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Mayaro virus (MAYV) is transmitted by Haemagogus spp. mosquitoes and has been circulating in Amazon areas in the North and Central West regions of Brazil since the 1980s, with an increase in human case notifications in the last 10 years. MAYV introduction in urban areas is a public health concern as infections can cause severe symptoms similar to other alphaviruses. Studies with Aedes aegypti have demonstrated the potential vector competence of the species and the detection of MAYV in urban populations of mosquitoes. Considering the two most abundant urban mosquito species in Brazil, we investigated the dynamics of MAYV transmission by Ae. aegypti and Culex quinquefasciatus in a mice model. Mosquito colonies were artificially fed with blood containing MAYV and infection (IR) and dissemination rates (DR) were evaluated. On the 7th day post-infection (dpi), IFNAR BL/6 mice were made available as a blood source to both mosquito species. After the appearance of clinical signs of infection, a second blood feeding was performed with a new group of non-infected mosquitoes. RT-qPCR and plaque assays were carried out with animal and mosquito tissues to determine IR and DR. For Ae. aegypti, we found an IR of 97.5–100% and a DR reached 100% in both 7 and 14 dpi. While IR and DR for Cx. quinquefasciatus was 13.1–14.81% and 60% to 80%, respectively. A total of 18 mice were used (test = 12 and control = 6) for Ae. aegypti and 12 (test = 8 and control = 4) for Cx. quinquefasciatus to evaluate the mosquito–mice transmission rate. All mice that were bitten by infected Ae. aegypti showed clinical signs of infection while all mice exposed to infected Cx. quinquefasciatus mosquitoes remained healthy. Viremia in the mice from Ae. aegypti group ranged from 2.5 × 108 to 5 × 109 PFU/mL. Ae. aegypti from the second blood feeding showed a 50% IR. Our study showed the applicability of an efficient model to complete arbovirus transmission cycle studies and suggests that the Ae. aegypti population evaluated is a competent vector for MAYV, while highlighting the vectorial capacity of Ae. aegypti and the possible introduction into urban areas. The mice model employed here is an important tool for arthropod–vector transmission studies with laboratory and field mosquito populations, as well as with other arboviruses.

The West Nile Virus (WNV) and Sindbis virus (SINV) are avian-hosted mosquito-borne zoonotic viruses that co-circulate in some geographical areas and share vector species such as Culex pipiens and Culex torrentium. These are widespread in Europe, including northern parts and Finland, where SINV is endemic, but WNV is currently not. As WNV is spreading northwards in Europe, we wanted to assess the experimental vector competence of Finnish Culex pipiens and Culex torrentium mosquitoes to WNV and SINV in different temperature profiles. Both mosquito species were found susceptible to both viruses and got infected via infectious blood meal at a mean temperature of 18 °C. WNV-positive saliva was detected at a mean temperature of 24 °C, whereas SINV-positive saliva was detected already at a mean temperature of 18 °C. Cx. torrentium was found to be a more efficient vector for WNV and SINV over Cx. pipiens. Overall, the results were in line with the previous studies performed with more southern vector populations. The current climate does not seem optimal for WNV circulation in Finland, but temporary summertime transmission could occur in the future if all other essential factors are in place. More field data would be needed for monitoring and understanding the northward spreading of WNV in Europe.

Background Japanese encephalitis virus (JEV) is the principal cause of mosquito-borne encephalitis in human populations within Asia. If introduced into new geographic areas, it could have further implications for public and animal health. However, potential mosquito vectors for virus transmission have not been fully investigated. The Asian tiger mosquito, Aedes albopictus , has emerged in Europe and is now expanding its geographical range into more northerly latitudes. Culex quinquefasciatus , although absent from Europe, has been detected in Turkey, a country with territory in Europe, and could act as a vector for JEV in other regions. To assess the risk of these invasive species acting as vectors for JEV and therefore potentially contributing to its geographical expansion, we have investigated the vector competence of Ae. albopictus and Cx. quinquefasciatus . Methods Two colonised lines of Ae. albopictus (Italy and Spain) and a line of Cx. quinquefasciatus (Tanzania) were compared for susceptibility to infection by oral feeding with JEV strain SA-14, genotype III at 10 6  PFU/ml and maintained at 25 °C. Specimens were processed at 7 and 14 days post-inoculation (dpi). Rates of infection, dissemination and transmission were assessed through detection of viral RNA by real-time polymerase chain reaction (RT-PCR) in mosquito body, legs and saliva, respectively, at each time point. Where possible, infection and dissemination were confirmed by immunohistochemical (IHC) detection of the JEV envelope protein. Results Aedes albopictus from Italy showed no susceptibility to infection with JEV strain SA-14. Conversely, Ae. albopictus colonised in Spain was susceptible and 100% of infected mosquitoes that were subjected to saliva screening expressed viral RNA at 14 dpi. Culex quinquefasciatus was highly susceptible to infection as early as 7 dpi and 50% of infected mosquitoes that were subjected to saliva screening expressed viral RNA at 14 dpi. Infection and dissemination were confirmed in Cx. quinquefasciatus by IHC detection of JEV envelope protein in both the mid-gut and salivary glands. Conclusions Aedes albopictus from two different locations in Europe range from being susceptible to JEV and capable of transmission through to being resistant. Culex quinquefasciatus also appears highly susceptible; therefore, both species could potentially act as vectors for JEV and facilitate the emergence of JEV into new regions. Graphical Abstract

Background Usutu virus (USUV) has been detected annually in the southeast of England since 2020. USUV RNA has been identified in wild birds and mosquito populations, and exposure of captive birds to USUV has also been confirmed in the UK. Since its first detection in London, USUV’s distribution has expanded across the South East, highlighting necessity to understand USUV transmission dynamics in the UK. The primary vectors of USUV in the UK are likely Culex pipiens mosquitoes. Culex pipiens molestus is one biotype which shows no restriction in host preference and may play an important role in transmitting USUV from birds to humans. Methods A laboratory colony of Cx. pipiens molestus mosquitoes were orally infected with the London strain of USUV and incubated at 22 ℃, 20 ℃ and 18 ℃ for up to 28 days. Body samples and mosquito saliva samples were collected and analysed using a quantitative real-time reverse transcription PCR to determine infection and transmission potential, respectively. Results USUV RNA was detected in all sample times at all temperatures assessed, with the 22 ℃ showing the greatest proportion of saliva and body positive samples. At this temperature, there was also an eight-fold increase in the relative viral copy number in the mosquito bodies, which was unobserved at other experimental temperatures. When a more sensitive PCR assay was used at the lowest experimental temperature used (18 ℃), USUV RNA was present in the mosquito saliva and body samples for longer and showed a greater proportion of positive samples compared to 20 ℃. Conclusions This study has demonstrated that Cx. pipiens molestus may be able to transmit USUV at 22 ℃. Active replication of USUV was identified in the mosquito bodies at 22 ℃ but not at lower temperatures, suggesting that 20 ℃ to 22 ℃ may be an important threshold in USUV replication and transmission. Utilisation of a more sensitive assay for the lower experimental temperatures revealed that USUV was detectable at 18 ℃. Therefore, when conducting infection studies on temperate mosquito-borne viruses, it is important to consider assay sensitivity. Graphical Abstract

RNA interference (RNAi) is triggered in eukaryotic organisms by double-stranded RNA (dsRNA), and it destroys any mRNA that has sequence identity with the dsRNA trigger. The RNAi pathway in Anopheles gambiae can be silenced by transfecting cells with dsRNA derived from exon sequence of the A. gambiae Argonaute2 (AgAgo2) gene. We hypothesized that RNAi may also act as an antagonist to alphavirus replication in A. gambiae because RNA viruses form dsRNA during replication. Silencing AgAgo2 expression would make A. gambiae mosquitoes more permissive to virus infection. To determine whether RNAi conditions the vector competence of A. gambiae for O'nyong-nyong virus (ONNV), we engineered a genetically modified ONNV that expresses enhanced GFP (eGFP) as a marker. After intrathoracic injection, ONNV-eGFP slowly spread to other A. gambiae tissues over a 9-day incubation period. Mosquitoes were then coinjected with virus and either control beta-galactosidase dsRNA (ds(beta)gal; note that \"ds\" is used as a prefix to indicate the dsRNA derived from a given gene throughout) or ONNV dsnsP3. Treatment with dsnsP3 inhibited virus spread significantly, as determined by eGFP expression patterns. ONNV-eGFP titers from mosquitoes coinjected with dsnsP3 were significantly lower at 3 and 6 days after injection than in mosquitoes coinjected with ds(beta)gal. Mosquitoes were then coinjected with ONNV-eGFP and dsAgAgo2. Mosquitoes coinjected with virus and AgAgo2 dsRNA displayed widespread eGFP expression and virus titers 16-fold higher than ds(beta)gal controls after 3 or 6 days after injection. These observations provide direct evidence that RNAi is an antagonist of ONNV replication in A. gambiae, and they suggest that the innate immune response conditions vector competence.

Anopheles stephensi mosquitoes, efficient vectors in parts of Asia and Africa, were found in 75.3% of water sources surveyed and contributed to 80.9% of wild-caught Anopheles mosquitoes in Awash Sebat Kilo, Ethiopia. High susceptibility of these mosquitoes to Plasmodium falciparum and vivax infection presents a challenge for malaria control in the Horn of Africa.

Since its first report in Anopheles mosquitoes in 1950s, insecticide resistance has spread very fast to most sub-Saharan African malaria-endemic countries, where it is predicted to seriously jeopardize the success of vector control efforts, leading to rebound of disease cases. Supported mainly by four mechanisms (metabolic resistance, target site resistance, cuticular resistance, and behavioural resistance), this phenomenon is associated with intrinsic changes in the resistant insect vectors that could influence development of invading Plasmodium parasites. A literature review was undertaken using Pubmed database to collect articles evaluating directly or indiretly the impact of insecticide resistance and the associated mechanisms on key determinants of malaria vector competence including sialome composition, anti- Plasmodium immunity, intestinal commensal microbiota, and mosquito longevity. Globally, the evidence gathered is contradictory even though the insecticide resistant vectors seem to be more permissive to Plasmodium infections. The actual body of knowledge on key factors to vectorial competence, such as the immunity and microbiota communities of the insecticide resistant vector is still very insufficient to definitively infer on the epidemiological importance of these vectors against the susceptible counterparts. More studies are needed to fill important knowledge gaps that could help predicting malaria epidemiology in a context where the selection and spread of insecticide resistant vectors is ongoing.

Dengue is prevalent in tropical and subtropical regions. As an arbovirus disease, it is mainly transmitted by Aedes aegypti and Aedes albopictus . According to the previous studies, temperature is closely related to the survival of Aedes mosquitoes, the proliferation of dengue virus (DENV) and the vector competence of Aedes to transmit DENV. This review describes the correlations between temperature and dengue epidemics, and explores the potential reasons including the distribution and development of Aedes mosquitoes, the structure of DENV, and the vector competence of Aedes mosquitoes. In addition, the immune and metabolic mechanism are discussed on how temperature affects the vector competence of Aedes mosquitoes to transmit DENV.

Culex gelidus Theobald has emerged as a major vector of Japanese encephalitis virus (JEV) in India, Southeast Asian countries and Australia. The species has expanded its geographic distribution from the Indian subcontinent to Japan, China, other Southeast Asian countries, Island nations in Australasian region and Australia. In recent years, a sudden increase in its population especially in the urban and sub-urban areas has been observed in several countries, thus, becoming a dominant mosquito species. The virus has been repeatedly isolated from from different geographical locations making it one of the most important vectors of JEV. Apart from JEV, other viruses of public health importance, viz. Getah, Ross River (RRV), Sindbis and Tembusu have been isolated from the mosquito. Experimental studies have shown that the mosquito Cx. qelidus is highly competent to transmit West Nile, Kunjin and Murray valley encephalitis viruses with infection and transmission rates of >80 and >50%, respectively for each virus. The species is also found competent to transmit RRV, but at a lower rate. Experimental studies have shown that the species is susceptible to chikungunya, Chandipura and Chittoor (Batai) viruses. Development of resistance to DDT and malathion has also been detected in the species recently. The invasive nature, ability to breed both in fresh and dirty waters, development of resistance to insecticides, high anthropophily and its potential to transmit important human viruses pose an increased threat of viral encephalitis in India and Oriental region especially in the light of explosive increase in its population.

Culex quinquefasciatus is one of the principal vectors of West Nile virus (WNV). The mosquito also acts as a bridge vector as it feeds on both birds and humans. In the background of the recent reports of WNV activity in Kerala and Assam with fatalities, a study was initiated to determine the growth kinetics and transmission mechanisms of three strains of WNV in two populations of Cx. quinquefasciatus. Mosquitoes were infected by oral feeding and growth on different post-infection days was determined with the three strains. Horizontal transmission was determined by confirming sickness and mortality in infant mice after infected mosquito bite. F1 generation eggs, larvae, pupae and adults of experimentally infected mosquitoes were screened for WNV to determine vertical (transovarial) transmission. Trans-stadial transmission was determined by detecting WNV in adult mosquitoes emerged from infected larvae. Both the mosquito populations replicated and maintained WNV for a prolonged period with high titers (≥ 5log10 PFU/ml). WNV could be detected in saliva from Days 2 to 32 post-infection. Horizontal transmission by both the populations could be established but no vertical transmission was observed. However, parenterally infected larvae transmitted WNV to adults. WNV has been isolated from >10 mosquito species from India, however, vector competence of none of the species has been studied. The present study demonstrates efficient transmission of WNV by Cx. quinquefasciatus mosquitoes. With its country wide prevalence and high vector competence, the mosquitoes could create grave consequences especially when virulent strains with potential to cause acute flaccid paralysis and death are circulating.