Entomological Investigation Following a Zika Outbreak in Brownsville, Texas

ABSTRACT In November and December 2016, an outbreak of locally transmitted Zika occurred in Brownsville, TX. The Texas Department of State Health Services requested for a Centers for Disease Control and Prevention (CDC) Epi Aid, and as part of that Epi Aid a team of CDC entomologists was deployed in January 2017. The mission was to improve mosquito-based arbovirus surveillance and evaluate the possibility of continuing local Zika virus (ZIKV) transmission in the city. The mosquito-based arbovirus surveillance program was expanded from 4 to 40 BG-Sentinel traps evenly distributed throughout the city. Over a 2-wk period, 15 mosquito species were detected; the most abundant species were Culex quinquefasciatus, Aedes aegypti, and Ae. albopictus, which accounted for 66.7%, 16.2%, and 5.7% of the total mosquito collection, respectively. The relative abundance of Ae. aegypti (1.0 mosquitoes/trap/day) and Ae. albopictus (0.4 mosquitoes/trap/day) was very low and unlikely to initiate and/or sustain ZIKV transmission. Zika virus was not detected in the mosquitoes collected, suggesting no or extremely low ZIKV transmission at that time.

Molecular epidemiology of mosquito-borne viruses at the China–Myanmar border: discovery of a potential epidemic focus of Japanese encephalitis

Background Mosquito-based arbovirus surveillance can serve as an early warning in evaluating the status of mosquito-borne virus prevalence and thus prevent local outbreaks. Although Tengchong County in Yunnan Province—which borders Myanmar—is abundant and diverse in mosquitoes, very few mosquito-based arbovirus investigations have been conducted in the recent decade. Herein, this study aims to evaluate the presence and the diffusion of mosquito-borne pathogens, currently prevalent in this region. Methods We collected 9486 mosquitoes, representing eight species, with Culex tritaeniorhynchus and Anopheles sinensis as the dominant species, during high mosquito activity seasons (July–October) in Tengchong, in 2018. Samples collected from 342 pools were tested using reverse-transcription PCR to determine the species, distribution, and infection rates of virus and parasite, and further analyze their genotypes, phylogenetic relationships, infection rate, and potential pathogenicity. Results Fifteen Japanese encephalitis virus (JEV) strains from Cx. tritaeniorhynchus pools were detected. Seven strains of insect-specific flaviviruses (ISFVs), including two Aedes flavivirus (AeFV) and Yunnan Culex flavivirus strains each, one Culex theileri flavivirus, Yamadai flavivirus (YDFV) and Anopheles-associated flavivirus (AAFV) strains each were detected in Aedes albopictus, Cx. tritaeniorhynchus, Cx. vagans, Cx. pseudovihnui, and An. sinensis pools, respectively. The whole-genome was successfully amplified in one strain of JEV and AeFV each. Phylogenetic analysis using the E gene placed all the newly detected JEV strains into the GI-b genotype. They showed highly nucleotide identities, and were most closely related to the strain detected in Tengchong in 2010. The comparison of the E protein of JEV strains and vaccine-derived strain, showed six amino residue differences. The bias-corrected maximum likelihood estimation values (and 95% confidence interval) for JEV in Cx. tritaeniorhynchus collected in Tengchong in 2018 were 2.4 (1.4–3.9). Conclusions A potential Japanese encephalitis epidemic focus with the abundance of host mosquitoes and high JEV infection rate was observed in Tengchong. In addition, at least five species of ISFVs co-circulate in this area. This study highlights the importance of widespread and sustained mosquito-based arbovirus surveillance in local areas to prevent the transmission of JEV, and other emerging/re-emerging mosquito-borne pathogens. Graphic Abstract

Fighting mosquito bite during a crisis: capabilities of Florida mosquito control districts during the COVID-19 pandemic

Background The stay-at-home orders imposed in early April 2020 due to the COVID-19 pandemic in various states complicated mosquito control activities across the United States (US), and Florida was no exception. Mosquito control programs are the first line of defense against mosquito-borne pathogens. The purpose of this study was to examine the capabilities of Florida mosquito programs to implement key mosquito measures during the COVID-19 pandemic lockdown. Methods Using a self-administered online survey, we examined the capabilities of all Florida mosquito control programs (both state-approved mosquito districts, N = 63; and open programs, N = 27) at a time when the state of Florida was still under heightened awareness of, stay-at-home orders and planning a phase 1 reopening over the COVID-19 pandemic (June to July 2020). The final sample included mosquito control programs structured as the Board of County Commissioners (BOCC) (n = 42), independent tax district (n = 16), municipal (n = 10), and health or emergency department (n = 5). We used descriptive statistics to summarize information about the characteristics of responding programs, their implemented mosquito control and surveillance activities. wWe used bivariate analysis to compare the characteristics of responding programs and the self-reported mosquito measures. Results Of the recruited mosquito control programs, 73 completed the survey (81.1% response rate; 73/90). Of these, 57.5% (n = 42) were Board of County Commissioners (BOCC) mosquito control programs, 21.9% (n = 16) were independent tax district programs, 13.7% (n = 10) were municipal mosquito control programs, and only 6.8% (n = 5) were either health or emergency department mosquito control programs. Except for arbovirus surveillance, most programs either fully or partially performed larval (61.8%) and adult (78.9%) surveillance; most programs conducted species-specific control for Aedes aegypti (85.2%, n = 54), Aedes albopictus (87.3%, n = 55), Culex quinquefasciatus (92.1%, n = 58), and Culex nigripalpus (91.9%, n = 57). Conclusions Findings underscore the importance of ongoing mosquito control activities, and suggest that Florida mosquito control programs are vigilant and have significant capability to handle potential mosquito-borne disease threats, but arbovirus surveillance systems (laboratory testing of mosquito pools and testing of human and nonhuman specimens for arboviruses) are needed during pandemics as well.

High throughput estimates of Wolbachia, Zika and chikungunya infection in Aedes aegypti by near-infrared spectroscopy to improve arbovirus surveillance

Deployment of Wolbachia to mitigate dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) transmission is ongoing in 12 countries. One way to assess the efficacy of Wolbachia releases is to determine invasion rates within the wild population of Aedes aegypti following their release. Herein we evaluated the accuracy, sensitivity and specificity of the Near Infrared Spectroscopy (NIRS) in estimating the time post death, ZIKV-, CHIKV-, and Wolbachia-infection in trapped dead female Ae. aegypti mosquitoes over a period of 7 days. Regardless of the infection type, time post-death of mosquitoes was accurately predicted into four categories (fresh, 1 day old, 2–4 days old and 5–7 days old). Overall accuracies of 93.2, 97 and 90.3% were observed when NIRS was used to detect ZIKV, CHIKV and Wolbachia in dead Ae. aegypti female mosquitoes indicating NIRS could be potentially applied as a rapid and cost-effective arbovirus surveillance tool. However, field data is required to demonstrate the full capacity of NIRS for detecting these infections under field conditions.

Fighting the Bite during a Crisis: Capabilities of Florida Mosquito Control Districts during the COVID-19 Pandemic

Background: The national wide lockdown order imposed in early April 2020 due to the COVID-19 pandemic complicated mosquito control activities across the United States (US), and Florida is no exception. Mosquito control programs are the first line of defense against mosquitoes-borne pathogens in the state of Florida. The purpose of this study was to examine the capabilities of Florida mosquito programs to implement key mosquito measures during the COVID-19 pandemic.Methods: In a self-administered online survey, we examined capabilities of all Florida mosquito control programs during the COVID-19 pandemic (both state-approved mosquito districts (N=63) and open programs (N=27). Descriptive statistics were used to summarize information about the characteristics of responding programs, as well as the implemented mosquito control and surveillance activities. We used bivariate analysis to compare the characteristics of these responding programs and the self-reported mosquito measures.Results: Of the recruited mosquito control programs, 77 completed the survey (85.6% response rate; 77/90). Of these, 57.5% (n=42) were Board of County Commissioners (BOCC) mosquito control programs, 21.9% (n=16) were independent tax district programs, 13.7% (n=10) were municipal mosquito control programs, and only 6.8% (n=5) were either health or emergency department mosquito control programs. Except for arbovirus surveillance, most programs either fully or partially performed larval (61.8%) and adult (78.9%) surveillance; and for Aedes aegypti (85.2%, n=54), Aedes albopictus (87.3%, n=55), Culex quinquefasciatus (92.1%, n=58), and Culex nigripalpus (91.9%, n=57).Conclusions: Findings underscore the importance of ongoing mosquito control activities and suggests that Florida mosquito control programs are vigilant and have significant capability to handle potential mosquito-borne disease threats, but arbovirus surveillance systems; laboratory testing of mosquito pools and testing of human and nonhuman specimens for arboviruses are needed during pandemics as well.

Metagenomic Analysis of the Virome of Mosquito Excreta

ABSTRACT Traditional screening for arboviruses in mosquitoes requires a priori knowledge and the utilization of appropriate assays for their detection. Mosquitoes can also provide other valuable information, including unexpected or novel arboviruses, nonarboviral pathogens ingested from hosts they feed on, and their own genetic material. Metagenomic analysis using next-generation sequencing (NGS) is a rapidly advancing technology that allows us to potentially obtain all this information from a mosquito sample without any prior knowledge of virus, host, or vector. Moreover, it has been recently demonstrated that pathogens, including arboviruses and parasites, can be detected in mosquito excreta by molecular methods. In this study, we investigated whether RNA viruses could be detected in mosquito excreta by NGS. Excreta samples were collected from Aedes vigilax and Culex annulirostris experimentally exposed to either Ross River or West Nile viruses and from field mosquitoes collected across Queensland, Australia. Total RNA was extracted from the excreta samples, reverse transcribed to cDNA, and sequenced using the Illumina NextSeq 500 platform. Bioinformatic analyses from the generated reads demonstrate that mosquito excreta provide sufficient RNA for NGS, allowing the assembly of near-full-length viral genomes. We detected Australian Anopheles totivirus, Wuhan insect virus 33, and Hubei odonate virus 5 and identified seven potentially novel viruses closely related to members of the order Picornavirales (2/7) and to previously described, but unclassified, RNA viruses (5/7). Our results suggest that metagenomic analysis of mosquito excreta has great potential for virus discovery and for unbiased arbovirus surveillance in the near future. IMPORTANCE When a mosquito feeds on a host, it ingests not only its blood meal but also an assortment of microorganisms that are present in the blood, thus acting as an environmental sampler. By using specific tests, it is possible to detect arthropod-borne viruses (arboviruses) like dengue and West Nile viruses in mosquito excreta. Here, we explored the use of next-generation sequencing (NGS) for unbiased detection of RNA viruses present in excreta from experimentally infected and field-collected mosquitoes. We have demonstrated that mosquito excreta provide a suitable template for NGS and that it is possible to recover and assemble near-full-length genomes of both arboviruses and insect-borne viruses, including potentially novel ones. These results importantly show the direct practicality of the use of mosquito excreta for NGS, which in the future could be used for virus discovery, environmental virome sampling, and arbovirus surveillance.

Fighting the Bite during Pandemics: Florida Mosquito Control District Capabilities during the COVID-19 crisis

Background The national wide lockdown order imposed in early April 2020 due to the COVID-19 outbreak has complicated mosquito control activities across the United States (US), and Florida is no exception. Mosquito control districts and public health programs are the first line of defense against mosquitoes-borne pathogens in the state of Florida. The purpose of study is to understand how the COVID-19 outbreak has impact the capabilities of mosquito programs to implement key mosquito measures to mitigate emergence and/or re-emergence of arthropod-borne arboviral diseases. Methods In a self-administered online survey, we examined capabilities of all Florida mosquito control programs during the COVID-19 outbreak (both state-approved mosquito districts (N = 63) and public health programs (N = 27). Descriptive statistics were used to summarize information about characteristics of responding mosquito control districts and programs, implemented mosquito control and surveillance activities. We used Bivariate analysis to compare the characteristics of responding mosquito control districts and programs and the self-reported mosquito measures. Results Of the recruited programs, 77 completed the survey (85.6% response rate; 77/90). Of the responding programs, 57.5% (n = 42) were Board of County Commissioners (BOCC) programs, 21.9% (n = 16) were independent tax districts, 13.7% (n = 10) were municipal programs, and only 6.8% (n = 5) were either health or emergency departments. Except for arbovirus surveillance, most programs either fully or partially performed larval (61.8%) and adult (78.9%) surveillance; and for Aedes aegypti (71.9%, n = 46), Aedes albopictus (85.9%, n = 55), Culex quinquefasciatus (88.2%, n = 60), and Culex nigripalpus (90.5%, n = 57). Conclusions Findings underscore the importance of ongoing mosquito control activities and suggests that Florida mosquito control programs are vigilant and have significant capability to handle potential mosquito-borne disease threats, but arbovirus surveillance systems -- laboratory testing of mosquito pools and testing of human and nonhuman specimens for arboviruses are needed during pandemics as well.

Sensitivity and specificity of metatranscriptomics as an arbovirus surveillance tool

The ability to identify all the viruses within a sample makes metatranscriptomic sequencing an attractive tool to screen mosquitoes for arboviruses. Practical application of this technique, however, requires a clear understanding of its analytical sensitivity and specificity. To assess this, five dilutions (1:1, 1:20, 1:400, 1:8,000 and 1:160,000) of Ross River virus (RRV) and Umatilla virus (UMAV) isolates were spiked into subsamples of a pool of 100 Culex australicus mosquitoes. The 1:1 dilution represented the viral load of one RRV-infected mosquito in a pool of 100 mosquitoes. The subsamples underwent nucleic acid extraction, mosquito-specific ribosomal RNA depletion, and Illumina HiSeq sequencing. The viral load of the subsamples was also measured using reverse transcription droplet digital PCR (RT-ddPCR) and quantitative PCR (RT-qPCR). Metatranscriptomic sequencing detected both RRV and UMAV in the 1:1, 1:20 and 1:400 subsamples. A high specificity was achieved, with 100% of RRV and 99.6% of UMAV assembled contigs correctly identified. Metatranscriptomic sequencing was not as sensitive as RT-qPCR or RT-ddPCR; however, it recovered whole genome information and detected 19 other viruses, including four first detections for Australia. These findings will assist arbovirus surveillance programs in utilising metatranscriptomics in routine surveillance activities to enhance arbovirus detection.