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Mosquito-specific viruses (MSVs) are a subset of insect-specific viruses that are found to infect mosquitoes or mosquito derived cells. There has been an increase in discoveries of novel MSVs in recent years. This has expanded our understanding of viral diversity and evolution but has also sparked questions concerning the transmission of these viruses and interactions with their hosts and its microbiome. In fact, there is already evidence that MSVs interact with the immune system of their host. This is especially interesting, since mosquitoes can be infected with both MSVs and arthropod-borne (arbo) viruses of public health concern. In this review, we give an update on the different MSVs discovered so far and describe current data on their transmission and interaction with the mosquito immune system as well as the effect MSVs could have on an arboviruses-co-infection. Lastly, we discuss potential uses of these viruses, including vector and transmission control.

Mosquitoes not only transmit human and veterinary pathogens called arboviruses (arthropod-borne viruses) but also harbor mosquito-associated insect-specific viruses (mosquito viruses) that cannot infect vertebrates. In the past, studies investigating mosquito viruses mainly focused on highly pathogenic interactions that were easier to detect than those without visible symptoms. However, the recent advances in viral metagenomics have highlighted the abundance and diversity of viruses which do not generate mass mortality in host populations. Over the last decade, this has facilitated the rapid growth of virus discovery in mosquitoes. The circumstances around the discovery of mosquito viruses greatly affected how they have been studied so far. While earlier research mainly focused on the pathogenesis caused by DNA and some double-stranded RNA viruses during larval stages, more recently discovered single-stranded RNA mosquito viruses were heavily studied for their putative interference with arboviruses in female adults. Thus, many aspects of mosquito virus interactions with their hosts and host-microbiota are still unknown. In this context, considering mosquito viruses as endosymbionts can help to identify novel research areas, in particular in relation to their long-term interactions with their hosts (e.g. relationships during all life stages, the stability of the associations at evolutionary scales, transmission routes and virulence evolution) and the possible context-dependent range of interactions (i.e. beneficial to antagonistic). Here, we review the symbiotic interactions of mosquito viruses considering different aspects of their ecology, such as transmission, host specificity, host immune system and interactions with other symbionts within the host cellular arena. Finally, we highlight related research gaps in mosquito virus research.

Culex pipiens mosquitoes are infected with w Pip. These endosymbionts induce a conditional sterility called CI resulting from embryonic deaths, which constitutes a cornerstone for Wolbachia antivectorial methods. In arthropods, Wolbachia endosymbionts induce conditional sterility, called cytoplasmic incompatibility (CI), resulting from embryonic lethality. CI penetrance (i.e., embryonic death rate) varies depending on host species and Wolbachia strains involved. All Culex pipiens mosquitoes are infected by the endosymbiotic alphaproteobacteria Wolbachia w Pip. CI in Culex , characterized as a binary “compatible/incompatible” phenomenon, revealed an unparalleled diversity of patterns linked to the amplification-diversification of cidA and cidB genes. Here, we accurately studied CI penetrance variations in the light of cid genes divergence by generating a C. pipiens compatibility matrix between 11 lines hosting different phylogenetic w Pip groups and exhibiting distinct cid gene repertoires. We showed, as expected, that crosses involving w Pip from the same group were mostly compatible. In contrast, only 22% of the crosses involving different w Pip groups were compatible, while 54% were fully incompatible. For the remaining 24% of the crosses, “intermediate” compatibilities were reported, and a cytological observation of the first zygotic division confirmed the occurrence of “canonical” CI phenotypes in a fraction of the eggs. Backcross experiments demonstrated that intermediate compatibilities were not linked to host genetic background but to the Wolbachia strains involved. This previously unstudied intermediate penetrance CI was more severe and frequent in crosses involving w Pip-IV strains exhibiting cid variants markedly divergent from other w Pip groups. Our data demonstrate that CI is not always a binary compatible/incompatible phenomenon in C. pipiens but that intermediate compatibilities putatively resulting from partial mismatch due to Cid proteins divergence exist in this species complex. IMPORTANCE Culex pipiens mosquitoes are infected with w Pip. These endosymbionts induce a conditional sterility called CI resulting from embryonic deaths, which constitutes a cornerstone for Wolbachia antivectorial methods. Recent studies revealed that (i) two genes, cidA and cidB , are central in Wolbachia -CI mechanisms, and (ii) compatibility versus incompatibility between mosquito lines depends on the w Pip phylogenetic groups at play. Here, we studied CI variations in relation to w Pip groups and cid genes divergence. We showed, as expected, that the crosses involving w Pip from the same group were compatible. In contrast, 78% of the crosses involving different w Pip groups were partially or fully incompatible. In such crosses, we reported defects during the first zygotic division, a hallmark of CI. We showed that CI was more severe and frequent in crosses involving w Pip-IV strains exhibiting cid variants, which markedly diverge from those of other w Pip groups.

Culex spp. mosquitoes are important vectors of viruses, such as West Nile virus, Eastern equine encephalitis virus and Rift valley fever virus. However, their interactions with innate antiviral immunity, especially RNA interference (RNAi), are not well known. Most research on RNAi pathways in mosquitoes is focused on the tropical vector mosquito Aedes aegypti. Here, we investigated the production of arbovirus-specific small RNAs in Cx. quinquefasciatus-derived HSU cells. Furthermore, by silencing RNAi-related proteins, we investigated the antiviral role of these proteins for two different arboviruses: Semliki Forest virus (SFV) and Bunyamwera orthobunyavirus (BUNV). Our results showed an expansion of Ago2 and Piwi6 in Cx. quinquefasciatus compared to Ae. aegypti. While silencing Ago2a and Ago2b increased BUNV replication, only Ago2b showed antiviral activity against SFV. Our results suggest differences in the function of Cx. quinquefasciatus and Ae. aegypti RNAi proteins and highlight the virus-specific function of these proteins in Cx. quinquefasciatus.

Background Wolbachia are maternally transmitted bacteria that can manipulate their hosts’ reproduction causing cytoplasmic incompatibility (CI). CI is a sperm-egg incompatibility resulting in embryonic death. Due to this sterilising effect on mosquitoes, Wolbachia are considered for vector control strategies. Important vectors for arboviruses, filarial nematodes and avian malaria, mosquitoes of Culex pipiens complex are suitable for Wolbachia- based vector control. They are infected with Wolbachia w Pip strains belonging to five genetically distinct groups ( w Pip-I to V) within the Wolbachia B supergroup. CI properties of w Pip strongly correlate with this genetic diversity: mosquitoes infected with w Pip strains from a different w Pip group are more likely to be incompatible with each other. Turkey is a critical spot for vector-borne diseases due to its unique geographical position as a natural bridge between Asia, Europe and Africa. However, general w Pip diversity, distribution and CI patterns in natural Cx. pipiens ( s . l .) populations in the region are unknown. In this study, we first identified w Pip diversity in Turkish Cx. pipiens ( s . l .) populations , by assigning them to one of the five groups within w Pip ( w Pip-Ito V). We further investigated CI properties between different w Pip strains from this region. Results We showed a w Pip fixation in Cx. pipiens ( s . l .) populations in Turkey by analysing 753 samples from 59 sampling sites. Three w Pip groups were detected in the region: w Pip-I , w Pip-II and w Pip-IV. The most dominant group was w Pip-II. While w Pip-IV was restricted to only two locations, w Pip-I and w Pip-II had wider distributions. Individuals infected with w Pip-II were found co-existing with individuals infected with w Pip-I or w Pip-IV in some sampling sites. Two mosquito isofemale lines harbouring either a w Pip-I or a w Pip-II strain were established from a population in northwestern Turkey. Reciprocal crosses between these lines showed that they were fully compatible with each other but bidirectionally incompatible with w Pip-IV Istanbul infected line. Conclusion Our findings reveal a high diversity of w Pip and CI properties in Cx. pipiens ( s . l .) populations in Turkey. Knowledge on naturally occurring CI patterns caused by w Pip diversity in Turkey might be useful for Cx. pipiens ( s . l .) control in the region.

Since its detection in 2015 in Brazil, Zika virus (ZIKV) has remained in the spotlight of international public health and research as an emerging arboviral pathogen. In addition to single infection, ZIKV may occur in co-infection with dengue (DENV) and chikungunya (CHIKV) viruses, with whom ZIKV shares geographic distribution and the mosquito Aedes aegypti as a vector. The main mosquito immune response against arboviruses is RNA interference (RNAi). It is unknown whether or not the dynamics of the RNAi response differ between single arboviral infections and co-infections. In this study, we investigated the interaction of ZIKV and DENV, as well as ZIKV and CHIKV co-infections with the RNAi response in Ae . aegypti . Using small RNA sequencing, we found that the efficiency of small RNA production against ZIKV -a hallmark of antiviral RNAi—was mostly similar when comparing single and co-infections with either DENV or CHIKV. Silencing of key antiviral RNAi proteins, showed no change in effect on ZIKV replication when the cell is co-infected with ZIKV and DENV or CHIKV. Interestingly, we observed a negative effect on ZIKV replication during CHIKV co-infection in the context of Ago2-knockout cells, though his effect was absent during DENV co-infection. Overall, this study provides evidence that ZIKV single or co-infections with CHIKV or DENV are equally controlled by RNAi responses. Thus, Ae . aegypti mosquitoes and derived cells support co-infections of ZIKV with either CHIKV or DENV to a similar level than single infections, as long as the RNAi response is functional.

Background Mosquito-specific viruses (MSVs) comprise a variety of different virus families, some of which are known to interfere with infections of medically important arboviruses. Viruses belonging to the family Mesoniviridae or taxon Negevirus harbor several insect-specific viruses, including MSVs, which are known for their wide geographical distribution and extensive host ranges. Although these viruses are regularly identified in mosquitoes all over the world, their presence in mosquitoes in Germany had not yet been reported. Methods A mix of three MSVs (Yichang virus [ Mesoniviridae ] and two negeviruses [Daeseongdong virus and Dezidougou virus]) in a sample that contained a pool of Coquillettidia richiardii mosquitoes collected in Germany was used to investigate the interaction of these viruses with different arboviruses in Culex -derived cells. In addition, small RNA sequencing and analysis of different mosquito-derived cells infected with this MSV mix were performed. Results A strain of Yichang virus ( Mesoniviridae ) and two negeviruses (Daeseongdong virus and Dezidougou virus) were identified in the Cq. richiardii mosquitoes sampled in Germany, expanding current knowledge of their circulation in central Europe. Infection of mosquito-derived cells with these three viruses revealed that they are targeted by the small interfering RNA (siRNA) pathway. In Culex -derived cells, co-infection by these three viruses had varying effects on the representative arboviruses from different virus families ( Togaviridae : Semliki forest virus [SFV]; Bunyavirales : Bunyamwera orthobunyavirus [BUNV]; or Flaviviridae : Usutu virus [USUV]). Specifically, persistent MSV co-infection inhibited BUNV infection, as well as USUV infection (but the latter only at specific time points). However, the impact on SFV infection was only noticeable at low multiplicity of infection (MOI 0.1) and at specific time points in combination with the infection status. Conclusions Taken together, these results are important findings that will lead to a better understanding of the complex interactions of MSVs, mosquitoes and arboviruses. Graphical Abstract

Mosquitoes are competent vectors for many important arthropod-borne viruses (arboviruses). In addition to arboviruses, insect-specific viruses (ISV) have also been discovered in mosquitoes. ISVs are viruses that replicate in insect hosts but are unable to infect and replicate in vertebrates. They have been shown to interfere with arbovirus replication in some cases. Despite the increase in studies on ISV–arbovirus interactions, ISV interactions with their hosts and how they are maintained in nature are still not well understood. In the present study, we investigated the infection and dissemination of the Agua Salud alphavirus (ASALV) in the important mosquito vector Aedes aegypti through different infection routes (per oral infection, intrathoracic injection) and its transmission. We show here that ASALV infects the female Ae. aegypti and replicates when mosquitoes are infected intrathoracically or orally. ASALV disseminated to different tissues, including the midgut, salivary glands and ovaries. However, we observed a higher virus load in the brain than in the salivary glands and carcasses, suggesting a tropism towards brain tissues. Our results show that ASALV is transmitted horizontally during adult and larval stages, although we did not observe vertical transmission. Understanding ISV infection and dissemination dynamics in Ae. aegypti and their transmission routes could help the use of ISVs as an arbovirus control strategy in the future.

Mosquitoes are efficient vectors for many arboviruses that cause emergent infectious diseases in humans. Many insect-specific viruses (ISVs) that can infect mosquitoes but cannot infect vertebrates have been discovered in the last decade. Arboviruses transmitted by mosquitoes are responsible for the death of millions of people each year. In addition to arboviruses, many insect-specific viruses (ISVs) have been discovered in mosquitoes in the last decade. ISVs, in contrast to arboviruses transmitted by mosquitoes to vertebrates, cannot replicate in vertebrate cells even when they are evolutionarily closely related to arboviruses. The alphavirus genus includes many arboviruses, although only a few ISVs have been discovered from this genus so far. Here, we investigate the interactions of a recently isolated insect-specific alphavirus, Agua Salud alphavirus (ASALV), with its mosquito host. RNA interference (RNAi) is one of the essential antiviral responses against arboviruses, although there is little knowledge on the interactions of RNAi with ISVs. Through the knockdown of transcripts of the different key RNAi pathway (small interfering RNA [siRNA], microRNA [miRNA], and P-element-induced wimpy testis [PIWI]-interacting RNA [piRNA]) proteins, we show the antiviral role of Ago2 (siRNA), Ago1 (miRNA), and Piwi4 proteins against ASALV in Aedes aegypti -derived cells. ASALV replication was increased in Dicer2 and Ago2 knockout cells, confirming the antiviral role of the siRNA pathway. In infected cells, mainly ASALV-specific siRNAs are produced, while piRNA-like small RNAs, with the characteristic nucleotide bias resulting from ping-pong amplification, are produced only in Dicer2 knockout cells. Taken together, ASALV interactions with the mosquito RNAi response differ from those of arthropod-borne alphaviruses in some aspects, although they also share some commonalities. Further research is needed to understand whether the identified differences can be generalized to other insect-specific alphaviruses. IMPORTANCE Mosquitoes are efficient vectors for many arboviruses that cause emergent infectious diseases in humans. Many insect-specific viruses (ISVs) that can infect mosquitoes but cannot infect vertebrates have been discovered in the last decade. ISVs have attracted great attention due to their potential use in mosquito or arbovirus control, by either decreasing mosquito fitness or restricting arbovirus replication and transmission to humans. However, ISV-mosquito interactions are not well understood. RNA interference (RNAi) is the most important innate immune response against many arboviruses, while it is unknown if it is antiviral against ISVs. Here, we investigate in detail the antiviral effect of the RNAi response in mosquitoes against an ISV for the first time. Using a recently isolated insect-specific alphavirus, we show that the regulation of virus replication was different from that for arthropod-borne alphaviruses despite some similarities. The differences in mosquito-virus interactions could drive the different transmission modes, which could eventually drive the evolution of arboviruses. Hence, an understanding of mosquito-ISV interactions can shed light on the ecology and evolution of both ISVs and the medically important arboviruses.

Arboviruses transmitted by mosquitoes pose a global health threat, causing diseases ranging from mild fevers to severe encephalitis and hemorrhagic fevers. Despite their growing impact, arbovirus research is hindered by biosafety constraints and the need of specialized BSL-3 insectariums. To circumvent these challenges, mosquito-derived cell lines have become indispensable tools for investigating virus-vector interactions. However, most available cell lines originate from Aedes and Anopheles spp., creating a critical research gap for other key vectors such as Culex spp. Although a few cell lines were previously established, they did not represent primary transmitters of West Nile virus (WNV) and other emerging arboviruses in Europe, such as Culex pipiens. To address this gap, the current study aimed to characterize two recently established Culex pipiens cell lines: CPE/LULS50 (Culex pipiens pipiens & molestus) and CPL/LULS56 (Culex pipiens molestus) in more detail including testing their virus susceptibility, antiviral RNAi response, and possible presence of insect-specific viruses. The replication of arboviruses from three clinically relevant families (Flaviviridae, Peribunyaviridae, and Togaviridae), as well as insect-specific viruses, was observed in both CPE/LULS50 and CPL/LULS56 cell lines. Furthermore, small RNA profiling revealed production of virus-specific small interfering RNA (siRNA) in both cell lines for all tested viruses. Interestingly, virus-specific PIWI-interacting RNA (piRNA) was only detected for the Peribunyaviridae. The current study demonstrates that the CPE/LULS50 and CPL/LULS56 cell lines are suitable candidates to facilitate research into Culex-specific virus-vector interactions, ultimately contributing to mitigation of the impact of Culex-borne arboviruses on public health.