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.

Fatal Case of Heartland Virus Disease Acquired in the Mid-Atlantic Region, United States.

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.

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.

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.

Genomic characterization of 99 viruses from the bunyavirus families Nairoviridae, Peribunyaviridae, and Phenuiviridae, including 35 previously unsequenced viruses.

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 relationships 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.

Powassan Virus Infection Likely Acquired Through Blood Transfusion Presenting as Encephalitis in a Kidney Transplant Recipient.

A kidney transplant patient without known tick exposure developed encephalitis 3 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. Here, we describe a probable instance of transfusion-transmitted Powassan virus infection.

Heartland Virus in Humans and Ticks, Illinois, USA, 2018-2019.

In 2018, Heartland disease virus infected 2 persons in Illinois, USA. In 2019, ticks were collected at potential tick bite exposure locations and tested for Heartland and Bourbon viruses. A Heartland virus-positive pool of adult male Amblyomma americanum ticks was found at 2 locations, 439 km apart, suggesting widespread distribution in Illinois.

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.

Focal amplification and suppression of West Nile virus transmission associated with communal bird roosts in northern Colorado.

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.

Surveillance for Heartland and Bourbon Viruses in Eastern Kansas, June 2016.

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.

Laboratory Evaluation of Commercially Available Platforms to Detect West Nile and Zika Viruses From Honey Cards.

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.

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.

Surveillance for Tick-Borne Viruses Near the Location of a Fatal Human Case of Bourbon Virus (Family Orthomyxoviridae: Genus Thogotovirus) in Eastern Kansas, 2015.

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 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.

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.

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.

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.