Southern California remains an important focus of West Nile virus (WNV) activity, with persistently elevated incidence after invasion by the virus in 2003 and subsequent amplification to epidemic levels in 2004. Eco-epidemiological studies of vectors-hosts-pathogen interactions are of paramount importance for better understanding of the transmission dynamics of WNV and other emerging mosquito-borne arboviruses. We investigated vector-host interactions and host-feeding patterns of 531 blood-engorged mosquitoes in four competent mosquito vectors by using a polymerase chain reaction (PCR) method targeting mitochondrial DNA to identify vertebrate hosts of blood-fed mosquitoes. Diagnostic testing by cell culture, real-time reverse transcriptase-PCR, and immunoassays were used to examine WNV infection in blood-fed mosquitoes, mosquito pools, dead birds, and mammals. Prevalence of WNV antibodies among wild birds was estimated by using a blocking enzyme-linked immunosorbent assay. Analyses of engorged Culex quinquefasciatus revealed that this mosquito species acquired 88.4% of the blood meals from avian and 11.6% from mammalian hosts, including humans. Similarly, Culex tarsalis fed 82% on birds and 18% on mammals. Culex erythrothorax fed on both birds (59%) and mammals (41%). In contrast, Culex stigmatosoma acquired all blood meals from avian hosts. House finches and a few other mostly passeriform birds served as the main hosts for the blood-seeking mosquitoes. Evidence of WNV infection was detected in mosquito pools, wild birds, dead birds, and mammals, including human fatalities during the study period. Our results emphasize the important role of house finches and several other passeriform birds in the maintenance and amplification of WNV in southern California, with Cx. quinquefasciatus acting as both the principal enzootic and “bridge vector” responsible for the spillover of WNV to humans. Other mosquito species, such as Cx. tarsalis and Cx. stigmatosoma, are important but less widely distributed, and also contribute to spatial and temporal transmission of WNV in southern California.

Eastern equine encephalitis (EEE) virus is a highly pathogenic mosquito-borne zoonosis that is responsible for outbreaks of severe disease in humans and equines, resulting in high mortality or severe neurological impairment in most survivors. In the northeastern United States, EEE virus is maintained in an enzootic cycle involving the ornithophilic mosquito , Culiseta melanura (Coquillett) and passerine birds in freshwater swamp habitats. To evaluate the role of Cs. melanura and Culiseta morsitans (Theobald) in recent episodes of EEE virus activity in Massachusetts, we collected blood-fed mosquitoes between June, 2007, and October, 2008, from virus foci in 6 counties, and identified the source of blood meals by PCR amplification of mitochondrial cytochrome b gene and sequencing. Analysis of 529 Cs. melanura and 25 Cs. morsitans revealed that nearly 99% and 96% of mosquitoes, respectively, acquired blood meals solely from avian hosts. American Robin, Turdus migratorius Linnaeus was identified as the most common vertebrate host for Cs. melanura (21.7%, n =115), followed by Tufted Titmouse, Baeolophus bicolor (L.) (8.7%, n =46), Black-capped Chickadee, Poecile atricapillus (L.) (8.5%, n =45), Scarlet Tanager, Piranga olivacea (Gmelin) (6.8%, n =36), Field Sparrow, Spizella pusilla (Wilson) (6.2%, n =33), Northern Cardinal, Cardinalis cardinalis (L.) (5.7%, n =30), and other mostly Passeriformes birds. Mammalian-derived blood meals were identified as white-tailed deer, Odocoileus virginianus Zimmermann, domestic cow, Bos taurus L., and human, Homo sapiens L. There were 4 isolations of EEE virus, West Nile virus, and Highland J virus from Cs. melanura . Our results in conjunction with other lines of evidence, including reservoir competency, prevalence of antibody, and infection in nature, suggest that the American Robin, Tufted Titmouse, Black-capped Chickadee, and a few other passerine birds may play key roles in supporting EEE virus transmission in Massachusetts. Infrequent blood feeding of Cs. melanura on mammalian hosts, including humans, also indicates that this mosquito may occasionally contribute to epidemic/epizootic transmission of EEE virus in this region.

Native and invasive tick species pose a serious public health concern in the United States. Range expansion of several medically important tick species has resulted in an increasing number of communities at risk for exposure to ticks and tick-borne pathogens.

Eastern equine encephalitis (EEE) virus (Togaviridae, Alphavirus) is a highly pathogenic mosquito-borne zoonosis that is responsible for occasional outbreaks of severe disease in humans and equines, resulting in high mortality and neurological impairment in most survivors. In the past, human disease outbreaks in the northeastern U.S. have occurred intermittently with no apparent pattern; however, during the last decade we have witnessed recurring annual emergence where EEE virus activity had been historically rare, and expansion into northern New England where the virus had been previously unknown. In the northeastern U.S., EEE virus is maintained in an enzootic cycle involving the ornithophagic mosquito, Culiseta melanura, and wild passerine (perching) birds in freshwater hardwood swamps. However, the identity of key avian species that serve as principal virus reservoir and amplification hosts has not been established. The efficiency with which pathogen transmission occurs within an avian community is largely determined by the relative reservoir competence of each species and by ecological factors that influence contact rates between these avian hosts and mosquito vectors. Contacts between vector mosquitoes and potential avian hosts may be directly quantified by analyzing the blood meal contents of field-collected specimens. We used PCR-based molecular methods and direct sequencing of the mitochondrial cytochrome b gene for profiling of blood meals in Cs. melanura, in an effort to quantify its feeding behavior on specific vertebrate hosts, and to infer epidemiologic implications in four historic EEE virus foci in the northeastern U.S. Avian point count surveys were conducted to determine spatiotemporal host community composition. Of 1,127 blood meals successfully identified to species level, >99% of blood meals were from 65 avian hosts in 27 families and 11 orders, and only seven were from mammalian hosts representing three species. We developed an empirically informed mathematical model for EEE virus transmission using Cs. melanura abundance and preferred and non-preferred avian hosts. To our knowledge this is the first mathematical model for EEE virus, a pathogen with many potential hosts, in the northeastern U.S. We measured strong feeding preferences for a number of avian species based on the proportion of mosquito blood meals identified from these bird species in relation to their observed frequencies. These included: American Robin, Tufted Titmouse, Common Grackle, Wood Thrush, Chipping Sparrow, Black-capped Chickadee, Northern Cardinal, and Warbling Vireo. We found that these bird species, most notably Wood Thrush, play a dominant role in supporting EEE virus amplification. It is also noteworthy that the competence of some of the aforementioned avian species for EEE virus has not been established. Our findings indicate that heterogeneity induced by mosquito host preference, is a key mediator of the epizootic transmission of vector-borne pathogens. Detailed knowledge of the vector-host interactions of mosquito populations in nature is essential for evaluating their vectorial capacity and for assessing the role of individual vertebrates as reservoir hosts involved in the maintenance and amplification of zoonotic agents of human diseases. Our study clarifies the host associations of Cs. melanura in four EEE virus foci in the northeastern U.S., identifies vector host preferences as the most important transmission parameter, and quantifies the contribution of preference-induced contact heterogeneity to enzootic transmission. Our study identifies Wood Thrush, American Robin and a few avian species that may serve as superspreaders of EEE virus. Our study elucidates spatiotemporal host species utilization by Cs. melanura in relation to avian host community. This research provides a basis to better understand the involvement of Cs. melanura and avian hosts in the transmission and ecology of EEE virus and the risk of human infection in virus foci. Eastern equine encephalitis (EEE) is a highly pathogenic mosquito-borne virus responsible for outbreaks of severe disease in humans and equines, causing high mortality and neurological impairment in most survivors. In the past, human disease outbreaks in the northeastern U.S. occurred sporadically with no apparent pattern; however, during the last decade, this region has experienced changes in the frequency of EEE virus activity with expansion into new localities. We studied vector-host interaction using molecular methods in order to: 1) quantify vector host-feeding preferences of Culiseta melanura, 2) identify key bird species that serve as frequent hosts for Cs. melanura and reservoir hosts for the virus, and 3) determine the extent to which these preferences shape virus transmission dynamics in four historic foci of EEE virus activity in Connecticut. We also examined avian population density and dynamics to determine spatiotemporal host community composition experienced by Cs. melanura and EEE virus. We developed an empirically informed mathematical model to describe enzootic transmission of EEE virus in a community of multiple avian hosts and the primary mosquito vector. To our knowledge this is the first mathematical model for EEE virus, a pathogen with many potential hosts, in the northeastern U.S. Our study clarifies the host associations of Cs. melanura in four foci of virus activity in the northeastern U.S., identifies vector host preferences as the most important transmission parameter, and quantifies the contribution of preference-induced contact heterogeneity to enzootic transmission. Our study also elucidates spatiotemporal host species utilization by Cs. melanura in relation to avian host abundance. We identified eight bird species (Wood Thrush, American Robin, Tufted Titmouse, Common Grackle, Chipping Sparrow, Black-capped Chickadee, Northern Cardinal, and Warbling Vireo) with a larger feeding index than the remaining birds, indicating these species were fed upon by Cs. melanura more frequently, and therefore are important in maintenance of EEE virus at these four foci. This research provides a basis for better understanding the involvement of Cs. melanura in the transmission and ecology of EEE virus, and the risk of human infection in virus foci.

Eastern equine encephalomyelitis virus (EEEV) causes a highly pathogenic zoonosis that circulates in an enzootic cycle involving the ornithophagic mosquito, Culiseta melanura, and wild passerine birds in freshwater hardwood swamps in the northeastern U.S. Epidemic/epizootic transmission to humans/equines typically occurs towards the end of the transmission season and is generally assumed to be mediated by locally abundant and contiguous mammalophagic “bridge vector” mosquitoes. Engorged mosquitoes were collected using CDC light, resting box, and gravid traps during epidemic transmission of EEEV in 2012 in Addison and Rutland counties, Vermont. Mosquitoes were identified to species and blood meal analysis performed by sequencing mitochondrial cytochrome b gene polymerase chain reaction products. Infection status with EEEV in mosquitoes was determined using cell culture and RT-PCR assays, and all viral isolates were sequenced and compared to other EEEV strains by phylogenetic analysis. The host choices of 574 engorged mosquitoes were as follows: Cs. melanura (n = 331, 94.3 % avian-derived, 5.7 % mammalian-derived); Anopheles quadrimaculatus (n = 164, 3.0 % avian, 97.0 % mammalian); An. punctipennis (n = 56, 7.2 % avian, 92.8 % mammalian), Aedes vexans (n = 9, 22.2 % avian, 77.8 % mammalian); Culex pipiens s.l. n = 6, 100 % avian); Coquillettidia perturbans (n = 4, 25.0 % avian, 75.0 % mammalian); and Cs. morsitans (n = 4, 100 % avian). A seasonal shift in blood feeding by Cs. melanura from Green Heron towards other avian species was observed. EEEV was successfully isolated from blood-fed Cs. melanura and analyzed by phylogenetic analysis. Vermont strains from 2012 clustered with viral strains previously isolated in Virginia yet were genetically distinct from an earlier EEEV isolate from Vermont during 2011. Culiseta melanura acquired blood meals primarily from birds and focused feeding activity on several competent species capable of supporting EEEV transmission. Culiseta melanura also occasionally obtained blood meals from mammalian hosts including humans. This mosquito species serves as the primary vector of EEEV among wild bird species, but also is capable of occasionally contributing to epidemic/epizootic transmission of EEEV to humans/equines. Other mosquito species including Cq. perturbans that feed more opportunistically on both avian and mammalian hosts may be important in epidemic/epizootic transmission under certain conditions. Phylogenetic analyses suggest that EEEV was independently introduced into Vermont on at least two separate occasions.