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.

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.

Cache Valley virus: an emerging arbovirus of public and veterinary health importance.

Cache Valley virus (CVV) is a mosquito-borne virus in the genus Orthobunyavirus (Bunyavirales: Peribunyaviridae) that has been identified as a teratogen in ruminants causing fetal death and severe malformations during epizootics in the U.S. CVV has recently emerged as a viral pathogen causing severe disease in humans. Despite its emergence as a public health and agricultural concern, CVV has yet to be significantly studied by the scientific community. Limited information exists on CVV's geographic distribution, ecological cycle, seroprevalence in humans and animals, and spectrum of disease, including its potential as a human teratogen. Here, we present what is known of CVV's virology, ecology, and clinical disease in ruminants and humans. We discuss the current diagnostic techniques available and highlight gaps in our current knowledge and considerations for future research.

Laboratory Validation of a Real-Time RT-PCR Assay for the Detection of Jamestown Canyon Virus.

The neuroinvasive disease caused by Jamestown Canyon virus (JCV) infection is rare. However, increasing incidence and widespread occurrence of the infection make JCV a growing public health concern. Presently, clinical diagnosis is achieved through serological testing, and mosquito pool surveillance requires virus isolation and identification. A rapid molecular detection test, such as real-time RT-PCR, for diagnosis and surveillance of JCV has not been widely utilized. To enhance testing and surveillance, here, we describe the development and validation of a real-time RT-PCR test for the detection of JCV RNA. Three primer and probe sets were evaluated for analytical sensitivity and specificity. One probe set, JCV132FAM, was found to be the most sensitive test detecting 7.2 genomic equivalents/µL. While less sensitive, a second probe set JCV231cFAM was the most specific test with limited detection of Keystone virus at high RNA loads. Taken together, these data indicate both probe sets can be utilized for a primary sensitive screening assay and a secondary specific confirmatory assay. While both primer and probe sets detected high viral loads of Keystone virus, these assays did not detect any virus in the California encephalitis virus clade, including negative detection of the medically important La Crosse virus (LACV) and snowshoe hare virus (SSHV). The real-time RT-PCR assay described herein could be utilized in diagnosis and surveillance in regions with co-circulation of JCV and LACV or SSHV to inform public health action.

The Effect of Fluctuating Incubation Temperatures on West Nile Virus Infection in Culex Mosquitoes.

Temperature plays a significant role in the vector competence, extrinsic incubation period, and intensity of infection of arboviruses within mosquito vectors. Most laboratory infection studies use static incubation temperatures that may not accurately reflect daily temperature ranges (DTR) to which mosquitoes are exposed. This could potentially compromise the application of results to real world scenarios. We evaluated the effect of fluctuating DTR versus static temperature treatments on the infection, dissemination, and transmission rates and viral titers of Culex tarsalis and Culex quinquefasciatus mosquitoes for West Nile virus. Two DTR regimens were tested including an 11 and 15 °C range, both fluctuating around an average temperature of 28 °C. Overall, no significant differences were found between DTR and static treatments for infection, dissemination, or transmission rates for either species. However, significant treatment differences were identified for both Cx. tarsalis and Cx. quinquefasciatus viral titers. These effects were species-specific and most prominent later in the infection. These results indicate that future studies on WNV infections in Culex mosquitoes should consider employing realistic DTRs to reflect interactions most accurately between the virus, vector, and environment.

Infection, Dissemination, and Transmission Potential of North American Culex quinquefasciatus, Culex tarsalis, and Culicoides sonorensis for Oropouche Virus.

Oropouche virus (OROV), a vector-borne Orthobunyavirus circulating in South and Central America, causes a febrile illness with high rates of morbidity but with no documented fatalities. Oropouche virus is transmitted by numerous vectors, including multiple genera of mosquitoes and Culicoides biting midges in South America. This study investigated the vector competence of three North American vectors, Culex tarsalis, Culex quinquefasciatus, and Culicoides sonorensis, for OROV. Cohorts of each species were fed an infectious blood meal containing 6.5 log10 PFU/mL OROV and incubated for 10 or 14 days. Culex tarsalis demonstrated infection (3.13%) but not dissemination or transmission potential at 10 days post infection (DPI). At 10 and 14 DPI, Cx. quinquefasciatus demonstrated 9.71% and 19.3% infection, 2.91% and 1.23% dissemination, and 0.97% and 0.82% transmission potential, respectively. Culicoides sonorensis demonstrated 86.63% infection, 83.14% dissemination, and 19.77% transmission potential at 14 DPI. Based on these data, Cx. tarsalis is unlikely to be a competent vector for OROV. Culex quinquefasciatus demonstrated infection, dissemination, and transmission potential, although at relatively low rates. Culicoides sonorensis demonstrated high infection and dissemination but may have a salivary gland barrier to the virus. These data have implications for the spread of OROV in the event of a North American introduction.

Eastern Equine Encephalitis Virus Seroprevalence in Maine Cervids, 2012-2017.

Eastern equine encephalitis virus (EEEV) first emerged in Maine in the early 2000s and resulted in an epizootic outbreak in 2009. Since 2009, serum samples from cervids throughout Maine have been collected and assessed for the presence of neutralizing antibodies to EEEV to assess EEEV activity throughout the state. We tested 1,119 Odocoileus virginianus (white-tailed deer) and 982 Alces americanus (moose) serum samples collected at tagging stations during the hunting seasons from 2012 to 2017 throughout the state of Maine. Odocoileus virginianus from all 16 counties were EEEV seropositive, whereas A. americanus were seropositive in the northwestern counties of Aroostook, Somerset, Piscataquis, and Franklin counties. Seroprevalence in O. virginianus ranged from 6.6% to 21.2% and in A. americanus from 6.6% to 10.1%. Data from this report in conjunction with findings previously reported from 2009 to 2011 indicate that EEEV is endemic throughout Maine.

Mosquito Control Activities during Local Transmission of Zika Virus, Miami-Dade County, Florida, USA, 2016.

In 2016, four clusters of local mosquitoborne Zika virus transmission were identified in Miami-Dade County, Florida, USA, generating "red zones" (areas into which pregnant women were advised against traveling). The Miami-Dade County Mosquito Control Division initiated intensive control activities, including property inspections, community education, and handheld sprayer applications of larvicides and adulticides. For the first time, the Mosquito Control Division used a combination of areawide ultralow-volume adulticide and low-volume larvicide spraying to effectively control Aedes aegypti mosquitoes, the primary Zika virus vector within the county. The number of mosquitoes rapidly decreased, and Zika virus transmission was interrupted within the red zones immediately after the combination of adulticide and larvicide spraying.

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.

Rapid Screening of Aedes aegypti Mosquitoes for Susceptibility to Insecticides as Part of Zika Emergency Response, Puerto Rico.

In response to the 2016 Zika outbreak, Aedes aegypti mosquitoes from 38 locations across Puerto Rico were screened using Centers for Disease Control and Prevention bottle bioassays for sensitivity to insecticides used for mosquito control. All populations were resistant to pyrethroids. Naled, an organophosphate, was the most effective insecticide, killing all mosquitoes tested.

Susceptibility and Vectorial Capacity of American Aedes albopictus and Aedes aegypti (Diptera: Culicidae) to American Zika Virus Strains.

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 Abstract: La rápida expansión del virus Zika, poco después de las epidemias de chikungunya, rebaso la infraestructura de salud pública en muchos países y sorprendió a muchos en la comunidad científica. Notablemente, Aedes aegypti y Aedes albopictus transmiten estos patógenos además 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 generación reciente. Encontramos altos porcentajes de infección y diseminación en ambas especies (95%). Se reporta, la transmisión viral en ambas especies (74 y 33%, para Aedes aegypti and Aedes albopictus, respectivamente) y una probable barrera a nivel de glándulas salivarías. Finalmente, calculamos la capacidad vectorial para ambas especies.

Restriction of Zika virus infection and transmission in Aedes aegypti mediated by an insect-specific flavivirus.

Previous studies demonstrated an insect-specific flavivirus, Nhumirim virus (NHUV), can suppress growth of West Nile virus (WNV) and decrease transmission rates in NHUV/WNV co-inoculated Culex quinquefasciatus. To assess whether NHUV might interfere with transmission of other medically important flaviviruses, the ability of NHUV to suppress viral growth of Zika virus (ZIKV) and dengue-2 virus (DENV-2) was assessed in Aedes albopictus cells. Significant reductions in ZIKV (100,000-fold) and DENV-2 (10,000-fold) were observed in either cells concurrently inoculated with NHUV or pre-inoculated with NHUV. In contrast, only a transient 10-fold titer reduction was observed with an alphavirus, chikungunya virus. Additionally, restricted in vitro mosquito growth of ZIKV was associated with lowered levels of intracellular ZIKV RNA in NHUV co-inoculated cultures. To assess whether NHUV could modulate vector competence for ZIKV, NHUV-inoculated Aedes aegypti were orally exposed to ZIKV. NHUV-inoculated mosquitoes demonstrated significantly lower ZIKV infection rates (18%) compared to NHUV unexposed mosquitoes (51%) (p < 0.002). Similarly, lower ZIKV transmission rates were observed for NHUV/ZIKV dually intrathoracically inoculated mosquitoes (41%) compared to ZIKV only inoculated mosquitoes (78%) (p < 0.0001), suggesting that NHUV can interfere with both midgut infection and salivary gland infection of ZIKV in Ae. aegypti. These results indicate NHUV could be utilized to model superinfection exclusion mechanism(s) and to study the potential for the mosquito virome to impact transmission of medically important flaviviruses.

Generation of a Lineage II Powassan Virus (Deer Tick Virus) cDNA Clone: Assessment of Flaviviral Genetic Determinants of Tick and Mosquito Vector Competence.

The Flavivirus genus comprises a diverse group of viruses that utilize a wide range of vertebrate hosts and arthropod vectors. The genus includes viruses that are transmitted solely by mosquitoes or vertebrate hosts as well as viruses that alternate transmission between mosquitoes or ticks and vertebrates. Nevertheless, the viral genetic determinants that dictate these unique flaviviral host and vector specificities have been poorly characterized. In this report, a cDNA clone of a flavivirus that is transmitted between ticks and vertebrates (Powassan lineage II, deer tick virus [DTV]) was generated and chimeric viruses between the mosquito/vertebrate flavivirus, West Nile virus (WNV), were constructed. These chimeric viruses expressed the prM and E genes of either WNV or DTV in the heterologous nonstructural (NS) backbone. Recombinant chimeric viruses rescued from cDNAs were characterized for their capacity to grow in vertebrate and arthropod (mosquito and tick) cells as well as for in vivo vector competence in mosquitoes and ticks. Results demonstrated that the NS elements were insufficient to impart the complete mosquito or tick growth phenotypes of parental viruses; however, these NS genetic elements did contribute to a 100- and 100,000-fold increase in viral growth in vitro in tick and mosquito cells, respectively. Mosquito competence was observed only with parental WNV, while infection and transmission potential by ticks were observed with both DTV and WNV-prME/DTV chimeric viruses. These data indicate that NS genetic elements play a significant, but not exclusive, role for vector usage of mosquito- and tick-borne flaviviruses.

Zika Virus MB16-23 in Mosquitoes, Miami-Dade County, Florida, USA, 2016.

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.

Transmission Incompetence of Culex quinquefasciatus and Culex pipiens pipiens from North America for Zika Virus.

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.

Entomological Investigations During Early Stages of A Chikungunya Outbreak In the United States Virgin Islands, 2014.

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.

Seasonal Patterns in Eastern Equine Encephalitis Virus Antibody in Songbirds in Southern Maine.

The intent of this study was to assess passerine eastern equine encephalitis virus (EEEv) seroprevalence during the breeding season in southern Maine by testing songbird species identified in the literature as amplifying hosts of this virus. In 2013 and 2014, we collected serum samples from songbirds at a mainland site and an offshore island migratory stopover site, and screened samples for EEEv antibodies using plaque reduction neutralization tests. We compared seasonal changes in EEEv antibody seroprevalence in young (hatched in year of capture) and adult birds at the mainland site, and also compared early season seroprevalence in mainland versus offshore adult birds. EEEv seroprevalence did not differ significantly between years at either site. During the early season (May), EEEv antibody seroprevalence was substantially lower (9.6%) in the island migrant adults than in mainland adults (42.9%), 2013-2014. On the mainland, EEEv antibody seroprevalence in young birds increased from 12.9% in midseason (June-August) to 45.6% in late season (September/October), 2013-2014. Seroprevalence in adult birds did not differ between seasons (48.8% vs. 53.3%). EEEv activity in Maine has increased in the past decade as measured by increased virus detection in mosquitoes and veterinary cases. High EEEv seroprevalence in young birds-as compared to that of young birds in other studies-corresponded with two consecutive active EEEv years in Maine. We suggest that young, locally hatched songbirds be sampled as a part of long-term EEEv surveillance, and provide a list of suggested species to sample, including EEEv "superspreaders."

MicroRNA reduction of neuronal West Nile virus replication attenuates and affords a protective immune response in mice.

West Nile virus (WNV) is an important agent of human encephalitis that has quickly become endemic across much of the United States since its identification in North America in 1999. While the majority (∼75%) of infections are subclinical, neurologic disease can occur in a subset of cases, with outcomes including permanent neurologic damage and death. Currently, there are no WNV vaccines approved for use in humans. This study introduces a novel vaccine platform for WNV to reduce viral replication in the central nervous system while maintaining peripheral replication to elicit strong neutralizing antibody titers. Vaccine candidates were engineered to incorporate microRNA (miRNA) target sequences for a cognate miRNA expressed only in neurons, allowing the host miRNAs to target viral transcription through endogenous RNA silencing. To maintain stability, these targets were incorporated in multiple locations within the 3'-untranslated region, flanking sequences essential for viral replication without affecting the viral open reading frame. All candidates replicated comparably to wild type WNV in vitro within cells that did not express the cognate miRNA. Insertional control viruses were also capable of neuroinvasion and neurovirulence in vivo in CD-1 mice. Vaccine viruses were safe at all doses tested and did not demonstrate mutations associated with a reversion to virulence when serially passaged in mice. All vaccine constructs were protective from lethal challenge in mice, producing 93-100% protection at the highest dose tested. Overall, this is a safe and effective attenuation strategy with broad potential application for vaccine development.

Potential for Co-Infection of a Mosquito-Specific Flavivirus, Nhumirim Virus, to Block West Nile Virus Transmission in Mosquitoes.

Nhumirim virus (NHUV) is an insect-specific virus that phylogenetically affiliates with dual-host mosquito-borne flaviviruses. Previous in vitro co-infection experiments demonstrated prior or concurrent infection of Aedes albopictus C6/36 mosquito cells with NHUV resulted in a 10,000-fold reduction in viral production of West Nile virus (WNV). This interference between WNV and NHUV was observed herein in an additional Ae. albopictus mosquito cell line, C7-10. A WNV 2K peptide (V9M) mutant capable of superinfection with a pre-established WNV infection demonstrated a comparable level of interference from NHUV as the parental WNV strain in C6/36 and C7-10 cells. Culex quinquefasciatus and Culex pipiens mosquitoes intrathoracically inoculated with NHUV and WNV, or solely with WNV as a control, were allowed to extrinsically incubate the viruses up to nine and 14 days, respectively, and transmissibility and replication of WNV was determined. The proportion of Cx. quinquefasciatus mosquitoes capable of transmitting WNV was significantly lower for the WNV/NHUV group than the WNV control at seven and nine days post inoculation (dpi), while no differences were observed in the Cx. pipiens inoculation group. By dpi nine, a 40% reduction in transmissibility in mosquitoes from the dual inoculation group was observed compared to the WNV-only control. These data indicate the potential that infection of some Culex spp. vectors with NHUV could serve as a barrier for efficient transmissibility of flaviviruses associated with human disease.

The first outbreak of eastern equine encephalitis in Vermont: outbreak description and phylogenetic relationships of the virus isolate.

The first known outbreak of eastern equine encephalitis (EEE) in Vermont occurred on an emu farm in Rutland County in 2011. The first isolation of EEE virus (EEEV) in Vermont (VT11) was during this outbreak. Phylogenetic analysis revealed that VT11 was most closely related to FL01, a strain from Florida isolated in 2001, which is both geographically and temporally distinct from VT11. EEEV RNA was not detected in any of the 3,905 mosquito specimens tested, and the specific vectors associated with this outbreak are undetermined.