We report a patient in North Carolina, USA, with Heartland virus infection whose diagnosis was complicated by previous Ehrlichia chaffeensis infection. We identified E. ewingii-infected and Bourbon virus-infected tick pools at the patient's residence. Healthcare providers should consider testing for tickborne viruses if ehrlichiosis is suspected.

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.

Background: La Crosse virus is an important endemic public health concern in the North Carolina Appalachian Mountains; however, human incidence is not commonly noted in this region on the South Carolina side of the mountain range border. No relevant studies have been performed in South Carolina evaluating mosquito vector populations for La Crosse virus (LACV) infection; thus, a pilot mosquito surveillance study was executed in summer 2020. Material and Methods: Vector surveillance occurred at five South Carolina state parks bordering neighboring state endemic counties from May to August 2020. Collections were approved by the state park authority, as noted in Permit No. N-8-20. Results: All three competent mosquito vectors were collected during the study duration; however, these vectors were collected in low abundance: Aedes triseriatus (4.5% of all collected mosquitos); Aedes albopictus (2.0%); Aedes japonicus (1.4%). Principal mosquito vector specimens, Ae. triseriatus, were sent to Centers for Disease Control and Prevention for testing of LACV by real-time reverse transcription PCR-all were negative. Discussion: While entomologic evidence suggests low transmission risk for this arbovirus in the South Carolina Appalachian Mountain region, further eco-epidemiologic investigations are warranted to understand this endemicity variance within a relatively small geographic area.

Heartland virus (HRTV) disease is an emerging tickborne illness in the midwestern and southern United States. We describe a reported fatal case of HRTV infection in the Maryland and Virginia region, states not widely recognized to have human HRTV disease cases. The range of HRTV could be expanding in the United States.

Although the specific cDNA amplification mechanisms of reverse-transcriptase polymerase chain reaction (RT-PCR) and RT loop-mediated isothermal amplification (RT-LAMP) are very different, both molecular assays serve as options to detect arboviral RNA in mosquito pools. Like RT-PCR, RT-LAMP uses a reverse transcription step to synthesize complementary DNA (cDNA) from an RNA template and then uses target-specific primers to amplify cDNA to detectable levels in a single-tube reaction. Using laboratory-generated West Nile virus (WNV) samples and field-collected mosquito pools, we evaluated the sensitivity and specificity of a commercially available WNV real-time RT-LAMP assay (Pro-AmpRT™ WNV; Pro-Lab Diagnostics, Inc., Round Rock, Texas) and compared the results to a validated real-time RT-PCR assay. Laboratory generated virus stock samples containing ≥ 2.3 log10 plaque-forming units (PFU)/ml and intrathoracically inoculated mosquitoes containing ≥ 2.4 log10 PFU/ml produced positive results in the Pro-AmpRT WNV assay. Of field-collected pools that were WNV positive by real-time RT-PCR, 74.5% (70 of 94) were also positive by the Pro-AmpRT WNV assay, resulting in an overall Cohen's kappa agreement of 79.4% between the 2 tests. The Pro-AmpRT WNV assay shows promise as a suitable virus screening tool for vector surveillance programs provided agencies are aware of its characteristics and limitations.

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.

We report the results of a laboratory sensitivity and specificity evaluation of the Rapid Analyte Measurement Platform (RAMP®) Dengue Virus (DENV) antigen detection assay, which is designed to detect all serotypes of DENV in mosquito pools. The RAMP DENV assay was able to detect geographically distinct strains of all 4 DENV serotypes in virus-spiked mosquito pools that contained at least 4.3 log10 plaque forming units/ml, although discrete sensitivity limits varied slightly for each serotype. The RAMP DENV assay also detected DENV 1-4 in mosquito pools containing a single infected mosquito and 24 laboratory-reared uninfected mosquitoes. No false positives were detected in negative control mosquito pools or in samples containing high titers of nontarget arboviruses. We found that while the kit-supplied RAMP buffer reduced the infectious titer of DENV, it did not completely inactivate all serotypes. We recommend adding a detergent, Triton X-100, to the buffer to ensure complete inactivation of DENV if the assay is to be conducted at a lower biosafety level than required for DENV handling.

This paper describes the development and implementation of a robust lesbian, gay, bisexual, transgender, and queer (LGBTQ) cultural competence curriculum for training health and human service providers across New York State. Between 2013-2018, The National LGBT Cancer Network developed and published Best Practices in Creating and Delivering LGBTQ Cultural Competency Trainings for Health and Social Service Agencies and designed a training curriculum. They also conducted Train the Trainer sessions, and evaluated pre- and post- curriculum knowledge, attitudes, self-efficacy and intentions of individuals who attended educational sessions conducted by the certified trainers. Most respondents improved from pretest to posttest, with significant improvements in knowledge, attitudes, self-efficacy and intentions. An increase in self-efficacy was significantly associated with pre- to posttest improvement in respondent intention. Future research should focus on what components specifically bolster self-efficacy and intention. Increasing the number of health and human service providers who are trained to address the needs of this population is an important step toward providing culturally competent care.

Following the recent discovery of Bourbon virus (BRBV) as a human pathogen, and the isolation of the virus from Amblyomma americanum (L.) collected near the location of a fatal human case, we undertook a series of experiments to assess the laboratory vector competence of this tick species for BRBV. Larval ticks were infected using an immersion technique, and transstadial transmission of virus to the nymphal and then to the adult stages was demonstrated. Transstadially infected nymphs transmitted virus to adult ticks at very high rates during cofeeding, indicating the presence of infectious virus in the saliva of engorging ticks. Vertical transmission by transstadially infected females to their progeny occurred, but at a low rate. Rabbits fed on by infected ticks of all active life stages developed high titers of antibody to the virus, demonstrating host exposure to BRBV antigens/live virus during tick blood feeding. These results demonstrate that A. americanum is a competent vector of BRBV and indicate that cofeeding could be critical for enzootic maintenance.

Bunyaviruses (Negarnaviricota: Bunyavirales) are a large and diverse group of viruses that include important human, veterinary, and plant pathogens. The rapid characterization of known and new emerging pathogens depends on the availability of comprehensive reference sequence databases that can be used to match unknowns, infer evolutionary and pathogenic potential, and make response decisions in an evidence-based manner. In this study, we determined the coding-complete genome sequences of 99 bunyaviruses in the Centers for Disease Control and Prevention's Arbovirus Reference Collection, focusing on orthonairoviruses (family Nairoviridae), orthobunyaviruses (Peribunyaviridae), and phleboviruses (Phenuiviridae) that either completely or partially lacked genome sequences. These viruses had been collected over 66 years from 27 countries from vertebrates and arthropods representing 37 genera. Many of the viruses had been characterized serologically and through experimental infection of animals but were isolated in the pre-sequencing era. We took advantage of our unusually large sample size to systematically evaluate genomic characteristics of these viruses, including reassortment, and co-infection. We corroborated our findings using several independent molecular and virologic approaches, including Sanger sequencing of 197 genome segments, and plaque isolation of viruses from putative co-infected virus stocks. This study contributes to the described genetic diversity of bunyaviruses and will enhance the capacity to characterize emerging human pathogenic bunyaviruses.

A kidney transplant patient without known tick exposure developed encephalitis three weeks after transplantation. During the transplant hospitalization, the patient had received a blood transfusion from an asymptomatic donor later discovered to have been infected with Powassan virus. This report describes a probable instance of transfusion-transmitted Powassan virus infection.

In August 2018, two Oregon patients with diagnosed Salmonella infection were interviewed using a standard enteric illness questionnaire; both patients reported having eaten raw cake mix. Standardized interview questionnaire data collected from 207 Oregon patients with salmonellosis in 2017 indicated a 5% rate of consumption of raw “cake mix or cornbread mix” (Oregon Health Authority, unpublished data, 2017). The binomial probability that both 2018 patients were exposed to raw cake mix by chance was determined to be 0.003, prompting the Oregon Health Authority (OHA) to collect and test the contents of 43 boxes of unopened cake mix of various brands from six retail locations. OHA sent samples to the Institute for Environmental Health Laboratories in Lake Forest Park, Washington, for pathogen testing. Salmonella Agbeni was isolated from an unopened box of white cake mix from manufacturer A, and whole genome sequencing (WGS) data describing the isolate were uploaded to the U.S. National Library of Medicine’s National Center for Biotechnology Information (NCBI) website (https://www.ncbi.nlm.nih.gov/pathogensexternal icon). OHA used the NCBI database to compare sequence data with the cake mix isolate (PNUSAS056022) and then consulted CDC’s System for Enteric Disease Response, Investigation, and Coordination (SEDRIC), a web-based, outbreak investigation tool designed for collaborative, multistate investigations of enteric disease outbreaks.* On October 19, OHA determined that clinical isolates from four patients from Maryland, Ohio, and Wisconsin, with specimen isolation dates ranging from June to September 2018, were genetically related to the Salmonella Agbeni isolate from the unopened box of white cake mix, within four single nucleotide polymorphisms (SNPs).

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.

La Crosse virus (LACV) is the most common cause of pediatric arthropod-borne viral (arboviral) encephalitis in the United States (1). It is a California serogroup bunyavirus primarily transmitted by the eastern tree-hole mosquito (Aedes triseriatus) (2). LACV encephalitis is a reportable condition in North Carolina and is a nationally notifiable disease. In North Carolina, LACV encephalitis is the most common endemic arboviral disease reported in humans, with seven western counties accounting for approximately 80% of confirmed cases since 2003 (3). The fatality rate for LACV encephalitis is <1%, with most patients recovering without overt clinical sequelae; however, long-term neurologic sequelae reported in some patients include recurrent seizures, hemiparesis, and cognitive and neurobehavioral abnormalities (4).

The rapid expansion of Zika virus (ZIKV), following the recent outbreaks of Chikungunya virus, overwhelmed the public health infrastructure in many countries and alarmed many in the scientific community. Aedes aegypti (L.) (Diptera: Culicidae) and Aedes albopictus (Skuse) (Diptera: Culicidae) have previously been incriminated as the vectors of these pathogens in addition to dengue virus. In our study, we challenged low generation Ae. aegypti (Chiapas, Mexico) and Ae. albopictus (North Carolina, Mississippi), with three strains of ZIKV, Puerto Rico (GenBank: KU501215), Honduras (GenBank: KX694534), and Miami (GenBank: MF988743). Following an oral challenge with 107.5 PFU/ml of the Puerto Rico strain, we observed high infection and dissemination rates in both species (95%). We report estimated transmission rates for both species (74 and 33%, for Ae. aegypti (L.) (Diptera: Culicidae) and Ae. albopictus (Skuse) (Diptera: Culicidae), respectively), and the presence of a probable salivary gland barrier in Ae. albopictus to Zika virus. Finally, we calculated vectorial capacity for both species and found that Ae. albopictus had a slightly lower vectorial capacity when compared with Ae. aegypti.Second Language La rapida expansion del virus Zika, poco despues de las epidemias de chikungunya, rebaso la infraestructura de salud publica en muchos paises y sorprendio a muchos en la comunidad cientifica. Notablemente, Aedes aegypti y Aedes albopictus transmiten estos patogenos ademas del virus del dengue. En este estudio se expusieron con tres cepas americanas de virus Zika a grupos de Aedes aegypti y Aedes albopictus de generacion reciente. Encontramos altos porcentajes de infeccion y diseminacion en ambas especies (95%). Se reporta, la transmision viral en ambas especies (74 y 33%, para Aedes aegypti and Aedes albopictus, respectivamente) y una probable barrera a nivel de glandulas salivarias. Finalmente, calculamos la capacidad vectorial para ambas especies.

In June 2016, we continued surveillance for tick-borne viruses in eastern Kansas following upon a larger surveillance program initiated in 2015 in response to a fatal human case of Bourbon virus (BRBV) (Family Orthomyxoviridae: Genus Thogotovirus). In 4 d, we collected 14,193 ticks representing four species from four sites. Amblyomma americanum (L.) (Acari: Ixodidae) accounted for nearly all ticks collected (n = 14,116, 99.5%), and the only other species identified were Amblyomma maculatum Koch (Acari: Ixodidae), Dermacentor variabilis (Say) (Acari: Ixodidae) and Ixodes scapularis Say (Acari: Ixodidae). All ticks were tested for both BRBV and Heartland virus (Family Bunyaviridae: Genus Phlebovirus) in 964 pools. Five Heartland virus positive tick pools were detected and confirmed by real-time reverse transcription PCR (rRT-PCR), while all pools tested negative for BRBV. Each Heartland positive pool was composed of 25 A. americanum nymphs with positive pools collected at three different sites in Bourbon County. A. americanum is believed to be the primary vector of both Heartland and BRBVs to humans based upon multiple detections of virus in field-collected ticks, its abundance, and its aggressive feeding behavior on mammals including humans. However, it is possible that A. americanum encounters viremic vertebrate hosts of BRBV less frequently than viremic hosts of Heartland virus, or that BRBV is less efficiently passed among ticks by co-feeding, or less efficiently passed vertically from infected female ticks to their offspring resulting in lower field infection rates.

We isolated a strain of Zika virus, MB16-23, from Aedes aegypti mosquitoes collected in Miami Beach, Florida, USA, on September 2, 2016. Phylogenetic analysis suggests that MB16-23 most likely originated from the Caribbean 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.

Commercially available assays utilizing antigen or nucleic acid detection chemistries provide options for mosquito control districts to screen their mosquito populations for arboviruses and make timely operational decisions regarding vector control. These assays may be utilized even more advantageously when combined with honey-soaked nucleic acid preservation substrate ('honey card') testing by reducing or replacing the time- and labor-intensive efforts of identifying and processing mosquito pools. We tested artificially inoculated honey cards and cards fed upon individually by West Nile virus (WNV) and Zika virus (ZIKV)-infected mosquitoes with three assays to compare detection rates and the limit of detection for each platform with respect to virus detection of a single infected mosquito and quantify the time interval of virus preservation on the cards. Assays evaluated included CDC protocols for real-time reverse transcriptase polymerase chain reaction (RT-PCR) for WNV and ZIKV, Pro-Lab Diagnostics ProAmpRT WNV loop-mediated amplification (LAMP) and ZIKV LAMP assays, and the Rapid Analyte Measurement Platform (RAMP) WNV assay. Real-time RT-PCR was the most sensitive assay and the most robust to viral RNA degradation over time. To maximize the detection of virus, honey cards should be left in the traps </=1 d if using LAMP assays and </=3 d if using real-time RT-PCR to detect viruses from field samples. The WNV RAMP assay, although effective for pool screening, lacks sensitivity required for honey card surveillance. Future studies may determine the minimum number of infectious mosquitoes required to feed on a honey card that would be reliably detected by the LAMP or RAMP assays.

Bourbon virus (Family Orthomyxoviridae: Genus Thogotovirus) was first isolated from a human case-patient residing in Bourbon County, Kansas, who subsequently died. Before becoming ill in late spring of 2014, the patient reported several tick bites. In response, we initiated tick surveillance in Bourbon County and adjacent southern Linn County during spring and summer of 2015. We collected 20,639 host-seeking ticks representing four species from 12 sites. Amblyomma americanum (L.) (Acari: Ixodidae) and Dermacentor variabilis (Say) (Acari: Ixodidae) accounted for nearly all ticks collected (99.99%). Three tick pools, all composed of adult A. americanum ticks collected in Bourbon County, were virus positive. Two pools were Heartland virus (Family Bunyaviridae: Genus Phlebovirus) positive, and one was Bourbon virus positive. The Bourbon virus positive tick pool was composed of five adult females collected on a private recreational property on June 5. Detection of Bourbon virus in the abundant and aggressive human-biting tick A. americanum in Bourbon County supports the contention that A. americanum is a vector of Bourbon virus to humans. The current data combined with virus detections in Missouri suggest that Bourbon virus is transmitted to humans by A. americanum ticks, including both the nymphal and adult stages, that ticks of this species become infected as either larvae, nymphs or both, perhaps by feeding on viremic vertebrate hosts, by cofeeding with infected ticks, or both, and that Bourbon virus is transstadially transmitted. Multiple detections of Heartland virus and Bourbon virus in A. americanum ticks suggest that these viruses share important components of their transmission cycles.

Bourbon virus (BRBV) was first isolated in 2014 from a resident of Bourbon County, Kansas, USA, who died of the infection. In 2015, an ill Payne County, Oklahoma, resident tested positive for antibodies to BRBV, before fully recovering. We retrospectively tested for BRBV in 39,096 ticks from northwestern Missouri, located 240 km from Bourbon County, Kansas. We detected BRBV in 3 pools of Amblyomma americanum (L.) ticks: 1 pool of male adults and 2 pools of nymphs. Detection of BRBV in A. americanum, a species that is aggressive, feeds on humans, and is abundant in Kansas and Oklahoma, supports the premise that A. americanum is a vector of BRBV to humans. BRBV has not been detected in nonhuman vertebrates, and its natural history remains largely unknown.

During the 2014 chikungunya (CHIK) outbreak in the Caribbean, we performed entomological surveys on 3 United States Virgin Islands (USVI): St. Croix, St. Thomas, and St. John. We aimed to evaluate the potential for chikungunya virus (CHIKV) transmission in the USVI. The surveys took place between June 19, 2014, and June 29, 2014, during the dry season in USVI. A total of 1,929 adult mosquitoes belonging to 4 species - Culex quinquefasciatus (68.4%), Aedes aegypti (29.7%), Ae. mediovittatus (1.3%), and Ae. sollicitans (<1%) - were detected. Environmental investigations showed that between 73% and 87% of the homes had containers that could serve as mosquito larval habitats. In addition, 47% of the homes did not have air conditioning and between 69% and 79% of homes showed evidence of frequent outdoor activity exhibited by residents. Taken together, these observations suggest a high potential for CHIKV transmission in USVI. The relative abundance of Ae. aegypti on St. John's, St. Thomas, and St. Croix was 21.0, 11.0, and 3.0 mosquitoes/trap per day, respectively, suggesting that the former 2 islands were at the highest risk of CHIKV outbreaks. Insecticide resistance testing detected high levels of resistance to malathion and permethrin in several local populations of Ae. aegypti on St. Croix Island, which suggested that these 2 insecticides should not be used during CHIK outbreaks.

AbstractIn late 2014, Zika virus (ZIKV; Flaviviridae, Flavivirus) emerged as a significant arboviral disease threat in the Western hemisphere. Aedes aegypti and Aedes albopictus have been considered the principal vectors of ZIKV in the New World due to viral isolation frequency and vector competence assessments. Limited reports of Culex transmission potential have highlighted the need for additional vector competence assessments of North American Culex species. Accordingly, North American Culex pipiens and Culex quinquefasciatus were orally exposed and intrathoracically inoculated with the African prototype ZIKV strain and currently circulating Asian lineage ZIKV strains to assess infection, dissemination, and transmission potential. Results indicated that these two North American Culex mosquito species were highly refractory to oral infection with no dissemination or transmission observed with any ZIKV strains assessed. Furthermore, both Culex mosquito species intrathoracically inoculated with either Asian or African lineage ZIKVs failed to expectorate virus in saliva. These in vivo results were further supported by the observation that multiple mosquito cell lines of Culex species origin demonstrated significant growth restriction of ZIKV strains compared with Aedes-derived cell lines. In summation, no evidence for the potential of Cx. pipiens or Cx. quinquefasciatus to serve as a competent vector for ZIKV transmission in North America was observed.

We assayed Zika virus-infected mosquitoes stored at room temperature for <30 days for live virus by using plaque assay and virus RNA by using real-time reverse transcription PCR. Viable virus was detected in samples stored <10 days, and virus RNA was detected in samples held for 30 days.

Heartland virus (HRTV; Bunyaviridae: Phlebovirus) is a recently described cause of human illness in the United States. After field studies conducted in 2012 implicated Amblyomma americanum (L.) as a vector of HRTV, we initiated experiments to assess the vector competence of A. americanum Larval and nymphal ticks were immersed in high-titered suspensions of HRTV, and then tested for virus at various intervals postimmersion. In a later trial larval ticks were immersed in HRTV, followed by engorgement on a rabbit. A subset of postmolt nymphs was tested for HRTV to document transstadial transmission. Putatively infected nymphs were cofed with uninfected colony larvae to assess nonviremic transmission. In another trial, nymphs were fed on a rabbit and allowed to molt to the adult stage. Male and female ticks fed and mated upon a rabbit, and females were allowed to oviposit. Male and spent female ticks were tested for HRTV, and offspring of infected females were tested to assess vertical transmission. Infection rates of ≤50% were observed in immersed larvae and nymphs tested at intervals following immersion. Transstadial transmission from larvae to nymphs and then to adults was documented. HRTV was detected in a pool of nymphs molted from uninfected larvae cofed with infected nymphs. Vertical transmission of HRTV was observed in progeny of infected females. Infected females took longer to oviposit and produced fewer offspring. Serologic conversions (without viremia) in rabbits fed upon by immersed larvae or transstadially infected ticks indicate horizontal transmission of HRTV.

We evaluated the ability of the Rapid Analyte Measurement Platform (RAMP(R)) mosquito-grinding buffer to inactivate West Nile virus (WNV) by subjecting WNV-positive samples ground in RAMP buffer to incubation intervals ranging from 5 min to 60 min. At each time point an aliquot was removed and serially diluted in bovine albumin (BA)-1 cell culture media to stop the inactivation process by RAMP buffer. Each BA-1 sample was tested for viable virus using Vero 6-well cell culture plaque assay and observed for plaques. We observed very limited inactivation of WNV (1-2 log10 plaque-forming units/ml) by RAMP buffer. Concerned for RAMP operators who may be using this assay in low-level biocontainment facilities, we developed an alternate sample homogenization protocol using Triton X-100 detergent that ensures complete WNV inactivation without compromising the performance of the RAMP assay.

During 2013, we collected and tested ticks for Heartland virus (HRTV), a recently described human pathogen in the genus Phlebovirus (Bunyaviridae), from six sites in northwestern Missouri. Five sites were properties owned by HRTV patients, and the sixth was a conservation area that yielded virus in ticks during 2012. We collected 39,096 ticks representing five species; however, two species, Amblyomma americanum (L.) (97.6%) and Dermacentor variabilis (Say) (2.3%), accounted for nearly all ticks collected. We detected 60 HRTV-positive tick pools and all were composed of A. americanum: 53 pools of nymphs, six pools of male adults, and one pool of female adults. This is the first record of HRTV in adult ticks. Virus was detected at five properties that yielded A. americanum ticks, including properties owned by four of five patients. Virus was detected at two sites that yielded virus in 2012. Detection of virus in multiple years indicates that the virus persists in ticks within a relatively small geographic area, although infection rates (IR) may vary greatly among sites and between years at a site. IR per 1,000 A. americanumin northwestern Missouri during the April-July 2013 study period were as follows: all adults, IR = 1.13; adult females, IR = 0.33; adult males, IR = 1.90; and nymphs, IR = 1.79. The IR in nymphs, the stage with the largest data set, corresponds to 1/559 infected ticks. Having robust estimates of IR in various stages for A. americanum should lead to more accurate public health messaging and a better understanding of virus transmission.

West Nile virus (WNV) is enzootic in northern Colorado. Annual surveillance activities in Fort Collins, CO, include collecting female Culex mosquitoes and testing them for the presence of WNV RNA in order to calculate 1) Culex female abundance, 2) WNV infection rate, and 3) the vector index (VI). These entomological risk indices inform public policy regarding the need for emergency adulticiding. Currently, these are calculated on a city-wide basis. In this study, we present descriptive data from historical surveillance records spanning 2006-2013 to discern seasonal and yearly patterns of entomological risk for WNV infection. Also, we retrospectively test the hypothesis that entomological risk is correlated with human transmission risk and is heterogeneous within the City of Fort Collins. Four logistically relevant zones within the city were established and used to test this hypothesis. Zones in the eastern portion of the city consistently had significantly higher Culex abundance and VI compared with zones in the west, leading to higher entomological risk indicators for human WNV infection in the east. Moreover, the relative risk of a reported human case of WNV infection was significantly higher in the eastern zones of the city. Our results suggest that a more spatially targeted WNV management program may better mitigate human risk for WNV infection in Fort Collins, and possibly other cities where transmission is enzootic, while at the same time reducing pesticide use.

Sandfly fever group viruses in the genus Phlebovirus (family Bunyaviridae) are widely distributed across the globe and are a cause of disease in military troops and indigenous peoples. We assessed the laboratory sensitivity and specificity of the Sand Fly Fever Virus Antigen Assay, a rapid dipstick assay designed to detect sandfly fever Naples virus (SFNV) and Toscana virus (TOSV) against a panel of phleboviruses. The assay detected SFNV and TOSV, as well as other phleboviruses including Aguacate, Anahanga, Arumowot, Chagres, and Punta Toro viruses. It did not detect sandfly fever Sicilian, Heartland, Rio Grande, or Rift Valley fever viruses. It did not produce false positive results in the presence of uninfected sand flies (Lutzomyia longipalpis) or Cache Valley virus, a distantly related bunyavirus. Results from this laboratory evaluation suggest that this assay may be used as a rapid field-deployable assay to detect sand flies infected with TOSV and SFNV, as well as an assortment of other phleboviruses.

We evaluated the utility of 2 types of commercially available antigens as positive controls in the Rapid Analyte Measurement Platform (RAMP(R)) West Nile virus (WNV) assay. Purified recombinant WNV envelope antigens and whole killed virus antigens produced positive RAMP results and either type would be useful as a positive control. Killed virus antigens provide operational and economic advantages and we recommend their use over purified recombinant antigens. We also offer practical applications for RAMP positive controls and recommendations for preparing them.