Nonretroviral integrated RNA viruses in arthropod vectors: an occasional event or something more?

Accepted Manuscript Title: Nonretroviral integrated RNA viruses in arthropod vectors: an occasional event or something more?Nonretroviral integrated RNA virus sequences in vectors–> Authors: Ken E Olson, Mariangela Bonizzoni PII: DOI: Reference:

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Nonretroviral integrated RNA viruses in arthropod vectors: an occasional event or something more?

Short title: Nonretroviral integrated RNA virus sequences in vectors

Ken E. Olson1 and Mariangela Bonizzoni2* 1

Department of Microbiology, Immunology and Pathology, Arthropod-borne and infectious disease

laboratory, Colorado State University, Fort Collins, CO, USA; 2Department of Biology and Biotechnology, University of Pavia, Pavia, Italy, corresponding author: Bonizzoni, Mariangela ([email protected])

Highlights  Viral DNA fragments of arboviruses are found in mosquito cells and mosquitoes adults at early stages of infection as episomal DNA forms  Metagenomic analyses detected nonretroviral integrated RNA viruses sequences (NIRVS) in vector genomes  Episomal vDNAs and NIRVS are an additional virus control strategy intercalated within the piRNA pathway  Being vertically-trasmitted, NIRVS have the potentials to be a novel mechanism of acquired adaptive immunity, functionally analogous the prokaryotic CRISPR-Cas system Abstract With few exceptions, all arthropod-borne viruses (arboviruses) are RNA viruses. Arbovirus RNA genomes are distinct from retrovirus RNA genomes in that they do not encode reverse transcriptases and integrases to integrate DNA intermediates of their RNA into the host genome. However, viral DNA (vDNA) fragments of nonretroviral RNA virus sequences (including arboviral vDNA) are detected with remarkable frequency in mosquito cells and mosquitoes. Arboviral vDNAs are detected in mosquito cells and adults at early stages of infection as episomal DNA forms. Additionally, next generation sequencing and bioinformatics analyses have convincingly shown vDNA from nonretroviral RNA viruses (NRVs) integrated in vector genomes. Although the biological relevance of the vDNA’s arising from NRVs such as arboviruses is unknown, we hypothesize their role may be linked to arbovirus immunity and persistence in the vector. Key

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questions remain about nonretroviral integrated RNA virus sequences (NIRVS) in mosquitoes such as what is driving vDNA synthesis from arboviral RNA, how does integration of vDNA occur and what is their biological function (if any). Here we review current knowledge about NIRVS, including hypotheses on mechanisms of NIRVS formation and their impact on mosquito biology. We highlight connections between NIRVS, host immunity and virus-vector co-evolution and we suggest directions for future research.

Introduction Arthropod-borne viruses (arboviruses) include a wide range of virus taxa, several of which contain viruses that are emerging or resurging public health threats [1*]. Despite having different genome structures and replication strategies, all arboviruses have two common features: 1) they do not code for reverse transcriptase and integrase; 2) they are maintained in a natural cycle involving transmission by the bite of an infected hematophagous arthropod (vector) to a vertebrate host [2]. Once infected with an arbovirus, the vector becomes persistently infected for life. The virus spends the majority of its life cycle in the vector, but is dependent on a vertebrate host having sufficient viremia to infect vectors with the next bite. The absence of vaccines for most arboviruses has led to arboviral disease control strategies that suppress vector populations. Historically, mosquitoes of the Culicidae family, such as Aedes aegypti and Aedes albopictus, have been important targets of vector control to suppress transmission of epidemiologically-relevant arboviruses (Zika virus, Dengue viruses [DENV] 1-4, and chikungunya virus [CHIKV]) [3-6]. Classical vector control efforts use insecticides, but these efforts have led to environmental concerns and emergence of widespread insecticide resistance in vector populations [7]. Geneticbased strategies of vector control, including those that alter the reproductive capacity of mosquitoes (population suppression strategies) or those that prevent viral transmission by mosquitoes (population replacement strategies) are emerging as much needed complementary approaches to the use of insecticides [7-9]. In the last few decades, the search for novel genetic-based vector control strategies has led

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to a deeper understanding of virus-vector interactions, vector genomics and transcriptomics, vector innate immunity, and genome evolution of both arboviruses and vectors. From these studies, RNA interference (RNAi) has emerged as the primary antiviral defence system of mosquitoes [10-13] and RNAi has been widely exploited as a functional tool to suppress expression of essential mosquito genes (i.e. genes that activate apoptosis and innate immunity, genes involved in olfaction, reproduction and embryogenesis) [14]. RNAi consists of three pathways termed the small interfering (si), the micro (mi) and the piwi interacting (pi) RNA pathways. These pathways follow a similar operational strategy: they use small RNAs to guide a protein-effector complex to target RNA based on sequence-complementarity, but differ based on biogenesis of the small RNAs and the proteins that constitute the effector complex [10-13]. While the siRNA pathway restricts arboviral infection in the vector, the miRNA pathway is important for post-translational regulation of host and viral gene expression. The biogenesis and biological function of siRNA and miRNA pathways have been amply documented across insect taxa [10-13]. The piRNA pathway has a central role in maintaining genome integrity by suppressing transposable element (TE) activity in eukaryotes. In drosophila, the biogenesis of piRNAs begins with nuclear transcripts with antisense polarity piRNA from discrete genomic loci called piRNA clusters. These clusters are derived from defective TE sequences and utilized to generate piRNAs that suppress TEs. The biogenesis of virus-derived piRNAs (vpiRNAs) is not as well understood, however, evidence is mounting that vpiRNAs have a role in suppressing virus infections in vectors [15**]. This antiviral activity is not consistent among insects in that the piRNA pathway appears to have no antiviral activity in the model insect Drosophila menogaster [16**], but the pathway restricts arboviral infections in Culicidae mosquitoes where vpiRNAs arise from both the mosquito and the infecting arbovirus genomes [10-13,15**]. In recent years, a new group of RNA viruses termed insect-specific viruses (ISVs) have been identified that are phylogenetically-related to arboviruses of the Flaviviridae, Bunyaviridae, Togaviridae and Rhabdovirdae families, but unlike arboviruses, ISVs do not replicate in vertebrate cells or hosts [17**] and may be a good model for determining a biological role for NIRVS. ISVs are NRVs that persistently infect arthropod vectors and are likely vertically (transovarially) transmitted

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within a vector population [17**-19]. Additionally, recent phylogenetic studies indicate that many ISVs, which are genetically related to arboviruses, tend to be ancestral and may be considered precursors of arboviruses within respective families [18-20]. The degree of competition between ISVs and cognate arboviruses in vectors is still controversial [21-22]. Interestingly, analyses of vector genomes have revealed sequences of ISVs and, to a lesser extent, arboviruses integrated into the vector’s genome [23-32]. In this review we discuss current literature highlighting the connections between NIRVS in the vector’s genome, vector immunity and evolution of both viral and host genomes. It is important to understand circumstances under which integration of viral sequences occurs and whether integrations are lineage-specific. This knowledge can elucidate poorly understood aspects of viral immunity in mosquitoes and would be useful for development of applications of these viruses in future gene therapy applications [33**]. Likewise it is essential to verify whether NIRVS affect susceptibility to subsequent infections with cognate arboviruses or allow persistent infections in order to aid in the development of genetic-based transmission blocking strategies based on alterations of virus-vector interactions.

Nonretroviral Integrated RNA Virus Sequences (NIRVS) in mosquito genomes Viral sequences that integrate into host genomes are called Endogenous Viral Elements (EVEs) [34]. EVEs from NRVs, such as ISVs and arboviruses are NIRVS [35-37]. A list of NIRVS presently described in mosquito genomes is shown in Table 1. The discovery of NIRVS in mosquitoes initially occurred using DNA amplification techniques to characterize vector genomes. As an example, in 2004 four NIRVS were identified as an unexpected amplification product using Flavivirus degenerate primers from DNA of Ae. aegypti and Ae. albopictus cells and adults [23*]. These integrations were initially termed Cell Silent Agents (CSAs) because they had sequence homology to flavivirus ISV’s (ISF’s) such as Cell Fusing Agent (CFA) and Kamiti River Virus (KRV) [23*]. One of the CSA integrations encompassed an uninterrupted Open Reading Frame (ORF) of 1557 aa corresponding to the NS1-NS4 coding region of the flavivirus RNA genome. The CSA sequence was transcriptionally active in C6/36 cells [23*]. Other ISF sequences have been identified as DNA fragments in Ae. albopictus, Aedes vexans and Ochlerotatus geniculatus

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mosquitoes [24-25]. The ISF DNA fragments were assumed to be integrated in the vector genome due to the absence of active ISF infection and the presence of point mutations that would have disrupted the ISF ORF [24-25]. A significant leap forward towards identifying NIRVS in mosquitoes was the use of nextgeneration sequencing and bioinformatics analyses. In silico screening of NIRVS included a twostep process starting with a Blast-based search of the genome/s under investigation followed by further analyses, including search for viral ORFs and molecular validation of bioinformatics data, to reduce false positives resulting from long stretches of unspecific short tandem repeats or homopolymers that are present in both viral and eukaryotic genomes [26-32,38-39]. In silico screening strategies revealed that mosquito genomes clearly contain NIRVS originating from flavivirus and rhabdovirus RNA genomes [27-29,30,32]. Specifically, integrations of ISF DNA sequences were detected in the genomes of Ae. aegypti, Ae. albopictus, Anopheles sinensis and Anopheles mininums [26,29-29,30,32]. ISF Integrations included one or more complete viral ORFs that were actively transcribed [26,29,32]. In Ae. aegypti and Cx. quinquefasciatus, but not An. gambiae, NIRVS with similarities to rhabdoviruses were detected and some NIRVS had sequence variability across geographic populations of the vector [26-27,30,32**]. Despite not following the same screening pipeline and not including the same mosquito genomes or viral species, all current in silico analyses describing NIRVS in mosquitoes were concordant in describing a larger number of NIRVS in Ae. aegypti and Ae. albopictus than in Cx. quinquefasciatus and Anophelinae mosquitoes [26-29,32**]. Data showing the widespread distribution of NIRVS across mosquito genomes are still fragmented and the bioinformatics-based searches have been mainly focused on integrations from flaviviruses and ISF sequences obtained from vector genome databases, often with limited analyses of NIRVS variability across mosquito populations [26-29,31]. Despite limited knowledge of NIRVS sequence variability in vector populations or how widespread NIRVS are in natural mosquito populations, current data suggest that exposure to environmental viruses is not a strong determinant of NIRVS formation, but infection intensity and prevalence may be. For instance, several mosquito species sampled from the same region (i.e. South East Asia) and whose genomes were analyzed for flavivirus integrations, showed differing levels of integration.

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The competent flavivirus vector Ae. albopictus showed tens of viral integrations [28,32**]. However, less than 10 viral integrations were detected in Anophelinae, which are poor vectors of flaviviruses, but may be infected with ISFs [29,32,40]. Additionally, a comprehensive analyses of viral integrations from 425 nonretroviral RNA virus species characterised sequences with similarities to only Flaviviridae, Rhabdoviridae, Bunyaviridae and Reoviridae, suggesting species-

specific interactions between RNA viruses and mosquitoes [32**]. Taken together the above results indicate that the co-evolution between NRVs and mosquitoes is a more complex interaction than previously thought. More exhaustive and comparative analyses of viral integrations in vectors, including those from recently characterized ISVs and RNA viruses [41-42**] and geographic vector populations are essential to clarify if NIRVS are lineage-specific, how NIRVS are distributed in natural vector populations and whether they are active in those populations.

Mechanisms of NIRVS formation: transposons and RNAi Formation of NIRVS requires the reverse transcription of RNA into cDNA, followed by integration of the cDNA into host genomes (Fig. 1). Reverse transcription of RNA into cDNA is catalysed by reverse transcriptase (RT) encoded by endogenous retroviruses and retrotransposons [43]. Retrotransposons are ubiquitous genetic elements in eukaryotic DNA that amplify themselves in host genomes including those of vectors. Retrotransposons express genes encoding reverse transcriptase and are most likely key players for initiating DNA forms of West Nile virus (WNV) in Culex tarsalis cells and of DENV, CHIKV and La Crosse virus (Bunyavirus) in Ae. albopictus and/or Ae. aegypti cells and adult mosquitoes [44**-45]. Viral-derived cDNA forms (vDNAs) are detected as early as 6 hours post infection in mosquito cells, but the frequency of occurrence with respect to the initial viral inoculum is unknown [44-45]. Researchers have shown that vDNA synthesis in vectors is dependent on the trans activity of endogenous RTs by inhibiting RT and vDNA formation in a dose-dependent manner with the addition of azidothymidine (AZT), a RT inhibitor [45-46**]. The observed vDNA fragments do not span the entire viral genome and tend to be linked to long terminal repeat (LTR) retrotransposons, retroviruses and repetitive sequences,

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suggesting ectopic recombination between viral RNA and transposons occurs during reverse transcription possibly by a copy choice strategy [44-47**]. Recent data on NIRVS distribution in Aedes mosquitoes support a close association between NIRVS and TEs, particularly Ty3_gypsy and Pao Bell retrotransposons [30,32**]. In adult mosquitoes, vDNA fragments are detected in lysates associated with various body parts, including legs and wings, following virus dissemination, suggesting either vDNAs are formed in all infected cells or alternatively are released from cells and disseminate throughout the mosquito [45]. Arboviruses acquire an outer envelope from host cell membranes during their budding stage, thus a possible strategy is that host cell membrane remodelling drives the formation of extracellular vesicles that contain vDNAs and favour antiviral intracellular communication [45,48-49]. vDNAs are reverse transcribed in the cytoplasm and must be deposited in the nucleus prior to integration. Access of viral nucleic acids to the nucleus could occur during mitosis, when the nuclear envelope is temporarily disassembled [50]. Alternatively, several viral and mosquito immunity proteins have been described with DNA-binding motifs and have been shown to localize both to the cytoplasm and the nucleus, suggesting active transport of vDNA [50-53]. For instance, nucleocapsid proteins may transiently localise in the nucleus of host cells, early in the infectious cycle [51]. Nucleocapsid proteins interact in the cytoplasm with viral genomic RNA, other structural proteins and host proteins to drive the formation of the replicative/transcriptive unit and promote assembly and packaging of new viral particles [51]. In the nucleus, capsid proteins interfere with host immune response and inhibit host transcription to free cellular machinery for viral RNA synthesis [51]. However, capsid proteins have been shown also to block nuclear pores, potentially limiting their role in transporting viral RNA or vDNA into the nucleus [52]. Transport of vDNAs into the nucleus and integration into mosquito genome may also result from an interaction between vDNAs and the mosquito RNAi machinery, particularly the piRNA pathway (Figure 1) [45,53-54]. In Ae. aegypti and Ae. albopictus, the piRNA pathway has antiviral activity in addition to its canonical function of preserving genome integrity [15**]. The piRNA pathway requires interaction among small RNAs, which are generated from piRNA clusters, and Piwi-class Argonaut proteins (PIWI proteins) to repress in trans nucleic acids based on sequence-

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complementarity [15**]. The PIWI family of proteins has expanded in Aedes when compared to D. melanogaster and Anophelinae mosquitoes, with some proteins showing functional specialization towards interacting with viruses or transposons [54**-56**]. In CHIKV-infected Ae. aegypti cells and adult mosquitoes, vDNAs contribute to the production of both vpiRNAs and virus-specific siRNA and prime cell immunity without restricting viral replication, thus favouring persistent infection [45]. Additionally, piRNAs map to viral-derived sequences adjacent to transposon bearing loci in Ae. aegypti [57**] and vpiRNAs are produced following alphavirus and flavivirus infections, even if their distribution along the corresponding viral genome is strikingly different [15**]. Albeit clear differences among virus and mosquito species, these observations support a close functional relationship between immunity mechanisms acting against RNA viruses and TEs. This relationship may originate in the evolutionary history of these two entities: like other NRVs, arboviruses presumably evolved through frequent gene exchange and reshuffling from prokaryotic Group II elements (self-splicing introns) also giving rise to retroelements [58**].

NIRVs biological role Figure 2 summarizes current experimental data and highlights knowledge gaps on NIRVS biological role. Transcription was observed from several NIRVS characterized from the Ae. albopictus, Ae. aegypti, An. sinensis and An. minimums genomes [23,27,29,31-32]. NIRVS may encode and express viral proteins that compete in trans with gene products from infecting viruses and block viral replication, transcription or assembly of mature particles [32,60]. A second mechanism of NIRVS action is that transcriptionally active NIRVS prime piRNA-based antiviral response by generating primary piRNAs transcripts similar to the nuclear primary piRNA transcripts that suppress transposon activity [15,55**]. These two mechanisms are not mutually exclusive, but could cooperate because gene products encoded by NIRVS may lead to defective interfering viral particles, which are commonly found following infections by arboviruses [60-61]. The genome of these defective particles may enter RNAi pathways more efficiently than dsRNA of viral replicative intermediates [47, 60-61]. Depending on the targeted viral gene and the timing of activation, NIRV-based stimulation of RNAi and/or trans competition with viral products may result

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in immunity to subsequent infection and/or favour the establishment of persistent infection, a phenomenon called “viral accommodation” [62-64]. A model was proposed recently whereby when a NRVs, such as a +sense RNA virus like DENV, infects a mosquito, its genome is targeted by the primary, antigenome –sense piRNAs likely transcribed from NIRVS or newly-generated episomal vDNAs. The primary piRNAs recognize the viral + sense genome leading to production of mostly positive sense secondary vpiRNAs[54**]. As infection becomes persistent and the rate of viral genome synthesis decreases, so is the source for secondary vpiRNAs, thus the predominant population of vpiRNAs becomes more negative in polarity [54**]. Recent findings showing limited mRNA production from NIRVS, but production of primary and, in some cases, also secondary piRNAs, support this hypothesis [32**]. A number of details of this process must be clarified, for instance what is the role of vpiRNAs from NIRVS: do they keep viral replication at a level that does not have a negative impact on mosquito fitness or do they keep immunity primed to block novel infections with cognate viruses? What would be the sequence-complementarity for NIRVs activation? What is the role of the different proteins of the piRNA pathway in this process? It is important to note that NIRVS identified from in silico analyses of genome databases represent only those integrations that occurred in the germ-line and were maintained. These are probably a small number with respect to integrations that may occur in somatic cells during an arbovirus infection, given the functional connection among the piRNA pathway, vDNAs and TEs. However, these integrations will be vertically-transmitted thus could have an evolutionary impact on vector competence. A large number of vDNAs may be transcriptionally active as episomal DNAs, thus it will be important to understand how stable and transmissible these episomal forms are. Despite numerous knowledge gaps, these data indicate that co-evolution between arbovirus and vector genomes may be a more fluid and dynamic process than previously thought. It is of utmost importance to expand our knowledge of the interplay between arboviruses, RNAi, NIRVS and TEs, particularly to inform novel avenues for the development of transmission-blocking control strategies based on manipulation of virus-vector interactions.

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Conclusions and Outlook The transfer of genetic material between separate evolutionary lineages (horizontal transfer) has had an important role in the evolution of genomes. Despite being overshadowed by the higher prevalence and more understood gene transfer among prokaryotes, the exchange of genetic material from DNA viruses and retroviruses to eukaryotic cells is a well-recognised phenomenon [65]. Recent discoveries show the existence of fragments from NRVs, including recentlycharacterised RNA viruses, ISVs and arboviruses both existing as episomal vDNAs and integrated vDNAs in the genome of mosquito vectors. Whether this phenomenon is lineage specific and widespread in natural populations is still unclear. However, mounting experimental evidence suggests that NIRVS may be an additional virus control strategy intercalated within the piRNA pathway to control virus replication and either favour persistent infection or prevent further infection with cognate viruses [66]. Genome integration of viral sequences provides for a permanent change that if involving the germline is transferred to the progeny and may precondition it to either be more or less susceptible to an infection with cognate viruses. ISVs have access to germline since they are transmitted transovarially [17]. Some arboviruses are also transmitted transovarially albet at a lower frequency than ISVs [67]. The different frequency of transovarial transmission between ISVs and arboviruses may explain the higher incidence of ISV vDNA in the genome than arbovirus vDNAs. vDNA- and NIRVS-dependent arboviral immunity would be a novel mechanism of acquired adaptive immunity, functionally analogous to the prokaryotic CRISPR-Cas system [68]. NIRVS formation and expression as a response to arbovirus infections of vectors could be an important mechanism contributing to the observed variability in vector competence across geographic populations. Depending on the sequence-similarity required for NIRVS to affect the outcome of subsequent infections with cognate viruses and NIRVS impact in either favouring persistent infection or blocking additional infections, this phenomenon could have also epidemiological consequences because it could favour the initial sweep of viral serotypes followed by a lowering of prevalence later. In this framework, we need to develop a deeper understanding of where NIRVS sequences originate in arboviral genomes, and what level of sequence specificity is required for NIRVS-arbovirus interaction to occur, particularly given that both heterologous and homologous

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interference has been described in mosquitoes [68]. Additionally, considering that vector competence is mediated by components that are specific to the genomes of the virus and vector [69-70], it is important to understand if NIRVS formation is a mechanism that vectors evolved as a general response to any infecting NRVs.

Acknowledgment

We are grateful to Gennaro Della Gala for images and to professors Anthony A. James and Carol Blair for critical reading of the manuscript. K. Olson research has been funded by NIH grants AI34014, AI48740, and Grand Challenges in Global Health through the Foundation for NIH. M. Bonizzoni research is supported by the ERC-Co 682394. Funding sources had no role in study design; in the collection, analysis, and interpretation of data; in the writing of this review; and in the decision to submit the manuscript.

Abbreviations

AZT = azidothymidine CHIKV = chikungunya CFA = Cell Fusing Agent CSA = Cell Silent Agent DENV = Dengue viruses EVEs = endogenous viral elements ISF = insect specific Flavivirus ISVs = insect specific viruses KRV = Kamiti River Virus LTR retrotransposon = long terminal repeat retrotransposon miRNA = micro RNA NIRVS = nonretroviral RNA virus sequences

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NRVs = nonretroviral RNA viruses ORF = Open Reading Frame piRNA = piwi interacting RNA RNAi = RNA interference RT = Reverse Transcriptase siRNA = small interferring RNA TE = transposable element vDNA = viral-derived cDNA vpiRNAs = virus-derived piRNAs WNV = West Nile virus

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Figure Legend

Fig. 1 Proposed interaction between NIRVS and mosquito RNAi antiviral mechanisms. Fig. 2 This figure aims to A) summarize current experimental data regarding possible routes of NIRVS formation, which requires reverse-transcription of RNA-based viral genomes and their nuclear import and B) highlight knowledge gaps and propose future research endeavours.

NIRVS

vDNA transcription

vDNA

Nucleus

Cytoplasm primary piRNA

Reverse transcriptase from retrotransposon

? R2D2

Other Piwi proteins? Dcr2

siRNA

Ago2 (siRISC)

Primary piRNAs 3’ 5’

5’

dsRNA

3’

Viral RDRP 3’

Ago3

Ping-pong amplification loop

5’

5’ 3’

5’

3’

Viral RNA 3’ 5’

19

NIRVS FORMATION

Infecting arbovirus (RNA genome)

Reverse transcription and nuclear import

NIRVS BIOLOGIAL IMPACT

Integration into mosquito genome

A. Experimental Evidences • Detection of viral cDNA (vDNAs) fragments upon arboviral infections in mosquitoes • vDNAs appear as early as 6 hour post infection and are still detected 9 days post infection • vDNAs do not form in presence of AZT • CHIKV-derived vDNAs affect piRNA profiles of U4.4 cells and mosquitoes, mainly at early stages of infection

• Which reverse transcriptase/s acts on arboviruses? • vDNA distribution across arbvirus genomes: conserved hotspots? • Do vDNAs interact with Argonoute proteins? Or viral proteins? • Are vDNAs disseminated through extracellular vescicles? • Are vDNAs imported into the nucleus?

• Flavivirus-like NIRVS in Ae. albopictus, Ae. aegypti, An. minimus and An. sinensis • Rhabdovirus-like NIRVs in Ae. aegypti and Cx. quinquefascatus

• Are episomal vDNAs transcribed and/or integrated? • Are NIRVS lineage-specific? • Where do NIRVS map in mosquito genomes? Do they associate with any TE? • Are there differences across mosquito species and among populations in NIRVS distribution and variability?

Maintenance and biological activity • mRNA expression from NIRVS • Ae. aegypti piRNA clusters harbor viral-like sequences

Immunity to new infections?

B. Open Questions and Future Perspectives

• Are NIRVS-derived mRNA translated? • NIRVS-piRNA profile with/without infection and tissue-specificity • Are NIRVS stable across generations? • Sequence complementarity between NIRVS product (s) and cognate viruses • NIRVS genetic manipulation

Persistance of viral infection?

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Table 1. Summary of currently-known NIRVS in mosquito genomes. Studies describing NIRVS in mosquitoes were classified based on the primary strategy used, either PCR amplification of mosquito DNA (A) or in silico analyses of genome databases (B).

Study (ref.)

Viruses: N. genera, N. species 1 (Flavivirus) 2 (Alphavirus, Flavivirus) 1 (Flavivirus)

Mosquito species Aedes aegypti, Aedes albopictus Ae. albopictus Ae. albopictus, Aedes vexans, Ochlerotatus genicolatus

N. NIRVs x41 12 93

A

Crochu et al., 2004 [23] Roiz et al., 2009 [24] Rizzo et al., 2014 [25]

B

Katzourakis and Gifford, 2010 [26] Fort et al., 2012 [27] Chen et al., 2015 [28]

48, 484

Ae. aegypti, Anopheles gambiae, Culex quinquefasciatus

585

9, 346 1 (Flavivirus), 261

627 248

Lequime and Lambrechts, 2017 [29] Whitfield et al., 2017 [30] Suzuki et al., 2017 [31] Palatini et al., 2017 [32]

1 (Flavivirus), 50

Ae. aegypti, An. gambiae, Cx. quinquefasciatus Ae. albopictus, Ae. aegypti, An. gambiae, Cx. quinquefasciatus currently-available genome sequences of 24 Anophelinae

several 1 (Flavivirus) 11, 425

Ae. aegypti Aag2 Cell line Ae. aegypti and Ae. albopictus, genome and wild populations All currently-available mosquito genome sequences

368 410 24211

39

1one

NIRVS was identified in Ae. aegypti, 3 in Ae. albopictus. One NIRVS had an uninterrupted viral ORF corresponding to NS1-NS4A genes and was transcribed. 2one NIRVS with similarities to ISFs 3Integrations sites were not characterised 4one viral species per genus was tested. Among genera with arboviruses, the following viruses were chosen as representative of the corresponding genus: Yellow Fever virus (Flavivirus), Bunyamwera virus (Orthobunyavirus), Rift Valley fever virus (Phlebovirus), Sindbis virus (Alphavirus), Bluetongue virus (Orbivirus), Vesicular stomatitis Indiana virus (Vesiculovirus) 557 NIRVS were identified in Ae. aegypti: 28 had similarities to vesciculoviruses, one to Lianning virus segment 5 (Seadornavirus) and an additional 28 to ISFs; one NIRVS with similatiry to vesciculoviruses was identified in Cx. quinquefasciatus, no NIRVS in An. gambiae 6All tested viruses belonged to the Monomegavirales order and included 6 genus-unclassified species 761 NIRVs were idenitifed in Ae. aegypti, one in Cx. quinquefasciatus and none in An. gambiae 8A total of 24 NIRVs encompassing complete or partial Flavivirus-like ORFs were described in Ae. albopcitus; additional uncharacterised sequences with similarities to recently-identified RNA viruses were also found. No Flavivirus-like integrations were detected in An. gambiae and Cx. quinquefasciatus. 21

9one

NIRVS with an ORF with no stop codons spanning NS4A/B and part of NS5 was identified in Anopheles minimus; this NIRVS is transcribed. Two NIRVS were identified in Anopheles sinensis, one with smilarities to ISFs and spanning a partial NS3 ORF and one with similarity to Xincheng mosquito virus, a recently-characterised negative-sense, single-stranded virus [38]. The Flavivirus-like NIRVS of An. sinensis was transcribed at low levels. 10Analyses is limited to the four viral integrations first described by Crochu et al., 2004. Their variability is described across geographic populations. 11Viral integrations were not evenly distributed across mosquito species and were enriched in piRNA clusters in the genome of the arboviral vectors Ae. aegypti and Ae. albopictus.

22