Complex Epidemiological Dynamics of Eastern Equine Encephalitis Virus in Florida.

Eastern equine encephalitis virus (EEEV) infection results in high mortality in infected horses and humans. Florida has been identified as an important source of EEEV epidemics to other states in the United States. In this study, we further characterized the epidemiological and evolutionary dynamics of EEEV in Florida. Epidemiological analysis of sentinel chicken seroconversion rates to EEEV infections during 2005-2016 suggested significant seasonality of EEEV activity in Florida. We observed significant annual activity of EEEV in the North and North Central regions, with little significant seasonality in the Panhandle region. Phylogenetic analysis of complete EEEV genome sequences from different host sources and regions in Florida during 1986-2014 revealed extensive genetic diversity and spatial dispersal of the virus within Florida and relatively more clustering of the viruses in the Panhandle region. We found no significant association between EEEV genetic variation and host source. Overall, our study revealed a complex epidemiological dynamic of EEEV within Florida, implicating the Panhandle region as a possible source of the virus with sustained year-round transmission. These findings will help in implementing targeted control measures that can have the most impact in reducing or eliminating EEEV and other mosquito-borne viral infections within Florida and in the rest of the United States.

Risk of exposure to eastern equine encephalomyelitis virus increases with the density of northern cardinals.

For a variety of infectious diseases, the richness of the community of potential host species has emerged as an important factor in pathogen transmission, whereby a higher richness of host species is associated with a lowered disease risk. The proposed mechanism driving this pattern is an increased likelihood in species-rich communities that infectious individuals will encounter dead-end hosts. Mosquito-borne pathogen systems potentially are exceptions to such "dilution effects" because mosquitoes vary their rates of use of vertebrate host species as bloodmeal sources relative to host availabilities. Such preferences may violate basic assumptions underlying the hypothesis of a dilution effect in pathogen systems. Here, we describe development of a model to predict exposure risk of sentinel chickens to eastern equine encephalitis virus (EEEV) in Walton County, Florida between 2009 and 2010 using avian species richness as well as densities of individual host species potentially important to EEEV transmission as candidate predictor variables. We found the highest support for the model that included the density of northern cardinals, a highly preferred host of mosquito vectors of EEEV, as a predictor variable. The highest-ranking model also included Culiseta melanura abundance as a predictor variable. These results suggest that mosquito preferences for vertebrate hosts influence pathogen transmission.

Spatial epidemiology of eastern equine encephalitis in Florida.

BACKGROUND: Eastern Equine Encephalitis virus (EEEV) is an alphavirus with high pathogenicity in both humans and horses. Florida continues to have the highest occurrence of human cases in the USA, with four fatalities recorded in 2010. Unlike other states, Florida supports year-round EEEV transmission. This research uses GIS to examine spatial patterns of documented horse cases during 2005-2010 in order to understand the relationships between habitat and transmission intensity of EEEV in Florida. METHODS: Cumulative incidence rates of EEE in horses were calculated for each county. Two cluster analyses were performed using density-based spatial clustering of applications with noise (DBSCAN). The first analysis was based on regional clustering while the second focused on local clustering. Ecological associations of EEEV were examined using compositional analysis and Euclidean distance analysis to determine if the proportion or proximity of certain habitats played a role in transmission. RESULTS: The DBSCAN algorithm identified five distinct regional spatial clusters that contained 360 of the 438 horse cases. The local clustering resulted in 18 separate clusters containing 105 of the 438 cases. Both the compositional analysis and Euclidean distance analysis indicated that the top five habitats positively associated with horse cases were rural residential areas, crop and pastureland, upland hardwood forests, vegetated non-forested wetlands, and tree plantations. CONCLUSIONS: This study demonstrates that in Florida tree plantations are a focus for epizootic transmission of EEEV. It appears both the abundance and proximity of tree plantations are factors associated with increased risk of EEE in horses and therefore humans. This association helps to explain why there is are spatially distinct differences in the amount of EEE horse cases across Florida.

Habitat associations of eastern equine encephalitis transmission in Walton County Florida.

Eastern Equine Encephalitis virus (EEEV; family Togaviridae, genus Alphavirus) a highly pathogenic mosquito-borne virus is endemic to eastern North America. The ecology of EEEV in Florida differs from that in other parts of the United States; EEEV in the northeastern United States is historically associated with freshwater wetlands. No formal test of habitat associations of EEEV in Florida has been reported. Geographical Information Sciences (GIS) was used in conjunction with sentinel chicken EEEV seroconversion rate data as a means to examine landscape features associated with EEEV transmission in Walton County, FL. Sentinel sites were categorized as enzootic, periodically enzootic, and negative based on the number of chicken seroconversions to EEEV from 2005 to 2009. EEEV transmission was then categorized by land cover usage using Arc GIS 9.3. The land classification data were analyzed using the Kruskal-Wallis test for each land use class to determine which habitats may be associated with virus transmission as measured by sentinel chicken seroconversion rates. The habitat class found to be most significantly associated with EEEV transmission was tree plantations. The ecological factor most commonly associated with reduced levels of EEEV transmission was vegetated nonforest wetlands. Culiseta melanura (Coquillett), the species generally considered to be the major enzootic EEEV vector, was relatively evenly distributed across all habitat classes, while Aedes vexans (Meigen) and Anopheles crucians Weidemann were most commonly associated with tree plantation habitats.

Phylogenetic analysis of eastern equine encephalitis virus isolates from Florida.

Florida has the highest degree of endemicity for eastern equine encephalitis virus (EEEV) of any state in the United States and is the only state with year-round transmission of EEEV. To further understand the viral population dynamics in Florida, the genome sequence of six EEEV isolates from central Florida were determined. These data were used to identify the most polymorphic regions of the EEEV genome from viruses isolated in Florida. The sequence of these polymorphic regions was then determined for 18 additional Florida isolates collected in four geographically distinct regions over a 20-year period. Phylogenetic analyses of these data suggested a rough temporal association of the Florida isolates, but no clustering by region or by source of the isolate. Some clustering of northeastern isolates with Florida isolates was seen, providing support for the hypothesis that Florida serves as a reservoir for the periodic introduction of EEEV into the northeastern United States.

Removal of species constraints in antibody detection.

Serum antibodies from myriad species, particularly birds, can provide key information regarding the transmission and the expansion of the territory of emerging pathogens. Expedient antibody analysis is constrained by a lack of species-specific reagents, a deficiency potentially highlighted by the recent swine-origin influenza A virus (H1N1) outbreak. Available methodologies present difficulties that discourage thorough serologic monitoring of potential disease vectors or hosts. Rapid high-throughput procedures that combined serum amine labeling via biotinylation, contaminant removal, and microsphere-based immunoassays for antibodies to three arboviruses were developed. Agent-specific adaptations of this simple format should facilitate expanded surveillance and diagnostic capabilities regarding pathogens of human and veterinary importance.

Correlations between microbial indicators, pathogens, and environmental factors in a subtropical estuary.

The objective of this study was to evaluate whether indicator microbes and physical-chemical parameters were correlated with pathogens within a tidally influenced Estuary. Measurements included the analysis of physical-chemical parameters (pH, salinity, temperature, and turbidity), measurements of bacterial indicators (enterococci, fecal coliform, Escherichia coli, and total coliform), viral indicators (somatic and MS2 coliphage), viral pathogens (enterovirus by culture), and protozoan pathogens (Cryptosporidium and Giardia). All pathogen results were negative with the exception of one sample which tested positive for culturable reovirus (8.5MPN/100L). Notable physical-chemical parameters for this sample included low salinity (<1ppt) and high water temperature (31 degrees C). Indicator bacteria and indicator virus levels for this sample were within average values typically measured within the study site and were low in comparison with levels observed in other freshwater environments. Overall results suggest that high levels of bacterial and viral indicators were associated with low salinity sites.

Isolation of genotype V St. Louis encephalitis virus in Florida.

We isolated and characterized St. Louis encephalitis virus (SLEV) from cloacal swabs of naturally exposed adult sentinel chickens in 2006. Phylogenetic analysis of SLEV strains isolated in Florida indicated that Brazilian SLEV circulated in 1972 and 2006; lineages were VA and VB.

Phylogenetic analysis of North American West Nile virus isolates, 2001-2004: evidence for the emergence of a dominant genotype.

The distribution of West Nile virus has expanded in the past 6 years to include the 48 contiguous United States and seven Canadian provinces, as well as Mexico, the Caribbean islands, and Colombia. The suggestion of the emergence of a dominant genetic variant has led to an intensive analysis of isolates made across North America. We have sequenced the pre-membrane and envelope genes of 74 isolates and the complete genomes of 25 isolates in order to determine if a dominant genotype has arisen and to better understand how the virus has evolved as its distribution has expanded. Phylogenetic analyses revealed the continued presence of genetic variants that group in a temporally and geographically dependent manner and provide evidence that a dominant variant has emerged across much of North America. The implications of these findings are discussed as they relate to transmission and spread of the virus in the Western Hemisphere.

Persistence of antibodies to West Nile virus in naturally infected rock pigeons (Columba livia).

Wild caught rock pigeons (Columba livia) with antibodies to West Nile virus were monitored for 15 months to determine antibody persistence and compare results of three serologic techniques. Antibodies persisted for the entire study as detected by epitope-blocking enzyme-linked immunosorbent assay and plaque reduction neutralization test. Maternal antibodies in squabs derived from seropositive birds persisted for an average of 27 days.

Arboviral infection in two species of wild jays (Aves: Corvidae): evidence for population impacts.

We examined the prevalence of antibodies to three mosquito-borne arboviruses in blue jays, Cyanocitta cristata, and Florida scrub-jays, Aphelocoma coerulescens, to identify the effects on host survival, the influence of sex and age on infection, and the temporal patterns of antibody prevalence. Blood samples from 306 blue jays and 219 Florida scrub-jays were collected at Archbold Biological Station (Lake Placid, FL) from April 1994 through December 1995. Sera were analyzed for hemagglutination-inhibition antibody to eastern equine encephalitis (EEE) and St. Louis encephalitis (SLE) viruses, and neutralizing antibodies to EEE, Highlands J (HJ), and SLE viruses. Overall, 31.4% of blue jay samples and 22.1% of scrub-jay samples had antibodies to EEE. Antibodies to HJ were detected in slightly >15% of samples in each jay species, and SLE was detected in <3% of the samples in each jay species. A single EEE virus isolation was made from the blood of an 11-d-old scrub-jay nestling. Survival of adult blue jays seropositive to EEE was significantly lower than that of seronegative birds based on resight rates, but infection did not seem to affect survival of adult or juvenile Florida scrub-jays.

Surveillance results from the first West Nile virus transmission season in Florida, 2001.

After West Nile virus (WNV) was first detected in Florida in July 2001, intensive surveillance efforts over the following five months uncovered virus activity in 65 of the state's 67 counties with 1,106 wild birds, 492 horses, 194 sentinel chickens, and 12 people found infected with the virus. Thirteen of 28 mosquito isolations came from Culex mosquitoes. As seen in the northeastern United States, wild bird mortality was the most sensitive surveillance method. However, unlike the predominantly urban 1999 and 2000 epizootics, the Florida transmission foci were rural with most activity detected in the northern part of the state. All human cases were preceded by the detection of WNV in animals; however, only eight of the twelve cases were preceded by reports of WNV activity in the county of residence. West Nile virus-positive animals detected by multiple surveillance systems preceded seven of these cases by two weeks or more.

West Nile virus infection rates in Culex nigripalpus (Diptera: Culicidae) do not reflect transmission rates in Florida.

We describe the first documented field transmission of West Nile (WN) virus by a North American mosquito. WN was first detected in northern Florida in 2001. An intensive mosquito trapping and surveillance program was conducted in this region for four nights to assess mosquito transmission of WN. Four mosquito traps, each with a single sentinel chicken, were placed at five different locations on each of four nights. A total of 11,948 mosquitoes was collected, and 14 mosquito pools were found to contain WN, giving a minimum infection rate between 1.08 and 7.54 per 1,000. Only one of the 80 sentinel chickens seroconverted to WN, demonstrating a single mosquito transmission event during the study and a mosquito transmission rate of between 0.8 and 1 per 1,000. Culex nigripalpus Theobald was responsible for WN transmission to the sentinel chicken, although both Cx. nigripalpus and Culex quinquefasciatus Say were found infected with WN. Mosquito transmission rates are reported in this study for the first time for a WN outbreak. This information is essential to determine risk of human and animal infection.

Mosquitoes infected with West Nile virus in the Florida Keys, Monroe County, Florida, USA.

More than 30,000 mosquitoes in 22 species or species groups were collected from the Florida Keys, Monroe County, FL, USA, in dry ice-baited light and gravid traps. Dry ice-baited traps collected more mosquitoes than did gravid traps. West Nile virus was detected in pools of Anopheles atropos Dyar & Knab, Deinocerites cancer Theobald, and Ochlerotatus taeniorhynchus (Wiedemann).