Rift Valley fever is a mosquito-borne zoonotic disease that affects domestic ruminants and humans. Culex flavivirus is an insect-specific flavivirus that naturally exists in field mosquito populations. The influence of Culex flavivirus on Rift Valley fever phlebovirus (RVFV) vector competence of Culex pipiens has not been investigated. Culex flavivirus infection in a Cx. pipiens colony was studied by Culex flavivirus oral feeding and intrathoracical inoculation. Similarly, vector competence of Cx. pipiens infected with Culex flavivirus was evaluated for RVFV. Infection, dissemination, transmission rates and transmission efficiency of Culex flavivirus-infected and non-infected Cx. pipiens artificially fed with RVFV infected blood were assessed. Culex flavivirus was able to infect Cx. pipiens after intrathoracically inoculation in Cx. pipiens mosquitos but not after Culex flavivirus oral feeding. Culex flavivirus did not affect RVFV infection, dissemination and transmission in Cx. pipiens mosquitoes. RVFV could be detected from saliva of both the Culex flavivirus-positive and negative Cx. pipiens females without significant differences. Moreover, RVFV did not interfere with the Culex flavivirus infection in Cx. pipiens mosquitoes. Culex flavivirus infected and non-infected Cx. pipiens transmit RVFV. Culex flavivirus existing in field-collected Cx. pipiens populations does not affect their vector competence for RVFV. Culex flavivirus may not be an efficient tool for RVFV control in mosquitoes.

Background: Aedes vexans(Meigen) is considered a nuisance species in central Europe and the Mediterranean region. It is an anthropophilic and mammalophilic floodwater mosquito involved in the transmission of several arboviruses. Rift Valley fever (RVF) is a relevant mosquito-borne zoonosis, affecting mainly humans and ruminants, that causes severe impact in public health and economic loses. Due to globalization and climate change, the European continent is threatened by its introduction. The main purpose of the present study was to evaluate the vector competence of a European field-collectedAe. vexanspopulation.Methods: Aedes vexansfield-collected larvae were reared in the laboratory under field-simulated conditions. To assess the vector competence for Rift Valley fever phlebovirus (RVFV) transmission, adult F0 females were exposed to infectious blood meals containing the 56/74 RVFV strain. Additionally, intrathoracic inoculations with the same virus strain were performed to evaluate the relevance of the salivary gland barriers. Natural circulation of alphavirus, flavivirus and phlebovirus was also tested.Results: To our knowledge, an autochthonousAe. vexanspopulation was experimentally confirmed as a competent vector for RVFV for the first time. This virus was capable of infecting and disseminating within the studiedAe. vexansmosquitoes. Moreover, infectious virus was isolated from the saliva of disseminated specimens, showing their capacity to transmit the virus. Additionally, a natural infection with a circulating Mosquito flavivirus was detected. The co-infection with the Mosquito flavivirus seemed to modulate RVFV infection susceptibility in field-collectedAe. vexans, but further studies are needed to confirm its potential interference in RVFV transmission.Conclusions: Our results show that field-collected EuropeanAe. vexanswould be able to transmit RVFV in case of introduction into the continent. This should be taken into consideration in the design of surveillance and control programmes.

Worldwide, emerging and re-emerging infectious diseases (EIDs) are a major burden on public and animal health. Arthropod vectors, with mosquitoes being the main contributors of global disease, transmit more than 70% of the recognized EIDs. To assess new alternatives for arthropod-borne viral diseases surveillance, and for the detection of new viruses, honey-baited Flinders Technology Associates (FTA) cards were used as sugar bait in mosquito traps during entomological surveys at the Llobregat River Delta (Catalonia, Spain). Next generation sequencing (NGS) metagenomics analysis was applied on honey-baited FTA cards, which had been exposed to field-captured mosquitoes to characterize their associated virome. Arthropod- and plant-infecting viruses governed the virome profile on FTA cards. Twelve near-complete viral genomes were successfully obtained, suggesting good quality preservation of viral RNAs. Mosquito pools linked to the FTA cards were screened for the detection of mosquito-associated viruses by specific RT-PCRs to confirm the presence of these viruses. The circulation of viruses related to Alphamesonivirus, Quaranjavirus and unclassified Bunyavirales was detected in mosquitoes, and phylogenetic analyses revealed their similarities to viruses previously reported in other continents. To the best our knowledge, our findings constitute the first distribution record of these viruses in European mosquitoes and the first hint of insect-specific viruses in mosquitoes’ saliva in field conditions, demonstrating the feasibility of this approach to monitor the transmissible fraction of the mosquitoes’ virome. In conclusion, this pilot viromics study on honey-baited FTA cards was shown to be a valid approach for the detection of viruses circulating in mosquitoes, thereby setting up an alternative tool for arbovirus surveillance and control programs.

Background: Historically,Anopheles atroparvushas been considered one of the most important malaria vectors in Europe. Since malaria was eradicated from the European continent, the interest in studying its vectors reduced significantly. Currently, to better assess the potential risk of malaria resurgence on the continent, there is a growing need to update the data on susceptibility of indigenousAnophelespopulations to importedPlasmodiumspecies. In order to do this, as a first step, an adequate laboratory colony ofAn. atroparvusis needed.Methods: Anopheles atroparvusmosquitoes were captured in rice fields from the Ebro Delta (Spain). Field-caught specimens were maintained in the laboratory under simulated field-summer conditions. Adult females were artificially blood-fed on fresh whole rabbit blood for oviposition. First- to fourth-instar larvae were fed on pulverized fish and turtle food. Adults were maintained with a 10% sucrose solutionad libitum.Results: AnAn. atroparvuspopulation from the Ebro Delta was successfully established in the laboratory. During the colonization process, feeding and hatching rates increased, while a reduction in larval mortality rate was observed.Conclusions: The present study provides a detailed rearing and maintenance protocol forAn. atroparvusand a publicly available reference mosquito strain within the INFRAVEC2 project for further research studies involving vector-parasite interactions.

Aedes caspius (Pallas, 1771) is a floodwater mosquito species widely distributed in the Western Palaearctic. As an anthropophilic species, its role as an arbovirus vector may be the key for understanding the transmission cycle of certain diseases in Europe such as Zika virus (ZIKV). Concerning vector competence for ZIKV, studies related to Ae. caspius are still scarce. ZIKV is an arbovirus that has provoked a widespread epidemic in the Pacific region (2007–2013) and in the Americas (2015–2016). ZIKV is associated with serious neurological injuries (e.g. microcephaly) and Guillain-Barré syndrome. Due to the ZIKV epidemics in the American continent, some viraemic travellers coming from endemic countries have been reported in Europe. More knowledge is therefore required to define the susceptibility of autochthonous mosquito species such as Ae. caspius for ZIKV in order to improve arbovirus surveillance and control programmes. In the present study, the vector competence of a European population of Ae. caspius was evaluated for two ZIKV lineages, the Suriname ZIKV strain (Asian lineage) and the MR766 ZIKV strain (African I lineage). Females were tested at 7, 14 and 21 days post-exposure (dpe) to infectious blood meals. An Ae. aegypti PAEA strain was used as a positive control. Aedes caspius presented low susceptibility to ZIKV infection and the virus was only detected by RT-qPCR in body samples. Low viral loads were detected for the MR766 strain at 7 dpe and for the Suriname strain at 14 and 21 dpe. Aedes caspius was unable to produce a disseminated infection and virus transmission at any of the tested time points. Using Ae. aegypti PAEA strain, infection, dissemination and transmission rates were calculated for the Suriname ZIKV strain (Asian lineage) at each time point. For the MR766 ZIKV strain (African I lineage), while only infection rates were estimated at each time point, no dissemination or transmission were detected in either species. The results of the present study reveal that the tested Ae. caspius population has a strong midgut escape barrier that limits the dissemination or transmission of the virus. As such, it seems unlikely that European Ae. caspius mosquitoes could be involved in ZIKV transmission if ZIKV was introduced into Europe. This information may help in designing a better strategy to European surveillance and control programmes for ZIKV.

Even though malaria was eradicated from Europe after the mid-20th century, in 2017, more than 8000 imported cases were reported in the continent. Due to travel routes to endemic areas, climate change, and the presence of native vector mosquitoes (genus Anopheles), the re-establishment of autochthonous malaria transmission is a current concern. Anopheles atroparvus (Van Thiel, 1972) is one of the 11 sibling species within the Palearctic Anopheles maculipennis complex, which formerly were considered the main vectors of the disease in the European continent. The microbiota (bacteria and viruses) of vector species has been demonstrated to play a significant role in the biology of these organisms, including their infection susceptibility and their capacity to transmit disease-causing agents. Recently, with the improvement of metagenomics techniques, several viruses that naturally infect vector mosquitoes have been identified. The purpose of the present study was to characterize, for the first time, the virome present in An. atroparvus from the Ebro Delta and assess its evolution after ten generations in the laboratory. Small RNA sequencing was used to characterize the virome from wild-caught An. atroparvus females and from the tenth generation produced under controlled laboratory conditions. Through this approach, we were able to identify viral linages previously reported in other invertebrates, such as Chaq virus and several Partiti-like viruses. A reduction in the viral composition was observed during the colonization process. The present study contributes to the understanding of the viral diversity of a medically relevant vector species in its natural setting and under confinement, and sets a baseline for further studies to assess the potential implications of these viruses in the transmission of pathogens.