Agricultural insects play a crucial role in transmitting plant viruses and host a considerable number of insect-specific viruses (ISVs). Among these insects, the white-backed planthoppers (WBPH; Sogatella furcifera, Hemiptera: Delphacidae) are noteworthy rice pests and are responsible for disseminating the southern rice black-streaked dwarf virus (SRBSDV), a significant rice virus. In this study, we analyzed WBPH transcriptome data from public sources and identified three novel viruses. These newly discovered viruses belong to the plant-associated viral family Solemoviridae and were tentatively named Sogatella furcifera solemo-like virus 1-3 (SFSolV1-3). Among them, SFSolV1 exhibited a prevalent existence in different laboratory populations, and its complete genome sequence was obtained using rapid amplification of cDNA ends (RACE) approaches. To investigate the antiviral RNA interference (RNAi) response in WBPH, we conducted an analysis of virus-derived small interfering RNAs (vsiRNAs). The vsiRNAs of SFSolV1 and -2 exhibited typical patterns associated with the host\'s siRNA-mediated antiviral immunity, with a preference for 21- and 22-nt vsiRNAs derived equally from both the sense and antisense genomic strands. Furthermore, we examined SFSolV1 infection and distribution in WBPH, revealing a significantly higher viral load of SFSolV1 in nymphs\' hemolymph compared to other tissues. Additionally, in adult insects, SFSolV1 exhibited higher abundance in male adults than in female adults.

BACKGROUND: Vertical transmission (VT) of arboviruses (arthropod-borne viruses) can serve as an essential link in the transmission cycle during adverse environmental conditions. The extent of VT among mosquito-borne arboviruses can vary significantly among different virus families and even among different viruses within the same genus. For example, orthobunyaviruses exhibit a higher VT rate than orthoflaviviruses and alphaviruses. Mosquitoes are also the natural hosts of a large number of insect-specific viruses (ISV) that belong to several virus families, including Bunyaviridae, Flaviviridae, and Togaviridae. Cell fusing agent virus (CFAV), an insect-specific orthoflavivirus, displays higher VT rates than other dual-host orthoflaviviruses, such as Zika and dengue viruses. High VT rates require establishment of stabilized infections in the germinal tissues of female vectors. To delve deeper into understanding the mechanisms governing these differences in VT rates and the establishment of stabilized infections, the ovary infection patterns and VT of Zika virus (ZIKV) and CFAV were compared.
METHODS: Laboratory colonized Aedes aegypti females were infected with either ZIKV or CFAV by intrathoracic injection. Ovary infection patterns were monitored by in situ hybridization using virus-specific probes, and VT was determined by detecting the presence of the virus among the progeny, using a reverse-transcription quantitative polymerase chain reaction (PCR) assay.
RESULTS: Both ZIKV and CFAV infect mosquito ovaries after intrathoracic injection. Infections then become widespread following a non-infectious blood meal. VT rates of ZIKV are similar to previously reported results (3.33%). CFAV, on the contrary transmits vertically very rarely. VT was not observed in the first gonotrophic cycle following intrathoracic injection, and only rarely in the second gonotrophic cycle. VT of CFAV is mosquito population independent, since similar results were obtained with Aedes aegypti collected from two different geographic locations.
CONCLUSIONS: Although CFAV infects mosquito ovaries, the occurrence of VT remains infrequent in artificially infected Ae. aegypti, despite the observation of high VT rates in field-collected mosquitoes. These results suggest that infections of insect-specific viruses are stabilized in mosquitoes by some as yet unidentified mechanisms.

How non-retroviral endogenous viral elements (EVEs) are established is a long-standing question. Viral DNA (vDNA) forms of RNA viruses are likely to be EVE precursors. Cell-fusing agent virus (CFAV) is a major insect-specific virus (ISV) in the Aedes aegypti mosquitoes and one of the few existing non-retroviral RNA viruses found as EVEs. We characterized CFAV-derived vDNA in the cell line to understand the mechanism of why current viruses are rarely endogenized. vDNA production was affected by cell culture media independent of CFAV replication. vDNAs that correspond to different regions covering the entire viral genome were detected, implying multiple initiation sites exist. A considerable proportion of vDNAs corresponded to ssDNA. Higher vDNA copies were detected in the cytoplasm than the nucleus. Our findings provide valuable insights into the intracellular characteristics of ISV-derived vDNAs, which will aid in understanding the underlying mechanisms of non-retroviral EVE formation.

Aedes mosquitoes are well-known vectors of arthropod-borne viruses (arboviruses). Mosquitoes are more frequently infected with insect-specific viruses (ISVs) that cannot infect vertebrates. Some ISVs interfere with arbovirus replication in mosquito vectors, which has gained attention for potential use against arbovirus transmission. Cell-fusing agent virus (CFAV), a widespread ISV, can reduce arbovirus dissemination in Ae. aegypti. However, vectorial capacity is largely governed by other parameters than pathogen load, including mosquito survival and biting behaviour. Understanding how ISVs impact these mosquito fitness-related traits is critical to assess the potential risk of using ISVs as biological agents. Here, we examined the effects of CFAV infection on Ae. aegypti mosquito fitness. We found no significant reduction in mosquito survival, blood-feeding behaviour and reproduction, suggesting that Ae. aegypti is tolerant to CFAV. The only detectable effect was a slight increase in human attraction of CFAV-infected females in one out of eight trials. Viral tolerance is beneficial for introducing CFAV into natural mosquito populations, whereas the potential increase in biting activity must be further investigated. Our results provide the first insight into the link between ISVs and Aedes mosquito fitness and highlight the importance of considering all aspects of vectorial capacity for arbovirus control using ISVs.

Anopheles mosquitoes are the vectors of Plasmodium, the etiological agent of malaria. In addition, Anopheles funestus and Anopheles gambiae are the main vectors of the O\'nyong-nyong virus. However, research on the viruses carried by Anopheles is scarce; thus, the possible transmission of viruses by Anopheles is still unexplored. This systematic review was carried out to identify studies that report viruses in natural populations of Anopheles or virus infection and transmission in laboratory-reared mosquitoes. The databases reviewed were EBSCO-Host, Google Scholar, Science Direct, Scopus and PubMed. After the identification and screening of candidate articles, a total of 203 original studies were included that reported on a variety of viruses detected in Anopheles natural populations. In total, 161 viruses in 54 species from 41 countries worldwide were registered. In laboratory studies, 28 viruses in 15 Anopheles species were evaluated for mosquito viral transmission capacity or viral infection. The viruses reported in Anopheles encompassed 25 viral families and included arboviruses, probable arboviruses and Insect-Specific Viruses (ISVs). Insights after performing this review include the need for (1) a better understanding of Anopheles-viral interactions, (2) characterizing the Anopheles virome-considering the public health importance of the viruses potentially transmitted by Anopheles and the significance of finding viruses with biological control activity-and (3) performing virological surveillance in natural populations of Anopheles, especially in the current context of environmental modifications that may potentiate the expansion of the Anopheles species distribution.

Previously unknown pathogens often emerge from primary ecosystems, but there is little knowledge on the mechanisms of emergence. Most studies analyzing the influence of land-use change on pathogen emergence focus on a single host-pathogen system and often observe contradictory effects. Here, we studied virus diversity and prevalence patterns in natural and disturbed ecosystems using a multi-host and multi-taxa approach. Mosquitoes sampled along a disturbance gradient in Côte d\'Ivoire were tested by generic RT-PCR assays established for all major arbovirus and insect-specific virus taxa including novel viruses previously discovered in these samples based on cell culture isolates enabling an unbiased and comprehensive approach. The taxonomic composition of detected viruses was characterized and viral infection rates according to habitat and host were analyzed. We detected 331 viral sequences pertaining to 34 novel and 15 previously identified viruses of the families Flavi-, Rhabdo-, Reo-, Toga-, Mesoni- and Iflaviridae and the order Bunyavirales. Highest host and virus diversity was observed in pristine and intermediately disturbed habitats. The majority of the 49 viruses was detected with low prevalence. However, nine viruses were found frequently across different habitats of which five viruses increased in prevalence towards disturbed habitats, in congruence with the dilution effect hypothesis. These viruses were mainly associated with one specific mosquito species (Culex nebulosus), which increased in relative abundance from pristine (3%) to disturbed habitats (38%). Interestingly, the observed increased prevalence of these five viruses in disturbed habitats was not caused by higher host infection rates but by increased host abundance, an effect tentatively named abundance effect. Our data show that host species composition is critical for virus abundance. Environmental changes that lead to an uneven host community composition and to more individuals of a single species are a key driver of virus emergence.

West Nile virus (WNV) is the leading cause of mosquito-borne illness in the United States. There are currently no human vaccines or therapies available for WNV, and vector control is the primary strategy used to control WNV transmission. The WNV vector Culex tarsalis is also a competent host for the insect-specific virus (ISV) Eilat virus (EILV). ISVs such as EILV can interact with and cause superinfection exclusion (SIE) against human pathogenic viruses in their shared mosquito host, altering vector competence for these pathogenic viruses. The ability to cause SIE and their host restriction make ISVs a potentially safe tool to target mosquito-borne pathogenic viruses. In the present study, we tested whether EILV causes SIE against WNV in mosquito C6/36 cells and Culex tarsalis mosquitoes. The titers of both WNV strains-WN02-1956 and NY99-were suppressed by EILV in C6/36 cells as early as 48-72 h post superinfection at both multiplicity of infections (MOIs) tested in our study. The titers of WN02-1956 at both MOIs remained suppressed in C6/36 cells, whereas those of NY99 showed some recovery towards the final timepoint. The mechanism of SIE remains unknown, but EILV was found to interfere with NY99 attachment in C6/36 cells, potentially contributing to the suppression of NY99 titers. However, EILV had no effect on the attachment of WN02-1956 or internalization of either WNV strain under superinfection conditions. In Cx. tarsalis, EILV did not affect the infection rate of either WNV strain at either timepoint. However, in mosquitoes, EILV enhanced NY99 infection titers at 3 days post superinfection, but this effect disappeared at 7 days post superinfection. In contrast, WN02-1956 infection titers were suppressed by EILV at 7 days post-superinfection. The dissemination and transmission of both WNV strains were not affected by superinfection with EILV at either timepoint. Overall, EILV caused SIE against both WNV strains in C6/36 cells; however, in Cx. tarsalis, SIE caused by EILV was strain specific potentially owing to differences in the rate of depletion of shared resources by the individual WNV strains.

Eilat virus (EILV) is an insect-specific alphavirus that has the potential to be developed into a tool to combat mosquito-borne pathogens. However, its mosquito host range and transmission routes are not well understood. Here, we fill this gap by investigating EILV\'s host competence and tissue tropism in five mosquito species: Aedes aegypti, Culex tarsalis, Anopheles gambiae, Anopheles stephensi, and Anopheles albimanus. Of the tested species, C. tarsalis was the most competent host for EILV. The virus was found in C. tarsalis ovaries, but no vertical or venereal transmission was observed. Culex tarsalis also transmitted EILV via saliva, suggesting the potential for horizontal transmission between an unknown vertebrate or invertebrate host. We found that reptile (turtle and snake) cell lines were not competent for EILV infection. We tested a potential invertebrate host (Manduca sexta caterpillars) but found they were not susceptible to EILV infection. Together, our results suggest that EILV could be developed as a tool to target pathogenic viruses that use Culex tarsalis as a vector. Our work sheds light on the infection and transmission dynamics of a poorly understood insect-specific virus and reveals it may infect a broader range of mosquito species than previously recognized. IMPORTANCE The recent discovery of insect-specific alphaviruses presents opportunities both to study the biology of virus host range and to develop them into tools against pathogenic arboviruses. Here, we characterize the host range and transmission of Eilat virus in five mosquito species. We find that Culex tarsalis-a vector of harmful human pathogens, including West Nile virus-is a competent host of Eilat virus. However, how this virus is transmitted between mosquitoes remains unclear. We find that Eilat virus infects the tissues necessary for both vertical and horizontal transmission-a crucial step in discerning how Eilat virus maintains itself in nature.

Mosquitoes are vectors of various pathogens that cause diseases in humans and animals. To prevent the outbreak of mosquito-borne diseases, it is essential to control vector populations, as treatment or vaccination for mosquito-borne diseases are often unavailable. Insect-specific viruses (ISVs) have previously been described as being potentially helpful against arboviral disease outbreaks. In this study, we present the first in vivo characterization of the ISV Culex Y virus (CYV). CYV was first isolated from free-living Culex pipiens mosquitoes in 2010; then, it was found in several mosquito cell lines in a further study in 2018. For mammalian cells, we were able to confirm that CYV does not replicate as it was previously described. Additionally, we found that CYV does not replicate in honey bees or locusts. However, we detected replication in the Culex pipiens biotype molestus, Aedes albopictus, and Drosophila melanogaster, thus indicating dipteran specificity. We detected significantly higher mortality in Culex pipiens biotype molestus males and Drosophila melanogaster, but not in Aedes albopictus and female Culex pipiens biotype molestus. CYV could not be transmitted transovarially to offspring, but we detected venereal transmission as well as CYV in mosquitos\' saliva, indicating that an oral route of infection would also be possible. CYV\'s dipteran specificity, transmission routes, and killing effect with respect to Culex males may be used as powerful tools with which to destabilize arbovirus vector populations in the future.

Insect-specific virus (ISV) is one of the most promising agents for the biological control of insects, which is abundantly distributed in hematophagous insects. However, few ISVs have been reported in Riptortus pedestris (Fabricius), one of the major pests threatening soybeans and causing great losses in yield and quality. In this work, field Riptortus pedestris was collected from six soybean-producing regions in China, and their virome was analyzed with the metatranscriptomic approach. Altogether, seven new insect RNA viruses were identified, three of which had complete RNA-dependent RNA polymerase (RdRp) and nearly full-length genome sequences, which were named Riptortus pedestris alphadrosrha-like virus 1 (RpALv1), Riptortus pedestris alphadrosrha-like virus 2 (RpALv2) and Riptortus pedestris almendra-like virus (RiALv). The three identified novel ISVs belonged to the family Rhabdoviridae, and phylogenetic tree analysis indicated that they were clustered into new distinct clades. Interestingly, the analysis of virus-derived small-interfering RNAs (vsiRNAs) indicated that only RiALv-derived siRNAs exhibited 22 nt length preference, whereas no clear 21 or 22 nt peaks were observed for RpALv1 and RpALv2, suggesting the complexity of siRNA-based antiviral immunity in R. pedestris. In conclusion, this study contributes to a better understanding of the microenvironment in R. pedestris and provides viral information for the development of potential soybean insect-specific biocontrol agents.