Infectious blood meal experiments have been frequently performed with different virus-vector combinations to assess the transmission potential of arthropod-borne (arbo)viruses. A wide variety of host blood sources have been used to deliver arboviruses to their arthropod vectors in laboratory studies. The type of blood used during vector competence experiments does not always reflect the blood from the viremic vertebrate hosts in the field, but little is known about the effect of blood source on the experimental outcome of vector competence studies. Here we investigated the effect of avian versus human blood on the infection and transmission rates of the zoonotic Usutu virus (USUV) in its primary mosquito vector Culex pipiens.

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus of the genus Phlebovirus that is highly pathogenic to ruminants and humans. The disease is currently confined to Africa and the Arabian Peninsula, but globalization and climate change may facilitate introductions of the virus into currently unaffected areas via infected animals or mosquitoes. The consequences of such an introduction will depend on environmental factors, the availability of susceptible ruminants and the capacity of local mosquitoes to transmit the virus. We have previously demonstrated that lambs native to the Netherlands are highly susceptible to RVFV and we here report the vector competence of Culex (Cx.) pipiens, the most abundant and widespread mosquito species in the country. Vector competence was first determined after artificial blood feeding of laboratory-reared mosquitoes using the attenuated Clone 13 strain. Subsequently, experiments with wild-type RVFV and mosquitoes hatched from field-collected eggs were performed. Finally, the transmission of RVFV from viremic lambs to mosquitoes was studied.

Zika virus (ZIKV) is a mosquito-borne pathogen that caused a large outbreak in the Americas in 2015 and 2016. The virus is currently present in tropical areas around the globe and can cause severe disease in humans, including Guillain-Barré syndrome and congenital microcephaly. The tropical yellow fever mosquito, Aedes aegypti, is the main vector in the urban transmission cycles of ZIKV. The discovery of ZIKV in wild-caught Culex mosquitoes and the ability of Culex quinquefasciatus mosquitoes to transmit ZIKV in the laboratory raised the question of whether the common house mosquito Culex pipiens, which is abundantly present in temperate regions in North America, Asia and Europe, could also be involved in ZIKV transmission. In this study, we investigated the vector competence of Cx. pipiens (biotypes molestus and pipiens) from the Netherlands for ZIKV, using Usutu virus as a control. After an infectious blood meal containing ZIKV, none of the tested mosquitoes accumulated ZIKV in the saliva, although 2% of the Cx. pipiens pipiens mosquitoes showed ZIKV-positive bodies. To test the barrier function of the mosquito midgut on virus transmission, ZIKV was forced into Cx. pipiens mosquitoes by intrathoracic injection, resulting in 74% (molestus) and 78% (pipiens) ZIKV-positive bodies. Strikingly, 14% (molestus) and 7% (pipiens) of the tested mosquitoes accumulated ZIKV in the saliva after injection. This is the first demonstration of ZIKV accumulation in the saliva of Cx. pipiens upon forced infection. Nevertheless, a strong midgut barrier restricted virus dissemination in the mosquito after oral exposure and we, therefore, consider Cx. pipiens as a highly inefficient vector for ZIKV.

Usutu virus (USUV) is a mosquito-borne flavivirus of African origin. Over the past decades, USUV has spread through Europe causing mass die-offs among multiple bird species. The natural transmission cycle of USUV involves Culex spp. mosquitoes as vectors and birds as amplifying hosts. Next to birds and mosquitoes, USUV has also been isolated from multiple mammalian species, including humans, which are considered dead-end hosts. USUV isolates are phylogenetically classified into an African and European branch, subdivided into eight genetic lineages (Africa 1, 2, and 3 and Europe 1, 2, 3, 4, and 5 lineages). Currently, multiple African and European lineages are co-circulating in Europe. Despite increased knowledge of the epidemiology and pathogenicity of the different lineages, the effects of co-infection and transmission efficacy of the co-circulating USUV strains remain unclear. In this study, we report a comparative study between two USUV isolates as follows: a Dutch isolate (USUV-NL, Africa lineage 3) and an Italian isolate (USUV-IT, Europe lineage 2). Upon co-infection, USUV-NL was consistently outcompeted by USUV-IT in mosquito, mammalian, and avian cell lines. In mosquito cells, the fitness advantage of USUV-IT was most prominently observed in comparison to the mammalian or avian cell lines. When Culex pipiens mosquitoes were orally infected with the different isolates, no overall differences in vector competence for USUV-IT and USUV-NL were observed. However, during the in vivo co-infection assay, it was observed that USUV-NL infectivity and transmission were negatively affected by USUV-IT but not vice versa.

Originating from Africa, Usutu virus (USUV) first emerged in Europe in 2001. This mosquito-borne flavivirus caused high mortality rates in its bird reservoirs, which strongly resembled the introduction of West Nile virus (WNV) in 1999 in the United States. Mosquitoes infected with USUV incidentally transmit the virus to other vertebrates, including humans, which can result in neuroinvasive disease. USUV and WNV co-circulate in parts of southern Europe, but the distribution of USUV extends into central and northwestern Europe. In the field, both viruses have been detected in the northern house mosquito Culex pipiens, of which the potential for USUV transmission is unknown. To understand the transmission dynamics and assess the potential spread of USUV, we determined the vector competence of C. pipiens for USUV and compared it with the well characterized WNV. We show for the first time that northwestern European mosquitoes are highly effective vectors for USUV, with infection rates of 11% at 18 °C and 53% at 23 °C, which are comparable with values obtained for WNV. Interestingly, at a high temperature of 28 °C, mosquitoes became more effectively infected with USUV (90%) than with WNV (58%), which could be attributed to barriers in the mosquito midgut. Small RNA deep sequencing of infected mosquitoes showed for both viruses a strong bias for 21-nucleotide small interfering (si)RNAs, which map across the entire viral genome both on the sense and antisense strand. No evidence for viral PIWI-associated RNA (piRNA) was found, suggesting that the siRNA pathway is the major small RNA pathway that targets USUV and WNV infection in C. pipiens mosquitoes.

Usutu virus (USUV) and West Nile virus (WNV) are closely related mosquito-borne flaviviruses that are mainly transmitted between bird hosts by vector mosquitoes. Infections in humans are incidental but can cause severe disease. USUV is endemic in large parts of Europe, while WNV mainly circulates in Southern Europe. In recent years, WNV is also frequently detected in Northern Europe, thereby expanding the area where both viruses co-circulate. However, it remains unclear how USUV may affect the future spread of WNV and the likelihood of human co-infection. Here we investigated whether co-infections with both viruses in cell lines and their primary mosquito vector, Culex pipiens, affect virus replication and transmission dynamics. We show that USUV is outcompeted by WNV in mammalian, avian and mosquito cells during co-infection. Mosquitoes that were exposed to both viruses simultaneously via infectious blood meal displayed significantly reduced USUV transmission compared to mosquitoes that were only exposed to USUV (from 15% to 3%), while the infection and transmission of WNV was unaffected. In contrast, when mosquitoes were pre-infected with USUV via infectious blood meal, WNV transmission was significantly reduced (from 44% to 17%). Injection experiments established the involvement of the midgut in the observed USUV-mediated WNV inhibition. The competition between USUV and WNV during co-infection clearly indicates that the chance of concurrent USUV and WNV transmission via a single mosquito bite is low. The competitive relation between USUV and WNV may impact virus transmission dynamics in the field and affect the epidemiology of WNV in Europe.

Small RNA mediated responses are essential for antiviral defence in mosquitoes, however, they appear to differ per virus-vector combination. To further investigate the diversity of small RNA responses against viruses in mosquitoes, we applied a small RNA deep sequencing approach on five mosquito cell lines: Culex tarsalis CT cells, Aedes albopictus U4.4 and C6/36 cells, Ae. aegypti Aag2 cells (cleared from cell fusing agent virus and Culex Y virus (CYV) by repetitive dsRNA transfections) and Ae. pseudoscutellaris AP-61 cells. De novo assembly of small RNAs revealed the presence of Phasi Charoen-like virus (PCLV), Calbertado virus, Flock House virus and a novel narnavirus in CT cells, CYV in U4.4 cells, and PCLV in Aag2 cells, whereas no insect-specific viruses (ISVs) were detected in C6/36 and AP-61 cells. Next, we investigated the small RNA responses to the identified ISVs and to acute infection with the arthropod-borne West Nile virus (WNV). We demonstrate that AP-61 and C6/36 cells do not produce siRNAs to WNV infection, suggesting that AP-61, like C6/36, are Dicer-2 deficient. CT cells produced a strong siRNA response to the persistent ISVs and acute WNV infection. Interestingly, CT cells also produced viral PIWI-interacting (pi)RNAs to PCLV, but not to WNV or any of the other ISVs. In contrast, in U4.4 and Aag2 cells, WNV siRNAs, and pi-like RNAs without typical ping-pong piRNA signature were observed, while this signature was present in PCLV piRNAs in Aag2 cells. Together, our results demonstrate that mosquito small RNA responses are strongly dependent on both the mosquito cell type and/or the mosquito species and family of the infecting virus.

Outbreaks of West Nile virus (WNV) have not occurred in northern Europe despite nearby circulation of WNV in the southern part of the continent. The main vector for WNV, the mosquito Culex (Cx.) pipiens, consists of two behaviorally distinct biotypes, pipiens and molestus, which can form hybrids. Although temperature has been shown to influence vector competence of Cx. pipiens for WNV and biotypes are differentially susceptible towards infection, the interaction between the two has not been elucidated.

RNA interference (RNAi) is a crucial antiviral defense mechanism in insects, including the major mosquito species that transmit important human viruses. To counteract the potent antiviral RNAi pathway, insect viruses encode RNAi suppressors. However, whether mosquito-specific viruses suppress RNAi remains unclear. We therefore set out to study RNAi suppression by Culex Y virus (CYV), a mosquito-specific virus of the Birnaviridae family that was recently isolated from Culex pipiens mosquitoes. We found that the Culex RNAi machinery processes CYV double-stranded RNA (dsRNA) into viral small interfering RNAs (vsiRNAs). Furthermore, we show that RNAi is suppressed in CYV-infected cells and that the viral VP3 protein is responsible for RNAi antagonism. We demonstrate that VP3 can functionally replace B2, the well-characterized RNAi suppressor of Flock House virus. VP3 was found to bind long dsRNA as well as siRNAs and interfered with Dicer-2-mediated cleavage of long dsRNA into siRNAs. Slicing of target RNAs by pre-assembled RNA-induced silencing complexes was not affected by VP3. Finally, we show that the RNAi-suppressive activity of VP3 is conserved in Drosophila X virus, a birnavirus that persistently infects Drosophila cell cultures. Together, our data indicate that mosquito-specific viruses may encode RNAi antagonists to suppress antiviral RNAi.

West Nile virus (WNV) is a highly pathogenic flavivirus transmitted by Culex spp. mosquitoes. In North America (NA), lineage 1 WNV caused the largest outbreak of neuroinvasive disease to date, while a novel pathogenic lineage 2 strain circulates in southern Europe. To estimate WNV lineage 2 epidemic potential it is paramount to know if mosquitoes from currently WNV-free areas can support further spread of this epidemic.

First identified in 1947, Zika virus took roughly 70 years to cause a pandemic unusually associated with virus-induced brain damage in newborns. Zika virus is transmitted by mosquitoes, mainly Aedes aegypti, and secondarily, Aedes albopictus, both colonizing a large strip encompassing tropical and temperate regions. As part of the international project ZIKAlliance initiated in 2016, 50 mosquito populations from six species collected in 12 countries were experimentally infected with different Zika viruses. Here, we show that Ae. aegypti is mainly responsible for Zika virus transmission having the highest susceptibility to viral infections. Other species play a secondary role in transmission while Culex mosquitoes are largely non-susceptible. Zika strain is expected to significantly modulate transmission efficiency with African strains being more likely to cause an outbreak. As the distribution of Ae. aegypti will doubtless expand with climate change and without new marketed vaccines, all the ingredients are in place to relive a new pandemic of Zika.

Chikungunya virus (CHIKV) is an arthritogenic alphavirus (family Togaviridae), transmitted by Aedes species mosquitoes. CHIKV re-emerged in 2004 with multiple outbreaks worldwide and recently reached the Americas where it has infected over a million individuals in a rapidly expanding epidemic. While alphavirus replication is well understood in general, the specific function (s) of non-structural protein nsP3 remain elusive. CHIKV nsP3 modulates the mammalian stress response by preventing stress granule formation through sequestration of G3BP. In mosquitoes, nsP3 is a determinant of vector specificity, but its functional interaction with mosquito proteins is unclear.