Evolutionary patterns of eastern equine encephalitis virus in North versus South America suggest ecological differences and taxonomic revision.

The eastern equine encephalitis (EEE) complex consists of four distinct genetic lineages: one that circulates in North America (NA EEEV) and the Caribbean and three that circulate in Central and South America (SA EEEV). Differences in their geographic, pathogenic, and epidemiologic profiles prompted evaluation of their genetic diversity and evolutionary histories. The structural polyprotein open reading frames of all available SA EEEV and recent NA EEEV isolates were sequenced and used in evolutionary and phylogenetic analyses. The nucleotide substitution rate per year for SA EEEV (1.2 x 10(-4)) was lower and more consistent than that for NA EEEV (2.7 x 10(-4)), which exhibited considerable rate variation among constituent clades. Estimates of time since divergence varied widely depending upon the sequences used, with NA and SA EEEV diverging ca. 922 to 4,856 years ago and the two main SA EEEV lineages diverging ca. 577 to 2,927 years ago. The single, monophyletic NA EEEV lineage exhibited mainly temporally associated relationships and was highly conserved throughout its geographic range. In contrast, SA EEEV comprised three divergent lineages, two consisting of highly conserved geographic groupings that completely lacked temporal associations. A phylogenetic comparison of SA EEEV and Venezuelan equine encephalitis viruses (VEEV) demonstrated similar genetic and evolutionary patterns, consistent with the well-documented use of mammalian reservoir hosts by VEEV. Our results emphasize the evolutionary and genetic divergences between members of the NA and SA EEEV lineages, consistent with major differences in pathogenicity and ecology, and propose that NA and SA EEEV be reclassified as distinct species in the EEE complex.

Cotton rats and house sparrows as hosts for North and South American strains of eastern equine encephalitis virus.

Eastern equine encephalitis virus (EEEV; family Togaviridae, genus Alphavirus) is an arbovirus that causes severe disease in humans in North America and in equids throughout the Americas. The enzootic transmission cycle of EEEV in North America involves passerine birds and the ornithophilic mosquito vector, Culiseta melanura, in freshwater swamp habitats. However, the ecology of EEEV in South America is not well understood. Culex (Melanoconion) spp. mosquitoes are considered the principal vectors in Central and South America; however, a primary vertebrate host for EEEV in South America has not yet been identified. Therefore, to further assess the reservoir host potential of wild rodents and wild birds, we compared the infection dynamics of North American and South American EEEV in cotton rats (Sigmodon hispidus) and house sparrows (Passer domesticus). Our findings suggested that each species has the potential to serve as amplification hosts for North and South America EEEVs.

Experimental infection of Aedes sollicitans and Aedes taeniorhynchus with two chimeric Sindbis/Eastern equine encephalitis virus vaccine candidates.

Two chimeric vaccine candidates for Eastern equine encephalitis virus (EEEV) were developed by inserting the structural protein genes of either a North American (NA) or South American (SA) EEEV into a Sindbis virus (SINV) backbone. To assess the effect of chimerization on mosquito infectivity, experimental infections of two potential North American bridge vectors of EEEV, Aedes sollicitans and Ae. taeniorhynchus, were attempted. Both species were susceptible to oral infection with all viruses after ingestion of high titer blood meals of ca. 7.0 log(10) plaque-forming units/mL. Dissemination rates for SIN/NAEEEV (0 of 56) and SIN/SAEEEV (1 of 54) were low in Ae. taeniorhynchus and no evidence of transmission potential was observed. In contrast, the chimeras disseminated more efficiently in Ae. sollicitans (19 of 68 and 13 of 57, respectively) and were occasionally detected in the saliva of this species. These results indicate that chimerization of the vaccine candidates reduces infectivity. However, its impact on dissemination and potential transmission is mosquito species-specific.

Infection and dissemination of Venezuelan equine encephalitis virus in the epidemic mosquito vector, Aedes taeniorhynchus.

The mosquito Aedes taeniorhynchus is an important epidemic vector of Venezuelan equine encephalitis virus (VEEV), but detailed studies of its infection are lacking. We compared infection by an epidemic VEEV strain to that by an enzootic strain using virus titrations, immunohistochemistry, and a virus expressing the green fluorescent protein. Ae. taeniorhynchus was more susceptible to the epidemic strain, which initially infected the posterior midgut and occasionally the anterior midgut and cardia. Once dissemination beyond the midgut occurred, virus was present in nearly all tissues. Transmission of the epidemic strain to mice was first detected 4 days after infection. In contrast, the enzootic strain did not efficiently infect midgut cells but replicated in muscles and nervous tissue on dissemination. Because VEEV emergence can depend on adaptation to epidemic vectors, these results show that epidemic/enzootic strain comparisons not only comprise a useful model system to study alphavirus transmission by mosquitoes, but also have important public health implications.

A strategy to estimate unknown viral diversity in mammals.

UNLABELLED: The majority of emerging zoonoses originate in wildlife, and many are caused by viruses. However, there are no rigorous estimates of total viral diversity (here termed "virodiversity") for any wildlife species, despite the utility of this to future surveillance and control of emerging zoonoses. In this case study, we repeatedly sampled a mammalian wildlife host known to harbor emerging zoonotic pathogens (the Indian Flying Fox, Pteropus giganteus) and used PCR with degenerate viral family-level primers to discover and analyze the occurrence patterns of 55 viruses from nine viral families. We then adapted statistical techniques used to estimate biodiversity in vertebrates and plants and estimated the total viral richness of these nine families in P. giganteus to be 58 viruses. Our analyses demonstrate proof-of-concept of a strategy for estimating viral richness and provide the first statistically supported estimate of the number of undiscovered viruses in a mammalian host. We used a simple extrapolation to estimate that there are a minimum of 320,000 mammalian viruses awaiting discovery within these nine families, assuming all species harbor a similar number of viruses, with minimal turnover between host species. We estimate the cost of discovering these viruses to be ~$6.3 billion (or ~$1.4 billion for 85% of the total diversity), which if annualized over a 10-year study time frame would represent a small fraction of the cost of many pandemic zoonoses. IMPORTANCE: Recent years have seen a dramatic increase in viral discovery efforts. However, most lack rigorous systematic design, which limits our ability to understand viral diversity and its ecological drivers and reduces their value to public health intervention. Here, we present a new framework for the discovery of novel viruses in wildlife and use it to make the first-ever estimate of the number of viruses that exist in a mammalian host. As pathogens continue to emerge from wildlife, this estimate allows us to put preliminary bounds around the potential size of the total zoonotic pool and facilitates a better understanding of where best to allocate resources for the subsequent discovery of global viral diversity.

Isolation and characterization of sylvatic mosquito-borne viruses in Trinidad: enzootic transmission and a new potential vector of Mucambo virus.

Mosquito surveillance was carried out in three forested regions of Trinidad during July 2007-March 2009. A total of 185,397 mosquitoes representing at least 46 species was collected, divided into pools of 1-50 mosquitoes according to species and sex, and screened for arboviruses using cytopathic effect assays on Vero cell monolayers. Eighty-five viruses were isolated, including members of the genera Alphavirus (Mucambo virus; MUCV) and Orthobunyavirus (Caraparu, Oriboca, Bimiti, and Wyeomyia viruses). Species of the Culex subgenus Melanoconion accounted for 56% of the total number of mosquitoes collected and 97% of the viruses isolated; Cx. (Mel.) portesi accounted for 92% of virus isolations. Our results also implicate for the first time Aedes (Ochlerotatus) hortator as a potential vector of MUCV. Phylogenetic analyses of 43 MUCV strains suggest population subdivision within Trinidad, consistent with the hypothesis of enzootic maintenance in localized rodent populations.

Isolation and phylogenetic analysis of Mucambo virus (Venezuelan equine encephalitis complex subtype IIIA) in Trinidad.

In the 1950s and 1960s, alphaviruses in the Venezuelan equine encephalitis (VEE) antigenic complex were the most frequently isolated arboviruses in Trinidad. Since then, there has been very little research performed with these viruses. Herein, we report on the isolation, sequencing, and phylogenetic analyses of Mucambo virus (MUCV; VEE complex subtype IIIA), including 6 recently isolated from Culex (Melanoconion) portesi mosquitoes and 11 previously isolated in Trinidad and Brazil. Results show that nucleotide and amino acid identities across the complete structural polyprotein for the MUCV isolates were 96.6-100% and 98.7-100%, respectively, and the phylogenetic tree inferred for MUCV was highly geographically- and temporally-structured. Bayesian analyses suggest that the sampled MUCV lineages have a recent common ancestry of approximately 198 years (with a 95% highest posterior density (HPD) interval of 63-448 years) prior to 2007, and an overall rate of evolution of 1.28 x 10(-4) substitutions/site/yr.