February 2018

This Word module should be used for all taxonomic proposals. Please complete Part 1 and:

either Part 3 for proposals to create new taxa or change existing taxa or Part 2 for proposals of a general nature.

Submit the completed Word module, together with the accompanying Excel module named in Part 3, to the appropriate ICTV Subcommittee Chair.

For guidance, see the notes written in blue, below, and the help notes in file Taxonomic_Proposals_Help_2018.

Part 1: TITLE, AUTHORS, etc

Code assigned: 2018.031P (to be completed by ICTV officers)

Short title: Recognizing the family Endornaviridae as a taxon of positive-sense RNA viruses in the ICTV framework

Author(s):Sead Sabanadzovic Nina Aboughanem-Sabanadzovic Valerian V. Dolja Mart KrupovicEugene V. KooninRodrigo A. Valverde

Corresponding author with e-mail address:Sead Sabanadzovic ss501@msstate.edu

List the ICTV study group(s) that have seen this proposal:A list of study groups and contacts is provided at http://www.ictvonline.org/subcommittees.asp . If in doubt, contact the appropriate subcommittee chair (there are six virus subcommittees: animal DNA and retroviruses, animal ssRNA-, animal ssRNA+, fungal and protist, plant, bacterial and archaeal)

No SGs, but the Fungal Virus SC Chair has seen it and is in agreement with the proposal

ICTV Study Group comments (if any) and response of the proposer:     

Date first submitted to ICTV: June 6th, 2018Date of this revision (if different to above): September 30th , 2018

ICTV-EC comments and response of the proposer:     

Part 2: NON-STANDARDTemplate for any proposal regarding ICTV procedures, rules or policy, not involving the creation of new taxonomy.

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February 2018

Text of proposal:

Background

Endornaviruses, originally described as cellular high molecular weight dsRNAs from French and broad beans (Grill and Garger, 1981; Wakarchuk and Hamilton, 1985), have been reported from plants, fungi and oomycetes. However, results of recent metagenomic (Shi et al., 2016) and “in silico cloning” studies (Liu et al., 2012) suggest a much broader host range.

With the exception of Vicia faba endornavirus (VfEV; Grill and Garger, 1981) and Helicobasidium mompa endornavirus 1 (HmEV-1; Osaki et al., 2006), these viruses are generally associated with persistent and symptomless infections in their respective hosts (Dolja and Koonin, 2012; Fukuhara and Gibbs, 2012; Roossinck et al., 2011). Unlike the majority of viruses, endornaviruses do not form typical virions (i.e., they are capsidless replicons), and do not move from cell to cell. Furthermore, plant-infecting endornaviruses do not move horizontally by grafting, mechanical inoculation, or via vector involvement. However, they are all efficiently transmitted vertically via seeds or spores.

History and a current status of the endornavirus taxonomy

Formal classification of large endogenous dsRNAs within the ICTV framework was prompted at the dawn of 21st Century as the first nucleotide sequences of these replicons became available. Here is a brief historic overview of the endornavirus taxonomy as well as its current status.

2000 - Gibbs and colleagues suggested classification of four “large double stranded RNA replicons from plants” as viruses based on observation that they encode helicase and RNA-dependent RNA polymerase domains related to those of viruses in the alphavirus-like supergroup (Gibbs et al., 2000). In the same paper, the authors proposed names Endornavirus and Endoviridae for the new genus and family, respectively.

2003 – A formal proposal for creation of a new floating genus named Endornavirus (authors T. Fukuhara, P. Pfeiffer and M.J. Gibbs) containing four species submitted to the ICTV Executive Committee for evaluation. The proposal was approved and ratified in 2004, making this taxon part of the official ICTV taxonomy.

2005 – New genus Endornavirus included in the Eight ICTV Report (Section ‘dsRNA viruses’)

2006 – Formal Taxonomic Proposal for creation of a new family Endornaviridae to embrace existing genus Endornavirus, coauthored by T. Fukuhara and M.J. Gibbs, submitted to the ICTV Executive Committee. Following an e-mail ratification vote, the family was officially recognized in 2008.

2012 – Family Endornaviridae (comprising a single genus Endornavirus) included in the section ‘dsRNA viruses” in the Ninth ICTV Report (Figure 1A) (Fukuhara and Gibbs, 2012)

2016 – Two taxonomic proposals for reorganization of the family (renaming the existing genus Endornavirus to Alphaendornavirus, establishing a new genus Betaendornavirus recognition of 10 new species) submitted on behalf of the Endornaviridae SG. Despite proposing major updates of the Endornaviridae taxonomy, the proposals did not address origin and nature of endornaviruses. These changes were approved in 2017.

2018 – Master Species List 2017 v1.0, released in March 2018, reports members of the family

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Text of proposal:Endornaviridae as ‘dsRNA viruses’ (Figure 1 B)

In summary, in the ICTV-recognized taxonomy, endornaviruses have been historically, and are currently, classified as dsRNA viruses.

Reasons for the proposal for recognition of viruses in the family Endornaviridae as positive sense RNA viruses

All currently recognized endornaviruses possess a monocistronic RNA genome ranging from 9.8 (Tuber aestivum endornavirus) to 17.6 kb (Vicia faba endornavirus) in size. Several endornaviruses have a 5’-proximal, position-specific nick in the positive RNA strand (Fukuhara and Moriyama, 2008; Okada et al., 2011; Okada et al., 2013). A large ORF, encompassing more than 95% of the endornavirus genomes, codes for a large protein that consists of several functional domains, with some variation of the domain composition depending on the specific virus (Figure 2). Hereinafter, we briefly discus three highly conserved replication-associated genes, namely, methyltransferase (MTR), superfamily 1 helicase (S1H) and RNA-dependent RNA polymerase (RdRp), which are characteristic of the alpha-like superfamily of positive-sense RNA viruses (Koonin and Dolja, 1993; Koonin et al., 2015) and are of particular relevance for this proposal.

The RdRp is the only domain that is present in all endornaviruses (Figure 2). The results of phylogenetic analyses of endornaviral RdRps leave no doubt about their evolutionary relationships with alphavirus-like superfamily of viruses (Dolja and Koonin, 2012; 2018; Gibbs et al., 2000; Koonin et al, 2015; Roossinck et al., 2011; Sabanadzovic et al., 2016), and in particular, with plant-infecting members of the families Virgaviridae, Bromoviridae and Closteroviridae. The relationship of endornaviral RdRp with alpha-like viruses is in part presented in Figure 3.

Furthermore, all but one of 24 recognized endornaviruses encode for S1H, another highly conserved domain in replication-associated proteins of the alphavirus-like supergroup members. In addition to S1H, three members of the genus Betaendornavirus code for Superfamily 2 helicase (S2H), the helicase family present in flavi-, poty- and hypoviruses (Koonin and Dolja, 1993; 2014). Tuber aestivum endornavirus (TaEV) is the only endornavirus that lacks S1H, but possesses S2H instead (Stielow et al., 2011). S1H domains of endornaviruses are phylogenetically related to orthologs in alpha-like viruses (Gibbs et al, 2000; Dolja and Koonin 2012; Koonin et al., 2015; Roossinck et al., 2011).

The third hallmark domain present in all alphavirus-like viruses, methyltransferase, is encoded by all 5 currently recognized betaendornaviruses, whereas it is identifiable only in a subset of the members of the genus Alphaendornavirus. Nevertheless, in-depth analyses suggest presence of a highly diverged form of this domain (non-detectable by BLAST searches in CDD, SMART and Pfam databases) in polyproteins encoded by alphaendornaviruses (S. Sabanadzovic, unpublished data). The MTRs of endornaviruses are evolutionarily related to the counterparts in positive-sense RNA viruses, in particular to those of members of the family Virgaviridae.

In summary, the majority of currently characterized endornaviruses possess an array of domains that are signatures of the replication-associated module in alphavirus-like supergroup of positive-strand RNA viruses: viral MTR, S1H and RdRp (RdRpP_2, PF00978). Furthermore, phylogenetic analyses of all three genes undeniably prove evolutionary affinity between extant endornaviruses and alpha-like viruses (Dolja and Koonin, 2012; Koonin and Dolja, 2014; Koonin et al., 2015; Roossinck et al., 2011). Therefore, the proposed evolutionary scenario in

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Text of proposal:which an ancestral endornavirus originated from an alphavirus-like virus via capsid loss seems highly plausible (Dolja and Koonin, 2012; 2018; Koonin and Dolja, 2014; Koonin et al., 2015).

From the above data, it seems obvious that the current position of endornaviruses within the ICTV-approved taxonomy framework as dsRNA viruses is not justified and they should be re-classified in the group of positive-strand RNA viruses, similar to the recent reassignment of the family Hypoviridae, another group of capsidless viruses that have been historically considered dsRNA viruses. The fact that most known endornaviruses have been characterized through analysis of dsRNAs, which are relatively stable and easy to isolate due to their abundance in the infected host, cannot justify their present classification as dsRNA viruses. Indeed, many of the positive-sense RNA fungal or plant viruses, and/or capsidless RNA replicons, generate relatively large amounts of dsRNA (replicative intermediates) during their replication cycle (Valverde et al., 1990; Valverde and De La Torre-Almaraz, 2017).

Accordingly, we propose to formally change the position of the family Endornaviridae in the ICTV taxonomy framework by moving it to the positive-strand RNA viruses, in order to reflect their evolutionary relationships with alpha-like positive-sense RNA viruses.

Part 3: PROPOSED TAXONOMY

Name of accompanying Excel module: NOT APPLICABLE

The taxonomic changes you are proposing should be presented on an accompanying Excel module, 2017_TP_Template_Excel_module. Please enter the file name of the completed module in this box.

Supporting material:additional material in support of this proposal

Please explain the reasons for the taxonomic changes you are proposing and provide evidence to support them. The following information should be provided, where relevant: Species demarcation criteria: Explain how new species differ from others in the genus and

demonstrate that these differences meet the criteria previously established for demarcating between species. If no criteria have previously been established, and if there will now be more than one species in the genus, please state the demarcation criteria you are proposing.

Higher taxa: o There is no formal requirement to state demarcation criteria when proposing new genera

or other higher taxa. However, a similar concept should apply in pursuit of a rational and consistent virus taxonomy.

o Please indicate the origin of names assigned to new taxa at genus level and above.o For each new genus a type species must be designated to represent it. Please explain

your choice. Supporting evidence: The use of Figures and Tables is strongly recommended (note that

copying from publications will require permission from the copyright holder). For phylogenetic analysis, try to provide a tree where branch length is related to genetic distance.

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Figure 1. Current position of family Endornaviridae within the ICTV taxonomy. A. Table reported in the 9th ICTV Report (King et al., 2012); B. Currently Master Species List. In both sources endornaviruses are listed as dsRNA viruses.

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Figure 2. Schematic comparison of genomes of some members of the family Endornaviridae (A) with tobacco mosaic virus (TMV), a plant-infecting alpha-like virus in the genus Tobamovirus, family Virgaviridae (B). Genomes of the following endornaviruses are depicted (from top to bottom of the figure): Oryza sativa endornavirus (OsEV), bell pepper endornavirus (BPEV), Sclerotinia sclerotiorum endornavirus 1 (SsEV1) and Gremmeniella abietina type B RNA virus XL (GaBRV-XL). Abbreviation of functional domains: MTR = methyltransferase, S1H = Helicase Superfamily 1, S2H = Helicase Superfamily 2, CPS = capsular polysaccharide synthase, GT = glycosyl transferase, S7 = phytoreo S7, RdRp = RNA-directed RNA polymerase, MP = movement protein, CP = coat protein. Figure is not to scale. Notice the presence of functional domains harbored in endornavirus-encoded polyproteins.

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Figure 3. Phylogenetic tree reconstructed using the Maximum Likelihood method implemented in the PhyML program (v. 3.1/3.0 aLRT) (Guindon & Gascuel, 2003). Sequences were aligned with MUSCLE (Edgar, 2004) configured for highest accuracy and positions with gaps were removed from the alignment. Reliability of internal branches was assessed using the aLRT test (SH-Like) (Anisimova et al., 2006). The analysis was performed on the Phylogeny.fr platform (http://www.phylogeny.fr/) (Dereeper et al., 2008). The tree was visualized with FigTree v. 1.4.3.

References:Anisimova, M. and Gascuel, O. (2006). Approximate likelihood ratio test for branches: A fast,

accurate and powerful alternative. Syst Biol 55:539-552.

Dereeper A., Guignon V., Blanc G., Audic S., Buffet S., Chevenet F., Dufayard J.F., Guindon S., Lefort V., Lescot M., Claverie J.M., Gascuel O (2008). Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 2008 Jul 1;36(Web Server issue): W465-9.

Dolja, V.V. and Koonin, E.V. (2012) Capsid‐Less RNA Viruses. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0023269]

Dolja, V.V. and Koonin, E.V (2018) Metagenomics reshapes the concepts of RNA virus evolution by revealing extensive horizontal virus transfer. Virus Res 244:36-52.

Edgar, R.C. (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32: 1792-1797.

Fukuhara, T. and Gibbs, M. J. (2012) Family Endornaviridae.In Virus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses, pp. 519–521. Edited by A. M. Q. King, M. J. Adams, E. B. Carstens and E. J. Lefkowitz. Elsevier Academic Press.

Fukuhara, T., and Moriyama, H. (2008) Endornavirus. In: Encyclopedia of virology 3rd ed, vol 2. Mahy BWJ, Van Regenmortel MHV, editors. Oxford: Elsevier. 109-116.

Gibbs, M. J., Koga, K., Moriyama, H., Pfeiffer, P., and Fukuhara, T. (2000) Phylogenetic analysis of some large double-stranded RNA replicons from plants suggests they evolved from a defective single-stranded RNA virus. J Gen Virol 81: 227–233.

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References:Grill, L.K., and Garger, S.J. (1981). Identification and characterization of double-stranded RNA

associated with cytoplasmic male sterility in Vicia faba. Proc Natl Acad Sci USA 78: 7043-7046.

Guindon, S. and Gascuel, O. (2003). A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696-704.

King, M.Q., Adams, M.J., Carstens E.B. and Lefkowitz E.J. (eds). (2012). Virus taxonomy, 9th ed. Elsevier Academic Press, Amsterdam, Netherlands.

Koonin, E.V., and Dolja, V.V. (1993) Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences. Critl Rev Biochem Mol Biol 28: 375-430.

Koonin, E.V., and Dolja, V.V. (2014) Virus world as an evolutionary network of viruses and capsidless selfish elements. Microbiol Mol Biol Rev 78:278–303.

Koonin, E.V., Dolja, V.V., and Krupovic, M. (2015) Origins and evolution of viruses of eukaryotes: the ultimate modularity. Virology 479–480: 2–25.

Liu, H., Fu Y., Xie J., Cheng J., Ghabria, l S.A., Li G, Yi. X., and Jiang, D. (2012) Discovery of novel dsRNA viral sequences by in silico cloning and implications for viral diversity, host range and evolution. PLoS One 7:e42147.

Okada, R., Kiyota, E., Sabanadzovic, S., Moriyama, H., Fukuhara, T., Saha, P., Roossinck M.J., Severin, A. and Valverde R.A. (2011) Bell pepper endornavirus: molecular and biological properties and occurrence in the genus Capsicum. J Gen Virol 92:2664-2673.

Okada, R., Yong, C.K., Valverde, R.A., Sabanadzovic, S., Aoki, N., Hotate S., Kiyota, E., Moriyama, H. and Fukuhara, T. (2013) Molecular characterization of two evolutionarily distinct endornaviruses co-infecting common bean (Phaseolus vulgaris). J Gen Virol 94:220-229.

Osaki, H., Nakamura, H., Sasaki, A., Matsumoto, N., and Yoshida, K. (2006) An endornavirus from a hypovirulent strain of the violet root rot fungus, Helicobasidium mompa. Virus Res 118:143-149.

Roossinck, M.J., Sabanadzovic, S., Okada, R. and Valverde, R.A. (2011) The remarkable evolutionary history of endornaviruses. J Gen Virol 92:2674-2678.

Sabanadzovic, S., Wintermantel, W.M., Valverde, R.A., McCreight, J.D. and Aboughanem-Sabanadzovic, N. (2016) Cucumis melo endornavirus: Genome organization, host range and co-divergence with the host. Virus Res 214:49-58.

Stielow, B., Klenk, H.-P., and Menzel, W. (2011) Complete genome sequence of the first endornavirus from the ascocarp of the ectomycorrhizal fungus Tuber aestivum Vittad. Arch Virol 156: 343–345.

Shi, M., Lin, X.D., Tian, J.H., Chen, L.J., Chen, X., Li, C.X., Qin, X.C., Li, J., Cao, J.P., Eden, J.S., Buchmann, J., Wang, W., Xu, J., Holmes, E.C. and Zhang, Y.Z. (2016) Redefining the invertebrate RNA virosphere. Nature 540:539–543.

Valverde, R.A., Nameth, S.T. and Jordan, R. (1990) Analysis of double-stranded RNA for plant virus diagnosis. Plant Disease 74: 255-258.

Valverde, R.A., and De La Torre-Almaraz, R. 2017. Extraction and purification of large dsRNAs from virus-infected plants and fungi: Applications in virus detection and identification. Revista Mexicana de Fitopatología 35:80-105.

Wakarchuk, D. A., and Hamilton, R. I. (1985) Cellular double-stranded RNA in Phaseolus vulgaris. Plant Mol Biol 5: 55-63.

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