic complex within the main clade of mosquito-borne alphaviruses. Electron microscopy revealed that, like other alphaviruses, EILV virions were spherical, 70 nm in diameter, and budded from the plasma membrane of mosquito cells in culture. EILV readily infected a variety of insect cells with little overt cytopathic effect. However, in contrast to typical mosquito-borne alphaviruses, EILV could not infect mammalian or avian cell lines, and viral as well as RNA replication could not be detected at 37 °C or 28 °C. Evolutionarily, these findings suggest that EILV lost its ability to infect vertebrate cells. Thus, EILV seems to be mosquito-specific and represents a previously undescribed complex within the genus Alphavirus. Reverse genetic studies of EILV may facilitate the discovery of determinants of alphavirus host range that mediate disease emergence.

Recombinant alphaviruses are safe and useful serological diagnostic tools.

Serological assays for diagnosis of Venezuelan equine encephalitis virus (VEEV) currently require bio-safety level 3 facilities and select agent certification to produce antigens, reference sera, or viral stocks. Rapid identification of VEEV infection is required to respond to human and equine outbreaks of encephalitis caused by that virus and can be useful for epidemiologic surveillance. Alphavirus (Sindbis)-based recombinant viruses that express VEEV structural proteins are attenuated in animal models, thus representing an alternative to the handling of virulent infectious virus. Virus and viral antigens from recombinant Sindbis/VEE constructs engineered to express structural proteins from multiple VEEV subtypes were evaluated as diagnostic reagents in VEEV-specific serological assays, e.g., plaque reduction neutralization test (PRNT), hemagglutination inhibition (HI) assay, and complement fixation (CF) test. Chimeric viruses were produced efficiently in cell culture and were as effective as the parental virus for identifying infection of humans, horses, and rodents in these serological assays.