Mosquito Pools Research Articles

Shotgun Metagenomic Sequencing Reveals Virome Composition of Mosquitoes from a Transition Ecosystem of North-Northeast Brazil.

A wide diversity of pathogenic mosquito-borne viruses circulate in the Brazilian Amazon, and the intense deforestation can contribute to the spread of these viruses. In this context, this study aimed to investigate the viral diversity in mosquitoes of the genera Aedes, Culex, Haemagogus, and Sabethes from a transition area between the Amazon, Cerrado, and Caatinga biomes in Brazil. Metagenomic high-throughput sequencing was used to characterize the virome of 20 mosquito pools. A total of 15 virus-like genomes were identified, comprising species genomically close to insect-specific viruses of the families Iflaviridae, Metaviridae, Lispiviridae, Rhabdoviridae, Xinmoviridae, and Parvoviridae and species of plant viruses of the families Solemoviridae, Virgaviridae, and Partitiviridae. However, sequences of viruses associated with human and animal diseases were not detected. Most of the recovered genomes were divergent from those previously described. These findings reveal that there are a large number of unknown viruses to be explored in the middle-north of Brazil.

Small RNA sequencing of field Culex mosquitoes identifies patterns of viral infection and the mosquito immune response

Mosquito-borne disease remains a significant burden on global health. In the United States, the major threat posed by mosquitoes is transmission of arboviruses, including West Nile virus by mosquitoes of the Culex genus. Virus metagenomic analysis of mosquito small RNA using deep sequencing and advanced bioinformatic tools enables the rapid detection of viruses and other infecting organisms, both pathogenic and non-pathogenic to humans, without any precedent knowledge. In this study, we sequenced small RNA samples from over 60 pools of Culex mosquitoes from two major areas of Southern California from 2017 to 2019 to elucidate the virome and immune responses of Culex. Our results demonstrated that small RNAs not only allowed the detection of viruses but also revealed distinct patterns of viral infection based on location, Culex species, and time. We also identified miRNAs that are most likely involved in Culex immune responses to viruses and Wolbachia bacteria, and show the utility of using small RNA to detect antiviral immune pathways including piRNAs against some pathogens. Collectively, these findings show that deep sequencing of small RNA can be used for virus discovery and surveillance. One could also conceive that such work could be accomplished in various locations across the world and over time to better understand patterns of mosquito infection and immune response to many vector-borne diseases in field samples.

Identification and Molecular Characteristics of a Novel Single-Stranded RNA Virus Isolated from Culex tritaeniorhynchus in China.

Hubei mosquito virus 2 (HMV2) is a novel mosquito virus that was first identified in 2016 in Hubei Province, China. Until now, HMV2 has been shown to be endemic in some areas of China and Japan, but its biological characteristics, epidemiology, and pathogenicity are not yet known. This report describes the detection of HMV2 in mosquitoes that were collected in Shandong Province in 2019 and presents the first isolation and molecular characterization of the virus. In this study, a total of 2,813 mosquitoes were collected and then divided into 57 pools, according to location and species. qRT-PCR and nested PCR were performed to confirm the presence of HMV2, and its genomic features, phylogenetic relationships, growth characteristics, and potential pathogenicity were further analyzed. The results showed that HMV2 was detected in 28 of the 57 mosquito pools and that the minimum infection rate (MIR) for HMV2 was 1.00% (28/2,813). A HMV2 strain and 14 viral partial sequences were obtained from the HMV2-positive pools, including one complete genome sequence. A phylogenetic analysis revealed that HMV2 from Shandong Province shared over 90% identity with other reported isolates and was closely related to the Culex inatomii luteo-like virus. IMPORTANCE Our study provided important epidemiological evidence for the epidemic of HMV2 in Shandong Province. Here, we report the first isolation and molecular characteristics of this virus and enrich our knowledge of the distribution of HMV2 in China.

Diversity and Genetic Reassortment of Keystone Virus in Mosquito Populations in Florida.

Keystone orthobunyavirus (KEYV), a member of the genus Orthobunyavirus, was first isolated in 1964 from mosquitoes in Keystone, Florida. Although data on human infections are limited, the virus has been linked to a fever/rash syndrome and, possibly, encephalitis, with early studies suggesting that 20% of persons in the Tampa, Florida, region had antibodies to KEYV. To assess the distribution and diversity of KEYV in other regions of Florida, we collected >6,000 mosquitoes from 43 sampling sites in St. Johns County between June 2019 and April 2020. Mosquitoes were separated into pools by species and collection date and site. All pools with Aedes spp. (293 pools, 2,171 mosquitoes) were screened with a real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assay that identifies KEYV and other closely related virus species of what was previously designated as the California encephalitis serogroup. In 2020, screening for KEYV was expanded to include 211 pools of Culex mosquitoes from sites where KEYV-positive Aedes spp. had been identified. rRT-PCR-positive samples were inoculated into cell cultures, and five KEYV isolates from Aedes atlanticus pools were isolated and sequenced. Analyses of the KEYV large genome segment sequences revealed two distinct KEYV clades, whereas analyses of the medium and small genome segments uncovered past reassortment events. Our data documented the ongoing seasonal circulation of multiple KEYV clades within Ae. atlanticus mosquito populations along the east coast of Florida, highlighting the need for further studies of the impact of this virus on human health.

Dengue Virus Serotypes Circulating among Aedes Mosquitoes in the Lucknow District of North India: Molecular Identification and Characterization

Dengue is one of the most critical diseases, caused by Dengue virus (DENV) serotypes (DENV-1 to DENV-4). Study aims to detect DENV in natural A. aegypti from endemic regions of Uttar Pradesh. From 2010- 2013, mosquitoes collected from Uttar Pradesh tested for Dengue virus serotypes by capsid-premembrane gene-based PCR analyzed for genotypes sequencing of the C-prM junction of DENV genome. A total of 4731, 53.54% (n=2671) A. aegypti and 46 % (n=2060) A. albopictus mosquitoes were collected. Of 226 mosquito pools, 10 pools of A. aegypti and 14 pools of A. albopictus were positive for DENV by PCR. All 24 isolates identified as DENV-I; Genotype (G)-III (n=8), G-V (n=1); DENV-2; G-IV (n=4); DENV-3; G-III (n=11). The overall minimum infection rate was much higher in A. albopictus mosquitoes and presence of MIR in male mosquitoes is an indicating natural vertical transmission and important observation in geographical area indicating natural vertical transmission.

Long-distance spread of Tembusu virus, and its dispersal in local mosquitoes and domestic poultry in Chongming Island, China

BackgroundChongming Island in China serves as a breeding and shelter point on the East Asian–Australasian Flyway. The resting frequency of migratory birds, abundance of mosquito populations, and the popular domestic poultry industry pose a potential risk of mosquito-borne zoonotic diseases. The aim of this study is to explore the role of migratory birds in the spread of mosquito-borne pathogens and their prevalent status on the island.MethodsWe conducted a mosquito-borne pathogen surveillance in 2021, in Chongming, Shanghai, China. Approximately 67,800 adult mosquitoes belonging to ten species were collected to investigate the presence of flaviviruses, alphaviruses, and orthobunyaviruses by RT-PCR. Genetic and phylogenetic analyses were conducted to explore the virus genotype and potential nature source. Serological survey was performed by ELISA to characterize Tembusu virus (TMUV) infection among domestic poultry.ResultsTwo strains of TMUV and Chaoyang virus (CHAOV) and 47 strains of Quang Binh virus (QBV) were detected in 412 mosquito pools, with the infection rate of 0.16, 0.16, and 3.92 per 1000 Culex tritaeniorhynchus, respectively. Furthermore, TMUVs viral RNA was found in serum samples of domestic chickens and faecal samples of migratory birds. Antibodies against TMUV were detected in domestic avian serum samples, generally ranging from 44.07% in pigeons to 55.71% in ducks. Phylogenetic analyses indicated that the TMUV detected in Chongming belonged to Cluster 3, Southeast Asia origin, and most closely related to the CTLN strain, which caused a TMUV outbreak in chickens in Guangdong Province in 2020, but distant from strains obtained previously in Shanghai, which were involved in the 2010 TMUV outbreak in China.ConclusionsWe speculate that the TMUV was imported to Chongming Island through long-distance spreading by migratory birds from Southeast Asia, followed by spill over and transmission in mosquitoes and domestic avian species, threatening the local domestic poultry. In addition, the expansion and prevalence of insect-specific flaviviruses and its simultaneous circulation with mosquito-borne virus are worthy of close attention and further study.

Genomic Characterization of Mosquito Isolates of Chikungunya Virus (Outbreak Strains 2022) Using Next-Generation Sequencing.

Background: A massive outbreak of dengue-like illness was reported from Pune district of Maharashtra, India during May-June 2022. Isolation and characterization of the etiological agent at genomic level for possible mutations that led to higher transmissibility is the topic of the study. Methods: Entomological investigations were carried out by ICMR-National Institute of Virology (Pune, India); Aedes aegypti mosquitoes were collected and processed for virus detection by molecular techniques. Positive mosquito pools were processed for virus isolation in cell culture. Sanger sequencing and whole-genome sequencing (WGS) using Oxford Nanopore Technology platform were used for genomic characterization. Results: Reverse transcriptase RT-PCR and qRT-PCR analysis detected chikungunya virus (CHIKV) in mosquito samples. Six CHIKV isolates were obtained. WGS revealed four nonsynonymous mutations in the structural polyprotein region, and five in the nonstructural polyprotein encoding region when compared with Yawat-2000 and Shivane-2016 strains. Sixty-four nucleotide changes in the nonstructural polyprotein region and 35 in the structural polyprotein region were detected. One isolate had an exclusive amino acid change, T1123I, in the nsP2 (protease) region. Conclusion: Abundant Ae. aegypti breeding and detection of CHIKV RNA in mosquitoes confirmed it as a chikungunya outbreak. Novel mutations detected in the epidemic strain warrants investigations to address their role in disease severity, transmission, and fitness.

Density of Aedes aegypti (Diptera: Culicidae) in a low-income Brazilian urban community where dengue, Zika, and chikungunya viruses co-circulate

BackgroundLow-income urban communities in the tropics often lack sanitary infrastructure and are overcrowded, favoring Aedes aegypti proliferation and arboviral transmission. However, as Ae. aegypti density is not spatially homogeneous, understanding the role of specific environmental characteristics in determining vector distribution is critical for planning control interventions. The objectives of this study were to identify the main habitat types for Ae. Aegypti, assess their spatial densities to identify major hotspots of arbovirus transmission over time and investigate underlying factors in a low-income urban community in Salvador, Brazil. We also tested the field-collected mosquitoes for arboviruses.MethodsA series of four entomological and socio-environmental surveys was conducted in a random sample of 149 households and their surroundings between September 2019 and April 2021. The surveys included searching for potential breeding sites (water-containing habitats) and for Ae. aegypti immatures in them, capturing adult mosquitoes and installing ovitraps. The spatial distribution of Ae. aegypti density indices were plotted using kernel density-ratio maps, and the spatial autocorrelation was assessed for each index. Visual differences on the spatial distribution of the Ae. aegypti hotspots were compared over time. The association of entomological findings with socio-ecological characteristics was examined. Pools of female Ae. aegypti were tested for dengue, Zika and chikungunya virus infection.ResultsOverall, 316 potential breeding sites were found within the study households and 186 in the surrounding public spaces. Of these, 18 (5.7%) and 7 (3.7%) harbored a total of 595 and 283 Ae. aegypti immatures, respectively. The most productive breeding sites were water storage containers within the households and puddles and waste materials in public areas. Potential breeding sites without cover, surrounded by vegetation and containing organic matter were significantly associated with the presence of immatures, as were households that had water storage containers. None of the entomological indices, whether based on immatures, eggs or adults, detected a consistent pattern of vector clustering in the same areas over time. All the mosquito pools were negative for the tested arboviruses.ConclusionsThis low-income community displayed high diversity of Ae. aegypti habitats and a high degree of heterogeneity of vector abundance in both space and time, a scenario that likely reflects other low-income communities. Improving basic sanitation in low-income urban communities through the regular water supply, proper management of solid wastes and drainage may reduce water storage and the formation of puddles, minimizing opportunities for Ae. aegypti proliferation in such settings.Graphical

IN-HOUSE TESTING OF MOSQUITO POOLS FOR WEST NILE VIRUS USING COMMERCIALLY AVAILABLE IMMUNOASSAY AND REAL-TIME REVERSE-TRANSCRIPTASE POLYMERASE CHAIN REACTION KITS

Arbovirus surveillance methods are an integral part of integrated mosquito management programs, providing information on arboviral presence, location, and transmission potential. For many vector control agencies, surveillance entails collecting vector mosquito species from the field and testing representative mosquito pools using commercially available immunoassays or real-time reverse transcriptase polymerase chain reaction test kits. In 2016, the Collier Mosquito Control District established an in-house arbovirus surveillance program to screen mosquitoes for a variety of diseases, including the endemic arbovirus, West Nile virus (WNV). Although guidance on interpreting test results is provided by the manufacturer, end users of commercial test kits are encouraged to establish their own cut-off values signifying an arbovirus positive mosquito pool. Here we report the Collier Mosquito Control District’s efforts to develop cut-off values for mosquito pools using two commercially available WNV test kits.

West Nile virus: The current situation in Egypt.

Due to climatic changes, arthropod-borne viruses have become a global health concern. In Egypt, West Nile virus (WNV) was initially detected in humans in 1950 and then in 1951, 1954, 1968, and 1989. Although WNV infection has been recorded in numerous Middle Eastern countries, its prevalence among the equine population in Egypt is unknown. This study aimed to investigate the current situation of vector-borne WNV in Egypt, estimate its seroprevalence, and assess the associated risk factors. We screened 1100 sera samples and nasal swabs from the same equids, 156 mosquito pools, and 336 oropharyngeal and cloacal swabs from migratory birds for WNV. The sera were investigated for the presence of immunoglobulin G (IgG) and immunoglobulin M (IgM) against WNV-prE. Real-time reverse transcription-polymerase chain reaction was used to detect WNV RNA in the nasal swab samples, mosquito pools, and migratory birds' oropharyngeal and cloacal swabs. The seroprevalence showed positive IgG in sera samples collected from different districts. The data showed that horses were 1.65-fold more susceptible than donkeys, with male being 1.45 times more susceptible than females. Moreover, the tested equids samples were divided into three groups based on their age: <5 years, 5-10 years, and >10 years. The 5-10-year group was 1.1 and 1.61 times more vulnerable to infection than the <5- and >10 year groups. All the sera samples were negative for IgM. The nasal swabs from equids, oropharyngeal and cloacal swabs from migratory birds, and mosquito samples tested negative for WNV by molecular detection. Based on the obtained data, we recommend that effective control programs should be implemented to enable epidemiological investigations and understand the current situation of WNV in Egypt.

Report of natural Mayaro virus infection in Mansonia humeralis (Dyar & Knab, Diptera: Culicidae)

BackgroundArboviruses are a group of viruses transmitted to vertebrate hosts by certain blood-feeding arthropods. Among urban vectors of arboviruses, mosquitoes of the genus Aedes are the most common. However, other mosquitoes may be susceptible to infection and involved in the transmission, such as Mansonia spp. Therefore, this study aimed to investigate whether Mansonia humeralis can be infected with the Mayaro virus (MAYV).MethodsThese insects were collected from 2018 to 2020 in chicken coops of rural communities in Jaci Paraná in Porto Velho, Rondônia, Brazil, while performing blood-feeding on roosters. The mosquitoes were randomly grouped in pools from which the head and thorax were macerated and checked for the presence of MAYV by quantitative reverse transcription polymerase chain reaction (RT-qPCR). The positive pools were used to infect the C6/36 cell line, and on different days post-infection, the supernatant of the infected cells was subjected to viral detection by RT-qPCR.ResultsA total of 183 pools of female mosquitoes were tested, of which 18% were positive for MAYV; some samples from insect pools inoculated into C6/36 cells showed in vitro multiplication capacity between 3 and 7 days post-infection.ConclusionsThis is the first report of Ma. humeralis mosquitoes that are naturally infected by MAYV, indicating that these vectors may be potential transmitting agents of this arbovirus.Graphical

Circulation of West Nile virus in mosquitoes approximate to the migratory bird stopover in West Coast Malaysia.

Being a tropical country with a conducive environment for mosquitoes, mosquito-borne illnesses such as dengue, chikungunya, lymphatic filariasis, malaria, and Japanese encephalitis are prevalent in Malaysia. Recent studies reported asymptomatic infection of West Nile virus (WNV) in animals and humans, but none of the studies included mosquitoes, except for one report made half a century ago. Considering the scarcity of information, our study sampled mosquitoes near migratory bird stopover wetland areas of West Coast Malaysia located in the Kuala Gula Bird Sanctuary and Kapar Energy Venture, during the southward migration period in October 2017 and September 2018. Our previous publication reported that migratory birds were positive for WNV antibody and RNA. Using a nested RT-PCR analysis, WNV RNA was detected in 35 (12.8%) out of 285 mosquito pools consisting of 2,635 mosquitoes, most of which were Culex spp. (species). Sanger sequencing and phylogenetic analysis revealed that the sequences grouped within lineage 2 and shared 90.12%-97.01% similarity with sequences found locally as well as those from Africa, Germany, Romania, Italy, and Israel. Evidence of WNV in the mosquitoes substantiates the need for continued surveillance of WNV in Malaysia.

The first molecular confirmation of Culex pipiens complex as potential natural vectors of Dirofilaria immitis in Myanmar.

Dirofilariosis, known as one of the most widespread vector-borne zoonotic diseases, is caused by several different species of the nematodes of the genus Dirofilaria, which can be transmitted by Culex, Anopheles and Aedes mosquito vectors. In order to identify key vector mosquitoes of filarial parasites in Myanmar, mosquitoes were collected during three different seasons (summer, rainy and winter) in three townships in Nay Pyi Taw area, Myanmar. DNA extraction and polymerase chain reaction (PCR) analyses were conducted for 185 mosquito pools, with each pool containing 1-10 mosquitoes. Dirofilaria immitis was detected in 20 pools of Culex pipiens complex mosquitoes. The minimum infection rate of mosquitoes was found to be 16.33. The small subunit ribosomal RNA (12S rDNA) gene targeted PCR revealed that the sequences obtained were completely identical to the sequences of D. immitis derived from dogs in China, Brazil and France. The sequences obtained from mitochondrial cytochrome oxidase subunit I (COI) gene PCR exhibited 100% identity with the sequences of D. immitis derived from dogs in Bangladesh, Iran, Japan and Thailand, as well as humans in Iran and Thailand, and mosquitoes in Germany and Hungary. The findings of this study demonstrated that the mosquito species of Cx. pipiens complex are potential mosquito vectors for dirofilariosis in Myanmar.

The pathology of co-infection with Usutu virus and Plasmodium spp. in naturally infected Eurasian blackbirds (Turdus merula)

Usutu virus (USUV) is a mosquito-borne zoonotic flavivirus causing mortality in Eurasian blackbirds (Turdus merula) in Europe. In dead blackbirds, avian malaria co-infection due to mosquito-borne hemosporidians (e.g., Plasmodium spp.) has been reported. In humans, a similar co-infection of a flavivirus, Dengue virus, and Plasmodium spp. is causing increased severity of clinical disease. Currently, the effects of co-infection of arboviruses and hemosporidians in blackbirds remain unclear. This study investigates the rate of USUV and Plasmodium spp. co-infection in found-dead blackbirds (n = 203) from 2016 to 2020 in the Netherlands. Presence of Plasmodium spp. was evaluated by cytology (43/203; 21,2%), histopathology (94/186; 50,5%) and qPCR (179/203; 88,1%). The severity of histological lesions in USUV and Plasmodium spp. co-infected dead blackbirds (121/203; 59,6%) were compared with those in Plasmodium spp. single-infected cases. Additionally, since no knowledge is present on the infection rate on live birds and mosquitoes in the Netherlands, a small group of live blackbirds (n = 12) and selected in the field-collected mosquito pools (n = 96) in 2020 were tested for the presence of Plasmodium spp. The latter was detected in the tested live blackbirds by qPCR (8/10; 80%), and cytology (3/11; 27,3%) and in the mosquito pools by qPCR (18/96; 18,7%). For this study, co-infection between USUV and Plasmodium spp. was observed only in the dead blackbirds. The high Plasmodium spp. presence, associated with lower lesions score, in single infected found dead birds suggest a predominantly smaller pathogenic role as single agent. On the other hand, the higher histological lesion scores observed in USUV and Plasmodium spp. co-infected birds suggests a major pathogenic role for the virus or an increased severity of the lesions due to a possible interplay of the two agents.

Metagenomic analysis of coastal Kenya female Aedes aegypti mosquito RNA metaviromes reveal presence of diverse insect specific viruses

Background: Coastal Kenya has experienced repeated outbreaks of Chikungunya (CHIKV) and Dengue (DENV) viruses mediated by competent Aedes aegypti mosquito populations. These mosquitoes harbor insect specific viruses (ISVs), some of which can prevent arboviral transmission. However, there has been no systematic molecular entomological surveillance in coastal Kenya and the diversity of viruses in local Aedes aegypti populations remains largely unknown. Methods: To obtain a snapshot of the Aedes aegypti viromes from coastal Kenya, we took advantage of a cross-sectional survey of mosquitoes to determine the prevalence of Zika virus. We collected adult mosquitoes using lured Biogent’s sentinel traps at 16 different localities along the Kenyan coast between May to September 2017. Pools of 20 female Aedes aegypti mosquitoes were generated following grouping by morphological characteristics. Presence of arboviruses in the mosquito pools was determined using virus-specific and genera-specific primers with real-time PCR. Metagenomic next generation sequencing (mNGS) on Illumina Miseq and analysis was used to characterize the virome. Results: A total of 16,520 female Aedes aegypti grouped into 826 pools were analysed. Flaviviruses were detected in 69/826 (8.4%) pools by real time PCR. Sequencing generated 8,459/971,754 (0.87%) clean reads that were taxonomically assigned to 16 and 28 viral families and species, respectively. The family Phenuiviridae represented by Phasi Charoen-like phasivirus (PCLV) species was the most prevalent, detected in 64/73 (87%) mosquito pools. No pathogenic viruses were identified by mNGS. Phylogenetic analysis revealed local PCLV and Cell fusing agent virus (CFAV) were distinct from global sequences. Conclusions: Our data provides information about virus diversity and composition of the Aedes aegypti mosquitoes from coastal Kenya and contributes to the body of knowledge of the Aedes aegypti virome. To the best of our knowledge, this is the first study to provide this information from this region.

Field Evaluation of Commonly Used Adult Mosquito Traps in Greece.

Background: Entomological monitoring activities are a major part of mosquito and mosquito-borne diseases surveillance. Several trapping methods are implemented worldwide, aiming to gather data on species composition and their abundance in various study areas. Methods: Several methodological modifications, such as trapping systems baited with attractants or carbon dioxide, have been proposed to increase trap efficiency. The aim of this study was to test different trap types, commonly used in Greece to collect mosquitoes, with the addition of the Biogents Sentinel lure. Moreover, traps were placed in two distinct land types and two different heights above the ground to compare their efficacy. West Nile Virus is endemic in Greece, so we also aimed to detect viral presence and circulation in selected mosquito pools. Results: Adult mosquitoes of Aedes albopictus, Culex pipiens s.l., and Culiseta longiareolata were collected in both study areas. The trap type had a significant impact on the total collections, while the trap position and interaction between trap and position did not significantly affect mosquito catches. WNV was detected in Cx. pipiens s.l. pools examined from the two study areas. Conclusion: This study emphasizes the role of trapping methods as a key component for monitoring and surveillance of adult mosquito populations, reflecting that several trap types present with different mosquito species capture and catch rates.

Comparison of mosquito and fly derived DNA as a tool for sampling vertebrate biodiversity in suburban forests in Berlin, Germany

AbstractThe use of invertebrate‐derived DNA (iDNA) is a promising non‐invasive tool to monitor wildlife. While most studies have been carried out in dense tropical and sub‐tropical forests and have focused on the use of a single category of invertebrates, this study compares the use of flies and mosquitoes‐derived DNA to assess vertebrate diversity in semi‐urban environments. We conducted our sampling in four different forest plots in Berlin, Germany. Pools of flies and non‐bloodfed mosquitoes were metabarcoded using 108‐bp vertebrate‐specific 12 S rRNA (12 S‐V5) and 94‐bp mammal‐specific 16 S rRNA (16Smam) mitochondrial markers, and individual bloodfed mosquitoes were sequenced using the 340‐bp vertebrate‐specific 12 S rRNA fragment (Mam‐12 S‐340). Most sequencing was only successful for mammal species. From the fly pools, we detected 10 mammal species using 16Smam, and six species using 12 S‐V5. From the non‐bloodfed mosquito pools, we only amplified putative contaminant DNA, indicating that mosquito females without visual signs of a blood meal carry no traces of vertebrate DNA. Finally, in the bloodfed mosquitoes, we identified four mammal species. We did not find significant differences in the proportion of mammal species detected regarding the total available number of species between sampling localities. Fly samples were easier to obtain and more abundant over the sampled localities compared to mosquito samples. We conclude that, while there are a few advantages in using mosquito blood meals, the use of flies in the detection of wildlife in a suburban environment is more effective in terms of collection of samples and detection of vertebrates, although this technique is limited to few mammal species in the urban environment.

West Nile, Sindbis and Usutu Viruses: Evidence of Circulation in Mosquitoes and Horses in Tunisia.

Mosquito-borne diseases have a significant impact on humans and animals and this impact is exacerbated by environmental changes. However, in Tunisia, surveillance of the West Nile virus (WNV) is based solely on the surveillance of human neuroinvasive infections and no study has reported mosquito-borne viruses (MBVs), nor has there been any thorough serological investigation of anti-MBV antibodies in horses. This study therefore sought to investigate the presence of MBVs in Tunisia. Among tested mosquito pools, infections by WNV, Usutu virus (USUV), and Sindbis virus (SINV) were identified in Cx. perexiguus. The serosurvey showed that 146 of 369 surveyed horses were positive for flavivirus antibodies using the cELISA test. The microsphere immunoassay (MIA) showed that 74 of 104 flavivirus cELISA-positive horses were positive for WNV, 8 were positive for USUV, 7 were positive for undetermined flaviviruses, and 2 were positive for tick-borne encephalitis virus (TBEV). Virus neutralization tests and MIA results correlated well. This study is the first to report the detection of WNV, USUV and SINV in Cx. perexiguus in Tunisia. Besides, it has shown that there is a significant circulation of WNV and USUV among horses, which is likely to cause future sporadic outbreaks. An integrated arbovirus surveillance system that includes entomological surveillance as an early alert system is of major epidemiological importance.

Alphaviruses Detected in Mosquitoes in the North-Eastern Regions of South Africa, 2014 to 2018.

The prevalence and distribution of African alphaviruses such as chikungunya have increased in recent years. Therefore, a better understanding of the local distribution of alphaviruses in vectors across the African continent is important. Here, entomological surveillance was performed from 2014 to 2018 at selected sites in north-eastern parts of South Africa where alphaviruses have been identified during outbreaks in humans and animals in the past. Mosquitoes were collected using a net, CDC-light, and BG-traps. An alphavirus genus-specific nested RT-PCR was used for screening, and positive pools were confirmed by sequencing and phylogenetic analysis. We collected 64,603 mosquitoes from 11 genera, of which 39,035 females were tested. Overall, 1462 mosquito pools were tested, of which 21 were positive for alphaviruses. Sindbis (61.9%, N = 13) and Middelburg (28.6%, N = 6) viruses were the most prevalent. Ndumu virus was detected in two pools (9.5%, N = 2). No chikungunya positive pools were identified. Arboviral activity was concentrated in peri-urban, rural, and conservation areas. A range of Culicidae species, including Culex univittatus, Cx. pipiens s.l., Aedes durbanensis, and the Ae. dentatus group, were identified as potential vectors. These findings confirm the active circulation and distribution of alphaviruses in regions where human or animal infections were identified in South Africa.

Genomic Analysis of West Nile Virus Lineage 1 Detected in Mosquitoes during the 2020-2021 Outbreaks in Andalusia, Spain.

Emerging infectious diseases are one of the most important global health challenges because of their impact on human and animal health. The vector-borne West Nile virus (WNV) is transmitted between birds by mosquitos, but it can also infect humans and horses causing disease. The local circulation of WNV in Spain has been known for decades, and since 2010, there have been regular outbreaks in horses, although only six cases were reported in humans until 2019. In 2020, Spain experienced a major outbreak with 77 human cases, which was followed by 6 additional cases in 2021, most of them in the Andalusian region (southern Spain). This study aimed to characterize the genomes of the WNV circulating in wild-trapped mosquitoes during 2020 and 2021 in Andalusia. We sequenced the WNV consensus genome from two mosquito pools and carried out the phylogenetic analyses. We also compared the obtained genomes with those sequenced from human samples obtained during the outbreak and the genomes obtained previously in Spain from birds (2007 and 2017), mosquitoes (2008) and horses (2010) to better understand the eco-epidemiology of WNV in Spain. As expected, the WNV genomes recovered from mosquito pools in 2020 were closely related to those recovered from humans of the same outbreak. In addition, the strain of WNV circulating in 2021 was highly related to the WNV strain that caused the 2020 outbreak, suggesting that WNV is overwintering in the area. Consequently, future outbreaks of the same strain may occur in in the future.