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Home , Alphavirus, Anopheles sinensis, Benyvirus, Bracovirus, Dicistroviridae, Ichnovirus

The Widespread Occurrence and Potential Biological Roles of Endogenous Viral Elements in

Carol D. Blair1, Ken E. Olson1 and Mariangela Bonizzoni2*

1Arthropod-borne and Infectious Diseases , Department of , Immunology and Pathology, Colorado State University, Fort Collins, CO, USA. 2Department of and Biotechnology, University of Pavia, Pavia, Italy. *Correspondence: [email protected] htps://doi.org/10.21775/cimb.034.013

Abstract insect biology, particularly antiviral immunity, and Modern genomic sequencing and bioinformatics suggest directions for future research. approaches have detected numerous examples of DNA sequences derived from DNA and RNA genomes integrated into both and insect Introduction: defnition of genomes. encode RNA-dependent endogenous viral elements DNA (reverse transcriptases) and and the surprising discovery of integrases that convert their RNA viral genomes those from non-retroviral RNA into DNA and facilitate proviral DNA integration into the . Surprisingly, Interactions between viruses and their hosts occur DNA sequences derived from RNA viruses that at many levels. Viruses evolve rapidly through do not encode these also occur in host genetic drif and/or and can genomes. Non-retroviral integrated RNA virus infuence the genetic structures of host popula- sequences (NIRVS) occur at relatively high fre- tions, especially if viral infection results in disease quency in the genomes of the arboviral vectors and mortality. A classic example of virus and host and Aedes albopictus, are not distrib- co- is the natural experiment initiated uted randomly and possibly contribute to by the release of highly pathogenic myxoma virus antiviral immunity, suggesting these mosquitoes into the naive Australian wild rabbit population could serve as a model system for unravelling the in 1950, followed by rapid development of resist- function of NIRVS. Here we address the follow- ance in rabbits coincident with natural atenuation ing questions: What drives DNA synthesis from of the virus (Best and Kerr, 2000; Kerr, 2012). the genomes of non-retroviral RNA viruses? How Another mechanism through which viruses afect does integration of virus cDNA into host DNA the genetics of the host is viral genome integra- occur, and what is its biological function (if any)? tion. Integrations of viral sequences We review current knowledge of viral integrations can occur in both somatic and cells. in insect genomes, hypothesize mechanisms of Somatic integrations are frequently associated with NIRVS formation and their potential impact on host genome instability (e.g. Chen et al., 2014).

Curr. Issues Mol. Biol. (2020) Vol. 34 caister.com/cimb 14 | Blair et al.

Additionally, complete viral sequence integration 2013; Ballinger et al., 2014; Olson and Bonizzoni, into host genomes can favour the persistence of the 2017; Palatini et al., 2017). infection (Cohn et al., 2015). If integrations occur Here we describe current knowledge on the in germline cells, they can be inherited by the next origin, widespread occurrence and genomic context generation. Te persistence and outcome of these of EVEs in insect genomes. Additionally, we report integrations in host populations depend on their examples of the infuence of EVEs on both verte- efects on host ftness. If deleterious, integrations brate and insect host physiology and immunity. are likely lost. Alternatively, viral sequences can be functionally adopted by the host and exert benefcial functions (Frank and Feschote, 2017). Integration of genomes from For instance, the product of the murine DNA viruses restriction , Fv1, protects mice against infec- (PDV) provide a well-known exam- tion with murine leukaemia virus (MLV). Fv1 ple of DNA virus integration into insect genomes in Mus musculus and other Mus spp. were (Table 2.1A; for a complete, authoritative review derived from the gag genes of ancient endogenous of polydnaviruses and their interactions with their retroviruses, some of which are distantly related insect hosts, see Chapter 8). PDV are recognized to MLV, and protect against other retroviruses, by the International Commitee on of including lentiviruses and spumaviruses (Yap et al., Viruses (ICTV; htps://talk.ictvonline.org/ictv- 2014). A third outcome is found when integrations reports/ictv_9th_report/dsdna-viruses-2011/w/ are in chromosomal locations that are not tran- dsdna_viruses/127/polydnaviridae) as the family scribed or lack regulatory regions. In this case, viral Polydnaviridae, divided into the genera integrations may persist but accumulate (BV) and (IV), which are associated at the host rate, which is typically much slower with braconid and ichneumonid , than that of exogenous viruses. Tese endogenous respectively (Strand and Burke, 2015; Gauthier et viral elements (EVEs) thus represent a magnifying al., 2018). have been domesticated by lens into the past, allowing researchers to more wasps, and their genomes are integrated as proviral closely examine the evolutionary history of viruses segments dispersed throughout the genome in and virus–host co-evolution (Aiewsakun and Kat- every of both female and male wasps (Desjar- sourakis, 2015; Katzourakis, 2017). dins et al., 2008). Tey replicate by producing Integration of genome sequences from DNA defective virus-like particles (VLPs) in the calyx of viruses and retroviruses is a relatively common the ovary. Bracovirus genomes in VLPs con- phenomenon, as evidenced by the abundance sist of multiple, diverse, circular dsDNAs depleted of virus-derived sequences in the genomes of of replication genes, with one DNA molecule per various . For instance, from VLP. Parasitoid wasps reproduce by depositing their retroviruses constitutes about 8% of the human eggs in or on other , which are consumed by genome (Grifths et al., 2001). Genome integra- juvenile stages of the wasp as they develop (Burke tions are essential events in the retrovirus cycle. et al., 2014). Te defective VLPs are deposited into Because non-retroviral RNA viruses lack coding the wasp’s hemipteran or dipteran host along with for , the machinery needed its eggs, their defective genomes migrate to the host for successful conversion to DNA and integration , where they subsequently express viru- into host genomes, their potential to endogenize lence factors that alter host physiology and allow has been considered minimal. However, an increas- the development of wasp progeny (Strand and ing number of studies show genome integrations Burke, 2015; Gauthier et al., 2018). PDVs cannot into both somatic and germline host cells from be transmited horizontally; rather, the integrated non-retroviral RNA viruses, including both proviruses are transmited vertically in wasps. Tran- single-stranded (positive and negative) and double- script studies suggest that BVs evolved from a single stranded RNA viruses. Te acronym non-retroviral whole genome integration of an ancient ancestor integrated RNA virus sequences (NIRVS) has been of the insect virus family (Bezier et al., proposed to emphasize the unusual viral origin of 2009; Burke and Strand, 2012). Lineages of ichneu- these EVEs (Ballinger et al., 2012; Kondo et al., monid wasps with integrated IVs are polyphyletic

Curr. Issues Mol. Biol. (2020) Vol. 34 caister.com/cimb Endogenous Viral Elements in Insect Genomes | 15 and integrated IVs are diverse, suggesting multiple additional unexpected integrations from ISVs were evolutionary integration events (Gauthier et al., detected in wild-caught Aedes and 2018). genomes by PCR using favivirus-specifc primers Analyses of a recently discovered endogeniza- (Roiz et al., 2009; Rizzo et al., 2009; Sanchez-Seco tion involving an alphanudivirus and the braconid et al., 2010). wasp Fopius arisanus are shedding light on the Descriptions of RNA virus genome sequence mechanisms of integration. Alphanudiviruses are integrations into insect genomes increased with members of the family Nudiviridae with a single the advent of next-generation sequencing tech- circular dsDNA genome that usually cause virulent nologies and metagenomic analyses in the early infections in insects. Fopius arisanus belongs to the 2000s. Bioinformatics-based identifcation of viral Opiinae subfamily of . Wasp species integrations uses BLAST+ in the form of BLASTx in this subfamily usually lack integrations from or tBLASTn (Altschul et al., 1990). Researchers bracoviruses (Burke et al., 2018). Consequently, parse positive BLAST hits using cut-of values of the identifcation of integrated alphanudivirus between 10–3 and 10–6 (Kondo et al., 2019; Whit- sequences in the genome of F. arisanus suggests that feld et al., 2017; ter Horst et al., 2018). Additional this integration is a recent event. Tis event shows criteria (i.e. presence of viral open reading frames that endogenization is accompanied with gene-loss or minimum size restrictions) are applied to further in the viral genome, and that changes in its archi- reduce false positive results that occur primarily tecture occur before expression of the endogenized due to low-complexity reads with short tandem virus genes falls under the control of the wasp repeats or homopolymers. Te complexity of the genome (Burke et al., 2018). viral database used to search for viral integrations Genome endogenization has also been observed may infuence the number of identifed BLAST for the betanudivirus HzNV-1, which naturally hits. Researchers have used a number of virus infects the corn earworm Helicoverpa zea. HzNV-1 genome databases to identify viral integrations can exhibit a latent phase of infection during in host genomes. Tese viral sequence databases which its DNA is integrated into the host genome include sequences from single virus families (Fort (Gauthier et al., 2018). Moreover, HzNV-1 genome et al., 2012; Chen et al., 2015), from viruses integrations were observed in persistently infected transmited by insects (Cui and Holmes, 2012), all cells of other lepidopteran species (Trichoplusia ni known RNA viruses, including those most-recently and Spodoptera fugiperda). Interestingly, cells of identifed (i.e. Whitfeld et al., 2017; Palatini et the fall armyworm carrying endogenous HzNV-1 al., 2017) and from a mixture of DNA and RNA appeared resistant to superinfections (Lin et al., viruses (Katzourakis and Giford, 2010; ter Horst et 1999). al., 2018). Tese analyses provided the number of viral integrations in sequenced insect genomes and insight into the diversity and richness of their viral Integration of non-retroviral RNA origins. Te results were infuenced by the breadth virus genomes: discovery and of viral genomes analysed and the stringency of association with retroelements the criteria used to defne an insect sequence as a Te frst discovery of integrations of non-retroviral viral integration (i.e. cut of BLAST e-value; reverse RNA virus genome sequences in insect genomes BLAST; analyses of low complexity sequences; was the unexpected fnding in 2004 of four regions length of sequences; inclusion of newly discovered similar to of cell fusing agent (CFAV) and and unclassifed viruses). Kamiti River (KRV) viruses in the genomes of Currently-known insect genome viral integra- Aedes aegypti and Aedes albopictus. Tese sequences, tions are largely from non-retroviral RNA viruses which were amplifed using degenerate favivirus with sequences also derived from single-stranded primers, were called cell silent agents (CSA) DNA viruses, e.g. from densoviruses () because of their similarity to CFAV (Crochu et al., in the genome of Acyrthosiphon pisum (ter Horst 2004). Both CFAV and KRV are insect-specifc et al., 2018). NIRVS have been characterized from viruses (ISVs) that are genetically related to arbo- the genomes of both haematophagous and non- viruses but exclusively infect insect cells. In 2009, bloodsucking insects (Table 2.1B and C). A review

Curr. Issues Mol. Biol. (2020) Vol. 34 caister.com/cimb Table 2.1 Characteristics of EVEs that have been identifed in insect genomes including parasitic wasps (A), haematophagous species (B) and non-bloodsucking insects (C)

Host EVEs caister.com/cimb Family , species Viral oigin/R, DN Context Expression Discovery method References

A. Parasitic wasps Ichneumonid and braconid Polydna, D Full viral genome Dispersed Y Burke and Strand subfamilies within wasp (2012), Gauthier et al. genome (2018) Helicoverpa; Trichoplusia ni Betanudivirus HzNV, not c ND Lin et al. (1999) and Spodoptera frugiperda D cells Hymenoptera Fopius arisanus Alphanudivirus, D 55 Dispersed In silico screening; PCR Burke et al. (2018) within host genome

B. Haematophagous insects Culicidae Aedes albopictus, Aedes Flavi1, R 6 Repetitive Y PCR Crochu et al. (2004) aegypti, C6/36 cell lines DNA3 Culicidae Ae. albopictus Flavi2, R Not c ND ND PCR Roiz et al. (2009) Culicidae Ochlerotatus and Aedes Flavi, R Not c ND ND PCR Sanchez-Seco et al. (2009) Culicidae Ochlerotatus geniculatus, Flavi, R Not c ND ND PCR Rizzo et al. (2009) Aedes vexans Culicidae Ae. aegypti, Culex Rhabdo, R 143 ND ND In silico screening Katzourakis and quinquefasciatus Giford (2010) Reo, R 1 ND ND In silico screening Flavi, R 5 ND ND In silico screening Culicidae Ae. vexans, Och. caspius, Flavi, R not c ND ND PCR Vasquez et al. (2012) Och. detritus, Culiseta annulata Culicidae Cx. quinquefasciatus, Ae. Rhabdo, R 112 in Ae. aegypti, 1 in ND Y In silico screening; PCR Fort et al. (2012) aegypti, wild mosquitoes Cu. quinquefasciatus Culicidae Ae. aegypti Plant RNA viruses6, 2 Ace1 gene ND In silico screening Cui and Holmes R (2012) Curr. Issues Mol. Biol. (2020) Vol. 34

Hemiptera Rhodnius prolixus , R4 repetitive ND In silico screening Kondo et al. (2013) DNA7 Culicidae Aedes aegypti, Chu, Rhabdo8, 1–129 ND In silico screening, RNA-seq spp. Bunya, Phlebovirus-like, Li et al. (2015)10 , R Chu, Rhabdo8, R ND In silico screening, RNA-seq Hemiptera Rhodnius prolixus Chu, Bunya, ND In silico screening, RNA-seq MonoN11 Culicidae Anopheles minimus, Flavi, R 2 Repetitive Y In silico screening Lequime and Anopheles sinensis4 DNA Lambrechts (2017) Culicidae Ae. aegypti, Ae. albopictus, Flavi, R At least 8 Repetitive Y12 PCR; in silico screening Suzuki et al. (2017) wild mosquitoes DNA Culicidae Aedes, Culex and Anopheles Rhabdo, R Up to 88 in Aedine; 7 Repetitive Y14 In silico screening Palatini et al. (2017) spp. with genome sequence in Anophelinae DNA13 available Flavi, R Up to 32 in Aedine; 1 in Anophelinae Bunya, R 1 Reo, R 1 Culicidae Ae. aegypti Aag2 cell line Rhabdo, Flavi, Chu, 368 repetitive ND In silico screening Whitfeld et al. (2017) Virg, Bunya, R DNA13

C. Non-blood-sucking insects Hymenoptera Apis mellifera Discistro15, R1 Intergenic Y PCR Maori et al. (2007) Drosophilidae Various species16 , R < 5 per genome Repetitive Y18 In silico screening Ballinger et al. (2012) DNA17 Drosophilidae Various species16 Rhabdo, R 1 ND ND In silico screening Fort et al. (2012) Homoptera Acyrthosiphon pisum Rhabdo, R 1 ND ND In silico screening Fort et al. (2012) Hymenoptera; Various species19 Plant RNA viruses5, < 20 per genome Genes or ND ND In silico screening Cui and Holmes Diptera; R (2012) Drosophilidae Various species16 Bunya, R 2 Intergenic Y In silico screening Ballinger et al. (2014) Table 2.1 Characteristics of EVEs that have been identifed in insect genomes including parasitic wasps (A), haematophagous species (B) and non-bloodsucking insects (C)

Host EVEs Family Genus, species Viral oigin/R, DN Context Expression Discovery method References

A. Parasitic wasps Hymenoptera Ichneumonid and braconid Polydna, D Full viral genome Dispersed Y Burke and Strand subfamilies within wasp (2012), Gauthier et al. genome (2018) Lepidoptera Helicoverpa; Trichoplusia ni Betanudivirus HzNV, not c ND Lin et al. (1999) and Spodoptera frugiperda D cells Hymenoptera Fopius arisanus Alphanudivirus, D 55 Dispersed In silico screening; PCR Burke et al. (2018) within host genome

B. Haematophagous insects Culicidae Aedes albopictus, Aedes Flavi1, R 6 Repetitive Y PCR Crochu et al. (2004) aegypti, C6/36 cell lines DNA3 Culicidae Ae. albopictus Flavi2, R Not c ND ND PCR Roiz et al. (2009) Culicidae Ochlerotatus and Aedes Flavi, R Not c ND ND PCR Sanchez-Seco et al. (2009) Culicidae Ochlerotatus geniculatus, Flavi, R Not c ND ND PCR Rizzo et al. (2009) Aedes vexans Culicidae Ae. aegypti, Culex Rhabdo, R 143 ND ND In silico screening Katzourakis and quinquefasciatus Giford (2010) Reo, R 1 ND ND In silico screening Flavi, R 5 ND ND In silico screening Culicidae Ae. vexans, Och. caspius, Flavi, R not c ND ND PCR Vasquez et al. (2012) Och. detritus, Culiseta annulata Culicidae Cx. quinquefasciatus, Ae. Rhabdo, R 112 in Ae. aegypti, 1 in ND Y In silico screening; PCR Fort et al. (2012) aegypti, wild mosquitoes Cu. quinquefasciatus Culicidae Ae. aegypti Plant RNA viruses6, 2 Ace1 gene ND In silico screening Cui and Holmes R (2012)

Hemiptera Rhodnius prolixus Benyvirus, R4 repetitive ND In silico screening Kondo et al. (2013) DNA7 Culicidae Aedes aegypti, Anopheles Chu, Rhabdo8, 1–129 ND In silico screening, RNA-seq spp. Bunya, Phlebovirus-like, Li et al. (2015)10 Quaranjavirus, R caister.com/cimb Culex quinquefasciatus Chu, Rhabdo8, R ND In silico screening, RNA-seq Hemiptera Rhodnius prolixus Chu, Bunya, ND In silico screening, RNA-seq MonoN11 Culicidae Anopheles minimus, Flavi, R 2 Repetitive Y In silico screening Lequime and Anopheles sinensis4 DNA Lambrechts (2017) Culicidae Ae. aegypti, Ae. albopictus, Flavi, R At least 8 Repetitive Y12 PCR; in silico screening Suzuki et al. (2017) wild mosquitoes DNA Culicidae Aedes, Culex and Anopheles Rhabdo, R Up to 88 in Aedine; 7 Repetitive Y14 In silico screening Palatini et al. (2017) spp. with genome sequence in Anophelinae DNA13 available Flavi, R Up to 32 in Aedine; 1 in Anophelinae Bunya, R 1 Reo, R 1 Culicidae Ae. aegypti Aag2 cell line Rhabdo, Flavi, Chu, 368 repetitive ND In silico screening Whitfeld et al. (2017) Virg, Bunya, R DNA13

C. Non-blood-sucking insects Hymenoptera Apis mellifera Discistro15, R1 Intergenic Y PCR Maori et al. (2007) Drosophilidae Various species16 Sigmavirus, R < 5 per genome Repetitive Y18 In silico screening Ballinger et al. (2012) DNA17 Drosophilidae Various species16 Rhabdo, R 1 ND ND In silico screening Fort et al. (2012) Homoptera Acyrthosiphon pisum Rhabdo, R 1 ND ND In silico screening Fort et al. (2012) Hymenoptera; Various species19 Plant RNA viruses5, < 20 per genome Genes or ND ND In silico screening Cui and Holmes Diptera; R (2012) Drosophilidae Various species16 Bunya, R 2 Intergenic Y In silico screening Ballinger et al. (2014) Curr. Issues Mol. Biol. (2020) Vol. 34 Table 2.1 Continued caister.com/cimb

Host EVEs Family Genus, species Viral oigin/R, DN Context Expression Discovery method References Coleoptera Dendroctonus ponderosae Chu, Bunya, R In silico screening, RNA-seq Li et al. (2015) Tribolium castaneum Chu, R 3 ND In silico screening, RNA-seq Drosophilidae spp. Rhabdo8,20, Bunya, R ND In silico screening, RNA-seq Isoptera Zootermopsis nevadensis Chu, R ND In silico screening, RNA-seq Hemiptera Acyrthosiphon pisum Chu, Rhabdo8, 9 ND In silico screening, RNA-seq Phlebovirus-like; Quaranjavirus, MonoN11, R Hymenoptera Atta cephalotes MonoN9, R ND In silico screening, RNA-seq Acromyrmex echinatior Chu, MonoN11 8 ND In silico screening, RNA-seq Camponotus foridanus Chu, MonoN11, 3 ND In silico screening, RNA-seq Rhabdo8, R Harpegnathos saltator Chu, R 7 ND In silico screening, RNA-seq Linepithema humile Chu, R 11 ND In silico screening, RNA-seq Nasonia vitripennis, N. Chu, R 4–2021 ND In silico screening, RNA-seq giraulti, N. longicornis Pogonomyrmex barbarus Chu, R 23 ND In silico screening, RNA-seq Solenopsis invicta Chu, MonoN11, R 12 ND In silico screening, RNA-seq Lepidoptera Chu, Quaranjavirus, ND In silico screening, RNA-seq Rhabdo8, R Melitaea cinxia Rhabdo20, ND In silico screening, RNA-seq Quaranjavirus, R Plutella xylostella Rhabdo8, R ND In silico screening, RNA-seq Spodoptera frugiperda Phlebovirus-like, R ND In silico screening, RNA-seq Curr. Issues Mol. Biol. (2020) Vol. 34

Lepidoptera Spodoptera frugiperda cell Rhabdo22, R423 ND Y In silico screening Geisler and Jarvis lines, Bombyx mori (2016) Hymenoptera; Bombus terrestris and Virga/nege-like24, R < 10 per genome repetitive DNA ND In silico screening; PCR Kondo et al. (2017) Thysanoptera, B. ignites; Frankliniella Lepidoptera, occidentalis, Heliconius Diptera

Only data from peer-reviewed publications are reported. 1Cell fusing agent virus, Kamiti River virus. 2Aedes favivirus. 3Repetitive DNA: LTR- of the Pao/Ninja and Copia lineages and an An. gambiae putative gene. 4In silico screening was done across all 24 currently available Anophelinae genomes. 5Repetitive DNA, LTR-retrotransposons of the Copia class. 6Passion fruit (), pelargonium zonate spot virus (), carrot red leaf virus (Luteoviridae), beet yellow virus (), cardamine chlorotic feck virus (), bamboo mosaic virus (Alphafexiviridae), blueberry nectrotic ring blotch virus and citrus leprosis virus c (unclassifed). 7Repetitive DNA, transposable elements as mariner and mos. 8Dirmarhabdovirus. 9One NIRVS in A. arabiensis, An. epiroticus, An. atroparvus and An. nilii, two in An. quandriannulatus, three in An. funestus, An. farauti, An. dirus, An. gambiae and An. minimus; four in An. stephensi; 12 in Aedes aegypti, fve in Cx. Quinquefasciatus. 10NIRVS copy number was shown only for NIRVS from Chuvirus. 11Unclassifed Mononegavirus. 12mRNA expression was detected, but no . Production of small RNA was also observed. 13Repetitive DNA, non-LTR retroelements, primarily of the Ty3/Gypsy and Pao Bel lineages. 14Limited mRNA expression; piRNA production. 15Israeli acute paralysis virus. 16Various species within the Sophophora and Drosophila subgenera. 17Repetitive DNA, non-LTR retroelements, primarily of the Ty3/Gypsy lineage. 18Expression was verifed for one X-linked Sigma virus-P like NIRVS of Drosophila yakuba. 19NIRVS were identifed in 14 out of 53-tested insect genomes, including Drosophila rhopaloa, Dr. fcusphila, Dr. anannasse, Bombus terrestris, B. impatiens, Megachile rotundata, Pogonomyrmex barbatus, Acyrthosiphon pisum, Bombyx mori, Danaus plexippus, Nosonia longicornis, Nosonia vitripennis, Nosonia giraulti. 20Unclassifed rhabdovirus. 21Four NIRVS in Nasonia giraulti, fve in N. longcornis, 20 in N. vitripennis. 22Sf-rhabdovirus. 23Four integrations were found in the genome of S. frugiperda and three in that of B. mori. 24Virga/nege like viruses are newly-recognized insect-specifc viruses. Key to headings: context, genomic context of integration; D, DNA-based genome; N, number; R, RNA-based genome; viral or., viral origin. Key to table: Bunya, ; Chu, Chuviridae; Dicistro, ; Flavi, ; N, no; ND, not determined; not c, not certain; Polydna, Polydnaviridae; Reo, ; Rhabdo, ; Virg, Virgaviridae; Y, yes. Host EVEs Family Genus, species Viral oigin/R, DN Context Expression Discovery method References Coleoptera Dendroctonus ponderosae Chu, Bunya, R In silico screening, RNA-seq Li et al. (2015) Tribolium castaneum Chu, R 3 ND In silico screening, RNA-seq Drosophilidae Drosophila spp. Rhabdo8,20, Bunya, R ND In silico screening, RNA-seq Isoptera Zootermopsis nevadensis Chu, R ND In silico screening, RNA-seq Hemiptera Acyrthosiphon pisum Chu, Rhabdo8, 9 ND In silico screening, RNA-seq Phlebovirus-like; Quaranjavirus, MonoN11, R Hymenoptera Atta cephalotes MonoN9, R ND In silico screening, RNA-seq Acromyrmex echinatior Chu, MonoN11 8 ND In silico screening, RNA-seq Camponotus foridanus Chu, MonoN11, 3 ND In silico screening, RNA-seq Rhabdo8, R Harpegnathos saltator Chu, R 7 ND In silico screening, RNA-seq Linepithema humile Chu, R 11 ND In silico screening, RNA-seq Nasonia vitripennis, N. Chu, R 4–2021 ND In silico screening, RNA-seq giraulti, N. longicornis Pogonomyrmex barbarus Chu, R 23 ND In silico screening, RNA-seq Solenopsis invicta Chu, MonoN11, R 12 ND In silico screening, RNA-seq Lepidoptera Bombyx mori Chu, Quaranjavirus, ND In silico screening, RNA-seq Rhabdo8, R Melitaea cinxia Rhabdo20, ND In silico screening, RNA-seq Quaranjavirus, R Plutella xylostella Rhabdo8, R ND In silico screening, RNA-seq Spodoptera frugiperda Phlebovirus-like, R ND In silico screening, RNA-seq

Lepidoptera Spodoptera frugiperda cell Rhabdo22, R423 ND Y In silico screening Geisler and Jarvis lines, Bombyx mori (2016) Hymenoptera; Bombus terrestris and Virga/nege-like24, R < 10 per genome repetitive DNA ND In silico screening; PCR Kondo et al. (2017) Thysanoptera, B. ignites; Frankliniella Lepidoptera, occidentalis, Heliconius Diptera caister.com/cimb

Only data from peer-reviewed publications are reported. 1Cell fusing agent virus, Kamiti River virus. 2Aedes favivirus. 3Repetitive DNA: LTR-retrotransposons of the Pao/Ninja and Copia lineages and an An. gambiae putative gene. 4In silico screening was done across all 24 currently available Anophelinae genomes. 5Repetitive DNA, LTR-retrotransposons of the Copia class. 6Passion fruit mosaic virus (Virgaviridae), pelargonium zonate spot virus (Bromoviridae), carrot red leaf virus (Luteoviridae), beet yellow virus (Closteroviridae), cardamine chlorotic feck virus (Tombusviridae), bamboo mosaic virus (Alphafexiviridae), blueberry nectrotic ring blotch virus and citrus leprosis virus c (unclassifed). 7Repetitive DNA, transposable elements as mariner and mos. 8Dirmarhabdovirus. 9One NIRVS in A. arabiensis, An. epiroticus, An. atroparvus and An. nilii, two in An. quandriannulatus, three in An. funestus, An. farauti, An. dirus, An. gambiae and An. minimus; four in An. stephensi; 12 in Aedes aegypti, fve in Cx. Quinquefasciatus. 10NIRVS copy number was shown only for NIRVS from Chuvirus. 11Unclassifed Mononegavirus. 12mRNA expression was detected, but no proteins. Production of small RNA was also observed. 13Repetitive DNA, non-LTR retroelements, primarily of the Ty3/Gypsy and Pao Bel lineages. 14Limited mRNA expression; piRNA production. 15Israeli acute paralysis virus. 16Various species within the Sophophora and Drosophila subgenera. 17Repetitive DNA, non-LTR retroelements, primarily of the Ty3/Gypsy lineage. 18Expression was verifed for one X-linked Sigma virus-P like NIRVS of Drosophila yakuba. 19NIRVS were identifed in 14 out of 53-tested insect genomes, including Drosophila rhopaloa, Dr. fcusphila, Dr. anannasse, Bombus terrestris, B. impatiens, Megachile rotundata, Pogonomyrmex barbatus, Acyrthosiphon pisum, Bombyx mori, Danaus plexippus, Nosonia longicornis, Nosonia vitripennis, Nosonia giraulti. 20Unclassifed rhabdovirus. 21Four NIRVS in Nasonia giraulti, fve in N. longcornis, 20 in N. vitripennis. 22Sf-rhabdovirus. 23Four integrations were found in the genome of S. frugiperda and three in that of B. mori. 24Virga/nege like viruses are newly-recognized insect-specifc viruses. Key to headings: context, genomic context of integration; D, DNA-based genome; N, number; R, RNA-based genome; viral or., viral origin. Key to table: Bunya, Peribunyaviridae; Chu, Chuviridae; Dicistro, Dicistroviridae; Flavi, Flaviviridae; N, no; ND, not determined; not c, not certain; Polydna, Polydnaviridae; Reo, Reoviridae; Rhabdo, Rhabdoviridae; Virg, Virgaviridae; Y, yes. Curr. Issues Mol. Biol. (2020) Vol. 34 20 | Blair et al.

of the widespread occurrence of NIRVS in insect contiguity between NIRVS and retroelements in genomes suggests similar mechanisms of biogen- genomes of insects has led to the hypothesis that esis and integration occurred across diferent insect integration events in insect genomes occur through species; however, striking diferences were found in non- between the the number of NIRVS and the viral origins of those genomes of infecting non-retroviral RNA viruses in Aedes mosquitoes relative to other insects. and LTR retroelements during their reverse tran- In contrast to endogenization of scription (Ballinger et al., 2012; Goic et al., 2013). sequences originating from entire rearranged viral Te hypothesis is supported by similar fndings in genomes, NIRVS consist of complete or inter- mammalian genomes (Geuking et al., 2009) and by rupted viral RNA open-reading frames (ORFs). the fact that LTR retroelement reverse Most NIRVS have similarity to coding regions of occurs in the , where most non-retroviral non-retroviral , glycoproteins, and/ RNA viruses complete their life-cycle (Havecker or RNA-dependent-RNA polymerases (Crochu et et al., 2004; Geuking et al., 2009; Horie et al., al., 2004; Katzourakis and Giford, 2010; Fort et al., 2010; Ballinger et al., 2012). Furthermore, LTR 2012; Ballinger et al., 2012; Cui and Holmes, 2012; retroelements are active in germline cells, which Kondo et al., 2013; Ballinger et al., 2014; Geisler could favour inheritance of newly acquired viral and Jarvis, 2016; Lequime and Lambrechts, 2017; sequences (Morozov et al., 2017). Suzuki et al., 2017; Palatini et al., 2017; Whitfeld Te association between NIRVS and retroele- et al., 2017). ments is also supported by the recent fnding that A striking similarity among NIRVS detected some retroelements harbour viral-like domains across diferent insect species and from diferent (Ballinger et al., 2012). Viral-like helicase domains virus families is that integrations are frequently of recently characterized ISVs were found as part found in regions of repetitive DNA, in association of TRS and Jockey non-LTR with long terminal repeat (LTR) retroelements. proteins in the genomes of Lepidoptera (i.e. Retrotransposons of the Pao/Ninja, BEL/Pao, Plutella xylostella), Hemiptera (i.e. Nilaparvata Copia and Ty3/Gypsy lineages and, less frequently, lugens, Homalodisca vitripennis) Orthoptera (spe- Tc1/mariner, fank NIRVS derived from faviviruses cies of the Ceutophilius genus), Hymenoptera and rhabdoviruses in both Culicidae and Droso- ( boulardi) and Culicidae (Ae. aegypti) philidae (Crochu et al., 2004; Ballinger et al., 2012; species (Lazareva et al., 2015; Shi et al., 2016; Goic et al., 2013; Lequime and Lambrechts, 2017; Morozov et al., 2017). Interestingly, expression of Palatini et al., 2017; Whitfeld et al., 2017). Retro- viral-insect fused proteins in some cases increased elements also fank NIRVS from plant RNA viruses the evolutionary ftness of the harbouring retro- and newly-characterized alphanudivirus-like ISVs elements by functioning as viral suppressors of in examples from Hemiptera, Hymenoptera, Ty- RNA silencing (Lazareva et al., 2015). Helicases sanoptera, Homoptera, Lepidoptera, and from containing a viral motif could stabilize retrotrans- Drosophilidae (Cui and Holmes, 2012; Kondo et poson transcripts and increase their transposition al., 2013, 2019). efciency. An excess of stabilizing helicase activity Te origin of NIRVS in insect genomes requires could also negatively impact non-retroviral RNA the formation of cDNA from the non-retroviral virus infection by altering the balance between RNA template with the help of reverse tran- virus transcription and replication (Morozov et al., scriptases. Since NIRVS come from viruses that lack 2017). this , retrotransposons are a likely source within insect cells. Naturally occurring cDNAs of non-retroviral RNA sequences, vDNAs, have been Integration of non-retroviral found in cells infected with these viruses (Goic et RNA virus genomes: viral origin al., 2013; Nag and Kramer, 2017). A reduction in and number of NIRVS in insect formation of virus (CHIKV; Alphavi- genomes rus) vDNA in Ae. albopictus treated with a reverse Because most current discoveries of NIRVS transcriptase inhibitor has been observed (Goic et come from in silico studies of sequenced insect al., 2016). Tis fnding and the observed physical genomes with limited data from natural specimens,

Curr. Issues Mol. Biol. (2020) Vol. 34 caister.com/cimb Endogenous Viral Elements in Insect Genomes | 21 knowledge of the widespread occurrence of thirty times greater than that detected in any other NIRVS in insect genomes is biased by comparisons culicine genome. Although the precise number of across species as compared to among populations F-NIRVS will vary with improvements in genome within species. With this limitation in mind, ISVs annotations and with sequencing of novel culicine and other viruses known to establish persistent genomes, the large number of F-NIRVS in these infections are well represented in the list of NIRVS two mosquito species was obtained by applying (Table 2.1B). the same conservative bioinformatics pipeline Currently known insect NIRVS are similar to across the genomes of Aedes, Culex and Anopheles the sequences of viruses from the , Beny- spp., and hence the analysis was methodologically virus, and Quaranjavirus genera; the Reoviridae, unbiased. Rhabdoviridae and Peribunyaviridae families and Current data, which come from studies using the recently characterized family Chuviridae and diferent bioinformatics approaches and analys- phlebovirus-like and virga/nege-like viruses. Chu- ing diferent insect species, show that NIRVS viridae is a newly identifed monophyletic family of from reovirus, bunyavirus, sigmavirus, benyvirus, negative-sense RNA viruses, with diverse genome orthomyxovirus and phlebovirus genomes are structures (i.e. unsegmented, bi-segmented and sporadic, as they are found in both limited numbers circular forms) (Li et al., 2015). Quaranjavirus and distribution across insect species (Table 2.1). and phlebovirus-like viruses have negative-sense, Even with the paucity of data currently available, it segmented RNA genomes, belong to the Ortho- is interesting to note that haematophagous species myxoviridae and families, respectively, like Ae. aegypti and the kissing bug Rhodnius pro- and have been recently identifed in (Li lixus harbour not only -derived NIRVS, et al., 2015). ‘Virga/nege-like viruses’ refers to a still but also NIRVS with similarity to the genomes of poorly characterized group of positive-strand RNA plant RNA viruses (Cui and Holmes, 2012; Kondo viruses that appear to belong to the -like et al., 2013). Non-blood-sucking insect species superfamily (Kondo et al., 2019). Virgaviridae is a harbour NIRVS primarily from Rhabdoviridae family of plant alpha-like viruses, while negevirus and Chuviridae, which are also in the genomes refers to newly recognized ISVs from sandfies and of haematophagous insects (Table 2.1). Te mosquitoes, which are distantly phylogenetically- Rhabdoviridae family includes viruses that infect related to plant cileviruses (Vasilakis et al., 2013; , invertebrates and and that are Carapeta et al., 2015; Nunes et al., 2017). extremely variable in their genomic organization NIRVS from faviviruses (F-NIRVS) have been (Dietzgen et al., 2017; Geoghegan et al., 2017). found almost exclusively in the genomes of culicine Te ecological diversity of rhabdoviruses is also mosquitoes (Table 2.1). Te Culicidae is a large refected in their frequent cross-species transmis- mosquito family that includes arboviral vectors sion between hosts (Geoghegan et al. 2017). such as Aedes spp., Ochlerotatus spp. and Culex spp. While the ecology of Chuviridae is not completely and protozoan vectors such as those in the sub- understood, viruses in this family were found in family Anophelinae (Chen et al., 2015). F-NIRVS species that share the same ecological are found in the genomes of medically important niche, indicative of possible horizontal transfer (Li arboviral vectors (Ae. aegypti and Ae. albopictus), et al., 2015). Integrations from Rhabdoviridae and in the genomes of the minor arboviral vectors Chuviridae may appear to be more frequent than Ochlerotatus spp. (i.e. Oc. caspius and Oc. detri- NIRVS from other viruses simply because these tus) and Ae. vexans, but not in the genome of Cx. viruses are ecologically diverse and have wide quinquefasciatus (Vasquez et al., 2012, Palatini et al., geographical distributions. Alternatively, the infec- 2017; Whitfeld et al., 2017; Blagrove et al., 2017). tion capabilities of these viruses and their capacity Among the 19 species of Anophelinae for which to transmit between host species, genera, and a genome sequence is available, F-NIRVS were even host kingdoms in the case of rhabdoviruses, identifed only in Anopheles minimus and Anopheles depending on their ecological and geographic prox- sinensis (Palatini et al., 2017; Lequime and Lambre- imity, could select for the of generalist chts, 2017). Interestingly, the number of F-NIRVS protection mechanisms, which may include viral in the Ae. aegypti and Ae. albopictus genomes is sequence integrations.

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Integration of RNA virus evolutionarily more recent than positive-strand genomes: focus on Aedes spp. faviviruses (Koonin et al., 2015). Among all the insect species in which NIRVS have been identifed to date, Ae. aegypti and Ae. albopictus are notable for their high number of Exogenous virus resistance NIRVS and the diversity of their viral origins. mediated by endogenous viral In the genomes of these two mosquito species, elements NIRVS derived from genomes of faviviruses, Te most comprehensive current data on rhabdoviruses, reoviruses, bunyaviruses, phlebo- the efects of NIRVS on host physiology are viruses, and quaranjavirus have been identifed from studies of endogenous bornavirus-like and NIRVS have been characterized from the Vir- -encoding sequences (EBLNs) in gaviridae and Chuviridae virus families. (Crochu human, mouse and squirrel genomes (Honda and et al., 2004; Roiz et al., 2009; Katzourakis and Tomonaga, 2016). A recombinant EBLN from the Giford, 2010; Fort et al., 2012; Cui and Holmes, ground squirrel genome was expressed in human 2012; Li et al., 2015; Suzuki et al., 2017; Palatini oligodendroglioma cells, where it co-localized et al., 2017; Whitfeld et al., 2017; ter Horst et with bornaviral sites of replication in the nucleus al., 2018). All these studies agree on hundreds of and inhibited the activity of infecting Bornavirus individual NIRVS in Aedes spp., although the exact (Fujino et al., 2014). Tis dominant number of NIRVS from each viral type difers negative efect was assumed to be dependent on among studies depending on the bioinformatics the relatively high-degree of amino-acid sequence parameters, sequencing and assembly method identity (77%) between the expressed from used, and source of data (i.e. genome sequence the integrated complete viral from the ground from the Aag2 cell line or adult mosquitoes). Tis squirrel genome and the nucleocapsid protein of richness and diversity of NIRVS in Ae. aegypti and the Bornavirus (Fujino et al., 2014). However, the Ae. albopictus is striking because these species are minimum degree of sequence-identity between among the most medically important vectors of the NIRVS-expressed protein and that of cognate such as dengue, Zika, viruses essential to exert competitive or dominant and chikungunya. Tis observation has prompted interaction was not established. ongoing research eforts to understand the dis- A transcript of an EBLN of the human genome tribution of NIRVS in natural populations and (i.e. hsEBLN-1) has been proposed to function as a whether NIRVS infuence mosquito physiology long non-coding RNA and control the expression and arbovirus infection and . of the neighbouring COMMD3 gene. COMMD3 NIRVS are not distributed randomly in the encodes a protein that inhibits the NFκB immune genomes of Ae. aegypti and Ae. albopictus, but pathway (Burstein et al., 2005; Sofuku et al., 2015). rather are statistically signifcantly enriched in As such, hsEBLN-1 is considered an epigenetic regions corresponding to PIWI-interacting RNA immune regulator (Sofuku et al., 2015; Honda and (piRNA)-generating loci, called piRNA clusters Tomonaga, 2016). Tis type of function has not (Palatini et al., 2017; Whitfeld et al., 2017). NIRVS been observed for insect NIRVS, likely because, in are also diferentially distributed in geographic addition to diferences between the insect and ver- populations (Pischedda et al., 2018). A diference tebrate antiviral immune pathways, insect NIRVS between F-NIRVS and NIRVS with similarity to are predominantly found in repetitive regions of rhabdoviruses (R-NIRVS) was seen in the frst the genome and correspond to partial viral ORFs. whole-genome survey of NIRVS across fve geo- Te frst evidence of a biological role for an graphical populations of Ae. albopictus: R-NIRVS insect NIRVS came when a ≈ 420 bp sequence from were more widespread and included more ancient the Israeli acute paralysis virus (IAPV) was found integrations based on accumulation of sequence integrated into the genome of (Apis mellifera), changes than F-NIRVS (Pischedda et al., 2018), an resulting in their resistance to subsequent infection interesting result considering that negative-strand with IAPV (Maori et al., 2007). Te molecular RNA viruses like rhabdoviruses are thought to be mechanisms underlying this phenomenon were not

Curr. Issues Mol. Biol. (2020) Vol. 34 caister.com/cimb Endogenous Viral Elements in Insect Genomes | 23 elucidated however (Maori et al., 2007; see Chapter R2D2, or Ago2 results in greatly increased arbovirus 3 for details of similar observations in Drosophila). replication, indicating their central role in siRNA- mediated antiviral defence (Keene et al., 2004; Sánchez-Vargas et al., 2009). In either Ae. aegypti Endogenous viral elements mosquitoes or Ae aegypti embryo-derived Aag2 and the piRNA pathway in cell cultures, infection with the favivirus dengue mosquitoes (DENV) results in production of DENV RNA- A possible biological function for NIRVS in mos- derived 21 nt siRNAs (Scot et al., 2010). However, quitos may be to enhance antiviral defence (Meisen in Ae albopictus-derived C6/36 cells, which have a et al., 2016b; Blair and Olson, 2017; Olson and single- deletion in their Dcr2 gene (Scot Bonizzoni, 2017). An antiviral function was sug- et al., 2010), arbovirus infection drives production gested by two observations. First, detection of of virus RNA-derived 27-nt piRNAs (Scot et al., RNA virus genome-derived piRNAs was shown 2010, Brackney et al., 2010). Te more robust repli- in both uninfected and infected mosquitoes and cation of DENV in exo-siRNA-defcient mosquito mosquito cells (Scot et al., 2010; Brackney et al., cells emphasizes the importance of this pathway in 2010; Arensburger et al., 2011; Morazzoni et al., antiviral defence in Aedes spp., but production of 2012; Vodovar et al., 2012; Schnetler et al., 2013; piRNAs suggests that this pathway might have a Miesen et al., 2016a,b). Second, integration of most secondary role in anti-arbovirus defence. NIRVS occurred in or near retroelements in piRNA In Drosophila, which has served as a model for clusters of Ae. aegypti and Ae. albopictus genomes. the mosquito RNAi system, production of mature Tese observations not only suggest a mechanism piRNAs is carried out by the three members by which non-retroviral RNA genomes were con- of the Piwi subfamily of the Argonaute protein verted to cDNA and inserted into host genomes, family, Piwi, Aubergine (Aub) and Ago3, and is but also that NIRVS function similarly to piRNA Dcr-independent (Brennecke, 2007, Gunawar- clusters, the genomic loci that express transposon- dane, 2007). Tese proteins are expressed in derived piRNAs implicated in defending host germline cells of the fy and their major function genome integrity by restricting transposon activity. is to protect the genome by silencing transposable Te small-interfering RNA (siRNA) pathway of element (TE) activity. Te substrates for produc- the RNA interference (RNAi) system is the major tion of primary piRNAs are nuclear transcripts antiviral defence system in acute arboviral infections from piRNA clusters in the genome that contain of Aedes spp. mosquitoes (Sánchez-Vargas et al., defective TE sequences. Te transcripts are pro- 2009; Scot et al., 2010; Blair, 2011). In mosquitoes, cessed in the nucleus to produce mature, antisense the siRNA pathway is a cell-autonomous, cytoplas- Piwi- or Aub-associated 24–27 nt piRNAs, known mic system in which the dsRNA-specifc RNase as piRNA-induced silencing complexes (piRISC), III family member Dicer 2 (Dcr2) recognizes viral that hybridize to and cleave complementary sense- dsRNA produced during replication. Dcr2 binds strand RNA in the cytoplasm. Tis ‘slicer’ action of the dsRNA and cleaves it into 21-nt siRNA duplexes the piRISC results in positive-sense, Ago3-bound with perfect base-pairing and 2-nt overhangs at piRNA that can initiate a ‘ping-pong’ amplifcation the 3′ ends. In complex with the dsRNA-binding loop (Siomi, 2011). protein R2D2, Dcr2 loads the siRNA duplex onto Te Piwi protein subfamily has undergone Argonaute 2 (Ago2), an endonuclease that is part of expansion to eight proteins, Ago3 and Piwi1–7, the multi-component RNA-induced silencing com- in Ae. aegypti (Campbell et al., 2008), suggesting plex (RISC). Ago2 cleaves and releases one strand additional functional roles of these proteins. Also, of the siRNA duplex, retaining the second strand the piRNA pathway is active in somatic as well as as a guide to hybridize to a complementary coding germline tissues of Ae. aegypti (Morazzani et al., sequence in viral genome/mRNA, which it then 2012). Further studies are needed to determine cleaves in the centre of the complementary region. which of the eight Aedes Piwi-family proteins may Transiently knocking down expression of Dcr2, be involved in binding, processing and cleavage of

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the piRNA precursor. Determining whether a par- Furthermore, germ-line infections and vertical ticular Piwi-family protein afects virus replication transmission of dengue viruses in Ae. aegypti prob- could depend on the mosquito tissue or cultured ably occur more frequently than previously thought cells analysed, the time post-infection at which anal- (Sánchez-Vargas et al., 2018). ysis is done, and the infecting arbovirus (favivirus or alphavirus) (Vodovar et al., 2012; Morazzani et al., 2012; Schnetler et al., 2013; Miesen et al., 2015, Concluding remarks and future 2016a; Goic et al., 2016). In addition to piRNAs directions derived from NIRVS in Aedes spp., newly gener- Te frst experimental evidence that non-retroviral ated piRNAs derived from the infecting arbovirus RNA viruses can integrate genome sequences genome occur during acute infections of Aedes spp. into host DNA genomes surfaced in the 1970s cells and mosquitoes by faviviruses, , (Zhdanov, 1975) but this evidence was ignored and bunyaviruses (Scot, 2010; Morazzani, 2012; until the development of powerful and inexpen- Léger, 2013; Schnetler, 2013; Miesen, 2015, sive next-generation sequencing technology that 2016a,b). However, their role in antiviral defence enabled the sequencing of many host genomes. is uncertain and the template for transcription of Analyses of genome data are revealing an increasing virus genome-derived piRNAs (vpiRNAs) and number of viral sequence integrations in diferent protein(s) involved in their biogenesis are not fully organisms including insects. Although current data understood. are mostly descriptive, preliminary observations Several studies have shown that Aedes mosqui- are already transforming our perception of virus– toes and mosquito cells are capable of synthesizing host co-evolution, allowing for the generation of short segments of cDNA (vDNA) from alphavirus, hypotheses about connections between virus evo- favivirus, or bunyavirus genome templates, using lution, insect ecology, insect genomic structure and endogenous reverse transcriptase (RT), likely insect antiviral immunity. through recombination between viral RNA and a Understanding the signifcance of the wide- retrotransposon transcript during reverse transcrip- spread occurrence of viral integrations within tion (Vodovar et al., 2012; Nag et al., 2016; Nag and insect genomes will require analysis of the genomes Kramer, 2017). By analogy to the proviral DNA of more species (Fig. 2.1). It would be benefcial form of retrovirus genomes, this DNA is for the search for – and characterization of – viral termed ‘proviral’. Proviral DNA is hypothesized to integrations to be standard aims during the anno- enter the nucleus where it is transcribed into pri- tation of insect genomes. Tis would help answer mary piRNA precursors from vDNA episomes or whether integration of viral sequences is a common afer integration into a piRNA cluster in the genome phenomenon within insects, or if it depends on the using the retroelement integrase. Although robust structure of the host genome (e.g. on the content of production of virus genome-derived piRNAs has LTR transposons). It will also be important to verify been observed in alphavirus-infected mosquitoes which viral species/genera can efciently integrate, (Morazzani et al., 2012), no alphavirus genome- especially as non-retroviral RNA viruses have been related NIRVS have been reported in mosquitoes. proposed as delivery tools for (Mogler Te evolutionary conservation of NIRVS and their and Kamrud, 2015). Tis type of medical treatment variation in diferent populations of Aedes spp. is based on the transient presence of non-retroviral provide support for their acquisition during acute RNA viruses in host cells. For safety, it is essential infection. NIRVS acquisition and any long-term to verify whether integrations into host genomes antiviral efects for a particular arbovirus would occur because of the properties of the host genome depend on infection of mosquito germ-line tissue or of the virus. Te use of long-read technologies and integration into the vector genome of NIRVS to refne the genome assembly of species where derived from the infecting viral RNA. Recent bio- NIRVS have been characterized will help resolve informatics analyses have detected NIRVS derived repetitive regions and unravel the landscape of from Dengue virus-1 in the genome of a distinct Ae. NIRVS, including their numbers and their posi- albopictus population from ; this may signify tioning within the genome (Whitfeld et al., 2017; a recent NIRVS acquisition (Chen et al., 2015). Mathews et al., 2018). Pertinent to this aim, a new,

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A-Discovery B- Biological Function C- Exploitation

Domestication virus ? Insect Host genome EVEs Viral NIRVS Analogous? biodiversity RNAi DNA

Host genome EVEs Host genome EVEs DNA DNA

• Widespread occurrence of NIRVS • Domestication of NIRVS in gene • Modification of the NIRVS landscape in insect genomes exons or engineeringdesign of single NIRVS for vector control • and dynamics of • ISV infections of cell lines as NIRVS in natural populations models for NIRVS biology and virus • NIRVS as molecular markers for persistance • Genomic context of NIRVS integrations • Interplay of NIRVS and TE control • NIRVS and the biogenesis/function of elements associated with piRNA piRNA clusters •Recent acquisitions of NIRVS in clusters natural vector populations following outbreaks

Figure 2.1 Current knowledge gaps and areas for future research on endogenous viral elements from non-retroviral RNA viruses (i.e. non-retroviral integrated RNA virus sequences or NIRVS) in insect genomes. (A) NIRVS Discovery, a better understanding of the widespread occurrence of NIRVS in insect genomes, the timing of integrations and the genomic context in which integrations occur will lead to understanding of the process of endogenization and its impact on insect genomes. (B) Biological Function, indirect evidence on the distribution of NIRVS in the genomes of the mosquitoes Ae. aegypti and Ae. albopictus and their viral origin point to an interplay with TE control elements associated with piRNA clusters and mainly ISVs, which could be experimentally addressed in cell-line based systems. Cases of NIRVS domestication could also have occurred given the fnding of several NIRVS as complete ORFs or part of annotated exons. (C) Exploitation, depending on the diferential distribution of NIRVS in insect populations, NIRVS could be co-opted as novel molecular markers to depict population genetic structure or trace recent invasions. Additionally, given that in Ae. aegypti and Ae. albopictus NIRVS are statistically signifcantly enriched in piRNA clusters, unravelling the process of endogenization could ofer insights on the biogenesis and functions of piRNA clusters in non-model organisms. Finally, NIRVS may be harnessed for vector control by modifying the landscape of NIRVS within the vector genome or using NIRVS sites as anchors to drive expression of efector molecules. In this regard, studies need to determine whether NIRVS have a direct role in anti-viral immunity, how the level of sequence-similarity between NIRVS and incoming viruses can attain antiviral activity, and if it is possible to genetically modify mosquitoes by introducing novel NIRVS into the host genome.

more complete and accurate annotation of the NIRVS or introduce novel viral sequences) may Ae. aegypti genome, designated AaegL5, has been be difcult. Examining the genomic landscape and released to resolve additional NIRVS and piRNA polymorphism of NIRVS in natural specimens will cluster sites (Mathews et al., 2018). Tis informa- enable exploration of the pervasiveness and micro- tion could enable testable predictions regarding the evolution of integrations. Finally, the similarity of mechanisms and entities involved in the integration most NIRVS to the genomes of ISVs, most of which process (e.g. by confrming NIRVS arrangements are easily cultivated in insect cells, point to the with respect to specifc TE lineages). At the same promise of using ISVs and insect hosts as ‘ time, elucidating the sites of integrations could help model’ systems to elucidate the endogenization design experiments to test for NIRVS functions. process and study the impact of NIRVS on exog- If integrations are numerous, if NIRVS are dupli- enous virus infections. cated and located primarily within piRNA clusters, Te major controller of acute arbovirus infec- which are composed of TE fragments, using genetic tion in the vector is the siRNA pathway of RNAi. engineering strategies that require unique anchor We (and others) have observed that when the RNAi sequences to modify NIRVS (i.e. knock-out inhibitor B2 (from Flock House virus) is expressed

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by the arbovirus Sindbis (genus Alphavirus), Sind- bunyavirid-like sequences in insect genomes. J. Virol. 88, 8783–8794. htps://doi.org/10.1128/JVI.00531-14 bis virus infection in the vector is greatly increased Best, S.M., and Kerr, P. (2000). of host and and the ftness of the vector is signifcantly lowered virus: the pathogenesis of virulent and atenuated strains (Myles et al., 2008; Cirimotich et al., 2009). We of myxoma virus in resistant and susceptible european hypothesize that NIRVS act by expressing virus rabbits. 267, 36–48. Bézier, A., Annaheim, M., Herbiniere, J., Weterwald, C., genome-derived piRNAs in the vector to target and Gyapay, G., Bernard-Samain, S., Wincker, P., Roditi, I., control infections. Tus, NIRVS may represent a Heller, M., Belghazi, M., et al. (2009). Polydnaviruses fortuitous co- between arboviruses and of braconid wasps derive from an ancestral . vectors (Blair and Olson, 2015; Olson and Blair, Science 323, 926–930. htps://doi.org/10.1126/ science.1166788. 2016; Palatini et al., 2017). NIRVS probably do not Blagrove, M.S., Sherlock, K., Chapman, G.E., Impoivil, D.E., prevent or ablate infections but help maintain virus McCall, P.J., Medlock, J.M., Lycet, G., Solomon, T., and persistence in the vectors. Baylis, M. (2017). Evaluation of vector competence of a We have developed RNAi- or -based native UK mosquito Ochlerotatus detritus (Aedes detritus) for dengue, Chikungunya and West Nile viruses. Parasit. antiviral efector genes. Integration and expression Vectors 9, 452. htps://doi.org/10.1186/s13071-016- of either of these efector genes in Ae. aegypti at the 1739-3 time of infection profoundly lowers or eliminates Blair, C.D. (2011). 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