Abundant and Diverse RNA Viruses in Insects Revealed by RNA-Seq Analysis: Ecological and Evolutionary Implications

Abundant and divergent RNA viruses identified in insects.We performed the virus discovery analyses on a curated collection of RNA-Seq data available publicly in databases. The data set contained 664 species spanning 32 orders of insects (see Data Set S1 in the supplemental material). The assembled contigs were subjected to BLAST analyses against the reference viral databases. This resulted in ∼16,990 virus-associated contigs from 648 insect species, of which 89.35% were associated with two or more virus species (Data Set S1). To elucidate the evolutionary aspect of the newly identified viruses, we extracted the putative viral contigs that had significant matches to the highly conserved viral RNA-dependent RNA polymerase (RdRp) domain and reconstructed the phylogenetic trees together with the recognized taxa of RNA viruses proposed by the International Committee on Taxonomy of Viruses (ICTV [https://talk.ictvonline.org/taxonomy/vmr/]), as well as some unclassified RNA viruses occupying important phylogenetic positions. A total of 1,213 contigs representing complete or partial viral genomes were recovered from the RNA-Seq data for 484 insect species (484/664, 72.89%) (see Fig. S1a in the supplemental material; see also Data Set S1). Phylogenetic analysis demonstrated that these putative RNA viruses fell within or in close proximity to a wide range of recognized viral taxa, including 40 viral families and 2 unclassified genera, as well as newly described RNA virus groups, such as Qinviridae and Chuviridae (Data Set S1). Nevertheless, BLAST analysis and pairwise comparisons revealed that the sequences of most of the newly identified RNA viruses were highly divergent from the previous reported viral sequences: almost 1,194 (91.01%) shared less than 70% amino acid (aa) identity with the most closely related viruses, while 471 (35.93%) shared less than 40% (Data Set S1; see also Fig. S1b).

The current official taxonomy could not recapitulate the extremely diverse RNA virus populations in insects accurately. Indeed, more than 40% of the novel viruses could not be precisely assigned to the current taxa or exhibited low confidence levels. Instead, we reclassified the RNA viruses into 26 clades as described for previous studies (Fig. 1a) (9, 33). These viral clades were abbreviated based on the representative members of each clade (e.g., “Picorna” is an abbreviation representing Picornavirales, Solinviviridae, and Caliciviridae). Although these viruses were highly divergent, their taxonomic status could be deduced based on the panoramic phylogenies. Newly discovered viruses were assigned into 22 viral clades, of which 13 were relatively ubiquitous as they were observed in more than 10 insect orders (Fig. 1b; see also Data Set S1). The others, including one putative RNA phage within the Levi clade, appeared to be infrequent. A clearly separated group which cannot be confidently assigned to any currently recognized taxon was found on the phylogenies (Fig. 1a). The novel clade comprised 36 viruses that were mostly identified in insect orders Psocodea (n = 21) and Hemiptera (n = 9). The closest relative of these viruses was Agaricus bisporus virus 16 (ABV16), a putative four-segmented virus found in mushroom (25.62% to 44.65% amino acid similarity for RdRp segment, Data Set S1) (34). These viruses might represent a novel viral family or order.

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FIG 1

Taxonomic diversity and distribution of RNA viruses in insects. (a) Taxonomic reassessment of the RNA viruses based on RdRp. For each phylogeny, branches are collapsed and are displayed as triangles filled with different colors (see legend). The name of each clade is abbreviated based on the collapsed viral families or orders. The number of viral contigs assigned to each clade is indicated by the Arabic numerals next to the clade name. (b) Percent relative abundance of RNA viruses across each insect order. Those insect orders with too few samples (<10 samples) analyzed in our data set are not listed individually. Astro, Astroviridae; Aspi, Aspiviridae; Hepe-Beny, Benyviridae, Hepeviridae, Alphatetraviridae, Togaviridae, Bastrovirus; Birna, Birnavirdae; Bunya, Bunyavirales, Arenaviridae; Cysto, Cystoviridae; Endorna, Endornaviridae; Flavi, Flaviviridae; Hypo, Hypoviridae; Levi, Leviviridae; Mononega, Mononegavirales, Jingchuvirales; Narna, Narnaviridae, Botourmiaviridae; Nido, Nidovirales; Partiti, Partitiviridae, Amalgaviridae, Picobirnaviridae; Orthomyxo, Orthomyxoviridae; Permutotetra, Permutotetraviridae; Poty, Potyviridae; Picorna, Picornavirales, Solinviviridaes, Caliciviridae, Marnaviridae; Qin, Qinviridae; Solemo, Solemoviridae, Luteoviridae, Barnaviridae, Alvernaviridae; Reo, Reoviridae; Tombus, Tombusviridae, Nodaviridae, Sinaivirus, Carmotetraviridae, Luteoviridae; Toti, Totiviridae, Chrysoviridae, Megabirnaviridae, Quadriviridae, Botybirnavirus; Tymo, Tymovirales; Virga, Virgaviridae, Togaviridae, Bromoviridae, Closteroviridae, Idaeovirus; Wei, Weivirus; NA, unclassified.

The detailed evolutionary landscape was reevaluated by phylogenetic analyses based on all available RNA viruses by clade. As indicated, viruses identified in insects were distributed throughout different phylogenies (Fig. 2; see also Fig. S2 to S6), implying a broader host range and much-more-diverse RNA virus communities in insects. The observed categories of RNA viruses could be comparable to those of the whole invertebrates, although some taxonomic groups appeared to be more highly represented in insects, including the recognized insect-specific iflaviruses, dicistroviruses, bunyaviruses, rhabdoviruses, flaviviruses, and negeviruses. A growing number of novel RNA viruses formed apparent insect-specific lineages within many other clades, such as Hepe-Beny, Orthomyxo, Tombus, Toti, Tymo, Solemo, Narna, Virga, Nido and Partiti (for definitions of the abbreviations of the virus designation that appear here and throughout, see the Fig. 1 legend). It should be noted that closely related species of plant viruses were frequently discovered in insects. These putative plant viruses might be transmitted by insect vectors and were most commonly found in clade Tymo, as well as some new members of Secoviridae, Virgaviridae, Partitiviridae, Aspiviridae, Botourmiaviridae, and other plant virus species (Data Set S1).

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FIG 2

Genetic diversity of RNA viruses in insects. The illustrated phylogenetic trees, which 19 major groups of RNA viruses, were inferred based on RdRp data using the maximum likelihood method and rerooted at midpoint. As indicated, a branch background in red shading represents viruses identified in this study, and a branch background in shading represents those described previously. Host groups are indicated by differently colored bars (see legend). Scale bars represent 0.5 amino acid substitutions per site. Detailed phylogenies are available in Fig. S2 to S6.

Novel positive-sense single-stranded RNA (+ssRNA) viruses.The picorna-like viruses were significantly overrepresented in insects (Fig. 1b), accounting for up to 31.08% (377/1,213) of the novel viruses, and were present in 33.73% (224/664) of the insect species. Novel picorna-like viruses were found to diversify into multiple different evolutionary lineages (Fig. S2), in which the insect-associated Iflaviridae and Dicistroviridae were the most abundant (73.7% of all picorna-like viruses). The family Secoviridae was the major group of plant picorna-like viruses (35); which six novel strains were identified with 48.3% to 99.0% aa identity to the most closely related plant virus species. Of those, two close relatives of nepoviruses (93.7% nucleotide [nt] pairwise identity) were identified in a pollinating bee (Halictus quadricinctus) and a predatory wasp (Crabro peltarius), respectively, and higher viral relative abundance was identified in Halictus quadricinctus (0.17% total reads). It might be expected that Halictus quadricinctus acted as a transmission vector for nepoviruses. Other plant virus-like sequences were scattered throughout the trees, such as the strains closely related to the plant-infecting Tomato matilda virus (TmaV) within Iflaviridae. In addition, some viruses were found to be evolutionarily related to vertebrate-associated species. For example, we observed a new picornavirus in Diplonychus rusticus that best matched Rhimavirus A (39.5% aa identity), a member of the proposed reptile- and amphibian-specific genus Rafivirus within the Picornaviridae (36). Another virus that was found in Hydrochara caraboides appeared to be closely related to Ampivirus A1 (83.0% aa identity), previously identified in a smooth newt (37). In the unclassified posavirus-like cluster, four novel members were found to be related to the isolates from humans, pigs, rats, and bats, though the identities of the natural hosts have not yet been clarified.

Twenty-five novel flavi-like viruses were identified, including nine in the newly described segmented Jingmenvirus (Fig. S3) (38). Novel jingmenviruses clustered together with Wuhan cricket virus (WHCV) and not with the tickborne Alongshan virus (ALSV) and Toxocara canis larva agent (TCLA). Notably, a novel jingmenvirus species was present in Ctenocephalides felis (cat flea), which might be implicated as a biological vector of Jingmenvirus, as previously described for blood-sucking ticks and mosquitoes (38–40). Another three viruses were clustered with Wenzhou shark flavivirus, representing a disparate evolutionary lineage from Flavivirus and Jingmenvirus. More viruses fell outside the Flavi-Jingmenvirus, Hepa-Pegivirus, and Pestivirus lineages. These viruses represented a highly divergent subset of unclassified viruses within the Flavi clade.

The newly identified tymovirus-like viruses comprised five major well-supported groups (Fig. S3), among which four groups were affiliated with known plant/fungus virus groups in the order Tymovirales. A total of 17 novel strains were recognized as plant viruses. These viruses demonstrated a close relationship with the well-known plant-infecting species, either with a high percentage of identity (70% nt identity with 90% coverage, Data Set S1) or resolved into a single monophyletic clade in the phylogenies. In addition, novel members within the Maculavirus were largely hosted by phytophagous or omnivorous insects, representing potential plant viruses (41). Fourteen novel viruses formed a deeply divergent branch distantly related to Deltaflexiviridae and were most frequently identified in insect order Psocodea (11/14, 78.57%). A BLASTX search revealed that the conserved RdRp domain shared low (26.58% to 34.36%) amino acid similarity with the most closely related viral sequences. The topologies of the phylogenetic trees demonstrated that these viruses belonged to a novel taxonomic group associated with insects, though the host range should be reconfirmed with more data. Within other plant/fungus-associated clades, such as Virga, Solemo, Narna, Tombus, and Hepe-Beny, extremely diverse RNA virus communities were also discovered (Fig. S3). In addition to the putative plant/fungus-infecting strains, most newly identified viruses formed genetically distinct evolutionary lineages, many of which are as yet unclassified. For example, the virgavirus/negevirus-like viruses represented a diverse group of viruses that were associated with insects and other invertebrate animals.

Insect-specific evolutionary lineages were also found in vertebrate-associated virus clades, such as the Nido, Astro, and Hepe clades (Fig. S3). Novel nidoviruses fell into two distinct insect-specific lineages, in which two strains belonged to the previously described Mesoniviridae. Another nido-like virus formed a well-supported monophyletic group with Wuhan insect virus 19 and Wuhan nido-like virus 1, representing an unclassified taxonomic group within Nidovirales. One distant relative of astrovirus was discovered in a terrestrial insect, Unaspis euonymi (Hemiptera: Diaspididae). The Unaspis euonymi astro-like virus clustered with two invertebrate-associated astroviruses (host: Myriapoda and Hirudinea) and fell in a position basal to the astroviruses of vertebrate on the phylogeny, possibly representing ancient divergence of astroviruses from invertebrates. To our knowledge, the Unaspis euonymi astro-like virus was the first astrovirus species identified in insect. One novel bastrovirus strain was also identified in a filter-feeding insect, Ephemera sp. HW-2014, and shared a moderate level of sequence similarity with two strains isolated from bat and sewage (60.14% and 61.53% aa identity, respectively). Other +ssRNA viruses included nine permutotetraviruses and one levi-like RNA phage (Fig. S3), among which the novel levi-like virus might be associated with the gut microbiota of the host Lepismachilis y-signata.

Novel negative-sense single-stranded RNA (−ssRNA) viruses.The mononega-like viruses were the most common −ssRNA viruses identified, presenting in more than 15% insects in this study. Altogether, newly identified mononega-like viruses diversified into nine major evolutionary groups, including six in Mononegavirales (Fig. S4). We observed two clearly separate groups within the Rhabdoviridae, of which one was related to plant-associated Nucleorhabdovirus and the other belonged to vertebrate-associated Almendravirus. It may be hypothesized that insect-associated rhabdoviruses arose independently. Other mononegaviruses included members within the Lispiviridae, Xinmoviridae, and Artoviridae lineages and a Borna-like lineage. Thirty-five viruses were assigned to the newly established Chuviridae lineage. These chuviruses were divided into three groups with huge differences in their genome architectures, indicating a possible relationship between the genome arrangement and diversity.

Sixty-nine bunya-like viruses were identified, of which most could be classified into three insect-specific groups, namely, Goukovirus, Orthophasmavirus, and an unclassified sister group to Phenuiviridae (Fig. S4). Within the Feravirus, two nearly identical strains (>99% nt identity) were identified in Trialeurodes vaporariorum and its parasitic counterpart Encarsia formosa, respectively, providing evidence of its importance with respect to feeding behavior in virus transmission. A novel tenuivirus strain was identified in Archaeopsylla erinacei and was found to be closely related to the Rice stripe tenuivirus (53.8% aa identity). Another strain obtained from a phytophagous insect, Larinus minutus, was distantly related to the plant-infecting Citrus concave gum-associated virus. These bunya-like viruses, including the arena-like virus identified in Heteropsilopus ingenuus, were assumed to be trisegmented, though the data have shown that some viral genomes lack one or two segments due to inadequate sequencing reads or extremely divergent features.

A remarkable diversity of orthomyxo-like viruses was revealed in insects (Fig. S4). These viruses formed multiple insect-specific lineages in Quaranjavirus. In addition, two thogotovirus variants were present in Plea minutissima and shared 51.64% amino acid sequence identity. Consistently, these viruses possessed five to six segments, although some genomic segments were missed and some might be redundant. Other −ssRNA viruses included six strains within the newly described bisegmented Qinvirus and one within the plant-infecting Ophiovirus (Fig. S4). The aspi-like virus was identified in a phytophagous insect, Panurgus dentipes, and was assigned to a new species in Ophiovirus with an unknown natural plant host.

Novel double-sense double-stranded RNA (dsRNA) viruses.A genetically diverse population was found in clade Partiti with a broad host range, including novel members within Partitiviridae and Amalgaviridae. More than 125 partitiviruses formed seven major groups, in which 31 novel strains were assigned to the recognized plant/fungus-infecting Alphapartitivirus and Betapartitivirus (Fig. S5). However, few were identified in genera Gammapartitivirus and Cryspovirus. Although a novel clade was identified adjacent to the Deltapartitivirus, these viruses were distantly related to the phytopathogenic virus species and might represent an invertebrate-specific lineage. Another four groups cannot be classified into any recognized taxa; one of the group was associated with the plant-infecting Maize-associated partiti-like virus. Members of the family Partitiviridae were believed to have two genomic segments. However, the capsid-encoded segment was rarely identified within these novel virus groups. It seemed that a much more diverse parititivirus community existed in insects, which might act as major reservoirs and/or transmission vectors for the parititivirus hosted by plants or fungi. In addition, three nonsegmented parititi-like viruses were found in omnivorous insects Thanasimus formicarius, Tetrodontophora bielanensis, and Haliplus fluviatilis. These novel viruses were phylogenetically related to the plant-infecting species within the Amalgaviridae and shared similar genome organizations, with two overlapping open reading frames (ORFs).

Three main virus groups within the Toti clade were found in insects (Fig. S5). These viruses showed limited sequence similarity with the recognized totiviruses. For example, 20 newly identified toti-like viruses clustered together with Hubei toti-like virus 22 and formed an apparently separate lineage distinct from those of fungus/protozoa giardiaviruses. Another group contained one plant virus strain, Persimmon latent virus, and could be regarded as a sister group to those noninsect viruses. However, novel viruses were occasionally found in the genera Victorivirus and Trichomonasvirus and one unclassified taxon associated with protozoa. Other toti-like viruses included botybirna- and chryso-like viruses. The botybirnavirus was found in a predatory insect, Grylloblatta bifratrilecta, with 30.7% aa identity with the most closely related viruses. The chryso-like viruses were identified in a phytophagous insect, Psyllopsis fraxinicola, and a parasitic wasp, Eurytoma brunniventris, with a typically four-segmented genome corresponding to those of members of the Chrysoviridae family.

Thirty-three reo-like viruses were identified, among which 11 strains were novel members of genus Orbivirus and 13 were related to the plant-associated Fijivirus (Fig. S5). Other strains were affiliated with different evolutionary lineages of the genera Coltivirus, Phytoreovirus, Seadornavirus, and Oryzavirus and four unclassified groups. However, none of the novel reo-like viruses was found in the insect-specific genus Cypovirus. Among them, two reo-like viruses were found to be identical to the previously reported strains isolated from Diaphorina citri and Nilaparvata lugens, respectively (>96% nt identity for all segments), implying wide distribution in the wild. However, most reo-like viruses were highly divergent from all known species; in particular, the multisegmented genome organization made them somewhat more variable.

One birnavirus was identified in a filter-feeding insect, Isonychia kiangsinensis. The genome of Isonychia kiangsinensis birna-like virus comprised two segments, of which the polymerase-encoded segment best matched the Drosophila X virus (42.8% aa identity). The birnavirus was divided into three evolutionary lineages (Fig. S5); one was associated with vertebrate animals, and the other two were associated with invertebrate animals (mostly insects). Hence, a genetically diverse population of birnaviruses might be present in insects and other invertebrate animals.

Great genome flexibility in insect viruses.Though the overall levels of sequence similarity were very low even within each virus clade, some genes were conserved among RNA viruses. The most ubiquitous and conserved motifs were associated with the viral polymerase (Fig. S7a). In general, the key conserved residues arrange themselves in an orderly manner and maintain structural integrity in the RNA polymerase domain, with the exception of few viruses (Fig. S7b). Highly conserved motif C (GDD) appeared to be lost in the polymerase active sites of birnaviruses or to be translocated upstream of motifs A and B in permutotetraviruses and negeviruses (A→B→C to C→A→B). Consensus motifs were also discovered in some essential viral proteins, including the RNA helicase, protease, methyltransferase, and coat proteins (Fig. 3). These viral proteins represent the most highly conserved genome components in the majority of RNA viruses, although some of them are alternatively encoded by different viruses.

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FIG 3

Genome architectures and comparison of representative viruses within major viral clades. The genomes are drawn as boxes and lines approximately to scale, representing open reading frames (ORFs) and noncoding regions, respectively. The predicted homologous genes are shown in colored boxes (see legend). The question marks (?) represent the missing genomic segments, and each asterisk (*) represents a novel hypothetical ORF on the minus strand of the virus genome.

Despite the subtle constraints imposed on RNA virus evolution, the insect viruses retained a substantial level of variability in genome size and architectures (Fig. 3; see also Fig. S2 to S6). The narnaviruses had the simplest genome organization of any RNA viruses, yielding to a single open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp). Nevertheless, an additional ORF was found in the complementary strand of the genome for some insect-derived narnaviruses, which encoded a hypothetical protein with unknown function or with no significant homology to known proteins. More differences were identified in virus genomes containing multiple ORFs for nonsegmented viruses or multiple-genome segments for segmented viruses. For example, the virga-like viruses encoded similar panels of nonstructural proteins, while the structural regions were very flexible and characterized by a complex genomic landscape (Fig. 3), which might be associated with a considerable capacity to accommodate different environments and hosts. Within the toti-like viruses, three different genome structures were observed across different evolutionary lineages (Fig. 3), including a single large open reading frame (ORF) or discontinuous ORFs characterized by overlapping or being separated. The difference in gene arrangements might reflect an alternative utilization of translation initiation by toti-like and other related viruses, including ribosomal frameshift by those viruses with overlapped ORFs and termination-reinitiation mechanism by those with separated ORFs.

The viral genomic heterogeneity was also enriched through genome rearrangement and segmentation. In picorna-like viruses, one of the most remarkable genetic differences was the gene modules’ inversion of the structural and nonstructural protein coding regions among different evolutionary groups (Fig. 3). Within the family Dicistroviridae, Ellipsidion picorna-like virus 2 and related strains were discovered to undergo an event of genome rearrangement, which likely resulted in the formation of a novel evolutionary lineage descended from Aparavirus (Fig. 3). Similarly, the unsegmented mononega-like viruses were characterized by the presence of three hallmark genes encoding the nucleoprotein (N), glycoprotein (G), and large polymerase (L) and were distinguished by a pool of hypothetical proteins with no known orthologues (Fig. 3). Nevertheless, the hallmark genes were not conserved in location in the corresponding genomes, characterized by an N-G-L module for Mononegavirales and a G-N-L or L-N-G module for Jingchuvirales. Although most mononegaviruses were believed to have a monopartite genome, some chuviruses were characterized by a bi- or trisegmented genome, including a putative circular form (Fig. 3). Genome segmentation was also found in flavi- and solemo-like viruses. In most cases, the segmented viruses were quite distinct from the nonsegmented counterparts. Apart from the conserved NS5 and NS3 segments, the gene repertoires possessed by the segmented jingmenviruses are quite different from those of the related nonsegmented flavi-like viruses and formed a well-supported monophyletic group. A major phylogenetic discrepancy was also observed between segmented and nonsegmented solemo-like viruses, although the encoded viral proteins were similar. Alternatively, the genomes of insect-associated beny-like viruses were not segmented as seen with the recognized Benyvirus. It seems that the segmented viruses might have originated from the primitive nonsegmented ones. The tight evolutionary link between segmented and nonsegmented viruses might represent the potential mechanisms that make possible the genome shuffling in some RNA viruses, such as the mononega- and picorna-like viruses.