Host-Feeding Behavior of Mosquitoes in the Florida Everglades.

Background: West Nile virus (WNV), Everglades virus (EVEV), and five species of Orthobunyavirus were isolated from mosquitoes collected in the Everglades in 2016-2017. Prior studies of blood meals of mosquitoes in southern Florida have related findings to acquisition and transmission of EVEV, St. Louis encephalitis virus, and WNV, but not the Orthobunyavirus viruses associated with the subgenus Melanoconion of the genus Culex. Materials and Methods: In the present study, blood-fed mosquitoes were collected in the Everglades in 2016, 2017, 2021, and 2022, and from an industrial site in Naples, FL in 2017. Blood meals were identified to host species by PCR assays using mitochondrial cytochrome b gene. Results: Blood meals were identified from Anopheles crucians complex and 11 mosquito species captured in the Florida Everglades and from 3 species collected from an industrial site. The largest numbers of blood-fed specimens were from Culex nigripalpus, Culex erraticus, Culex cedecei, and Aedes taeniorhynchus. Cx. erraticus fed on mammals, birds, and reptiles, particularly American alligator. This mosquito species could transmit WNV to American alligator in the wild. Cx. nigripalpus acquired blood meals primarily from birds and mammals and frequently fed on medium-sized mammals and white-tailed deer. Water and wading birds were the primary avian hosts for Cx. nigripalpus and Cx. erraticus in the Everglades. Wading birds are susceptible to WNV and could serve as reservoir hosts. Cx. cedecei fed on five species of rodents, particularly black and hispid cotton rats. EVEV and three different species of Orthobunyavirus have been isolated from the hispid cotton rat and Cx. cedecei in the Everglades. Cx. cedecei is likely acquiring and transmitting these viruses among hispid cotton rats and other rodents. The marsh rabbit was a frequent host for An. crucians complex. An. crucians complex, and other species could acquire Tensaw virus from rabbits. Conclusions: Our study contributes to a better understanding of the host and viral associations of mosquito species in southwestern Florida.

A ticking time bomb hidden in plain sight.

The deer tick transmits nearly half of the known tick-borne pathogens in the United States, and its expanding geographic range increases the risk of human infection. To decrease the abundance of and infection risk from deer ticks, approaches that include vaccines for human use and for animal hosts are desired.

Everglades virus: an underrecognized disease-causing subtype of Venezuelan equine encephalitis virus endemic to Florida, USA.

Everglades virus (EVEV) is subtype II of the Venezuelan equine encephalitis virus (VEEV) complex (Togaviridae: Alphavirus), endemic to Florida, USA. EVEV belongs to a clade that includes both enzootic and epizootic/epidemic VEEV subtypes. Like other enzootic VEEV subtypes, muroid rodents are important vertebrate hosts for EVEV and certain mosquitoes are important vectors. The hispid cotton rat Sigmodon hispidus and cotton mouse Peromyscus gossypinus are important EVEV hosts, based on natural infection (virus isolation and high seropositivity), host competence (experimental infections), and frequency of contact with the vector. The mosquito Culex (Melanoconion) cecedei is the only confirmed vector of EVEV based upon high natural infection rates, efficient vector competence, and frequent feeding upon muroid rodents. Human disease attributed to EVEV is considered rare. However, cases of meningitis and encephalitis are recorded from multiple sites, separated by 250 km or more. Phylogenetic analyses indicate that EVEV is evolving, possibly due to changes in the mammal community. Mutations in the EVEV genome are of concern, given that epidemic strains of VEEV (subtypes IAB and IC) are derived from enzootic subtype ID, the closest genetic relative of EVEV. Should epizootic mutations arise in EVEV, the abundance of Aedes taeniorhynchus and other epizootic VEEV vectors in southern Florida provides a conducive environment for widespread transmission. Other factors that will likely influence the distribution and frequency of EVEV transmission include the establishment of Culex panocossa in Florida, Everglades restoration, mammal community decline due to the Burmese python, land use alteration by humans, and climate change.

Seasonal Dynamics of Mosquito-Borne Viruses in the Southwestern Florida Everglades, 2016, 2017.

Mosquitoes were collected for 12 consecutive months beginning June 2016, from 11 locations in the Florida Everglades, Collier County, and tested for viruses by isolation in Vero cells and subsequent identification. One species complex and 31 species of mosquitoes were identified from 668,809 specimens. Ochlerotatus taeniorhynchus comprised 72.2% of the collection. Other notable species were Anopheles crucians complex, Culex nigripalpus, Cx. erraticus, and Cx. cedecei. Seven species of virus were identified from 110 isolations: Everglades, Gumbo Limbo, Mahogany Hammock, Pahayokee, Shark River, Tensaw, and West Nile viruses. Everglades, West Nile, Tensaw, and Mahogany Hammock viruses were most frequently isolated. Largest numbers of viruses were identified from Cx. cedecei, Cx. nigripalpus, and An. crucians complex. Five species of virus were isolated from Cx. cedecei. Viruses were isolated from mangrove, cypress swamp, hardwood hammock, and sawgrass habitats. West Nile virus was isolated August through October when Cx. nigripalpus was most abundant. Everglades virus was the most frequently isolated virus from nine species of mosquitoes collected from June through August. Tensaw virus was isolated primarily from Anopheles species. Isolations were made in July, August, January, February, and April, suggesting that this virus may be present in host-seeking mosquitoes throughout the year. Mahogany Hammock, Shark River, Gumbo Limbo, and Pahayokee viruses were isolated primarily from Cx. cedecei from June through December. Shotgun metagenomic sequencing was used to document that seven pools of Cx. cedecei were infected with two arboviruses. As communities expand into the Everglades, more humans will become exposed to arboviruses.

Emergence potential of mosquito-borne arboviruses from the Florida Everglades.

The Greater Everglades Region of South Florida is one of the largest natural wetlands and the only subtropical ecosystem found in the continental United States. Mosquitoes are seasonally abundant in the Everglades where several potentially pathogenic mosquito-borne arboviruses are maintained in natural transmission cycles involving vector-competent mosquitoes and reservoir-competent vertebrate hosts. The fragile nature of this ecosystem is vulnerable to many sources of environmental change, including a wetlands restoration project, climate change, invasive species and residential development. In this study, we obtained baseline data on the distribution and abundance of both mosquitos and arboviruses occurring in the southern Everglades region during the summer months of 2013, when water levels were high, and in 2014, when water levels were low. A total of 367,060 mosquitoes were collected with CO2-baited CDC light traps at 105 collection sites stratified among the major landscape features found in Everglades National Park, Big Cypress National Preserve, Fakahatchee State Park Preserve and Picayune State Forest, an area already undergoing restoration. A total of 2,010 pools of taxonomically identified mosquitoes were cultured for arbovirus isolation and identification. Seven vertebrate arboviruses were isolated: Everglades virus, Tensaw virus, Shark River virus, Gumbo Limbo virus, Mahogany Hammock virus, Keystone virus, and St. Louis encephalitis virus. Except for Tensaw virus, which was absent in 2013, the remaining viruses were found to be most prevalent in hardwood hammocks and in Fakahatchee, less prevalent in mangroves and pinelands, and absent in cypress and sawgrass. In contrast, in the summer of 2014 when water levels were lower, these arboviruses were far less prevalent and only found in hardwood hammocks, but Tensaw virus was present in cypress, sawgrass, pinelands, and a recently burned site. Major environmental changes are anticipated in the Everglades, many of which will result in increased water levels. How these might lead to the emergence of arboviruses potentially pathogenic to both humans and wildlife is discussed.

Characterization of Three New Insect-Specific Flaviviruses: Their Relationship to the Mosquito-Borne Flavivirus Pathogens.

Three novel insect-specific flaviviruses, isolated from mosquitoes collected in Peru, Malaysia (Sarawak), and the United States, are characterized. The new viruses, designated La Tina, Kampung Karu, and Long Pine Key, respectively, are antigenically and phylogenetically more similar to the mosquito-borne flavivirus pathogens, than to the classical insect-specific viruses like cell fusing agent and Culex flavivirus. The potential implications of this relationship and the possible uses of these and other arbovirus-related insect-specific flaviviruses are reviewed.

Human rickettsial pathogen modulates arthropod organic anion transporting polypeptide and tryptophan pathway for its survival in ticks.

The black-legged tick Ixodes scapularis transmits the human anaplasmosis agent, Anaplasma phagocytophilum. In this study, we show that A. phagocytophilum specifically up-regulates I. scapularis organic anion transporting polypeptide, isoatp4056 and kynurenine amino transferase (kat), a gene involved in the production of tryptophan metabolite xanthurenic acid (XA), for its survival in ticks. RNAi analysis revealed that knockdown of isoatp4056 expression had no effect on A. phagocytophilum acquisition from the murine host but affected the bacterial survival in tick cells. Knockdown of the expression of kat mRNA alone or in combination with isoatp4056 mRNA significantly affected A. phagocytophilum survival and isoatp4056 expression in tick cells. Exogenous addition of XA induces isoatp4056 expression and A. phagocytophilum burden in both tick salivary glands and tick cells. Electrophoretic mobility shift assays provide further evidence that A. phagocytophilum and XA influences isoatp4056 expression. Collectively, this study provides important novel information in understanding the interplay between molecular pathways manipulated by a rickettsial pathogen to survive in its arthropod vector.

Lyme disease ecology in a changing world: consensus, uncertainty and critical gaps for improving control.

Lyme disease is the most common tick-borne disease in temperate regions of North America, Europe and Asia, and the number of reported cases has increased in many regions as landscapes have been altered. Although there has been extensive work on the ecology and epidemiology of this disease in both Europe and North America, substantial uncertainty exists about fundamental aspects that determine spatial and temporal variation in both disease risk and human incidence, which hamper effective and efficient prevention and control. Here we describe areas of consensus that can be built on, identify areas of uncertainty and outline research needed to fill these gaps to facilitate predictive models of disease risk and the development of novel disease control strategies. Key areas of uncertainty include: (i) the precise influence of deer abundance on tick abundance, (ii) how tick populations are regulated, (iii) assembly of host communities and tick-feeding patterns across different habitats, (iv) reservoir competence of host species, and (v) pathogenicity for humans of different genotypes of Borrelia burgdorferi Filling these knowledge gaps will improve Lyme disease prevention and control and provide general insights into the drivers and dynamics of this emblematic multi-host-vector-borne zoonotic disease.This article is part of the themed issue 'Conservation, biodiversity and infectious disease: scientific evidence and policy implications'.

Ticks elicit variable fibrinogenolytic activities upon feeding on hosts with different immune backgrounds.

Ticks secrete several anti-hemostatic factors in their saliva to suppress the host innate and acquired immune defenses against infestations. Using Ixodes scapularis ticks and age-matched mice purchased from two independent commercial vendors with two different immune backgrounds as a model, we show that ticks fed on immunodeficient animals demonstrate decreased fibrinogenolytic activity in comparison to ticks fed on immunocompetent animals. Reduced levels of D-dimer (fibrin degradation product) were evident in ticks fed on immunodeficient animals in comparison to ticks fed on immunocompetent animals. Increased engorgement weights were noted for ticks fed on immunodeficient animals in comparison to ticks fed on immunocompetent animals. Furthermore, the LC-MS/MS and quantitative real-time-PCR analysis followed by inhibitor and antibody-blocking assays revealed that the arthropod HSP70-like molecule contributes to differential fibrinogenolysis during tick feeding. Collectively, these results not only indicate that ticks elicit variable fibrinogenolysis upon feeding on hosts with different immune backgrounds but also provide insights for the novel role of arthropod HSP70-like molecule in fibrinogenolysis during blood feeding.

Genetic characterization, molecular epidemiology, and phylogenetic relationships of insect-specific viruses in the taxon Negevirus.

The recently described taxon Negevirus is comprised of a diverse group of insect-specific viruses isolated from mosquitoes and phlebotomine sandflies. In this study, a comprehensive genetic characterization, molecular, epidemiological and evolutionary analyses were conducted on nearly full-length sequences of 91 new negevirus isolates obtained in Brazil, Colombia, Peru, Panama, USA and Nepal. We demonstrated that these arthropod restricted viruses are clustered in two major phylogenetic groups with origins related to three plant virus genera (Cilevirus, Higrevirus and Blunevirus). Molecular analyses demonstrated that specific host correlations are not present with most negeviruses; instead, high genetic variability, wide host-range, and cross-species transmission were noted. The data presented here also revealed the existence of five novel insect-specific viruses falling into two arthropod-restrictive virus taxa, previously proposed as distinct genera, designated Nelorpivirus and Sandewavirus. Our results provide a better understanding of the molecular epidemiology, evolution, taxonomy and stability of this group of insect-restricted viruses.

Closely-related Borrelia burgdorferi (sensu stricto) strains exhibit similar fitness in single infections and asymmetric competition in multiple infections.

BACKGROUND: Wild hosts are commonly co-infected with complex, genetically diverse, pathogen communities. Competition is expected between genetically or ecologically similar pathogen strains which may influence patterns of coexistence. However, there is little data on how specific strains of these diverse pathogen species interact within the host and how this impacts pathogen persistence in nature. Ticks are the most common disease vector in temperate regions with Borrelia burgdorferi, the causative agent of Lyme disease, being the most common vector-borne pathogen in North America. Borrelia burgdorferi is a pathogen of high public health concern and there is significant variation in infection phenotype between strains, which influences predictions of pathogen dynamics and spread. METHODS: In a laboratory experiment, we investigated whether two closely-related strains of B. burgdorferi (sensu stricto) showed similar transmission phenotypes, how the transmission of these strains changed when a host was infected with one strain, re-infected with the same strain, or co-infected with two strains. Ixodes scapularis, the black-legged tick, nymphs were used to sequentially infect laboratory-bred Peromyscus leucopus, white-footed mice, with one strain only, homologous infection with the same stain, or heterologous infection with both strains. We used the results of this laboratory experiment to simulate long-term persistence and maintenance of each strain in a simple simulation model. RESULTS: Strain LG734 was more competitive than BL206, showing no difference in transmission between the heterologous infection groups and single-infection controls, while strain BL206 transmission was significantly reduced when strain LG734 infected first. The results of the model show that this asymmetry in competition could lead to extinction of strain BL206 unless there was a tick-to-host transmission advantage to this less competitive strain. CONCLUSIONS: This asymmetric competitive interaction suggests that strain identity and the biotic context of co-infection is important to predict strain dynamics and persistence.

Assessment of synthetic floral-based attractants and sugar baits to capture male and female Aedes aegypti (Diptera: Culicidae).

BACKGROUND: The viruses transmitted by Aedes aegypti, including dengue and Zika viruses, are rapidly expanding in geographic range and as a threat to public health. In response, control programs are increasingly turning to the use of sterile insect techniques resulting in a need to trap male Ae. aegypti to monitor the efficacy of the intervention. However, there is a lack of effective and cheap methods for trapping males. Thus, we attempted to exploit the physiological need to obtain energy from sugar feeding in order to passively capture male and female Ae. aegypti (nulliparous and gravid) in free-flight attraction assays. Candidate lures included previously identified floral-based (phenylacetaldehyde, linalool oxide, phenylethyl alcohol, and acetophenone) attractants and an attractive toxic sugar bait-based (ATSB) solution of guava and mango nectars. A free-flight attraction assay assessed the number of mosquitoes attracted to each candidate lure displayed individually. Then, a choice test was performed between the best-performing lure and a water control displayed in Gravid Aedes Traps (GAT). RESULTS: Results from the attraction assays indicated that the ATSB solution of guava and mango nectars was the most promising lure candidate for males; unlike the floral-based attractants tested, it performed significantly better than the water control. Nulliparous and gravid females demonstrated no preference among the lures and water controls indicating a lack of attraction to floral-based attractants and sugar baits in a larger setting. Although the guava-mango ATSB lure was moderately attractive to males when presented directly (i.e. no need to enter a trap or other confinement), it failed to attract significantly more male, nulliparous female, or gravid female Ae. aegypti than water controls when presented inside a Gravid Aedes Trap. CONCLUSIONS: Our findings suggest that the use of volatile floral-based attractants and sugar mixtures that have been identified in the literature is not an effective lure by which to kill Ae. aegypti at ATSB stations nor capture them in the GAT. Future trapping efforts would likely be more successful if focused on more promising methods for capturing male and female Ae. aegypti.

Almendravirus: A Proposed New Genus of Rhabdoviruses Isolated from Mosquitoes in Tropical Regions of the Americas.

The Rhabdoviridae is a diverse family of negative-sense single-stranded RNA viruses, many of which infect vertebrate hosts and are transmitted by hematophagous arthropods. Others appear to be arthropod specific, circulating only within arthropod populations. Herein, we report the isolation and characterization of three novel viruses from mosquitoes collected from the Americas. Coot Bay virus was isolated from Anopheles quadrimaculatus mosquitoes collected in the Everglades National Park, Florida; Rio Chico virus was isolated from Anopheles triannulatus mosquitoes collected in Panama; and Balsa virus was isolated from two pools of Culex erraticus mosquitoes collected in Colombia. Sequence analysis indicated that the viruses share a similar genome organization to Arboretum virus and Puerto Almendras virus that had previously been isolated from mosquitoes collected in Peru. Each genome features the five canonical rhabdovirus structural protein genes as well as a gene encoding a class 1A viroporin-like protein (U1) located between the G and L genes (3'-N-P-M-G-U1-L-5'). Phylogenetic analysis of complete L protein sequences indicated that all five viruses cluster in a unique clade that is relatively deeply rooted in the ancestry of animal rhabdoviruses. The failure of all viruses in this clade to grow in newborn mice or vertebrate cells in culture suggests that they may be poorly adapted to replication in vertebrates.

A Cost-Effectiveness Tool for Informing Policies on Zika Virus Control.

BACKGROUND: As Zika virus continues to spread, decisions regarding resource allocations to control the outbreak underscore the need for a tool to weigh policies according to their cost and the health burden they could avert. For example, to combat the current Zika outbreak the US President requested the allocation of $1.8 billion from Congress in February 2016. METHODOLOGY/PRINCIPAL FINDINGS: Illustrated through an interactive tool, we evaluated how the number of Zika cases averted, the period during pregnancy in which Zika infection poses a risk of microcephaly, and probabilities of microcephaly and Guillain-Barré Syndrome (GBS) impact the cost at which an intervention is cost-effective. From Northeast Brazilian microcephaly incidence data, we estimated the probability of microcephaly in infants born to Zika-infected women (0.49% to 2.10%). We also estimated the probability of GBS arising from Zika infections in Brazil (0.02% to 0.06%) and Colombia (0.08%). We calculated that each microcephaly and GBS case incurs the loss of 29.95 DALYs and 1.25 DALYs per case, as well as direct medical costs for Latin America and the Caribbean of $91,102 and $28,818, respectively. We demonstrated the utility of our cost-effectiveness tool with examples evaluating funding commitments by Costa Rica and Brazil, the US presidential proposal, and the novel approach of genetically modified mosquitoes. Our analyses indicate that the commitments and the proposal are likely to be cost-effective, whereas the cost-effectiveness of genetically modified mosquitoes depends on the country of implementation. CONCLUSIONS/SIGNIFICANCE: Current estimates from our tool suggest that the health burden from microcephaly and GBS warrants substantial expenditures focused on Zika virus control. Our results justify the funding committed in Costa Rica and Brazil and many aspects of the budget outlined in the US president's proposal. As data continue to be collected, new parameter estimates can be customized in real-time within our user-friendly tool to provide updated estimates on cost-effectiveness of interventions and inform policy decisions in country-specific settings.

Evaluating the effectiveness of localized control strategies to curtail chikungunya.

Chikungunya, a re-emerging arbovirus transmitted to humans by Aedes aegypti and Ae. albopictus mosquitoes, causes debilitating disease characterized by an acute febrile phase and chronic joint pain. Chikungunya has recently spread to the island of St. Martin and subsequently throughout the Americas. The disease is now affecting 42 countries and territories throughout the Americas. While chikungunya is mainly a tropical disease, the recent introduction and subsequent spread of Ae. albopictus into temperate regions has increased the threat of chikungunya outbreaks beyond the tropics. Given that there are currently no vaccines or treatments for chikungunya, vector control remains the primary measure to curtail transmission. To investigate the effectiveness of a containment strategy that combines disease surveillance, localized vector control and transmission reduction measures, we developed a model of chikungunya transmission dynamics within a large residential neighborhood, explicitly accounting for human and mosquito movement. Our findings indicate that prompt targeted vector control efforts combined with measures to reduce transmission from symptomatic cases to mosquitoes may be highly effective approaches for controlling outbreaks of chikungunya, provided that sufficient detection of chikungunya cases can be achieved.

Spatial and Temporal Clustering of Chikungunya Virus Transmission in Dominica.

Using geo-referenced case data, we present spatial and spatio-temporal cluster analyses of the early spread of the 2013-2015 chikungunya virus (CHIKV) in Dominica, an island in the Caribbean. Spatial coordinates of the locations of the first 417 reported cases observed between December 15th, 2013 and March 11th, 2014, were captured using the Global Positioning System (GPS). We observed a preponderance of female cases, which has been reported for CHIKV outbreaks in other regions. We also noted statistically significant spatial and spatio-temporal clusters in highly populated areas and observed major clusters prior to implementation of intensive vector control programs suggesting early vector control measures, and education had an impact on the spread of the CHIKV epidemic in Dominica. A dynamical identification of clusters can lead to local assessment of risk and provide opportunities for targeted control efforts for nations experiencing CHIKV outbreaks.

Response to Esteve-Gassent et al.: flaB sequences obtained from Texas PCR products are identical to the positive control strain Borrelia burgdorferi B31.

Feria-Arroyo et al. had reported previously that, based on PCR analysis, 45% of Ixodes scapularis ticks collected in Texas and Mexico were infected with the Lyme disease spirochete Borrelia burgdorferi (Parasit. Vectors 2014, 7:199). However, our analyses of their initial data (Parasit. Vectors 2014, 7:467) and a recent response by Esteve-Gassent et al. (Parasit. Vectors 2015, 8:129) provide evidence that the positive PCR results obtained from both ribosomal RNA intergenic sequences and the flagellin gene flaB are highly likely due to contamination by the B. burgdorferi B31 positive control strain.