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Background: In recent years the Asian bush mosquito Aedes japonicus has invaded Europe, including the Netherlands. This species is a known vector for a range of arboviruses, possibly including West Nile virus (WNV). As WNV emerged in the Netherlands in 2020, it is important to investigate the vectorial capacity of mosquito species present in the Netherlands to estimate the risk of future outbreaks and further spread of the virus. Therefore, this study evaluates the potential role of Ae. japonicus in WNV transmission and spillover from birds to dead-end hosts in the Netherlands., Methods: We conducted human landing collections in allotment gardens (Lelystad, the Netherlands) in June, August and September 2021 to study the diurnal and seasonal host-seeking behaviour of Ae. japonicus. Furthermore, their host preference in relation to birds using live chicken-baited traps was investigated. Vector competence of field-collected Ae. japonicus mosquitoes for two isolates of WNV at two different temperatures was determined. Based on the data generated from these studies, we developed a Susceptible-Exposed-Infectious-Recovered (SEIR) model to calculate the risk of WNV spillover from birds to humans via Ae. japonicus, under the condition that the virus is introduced and circulates in an enzootic cycle in a given area., Results: Our results show that Ae. japonicus mosquitoes are actively host seeking throughout the day, with peaks in activity in the morning and evening. Their abundance in August was higher than in June and September. For the host-preference experiment, we documented a small number of mosquitoes feeding on birds: only six blood-fed females were caught over 4 full days of sampling. Finally, our vector competence experiments with Ae. japonicus compared to its natural vector Culex pipiens showed a higher infection and transmission rate when infected with a local, Dutch, WNV isolate compared to a Greek isolate of the virus. Interestingly, we also found a small number of infected Cx. pipiens males with virus-positive leg and saliva samples., Conclusions: Combining the field and laboratory derived data, our model predicts that Ae. japonicus could act as a spillover vector for WNV and could be responsible for a high initial invasion risk of WNV when present in large numbers., (© 2024. The Author(s).)

Background: 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., Methods: Cx. pipiens biotypes (pipiens and molestus) were orally infected with USUV through infectious blood meals containing either chicken or human whole blood. The USUV infection and transmission rates were determined by checking mosquito bodies and saliva for USUV presence after 14 days of incubation at 28 °C. In addition, viral titers were determined for USUV-positive mosquito bodies and saliva., Results: Human and chicken blood lead to similar USUV transmission rates for Cx. pipiens biotype pipiens (18% and 15%, respectively), while human blood moderately but not significantly increased the transmission rate (30%) compared to chicken blood (17%) for biotype molestus. USUV infection rates with human blood were consistently higher in both Cx. pipiens biotypes compared to chicken blood. In virus-positive mosquitoes, USUV body and saliva titers did not differ between mosquitoes taking either human or chicken blood. Importantly, biotype molestus had much lower USUV saliva titers compared to biotype pipiens, regardless of which blood was offered., Conclusions: Infection of mosquitoes with human blood led to higher USUV infection rates as compared to chicken blood. However, the blood source had no effect on the vector competence for USUV. Interestingly, biotype molestus is less likely to transmit USUV compared to biotype pipiens due to very low virus titers in the saliva.

Background: 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., Methods: We determined vector competence of the Cx. pipiens biotypes and hybrids, after 14 days of incubation at 18, 23 and 28 °C. Mosquitoes were orally infected by providing an infectious blood meal or by injecting WNV directly in the thorax. Infection and transmission rates were determined by testing the bodies and saliva for WNV presence. In addition, titers of mosquitoes with WNV-positive bodies and saliva samples were determined., Results: Orally infected biotype pipiens and hybrids showed significantly increased transmission rates with higher temperatures, up to 32 and 14 %, respectively. In contrast, the molestus biotype had an overall transmission rate of 10 %, which did not increase with temperature. All mosquitoes that were infected via WNV injections had (close to) 100 % infection and transmission rates, suggesting an important role of the mosquito midgut barrier. We found no effect of increasing temperature on viral titers., Conclusions: Temperature differentially affected vector competence of the Cx. pipiens biotypes. This shows the importance of accounting for biotype-by-temperature interactions, which influence the outcomes of vector competence studies. Vector competence studies with Cx. pipiens mosquitoes differentiated to the biotype level are essential for proper WNV risk assessments.

Background: 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., Methods: In this research we studied the domains required for localization of CHIKV nsP3 in insect cells and demonstrated its molecular interaction with Rasputin (Rin), the mosquito homologue of G3BP. The biological involvement of Rin in CHIKV infection was investigated in live Ae. albopictus mosquitoes., Results: In insect cells, nsP3 localized as cytoplasmic granules, which was dependent on the central domain and the C-terminal variable region but independent of the N-terminal macrodomain. Ae. albopictus Rin displayed a diffuse, cytoplasmic localization, but was effectively sequestered into nsP3-granules upon nsP3 co-expression. Site-directed mutagenesis showed that the Rin-nsP3 interaction involved the NTF2-like domain of Rin and two conserved TFGD repeats in the C-terminal variable domain of nsP3. Although in vitro silencing of Rin did not impact nsP3 localization or CHIKV replication in cell culture, Rin depletion in vivo significantly decreased the CHIKV infection rate and transmissibility in Ae.albopictus., Conclusions: We identified the nsP3 hypervariable C-terminal domain as a critical factor for granular localization and sequestration of mosquito Rin. Our study offers novel insight into a conserved virus-mosquito interaction at the molecular level, and reveals a strong proviral role for G3BP homologue Rin in live mosquitoes, making the nsP3-Rin interaction a putative target to interfere with the CHIKV transmission cycle.

Background: Chikungunya virus (CHIKV) is a mosquito-borne, arthrogenic Alphavirus that causes large epidemics in Africa, South-East Asia and India. Recently, CHIKV has been transmitted to humans in Southern Europe by invading and now established Asian tiger mosquitoes. To study the processing of envelope proteins E1 and E2 and to develop a CHIKV subunit vaccine, C-terminally his-tagged E1 and E2 envelope glycoproteins were produced at high levels in insect cells with baculovirus vectors using their native signal peptides located in CHIKV 6K and E3, respectively., Results: Expression in the presence of either tunicamycin or furin inhibitor showed that a substantial portion of recombinant intracellular E1 and precursor E3E2 was glycosylated, but that a smaller fraction of E3E2 was processed by furin into mature E3 and E2. Deletion of the C-terminal transmembrane domains of E1 and E2 enabled secretion of furin-cleaved, fully processed E1 and E2 subunits, which could then be efficiently purified from cell culture fluid via metal affinity chromatography. Confocal laser scanning microscopy on living baculovirus-infected Sf21 cells revealed that full-length E1 and E2 translocated to the plasma membrane, suggesting similar posttranslational processing of E1 and E2, as in a natural CHIKV infection. Baculovirus-directed expression of E1 displayed fusogenic activity as concluded from syncytia formation. CHIKV-E2 was able to induce neutralizing antibodies in rabbits., Conclusions: Chikungunya virus glycoproteins could be functionally expressed at high levels in insect cells and are properly glycosylated and cleaved by furin. The ability of purified, secreted CHIKV-E2 to induce neutralizing antibodies in rabbits underscores the potential use of E2 in a subunit vaccine to prevent CHIKV infections.