Mosquito Virus Research Articles

Arbovirus Screening in Mosquitoes in Emilia-Romagna (Italy, 2021) and Isolation of Tahyna Virus.

ABSTRACTSeveral viruses can be transmitted by mosquitoes. We searched some of these viruses in 20,778 mosquitoes, collected in 95 traps on the plains of Emilia-Romagna (North of Italy) in 2021. We detected West Nile virus (WNV) and Usutu virus (USUV) in pools of Culex (Cx.) pipiens. In addition, we detected two insect-specific flaviviruses in three pools of Aedes (Ae.) caspius and in two of Ae. vexans. Tahyna virus (TAHV) was detected in six pools, three of Ae. caspius and three of Cx. pipiens, and one isolated strain was obtained from one of the Ae. caspius pools. Moreover, we detected TAHV in pools of several mosquito species (Ae. caspius, Ae. vexans, Ae. albopictus, Anopheles maculipennis s.l.) collected in the previous year of surveillance. Our data indicate Ae. caspius as the species most infected with TAHV in the surveyed area. Together with the likely plasticity of the cycle, we reported strong genome stability of the TAHV, probably linked to a successful adaptation of the virus to its ecological niche. Interestingly, in six pools of Cx. pipiens we detected two associated viruses among USUV, WNV, TAHV and all the three viruses in two pools. This result allows us to assume the presence of particular conditions that prompt the circulation of arboviruses, creating the conditions for viral hot spots. While no human diseases related to Tahyna virus were reported in Italy, its detection over the years suggests that it is worth investigating this virus as a potential cause of disease in humans in order to assess its health burden.IMPORTANCE We reported in this work the detection of three Arboviruses (Arthropod-borne viruses) in mosquitoes collected in Emilia-Romagna in 2021. In addition to West Nile and Usutu viruses, which were reported from more than 10 years in the study area, we detected and isolated Tahyna virus (TAHV). We also reported detections of TAHV obtained in previous years of surveillance in different species of mosquitoes. TAHV is the potential causative agent of summer influenza-like diseases and also of meningitis. Even if human cases of disease referable to this virus are not reported in Italy, its relevant presence in mosquitoes suggests investigating the possibility they could.

Arbovirus detection in synanthropic mosquitoes from the Brazilian Amazon and in mosquito saliva using Flinders Technology Associates cards

Although arbovirus transmission and identifying target vectors may provide a baseline for planning disease control strategies, there are many gaps in knowledge regarding these mosquitoes and viral species in urban, rural, or sylvatic habitats in the Brazilian Amazon. Our goal was to screen for dengue, chikungunya, and Zika viruses in synanthropic mosquitoes and with Flinders Technology Associates (FTA) cards using insect saliva. Mosquitoes were caught using ovitraps and aspirators in the city of Porto Velho, Rondônia, Brazil. Honey-baited FTA cards were placed in mosquito cages for 15 days; whole mosquitoes and FTA cards were analysed for viral RNA using RT-qPCR assays. One pool of Aedes aegypti females was found to be infected with the Zika virus and one male mosquito was infected with dengue-4, suggesting natural vertical/venereal transmission. Our study also reported evidence of vertical/venereal transmission of ZIKV in Culex quinquefasciatus males for the first time in the Brazilian Amazon, and the feasibility of using FTA cards to detect arboviruses in the saliva of field-collected mosquitoes. Vertical/venereal transmission of viruses by atypical mosquito species reinforces the need for combined viral and entomological screening in arbovirus surveillance programs.

Bidirectional Interactions between Arboviruses and the Bacterial and Viral Microbiota in Aedes aegypti and Culex quinquefasciatus.

ABSTRACTMosquitoes are important vectors for many arboviruses. It is becoming increasingly clear that various symbiotic microorganisms (including bacteria and insect-specific viruses; ISVs) in mosquitoes have the potential to modulate the ability of mosquitoes to transmit arboviruses. In this study, we compared the bacteriome and virome (both eukaryotic viruses and bacteriophages) of female adult Aedes aegypti and Culex quinquefasciatus mosquitoes fed with sucrose/water, blood, or blood spiked with Zika virus (ZIKV) or West Nile virus (WNV), respectively. Furthermore, we investigated associations between the microbiota and vector competence. We show that the influence of arboviruses on the mosquito microbiome—and vice versa—is distinct for each combination of arbovirus/mosquito species. The presence of ZIKV resulted in a temporarily increased Aedes ISV diversity. However, this effect was distinct for different ISVs: some ISVs decreased following the blood meal (Aedes aegypti totivirus), whereas other ISVs increased only when the blood contained ZIKV (Guadeloupe mosquito virus). Also, the diversity of the Aedes bacteriome depended on the diet and the presence of ZIKV, with a lower diversity observed for mosquitoes receiving blood without ZIKV. In Cx. quinquefasciatus, some ISVs increased in WNV-infected mosquitoes (Guadeloupe Culex tymo-like virus). Particularly, the presence of Wenzhou sobemo-like virus 3 (WSLV3) was associated with the absence of infectious WNV in mosquito heads, suggesting that WSLV3 might affect vector competence for WNV. Distinct profiles of bacteriophages were identified in Culex mosquitoes depending on diet, despite the lack of clear changes in the bacteriome. Overall, our data demonstrate a complex three-way interaction among arboviruses, resident microbiota, and the host, which is distinct for different arbovirus–mosquito combinations. A better understanding of these interactions may lead to the identification of microbiota able to suppress the ability of arbovirus transmission to humans, and hence improved arbovirus control measures.

Arthropod-Borne Virus Surveillance as a Tool to Study the Australian Mosquito Virome

Mosquitoes (n = 4381 in 198 pools) were collected in March and April 2018 to survey the presence of West Nile virus Kunjin strain in mosquito populations around crocodile farms in the Darwin region of the Northern Territory (NT) of Australia. While no Kunjin virus was detected in these mosquitoes, we applied our viral replicative intermediates screening system termed monoclonal antibodies to viral RNA intermediates in cells or MAVRIC to this set of samples. This resulted in the detection of 28 pools with virus replicating in C6/36 mosquito cells and the identification of three insect viruses from three distinct virus classes. We demonstrate the persistence of the insect-specific flavivirus Palm Creek virus in Coquillettidia xanthogaster mosquitoes from Darwin over almost a decade, with limited genetic drift. We also detected a novel Hubei macula-like virus 3 strain in samples from two mosquito genera, suggesting the virus, for which the sequence was originally detected in spiders and soybean thrips, might be involved in a horizontal transmission cycle between arthropods and plants. Overall, these data demonstrate the strength of the optimized MAVRIC system and contribute to our general knowledge of the mosquito virome and insect viruses.

Studies on the fitness characteristics of wMel- and wAlbB-introgressed Aedes aegypti (Pud) lines in comparison with wMel- and wAlbB-transinfected Aedes aegypti (Aus) and wild-type Aedes aegypti (Pud) lines.

Wolbachia, an intracellular maternally transmitted endosymbiont, has been shown to interfere with the replication of dengue virus in Aedes aegypti mosquitoes. The Wolbachia-transinfected Ae. aegypti has been currently released in many countries to test its effectiveness in preventing the transmission of dengue virus. ICMR-Vector Control Research Centre in collaboration with World Mosquito Program Monash University, Australia, has generated two new Wolbachia-introgressed Ae. aegypti Puducherry (Pud) lines via backcrossing Ae. aegypti females of Australian (Aus) strains, infected with wMel and wAlbB Wolbachia with wild-type Ae. aegypti Puducherry (Pud) males. Wolbachia infections are known to induce a fitness cost and confer benefit on the host mosquito populations that will influence spread of the Wolbachia into native wild mosquito populations during the field release. Hence, the induced fitness cost or benefit/advantage in the two newly generated Ae. aegypti (Pud) lines was assessed in the laboratory in comparison with the wild-type Ae. aegypti (Pud) strain. In addition, maternal transmission (MT) efficiency, induced cytoplasmic incompatibility (CI), and insecticide resistance status of the two (Pud) lines were determined to assess the likely frequency of wMel and wAlbB infections in the native wild population after field invasion. The study shows that wMel and wAlbB infections did not induce any fitness cost on the two newly generated (Pud) lines. Rather, in terms of wing length, fecundity, egg hatch rate, and adult survival, the Wolbachia introgression conferred fitness benefits on the (Pud) lines compared to uninfected Wolbachia free wild Ae. aegypti population. wMel and wAlbB exhibited a high maternal transmission (99–100%) and induced nearly complete (98–100%) cytoplasmic incompatibility. Both the (Pud) lines were resistant to deltamethrin, malathion, DDT, and temephos, and the level of resistance was almost the same between the two lines as in the wild type. Overall, the stable association of wMel and wAlbB established with Ae. aegypti and the reproductive advantages of the (Pud) lines encourage a pilot release in the field for population replacement potential.

Perbandingan Hasil Radiografi Efusi Pleura Pada Proyeksi Right Lateral Decubitus (RLD) Dan Left Lateral Decubitus (LLD) Pada Klinis Dengue Haemoragic Fever (DHF) Di RSU. Wisata Universitas Indonesia Timur

Dengue Hemorrhagic Fever (DHF) is an infectious disease that can be fatal in a relatively short time. This disease is classified as "difficult to differentiate" from other dengue fever diseases. If without quick and appropriate treatment, the sufferer could fall into an even more fatal condition. And generally the supporting action taken is an X-ray examination of the chest to assess the presence of fluid in the pleura as a result of the Aedes Aegypti mosquito virus. The purpose of this research is to know the Comparison of Radiographic Results of Pleura Effusion on Right Lateral Decubitus Projection and LLD ( Left Lateral Decubitus ) in DHF ( Dengue Haemoragic Fever ) case. This research was conducted in RSU.Wisata Radiology installation of Universitas Indonesia Timur with quantitative research method with experimental design approach. Data collection is done by finding literature, observation, interview, and documentation. From the results of the research that the image of pleural effusion with RLD projection ( Right Lateral Decubitus ) shows better radiographic picture in assessing the level of pleural effusion with clinical DHF ( Dengue Haemoragic Fever ).

Prevalence of Japanese encephalitis (JE) virus in mosquitoes and animals of the Asian continent: A systematic review and meta-analysis.

BackgroundJapanese encephalitis (JE) is a viral zoonotic disease that has been found in several countries of Asia and is responsible for high mortality and morbidity of men and animals in rural and sub-urban endemic areas due to the virus re-circulation among diverse hosts and vectors. The present study estimates the prevalence of the JE virus in the vector and animal population of the Asian continent using a systematic review and meta-analysis. MethodsThe Cochran collaborators' Preferred Reporting Items for Systematic Reviews and Meta-Analysis [PRISMA] guidelines were used for systematic review and meta-analysis. The heterogeneity was observed in meta-regression analysis due to several factors including region, species, and different diagnostic assays used in various studies. Thus we did sensitivity and subgroup analysis. ResultsThe prevalence of the JE virus was calculated using a total sample size of 47,391. Subgroup analysis revealed the JE virus prevalence of 39% in the Southeast Asia region, followed by East Asia with 35% and South Asia with 15% prevalence. Hence, the overall pooled prevalence of the JE virus was 26% in the Asian continent. ConclusionsThe highest proportion of infection was found in pigs amongst all animals, reinforcing the fact that they can be used as sentinels to predict outbreaks in humans. The findings of this study will enable researchers and policymakers in better understanding the disease's spatial and temporal distribution, as well as in creating and implementing location-specific JE prevention and control measures.

Rapid molecular assays for the detection of the four dengue viruses in infected mosquitoes.

The pantropic emergence of severe dengue disease can partly be attributed to the co-circulation of different dengue viruses (DENVs) in the same geographical location. Effective monitoring for circulation of each of the four DENVs is critical to inform disease mitigation strategies. In low resource settings, this can be effectively achieved by utilizing inexpensive, rapid, sensitive and specific assays to detect viruses in mosquito populations. In this study, we developed four rapid DENV tests with direct applicability for low-resource virus surveillance in mosquitoes. The test protocols utilize a novel sample preparation step, a single-temperature isothermal amplification, and a simple lateral flow detection. Analytical sensitivity testing demonstrated tests could detect down to 1,000 copies/µL of virus-specific DENV RNA, and analytical specificity testing indicated tests were highly specific for their respective virus, and did not detect closely related flaviviruses. All four DENV tests showed excellent diagnostic specificity and sensitivity when used for detection of both individually infected mosquitoes and infected mosquitoes in pools of uninfected mosquitoes. With individually infected mosquitoes, the rapid DENV-1, -2 and -3 tests showed 100% diagnostic sensitivity (95% CI = 69% to 100%, n=8 for DENV-1; n=10 for DENV 2,3) and the DENV-4 test showed 92% diagnostic sensitivity (CI: 62% to 100%, n=12) along with 100% diagnostic specificity (CI: 48-100%) for all four tests. Testing infected mosquito pools, the rapid DENV-2, -3 and -4 tests showed 100% diagnostic sensitivity (95% CI = 69% to 100%, n=10) and the DENV-1 test showed 90% diagnostic sensitivity (55.50% to 99.75%, n=10) together with 100% diagnostic specificity (CI: 48-100%). Our tests reduce the operational time required to perform mosquito infection status surveillance testing from > two hours to only 35 minutes, and have potential to improve accessibility of mosquito screening, improving monitoring and control strategies in low-income countries most affected by dengue outbreaks.

Status Resistensi dan Keberadaan Virus Dengue pada Nyamuk Aedes Sp di Kelurahan Pinang Kencana, Kota Tanjungpinang, Kepulauan Riau

Tanjungpinang Timur Village is an endemic area, and every year there is a death rate due to dengue fever. This study aimed to map the resistance and presence of the dengue virus in Aedes sp mosquitoes in Pinang Kencana Village. This research was observational with a descriptive approach. The samples were mosquitoes bred in the Vector Laboratory of Poltekkes, Ministry of Health, Tanjungpinang, from eggs trapped in ovitraps distributed in the study area. A total of 78 ovitraps were placed inside the house, 45 were positive, while 45 positive ovitraps were placed outside the house out of a total of 84 ovitraps. Ovitrap index numbers outside (57.7%) and inside the house (53.6%) with an overall ovitrap index of 55.6%. The resistance test results using the CDC bottle bioassay method showed that Aedes sp mosquitoes from Pinang Kencana Village were resistant to the pesticide deltamethrin dose of 10 g/bottle with a diagnosis time of 30 minutes with a mosquito mortality percentage of 68.33%. The PCR results for dengue virus in the sample mosquitoes showed no presence of dengue virus in the mosquito samples, both DEN-1, DEN-2, DEN-3, and DEN-4 serotypes.

Detection of Dengue Virus From Aedes aegypti (Diptera, Culicidae) in Field-Caught Samples From Makkah Al-Mokarramah, Kingdom of Saudi Arabia, Using RT-PCR.

Dengue fever (DF) is endemic to Makkah and Jeddah, the Kingdom of Saudi Arabia (KSA). However, until recently, the circulation of dengue virus (DENV) in Aedes mosquitoes in these areas was unknown. Serological surveillance of DENV in Ae aegypti is a powerful tool for early detection of dengue outbreaks and essential for developing effective control strategies. Therefore, this research aimed to examine a sample of adult Ae aegypti mosquitoes from Makkah, KSA, to detect DENV. In total, 1295 Ae aegypti mosquitoes were collected from the field from target areas of Makkah with a high incidence and prevalence of DF. The samples were divided into 259 coded pools (five mosquitoes in each) and preserved in 1.5 mL plastic tubes. The tubes were labeled, capped, and stored at−86°C until use. RT-PCR was used to detect DENV in the samples. All positive pools were confirmed by RT-PCR. The RT-PCR products were analyzed by gel electrophoresis (1.5% agarose in Tris-acetate EDTA buffer), stained with ethidium bromide, and visualized. DENV was isolated from six female Ae Aegypti collected from six pools (out of 259 pools). No other viruses were detected. Only five of the nine target localities had positive pools. Samples from the remaining four localities yielded negative results. Four DENV-positive mosquitoes were collected at the aquatic stages, and two were collected at the adult stage. These results show the circulation of DENV in adult mosquitoes and offspring, indicating vertical transmission of DENV. In conclusion, this study found that, in Makkah, DENV is circulating in dengue vectors with a high significance rate, suggesting the possibility of a dengue outbreak in the future; therefore, a sensitive surveillance system is vital to predict the outbreak and for early intervention and control.

Defining virus-carrier networks that shape the composition of the mosquito core virome of a local ecosystem.

Mosquitoes are the most important vectors of emerging infectious diseases. During the past decade, our understanding of the diversity of viruses they carry has greatly expanded. Most of these viruses are considered mosquito-specific, but there is increasing evidence that these viruses may affect the vector competence of mosquitoes. Metagenomics approaches have focused on specific mosquito species for the identification of what is called the core virome. Despite the fact that, in most ecosystems, multiple species may participate in virus emergence and circulation, there is a lack of understanding of the virus-carrier/host network for both vector-borne and mosquito-specific viruses. Here, we studied the core virome of mosquitoes in a diverse local ecosystem that had 24 different mosquito species. The analysis of the viromes of these 24 mosquito species resulted in the identification of 34 viruses, which included 15 novel viruses, as determined according to the species demarcation criteria of the respective virus families. Most of the mosquito species had never been analysed previously, and a comparison of the individual viromes of the 24 mosquito species revealed novel relationships among mosquito species and virus families. Groups of related viruses and mosquito species from multiple genera formed a complex web in the local ecosystem. Furthermore, analyses of the virome of mixed-species pools of mosquitoes from representative traps of the local ecosystem showed almost complete overlap with the individual-species viromes identified in the study. Quantitative analysis of viruses’ relative abundance revealed a linear relationship to the abundance of the respective carrier/host mosquito species, supporting the theory of a stable core virome in the most abundant species of the local ecosystem. Finally, our study highlights the importance of using a holistic approach to investigating mosquito viromes relationships in rich and diverse ecosystems.

Detection of West Nile Virus in Overwintering Mosquitoes in the Volgograd Region

Introduction: West Nile fever is a zoonotic, vector-borne viral infection caused by West Nile virus. The possibility of persistence of West Nile virus in overwintering mosquitoes in regions with a temperate climate is of great importance for understanding the mechanisms of pathogen circulation. Objective: To detect West Nile virus in mosquitoes during the inter-epizootic period in the Volgograd Region. Materials and methods: In 2013–2021, we collected overwintering mosquitoes in different locations of the Volgograd Region using a battery-powered aspirator with a Krishtal’s trap to detect West Nile virus RNA in them using a real-time reverse transcription polymerase chain reaction. An isolate (WNV Volgograd_o16/19) was obtained from sample o16/19 (Cx. pipiens, collected on April 2, 2019) with detected West Nile virus RNA using a VERO cell culture. After that, total RNA was isolated from the filtered cell supernatant of that isolate. Metagenomic sequencing of the sample was performed using a high-throughput Illumina MiSeq sequencer, Illumina Inc., USA. Results: In total, we collected 4,070 mosquitoes in wintering shelters and tested 157 pools of the insects for West Nile virus RNA. The latter was detected in two pools of Culex pipiens and in one pool of Anopheles maculipennis complex. The phylogenetic analysis showed that the WNV Volgograd_о16/19 strain isolated from the pool of wintering mosquitoes belonged to lineage 2 of West Nile virus. We also established its belonging to the monophyletic clade of West Nile virus strains isolated in the Volgograd, Astrakhan, and Rostov regions in the years 2007 and 2018–2020. Conclusions: We were first to detect West Nile virus in overwintering mosquitoes in the Volgograd Region. Our findings confirm the hypothesis that lineage 2 strains of encephalitic West Nile virus persist in mosquitoes during the inter-epizootic period and can be transmitted from mosquito to bird in springtime as one of the mechanisms of forming autochthonous foci in WNV endemic areas of the Russian Federation in the absence of the annual import of this infection.

Concurrent Infection With Dengue and Chikungunya Viruses in Humans and Mosquitoes: A Field Survey in Lower Moshi, Tanzania

Introduction: Dengue and Chikungunya have re-emerged as important diseases of global concern. Co-infections with Dengue virus (DENV) and Chikungunya virus (CHIKV) could have serious outcomes if not diagnosed and managed optimally. However, the key focal points for the maintenance of CHIKV and DENV infections and the extent of their co-infection remain poorly understood in many geo-ecologically distinct parts of Tanzania. Objective: We aimed to comparatively examine the prevalence and factors for seropositivity to DENV and CHIKV and their infection rates in humans and mosquitoes Methods: A cross-sectional study was performed in the Lower Moshi area of the Kilimanjaro region from April to July 2020. DENV and CHIKV exposure was determined by detecting IgM to the viruses using enzyme linked immunosorbent assay whereas infection was determined by real time quantitative polymerase chain reaction (RT-qPCR) assay. Results: Insecticide Treated Bed Net (ITN) use (χ2=3.504; p< 0.05), being ≥7 individuals living in the same household (χ2=4.655; p<0.05) and a recent travel to an urban destination (χ2=3.39; p< 0.05) were the only factors associated with CHIKV seropositivity. ITN use was the only factor associated with CHIKV infection (χ2=5.204; p<0.05). A recent travel to an urban destination (χ2=4.401; p< 0.05) was the only factor associated with DENV seropositivity. Five (1.5%) Ae. aegypti pools were positive for CHIKV whereas 1 (0.3%) was positive for DENV. Two Cx. pipiens, pools (1.9%) were positive for CHIKV. None of the Cx. pipiens mosquitoes was positive for DENV. No associations between DENV and CHIKV seropositivity was observed in humans but DENV infection was strongly associated with CHIKV infection (χ2 = 238.45; p<0.01). CHIKV infection was observed to be consistently higher in both, humans and mosquitoes. Conclusion: Detection of DENV and CHIKV in both humans and vector mosquitoes confirms that both viruses are actively circulating in the Lower Moshi area of Kilimanjaro region in Tanzania. Our findings point out the Lower Moshi area as a potential focal point for the maintenance of the two viruses and possibly other vector borne viruses. We call upon sustained active surveillance of arboviruses and other re-emerging infections to be better prepared for possible outbreaks by the viruses.

Detection of Dengue Virus Transovarial Transmission in Dengue Hemorrhagic Fever Endemic Areas

Dengue virus is a group of RNA viruses that are highly pathogenic in humans and spread quickly through the bites of Aedes aegypti and Aedes albopictus mosquitoes, especially in tropical countries. More than half a billion out of 100 countries worldwide are at serious risk of dengue virus infection. Vector surveillance activities with Ovitrap and detection of dengue virus types in Aedes aegypti and Aedes albopictus have never been carried out in Pontianak City. It is important in early alert systems at transmission foci. The purpose of this study was to prove the transovarial transmission of dengue virus in Aedes aegypti and Aedes albopictus mosquitoes with a transovarial transmission index (TTI) in endemic areas in Pontianak City, West Kalimantan. The method used in this research is descriptive observational, viral examination method with immunocytochemistry streptavidin-biotin peroxidase complex (ISBPC) and Polymerase Chain Reaction Transcription Reaction (PCR) aimed at proving the presence of transovarial transmission of dengue virus in the same period. The conclusion in this study is that there is evidence of transovarial transmission of dengue virus in Aedes mosquitoes in endemic areas by 29.3% in Sungai Jawi Dalam sub-district, West Pontianak sub-district, and 39.6% in Batu Layang sub-district, North Pontianak sub-district, mosquito density from the results of the Ovitrap Index measurement (OI) in Batu Layang Village is denser, namely 41.3%, compared to Sungai Jawi Village, which is 38.22% and has succeeded in identifying the type of dengue virus, namely the Dengue virus strain, in the two research locations.

Development of Novel Dengue NS1 Multiplex Lateral Flow Immunoassay to Differentiate Serotypes in Serum of Acute Phase Patients and Infected Mosquitoes

Dengue is among the most rapidly spreading arboviral disease in the world. A low-cost, easy to use point-of-care diagnostic tool for the detection and differentiation of dengue virus serotypes could improve clinical management, disease prevention, epidemiological surveillance, and outbreak monitoring, particularly in regions where multiple serotypes co-circulate. Despite widespread deployment, no commercial dengue antigen diagnostic test has proven effective in differentiating among dengue virus serotypes. In the current study, we first established mAb pairs and developed a multiplex lateral flow immunoassay for the simultaneous detection of the dengue viral NS1 antigen and identification of serotype. The proposed system, called Dengue serotype NS1 Multiplex LFIA, provides high sensitivity and specificity. In testing for JEV, ZIKV, YFV, WNV, and CHIKV, the multiplex LFIA gave no indication of cross- reactivity with cell culture supernatants of other flaviviruses or chikungunya virus. In analyzing 187 samples from patients suspected of dengue infection, the detection sensitivity for serotype D1 to D4 was 90.0%, 88.24%, 82.61%, and 83.33% and serotype specificity was 98.74%, 96.13%, 99.39%, and 97.04%, respectively. Our multiplex LFIA can also identify mono- and co-infection of different serotype of dengue viruses in mosquitoes. The proposed Multiplex LFIA provides a simple tool for the rapid detection of dengue serotypes and in the differential diagnosis of fever patients in regions where medical resources are limited and/or multiple DENVs co-circulate.

Development and Validation of a Colorimetric Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay for Detection of Eastern Equine Encephalitis Virus in Mosquitoes.

Eastern equine encephalitis virus (EEEV) is a highly pathogenic alphavirus that causes periodic outbreaks in the eastern USA. Mosquito abatement programs are faced with various challenges with surveillance and control of EEEV and other mosquito-borne illnesses. Environmental sampling of mosquito populations can be technically complex. Here we report the identification of biomarkers, development and validation of a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of EEEV. Positive samples are easily visualized by a color change from pink to yellow. The assay was validated using EEEV from viral culture, experimentally spiked mosquito pools, and previously tested mosquito pools. The RT-LAMP assay detected viral titers down to approximately 10% of what would be present in a single infectious mosquito, based upon EEEV viral titers determined by previous competency studies. The RT-LAMP assay efficiently detected EEEV in combined aliquots from previously homogenized pools of mosquitoes, allowing up to 250 individual mosquitoes to be tested in a single reaction. No false positive results were obtained from RNA prepared from negative mosquito pools acquired from known and potential EEEV vectors. The colorimetric RT-LAMP assay is highly accurate, technically simple, and does not require sophisticated equipment, making it a cost-effective alternative to real time reverse transcriptase-polymerase chain reaction (RT-PCR) for vector surveillance.

LAMP Detection of Virus-Derived DNA of Zika Virus in Vector Mosquito

Detection of infectious viruses in mosquitoes is one of the prerequisite measures to monitor the prevalence of vector-borne viral diseases. Determining which mosquitoes are currently infected with arboviruses such as Zika, dengue, and chikungunya virus is not yet practical in endemic areas due to multiple causes including the difficulty of dealing with the virus’ unstable RNA. In this study, instead of handling viral RNA, virus-derived DNA (vDNA) was introduced as a target template for nucleic acid amplification. In combination with loop-mediated isothermal amplification (LAMP), we examined a LAMP-based vDNA detection assay (vDNA-LAMP) targeting Zika virus (ZIKV). The vDNA-LAMP reaction amplifying part of the NS3 region of ZIKV successfully detected its vDNA from crude DNA purified from artificially infected cultured cells and Aedes mosquitoes. This rapid, simple, and versatile method may provide a promising field-surveillance method for arbovirus circulation via vector mosquitoes.

Assessing Aedes aegypti candidate genes during viral infection and Wolbachia-mediated pathogen blocking.

One approach to control dengue virus transmission is the symbiont Wolbachia, which limits viral infection in mosquitoes. Despite plans for its widespread use in Aedes aegypti, Wolbachia's mode of action remains poorly understood. Many studies suggest that the mechanism is likely multifaceted, involving aspects of immunity, cellular stress and nutritional competition. A previous study from our group used artificial selection to identify a new mosquito candidate gene related to viral blocking; alpha‐mannosidase‐2a (alpha‐Mann‐2a) with a predicted role in protein glycosylation. Protein glycosylation pathways tend to be involved in complex host–viral interactions; however, the function of alpha‐mannosidases has not been described in mosquito–virus interactions. We examined alpha‐Mann‐2a expression in response to virus and Wolbachia infections and whether reduced gene expression, caused by RNA interference, affected viral loads. We show that dengue virus (DENV) infection affects the expression of alpha‐Mann‐2a in a tissue‐ and time‐dependent manner, whereas Wolbachia infection had no effect. In the midgut, DENV prevalence increased following knockdown of alpha‐Mann‐2a expression in Wolbachia‐free mosquitoes, suggesting that alpha‐Mann‐2a interferes with infection. Expression knockdown had the same effect on the togavirus chikungunya virus, indicating that alpha‐Mann‐2a may have broad antivirus effects in the midgut. Interestingly, we were unable to knockdown the expression in Wolbachia‐infected mosquitoes. We also provide evidence that alpha‐Mann‐2a may affect the transcriptional level of another gene predicted to be involved in viral blocking and cell adhesion; cadherin87a. These data support the hypothesis that glycosylation and adhesion pathways may broadly be involved in viral infection in Ae. aegypti.

A time-series meta-transcriptomic analysis reveals the seasonal, host, and gender structure of mosquito viromes.

Although metagenomic sequencing has revealed high numbers of viruses in mosquitoes sampled globally, our understanding of how their diversity and abundance varies in time and space as well as by host species and gender remains unclear. To address this, we collected 23,109 mosquitoes over the course of 12 months from a bat-dwelling cave and a nearby village in Yunnan province, China. These samples were organized by mosquito species, mosquito gender, and sampling time for meta-transcriptomic sequencing. A total of 162 eukaryotic virus species were identified, of which 101 were novel, including representatives of seventeen RNA virus multi-family supergroups and four species of DNA virus from the families Parvoviridae, Circoviridae, and Nudiviridae. In addition, two known vector-borne viruses—Japanese encephalitis virus and Banna virus—were found. Analyses of the entire virome revealed strikingly different viral compositions and abundance levels in warmer compared to colder months, a strong host structure at the level of mosquito species, and no substantial differences between those viruses harbored by male and female mosquitoes. At the scale of individual viruses, some were found to be ubiquitous throughout the year and across four mosquito species, while most of the other viruses were season and/or host specific. Collectively, this study reveals the diversity, dynamics, and evolution of the mosquito virome at a single location and sheds new lights on the ecology of these important vector animals.

Detection of Mayaro virus in Aedes aegypti mosquitoes circulating in Goiânia-Goiás-Brazil

The Arboviral diseases are caused by arthropod-borne viruses, such as Mayaro virus (MAYV), the etiological agent of Mayaro fever. This disease has been drawing the attention of the public health authorities for the increased number of cases likely due to virus adaptation for survival to urban areas as well as infection and multiplication in other vectors insects. Therefore, this work aimed to identify the MAYV infecting Aedes aegypti mosquitoes in Goiânia, the capital of state of Goiás, Brazil. For the development of study, the larvae of A. aegypti were collected in Basic Health Units from different regions of Goiânia then the larvae were grown to adult mosquitoes in controlled laboratory conditions. The female mosquitoes were submitted to the procedure of head and body separation. The RNAs obtained from these samples were analyzed by real-time PCR for identification of arboviruses. We only detect the presence of MAVY in the mosquitoes, in this sense our findings suggest that A. aegypti harbor MAYV in different anatomical sites, and potentially the process of vertical transmission of MAYV can occur in this vector.