Bourbon Virus in Wild and Domestic Animals, Missouri, USA, 2012-2013.

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%).

Flanders hapavirus in western North America.

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.

VectorTest™ West Nile Virus Antigen Assay in an Inhibition Platform as Field Screening Tool for Flavivirus Group-Specific Antibodies in Brazilian Equines.

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.

Heartland Virus Neutralizing Antibodies in Vertebrate Wildlife, United States, 2009-2014.

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.

Nhumirim virus, a novel flavivirus isolated from mosquitoes from the Pantanal, Brazil.

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.

Neutralising antibodies for Mayaro virus in Pantanal, Brazil.

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.

Methods for detection of West Nile virus antibodies in mosquito blood meals.

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.

Field-Collected Ticks From Benton County, Arkansas, and Prevalence of Associated Pathogens.

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.

Enhancing Eastern Equine Encephalitis Virus Surveillance in New Jersey: Optimized Collection of Culiseta melanura.

Eastern equine encephalitis virus (EEEV) causes the most clinically severe neuroinvasive arboviral disease in the United States. The virus is endemic in eastern and Gulf Coast states and the Great Lakes region, causing cases annually. To detect EEEV circulation in its enzootic cycle before the virus infects humans and other mammals, mosquito control agencies in New Jersey have conducted mosquito surveillance using a series of permanent wooden resting box sites since 1975. We conducted 2 field studies, 1 evaluating resting traps and 1 evaluating efficacy of CO2 lures, to optimize collection of Culiseta melanura, the primary enzootic vector of EEEV. Resulting mosquito samples were subjected to molecular analysis to determine EEEV infection rates. Corrugated plastic boxes trapped more bloodfed Cs. melanura than other resting trap types (resting boxes, Centers for Disease Control and Prevention [CDC] resting traps, or fiber pots) and were similar to resting boxes in total number of female Cs. melanura caught. Further, non-baited CDC light traps were more successful in trapping host-seeking Cs. melanura than those baited with dry ice, a CO2 lure. The EEEV RNA was identified in Cs. melanura, Aedes vexans, Anopheles quadrimaculatus, and Uranotaenia sapphirina. Our findings indicate that corrugated plastic boxes and non-CO2 baited traps could improve detection of Cs. melanura. Mosquito control agencies are encouraged to periodically assess their surveillance strategy for EEEV.

Predicted reduction in transmission from deployment of ivermectin-treated birdfeeders for local control of West Nile virus.

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.

Serological detection of West Nile virus in horses and chicken from Pantanal, Brazil.

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 (PRNT80). These results provide evidence of the circulation of WNV in chickens and horses in Pantanal.

Retrospective molecular investigation of Mayaro and Oropouche viruses at the human-animal interface in West-central Brazil, 2016-2018.

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 infection of birds, Mexico.

West Nile virus (WNV) has caused disease in humans, equids, and birds at lower frequency in Mexico than in the United States. We hypothesized that the seemingly reduced virulence in Mexico was caused by attenuation of the Tabasco strain from southeastern Mexico, resulting in lower viremia than that caused by the Tecate strain from the more northern location of Baja California. During 2006-2008, we tested this hypothesis in candidate avian amplifying hosts: domestic chickens, rock pigeons, house sparrows, great-tailed grackles, and clay-colored thrushes. Only great-tailed grackles and house sparrows were competent amplifying hosts for both strains, and deaths occurred in each species. Tecate strain viremia levels were higher for thrushes. Both strains produced low-level viremia in pigeons and chickens. Our results suggest that certain avian hosts within Mexico are competent for efficient amplification of both northern and southern WNV strains and that both strains likely contribute to bird deaths.

An enzootic vector-borne virus is amplified at epizootic levels by an invasive avian host.

Determining the effect of an invasive species on enzootic pathogen dynamics is critical for understanding both human epidemics and wildlife epizootics. Theoretical models suggest that when a naive species enters an established host-parasite system, the new host may either reduce ('dilute') or increase ('spillback') pathogen transmission to native hosts. There are few empirical data to evaluate these possibilities, especially for animal pathogens. Buggy Creek virus (BCRV) is an arthropod-borne alphavirus that is enzootically transmitted by the swallow bug (Oeciacus vicarius) to colonially nesting cliff swallows (Petrochelidon pyrrhonota). In western Nebraska, introduced house sparrows (Passer domesticus) invaded cliff swallow colonies approximately 40 years ago and were exposed to BCRV. We evaluated how the addition of house sparrows to this host-parasite system affected the prevalence and amplification of a bird-associated BCRV lineage. The infection prevalence in house sparrows was eight times that of cliff swallows. Nestling house sparrows in mixed-species colonies were significantly less likely to be infected than sparrows in single-species colonies. Infected house sparrows circulated BCRV at higher viraemia titres than cliff swallows. BCRV detected in bug vectors at a site was positively associated with virus prevalence in house sparrows but not with virus prevalence in cliff swallows. The addition of a highly susceptible invasive host species has led to perennial BCRV epizootics at cliff swallow colony sites. The native cliff swallow host confers a dilution advantage to invasive sparrow hosts in mixed colonies, while at the same sites house sparrows may increase the likelihood that swallows become infected.

Mosquito (Diptera: Culicidae) bloodmeal sources during a period of West Nile virus transmission in Puerto Rico.

Host bloodmeals of indigenous Caribbean mosquitoes have not been studied previously. We identified vertebrate DNA in 90 blood-engorged mosquitoes belonging to four genera (Aedes, Culex, Deinocerites, and Uranotaenia) and 12 species that were collected in Puerto Rico within a geographic and temporal focus of West Nile virus transmission in 2007. It was found that 62 (68.8%) bloodmeals were from reptiles, 18 (20.0%) from birds, and 10 (11.1%) from mammals. Only one bloodmeal of 18 derived from Culex (Culex) species was passerine, suggesting a preference for nonpasserine birds and other vertebrates (i.e., reptiles) among the candidate WNV vectors. We interpret the results with respect to vectorial capacity for West Nile virus, an emerging arbovirus throughout the Caribbean Basin.

Seroconversion for West Nile and St. Louis encephalitis viruses among sentinel horses in Colombia.

We prospectively sampled flavivirus-naïve horses in northern Colombia to detect West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) seroconversion events, which would indicate the current circulation of these viruses. Overall, 331 (34.1%) of the 971 horses screened were positive for past infection with flaviviruses upon initial sampling in July 2006. During the 12-month study from July 2006-June 2007, 33 WNV seroconversions and 14 SLEV seroconversions were detected, most of which occurred in the department of Bolivar. The seroconversion rates of horses in Bolivar for the period of March-June 2007 reached 12.4% for WNV and 6.7% for SLEV. These results comprise the first serologic evidence of SLEV circulation in Colombia. None of the horses sampled developed symptoms of encephalitis within three years of initial sampling. Using seroconversions in sentinel horses, we demonstrated an active circulation of WNV and SLEV in northern Colombia, particularly in the department of Bolivar. The absence of WNV-attributed equine or human disease in Colombia and elsewhere in the Caribbean Basin remains a topic of debate and speculation.

The Centers for Disease Control and Prevention resting trap: a novel device for collecting resting mosquitoes.

Commercially available wood-fiber pots used to collect resting mosquitoes were modified to improve sampling efficiency. The modified traps, called the Centers for Disease Control and Prevention resting traps, collected 16.0 and 5.2 times more adult Culex pipiens and Cx. tarsalis than the conventional wood-fiber pots. The resting trap increases the mean number of resting mosquitoes collected per trap-night and is useful for collecting blood-engorged mosquitoes.

Host selection pattern and flavivirus screening of mosquitoes in a disturbed Colombian rainforest.

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.

Surveillance for West Nile virus in American white pelicans, Montana, USA, 2006-2007.

West Nile virus (WNV)-associated deaths of American white pelican (Pelecanus erythrorhynchos) chicks have been recognized at various nesting colonies in the United States since 2002. We evaluated American white pelican nesting colonies in Sheridan County, Montana, USA, for an association between WNV-positive pelican carcasses and human West Nile neuroinvasive disease. Persons in counties hosting affected colonies had a 5x higher risk for disease than those in counties with unaffected colonies. We also investigated WNV infection and blood meal source among mosquitoes and pelican tissue type for greatest WNV detection efficacy in carcasses. WNV-infected Culex tarsalis mosquitoes were detected and blood-engorged Cx. tarsalis contained pelican DNA. Viral loads and detection consistency among pelican tissues were greatest in feather pulp, brain, heart, and skin. Given the risks posed to wildlife and human health, coordinated efforts among wildlife and public health authorities to monitor these pelican colonies for WNV activity are potentially useful.