Varroa destructor shapes the unique viral landscapes of the honey bee populations of the Azores archipelago.

The worldwide dispersal of the ectoparasitic mite Varroa destructor from its Asian origins has fundamentally transformed the relationship of the honey bee (Apis mellifera) with several of its viruses, via changes in transmission and/or host immunosuppression. The extent to which honey bee-virus relationships change after Varroa invasion is poorly understood for most viruses, in part because there are few places in the world with several geographically close but completely isolated honey bee populations that either have, or have not, been exposed long-term to Varroa, allowing for separate ecological, epidemiological, and adaptive relationships to develop between honey bees and their viruses, in relation to the mite's presence or absence. The Azores is one such place, as it contains islands with and without the mite. Here, we combined qPCR with meta-amplicon deep sequencing to uncover the relationship between Varroa presence, and the prevalence, load, diversity, and phylogeographic structure of eight honey bee viruses screened across the archipelago. Four viruses were not detected on any island (ABPV-Acute bee paralysis virus, KBV-Kashmir bee virus, IAPV-Israeli acute bee paralysis virus, BeeMLV-Bee macula-like virus); one (SBV-Sacbrood virus) was detected only on mite-infested islands; one (CBPV-Chronic bee paralysis virus) occurred on some islands, and two (BQCV-Black queen cell virus, LSV-Lake Sinai virus,) were present on every single island. This multi-virus screening builds upon a parallel survey of Deformed wing virus (DWV) strains that uncovered a remarkably heterogeneous viral landscape featuring Varroa-infested islands dominated by DWV-A and -B, Varroa-free islands naïve to DWV, and a refuge of the rare DWV-C dominating the easternmost Varroa-free islands. While all four detected viruses investigated here were affected by Varroa for one or two parameters (usually prevalence and/or the Richness component of ASV diversity), the strongest effect was observed for the multi-strain LSV. Varroa unambiguously led to elevated prevalence, load, and diversity (Richness and Shannon Index) of LSV, with these results largely shaped by LSV-2, a major LSV strain. Unprecedented insights into the mite-virus relationship were further gained from implementing a phylogeographic approach. In addition to enabling the identification of a novel LSV strain that dominated the unique viral landscape of the easternmost islands, this approach, in combination with the recovered diversity patterns, strongly suggests that Varroa is driving the evolutionary change of LSV in the Azores. This study greatly advances the current understanding of the effect of Varroa on the epidemiology and adaptive evolution of these less-studied viruses, whose relationship with Varroa has thus far been poorly defined.

Identification and Genome Characterization of a Novel Nege-like Virus Isolated from Aphids (Aphis gossypii) in Yunnan Province.

Negeviruses are insect-specific enveloped RNA viruses that exhibit a wide geographic distribution. A novel nege-like virus, tentatively named Aphis gossypii nege-like virus (AGNLV, GenBank: OR880429.1), was isolated from aphids (Aphis gossypii) in Lijiang City, Yunnan, China. AGNLV has a genome sequence of 9258 nt (excluding the polyA tail) encoding three open reading frames (ORFs). ORF1 (7149 nt) encodes a viral methyltransferase, a viral RNA helicase, and an RNA-dependent RNA polymerase. ORF2 (1422 nt) encodes a DiSB-ORF2_chro domain and ORF3 encodes an SP24 domain. The genome sequence of AGNLV shares the highest nucleotide identity of 60.0% and 59.5% with Wuhan house centipede virus 1 (WHCV1) and Astegopteryx formosana nege-like virus (AFNLV), respectively. Phylogenetic analysis based on the RNA-dependent RNA polymerase shows that AGNLV is clustered with other negeviruses and nege-like viruses discovered in aphids, forming a distinct "unclassified clade". Interestingly, AGNLV only encodes three ORFs, whereas AFNLV and WHCV1 have four ORFs. Structure and transmembrane domain predictions show the presence of eight alpha helices and five transmembrane helices in the AGNLV ORF3. Translational enhancement of the AGNLV 5' UTR was similar to that of the 5' UTR of plant viruses. Our findings provide evidence of the diversity and structure of nege-like viruses and are the first record of such a virus from a member of the genus Aphis.

Structural diversity and phylogenetic distribution of valyl tRNA-like structures in viruses.

Viruses commonly use specifically folded RNA elements that interact with both host and viral proteins to perform functions important for diverse viral processes. Examples are found at the 3' termini of certain positive-sense ssRNA virus genomes where they partially mimic tRNAs, including being aminoacylated by host cell enzymes. Valine-accepting tRNA-like structures (TLSVal) are an example that share some clear homology with canonical tRNAs but have several important structural differences. Although many examples of TLSVal have been identified, we lacked a full understanding of their structural diversity and phylogenetic distribution. To address this, we undertook an in-depth bioinformatic and biochemical investigation of these RNAs, guided by recent high-resolution structures of a TLSVal We cataloged many new examples in plant-infecting viruses but also in unrelated insect-specific viruses. Using biochemical and structural approaches, we verified the secondary structure of representative TLSVal substrates and tested their ability to be valylated, confirming previous observations of structural heterogeneity within this class. In a few cases, large stem-loop structures are inserted within variable regions located in an area of the TLS distal to known host cell factor binding sites. In addition, we identified one virus whose TLS has switched its anticodon away from valine, causing a loss of valylation activity; the implications of this remain unclear. These results refine our understanding of the structural and functional mechanistic details of tRNA mimicry and how this may be used in viral infection.

Enoxacin Shows Broad-Spectrum Antiviral Activity against Diverse Viruses by Enhancing Antiviral RNA Interference in Insects.

RNA interference (RNAi) functions as the major host antiviral defense in insects, while less is understood about how to utilize antiviral RNAi in controlling viral infection in insects. Enoxacin belongs to the family of synthetic antibacterial compounds based on a fluoroquinolone skeleton that has been previously found to enhance RNAi in mammalian cells. In this study, we show that enoxacin efficiently inhibited viral replication of Drosophila C virus (DCV) and cricket paralysis virus (CrPV) in cultured Drosophila cells. Enoxacin promoted the loading of Dicer-2-processed virus-derived small interfering RNA (siRNA) into the RNA-induced silencing complex, thereby enhancing the antiviral RNAi response in infected cells. Moreover, enoxacin treatment elicited RNAi-dependent in vivo protective efficacy against DCV or CrPV challenge in adult fruit flies. In addition, enoxacin also inhibited the replication of flaviviruses, including dengue virus and Zika virus, in Aedes mosquito cells in an RNAi-dependent manner. Together, our findings demonstrate that enoxacin can enhance RNAi in insects, and enhancing RNAi by enoxacin is an effective antiviral strategy against diverse viruses in insects, which may be exploited as a broad-spectrum antiviral agent to control the vector transmission of arboviruses or viral diseases in insect farming. IMPORTANCE RNAi has been widely recognized as one of the most broadly acting and robust antiviral mechanisms in insects. However, the application of antiviral RNAi in controlling viral infections in insects is less understood. Enoxacin is a fluoroquinolone compound that was previously found to enhance RNAi in mammalian cells, while its RNAi-enhancing activity has not been assessed in insects. Here, we show that enoxacin treatment inhibited viral replication of DCV and CrPV in Drosophila cells and adult fruit flies. Enoxacin promoted the loading of Dicer-generated virus-derived siRNA into the Ago2-incorporated RNA-induced silencing complex and in turn strengthened the antiviral RNAi response in the infected cells. Moreover, enoxacin displayed effective RNAi-dependent antiviral effects against flaviviruses, such as dengue virus and Zika virus, in mosquito cells. This study is the first to demonstrate that enhancing RNAi by enoxacin elicits potent antiviral effects against diverse viruses in insects.

The first complete genome sequence of an iflavirus from the endoparasitoid wasp Tetrastichus brontispae.

The complete genome sequence of a novel iflavirus isolated from the gregarious and koinobiont endoparasitoid Tetrastichus brontispae, tentatively named "Tetrastichus brontispae RNA virus 3" (TbRV-3), was determined by total RNA and Sanger sequencing. The complete genome is 9998 nucleotides in length, 8934 nt of which encodes a putative polyprotein of 2978 amino acids. TbRV-3 was found to have a similar genome organization and to contain conserved domains and motifs found in other iflaviruses, with some variations. Phylogenetic analysis based on deduced amino acid sequences of the RdRp domain showed that TbRV-3 clustered with Dinocampus coccinellae paralysis virus (DcPV). However, the percent amino acid sequence identity of the putative capsid proteins of TbRV-3 and DcPV determined using BLASTp was below the species demarcation threshold (90%), suggesting that TbRV-3 is a new iflavirus. This is the first virus of the family Iflaviridae to be isolated from a wasp of the family Eulophidae.

The Insect Virome: Opportunities and Challenges.

The insect virome is composed of a myriad of viruses. Both field populations and laboratory colonies of insects harbour diverse viruses, including viruses that infect the insect itself, viruses of microbes associated with the insect, and viruses associated with ingested materials. Metagenomics analysis for identification of virus-derived sequences has allowed for new appreciation of the extent and diversity of the insect virome. The complex interactions between insect viruses and host antiviral immune pathways (RNA interference and apoptosis), and between viruses and other members of the microbiome (e.g. Wolbachia) are becoming apparent. In this chapter, an overview of the diversity of viruses in insects and recent virus discovery research for specific insects and insect-derived cell lines is provided. The opportunities and challenges associated with the insect virome, including the potential impacts of viruses on both research and insect management programs are also addressed.

ICTV Virus Taxonomy Profile: Metaviridae.

Metaviridae is a family of retrotransposons and reverse-transcribing viruses with long terminal repeats belonging to the order Ortervirales. Members of the genera Errantivirus and Metavirus include, respectively, Saccharomyces cerevisiae Ty3 virus and its Gypsy-like relatives in drosophilids. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Metaviridae, which is available at ictv.global/report/metaviridae.

Effect of Wolbachia wAlbB on a positive-sense RNA negev-like virus: a novel virus persistently infecting Aedes albopictus mosquitoes and cells.

The Aedes aegypti mosquito is the primary vector of several medically important arboviruses. The endosymbiotic bacterium, Wolbachia pipientis, has emerged as a means of blocking transmission of arboviruses such as dengue and Zika viruses. One Wolbachia strain that has shown potential in field trials is wAlbB, a naturally occurring Wolbachia strain of the Asian tiger mosquito Aedes albopictus. When transinfected into Ae. aegypti, wAlbB exhibits strong virus inhibition. In addition to modulating arboviruses, Wolbachia also modulates some insect-specific viruses. Here, we explored the effect of Wolbachia on the virome of the Ae. albopictus cell line Aa23 naturally infected with wAlbB and also a stably transinfected recipient Ae. aegypti cell line (Aag2.wAlbB). RNA sequencing and bioinformatic analysis on both cell lines revealed an 11 kb genome of a single-stranded positive-sense RNA negev-like virus related to the recently proposed negevirus taxon. We denoted this novel virus as Aedes albopictus negev-like virus (AalNLV). Tetracycline clearance of Wolbachia from Aa23 cells did not significantly affect AalNLV levels, while in Aag2.wAlbB cells, a significant increase in virus genome RNA copies was observed. We further investigated the inhibitory effect of wAlbB on AalNLV and another positive-sense RNA virus, cell fusing agent virus, which is present in Aag2 cells and known to be suppressed by Wolbachia. wAlbB suppressed both viruses, with the effect on AalNLV being more striking. The findings from this study further supplement our understanding of the complex interaction between Wolbachia, host and virome.

Diaphorina citri densovirus is a persistently infecting virus with a hybrid genome organization and unique transcription strategy.

Diaphorina citri densovirus (DcDV) is an ambisense densovirus with a 5071 nt genome. Phylogenetic analysis places DcDV in an intermediate position between those in the Ambidensovirus and Iteradensovirus genera, a finding that is consistent with the observation that DcDV possesses an Iteradensoviris-like non-structural (NS) protein-gene cassette, but a capsid-protein (VP) gene cassette resembling those of other ambisense densoviruses. DcDV is maternally transmitted to 100 % of the progeny of infected female Diaphorina citri, and the progeny of infected females carry DcDV as a persistent infection without outward phenotypic effects. We were unable to infect naïve individuals by oral inoculation, however low levels of transient viral replication are detected following intrathoracic injection of DcDV virions into uninfected D. citri insects. Transcript mapping indicates that DcDV produces one transcript each from the NS and VP gene cassettes and that these transcripts are polyadenylated at internal sites to produce a ~2.2 kb transcript encoding the NS proteins and a ~2.4 kb transcript encoding the VP proteins. Additionally, we found that transcriptional readthrough leads to the production of longer non-canonical transcripts from both genomic strands.

Chronic bee paralysis virus (CBPV) in South American non-Apis bees.

Chronic bee paralysis virus (CBPV) is a positive single-stranded RNA virus that exhibits a worldwide distribution. Although the effects of this virus on honeybees' health are well known, its presence in other bee species has not been fully studied. In this work, CBPV was detected in several native bees from Argentina, including Bombus pauloensis, Halictillus amplilobus, Peponapis fervens, and members of the genus Xylocopa. Here, we report for the first time the presence of CBPV in native bees from South America.

A novel circular Rep-encoding single-stranded DNA virus detected in Agrotis ipsilon (Lepidoptera: Noctuidae) in China.

Long-distance migratory insects carry microorganisms that can potentially play a crucial role in the life cycles of their hosts. Here, we used Illumina and Sanger sequencing to determine the complete genome sequence of a novel circular Rep-encoding single-stranded (ss) DNA virus from an important migratory pest, Agrotis ipsilon (Hufnagel). The full genome of this new virus is about 2, 242 nt in length and shares 55-75% genome-wide pairwise sequence identity with members of the family Genomoviridae but 91% nucleotide sequence identity with finch-associated genomovirus 3 isolate S30P_D, which is tentatively abbreviated "FaGmV-3". Viral infection rates in A. ipsilon from Yantai, Langfang and Xinxiang were 4.5% (n = 88), 11.8% (n = 85) and 0% (n = 35), respectively. Phylogenetic analysis based on the deduced amino acid sequence of Rep indicated that the Agrotis ipsilon-associated virus is closely related to members of the genus Gemykibivirus, and we propose it to be a new member of this genus. Hence, it is tentatively named "Agrotis ipsilon-associated genomovirus" (AiGmV).

Isolation and characterization of a novel cripavirus, the first Dicistroviridae family member infecting the cotton mealybug Phenacoccus solenopsis.

A new virus belonging to the family Dicistroviridae was identified in the hibiscus-infesting cotton mealybug Phenacoccus solenopsis. Using high-throughput sequencing (HTS) on an Illumina HiSeq platform, a single contig of the complete genome sequence was assembled. The authenticity of the sequence obtained by HTS was validated by RT-PCR and Sanger sequencing of the amplicons, which was also employed for the 3' untranslated region (UTR). The 5' UTR was sequenced using a rapid amplification of cDNA ends kit. A large segment encompassing the whole genome was amplified by RT-PCR using viral RNA extracted from mealybugs. A whole-genome nucleotide sequence comparison showed 89% sequence identity to aphid lethal paralysis virus (ALPV), covering a short segment of 44 bp. Pairwise amino acid sequence comparisons of the protein encoded by open reading frame (ORF) 2 with its counterparts in the GenBank database, showed less than 40% identity to several members of the genus Cripavirus, including ALPV. Phylogenetic analysis based on the deduced amino acid sequence of the ORF 2 protein showed that the new virus grouped with members of the genus Cripavirus. The intergenic region (IGR) internal ribosome entry site (IRES) showed the conserved nucleotides of a type I IGR IRES and had two bulge sites, three pseudoknots, and two stem-loops. Virus morphology visualized by transmission electron microscopy demonstrated spherical particles with a diameter of ~30 nm. This virus was the only arthropod virus identified in the sampled mealybugs, and the purified virus was able to infect cotton mealybugs. To the best of our knowledge, this is the first report of a Dicistroviridae family member infecting P. solenopsis, and we have tentatively named this virus Phenacoccus solenopsis virus (PhSoV).

A novel negevirus isolated from Aedes vexans mosquitoes in Finland.

Negeviruses are insect-specific enveloped RNA viruses that have been detected in mosquitoes and sandflies from various geographical locations. Here, we describe a new negevirus from Northern Europe, isolated from pool of Aedes vexans mosquitoes collected in Finland, designated as Mekrijärvi negevirus (MEJNV). MEJNV had a typical negevirus genome organization, is 9,740 nucleotides in length, and has a GC content of 47.53%. The MEJNV genome contains three ORFs, each containing the following identified conserved domains: ORF1 (7,068 nt) encodes a viral methyltransferase, an FtsJ-like methyltransferase, a viral RNA helicase, and an RNA-dependent RNA polymerase, ORF2 (1,242 nt) encodes a putative virion glycoprotein, and ORF3 (660 nt) encodes a putative virion membrane protein. A distinctive feature relative to other currently known negeviruses is a 7-nucleotide-long overlap between ORF1 and ORF2. MEJNV shares the highest sequence identity with Ying Kou virus from China, with 67.71% nucleotide and 75.19% and 59.00% amino acid sequence identity in ORF 1 and ORF 2, respectively. ORF3 had the highest amino acid sequence similarity to Daeseongdong virus 1 and negevirus Nona 1, both with 77.61% identity, and to Ying Kou virus, with 71.22% identity. MEJNV is currently the northernmost negevirus described. Our report supports the view that negeviruses are a globally distributed, diverse group of viruses that can be found from mosquitoes in a wide range of terrestrial biomes from tropical to boreal forests.

Complete genome sequence of a novel rhabdo-like virus from the Chinese black cutworm Agrotis ipsilon (Lepidoptera: Noctuidae).

This work identified a novel rhabdo-like virus in a Chinese black cutworm (Agrotis ipsilon), which we tentatively named "Agrotis ipsilon virus" (AIpsV). The complete genome of AIpsV is 15,454 nucleotides in length and contains seven open reading frames, collectively encoding more than 160 amino acids. The AIpsV genome is predicted to encode three structural proteins, nucleoprotein (N), glycoprotein (G), and large polymerase protein (L), and four unknown proteins. Phylogenetic analysis revealed that the AIpsV clusters with Wuhan ant virus and Hubei rhabdo-like virus 1 within the rhabdo-like virus clade. The level of expression of AIpsV genes was found to be higher in the pupal and adult stages than in the egg and larval stages.

ICTV Virus Taxonomy Profile: Hytrosaviridae.

Hytrosaviridae is a family of large, rod-shaped, enveloped entomopathogenic viruses with dsDNA genomes of 120-190 kbp. Hytrosaviruses (also known as salivary gland hypertrophy viruses) primarily replicate in the salivary glands of adult dipteran flies. Hytrosaviruses infecting the haematophagous tsetse fly and the filth-feeding housefly are assigned to two genera, Glossinavirus and Muscavirus, respectively. Whereas muscavirus infections are only overt, glossinavirus infections can be either covert or overt. Overt infections are characterized by diagnostic salivary gland hypertrophy and cause either partial or complete infertility. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Hytrosaviridae, which is available at ictv.global/report/hytrosaviridae.

Aedes Anphevirus: an Insect-Specific Virus Distributed Worldwide in Aedes aegypti Mosquitoes That Has Complex Interplays with Wolbachia and Dengue Virus Infection in Cells.

Insect-specific viruses (ISVs) of the yellow fever mosquito Aedes aegypti have been demonstrated to modulate transmission of arboviruses such as dengue virus (DENV) and West Nile virus by the mosquito. The diversity and composition of the virome of A. aegypti, however, remains poorly understood. In this study, we characterized Aedes anphevirus (AeAV), a negative-sense RNA virus from the order Mononegavirales AeAV identified from Aedes cell lines was infectious to both A. aegypti and Aedes albopictus cells but not to three mammalian cell lines. To understand the incidence and genetic diversity of AeAV, we assembled 17 coding-complete and two partial genomes of AeAV from available transcriptome sequencing (RNA-Seq) data. AeAV appears to transmit vertically and be present in laboratory colonies, wild-caught mosquitoes, and cell lines worldwide. Phylogenetic analysis of AeAV strains indicates that as the A. aegypti mosquito has expanded into the Americas and Asia-Pacific, AeAV has evolved into monophyletic African, American, and Asia-Pacific lineages. The endosymbiotic bacterium Wolbachia pipientis restricts positive-sense RNA viruses in A. aegypti Reanalysis of a small RNA library of A. aegypti cells coinfected with AeAV and Wolbachia produces an abundant RNA interference (RNAi) response consistent with persistent virus replication. We found Wolbachia enhances replication of AeAV compared to a tetracycline-cleared cell line, and AeAV modestly reduces DENV replication in vitro The results from our study improve understanding of the diversity and evolution of the virome of A. aegypti and adds to previous evidence that shows Wolbachia does not restrict a range of negative-strand RNA viruses.IMPORTANCE The mosquito Aedes aegypti transmits a number of arthropod-borne viruses (arboviruses), such as dengue virus and Zika virus. Mosquitoes also harbor insect-specific viruses that may affect replication of pathogenic arboviruses in their body. Currently, however, there are only a few insect-specific viruses described from A. aegypti in the literature. Here, we characterize a novel negative-strand virus, AeAV. Meta-analysis of A. aegypti samples showed that it is present in A. aegypti mosquitoes worldwide and is vertically transmitted. Wolbachia-transinfected mosquitoes are currently being used in biocontrol, as they effectively block transmission of several positive-sense RNA viruses in mosquitoes. Our results demonstrate that Wolbachia enhances the replication of AeAV and modestly reduces dengue virus replication in a cell line model. This study expands our understanding of the virome in A. aegypti as well as providing insight into the complexity of the Wolbachia virus restriction phenotype.

Discovery of three RNA viruses using ant transcriptomic datasets.

Three novel RNA viruses, named Formica fusca virus 1 (GenBank accession no. MH477287), Lasius neglectus virus 2 (MH477288) and Myrmica scabrinodis virus 2 (MH477289), were discovered in ants collected in Cambridge, UK. The proposed virus names were given based on the hosts in which they were identified. The genome sequences were obtained using de novo transcriptome assembly of high-throughput RNA sequencing reads and confirmed by Sanger sequencing. Phylogenetic analysis showed that Formica fusca virus 1 grouped within the family Nyamiviridae, Lasius neglectus virus 2 grouped within the family Rhabdoviridae and Myrmica scabrinodis virus 2 belongs to the family Dicistroviridae. All three viruses are highly divergent from previously sequenced viruses.

Complete genome sequence of a novel negevirus isolated from Culex tritaeniorhynchus in China.

A novel negevirus, tentatively named Manglie virus (MaV), was isolated from Culex tritaeniorhynchus from the village of Manglie, Yunnan, China, in August 2011. It was identified by high-throughput sequencing of cell culture supernatants, and the complete genome was sequenced using an Illumina MiSeq sequencer. The complete MaV genome comprised 9,218 nt encoding three hypothetical proteins and had a poly(A) tail. BLASTn analysis showed that the genome had the greatest similarity to Ngewotan virus strain Nepal22, with query coverage of 100% and 79% identity. Genomic and phylogenetic analyses demonstrated that MaV should be considered a novel negevirus.

Isolation and genomic characterization of a cypovirus from the oleander hawk moth, Daphnis nerii.

The oleander hawk moth, Daphnis nerii, is a serious pest of plants belonging to the family Apocynaceae. Thus far, pathogen infection has not been reported in D. nerii. In this study, a new cytoplasmic polyhedrosis virus (cypovirus; CPV) was isolated from naturally diseased D. nerii larvae and named DnCPV-23. Virions were observed in ultrathin sections of DnCPV polyhedral bodies. Electrophoretic analysis revealed that the DnCPV genome consisted of 10 segments of double-stranded RNA (dsRNA). cDNA copies of these dsRNA segments were amplified using the method of full-length amplification of cDNAs (FLAC), cloned, and sequenced. Sequencing results showed that all segments contained one open reading frame (ORF); They shared the conserved terminal sequences AGUCAAA and AGC at 5' and 3' ends respectively, except segment 4, which is different from previously reported 22 cypoviruses. Phylogenetic analysis based on amino acid sequences of polyhedrin (encoded by segment 10) indicated that this CPV was closely related to CPV type 19. Altogether, DnCPV-23 is a new type of cypovirus.

The insect reservoir of biodiversity for viruses and for antiviral mechanisms.

Insects are the most diverse group of animals. They can be infected by an extraordinary diversity of viruses. Among them, arthropod-borne viruses (arboviruses) can be transmitted to humans. High-throughput sequencing of small RNAs from insects provides insight into their virome, which may help understand the dynamics of vector borne infectious diseases. Furthermore, investigating the mechanisms that restrict viral infections in insects points to genetic innovations that may inspire novel antiviral strategies.