1. Introduction

2. Materials and Methods

3. Results

Figure 1. Gene organization of the negevirus and selected negev-like viruses. The replicase protein (REP) is shown in pink (see text for details). SP24 protein is shown in brown. ORFs coding for DiSB-chro domain proteins are in green. Proteins with putative membrane-bound segments are shown in blue. Read-through of leaky terminator codon is shown by arrow above RdFV genome scheme.

Figure 1. Gene organization of the negevirus and selected negev-like viruses. The replicase protein (REP) is shown in pink (see text for details). SP24 protein is shown in brown. ORFs coding for DiSB-chro domain proteins are in green. Proteins with putative membrane-bound segments are shown in blue. Read-through of leaky terminator codon is shown by arrow above RdFV genome scheme.

3.1. Virus RNA genomes with two or three gene copies encoding SP24

In contrast to many other known negeviruses and negev-like viruses, the genome of a symbiotic virus RdFV infecting rice green zigzag leafhopper Recilia dorsalis (class Insecta, order Hemiptera, family Cicadellidae) contains two gene copies encoding SP24 [19] that may correlate with the ability of the virus to form filamentous particles and/or to spread by vertical transmission. In general, this virus encodes seven proteins (Figure 1). Replicase is encoded by ORF1, which has an internal leaky termination codon and may be translated to form two overlapping polypeptides [20]. Among non-replicative proteins of RdFV, only the SP24 polypeptides have significant similarity to proteins of annotated viruses. However, a search of the NCBI TSA database revealed that the transcriptome of insect Xestocephalus desertorum, which belongs to the same family Cicadellidae as zigzag leafhopper, contains a partial VLRA encoding ORF1 RdRp domain closely related to that of RdFV (e-value 1e-49), a protein homolog of RdFV ORF5 protein (e-value 1e-19) and three copies of SP24 (Table 1). Three copies of SP24 genes were also revealed in the annotated genomes of pyrrhocoris apterus virus 1 infecting insect Pyrrhocoris apterus (class Insecta, order Hemiptera, family Pyrrhocoridae) and wuhan heteroptera virus 1 isolated from mixed insects of the suborder Heteroptera (order Hemiptera) [5] (Figure 1) (Table 1). It should be noted that the RdRp domain encoded by ORF1 of RdFV, pyrrhocoris apterus virus 1 and wuhan heteroptera virus 1 are clustered together in the phylogenetic tree of Virgaviridae-like RdRp domains (Supplementary Figure S1).

Another cluster of RdRp domains of negev-like viruses encoding two SP24 gene copies includes, particularly, zeugodacus cucurbitae negev-like virus, broome virga-like virus 1, bactrocera oleae negev-like virus and bactrocera dorsalis negev-like virus isolate Bz infecting mosquitoes and invasive fruit flies from order Diptera (class Insecta) [21,22] (Table 1) (Supplementary Figure S1). Our search also revealed negev-like VLRAs encoding two SP24 gene copies in the transcriptomes of class Arachnida, specifically Scotophaeus scutulatus (family Gnaphosidae) and Neoscona scylloides (family Araneidae) (Table 1).

Table 1. Examples of the viruses and VLRAs encoding two or three SP24 gene copies.

Table 1. Examples of the viruses and VLRAs encoding two or three SP24 gene copies.

Virus and host species	Host Class/Order	Accession number
recilia dorsalis filamentous virus Recilia dorsalis	Insecta/Hemiptera	OP326514
pyrrhocoris apterus virus 1 Pyrrhocoris apterus	Insecta/Hemiptera	MK024711
VLRA Xestocephalus desertorum	Insecta/Hemiptera	GELC01067077
VLRA Neoscona scylloides	Arachnida/Araneae	ICMG01043884
VLRA Scotophaeus scutulatus	Arachnida/Araneae	ICQK01020075
atrato virga-like virus 6 Psorophora sp.	Insecta/Diptera	MN661118
atrato virga-like virus 7 Psorophora albipes	Insecta/Diptera	MN661129
wuhan heteroptera virus 1 mixed insects	Insecta/Hemiptera	KX883821
dougjudy virga-like virus Calliphora vicina	Insecta/Diptera	MT129773
bactrocera oleae negev-like virus Bactrocera oleae	Insecta/Diptera	MW310375
broome virga-like virus 1 Culex annulirostris	Insecta/Diptera	MT498833
bactrocera dorsalis negev-like virus Bactrocera zonata	Insecta/Diptera	MW310386
zeugodacus tau negev-like virus Zeugodacus tau	Insecta/Diptera	MW310340
zeugodacus cucurbitae negev-like virus Zeugodacus cucurbitae	Insecta/Diptera	MW310350
entomophthora virgavirus A* Entomophthora muscae	Entomophthoromycetes/ Entomophthorales	MK231110

*—infects fungi.

3.1.1. Unique fungal virus encoding SP24 gene

Interestingly, the RdRp phylogenetic tree cluster containing Diptera-infecting viruses (in particular, broome virga-like virus 1) also contains the fungal entomophthora virgavirus A (Supplementary Figure S1) with RNA genome encoding two SP24 gene copies (Figure 1) (Table 1). This virus genome has been found in transcriptome of the fungus Entomophthora muscae, whose hosts are mainly insects of the order Diptera [23]. It can be assumed that this virus has evolutionarily originated due to transfer of an insect virus from a dipteran organism to a fungus and its subsequent adaptation to replicate in fungal hyphal bodies. On the other hand, entomophthora virgavirus A can be a true insect virus, and the finding of virus genome in fungal cells is a consequence of frequent passive transfer of virus RNA from host insects.

3.2. Virus RNA genomes encoding SP24 and infecting arthropods of class Arachnida

The transcriptomes of more than 1000 species of spiders (class Arachnidae) [18] recently deposited in the NCBI TSA database have the potential to greatly increase the number of novel RNA viruses encoding SP24. First of all, our search of the NCBI TSA database revealed many spiders infected by solenopsis invicta virus 17 (SINV-17) or its closely related strains (Table 2). This monopartite RNA virus, first described in the red imported fire ant [24], encodes three genes, namely replicase, ORF2 with DiSB-ORF_chro domain and SP24 gene (Figure 1). SINV-17 RdRp domain is phylogenetically most closely related to hubei virga-like virus 8 from Myriapoda sub-philum [5] and VLRA from the insect Chrysoperla carnea, and more distantly related to sandewaviruses (Supplementary Figure S1).

Table 2. Examples of VLRAs with encoded proteins having >90% homology to SINV protein SP24.

Table 2. Examples of VLRAs with encoded proteins having >90% homology to SINV protein SP24.

VLRA origin and accession number	Host Class/Order	Identity to SINV (%) SP24 *
Acheta domesticus, GHUU01023074	Insecta/Orthoptera	100
Orestes mouhotii, GDZN01041390	Insecta/Phasmatodea	95
Polistes metricus, GDHQ01029178	Insecta/Hymenoptera	97
Karoophasma biedouwense, GINP01229017	Insecta/Mantophasmatodea	99
Austrophasmatidae sp., GDYW01015845	Insecta/Mantophasmatodea	98
Gryllus bimaculatus, GISW01177432	Insecta/Orthoptera	99
Nylanderia pubens, JP785474	Insecta/Hymenoptera	96
Bassaniana decorate, ICCJ01037167	Arachnida/Araneae	94
Weintrauboa contortipes, ICJX01013850	Arachnida/Araneae	94
Meotipa spiniventris, IBDD01006961	Arachnida/Araneae	94
Theridion zonulatum, IATV01002901	Arachnida/Araneae	94
Thiania bhamoensis, IAGH01040226	Arachnida/Araneae	94
Oxyopes macilentus, IAPY01004984	Arachnida/Araneae	94
Cyrtophora unicolor, IAYW01028924	Arachnida/Araneae	94
Argyrodes kumadai, IBBR01021510	Arachnida/Araneae	94
Mendoza elongate, ICIJ01021408	Arachnida/Araneae	89
Agelena labyrinthica, IBZQ01034950	Arachnida/Araneae	94
Tegecoelotes corasides, IASA01013670	Arachnida/Araneae	94
Octonoba sybotides, IAZZ01004659	Arachnida/Araneae	94
Leucauge celebesiana, IAWQ01030888	Arachnida/Araneae	94
Cyclosa mulmeinensis, ICEF01010633	Arachnida/Araneae	94
Heptathela higoensis, IBEA01018510	Arachnida/Araneae	94
Oxyopes sp., ICGS01044124	Arachnida/Araneae	94
Argiope minuta, ICGZ01029236	Arachnida/Araneae	94
Uloborus sp., IBJL01027593	Arachnida/Araneae	94
Chikunia albipes, IAUZ01010102	Arachnida/Araneae	94
Ebrechtella tricuspidata, IBYS01040384	Arachnida/Araneae	94
Rhomphaea hyrcana, IAOI01013840	Arachnida/Araneae	94
Latouchia swinhoei, IARH01001083	Arachnida/Araneae	94
Lycosa ishikariana, IBVL01017570	Arachnida/Araneae	94
Dolomedes pegasus, ICEZ01008221	Arachnida/Araneae	94
Araneus seminiger, IAIN01035359	Arachnida/Araneae	94
Thanatus bungei, IALY01037623	Arachnida/Araneae	94
Iwogumoa interuna, IBKG01028989	Arachnida/Araneae	94
Sinopoda okinawana, ICJY01003033	Arachnida/Araneae	94
Thelcticopis severa, ICJF01009625	Arachnida/Araneae	94
Heptathela higoensis, IBNB01017401	Arachnida/Araneae	94
Heptathela yanbaruensis, ICKJ01022680	Arachnida/Araneae	94
Mendoza elongata, ICIJ01011610	Arachnida/Araneae	94
Iwogumoa interuna, IBKG01014765	Arachnida/Araneae	94
Dolomedes triton, GGRN01030862	Arachnida/Araneae	94
Anolis carolinensis, GAFD01024809	Lepidosauria/Iguania	98
Sceloporus jarrovii, GIWZ010003422	Lepidosauria/Iguania	99
Protopterus annectens, GGXP01055242	Euteleostomi/Ceratodontiformes	97

* Percentage identity is shown for SP24 sequence assuming near 100% identity for replicase.

Spiders represent only a part of the arthropod hosts for SINV-17. Our transcriptome search revealed seven insect species that can be infected by SINV-17 (Table 2). Moreover, it is quite unexpected that sequences highly similar to SINV-17 were found in a search of TSA databases of vertebrate species, namely the West African the lungfish (Protopterus annectens), the Yarrow’s spiny lizard (Sceloporus jarrovii) and the lizard Anolis carolinensis (Table 2).

Additionally, we revealed many novel spider viruses encoding SP24 that are distinct from SINV-17 (Supplementary Figure S1). Previously, several viruses of this type have only been revealed in the golden orb-weaver spider (Nephila clavipes) and the spider mite (Tetranychus urticae) [25].

3.3. Negev-like virus RNA genomes infecting species of metazoan taxa outside insects

The finding of VLRAs highly similar to the negev-like SINV-17 genome in vertebrates (see above) prompted us to perform another comprehensive search of the transcriptomes of non-arthropod metazoan taxa to find new negev-like virus RNA genomes. Our search revealed that viruses only distantly related to SINV-17 infect at least three fish species (Table 3). These species are included in the infraclass Teleostei (teleost fishes), clade Acanthomorphata.

Table 3. Examples of negev-like virus RNA genomes infecting species of metazoan taxa outside arthropods *.

Table 3. Examples of negev-like virus RNA genomes infecting species of metazoan taxa outside arthropods *.

Host taxonomic position	Name (Virus and VLRA)	Accession number
Teleostei; Anabantiformes	Parargyrops edita VLRA	GICI01264178
Teleostei; Cyprinodontiformes	Aphanius dispar VLRA DN-106	GJEY01002386
Teleostei; Anabantiformes	Channa argus VLRA	GEML01178446
Teleostei; Cyprinodontiformes	Aphanius dispar VLRA DN-113	GJEY01012897
Porifera; Demospongiae	Halisarca dujardinii VLRA	GIFI01065598
Cnidaria; Actiniaria	beihai anemone virus 1	KX883744
Spiralia; Entoprocta	Loxosomella nordgaardi VLRA	GIMJ01025245
Spiralia; Mollusca	Octopus vulgaris VLRA	GKAX01007523
Spiralia; Mollusca	Charonia lampas VLRA	GIQZ01066080
Spiralia; Mollusca	Potamilus streckersoni VLRA	GJAA01000017
Spiralia; Platyhelminthes	dicrocoelium nege-like virius	OP548619
Spiralia; Platyhelminthes	Girardia sp. VLRA	GHOU01019505
Spiralia; Platyhelminthes	provittati virus	BK059742
Spiralia; Platyhelminthes	fasciogiga virus	BK059714
Spiralia; Platyhelminthes	fasciohepa virus 1	BK059715
Spiralia; Platyhelminthes	meterori virus	BK059677
Spiralia; Platyhelminthes	clonorsi virus 1	BK059702
Spiralia; Platyhelminthes	fasciohepa virus 3	BK059717
Spiralia; Platyhelminthes	clonorsi virus 2	BK059703
Spiralia; Platyhelminthes	fasciohepa virus 2	BK059716
Ecdysozoa; Onychophora	Principapillatus hitoyensis VLRA	GJGV01069003
Ecdysozoa; Nematoda	Nippostrongylus brasiliensis VLRA	VDL74358
Ecdysozoa; Nematoda	Anguina tritici VLRA	GKDZ01058772
Ecdysozoa; Nematoda	xingshan nematode virus 1	KX883837
Ecdysozoa; Nematoda	xingshan nematode virus 2	KX883836
Ecdysozoa; Nematoda	xinzhou nematode virus 1	KX883838
Ecdysozoa; Nematoda	Anisakis pegreffii	HBXC01106013

* Viruses and VLRAs do not include those nearly identical to SINV-17.

A VLRA of Crimson seabream (Parargyrops edita, order Anabantiformes) contains four ORFs (Figure 2). The incomplete ORF1 encodes the C-terminal part of the replicase with RNA helicase and RdRp domains. ORF2 codes for a protein of 224 amino acids with two long hydrophobic segments and no significant similarity to known viral proteins. ORF3 (221 codons) represents a SP24 gene, while ORF4 encodes a protein of 109 amino acids with distant similarity to the VP-2 capsid protein of jingmen tick virus [26]. Another fish virus encoding SP24 is represented by a VLRA from euryhaline fish (Aphanius dispar, order Cyprinodontiformes) (Table 3). This virus genome encodes five proteins (Figure 2). A replicase ORF is followed by overlapping ORF2 and ORF3, which encode hydrophobic proteins of 467 and 268 amino acids, respectively. ORF4 encodes an SP24 protein of 306 amino acid residues, while ORF5 encodes a small protein of 119 residues (Figure 2). This virus is moderately similar to another negev-like virus (Table 3), which also infects the fish A. dispar and encodes five proteins, namely replicase, ORF2 protein (492 residues long), ORF3 protein (382 amino acids), ORF4 protein (209 amino acids) representing SP24, and ORF5 (141 residues). It should be noted that RdRp domains encoded by ORF1 of VLRAs from Parargyrops edita and Aphanius dispar are clustered together with negev virus and nelorpiviruses in the phylogenetic tree of Virgaviridae-like RdRp domains (Supplementary Figure S1).

Figure 2. Gene organization of the selected fish VLRAs. See Figure 1 for details of ORF naming and shading.

Figure 2. Gene organization of the selected fish VLRAs. See Figure 1 for details of ORF naming and shading.

Among metazoan species in the clade Spiralia, negev-like viruses encoding SP24 have only been identified in flatworms (phylum Platyhelminthes) [17]. Our searches of the NCBI TSA databases revealed additional flatworm viruses that encode SP244-like proteins (Table 3). One more SP24-encoding negev-like virus was found by our search in the goblet worm (Loxosomella nordgaardi), which belongs to Entoprocta, another phylum of Spiralia [27,28] (Table 3). In addition, novel viruses encoding SP24 were found in species of phylum Mollusca (classes Bivalvia, Gastropoda and Cephalopoda). Specifically, these animals include Octopus vulgaris (class Cephalopoda, order Octopoda), Charonia lampas (triton shell, class Gastropoda, order Littorinimorpha) and Potamilus streckersoni (class Bivalvia, order Unionida) [28] (Table 3). The octopus SP24-encoding VLRA contains seven ORFs (Figure 3). A partial replicase ORF is followed by ORF2, which encodes a hydrophobic protein of 442 residues with a DiSB-like chro domain. ORF3 encodes a protein of 103 residues with a long predicted transmembrane region. ORF4 encodes a small protein (110 residues) and overlaps with ORF5 of the SP24 protein (304 residues), while ORF6 encodes a highly hydrophobic protein of 254 residues and overlaps ORF7 (285 residues) (Figure 3). In the RdRp phylogenetic tree, RdRp domains of ORF1 in VLRAs from Octopus vulgaris and Charonia lampas are positioned in a large cluster that is quite distant from a subtree containing RdRps from flatworm negev-like viruses (Supplementary Figure S1).

In the animal clade Ecdysozoa, species infected by negev-like viruses encoding SP24 were found not only in the phylum Arthropoda, but also in the phylum Nematoda (Table 3). Furthermore, we found that the velvet worm Principapillatus hitoyensis, which belongs to the phylum Onychophora, which is phylogenetically closest to Arthropoda [28], can be infected by a negev-like virus (Table 3). This velvet worm-specific virus encodes three proteins, the ORF1 replicase with viral methyltransferase domain, macro domain representing an ADP-ribose binding module, OTU domain family (cysteine proteases), superfamily 1 viral RNA helicase domain and the C-terminal RdRp domain; the ORF2 protein with DiSB-like chro domain; the ORF3 protein with SP24 domain (Figure 3).

Figure 3. Gene organization of the selected VLRAs. See Figure 1 for details of ORF naming and shading.

Figure 3. Gene organization of the selected VLRAs. See Figure 1 for details of ORF naming and shading.

The phylum Porifera (sponges) and the phylum Cnidaria contain species that are among the most primitive metazoan organisms [28]. In this study, we report for the first time a sponge VLRA that corresponds to a distant relative of negeviruses (Table 3). This sponge-specific virus infects Halisarca dujardinii (phylum Porifera, class Demospongiae, order Dendroceratida) and encodes four proteins (Figure 3). The ORF1-encoded replicase has three domains: a replicase with viral methyltransferase domain, a superfamily 1 viral RNA helicase domain, and the C-terminal RdRp domain. ORF1 is followed by ORF2, which encodes a hydrophobic protein of 524 amino acid residues. ORF3 encodes an SP24-like protein (200 residues), while ORF4 encodes a small protein of 136 residues (Figure 3). Previously, a transcriptome search of sea anemones (phylum Cnidaria, class Hexacorallia, order Actiniaria) has revealed a negev-like virus encoding SP24 [5]. This virus, named beihai anemone virus 1 (Table 3), encodes six proteins. ORF1 encodes a replicase with viral methyltransferase domain, macro domain representing an ADP-ribose binding module, superfamily 1 viral RNA helicase domain and the C-terminal RdRp domain. ORF2 encodes a protein (166 residues) that has no counterparts among known viral proteins and overlaps with ORF3, which encodes a highly hydrophobic protein (163 amino acids). ORF4 protein (438 amino acids) contains DiSB-like chro domain, while ORF5 (203 residues) encodes SP24 and ORF6 encodes a small protein (110 amino acids) with no similarity to known viral proteins. Phylogenetic analysis of the RdRp domain of beihai anemone virus 1 showed that the most similar virus is sanxia atyid shrimp virus 1 (SASV1), which infects shrimps of the family Atyidae (phylum Arthropoda, class Malacostraca) (Supplementary Figure S1). This shrimp-specific virus encodes 5 proteins. ORF1 encodes a replicase (2629 amino acids) with viral methyltransferase domain, macro domain, superfamily 1 viral RNA helicase domain and RdRp domain. ORF2 encodes a protein (128 residues) that has no counterparts among known viral proteins. ORF3 encodes a highly hydrophobic protein (164 amino acids). ORF4 protein (570 amino acids) contains the DiSB-like chro domain, while ORF5 (208 residues) encodes SP24. Our data show that several novel species of the class Malacostraca can be infected by negev-like viruses encoding SP24. Some of these viruses appear to be related to SASV1 (Supplementary Figure S1). In particular, eriocheir sinensis kita-like virus, which infects Chinese hairy crab (phylum Arthropoda, class Malacostraca, family Varunidae), encodes 3 proteins (Supplementary Figure S1): ORF1 protein is a replicase; ORF2 protein (665 amino acids) is a fusion of SASV1 ORF3 and ORF4 (DiSB) proteins; ORF3 (231 redidues) encodes SP24. However, some other negev-like viruses that infect the class Malacostraca are only distantly related to SASV1. For example, the RdRp domain of the SP24-encoding VLRA of the amphipod Gammarus fossarum (class Malacostraca, family Gammaridae) is phylogenetically more closely related to a protein encoded by a brown algae VLRA than to other negev-like viruses infecting class Malacostraca (Supplementary Figure S1).

3.4. SP24-encoding negev-like viruses infecting species of green plants and brown algae

The discovery of the SP24-encoding kitaviruses in plants [9] suggests that the SP24 gene may also be a genomic component of other plant viruses. Indeed, a novel negev-like RNA virus, fragaria vesca associated virus 1 (FVaV-1), has been found to contain the SP24 gene, and an SP-24-encoding gene has been found in the genome of recently annotated chrysanthemum kita-like virus (CKLV) [29]. Our search of plant transcriptomes revealed an additional plant VLRA encoding SP24 (Table 4). The phylogeny of the RdRp domains in FVaV-1, Triticum polonicum VLRA and Glycine dolichocarpa VLRA (Supplementary Figure S1) shows their rather close relationship to arthropod viruses. We hypothesize that these viruses may have emerged in virus evolution by a host change of an insect virus that was accompanied by an acquired ability to replicate or passively persist in plant cells (see also Section 3.1.1.).

Table 4. Negev-like virus RNA genomes infecting species of green plants and brown algae.

Table 4. Negev-like virus RNA genomes infecting species of green plants and brown algae.

Host taxonomic position	Name (Virus and VLRA)	Accession number
Magnoliopsida, Rosales	fragaria vesca associated virus 1	MN895062
Magnoliopsida, Asterales	chrysanthemum kita like virus	OP807956
Magnoliopsida, Caryophyllales	Amaranthus tuberculatus VLRA	GGGT01091955
Magnoliopsida, Lamiales	Linaria vulgaris VLRA*	HBXK01234259
Magnoliopsida, Caryophyllales	Silene latifolia VLRA	JO777742
Magnoliopsida, Poales	Hordeum vulgare VLRA	GGCQ01066052
Magnoliopsida, Poales	Alloteropsis semialata VLRA	GFYF01029264
Magnoliopsida, Ranunculales	Papaver nudicaule VLRA	GJOR01065662
Magnoliopsida, Ranunculales	Papaver armeniacum VLRA	GJOO01068528
Magnoliopsida, Ranunculales	Papaver pavoninum VLRA	GJOU01016337
Magnoliopsida, Fabales	Glycine dolichocarpa VLRA	GGIW01009300
Magnoliopsida, Oxalidales	Elaeocarpus photiniifolius VLRA	FX134396
Pinopsida, Pinales	Pseudotsuga menziesii VLRA	GFFY01083799
Magnoliopsida, Apiales	Panax ginseng VLRA	GDQW01045297
Phaeophyceae, Fucales	Sargassum vulgare VLRA	GEHA01041094

*—VLRAs of Linaria vulgaris and related virus strains are in bold.

VLRA from the common toadflax Linaria vulgaris (order Lamiales, family Plantaginaceae), although encoding SP24, differs significantly from other plant negev-like viruses in terms of RdRp phylogeny (Supplementary Figure S1) and gene organization. Unlike FVaV-1 and CKLV, which have four genes, Linaria vulgaris VLRA encodes six potential proteins (Figure 4). Replicase protein of more than 1865 residues is potentially encoded by two overlapping ORFs: ORF1a protein has single annotated domain of superfamily 1 viral RNA helicase, while ORF1b overlaps ORF1 by 555 nucleotides and encodes a protein part with RdRp domain. ORF2 protein (637 amino acids) is distantly related to the predicted DiSB domain-containing glycoprotein of astegopteryx formosana nege-like virus 1. ORF3 is completely embedded in ORF2 and encodes a hydrophobic protein with no similarity to known viral proteins. ORF4 protein (329 amino acids) includes a cysteine-rich region, while overlapping ORF5 coding for protein possessing the SP24 domain. ORF6 encodes a small protein of 69 amino acids (Figure 4). Our extensive search of the plant NCBI TSA database revealed that SP24-encoding virus of Linaria vulgaris can infect several other monocot and dicot plants, namely, Hordeum vulgare, Alloteropsis semialata, Papaver nudicaule, Papaver armeniacum, Papaver californicum, Papaver atlanticum and Silene latifolia (Table 4). The RdRp domain of Linaria vulgaris VLRA and its isolates specific for the listed above hosts forms a common cluster with virga-like viruses of brown algae in the PdRp phylogenetic tree (Supplementary Figure S1).

Figure 4. Gene organization of the selected VLRAs. See Figure 1 for details of ORF naming and shading.

Figure 4. Gene organization of the selected VLRAs. See Figure 1 for details of ORF naming and shading.

Strikingly, one of brown algae virga-like viruses infecting Sargassum vulgare (class Phaeophyceae, order Fucales) encodes SP24 (Table 4). This VLRA encodes 3 proteins typical for nege-like viruses (Figure 4). Incomplete ORF1 codes for replicase C-terminal part with superfamily 1 viral RNA helicase domain (pfam01443, e-value—2.74e-38) and RdRp domain (pfam00978, e-value—2.27e-85). ORF2 protein (490 residues) contains DiSB-like domain but has no significant sequence similarity to negevirus proteins. ORF3 encodes protein of 185 amino acids which possesses SP24 domain (e-value—1.06e-07).

3.5. NCBI conserved domain database search reveals horizontal transfer of SP24 protein genes to viruses of order Hepelivirales and insect genomes

The NCBI-conserved domain search tool [30] was used to find potential SP24 genes encoded by cellular genomes. Among the four SP24-producing genes, three belong to insects, namely, Macrosiphum euphorbiae, order Hemiptera (locus CAI6355448, protein size—206 amino acids), Drosophila melanogaster, order Diptera (locus ABC86319, protein size—202 amino acids), and Glossina morsitans morsitans, order Diptera (locus ADD20599, protein size—271 amino acids). Another gene is found in the nematode Nippostrongylus brasiliensis, order Strongylida (locus VDL74358, protein size—184 amino acids). Thus, insect and nematode genomes may acquire viral SP24 genes by putative horizontal transfer from negev-like viruses known to infect species of both taxa (see above).

Another result of our NCBI CDD search is the pioneering discovery of SP24 genes in the genomes of two insect RNA viruses belonging to the order Hepelivirales. The genome of hubei hepe-like virus 1 [5] encodes three proteins (accession KX883803). ORF1 encodes a replicase (1661 amino acids) with the C-terminal RdRp domain showing obvious similarity to RdRp of hepeviruses (Table 5). ORF2 encodes a protein (206 amino acids) with SP24 domain, whereas ORF3 encodes a longer protein (560 amino acids) with DiSB-chro domain. Thus, the general genome organization of this virus is different from that of negeviruses because of the inverse order of DiSB-chro and SP24 genes. Interestingly, although the replicase of sogatella furcifera hepe-like virus (1844 amino acids) is also related to viruses belonging to the order Hepelivirales [13] (Table 5), the genome cistron order in this case is identical to that of negev virus (Figure 1). ORF2 encodes a protein (622 amino acids) with DiSB-chro domain, and ORF3 encodes the SP24 protein (209 amino acids).

Table 5. Similarity of Hubei hepe-like virus 1 RdRp to sequences in NCBI protein database.

Table 5. Similarity of Hubei hepe-like virus 1 RdRp to sequences in NCBI protein database.

Virus and accession number	E-value	Identity to HHLV1 RdRp (%)
Astroviridae sp.; QYJ54474	2e-48	34.54
Hepelivirales sp.; WAY16503	9e-48	34.08
Sogatella furcifera hepe-like virus; YP_009553211	3e-47	34.53
Flumine bastrovirus 3; UQB75993	9e-47	32.84
Hubei sediment bastro-like virus; QYF50028	2e-45	33.80
Hepelivirales sp.; WAY16393	2e-44	32.34
Bastrovirus-like_virus/VietNam; YP_009333174	5e-44	33.42
Microbat bastrovirus; UBK24595	1e-42	31.91

4. Discussion

References

1. Zerbini, F.M.; Siddell, S.G.; Mushegian, A.R.; Walker, P.J.; Lefkowitz, E.J.; Adriaenssens, E.M.; Alfenas-Zerbini, P.; et al. Differentiating between viruses and virus species by writing their names correctly. Arch. Virol. 2022, 167, 1231-1234. [CrossRef]
2. Kuchibhatla, D.B.; Sherman, W.A.; Chung, B.Y.; Cook, S.; Schneider, G.; Eisenhaber, B.; Karlin, D.G. Powerful sequence similarity search methods and in-depth manual analyses can identify remote homologs in many apparently “orphan” viral proteins. J. Virol. 2014, 88, 10-20. [CrossRef]
3. Solovyev A.G.; Morozov, S.Y. Non-replicative integral membrane proteins encoded by plant alpha-like viruses: emergence of diverse orphan ORFs and movement protein genes. Front. Plant. Sci. 2017, 8, 1820. [CrossRef]
4. Chevin, A.; Coutard, B.; Blanchard, P.; Dabert-Gay, A.S.; Ribiere-Chabert, M.; Thiery, R. Characterization of structural proteins from Chronic Bee Paralysis Virus (CBPV) using mass spectrometry. Viruses 2015, 7, 3329-3344. [CrossRef]
5. Shi, M.; Lin, X-D.; Tian, J-H.; et al. Redefining the invertebrate RNA virosphere. Nature 2016, 540, 539–543. [CrossRef]
6. Chiapello, M.; Bosco, L.; Ottati, S.; et al. Complexity and local specificity of the virome associated with tospovirus-transmitting thrips species. J. Virol. 2021, 95, e00597. [CrossRef]
7. Parry, R.; James, M.E.; Asgari, S. Uncovering the worldwide diversity and evolution of the virome of the mosquitoes Aedes aegypti and Aedes albopictus. Microorganisms 2021, 9, 1653. [CrossRef]
8. Morozov, S.Y.; Lazareva, E.A.; Solovyev, A.G. Sequence relationships of RNA helicases and other proteins encoded by blunervirus RNAs highlight recombinant evolutionary origin of kitaviral genomes. Front. Microbiol. 2020, 11, 561092. [CrossRef]
9. Ramos-González, P. L.; Arena, G.D.; Tassi, A.D.; et al. Kitaviruses: a window to atypical plant viruses causing nonsystemic diseases. Ann. Rev Phytopathol. 2023, 61, 97-118. [CrossRef]
10. Dorrington, R.A.; Jiwaji, M.; Awando, J.A.; de Bruyn, M.M. Advances of tetravirus research: new insights into the infectious virus lifecycle and an expanding host range. Curr. Issues Mol. Biol. 2020, 34, 145-162. [CrossRef]
11. Kallies, R.; Kopp, A.; Zirkel, F.; et al. Genetic characterization of goutanap virus, a novel virus related to negeviruses, cileviruses and higreviruses. Viruses 2014, 6, 4346-4357. [CrossRef]
12. Nunes, M. R.; Contreras-Gutierrez, M. A.; Guzman, H.; et al. Genetic characterization, molecular epidemiology, and phylogenetic relationships of insect-specific viruses in the taxon Negevirus. Virology2017, 504, 152-167. [CrossRef]
13. Wu, N.; Zhang, P.; Liu,W.; Wang, X. Sogatella furcifera hepe-like virus: first member of a novel Hepeviridae clade identified in an insect. Virus Res. 2018, 250, 81-86. [CrossRef]
14. Kondo, H.; Chiba, S.; Maruyama, K.; Andika, I. B.; Suzuki, N. A novel insect-infecting virga/nege-like virus group and its pervasive endogenization into insect genomes. Virus Res. 2019, 262, 37–47. [CrossRef]
15. Kondo, H.; Fujita, M.; Hisano, H.; Hyodo, K.; Andika, I. B.; Suzuki, N. Virome Analysis of Aphid Populations That Infest the Barley Field: The Discovery of Two Novel Groups of Nege/Kita-Like Viruses and Other Novel RNA Viruses. Front. Microbiol. 2020, 11, 509. [CrossRef]
16. O’Brien, C.A.; McLean, B.J.; Colmant, A.M.G.; et al. Discovery and characterization of Castlerea virus, a new species of negevirus isolated in Australia. Evol. Bioinform. Online 2017, 13, 1176934317691269. [CrossRef]
17. Dheilly, N.M.; Lucas, P.; Blanchard, Y.; Rosario, K. A world of viruses nested within parasites: Unraveling viral diversity within parasitic flatworms (Platyhelminthes). Microbiol. Spectr. 2022, 10, e0013822. [CrossRef]
18. Arakawa, K.; Kono, N.; Malay, A.D.; et al. 1000 spider silkomes: Linking sequences to silk physical properties. Sci. Adv. 2022, 8, eabo6043. [CrossRef]
19. Wan, J.; Liang, Q.; Zhang, R.; Chen, Y.; et al. Arboviruses and symbiotic viruses cooperatively hijack insect sperm-specific proteins for paternal transmission. Nat. Commun. 2023, 14, 1289. [CrossRef]
20. Agranovsky, A. A. Where to stop: Occurrence and evolution of translational recording signals in RNA viruses of eukaryotes. Gene Expression 2023, 22, 240-249. [CrossRef]
21. Williams, S.H.; Levy, A.; Yates, R.A.; et al. The Diversity and Distribution of Viruses Associated with Culex annulirostris Mosquitoes from the Kimberley Region of Western Australia. Viruses 2020, 12, E717. [CrossRef]
22. Zhang, W.; Zhang, Y.C.; Wang, Z.G.; Gu, Q.Y.; Niu, J.Z.; Wang, J.J. The Diversity of Viral Community in Invasive Fruit Flies (Bactrocera and Zeugodacus) Revealed by Meta-transcriptomics. Microb. Ecol. 2022, 83, 739-752. [CrossRef]
23. Linnakoski, R.; Sutela, S.; Coetzee, M.P.A.; et al. Armillaria root rot fungi host single-stranded RNA viruses. Sci. Rep. 2021, 11, 7336. [CrossRef]
24. Xavier, C.A.D.; Allen, M.L.; Whitfield, A.E. Ever-increasing viral diversity associated with the red imported fire ant Solenopsis invicta (Formicidae: Hymenoptera). Virol. J. 2021, 18, 5. [CrossRef]
25. Niu, J.; Zhang, W.; Sun, Q.-Z.; Wang, J.-J. Three novel RNA viruses in the spider mite Tetranychus urticae and their possible interactions with the host RNA interference response. J. Invertebr. Pathol. 2019, 166, 107228. [CrossRef]
26. Qin, X.C.; Shi, M.; Tian, J.H.; et al. A tick-borne segmented RNA virus contains genome segments derived from unsegmented virus ancestors. Proc. Nat. Acad. Sci. USA 2014, 111, 6744-6749. [CrossRef]
27. Khalturin, K.; Shunatova, N.; Shchenkov, S.; Sasakura, Y.; Kawamitsu, M.; Satoh, N. Polyzoa is back: the effect of complete gene sets on the placement of Ectoprocta and Entoprocta. Sci. Adv. 2022, 8, 26, eabo4400. [CrossRef]
28. Dunn, C.W.; Giribet, G.; Edgecombe, G.D.; Hejnol, A. Animal phylogeny and its evolutionary implications. Ann. Rev. Ecol. Evol. Syst. 2014, 45, 371-395. [CrossRef]
29. Lenz, O.; Přibylová, J.; Fránová, J.; Koloniuk, I. Fragaria vesca-associated virus 1: a new virus related to negeviruses. Arch. Virol. 2020, 165, 1249-1252. [CrossRef]
30. Marchler-Bauer, A.; Bo, Y.; Han, L.; et al. CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res. 2017, 45, D200-D203. [CrossRef]
31. Tatineni, S.; Gowda, S.; Dawson, W.O. Heterologous minor coat proteins of Citrus tristeza virus strains affect encapsidation, but the coexpression of HSP70h and p61 restores encapsidation to wild-type levels. Virology 2010, 402, 262-270. [CrossRef]
32. Mostert, I.; Bester, R.; Burger, J.T.; Maree, H.J. Identification of Interactions between Proteins Encoded by Grapevine Leafroll-Associated Virus 3. Viruses 2023, 15, 208. [CrossRef]
33. Chen, S.; Yu, F.; Li, Z.; et al. Complete genome sequence of a novel virga-like virus infecting Hevea brasiliensis. Arch. Virol. 2022, 167, 965-968. [CrossRef]
34. Longdon, B.; Brockhurst, M.A.; Russell, C.A.; Welch, J.J.; Jiggins, F.M. The evolution and genetics of virus host shifts. PLoS Pathog. 2014, 10, e1004395. [CrossRef]
35. Schlappi, D.; Chejanovsky, N.; Yañez, O.; Neumann P. Virus transmission via honey bee prey and potential impact on cocoon-building in labyrinth spiders (Agelena labyrinthica). PLoS ONE 2023, 18, e0282353. [CrossRef]
36. Shelomi, M.; Lin, W.; Johnson, B. R.; Furlong, M.J.; Etebari, K. Detection of deformed wing virus (DWV) in the Vietnamese walking stick Medauroidea extradentata (Phasmatodea). Virus Res. 2021, 293, 198263. [CrossRef]
37. Lazareva, E.; Lezzhov, A.; Vassetzky, N.; Solovyev, A.; Morozov S. Acquisition of full-length viral helicase domains by insect retrotransposon-encoded polypeptides. Front. Microbiol. 2015, 6, 1447. [CrossRef]
38. Morozov, S. Y.; Lazareva, E. A.; Solovyev, A. G. RNA helicase domains of viral origin in proteins of insect retrotransposons: possible source for evolutionary advantages. PeerJ. 2017, 5, e3673. [CrossRef]
39. Gilbert, C.; Belliardo, C. The diversity of endogenous viral elements in insects. Curr. Opin. Insect Sci. 2022, 49, 48-55. [CrossRef]
40. Blair, C.D.; Olson, K.E.; Bonizzoni M. The Widespread Occurrence and Potential Biological Roles of Endogenous Viral Elements in Insect Genomes. Curr. Issues Mol. Biol. 2020, 34, 13-30. [CrossRef]
41. Wallau, G.L. RNA virus EVEs in insect genomes. Curr. Opin. Insect Sci. 2022, 49, 42-47. [CrossRef]
42. Morozov, S. Y.; Lezzhov, A.A.; Lazareva, E. A.; Erokhina, T.N.; Solovyev, A. G. Potential role of accessory domains in polyproteins encoded by retrotransposons in anti-viral defense of host cells. Front. Microbiol. 2019, 9, 3193. [CrossRef]