Chromosome-level assembly of the gray fox (Urocyon cinereoargenteus) confirms the basal loss of PRDM9 in Canidae.

Reference genome assemblies have been created from multiple lineages within the Canidae family; however, despite its phylogenetic relevance as a basal genus within the clade, there is currently no reference genome for the gray fox (Urocyon cinereoargenteus). Here, we present a chromosome-level assembly for the gray fox (U. cinereoargenteus), which represents the most contiguous, non-domestic canid reference genome available to date, with 90% of the genome contained in just 34 scaffolds and a contig N50 and scaffold N50 of 59.4 and 72.9 Megabases, respectively. Repeat analyses identified an increased number of simple repeats relative to other canids. Based on mitochondrial DNA, our Vermont sample clusters with other gray fox samples from the northeastern United States and contains slightly lower levels of heterozygosity than gray foxes on the west coast of California. This new assembly lays the groundwork for future studies to describe past and present population dynamics, including the delineation of evolutionarily significant units of management relevance. Importantly, the phylogenetic position of Urocyon allows us to verify the loss of PRDM9 functionality in the basal canid lineage, confirming that pseudogenization occurred at least 10 million years ago.

Culex erraticus (Diptera: Culicidae) utilizes gopher tortoise burrows for overwintering in North Central Florida.

Mosquito-borne diseases represent a significant threat to human and animal health in the United States. Several viruses, including West Nile, Saint Louis encephalitis, and Eastern equine encephalitis are endemic. In humans, the disease is typically detected during the summer months, but not during the winter months. The ability of these viruses to reemerge year after year is still not fully understood, but typically involves persistence in a reservoir host or vector during periods of low transmission. Mosquito species are known to overwinter at different life stages (adults, larvae, or eggs) in manufactured or natural sites. Gopher tortoise burrows are known to serve as refuge for many vertebrate and invertebrate species in pine savannas. In this study, we surveyed the interior of gopher tortoise burrows for overwintering mosquitoes. We identified 4 species (Anopheles crucians s.l., Culex erraticus, Mansonia dyari, and Uranotaenia sapphirina). Cx. erraticus was the most abundant, and its presence and abundance increased in winter months, implying that this species utilized gopher tortoise burrows for overwintering. Bloodfed Cx. erraticus and An. crucians s.l. females were detected. While An. crucians s.l. fed exclusively on the white-tailed deer, Cx. erraticus had a more diverse host range but fed primarily on the gopher tortoise. Tortoises and other long-lived reptiles like the American alligator have been shown to sustain high viremia following West Nile virus (WNV) and Eastern equine encephalitis virus (EEEV) infection and therefore could play a role in the maintenance of these viruses. In addition, Cx. erraticus is naturally infected with WNV and is a known bridge vector for EEEV. As such, these overwintering sites may play a role in perpetuating over-winter arboviral activity in Florida.

Current and Future Applications of Fluorescence-Guided Surgery in Head and Neck Cancer.

Despite improvements in medical management of head and neck cancers, positive surgical margin rates have remained relatively unchanged in the past 30 years, emphasizing a need for improved intraoperative imaging and tumor visualization. This review provides a detailed summary on preoperative anatomic imaging techniques, nonspecific fluorescence imaging modalities, the recent emergence of tumor-targeting fluorophores for intraoperative imaging, and the future of fluorescence-guided surgery in head and neck cancer.

Domestic Dogs as Sentinels for West Nile Virus but not Aedes-borne Flaviviruses, Mexico.

We tested 294 domestic pet dogs in Mexico for neutralizing antibodies for mosquito-borne flaviviruses. We found high (42.6%) exposure to West Nile virus in Reynosa (northern Mexico) and low (1.2%) exposure in Tuxtla Gutierrez (southern Mexico) but very limited exposure to Aedes-borne flaviviruses. Domestic dogs may be useful sentinels for West Nile virus.

The effects of habitat type and pathogen infection on tick host-seeking behaviour.

Tick-borne pathogens pose a significant risk to livestock, wildlife and public health. Host-seeking behaviours may depend on a combination of infection status and environmental factors. Here, we assessed the effects of habitat type and pathogen infection on host-seeking behaviour (questing) in the lone star tick, Amblyomma americanum. Ticks were collected using a tick drag from two different habitat types: xeric hammock and successional hardwood forests. Using a standardized assay, we recorded the likelihood of questing for each tick, the average height quested and total time spent questing and then tested each tick for the presence of Rickettsia spp. and Ehrlichia spp. using conventional polymerase chain reaction. We did not detect Ehrlichia in any ticks, although 30% tested positive for Rickettsia amblyommatis, a member of the Rickettsia spotted fever group. Ticks infected with R. amblyommatis spent less time questing compared to uninfected ticks, with infected ticks spending 85 s on average questing and uninfected ticks spending 112 s. Additionally, ticks collected from xeric hammock habitats spent over twice as long questing compared to ticks from successional hardwood forests. Ticks from xeric hammock spent 151 s on average questing while ticks from successional hardwood forest spent only 58 s during a 10-min observation period. These results demonstrate that habitat type and infection status can influence tick host-seeking behaviours, which can play a pivotal role in disease dynamics.

Variable coverage in an Autocidal Gravid Ovitrap intervention impacts efficacy of Aedes aegypti control.

Control of the arboviral disease vector Aedes aegypti has shown variable levels of efficacy around the globe. We evaluated an Autocidal Gravid Ovitrap (AGO) intervention as a stand-alone control tool for population suppression of A. aegypti in US communities bordering Mexico.We conducted a cluster randomized crossover trial with weekly mosquito surveillance of sentinel households from July 2017 to December 2018. The intervention took place from August to December of both years. Multilevel models (generalized linear and additive mixed models) were used to analyse the changes in population abundance of female A. aegypti.We observed that female populations were being suppressed 77% (2018) and four times lower outdoor female abundance when AGO coverage (number of intervention AGO traps that surrounded a sentinel home) was high (2.7 AGOs/house). However, we also observed that areas with low intervention AGO coverage resulted in no difference (2017) or slightly higher abundance compared to the control. These results suggest that coverage rate might play a critical role on how populations of female A. aegypti are being modulated in the field. The lack of larval source habitat reduction and the short duration of the intervention period might have limited the A. aegypti population suppression observed in this study. Synthesis and applications. The mosquito, A. aegypti, is a public health concern in most tropical and subtropical regions. With the rise of insecticide resistance, the evaluation of non-chemical tools has become pivotal in the fight against arboviral disease transmission. Our study shows that the AGO intervention, as a stand-alone control tool, is limited by its coverage in human settlements. Vector control programmes should consider, that if the target coverage rate is not achieved, measures will be ineffective unless coupled with other control approaches. Although our multilevel modelling was focused on A. aegypti and the AGO, the approach can be applied to other mosquito vector species.

Cell fusing agent virus (Flavivirus) infection in Aedes aegypti in Texas: seasonality, comparison by trap type, and individual viral loads.

South Texas has experienced local transmission of Zika virus and of other mosquito-borne viruses such as chikungunya virus and dengue virus in the last decades. Using a mosquito surveillance program in the Lower Rio Grande Valley (LRGV) and San Antonio, TX, from 2016 to 2018, we detected the presence of an insect-specific virus, cell fusing agent virus (CFAV), in the Aedes aegypti mosquito population. We tested 6,326 females and 1,249 males from the LRGV and 659 females from San Antonio for CFAV by RT-PCR using specific primers. Infection rates varied from 0 to 261 per 1,000 mosquitoes in the LRGV and 115 to 208 per 1,000 in San Antonio depending on the month of collection. Infection rates per 1,000 individuals appeared higher in females collected from BG Sentinel 2 traps compared to Autocidal Gravid Ovitraps, but the ratio of the percentage of infected pools did not differ by trap type. The natural viral load in individual males ranged from 1.25 x 102 to 5.50 x 106 RNA copies and in unfed females from 5.42 x 103 to 8.70 x 106 RNA copies. Gravid females were found to harbor fewer viral particles than males and unfed females.

High Rate of Non-Human Feeding by Aedes aegypti Reduces Zika Virus Transmission in South Texas.

Mosquito-borne viruses are emerging or re-emerging globally, afflicting millions of people around the world. Aedes aegypti, the yellow fever mosquito, is the principal vector of dengue, Zika, and chikungunya viruses, and has well-established populations across tropical and subtropical urban areas of the Americas, including the southern United States. While intense arboviral epidemics have occurred in Mexico and further south in the Americas, local transmission in the United States has been minimal. Here, we study Ae. aegypti and Culex quinquefasciatus host feeding patterns and vertebrate host communities in residential environments of South Texas to identify host-utilization relative to availability. Only 31% of Ae. aegypti blood meals were derived from humans, while 50% were from dogs and 19% from other wild and domestic animals. In Cx. quinquefasciatus, 67% of blood meals were derived from chicken, 22% came from dogs, 9% from various wild avian species, and 2% from other mammals including one human, one cat, and one pig. We developed a model for the reproductive number, R0, for Zika virus (ZIKV) in South Texas relative to northern Mexico using human disease data from Tamaulipas, Mexico. We show that ZIKV R0 in South Texas communities could be greater than one if the risk of human exposure to Ae. aegypti bites in these communities is at least 60% that of Northern Mexico communities. The high utilization of non-human vertebrates and low risk of human exposure in South Texas diminishes the outbreak potential for human-amplified urban arboviruses transmitted by Ae. aegypti.

Dispersal of female and male Aedes aegypti from discarded container habitats using a stable isotope mark-capture study design in South Texas.

Aedes aegypti is the main vector of arboviral diseases such as dengue, chikungunya and Zika. A key feature for disease transmission modeling and vector control planning is adult mosquito dispersal. We studied Ae aegypti adult dispersal by conducting a mark-capture study of naturally occurring Ae. aegypti from discarded containers found along a canal that divided two residential communities in Donna, Texas, USA. Stable isotopes were used to enrich containers with either 13C or 15N. Adult mosquitoes were collected outdoors in the yards of households throughout the communities with BG Sentinel 2 traps during a 12-week period. Marked mosquito pools with stable isotopes were used to estimate the mean distance travelled using three different approaches (Net, Strip or Circular) and the probability of detecting an isotopically marked adult at different distances from the larval habitat of origin. We consistently observed, using the three approaches that male (Net: 220 m, Strip: 255 m, Circular: 250 m) Ae. aegypti dispersed further in comparison to gravid (Net: 135 m, Strip: 176 m, Circular: 189 m) and unfed females (Net: 192 m, Strip: 213 m, Circular: 198 m). We also observed that marked male capture probability slightly increased with distance, while, for both unfed and gravid females, such probability decreased with distance. Using a unique study design documenting adult dispersal from natural larval habitat, our results suggest that Ae. aegypti adults disperse longer distances than previously reported. These results may help guide local vector control authorities in their fight against Ae. aegypti and the diseases it transmits, suggesting coverage of 200 m for the use of insecticides and innovative vector control tools.

Genetic Diversity of Culicoides stellifer (Diptera: Ceratopogonidae) in the Southeastern United States Compared With Sequences From Ontario, Canada.

Much of the bluetongue (BT) and epizootic hemorrhagic disease (EHD) research in North America focuses on white-tail deer and Culicoides sonorensis (Wirth & Jones) (Diptera: Ceratopogonidae), though several other biting midge species have been suggested as vectors. Culicoides stellifer (Coquillett) has been associated with hosts susceptible to hemorrhagic disease (HD), and more recently, specimens from Florida have tested positive for EHD and BT viral RNA. If C. stellifer is acting as a vector, this could have an impact on the distribution of HD in North America. To determine if gene flow is occurring across the range of C. stellifer within the southeast United States, a mitochondrial haplotype analysis was performed using the COI gene. Our haplotype network showed no population structure in C. stellifer from Florida, Texas, and South Carolina, as the overall genetic divergence between these sites was equal to the genetic divergence within each. We also compared these haplotypes to published sequences of C. stellifer collected in Ontario, Canada. Surprisingly, the genetic diversity of the flies from Ontario was two times greater than what was observed between the southeast U.S. collection sites. This considerable divergence could be evidence of a cryptic species. A better understanding of the connectivity between C. stellifer populations across all of North America will give insight into the distribution of HD. Our results show that gene flow is occurring between sites in the southeastern United States and potentially throughout the eastern distribution of the species.

Sugar Feeding Patterns for Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae) Mosquitoes in South Texas.

Effective mosquito surveillance and management depend on a thorough understanding of the biology and feeding patterns unique to species and sex. Given that a propensity to sugar feed is necessary for some mosquito surveillance and newer control strategies, we sought to document the amount of total sugar in wild Aedes aegypti (L.) and Culex quinquefasciatus (Say) captured from five different locations in the Lower Rio Grande Valley (LRGV) of South Texas over 2 yr. We used Biogents Sentinel 2 (BGS2) traps in year 1 and aspirators, BGS2, and CDC resting traps in years 2 and 3 to collect adult mosquitoes. The hot anthrone test was used to quantify total sugar content in each mosquito. Additionally, the cold and hot anthrone tests were used to distinguish fructose content from total sugars for mosquitoes captured in 2019. Overall, Ae. aegypti females had significantly lower total sugar content than Ae. aegypti males as well as both sexes of Cx. quinquefasciatus. However, the percentage of Ae. aegypti positive for fructose consumption was four to eightfold higher than Ae. aegypti previously reported in other regions. The difference between locations was significant for males of both species, but not for females. Seasonality and trapping method also revealed significant differences in sugar content of captured mosquitoes. Our results reinforce that sugar feeding in female Ae. aegypti is less than Cx. quinquefasciatus, although not absent. This study provides necessary data to evaluate the potential effectiveness of sugar baits in surveillance and control of both Ae. aegypti and Cx. quinquefasciatus mosquitoes.

Mosquito-Borne Viruses and Insect-Specific Viruses Revealed in Field-Collected Mosquitoes by a Monitoring Tool Adapted from a Microbial Detection Array.

Several mosquito-borne diseases affecting humans are emerging or reemerging in the United States. The early detection of pathogens in mosquito populations is essential to prevent and control the spread of these diseases. In this study, we tested the potential applicability of the Lawrence Livermore Microbial Detection Array (LLMDA) to enhance biosurveillance by detecting microbes present in Aedes aegypti, Aedes albopictus, and Culex mosquitoes, which are major vector species globally, including in Texas. The sensitivity and reproducibility of the LLMDA were tested in mosquito samples spiked with different concentrations of dengue virus (DENV), revealing a detection limit of >100 but <1,000 PFU/ml. Additionally, field-collected mosquitoes from Chicago, IL, and College Station, TX, of known infection status (West Nile virus [WNV] and Culex flavivirus [CxFLAV] positive) were tested on the LLMDA to confirm its efficiency. Mosquito field samples of unknown infection status, collected in San Antonio, TX, and the Lower Rio Grande Valley (LRGV), TX, were run on the LLMDA and further confirmed by PCR or quantitative PCR (qPCR). The analysis of the field samples with the LLMDA revealed the presence of cell-fusing agent virus (CFAV) in A. aegypti populations. Wolbachia was also detected in several of the field samples (A. albopictus and Culex spp.) by the LLMDA. Our findings demonstrated that the LLMDA can be used to detect multiple arboviruses of public health importance, including viruses that belong to the Flavivirus, Alphavirus, and Orthobunyavirus genera. Additionally, insect-specific viruses and bacteria were also detected in field-collected mosquitoes. Another strength of this array is its ability to detect multiple viruses in the same mosquito pool, allowing for the detection of cocirculating pathogens in an area and the identification of potential ecological associations between different viruses. This array can aid in the biosurveillance of mosquito-borne viruses circulating in specific geographical areas.IMPORTANCE Viruses associated with mosquitoes have made a large impact on public and veterinary health. In the United States, several viruses, including WNV, DENV, and chikungunya virus (CHIKV), are responsible for human disease. From 2015 to 2018, imported Zika cases were reported in the United States, and in 2016 to 2017, local Zika transmission occurred in the states of Texas and Florida. With globalization and a changing climate, the frequency of outbreaks linked to arboviruses will increase, revealing a need to better detect viruses in vector populations. With the capacity of the LLMDA to detect viruses, bacteria, and fungi, this study highlights its ability to broadly screen field-collected mosquitoes and contribute to the surveillance and management of arboviral diseases.

Reviewing the Potential Vectors and Hosts of African Swine Fever Virus Transmission in the United States.

African swine fever virus (ASFV) continues to threaten global animal health and agricultural biosecurity. Mitigating the establishment of ASFV in the United States (U.S.) is contingent on (1) the identification of arthropod vectors and vertebrate hosts that are capable of viral maintenance and transmission in the U.S. and (2) knowledge of vector-host associations that may permit transmission. We aggregated data on vector competence, host competence and tick-host associations by systematic review of published articles and collection records to identify species that may support the invasion of ASFV in the U.S. Three species of competent soft ticks occur in the U.S., Ornithodoros coriaceus, Ornithodoros turicata, and Ornithodoros puertoricensis, however, vector competence for the majority of soft ticks in the U.S. remains unknown. Three species of competent vertebrate hosts currently occur in the U.S.: domestic pigs (Sus scrofa domesticus), feral hogs (Sus scrofa), and common warthogs (Phacochoerus africanus). Hierarchical hazard categories based on vector competence, tick-host contact rates, and vector abundance were used to semiquantitatively rank U.S. soft tick species by their relative risk for contributing to ASFV transmission to identify which soft tick species are a priority for future studies. High-risk vector and host species identified in this study can be used to focus ASFV risk assessments in the U.S., guide targeted surveillance and control strategies, and proactively prepare for an ASFV incursion event. Results indicate O. coriaceus, O. turicata, and O. puertoricensis demonstrate the highest relative risk for contributing to ASFV transmission in the U.S., however, many gaps in knowledge exist preventing the full evaluation of at least 30 soft tick species in the U.S. Further study is required to identify soft tick vectors that interact with feral swine populations, elucidate vector competence, and further understand the biology of soft tick species.

Surveillance of Aedes aegypti indoors and outdoors using Autocidal Gravid Ovitraps in South Texas during local transmission of Zika virus, 2016 to 2018.

The yellow fever mosquito, Aedes aegypti, has facilitated the re-emergence of dengue virus (DENV) and emergence of chikungunya virus (CHIKV) and Zika virus (ZIKV) in the Americas and the Caribbean. The recent transmission of these arboviruses in the continental United States has been limited, to date, to South Florida and South Texas despite Ae. aegypti occurring over a much larger geographical region within the country. The main goal of our study was to provide the first long term longitudinal study of Ae. aegypti and enhance the knowledge about the indoor and outdoor relative abundance of Ae. aegypti as a proxy for mosquito-human contact in South Texas, a region of the United States that is at high risk for mosquito-borne virus transmission. Here, the relative abundance of indoors and outdoors mosquitoes of households in eight different communities was described. Surveillance was done weekly from September 2016 to April 2018 using the CDC Autocidal Gravid Ovitraps in low- and middle-income communities. A total of 69 houses were included in this survey among which 36 were in the low-income communities (n = 11 for Donna, n = 15 for Progresso, n = 5 for Mesquite, n = 5 for Chapa) and 33 in middle-income communities (n = 9 for La Feria, n = 8 for Weslaco, n = 11 for McAllen, and n = 5 for Rio Rico). Overall, Ae. aegypti was the dominant species (59.2% of collections, n = 7255) followed by Culex spp. mosquitoes (27.3% of collections, n = 3350). Furthermore, we demonstrated for Ae. aegypti that 1) outdoor relative abundance was higher compared to indoor relative abundance, 2) low-income communities were associated with an increase in mosquito relative abundance indoors when compared to middle-income communities, 3) no difference was observed in the number of mosquitoes collected outdoors between low-income and middle-income communities, and 4) warmer months were positively correlated with outdoor relative abundance whereas no seasonality was observed in the relative abundance of mosquitoes indoors. Additionally, Ae. aegypti mosquitoes collected in South Texas were tested using a specific ZIKV/CHIKV multiplex real-time PCR assay, however, none of the mosquitoes tested positive. Our data highlights the occurrence of mosquitoes indoors in the continental United States and that adults are collected nearly every week of the calendar year. These mosquito data, obtained concurrently with local ZIKV transmission of 10 locally acquired cases in nearby communities, represent a baseline for future studies in the Lower Rio Grande Valley (LRGV) including vector control interventions relying on the oviposition behavior to reduce mosquito populations and pathogen transmission.

Risk of African Swine Fever Virus Sylvatic Establishment and Spillover to Domestic Swine in the United States.

African swine fever virus (ASFV) causes a high-consequence foreign animal disease that has emerged along international trade routes. Owing to high lethality and resulting trade sanctions, establishment of this disease in the United States would have devastating economic consequences. ASFV can be transmitted by soft ticks in the genus Ornithodoros or directly between swine, including domestic, feral, and wild swine. Consequently, the spatial risk of ASFV establishment depends on where susceptible animals, with or without competent vectors, co-occur. We synthesized county-level historical records of soft tick occurrence, current maps of feral swine distribution, and domestic swine inventory to evaluate the risk of ASFV establishment and spillover in the United States. Areas of California, Florida, and much of the southwestern United States were classified as high risk for ASFV establishment and spillover should an introduction event occur. Our analyses indicate that California, Texas, Georgia, and Florida are high-priority candidates for proactive risk reduction strategies. Domestic swine are often produced in high-biosecurity environments, mitigating health risks associated with contacting infected hosts and vectors. However, small-scale and organic pig producers in much of the southern United States remain more vulnerable to disease emergence.

Culicoides species community composition and infection status with parasites in an urban environment of east central Texas, USA.

BACKGROUND: Despite their importance as vectors of zoonotic parasites that can impact human and animal health, Culicoides species distribution across different habitat types is largely unknown. Here we document the community composition of Culicoides found in an urban environment including developed and natural sites in east central Texas, a region of high vector diversity due to subtropical climates, and report their infection status with haemoparasites. RESULTS: A total of 251 individual Culicoides were collected from May to June 2016 representing ten Culicoides species, dominated by C. neopulicaris followed by C. crepuscularis. We deposited 63 sequences to GenBank among which 25 were the first deposition representative for six Culicoides species: C. arboricola (n = 1); C. nanus (n = 4); C. debilipalpis (n = 2); C. haematopotus (n = 14); C. edeni (n = 3); and C. hinmani (n = 1). We also record for the first time the presence of C. edeni in Texas, a species previously known to occur in the Bahamas, Florida and South Carolina. The urban environments with natural area (sites 2 and 4) had higher species richness than sites more densely populated or in a parking lot (sites 1 and 3) although a rarefaction analysis suggested at least two of these sites were not sampled sufficiently to characterize species richness. We detected a single C. crepuscularis positive for Onchocercidae gen. sp. DNA and another individual of the same species positive for Haemoproteus sacharovi DNA, yielding a 2.08% prevalence (n = 251) for both parasites in this species. CONCLUSIONS: We extend the knowledge of the Culicoides spp. community in an urban environment of Texas, USA, and contribute to novel sequence data for these species. Additionally, the presence of parasite DNA (Onchocercidae gen. sp. and H. sacharovi) from C. crepuscularis suggests the potential for this species to be a vector of these parasites.

Nurse-led active surveillance for prostate cancer is safe, effective and associated with high rates of patient satisfaction-results of an audit in the East of England.

INTRODUCTION: Active surveillance (AS) is an option in the management of men with low-stage, low-risk prostate cancer. These patients, who often require prolonged follow-up, can put a strain on outpatient resources. Nurses are ideally placed to develop advanced roles to help meet this increased demand-a model we have utilised since 2014. We set about to comprehensively evaluate our nurse-led AS (NLAS) programme. PATIENTS AND METHODS: An audit of patient notes was carried out to assess compliance with trust and national guidelines. A questionnaire was designed to capture patients' experiences of NLAS. This was piloted and then distributed to all patients in our NLAS programme. A second questionnaire was designed to assess the views of stakeholders within the department. RESULTS: Compliance with various aspects of local guidelines ranged from 88.8% to 100%. 143 patients are currently in the programme with a mean duration of AS of 37.03 months. 104 questionnaires were returned. Most of the patients were aware of the role of the nurse prior to their visit, and all were happy to meet with a nurse. All of the patients indicated their confidence in the nurse monitoring their prostate-specific antigen. Among those requiring further investigations, 85.3% were happy with the information they received prior to their tests. Overall, 96.2% were either very satisfied or satisfied with NLAS. All stakeholders held positive views about NLAS. CONCLUSIONS: NLAS is safe and effective. Patients and stakeholders alike held positive views of the programme.

Co-circulation of Flanders Virus and West Nile Virus in Culex Mosquitoes (Diptera: Culicidae) from Chicago, Illinois.

West Nile virus (WNV) and Flanders virus (FLAV) co-occur in regions of North America. Because both viruses are maintained in a transmission cycle involving Culex mosquitoes and birds, screening mosquitoes for FLAV has been suggested as an enhancement to WNV surveillance and epidemic prediction. Using samples collected in 2010 and 2012 in Chicago, IL, USA, we demonstrate the presence of FLAV in four out of 287 (1.4%) Culex pools. We estimated minimum infection rates for WNV and FLAV to be 5.66 and 1.22 in 2010 and 8.74 and 0.61 in 2012, respectively. FLAV occurred 1 and 3 wk prior to the peak of WNV transmission in 2010 and 2012, respectively. FLAV sequences from Chicago were genetically diverse and phylogenetically representative of lineage A viruses from across the United States.

Insights into the molecular evolution of Dengue virus type 4 in Puerto Rico over two decades of emergence.

Dengue has emerged globally as a major human health problem since the 1950s and is now the most important arboviral disease of humans, infecting nearly 400 million people annually. While some cases are asymptomatic, others can develop a febrile illness (dengue fever) or even progress to severe and fatal dengue. Dengue is caused by any of 4 closely related but distinct viruses, known as Dengue virus serotype 1 to 4 (DENV-1 to DENV-4) which are maintained in endemic transmission to humans in large urban centers of the tropics by Aedes mosquitoes. Since the early 1960s, Puerto Rico, a major metropolitan center in the Caribbean, has experienced increasingly larger and clinically more severe epidemics following the introduction of all four dengue serotypes. The first dengue hemorrhagic fever epidemic in 1986, and a particularly severe outbreak in 1998 were dominated by novel DENV-4 strains that evolved in Puerto Rico, replacing earlier strains and spreading throughout the region. Sequence characterization of 54 complete DENV-4 genomes and their comparative evolution against 74 previously published viral sequences from the region over several decades shows that DENV-4 strains from these periods were genetically distinct based on unique changes in the envelope and non-structural genes. Their replacement of earlier strains in Puerto Rico progressed rapidly, suggesting that strong natural selection played a role in their fixation. This study confirms that DENVs evolve through rapid lineage turnover driven in part by natural selection and genetic drift.

Characterization of Aedes aegypti innate-immune pathways that limit Chikungunya virus replication.

Replication of arboviruses in their arthropod vectors is controlled by innate immune responses. The RNA sequence-specific break down mechanism, RNA interference (RNAi), has been shown to be an important innate antiviral response in mosquitoes. In addition, immune signaling pathways have been reported to mediate arbovirus infections in mosquitoes; namely the JAK/STAT, immune deficiency (IMD) and Toll pathways. Very little is known about these pathways in response to chikungunya virus (CHIKV) infection, a mosquito-borne alphavirus (Togaviridae) transmitted by aedine species to humans resulting in a febrile and arthralgic disease. In this study, the contribution of several innate immune responses to control CHIKV replication was investigated. In vitro experiments identified the RNAi pathway as a key antiviral pathway. CHIKV was shown to repress the activity of the Toll signaling pathway in vitro but neither JAK/STAT, IMD nor Toll pathways were found to mediate antiviral activities. In vivo data further confirmed our in vitro identification of the vital role of RNAi in antiviral defence. Taken together these results indicate a complex interaction between CHIKV replication and mosquito innate immune responses and demonstrate similarities as well as differences in the control of alphaviruses and other arboviruses by mosquito immune pathways.