Abstract
Here, we report the detection and complete genome sequence of a novel potexvirus, tentatively named “Adenium obesum virus X” (AobVX), isolated from Adenium obesum, that was sent for virus screening at Australian Government post-entry quarantine (PEQ) facilities after being imported into Australia from China. The AobVX genome is 6781 nucleotides in length excluding the poly(A) tail and is predicted to encode conserved potexvirus proteins and sequence motifs across five open reading frames. The RNA-dependent RNA polymerase of this virus shares the highest amino acid sequence similarity with that of nerine potexvirus 1 (58.7% identity) and nerine virus X (58.58% identity). This is the first report of a positive-sense single-stranded RNA virus in A. obesum related to members of the genus Potexvirus in the family Alphaflexiviridae.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Adenium obesum (Forssk.) Roem. and Schult. (family Apocynaceae), commonly known as desert rose, is a perennial succulent shrub native to southeast Africa. It is a popular exotic ornamental that is gaining prominence due to its drought resistance, ease of maintenance, and the variety of colours and shapes of its abundant and long-lasting flowers [1]. It is a known host to some viruses, such as cucumber mosaic virus [2] and tomato spotted wilt virus [3], but not potexviruses. Potexviruses are characterized by non-enveloped flexuous filaments (470–580 × 13 nm) that contain a monopartite single-stranded RNA genome ranging between 5.9 and 7.0 kb in size. A typical potexvirus genome is composed of a 5’ cap structure, five open reading frames (ORFs), and a 3’ poly(A) tail. It encodes an RNA-dependent RNA polymerase (RdRp), triple gene block (TGB 1, 2, and 3) proteins, and a coat protein (CP). Potexviruses mainly infect herbaceous hosts and have no known vectors [4].
In November 2021, mottling symptoms were observed on leaves of a batch of imported A. obesum, which were growing in an approved arrangement at PEQ Nambour Queensland (Fig. 1A). The plants were tested by the Department of Agriculture, Forestry and Fisheries, using ImmunoStrip testing (Agdia), and were positive for cucumber mosaic virus (CMV). Transmission electron microscopy examination revealed that the sap from the infected plants also contained filamentous particles approximately 500 nm in length and 11–20 nm in diameter (Fig. 1B), morphologically similar to potexviruses.
Total RNA was extracted from a symptomatic plant, and a small RNA (sRNA)-Seq library was generated and sequenced as recommended by Lelwala et al. [6]. De novo assembly of the quality-filtered reads obtained followed by contig scaffolding using VirReport [7] enabled the detection of CMV, as well as the full genome reconstruction of a novel potexvirus. Mapping of sRNA reads onto the AobVX genome sequence resulted in 1,658,356 aligned reads (mean read depth, 244), of which 62.7%, 29.3%, and 0.6% were 21 nt, 22 nt, and 24 nt long, respectively (Fig. 1C). The high proportions of mapped 21-nt and 22-nt reads were consistent with them being derived from the plant antiviral RNAi silencing response [8]. The AobVX genome was recovered from seven additional Adenium plants showing similar symptoms. The nucleotide sequence has been deposited in the GenBank database under the accession number OR039325, and the raw data are available under BioProject PRJNA984418 (reviewer link available at https://dataview.ncbi.nlm.nih.gov/object/PRJNA984418?reviewer=vfrple83dh9oq2f6o04eieohhm).
The complete sequence of the sRNA-based genome assembly, including the 5’ and 3’ untranslated regions (UTRs), was independently confirmed in one of the Adenium specimens tested by coupling rapid amplification of cDNA ends (RACE) [9] and Oxford Nanopore Technology (ONT)-based rapid amplicon sequencing (Supplementary Table S1) as described previously [10]. The scaffold obtained was > 99% identical to the original sRNA-based genome assembly. Mapping of the ONT reads to the AobVX genome sequence using minimap2 [11] resulted in 6501 aligned reads (mean read depth, 235) (Fig. 1C).
The genome sequence of AobVX is 6781 nt long and predicted to contain five ORFs, which are flanked by a 91-nt 5’ UTR and a 50-nt 3’ UTR (Fig. 1D). The 5’ UTR begins with the conserved motif “GGAAAA” [12], and the 3’ UTR harbours the conserved hexamer “ACUUAA” [13]. The first and second ORFs are separated by a 12-nt-long intergenic region, while the last four ORFs (2–5) overlap slightly with one another. This genomic organization has been reported in other potexvirus members [14].
ORF1 (4833 nt) encodes an RdRp of 1610 aa that is predicted to contain the three conserved replicase domains of the “alphavirus-like” supergroup of RNA viruses: (a) an N-terminal putative methyltransferase domain, (b) a helicase domain beginning with the NTP-binding consensus motif “GX2GXGKS”, (c) and a C-terminal RdRp domain. The region between the putative methyltransferase and the helicase domains shows the most variation both in terms of length and sequence similarity, as has been observed in other potexviruses [15]. ORF2 (687 nt), ORF3 (344 nt), and ORF4 (261 nt) compose the TGB structure and encode polypeptides of 228 aa (TGB1), 114 aa (TGB2), and 86 aa (TGB3), respectively. TGB1 is predicted to also contain an NTPase/ helicase domain. ORF5 (807 nt) encodes a 268-aa CP, which contains the conserved hydrophobic sequence “FAAFDFFDAV” within the predicted coat protein domain (Fig. 1D, Supplementary Fig. S1).
Pairwise comparisons indicated that the predicted RdRP of AboVX showed the highest amino acid sequence similarity to that of nerine potexvirus 1 (58.7%) and nerine virus X (58.58%) (Supplementary Table S2). This is consistent with the results of phylogenetic analysis based on the RdRp sequences of AobVX and those of other representatives of the family Alphaflexiviridae, in which AobVX clusters with potexviruses and is most closely related to nerine potexvirus 1 and nerine virus X (Fig. 2). Based on its morphology, genome organization, sequence similarity, and phylogenetic clustering with previously reported potexviruses, the newly isolated virus AobVX is proposed to be a member of a distinct species in the genus Potexvirus.
References
Varella TL, Silva GM, Cruz KZCM, Mikovski AI, Nunes JRS, Carvalho IF, Silva ML (2015) In vitro germination of desert rose varieties. Ornam Hortic 21:227–234
Baker CA, Achor D, Adkins S (2003) Cucumber mosaic virus diagnosed in desert rose in Florida. Plant Dis 87:1007
Adkins S, Baker CA (2005) Tomato spotted wilt virus Identified in Desert Rose in Florida. Plant Dis 89:526
Kreuze JF, Vaira AM, Menzel W, Candresse T, Zavriev SK, Hammond J, Hyun Ryu K, ICTV Report Consortium (2020) ICTV virus taxonomy profile: Alphaflexiviridae. J Gen Virol 101:699–700
Leblanc Z, Gauthier ME, Lelwala R, Elliott C, McMaster C, Eichner R, Davis K, Liefting L, Thompson J, Dinsdale A, Whattam M, Pattemore J, Barrero RA (2022) Complete genome sequence of a novel potyvirus infecting Miscanthus sinensis (silver grass). Arch Virol 167:1701–1705
Lelwala RV, LeBlanc Z, Gauthier MEA, Elliott CE, Constable FE, Murphy G, Tyle C, Dinsdale A, Whattam M, Pattemore J, Barrero RA (2022) Implementation of GA-VirReport, a web-based bioinformatics toolkit for post-entry quarantine screening of virus and viroids in plants. Viruses 14:1480
Gauthier MEA, Lelwala RV, Elliott CE, Windell C, Fiorito S, Dinsdale A, Whattam M, Pattemore J, Barrero RA (2022) Side-by-side comparison of post-entry quarantine and high throughput sequencing methods for virus and viroid diagnosis. Biology 11:263
Llave C (2010) Virus-derived small interfering RNAs at the core of plant–virus interactions. Trends Plant Sci 15:701–707
Adamopoulos PG, Tsiakanikas P, Stolidi I, Scorilas A (2022) A versatile 5′ RACE-Seq methodology for the accurate identification of the 5′ termini of mRNAs. BMC Genomics 23:163
Abeynayake SW, Fiorito S, Dinsdale A, Whattam M, Crowe B, Sparks K, Campbell PR, Gambley C (2021) A Rapid and Cost-Effective Identification of Invertebrate Pests at the Borders Using MinION Sequencing of DNA Barcodes. Genes 12:1138
Li H (2021) New strategies to improve minimap2 alignment accuracy. Bioinformatics 37:4572–4574
Wong SM, Mahtani PH, Lee KC, Yu HH, Tan Y, Neo KK, Chan Y, Wu M, Chng CG (1997) Cymbidium mosaic potexvirus RNA: complete nucleotide sequence and phylogenetic analysis. Arch Virol 142:383–391
White KA, Bancroft JB, Mackie GA (1992) Mutagenesis of a hexanucleotide sequence conserved in potexvirus RNAs. Virology 189:817–820
Fuji S, Shinoda K, Furuya H, Naito H, Fukumoto F (2006) Complete nucleotide sequence of Nerine virus X (NVX-J) isolated from the African lily plant (Agapanthus campanulatus) in Japan. Arch Virol 151:205–208
Chen J, Shi YH, Adams MJ, Chen JP (2005) The complete sequence of the genomic RNA of an isolate of Lily virus X (genus Potexvirus). Arch Virol 150:825–832
Funding
Open Access funding was enabled and organized by CAUL and its member institutions. The project Improving plant industry access to new genetics through faster and more accurate diagnostics using Next Generation Sequencing", MT18005, was funded by Hort Innovation, using the Hort Innovation Citrus, Grape Tables, Rubus, Potato and Nursery research and development levy, co-investment from Queensland University of Technology and contributions from the Australian Government. Hort Innovation is a grower-owned, not-for-profit research and development corporation for Australian horticulture.
Open Access funding enabled and organized by CAUL and its Member Institutions
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Additional information
Communicated by Ioannis E. Tzanetakis.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic Supplementary Material
Below is the link to the electronic supplementary material
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Gauthier, ME.A., Abeynayake, S.W., Lelwala, R.V. et al. First detection and complete genome sequence of a new potexvirus naturally infecting Adenium obesum. Arch Virol 168, 244 (2023). https://doi.org/10.1007/s00705-023-05871-x
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s00705-023-05871-x