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Mosquito-borne arboviruses include several pathogens that are responsible for many diseases of significant public health burden. Mosquitoes also host many insect-specific viruses that cannot replicate in vertebrate cells. These insect-specific viruses persist in nature predominantly via vertical transmission (VT), and they exhibit high VT rates (VTRs). Cell-fusing agent virus (CFAV), an insect-specific orthoflavivirus, shows high VTRs in naturally infected mosquitoes but not in artificially infected mosquitoes. To determine whether the high VTRs are due to transovarial transmission, we investigated VT and ovary infection patterns in naturally CFAV-infected Aedes aegypti (Bangkok) mosquitoes. VT was monitored by detecting CFAV among the progeny by reverse-transcription polymerase chain reaction and ovary infection was determined by in situ hybridization using a virus-specific probe. We showed that in CFAV-positive mosquitoes, ovarian follicles were infected, suggesting that VT occurs by transovarial transmission in naturally infected mosquitoes. Additionally, mosquitoes harbored dormant, non-replicative CFAV that remained below the detection level. These results suggested that CFAV persists via VT in nature and has the potential to remain dormant in diapausing mosquitoes during unfavorable conditions. Understanding this VT mechanism is crucial for comprehending the persistence of insect-specific viruses (and potentially dual-host arboviruses) in their natural environment. [ABSTRACT FROM AUTHOR]

Mosquito-borne arboviruses include several pathogens that are responsible for many diseases of significant public health burden. Mosquitoes also host many insect-specific viruses that cannot replicate in vertebrate cells. These insect-specific viruses persist in nature predominantly via vertical transmission (VT), and they exhibit high VT rates (VTRs). Cell-fusing agent virus (CFAV), an insect-specific orthoflavivirus, shows high VTRs in naturally infected mosquitoes but not in artificially infected mosquitoes. To determine whether the high VTRs are due to transovarial transmission, we investigated VT and ovary infection patterns in naturally CFAV-infected Aedes aegypti (Bangkok) mosquitoes. VT was monitored by detecting CFAV among the progeny by reverse-transcription polymerase chain reaction and ovary infection was determined by in situ hybridization using a virus-specific probe. We showed that in CFAV-positive mosquitoes, ovarian follicles were infected, suggesting that VT occurs by transovarial transmission in naturally infected mosquitoes. Additionally, mosquitoes harbored dormant, non-replicative CFAV that remained below the detection level. These results suggested that CFAV persists via VT in nature and has the potential to remain dormant in diapausing mosquitoes during unfavorable conditions. Understanding this VT mechanism is crucial for comprehending the persistence of insect-specific viruses (and potentially dual-host arboviruses) in their natural environment.

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.

Several mosquito-borne diseases affecting humans are emerging or reemerging in the United States. The early detection of pathogens in mosquito populations is essential to prevent and control the spread of these diseases. In this study, we tested the potential applicability of the Lawrence Livermore Microbial Detection Array (LLMDA) to enhance biosurveillance by detecting microbes present in Aedes aegypti, Aedes albopictus, and Culex mosquitoes, which are major vector species globally, including in Texas. The sensitivity and reproducibility of the LLMDA were tested in mosquito samples spiked with different concentrations of dengue virus (DENV), revealing a detection limit of >100 but

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. • The efficiency of CFAV-derived vDNA production in Aag2 cells varied on culture media but did not rely on CFAV RNA level. • CFAV vDNA was produced as a fragmented form. • A large proportion of vDNA exists as single-stranded DNA. • VDNAs were slightly more abundant in the cytoplasm than nucleus. [ABSTRACT FROM AUTHOR]

Mosquitoes in the Aedes and Culex genera are considered the main vectors of pathogenic flaviviruses worldwide. Entomological surveillance using universal flavivirus sets of primers in mosquitoes can detect not only pathogenic viruses but also insect-specific ones. It is hypothesized that insect-specific flaviviruses, which naturally infect these mosquitoes, may influence their vector competence for zoonotic arboviruses. Here, entomological surveillance was performed between January 2014 and May 2018 in five different provinces in the northeastern parts of South Africa, with the aim of identifying circulating flaviviruses. Mosquitoes were sampled using different carbon dioxide trap types. Overall, 64,603 adult mosquitoes were collected, which were screened by RT-PCR and sequencing. In total, 17 pools were found positive for insect-specific Flaviviruses in the mosquito genera Aedes (12/17, 70.59%) and Anopheles (5/17, 29.41%). No insect-specific viruses were detected in Culex species. Cell-fusing agent viruses were detected in Aedes aegypti and Aedes caballus. A range of anopheline mosquitoes, including Anopheles coustani, An. squamosus and An. maculipalpis, were positive for Culex flavivirus-like and Anopheles flaviviruses. These results confirm the presence of insect-specific flaviviruses in mosquito populations in South Africa, expands their geographical range and indicates potential mosquito species as vector species.

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., Competing Interests: We declare we have no competing interests., (© 2024 The Authors.)

Highlights: [•] Cell-fusing agent virus, an insect-specific flavivirus, has been poorly studied. [•] We isolated 29 strains from male and female Aedes aegypti mosquitoes in Thailand. [•] Envelope and nonstructural proteins 3 and 5 genes were analyzed phylogenetically. [•] The present isolates were distinct from the previous strains from other countries. [•] Isolates were predicted to have the most recent common ancestor of 11–27years ago. [ABSTRACT FROM AUTHOR]

Mosquitoes in the Aedes and Culex genera are considered the main vectors of pathogenic flaviviruses worldwide. Entomological surveillance using universal flavivirus sets of primers in mosquitoes can detect not only pathogenic viruses but also insect-specific ones. It is hypothesized that insect-specific flaviviruses, which naturally infect these mosquitoes, may influence their vector competence for zoonotic arboviruses. Here, entomological surveillance was performed between January 2014 and May 2018 in five different provinces in the northeastern parts of South Africa, with the aim of identifying circulating flaviviruses. Mosquitoes were sampled using different carbon dioxide trap types. Overall, 64,603 adult mosquitoes were collected, which were screened by RT-PCR and sequencing. In total, 17 pools were found positive for insect-specific Flaviviruses in the mosquito genera Aedes (12/17, 70.59%) and Anopheles (5/17, 29.41%). No insect-specific viruses were detected in Culex species. Cell-fusing agent viruses were detected in Aedes aegypti and Aedes caballus . A range of anopheline mosquitoes, including Anopheles coustani , An. squamosus and An. maculipalpis , were positive for Culex flavivirus-like and Anopheles flaviviruses. These results confirm the presence of insect-specific flaviviruses in mosquito populations in South Africa, expands their geographical range and indicates potential mosquito species as vector species.

Endogenous viral elements (EVEs) are viral sequences integrated in host genomes. A large number of non-retroviral EVEs was recently detected in Aedes mosquito genomes, leading to the hypothesis that mosquito EVEs may control exogenous infections by closely related viruses. Here, we experimentally investigated the role of an EVE naturally found in Aedes aegypti populations and derived from the widespread insect-specific virus, cell-fusing agent virus (CFAV). Using CRISPR-Cas9 genome editing, we created an Ae. aegypti line lacking the CFAV EVE. Absence of the EVE resulted in increased CFAV replication in ovaries, possibly modulating vertical transmission of the virus. Viral replication was controlled by targeting of viral RNA by EVE-derived P-element-induced wimpy testis-interacting RNAs (piRNAs). Our results provide evidence that antiviral piRNAs are produced in the presence of a naturally occurring EVE and its cognate virus, demonstrating a functional link between non-retroviral EVEs and antiviral immunity in a natural insect-virus interaction. • Aedes aegypti harbors EVEs with high sequence identity to a contemporary RNA virus • EVE-derived piRNAs target genomic viral RNA in infected mosquitoes • Ablation of EVE results in increased viral replication in Aedes aegypti ovaries • piRNA pathway fulfills antiviral function in presence of EVE and cognate virus Suzuki, Baidaliuk et al. identify a new endogenous viral element (EVE) in the genome of Aedes aegypti mosquitoes that was derived from a currently circulating, insect-specific flavivirus. Using this naturally occurring interaction, they demonstrate that a non-retroviral EVE confers antiviral immunity against its cognate virus via the piRNA pathway. [ABSTRACT FROM AUTHOR]