The recovery of a Haemaphysalis longicornis Neumann (Acari: Ixodidae) tick from a dog in Benton County, Arkansas, in 2018 triggered a significant environmental sampling effort in Hobbs State Park Conservation Area. The objective of the investigation was to assess the tick population density and diversity, as well as identify potential tick-borne pathogens that could pose a risk to public health. During a week-long sampling period in August of 2018, a total of 6,154 ticks were collected, with the majority identified as Amblyomma americanum (L), (Acari: Ixodidae) commonly known as the lone star tick. No H. longicornis ticks were found despite the initial detection of this species in the area. This discrepancy highlights the importance of continued monitoring efforts to understand the dynamics of tick populations and their movements. The investigation also focused on pathogen detection, with ticks being pooled by species, age, and sex before being processed with various bioassays. The results revealed the presence of several tick-borne pathogens, including agents associated with ehrlichiosis (n = 12), tularemia (n = 2), and Bourbon virus (BRBV) disease (n = 1), as well as nonpathogenic rickettsial and anaplasmosis organisms. These findings emphasize the importance of public health messaging to raise awareness of the risks associated with exposure to tick-borne pathogens. Prevention measures, such as wearing protective clothing, using insect repellent, and conducting regular tick checks, should be emphasized to reduce the risk of tick-borne diseases. Continued surveillance efforts and research are also essential to improve our understanding of tick-borne disease epidemiology and develop effective control strategies.

We describe the isolation of a novel flavivirus, isolated from a pool of mosquitoes identified as Culex (Culex) chidesteri collected in 2010 in the Pantanal region of west-central Brazil. The virus is herein designated Nhumirim virus (NHUV) after the name of the ranch from which the mosquito pool was collected. Flavivirus RNA was detected by real-time RT-PCR of homogenized mosquitoes and from the corresponding C6/36 culture supernatant. Based on full-genome sequencing, the virus isolate was genetically distinct from but most closely related to Barkedji virus (BJV), a newly described flavivirus from Senegal. Phylogenetic analysis demonstrated that NHUV grouped with mosquito-borne flaviviruses forming a clade with BJV. This clade may be genetically intermediate between the Culex-borne flaviviruses amplified by birds and the insect-only flaviviruses.

Ivermectin (IVM)-treated birds provide the potential for targeted control of Culex mosquitoes to reduce West Nile virus (WNV) transmission. Ingestion of IVM increases mosquito mortality, which could reduce WNV transmission from birds to humans and in enzootic maintenance cycles affecting predominantly bird-feeding mosquitoes and from birds to humans. This strategy might also provide an alternative method for WNV control that is less hampered by insecticide resistance and the logistics of large-scale pesticide applications. Through a combination of field studies and modeling, we assessed the feasibility and impact of deploying IVM-treated birdfeed in residential neighborhoods to reduce WNV transmission. We first tracked 105 birds using radio telemetry and radio frequency identification to monitor their feeder usage and locations of nocturnal roosts in relation to five feeder sites in a neighborhood in Fort Collins, Colorado. Using these results, we then modified a compartmental model of WNV transmission to account for the impact of IVM on mosquito mortality and spatial movement of birds and mosquitoes on the neighborhood level. We found that, while the number of treated lots in a neighborhood strongly influenced the total transmission potential, the arrangement of treated lots in a neighborhood had little effect. Increasing the proportion of treated birds, regardless of the WNV competency status, resulted in a larger reduction in infection dynamics than only treating competent birds. Taken together, model results indicate that deployment of IVM-treated feeders could reduce local transmission throughout the WNV season, including reducing the enzootic transmission prior to the onset of human infections, with high spatial coverage and rates of IVM-induced mortality in mosquitoes. To improve predictions, more work is needed to refine estimates of daily mosquito movement in urban areas and rates of IVM-induced mortality. Our results can guide future field trials of this control strategy.

Mayaro virus (MAYV, Togaviridae) and Oropouche orthobunyavirus (OROV, Peribunyaviridae) are emerging enzootic arboviruses in Latin America. Outbreaks of febrile illness associated with MAYV and OROV have been reported among humans mainly in the northern region of Brazil since the 1980s, and recent data suggest these viruses have circulated also in more populated areas of western Brazil. MAYV shares mosquito vectors with yellow fever virus and it has been historically detected during yellow fever epidemics. Aiming to investigate the transmission of OROV and MAYV at the human-animal interface during a yellow fever, chikungunya and Zika outbreaks in Brazil, we conducted a retrospective molecular investigation in 810 wild and domestic animals, 106 febrile patients, and 22.931 vectors collected from 2016 to 2018 in Cuiaba and Campo Grande metropolitan regions, western Brazil. All samples tested negative for OROV and MAYV RNA by RT-qPCR. Findings presented here suggest no active circulation of MAYV and OROV in the sampled hosts. Active surveillance and retrospective investigations are instrumental approaches for the detection of cryptic and subclinical activity of enzootic arboviruses and together serve as a warning system to implement appropriate actions to prevent outbreaks.

West Nile virus activity was evaluated within an island waterbird nesting colony with > 1,250 birds at Riverside Reservoir, Weld County, Colorado, USA. To avoid disturbance of nesting birds, blood-engorged mosquitoes (Culex tarsalis) were used to sample blood indirectly from birds rather than capturing, sampling, and releasing live birds. Local virus activity was confirmed by West Nile virus-positive feather samples from 26% of 46 carcasses collected during monthly visits to the colony from June to September 2009, including American White Pelican (Pelecanus erythrorhynchos; n = 7), California Gull (Larus californicus; n = 1), Snowy Egret (Egretta thula; n = 2), and Cattle Egret (Bubulcus ibis; n = 2). Of 22 blood-engorged mosquitoes collected and the blood meal host identified to species, one West Nile virus infection was detected (putatively from a Snowy Egret), and West Nile virus-specific antibodies were detected in eight samples: Snowy Egret (n = 5), Great Blue Heron (Ardea herodias; n = 2), and American White Pelican (n = 1). The engorgement rate of female Culex tarsalis at the nesting colony was 34%, sixfold higher than that at a nearby mainland site of 5.3%. The utilization of mosquitoes for sampling blood from wild animals may have broader application, and potentially reduce human disturbance of sensitive nesting bird species. © 2021 The Waterbird Society. All rights reserved.

Studies on the feeding behavior of hematophagous insects, particularly those of medical importance, are relevant for tracking possible pathogen transmission routes and identifying biases in the choice of vertebrates. We evaluated host selection of blood-feeding mosquitoes in a disturbed forest in the Magdalena Medio valley in Colombia from March 2017 to April 2018, after the introduction of Zika virus to the Americas from the 2015-2016 outbreak. We estimated vertebrate diversity and collected blood-engorged female mosquitoes. Genomic DNA/RNA was extracted from the mosquito's abdomen for vertebrate host identification and pathogen detection. We performed conventional PCR and sequencing, using universal primers targeting vertebrate regions of the eukaryotic mitochondrial genome to determine bloodmeal host. Additionally, we tested for the presence of flaviviruses in all mosquito samples with RT-PCR. Based on the identity and quantity of detected bloodmeals, we performed mosquito-vertebrate interaction network analysis and estimated topology metrics. In total, we collected 292 engorged female mosquitoes representing 20 different species. Bloodmeal analyses identified 26 vertebrate species, the majority of which were mammals (N = 16; 61.5%). No flaviviruses of medical importance were detected from the samples. Although feeding patterns varied, network analyses showed a high degree of specialization by mosquitoes and revealed ecological and phylogenetic relationships among the host community. We conclude that host selection or preference by mosquitoes is species specific.

Bourbon virus (Thogotovirus, Orthomyxoviridae) was discovered in 2014 when a patient with history of multiple tick bites in Kansas died from an | unknown infection [1]. Human infections from Bourbon virus | have now been recognized in several states (i.e., Kansas, | Oklahoma, Missouri). The virus was detected in collections | of the lone star tick (Amblyomma americanum) in Missouri | [2]. A serosurvey of domestic and wild mammals in Missouri | noted the presence of Bourbon virus-neutralizing antibodies in serum samples collected from a variety of species, but | most frequently in white-tailed deer (Odocoileus virginianus) | and raccoon (Procyon lotor) [3]. We report here that neutralizing antibodies against Bourbon virus were detected in | white-tailed deer in North Carolina, suggesting that the virus | is present in the state. We screened 32 white-tailed deer | for the presence of Bourbon virus-specific neutralizing antibodies. Of 20 plasma samples that reacted with the virus, | 18 were confirmed with neutralizing antibody titers ranging | from 10 to ≥ 320 for a seroprevalence rate of 56% (95% confidence interval 39%–72%). The seropositive samples were | from deer killed during the 2014 hunting season from Stanly | and New Hanover counties

Zika virus (ZIKV) was first discovered in 1947 in Uganda but was not considered a public health threat until 2007 when it found to be the source of epidemic activity in Asia. Epidemic activity spread to Brazil in 2014 and continued to spread throughout the tropical and subtropical regions of the Americas. Despite ZIKV being zoonotic in origin, information about transmission, or even exposure of non-human vertebrates and mosquitoes to ZIKV in the Americas, is lacking. Accordingly, from February 2017 to March 2018, we sought evidence of sylvatic ZIKV transmission by sampling whole blood from approximately 2000 domestic and wild vertebrates of over 100 species in West-Central Brazil within the active human ZIKV transmission area. In addition, we collected over 24,300 mosquitoes of at least 17 genera and 62 species. We screened whole blood samples and mosquito pools for ZIKV RNA using pan-flavivirus primers in a real-time reverse-transcription polymerase chain reaction (RT-PCR) in a SYBR Green platform. Positives were confirmed using ZIKV-specific envelope gene real-time RT-PCR and nucleotide sequencing. Of the 2068 vertebrates tested, none were ZIKV positive. Of the 23,315 non-engorged mosquitoes consolidated into 1503 pools tested, 22 (1.5%) with full data available showed some degree of homology to insect-specific flaviviruses. To identify previous exposure to ZIKV, 1498 plasma samples representing 62 species of domestic and sylvatic vertebrates were tested for ZIKV-neutralizing antibodies by plaque reduction neutralization test (PRNT90). From these, 23 (1.5%) of seven species were seropositive for ZIKV and negative for dengue virus serotype 2, yellow fever virus, and West Nile virus, suggesting potential monotypic reaction for ZIKV. Results presented here suggest no active transmission of ZIKV in non-human vertebrate populations or in alternative vector candidates, but suggest that vertebrates around human populations have indeed been exposed to ZIKV in West-Central Brazil.

Since its recent discovery, Bourbon virus has been isolated from a human and ticks. To assess exposure of potential vertebrate reservoirs, we assayed banked serum and plasma samples from wildlife and domestic animals in Missouri, USA, for Bourbon virus-neutralizing antibodies. We detected high seroprevalence in raccoons (50%) and white-tailed deer (86%).

St. Louis encephalitis virus (SLEV) and West Nile virus (WNV) have recently emerged in the southwestern United States. Surveillance for arboviruses in Las Vegas, NV, detected a surge of SLEV activity in the southern house mosquito (Culex quinquefasciatus Say) during 2016. To identify candidate avian amplifiers, we assessed the identification, viral infection, and immune status of vertebrate hosts for 195 blood-engorged Cx. quinquefasciatus mosquitoes collected in August and September 2016. Bloodmeals were identified from 164 engorged abdomens, representing 19 species of birds and three species of mammals. No SLEV or WNV viremia was detected, but one mosquito tested positive for Culex flavivirus. House finch (Haemorhous mexicanus) (Muller) was the most common bloodmeal, followed by domestic chicken (Gallus gallus) (Linnaeus), American robin (Turdus migratorius) L., house sparrow (Passer domesticus) (L.), great-tailed grackle (Quiscalus mexicanus) (Gmelin), northern mockingbird (Mimus polyglottos) (L.) and mourning dove (Zenaida macroura) (L.). SLEV-reactive antibodies were detected in six identified bloodmeals and WNV-reactive antibodies were detected in 33. House sparrow and house finch were the most likely hosts to show previous exposure to SLEV and WNV, respectively. Over-utilization by Cx. quinquefasciatus for bloodmeal hosts was observed primarily among robin, finch and sparrow, all species that roost communally. House finch stands out as a candidate important amplifier for both SLEV and WNV because of its preference by mosquito vectors, and high competence for closely related virus strains. While implicated in previous outbreaks as an important mosquito vector, Cx. quinquefasciatus feeds infrequently on mammals in Las Vegas, indicating a low risk for bridge transmission to humans.

To explain the patchy distribution of West Nile virus (WNV), we propose that avian immunity encountered by Culex vectors regulates WNV transmission, particularly at communal bird roosts. To test this hypothesis, we selected two test sites with communally roosting American robins (Turdus migratorius) and two control sites that lacked communal roosts. The density of vector-vertebrate contacts, represented by engorged Culex pipiens, was 23-fold greater at test sites compared to control sites, and the density of blood-engorged Cx. pipiens measured in resting mosquito traps correlated positively with the presence of robins and negatively with the presence of other birds, confirming an attraction to robins for blood feeding. WNV transmission was alternately up-regulated (amplification) and down-regulated (suppression) at both test sites. At one test site, infection in resting Cx. pipiens surged from zero to 37.2 per thousand within four weeks, and robin immunity rose from 8.4% to 64% before reducing to 33%. At this site, ten potentially infectious contacts between vector and vertebrates (including nine robins and a mourning dove [Zenaida macroura]) were documented. Infectious vector-vertebrate contacts were absent from control sites. The use of infectious vector-vertebrate contacts, rather than infected mosquitoes, to evaluate a transmission focus is novel.

Flanders virus (FLAV; family Rhabdoviridae) is a mosquito-borne hapavirus with no known pathology that is frequently isolated during arbovirus surveillance programs. Here, we document the presence of FLAV in Culex tarsalis mosquitoes and a Canada goose (Branta canadensis) collected in western North America, outside of the currently recognized range of FLAV. Until now, FLAV-like viruses detected in the western United States were assumed to be Hart Park virus (HPV, family Rhabdoviridae), a closely related congener. A re-examination of archived viral isolates revealed that FLAV was circulating in California as early as 1963. FLAV also was isolated in Nebraska, Colorado, South Dakota, North Dakota, and Saskatchewan, Canada. Phylogenetic analysis of the U1 pseudogene for 117 taxa and eight nuclear genes for 15 taxa demonstrated no distinct clustering between western FLAV isolates. Assuming the range of FLAV has been expanding west, these results indicate that FLAV likely spread west following multiple invasion events. However, it remains to be determined if the detection of FLAV in western North America is due to expansion or is a result of enhanced arbovirus surveillance or diagnostic techniques. Currently, the impact of FLAV infection remains unknown.

Culex quinquefasciatus is the principal vector of West Nile virus (WNV) in the South Central United States, yet limited data on host utilization are available. We evaluated host utilization over a 3-month period in 2013 in a residential landscape in College Station, Texas. PCR sequencing of the mitochondrial cytochrome oxidase 1 gene permitted molecular identification of vertebrate bloodmeals to the species level. Forage ratio analysis identified bird species that were overutilized and underutilized by comparing community feeding index values to expected relative abundance values of bird species, derived from eBird data. Community feeding index values were also used in conjunction with reservoir competence data from the literature to generate reservoir capacity index values, a means of identifying relative importance of vertebrate reservoir hosts. Of 498 blood-engorged Cx. quinquefasciatus, 313 (62.9%) were identified to vertebrate species. The majority (95.5%) of bloodmeals originated from avian species with the remainder from mammals, but not humans. Northern mockingbird (Mimus polyglottos) was the principal host for mosquito feeding in June and July, but northern cardinal (Cardinalis cardinalis) became primary host in August. Forage ratio analysis revealed the overutilization of house finch (Haemorhous mexicanus), American robin (Turdus migratorius), northern mockingbird, northern cardinal, white-winged dove (Zenaida asiatica), and mourning dove (Zenaida macroura). Great-tailed grackle (Quiscalus mexicanus), blue jay (Cyanocitta cristata), and Carolina wren (Thryothorus ludovicianus) were under-utilized relative to availability. Reservoir capacity calculations suggested that northern mockingbird and northern cardinal were the principal amplifiers in the study area. These data identify the primary avian species contributing to the enzootic amplification of WNV in East-Central Texas and reveal that the heavy feeding on moderately competent hosts and no feeding on humans likely limit epidemics in this region.

Raptors are a target sentinel species for West Nile virus (WNV) because many are susceptible to WNV disease, they are easily sighted because of their large size, and they often occupy territories near human settlements. Sick and dead raptors accumulate at raptor and wildlife rehabilitation clinics. However, investigations into species selection and specimen type for efficient detection of WNV are lacking. Accordingly, we evaluated dead raptors from north-central Colorado and SE Wyoming over a 4-yr period. Nonvascular mature feathers ("quill"), vascular immature feathers ("pulp"), oropharyngeal swabs, cloacal swabs, and kidney samples were collected from raptor carcasses at the Rocky Mountain Raptor Program in Colorado from 2013 through 2016. We tested the samples using real-time reverse transcriptase-PCR. We found that 11% (53/482) of raptor carcasses tested positive for WNV infection. We consistently detected positive specimens during a 12-wk span between the second week of July and the third week of September across all years of the study. We detected WNV RNA most frequently in vascular feather pulp from Cooper's Hawk ( Accipiter cooperii). North American avian mortality surveillance for WNV using raptors can obviate necropsies by selecting Cooper's Hawk and Red-tailed Hawk ( Buteo jamaicensis) as sentinels and targeting feather pulp as a substrate for viral detection.

The Pantanal is a hotspot for arbovirus studies in South America. Various medically important flaviviruses and alphaviruses have been reported in domestic and wild animals in the region. To expand the knowledge of local arbovirus circulation, a serosurvey for 14 Brazilian orthobunyaviruses was conducted with equines, sheep and free-ranging caimans. Sera were tested for specific viral antibodies using plaque-reduction neutralization test (PRNT). Monotypic reactions were detected for Maguari, Xingu, Apeu, Guaroa, Murutucu, Oriboca, Oropouche and Nepuyo viruses. Despite the low titers for most of the orthobunyaviruses tested, the detection of monotypic reactions for eight orthobunyaviruses suggests the Pantanal as a region of great orthobunyavirus diversity. The present data, in conjunction with previous studies that detected a high diversity of other arboviruses, ratify the Pantanal as an important natural reservoir for sylvatic and medically important arboviruses in Brazil.

Current methods for detecting Flavivirus antibodies are enzyme-linked immunosorbent assays (ELISAs) and neutralization tests, both of which require laboratories and trained staff. We evaluated the VectorTest West Nile Virus Antigen Assay in an inhibition platform (VecTest-inhibition assay [VIA]) as a simpler screening method for detecting antibodies for a variety of flaviviruses among a population of equines from Brazil. We found that the VIA is a field-deployable rapid method with 100% sensitivity and 64% specificity compared with blocking ELISA for the detection of group-specific Flavivirus antibodies in equine serum samples. The VIA is a potentially useful field test for rapid field-based Flavivirus antibody detection in equine serum samples.

Genomic sequences are described from five novel viruses and divergent strains of Brejeira and Guaico Culex viruses from mosquitoes collected in Pantanal, Brazil, in 2010.

St. Louis encephalitis virus (SLEV) (Flavivirus) is a reemerging arbovirus in the southern cone of South America. In 2005, an outbreak of SLEV in central Argentina resulted in 47 human cases with 9 deaths. In Argentina, the ecology of SLEV is poorly understood. Because certain birds are the primary amplifiers in North America, we hypothesized that birds amplify SLEV in Argentina as well. We compared avian SLEV seroprevalence in a variety of ecosystems in and around Cordoba city from 2004 (before the epidemic) and 2005 (during the epidemic). We also explored spatial patterns to better understand the local ecology of SLEV transmission. Because West Nile virus (WNV) was also detected in Argentina in 2005, all analyses were also conducted for WNV. A total of 980 birds were sampled for detection of SLEV and WNV neutralizing antibodies. SLEV seroprevalence in birds increased 11-fold from 2004 to 2005. Our study demonstrated that a high proportion (99.3%) of local birds were susceptible to SLEV infection immediately prior to the 2005 outbreak, indicating that the vertebrate host population was primed to amplify SLEV. SLEV was found distributed in a variety of environments throughout the city of Cordoba. However, the force of viral transmission varied among sites. Fine scale differences in populations of vectors and vertebrate hosts would explain this variation. In summary, we showed that in 2005, both SLEV and to a lesser extent WNV circulated in the avian population. Eared Dove, Picui Ground-Dove and Great Kiskadee are strong candidates to amplify SLEV because of their exposure to the pathogen at the population level, and their widespread abundance. For the same reasons, Rufous Hornero may be an important maintenance host for WNV in central Argentina. Competence studies and vector feeding studies are needed to confirm these relationships.

Since its discovery in 2009, the tickborne Heartland virus (HRTV) has caused human illness in Missouri, Oklahoma, and Tennessee USA. To better assess the geographic distribution of HRTV, we used wildlife serology as an indicator. This retrospective evaluation determined that HRTV is widespread within the central and eastern United States.

Heartland virus (HRTV; Bunyaviridae, Phlebovirus) has recently emerged as the causative agent of human disease characterized by thrombocytopenia and leukopenia in the United States. The lone star tick (Amblyomma americanum L.) has been implicated as a vector. To identify candidate vertebrate amplification hosts associated with enzootic maintenance of the virus, sera and ticks were sampled from 160 mammals (8 species) and 139 birds (26 species) captured near two human case residences in Andrew and Nodaway Counties in northwest Missouri. HRTV-specific neutralizing antibodies were identified in northern raccoons (42.6%), horses (17.4%), white-tailed deer (14.3%), dogs (7.7%), and Virginia opossums (3.8%), but not in birds. Virus isolation attempts from sera and ticks failed to detect HRTV virus. The high antibody prevalence coupled with local abundance of white-tailed deer and raccoons indicates these species as candidate amplification hosts.

We describe and compare 2 qualitative serologic techniques for detecting West Nile virus (WNV)-specific antibodies in mosquito blood meals. The techniques are the biotin microsphere immunoassay (b-MIA) and the inhibition platform of the VectorTest WNV antigen assay (VecTest-inhibition). To demonstrate the ability of these tests to detect WNV-neutralizing antibodies, we experimentally exposed feeding mosquitoes to blood containing 5 concentrations of 6B6C-1, a flavivirus-neutralizing monoclonal antibody. Antibody concentrations were quantified using the 90% plaque-reduction neutralization test (PRNT90). After 24 h of blood-meal digestion at 22.5°C, the threshold PRNT90 titer of detection was ≤18 for b-MIA and ≤50 for VecTest-inhibition. Both tests reliably detected antibodies in 3 of 3 blood meals that had been digested for up to 30 h, or were about 25% digested. The b-MIA was also applied to mosquitoes that had engorged on avian blood in Arizona following a WNV epidemic in 2010. There was no significant difference in the WNV antibody prevalence determined by b-MIA (52% of 71 avian blood meals) compared to the WNV-neutralizing antibody prevalence in birds determined by direct sampling (49% of 234 birds). VecTest-inhibition requires fewer resources and may be used in the field without a laboratory, but consumes the entire blood meal and relies on subjective interpretation of results. The b-MIA requires a laboratory and sophisticated equipment and reagents. Results for b-MIA are analyzed objectively and can be applied to mosquito blood meals with greater confidence than the VecTest-inhibition method and thus can contribute substantially to research and surveillance programs that would benefit from the detection of specific WNV antibodies in mosquito blood meals.

The Pantanal hosts diverse wildlife species and therefore is a hotspot for arbovirus studies in South America. A serosurvey for Mayaro virus (MAYV), eastern (EEEV), western (WEEV) and Venezuelan (VEEV) equine encephalitis viruses was conducted with 237 sheep, 87 free-ranging caimans and 748 equids, including 37 collected from a ranch where a neurologic disorder outbreak had been recently reported. Sera were tested for specific viral antibodies using plaque-reduction neutralisation test. From a total of 748 equids, of which 264 were immunised with vaccine composed of EEEV and WEEV and 484 had no history of immunisation, 10 (1.3%) were seropositive for MAYV and two (0.3%) for VEEV using criteria of a ≥ 4-fold antibody titre difference. Among the 484 equids without history of immunisation, 48 (9.9%) were seropositive for EEEV and four (0.8%) for WEEV using the same criteria. Among the sheep, five were sero- positive for equine encephalitis alphaviruses, with one (0.4%) for EEEV, one (0.4%) for WEEV and three (1.3%) for VEEV. Regarding free-ranging caimans, one (1.1%) and three (3.4%), respectively, had low titres for neutralising antibodies to VEEV and undetermined alphaviruses. The neurological disorder outbreak could not be linked to the alphaviruses tested. Our findings represent strong evidence that MAYV and all equine encephalitis alphaviruses circulated in the Pantanal.

West Nile virus has caused several outbreaks among humans in the Phoenix metropolitan area (Arizona, southwest USA) within the last decade. Recent ecologic studies have implicated Culex quinquefasciatus and Culex tarsalis as the mosquito vectors and identified three abundant passerine birds-great-tailed grackle (Quiscalus mexicanus), house sparrow (Passer domesticus), and house finch (Haemorhous mexicanus)-as key amplifiers among vertebrates. Nocturnal congregations of certain species have been suggested as critical for late summer West Nile virus amplification. We evaluated the hypothesis that house sparrow (P. domesticus) and/or great-tailed grackle (Q. mexicanus) communal roost sites (n = 22 and n = 5, respectively) in a primarily suburban environment were spatially associated with West Nile virus transmission indices during the 2010 outbreak of human neurological disease in metropolitan Phoenix. Spatial associations between human case residences and communal roosts were non-significant for house sparrows, and were negative for great-tailed grackle. Several theories that explain these observations are discussed, including the possibility that grackle communal roosts are protective.

We describe the isolation of West Nile virus (WNV; Flaviviridae, Flavivirus) from blood of a Virginia opossum (Didelphis virginiana) collected in northwestern Missouri in August 2012. Sequencing determined that the virus was related to lineage 1a WNV02 strains. We discuss the role of wildlife in WNV disease epidemiology.

Serial samples from the same individuals may be required for certain virological studies, however, some small animals cannot easily be blood-sampled. Therefore, we evaluated the use of Culex quinquefasciatus Say and Aedes albopictus Skuse mosquitoes as "biological syringes" to draw blood for virus titer determinations in small vertebrates. Groups of chicks (Gallus gallus), hamsters (Mesocricetus auratus), and house sparrows (Passer domesticus) were experimentally infected with West Nile virus (WNV) or Highlands J virus (HJV). In general, good correlation was seen between mosquito- and syringe-derived blood samples at titers ≥5.0 log10 pfu/mL serum as compared with titers <5.0 log10 pfu/mL serum for chicks, hamsters, and sparrows. Ninety-two percent (24/26) of sparrows with virus titers >105 pfu/mL serum had mosquito- and syringe-derived titers within one log of each other. Sparrow viremia profiles generated from single mosquito blood meals and syringe were not significantly different (p>0.05). This technique is valuable for assessing the roles of small vertebrates in the ecologies of arboviruses, and could be used in applications beyond virology and infectious diseases, when <10 microL of whole blood is required.

A recent study reported neutralizing antibodies to West Nile virus (WNV) in horses from four ranches of southern Pantanal. To extend that study, a serosurvey for WNV and 11 Brazilian flaviviruses was conducted with 760 equines, 238 sheep and 61 caimans from 17 local cattle ranches. Among the tested equines, 32 were collected from a ranch where a neurologic disorder outbreak had been recently reported. The sera were initially screened by using a blocking ELISA and then titrated by 90% plaque-reduction neutralization test (PRNT90) for 12 flaviviruses. Employing the criterion of 4-fold greater titer, 78 (10.3%) equines were seropositive for Ilheus virus, 59 (7.8%) for Saint Louis encephalitis virus, 24 (3.2%) for WNV, two (0.3%) for Cacipacore virus and one (0.1%) for Rocio virus. No serological evidence was found linking the neurological disease that affected local equines to WNV. All caimans and sheep were negative by blocking ELISA for flaviviruses. There were no seropositive equines for Bussuquara, Iguape, Yellow fever and all four Dengue virus serotypes. The detection of WNV-seropositive equines in ten ranches and ILHV and SLEV-seropositive equines in fourteen ranches of two different sub-regions of Pantanal is strong evidence of widespread circulation of these flaviviruses in the region.

The Eurasian collared-dove (Streptopelia decaocto) has recently experienced a population explosion in North America. It is frequently infected with West Nile virus (WNV). To test the hypothesis that the Eurasian collared-dove is competent to transmit WNV, we experimentally infected two cohorts of doves with two different strains of WNV, CO08, and NY99, respectively. Both virus strains induced a low-level viremia, capable of infecting a small fraction of vector mosquitoes. We suggest that the Eurasian collared-dove plays a relatively insignificant role as an amplifying host for WNV, but it may be important where it is locally abundant.

The wetlands of the Brazilian Pantanal host large concentrations of diverse wildlife species and hematophagous arthropods, conditions that favor the circulation of zoonotic arboviruses. A recent study from the Nhecolandia sub-region of Pantanal reported serological evidence of various flaviviruses, including West Nile virus and Ilheus virus (ILHV). According to the age of seropositive horses, at least three flaviviruses, including ILHV, circulated in the Brazilian Pantanal between 2005 and 2009. To extend this study, we collected 3,234 adult mosquitoes of 16 species during 2009 and 2010 in the same sub-region. Mosquito pool homogenates were assayed for infectious virus on C6/36 and Vero cell monolayers and also tested for flaviviral RNA by a group-specific real-time RT-PCR. One pool containing 50 non-engorged female specimens of Aedes scapularis tested positive for ILHV by culture and for ILHV RNA by real-time RT-PCR, indicating a minimum infection rate of 2.5 per 1000. Full-length genomic sequence exhibited 95% identity to the only full genome sequence available for ILHV. The present data confirm the circulation of ILHV in the Brazilian Pantanal.

West Nile virus (WNV) causes sporadic outbreaks of human encephalitis in Phoenix, Arizona. To identify amplifying hosts of WNV in the Phoenix area, we blood-sampled resident birds and measured antibody prevalence following an outbreak in the East Valley of metropolitan Phoenix during summer, 2010. House sparrow (Passer domesticus), house finch (Haemorhous mexicanus), great-tailed grackle (Quiscalus mexicanus), and mourning dove (Zenaida macroura) accounted for most WNV infections among locally resident birds. These species roost communally after early summer breeding. In September 2010, Culex vector-avian host contact was 3-fold greater at communal bird roosts compared with control sites, as determined by densities of resting mosquitoes with previous vertebrate contact (i.e., blood-engorged or gravid mosquitoes). Because of the low competence of mourning doves, these were considered weak amplifiers but potentially effective free-ranging sentinels. Highly competent sparrows, finches, and grackles were predicted to be key amplifying hosts for WNV in suburban Phoenix.

In an effort to detect West Nile virus (WNV) in Brazil, we sampled serum from horses and chickens from the Pantanal region of the state of Mato Grosso and tested for flavivirus-reactive antibodies by blocking ELISA. The positive samples were further confirmed for serological evidence of WNV infection in three (8%) of the 38 horses and one (3.2%) of the 31 chickens using an 80% plaque-reduction neutralisation test (PRNT 80). These results provide evidence of the circulation of WNV in chickens and horses in Pantanal.