Consensus and uncertainty in the geographic range of Aedes aegypti and Aedes albopictus in the contiguous United States: Multi-model assessment and synthesis.

Aedes (Stegomyia) aegypti (L.) and Ae. (Stegomyia) albopictus (Skuse) mosquitoes can transmit dengue, chikungunya, yellow fever, and Zika viruses. Limited surveillance has led to uncertainty regarding the geographic ranges of these vectors globally, and particularly in regions at the present-day margins of habitat suitability such as the contiguous United States. Empirical habitat suitability models based on environmental conditions can augment surveillance gaps to describe the estimated potential species ranges, but model accuracy is unclear. We identified previously published regional and global habitat suitability models for Ae. aegypti (n = 6) and Ae. albopictus (n = 8) for which adequate information was available to reproduce the models for the contiguous U.S. Using a training subset of recently updated county-level surveillance records of Ae. aegypti and Ae. albopictus and records of counties conducting surveillance, we constructed accuracy-weighted, probabilistic ensemble models from these base models. To assess accuracy and uncertainty we compared individual and ensemble model predictions of species presence or absence to both training and testing data. The ensemble models were among the most accurate and also provided calibrated probabilities of presence for each species. The quantitative probabilistic framework enabled identification of areas with high uncertainty and model bias across the U.S. where improved models or additional data could be most beneficial. The results may be of immediate utility for counties considering surveillance and control programs for Ae. aegypti and Ae. albopictus. Moreover, the assessment framework can drive future efforts to provide validated quantitative estimates to support these programs at local, national, and international scales.

Detection of Zika Virus in Desiccated Mosquitoes by Real-Time Reverse Transcription PCR and Plaque Assay.

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.

Bourbon Virus in Field-Collected Ticks, Missouri, USA.

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.

Zika Virus Infection in Patient with No Known Risk Factors, Utah, USA, 2016.

In 2016, Zika virus disease developed in a man (patient A) who had no known risk factors beyond caring for a relative who died of this disease (index patient). We investigated the source of infection for patient A by surveying other family contacts, healthcare personnel, and community members, and testing samples for Zika virus. We identified 19 family contacts who had similar exposures to the index patient; 86 healthcare personnel had contact with the index patient, including 57 (66%) who had contact with body fluids. Of 218 community members interviewed, 28 (13%) reported signs/symptoms and 132 (61%) provided a sample. Except for patient A, no other persons tested had laboratory evidence of recent Zika virus infection. Of 5,875 mosquitoes collected, none were known vectors of Zika virus and all were negative for Zika virus. The mechanism of transmission to patient A remains unknown but was likely person-to-person contact with the index patient.

A Simple Modification to the Mosquito Homogenization Protocol Safely Inactivates West Nile Virus and Allows Virus Detection by the Rapid Analyte Measurement Platform (RAMP®) ASSAY.

We evaluated the ability of the Rapid Analyte Measurement Platform (RAMP(®)) 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.

Limited genomic divergence between intraspecific forms of Culex pipiens under different ecological pressures.

BACKGROUND: Divergent selection can be a major driver of ecological speciation. In insects of medical importance, understanding the speciation process is both of academic interest and public health importance. In the West Nile virus vector Culex pipiens, intraspecific pipiens and molestus forms vary in ecological and physiological traits. Populations of each form appear to share recent common ancestry but patterns of genetic differentiation across the genome remain unknown. Here, we undertook an AFLP genome scan on samples collected from both sympatric and allopatric populations from Europe and the USA to quantify the extent of genomic differentiation between the two forms. RESULTS: The forms were clearly differentiated but each exhibited major population sub-structuring between continents. Divergence between pipiens and molestus forms from USA was higher than in both inter- and intra-continental comparisons with European samples. The proportion of outlier loci between pipiens and molestus (≈3 %) was low but consistent in both continents, and similar to those observed between sibling species of other mosquito species which exhibit contemporary gene flow. Only two of the outlier loci were shared between inter-form comparisons made within Europe and USA. CONCLUSION: This study supports the molestus and pipiens status as distinct evolutionary entities with low genomic divergence. The low number of shared divergent loci between continents suggests a relatively limited number of genomic regions determining key typological traits likely to be driving incipient speciation and/or adaptation of molestus to anthropogenic habitats.

Evaluating the Use of Commercial West Nile Virus Antigens as Positive Controls in the Rapid Analyte Measurement Platform West Nile Virus Assay.

We evaluated the utility of 2 types of commercially available antigens as positive controls in the Rapid Analyte Measurement Platform (RAMP®) 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.

Evaluation of a rapid analyte measurement platform and real-time reverse-transcriptase polymerase chain reaction assay West Nile virus detection system in mosquito pools.

We evaluated the commercially available Rapid Analyte Measurement Platform (RAMP) West Nile virus (WNV) antigen detection test for sensitivity and consistency with real-time reverse transcriptase polymerase chain reaction (RT-PCR) confirmation testing. Panels of samples consisting of WNV-spiked mosquito pools and negative control pools were sent to 20 mosquito abatement districts (MADs) that processed the pools using the RAMP assay. The samples were then sent to the reference laboratories used by the MADs for confirmation by real-time RT-PCR. Positive pools with virus titers of roughly 1-3 log10 PFU/ml had RAMP scores above the RAMP test positive cutoff score of 30 RAMP units, but these virus-positive samples could not be reliably confirmed by real-time RT-PCR testing. Pools with virus titers > or =4 log10 PFU/ml scored > or =50 RAMP units. Real-time RT-PCR results varied among the confirmation laboratories. With few exceptions, pools returning a RAMP score of > or =100 were confirmed with real-time RT-PCR, while pools returning a RAMP score of 50-99 appeared to be at the limit of real-time RT-PCR detection. Therefore, we recommend using a positive cutoff of 50 RAMP units with no real-time RT-PCR confirmation to maximize speed, efficiency, and economy of the RAMP assay. A more conservative approach would be to implement a "gray zone" range of 50-100 RAMP units. Pools scoring within the gray zone could be submitted for real-time RT-PCR confirmation with the understanding that positive pools may not confirm due to the inhibitory effect of the RAMP buffer on the real-time RT-PCR assay. We also conducted a series of experiments using laboratory-prepared mosquito pools spiked with WNV to compare mosquito homogenization buffers, pool sizes, and grinding methods in order to determine how these variables affect the RAMP and real-time RT-PCR assay results.

Overwintering biology of Culex (Diptera: Culicidae) mosquitoes in the Sacramento Valley of California.

At temperate latitudes, Culex (Diptera: Culicidae) mosquitoes typically overwinter as adult females in reproductive arrest and also may serve as reservoir hosts for arboviruses when cold temperatures arrest viral replication. To evaluate their role in the persistence of West Nile virus (WNV) in the Sacramento Valley of California, the induction and termination of diapause were investigated for members of the Culex pipiens (L.) complex, Culex tarsalis Coquillett, and Culex stigmatosoma Dyar under field, seminatural, and experimental conditions. All Culex spp. remained vagile throughout winter, enabling the collection of 3,174 females and 1,706 males from diverse habitats during the winters of 2010-2012. Overwintering strategies included both quiescence and diapause. In addition, Cx. pipiens form molestus Forskäl females remained reproductively active in both underground and aboveground habitats. Some blood-fed, gravid, and parous Cx. tarsalis and Cx. pipiens complex females were collected throughout the winter period. Under both field and experimental conditions, Cx. tarsalis and Cx. stigmatosoma females exposed to autumnal conditions arrested primary follicular maturation at previtellogenic stage I, with primary to secondary follicular ratios <1.5 (indicative of a hormonally induced diapause). In contrast, most Cx. pipiens complex females did not enter reproductive diapause and ovarian follicles matured to >or=stage I-II (host-seeking arrest) or were found in various stages of degeneration. Diapause was initiated in the majority of Cx. tarsalis and Cx. stigmatosoma females by mid-late October and was terminated after the winter solstice, but host-seeking seemed limited by temperature. An accrual of 97.52 +/- 30.7 and 162.85 +/- 79.3 degree-days after the winter solstice was estimated to be necessary for diapause termination in Cx. tarsalis under field and seminatural conditions, respectively. An increase in the proportion of blood-fed Culex females in resting collections occurred concurrently with diapause termination in field populations based on ovarian morphometrics. WNV RNA was detected in one pool of 18 males and in a single blood-fed female Cx. tarsalis collected during winter. Therefore, both vertically and horizontally infected Culex females may persist through winter and possibly transmit WNV after diapause termination in late winter or early spring in the Sacramento Valley of California.

The Culex pipiens complex in the Mississippi River basin: identification, distribution, and bloodmeal hosts.

Members of the Culex pipiens complex are the primary vectors of St. Louis encephalitis virus and West Nile virus in the Mississippi River basin (MRB). The Cx. pipiens complex in the MRB is composed of 4 taxa: Cx. p. pipiens form pipiens, Cx. p. quinquefasciatus, hybrids between Cx. p. pipiens f. pipiens and Cx. p. quinquefasciatus, and Cx. p. pipiens form molestus. Three studies on bloodmeal hosts with large sample sizes have been conducted on members of the Cx. pipiens complex in the MRB including 1 each on Cx. p. quinquefasciatus from Louisiana, Cx. p. pipiens-quinquefasciatus hybrids from Tennessee, and Cx. p. pipiens from Illinois. The top 8 bloodmeal hosts from each of the 3 sites accounted for 68-92% of bloodmeals. Only 14 species accounted for the top 8 bloodmeal hosts at each of the 3 sites. The most often utilized bloodmeal hosts for members of the Culex pipiens complex within the MRB are the American robin, Northern cardinal, human, raccoon, common grackle, house sparrow, mourning dove, dog, Northern mockingbird, blue jay, opossum, domestic horse, house finch and European starling. Human feeding varied widely among sites from 1% to 15.7% of bloodmeals. The proportion of bloodmeals taken on humans is an important epidemiological variable and future studies are needed to define the primary genetic and environmental factors that influence host utilization by members of the Cx. pipiens complex.

A comparison of above-ground and below-ground populations of Culex pipiens pipiens in Chicago, Illinois, and New York City, New York, using 2 microsatellite assays.

Aboveground and belowground populations of the mosquito Culex pipiens pipiens are traditionally classified as form (f.) pipiens and f. molestus, respectively, and gene flow between forms is thought to be limited. Relatively few f. molestus populations have been found in the United States, which has hindered their study in North America. In this investigation, we used microsatellites to characterize a recently discovered population of f. molestus in Chicago, IL, and compared levels of genetic diversity and differentiation in above-ground and below-ground populations from Chicago and New York City, NY. Levels of genetic diversity were markedly lower in both f. molestus populations. Pairwise F(ST) values between populations indicated that f. molestus populations were highly divergent from each other, as well as from their associated aboveground populations. The most likely number of genetic clusters depended on the number of loci used; we began with a set of 8, and reanalyzed the specimens with 17. Using a panel of 17 loci, there were 4 clusters, 1 for each below-ground population, and 1 for each pair of above-ground populations. Our findings are supportive of the hypothesis that f. molestus populations in Chicago and New York City arose from local aboveground populations.

Development of multiplexed species specific polymerase chain reaction assays for identification of the Culex (Melanoconion) species (Diptera: Culicidae) of the southeastern United States based on rDNA.

Adult female mosquitoes within the subgenus Culex (Melanoconion) Theobald (Diptera: Culicidae) are difficult to identify to species using external morphological features. We present two multiplexed polymerase chain reaction assays that quickly and accurately identify specimens from the southeastern United States based on sequence differences in the internal transcribed spacers of the ribosomal DNA gene array. One assay identifies all species that occur only in Florida, whereas the second assay identifies species that may occur in other southeastern states. These assays require small amounts of DNA, such as DNA from two sonicated legs, or an individual specimen. These assays also may be run as multiple singleplex reactions to determine the mosquito species composition of virus-positive mosquito pools. Reaction volumes may be as low as 10 microl, which reduces assay cost.

Vector competence of the stable fly (Diptera: Muscidae) for West Nile virus.

In 2006-2007, stable flies, Stomoxys calcitrans (L.) (Diptera: Muscidae), were suspected of being enzootic vectors of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) during a die-off of American white pelicans (Pelecanus erythrorhynchos Gmelin) (Pelecanidae) in Montana, USA. WNV-positive stable flies were observed feeding en masse on incapacitated, WNV-positive pelicans, arousing suspicions that the flies could have been involved in WNV transmission among pelicans, and perhaps to livestock and humans. We assessed biological transmission by infecting stable flies intrathoracically with WNV and testing them at 2-d intervals over 20 d. Infectious WNV was detected in fly bodies in decreasing amounts over time for only the first 6 d postinfection, an indication that WNV did not replicate within fly tissues and that stable flies cannot biologically transmit WNV. We assessed mechanical transmission using a novel technique. Specifically, we fed WNV-infected blood to individual flies by using a cotton swab (i.e., artificial donor), and at intervals of 1 min-24 h, we allowed flies to refeed on a different swab saturated with WNV-negative blood (i.e., artificial recipient). Flies mechanically transmitted viable WNV from donor to recipient swabs for up to 6 h postinfection, with the majority of the transmission events occurring within the first hour. Flies mechanically transmitted WNV RNA to recipient swabs for up to 24 h, mostly within the first 6 h. Given its predilection to feed multiple times when disturbed, these findings support the possibility that the stable fly could mechanically transmit WNV.

Laboratory Evaluation of the Rapid Analyte Measurement Platform Assay to Detect Dengue Virus in Mosquito Pools.

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.

Laboratory and Field Evaluations of a Commercially Available Real-Time Loop-Mediated Isothermal Amplification Assay for the Detection of West Nile Virus in Mosquito Pools.

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.

Experimental Infection of Amblyomma americanum (Acari: Ixodidae) With Bourbon Virus (Orthomyxoviridae: Thogotovirus).

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.

A comparison of aboveground and belowground populations of Culex pipiens (Diptera: Culicidae) mosquitoes in Chicago, Illinois, and New York City, New York, using microsatellites.

Aboveground and belowground populations of the mosquito Culex pipiens L. are traditionally classified as form pipiens and form molestus, respectively, and gene flow between forms is thought to be limited. Relatively few f. molestus populations have been found in the United States, which has hindered their study in North America. In this study, we used microsatellites to characterize a newly discovered population of f. molestus in Chicago, IL, and compared levels of genetic diversity and differentiation in aboveground and belowground populations from Chicago and New York City, NY. Levels of genetic diversity, as measured by expected heterozygosity and allelic richness, were markedly lower in both f. molestus populations. Allele frequencies were distinctly different between the two f. molestus populations, and some alleles were present in one belowground population and not the other. Pairwise F(ST) values between populations indicated that f. molestus populations were highly divergent from each other, as well as from their associated aboveground populations. Cluster analysis suggested the most likely number of groups was three, with the four f. pipiens populations in one cluster, and each of the f. molestus populations in its own cluster. Admixture analysis detected a low number of hybrids, 8%, between forms. We also tested the efficacy of two assays purported to distinguish between the forms, the CQ11 assay and a restriction fragment-length polymorphism assay of the COI gene, and found neither assay reliable in this regard. Our findings support the hypothesis that f. molestus populations in Chicago and New York City arose from local aboveground populations.

Seasonality and time of host-seeking activity of Culex tarsalis and floodwater Aedes in northern Colorado, 2006-2007.

Effective and economical control of adult vector and pest mosquitoes requires knowledge of their seasonal abundance and host-seeking activity patterns. We conducted research in 2006-2007 to study these variables for Culex tarsalis, Aedes vexans, Ae. melanimon, and Ae. dorsalis in Larimer County, CO. Mosquitoes were collected with traps that segregated catches in 7 consecutive 2-h intervals initiating at 1730 h at 4 sites. Seasonal abundance varied for all species by site and year. Time of host-seeking activity was consistent for all species by site and year. Culex tarsalis counts were significantly higher 1.2-4.5 h after sunset than during the preceding time intervals. Maximum host-seeking activity of the 3 Aedes species occurred from 0.8 h before sunset to 6.5 h after. Host seeking by all species continued throughout the night. For optimal control of Cx. tarsalis adulticide application should start approximately 1 h after sunset, and control of Aedes species should begin soon after sunset, and for all species applications can continue throughout most of the night.

Bloodmeal, Host Selection, and Genetic Admixture Analyses of Culex pipiens Complex (Diptera: Culicidae) Mosquitoes in Chicago, IL.

The area in and around Chicago, IL, is a hotspot of West Nile virus activity. The discovery of a Culex pipiens form molestus Forskӓl population in Chicago in 2009 added to speculation that offspring from hybridization between Cx. pipiens f. pipiens L. and f. molestus could show a preference for feeding on humans. We collected blood-fed female mosquitoes (N = 1,023) from eight residential sites and one public park site in Chicago in July and August 2012. Bloodmeal analysis using the COI (cytochrome c oxidase subunit I) gene was performed to ascertain host choice. Almost all (99%) bloodmeals came from birds, with American Robins (Turdus migratorius L.) and House Sparrows (Passer domesticus L.) making up the largest percentage (74% combined). A forage ratio analysis comparing bird species fed upon and available bird species based on point count surveys indicated Northern Cardinals (Cardinalis cardinalis) and American Robins (Turdus migratorius) appeared to be over-utilized, whereas several species were under-utilized. Two human bloodmeals came from Culex pipiens complex mosquitoes. Admixture and population genetic analyses were conducted with 15 microsatellite loci on head and thorax DNA from the collected blood-fed mosquitoes. A modest amount of hybridization was detected between Cx. pipiens f. pipiens and f. molestus, as well as between f. pipiens and Cx. quinquefasciatus Say. Several pure Cx. quinquefasciatus individuals were noted at the two Trumbull Park sites. Our data suggest that Cx. pipiens complex mosquitoes in the Chicago area are not highly introgressed with f. molestus and appear to utilize avian hosts.