Identification and genetic characterization of a new totivirus from Bursera graveolens in western Ecuador

The complete genomic sequence of a previously uncharacterized virus provisionally named “Bursera graveolens associated totivirus 1” (BgTV-1) was obtained from Bursera graveolens (Kunth) Triana & Planch., a tree known as “palo santo” in Ecuador. The BgTV-1 genome is a monopartite double-stranded RNA (dsRNA) that is 4794 nucleotides (nt) long (GenBank accession number ON988291). Phylogenetic analysis of the capsid protein (CP) and RNA-dependent RNA polymerase (RdRp) placed BgTV-1 in a clade with other plant-associated totiviruses. Amino acid (aa) sequence comparisons of putative BgTV-1 proteins showed the highest sequence similarity to those of taro-associated totivirus L (QFS21890.1-QFS21891.1) and Panax notoginseng virus A (YP_009225664.1- YP_009225665.1), with 51.4% and 49.8% identity, respectively, in the CP and 56.4% and 55.2% identity, respectively, in the RdRp. BgTV-1 was not detected in total RNA from either of the two endophytic fungi cultured from BgTV-1-positive B. graveolens leaves, suggesting that BgTV-1 may be a plant-infecting totivirus. Based on its distinct host and the low aa sequence similarity between the CP of BgTV-1 and its counterparts from the closest relatives, the virus described in this study should be assigned as a new member of the genus Totivirus.

Bursera graveolens (Kunth) Triana & Planch. (Common names: palo santo ["holy stick"], sassafras, or incense tree) belongs to the family Burseraceae. It is one of the most common native trees in tropical dry forests of Ecuador, including the Galapagos Islands [1]. The distribution of B. graveolens extends from Mexico to Peru [2]. The wood, which has an intense and characteristic scent, is commonly dried and burned as a mosquito repellent. In addition, essential oils from B. graveolens are used to mitigate different types of pain from arthritis, atherosclerosis, and rheumatism [3].
Currently, there is no available information about diseases affecting B. graveolens trees involving potential viral pathogens. Instead, most research has focused on the properties of its essential oils and their medicinal use [4][5][6].
Members of the genus Totivirus have non-enveloped isometric virions about ~40 nm in diameter with genomes consisting of monopartite double-stranded RNA (dsRNA) of 4.5-7 kb with two open reading frames (ORFs). ORF 1 encodes a putative capsid protein (CP), and ORF 2 codes for a putative RNA-dependent RNA polymerase (RdRp). In some totiviruses, the RdRp is expressed as a CP-Pol fusion protein mediated by a −1 ribosomal frameshift (FS), which is mediated by a heptameric slippery site before the stop codon of ORF 1 and an essential pseudoknot structure [7]. In other totiviruses such as Helminthosporium victoriae 190S virus, the RdRp has been detected as a separate and nonfused polypeptide [12]. In this communication, we describe the genomic characterization and phylogenetic relatedness of a previously uncharacterized totivirus from B. graveolens.
In 2020, leaves from a B. graveolens tree located in Prosperina, a sector on the western side of Guayaquil, Ecuador (-2.151364, -79.953438), showing virus-like symptoms including chlorotic mottle and curling were collected for examination. These leaves were used for dsRNA extraction following the protocol described by Morris and Dodds [13], using fibrous cellulose (Cat. No. C6288 Sigma-Aldrich, USA) instead of the original and now discontinued Whatman CF11. The dsRNA was subjected to plant ribosomal RNA depletion using an Illumina Ribo-Zero Kit prior to cDNA library preparation using a TruSeq Library Prep Kit and sent for high-throughput sequencing (HTS). Sequencing was done on a NextSeq 500 Illumina platform as single 75-bp reads.
A total of 14,607,877 sequence reads were obtained using HTS. Sequence data were analyzed using the next-generation sequencing analysis tools available in Geneious Prime® 2022.0.1. Raw sequences were trimmed for adapter removal and quality using the BBDuk plugin and assembled de novo using SPAdes. A total of 2,418 contigs were assembled from the sequence set. A BLASTx search, performed using Geneious Prime, identified a 4,771-nt-long contig showing relevant sequence similarity to several members of the genus Totivirus. The totivirus-like contig was assembled from 92,171 reads, with a sequencing depth ranging from 23X at the terminal regions to 7,302X in the replicase region. No other virus-like contigs were identified in this sample.
To confirm the HTS sequencing results, primers were designed for re-sequencing (whole genome coverage) and viral detection. Total RNA extraction and reverse transcription (RT) were done as described by Halgren et al. [14]. Polymerase chain reaction (PCR) was performed using 2x GoTaq Green Master Mix (Promega, USA) in a 10-μl mixture containing 0.2 μl of each primer (40 μM), 2 μl of cDNA template, and 2.6 μl of DEPC-treated water. PCR parameters were as follows: 94 °C for 4 min, 40 cycles of 94 °C for 40 s, 55 °C for 40 s, and 72 °C for 60 s, and a final extension step of 5 min at 72 °C. PCR products were cloned using a pGEM-T-Easy Vector System (Promega, USA) and sequenced by the Sanger method in both directions. All primers used in this study were designed using Geneious Prime® 2022.0.1, and their sequences can be found in Online Resource 1. All PCR reactions were done in a MiniAmp Plus Thermal Cycler (Applied Biosystems), and PCR amplicons were visualized on 2% agarose gels using a Gel Doc XR UV transilluminator (Bio-Rad).
The 5´ and 3´ termini of the viral genome were obtained using a 5´/3´ RACE Kit, 2 nd Generation (Roche, Germany). RACE reactions were carried out using dsRNA as the initial template. To denature the dsRNA, a mixture containing the dsRNA and random primers was heated to 98°C for 10 min and immediately placed on ice. The denatured mixture was used to perform an RT reaction using a SuperScript III reverse transcription kit according to the manufacturer's instructions (Thermo Scientific). Amplicons were cloned and sequenced as described above.
Phylogenetic analysis was done using the amino acid sequences of the CP, the RdRp, or the putative CP-RdRp fusion protein of the tentatively new totivirus and representative homologues of the family Totiviridae available in the NCBI database. Multiple sequence alignments were done using the Multiple Sequence Comparison by Log-Expectation (MUSCLE) method [15]. A maximum-likelihood phylogenetic tree was constructed using MEGA X with the best-fit protein models (WAG+G+F), (LG+G+I+F), and (LG+G+I) for the CP, CP-RdRp, and RdRp, respectively, with 1,000 bootstrap replicates [16]. The tree topologies obtained by the three phylogenetic analyses were similar, showing the tentatively new totivirus in a clade with taroassociated totivirus L, Pterostylis totivirus, Panax notoginseng virus, loquat-associated totivirus 1, and peach-associated virus 2 (Fig. 2). The phylogenetic tree using the CP showed poor bootstrap support compared to the trees based on the CP-RdRp or RdRp, suggesting that the CP sequence is more diverse and may need additional homologues to confidently resolve the closest evolutionary relationships of the newly discovered Bursera graveolens totivirus.
According to the species demarcation criteria for the genus Totivirus, viruses with less than 50% aa sequence identity that are found in distinct hosts should be assigned to different species [7]. When both putative proteins of BgTV-1 were analyzed, the aa sequence identity to its closest relatives was in the 37-51% range for the CP and 55-58% for the RdRp (Online Resource 2). Based on the fact that the totivirus described here was found in a distinct, unrelated plant host, we infer that it should be considered a new member of the genus Totivirus, with taro-associated totivirus L, Panax notoginseng virus B, and Pterostylis totivirus as its closest relatives. The new virus was provisionally named "Bursera graveolens associated totivirus 1" (BgTV-1).
Twenty-two samples of B. graveolens collected in Prosperina near the area where it was first identified were screened for BgTV-1. The leaf surface of collected samples was sterilized with 5% sodium hypochlorite and 70% ethyl alcohol, subjected to total RNA extraction, and tested by RT-PCR for the virus as described above.
BgTV-1 was detected in 10 out of 22 B. graveolens trees tested in this study. Both symptomless and symptomatic plants were found to be positive for the virus (Online Resource 3). Hence, it was not possible to ascribe the observed symptoms to BgTV-1 infection.
In recent years, there have been numerous reports of totivirus-like sequences identified in plants [8,[17][18][19]. Nevertheless, the genus Totivirus is still considered a genus of fungus-infecting viruses by the ICTV [7]. To investigate the possible presence of an endophytic fungal host for BgTV-1, tissue from four BgTV-1-infected trees was collected, sanitized, cut into squares of one centimeter, placed in potato dextrose agar (PDA) medium supplemented with ampicillin, and kept under controlled conditions (28°C and a 12-h light/dark cycle for 6 days) to obtain fungal cultures. For the identification of fungal endophytes, grown mycelia were subjected to DNA extraction by the modified CTAB method [20] and PCR as described above, using the internal transcribed spacer (ITS) primers ITS1 and ITS4 [21]. The PCR test was validated using DNA from Trichoderma spp. as a positive control. PCR products were cloned and sequenced as described above. Only two fungal endophytes grew in the medium, and they were identified as Nigrospora sphaerica and Bipolaris sorokiniana. RT-PCR from total RNA extracted from both fungi failed to detect BgTV-1, suggesting the possibility that the virus is a plant-infecting totivirus, as has been suggested for a considerable number of newly discovered totiviruses [8,[17][18][19].
The genetic closeness between BgTV-1 and plant-associated totiviruses was demonstrated by the phylogenetic analysis and sequence comparisons presented in this study. This is the first report of a virus associated with B. graveolens. Further studies should be conducted to investigate the transmission, pathogenicity, and host range of this new totivirus. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. LG+G+I for the CP, CP-RdRp, and RdRp, respectively, and 1000 bootstrap replicates were used. The phylogenetic trees were con-structed using MEGA X. Bootstrap values are shown at the nodes. NCBI GenBank accession numbers are shown in the tree. Greenshaded boxes highlight clades containing plant-associated totiviruses, except tea-oil camellia-associated totivirus, a plant-associated totivirus that shares a most recent ancestor with red clover powdery mildew-associated totivirus, a fungal totivirus. The new Bursera graveolens associated totivirus 1 (BgTV-1) is highlighted in red.

Data availability
The complete genome sequence reported here is available in the GenBank database under accession number: ON988291

Declarations
Conflict of interest The authors declare that they have no conflict of interest.
Ethical approval This research does not include any experiments involving human participants or animals and was directed under Genetic Resource Access Permit # MAE-DNB-CM-2018-0098 granted by the Department of Biodiversity of the Ecuadorean Ministry of the Environment.