Plant viruses in the family Secoviridae (order Picornavirales) mainly infect dicotyledonous plant species and are naturally transmitted by nematodes or arthropods. The capsids of these viruses are icosahedral, 25- to 30-nm in diameter, and composed of 60 coat protein (CP) subunits [9, 10]. The secovirus genome can be monopartite or bipartite. Bipartite genomes are divided between two RNA segments (RNA-1 and -2), each modified by addition of a 3’-terminal poly(A) tail, and a covalently bound VPg protein at the 5’-end. RNA-1 ranges in length from 6 to 8 kb and encodes the proteins necessary for cytoplasmatic replication. In contrast, RNA-2 is smaller at 2 to 4 kb and encodes the movement protein (MP) and up to three coat proteins (CP) [7]. The Pro-Pol sequence on RNA-1 is used to infer phylogenetic relationships within the family and with other members of the order [7]. Currently, members of the Secoviridae family are classified into eight genera: Comovirus, Fabavirus, Nepovirus, Cheravirus, Sadwavirus, Torradovirus, Sequivirus, and Waikavirus [7, 10]. A ninth genus, “Stralarivirus”, and the division of the genus Sadwavirus into the three subgenera, “Stramovirus”, “Satsumavirus”, and “Cholivirus” was proposed in the revision taxonomy of the family Secoviridae [7].
Pineapple secovirus A (PSV-A), an unassigned member within the family Secoviridae, was first detected from an Ananas comosus germplasm accession (HANA 187) from the Pacific Basin Agricultural Research center (PBARC) in Hilo, Hawaii [5]. PSV-A survey was carried out in 2019 on the island of Oahu and revealed the presence of the virus in six out of twelve plants presenting mealybug wilt of pineapple (MWP) symptoms (reddening and wilting of the leaves) but not in any the 13 asymptomatic plants [5]. Various combinations of ampelovirus from the pineapple mealybug wilt-associated virus (PMWaV) complex of species were also detected in the 12 symptomatic plants (Larrea-Sarmiento et al, unpublished results).
To examinate for the presence of further undiscovered viruses infecting Ananas comosus, high-throughput sequencing (HTS) was done on the same field plants detailed in the study by Larrea-Sarmiento et al. (2020). Total RNAs were extracted from the basal portions of individual pineapple leaf samples using SpectrumTM total RNA kit (Sigma Aldrich) following the manufacturer’s instructions. Total RNA extracted from the twelve MWP symptomatic field samples and thirteen healthy-looking plants were pooled into two composite RNA samples followed by a ribodepletion step to remove the ribosomal RNA (rRNA). cDNA library synthesis was followed by HTS using Illumina® NovaSeq6000 to obtain paired-end reads (2 × 100 bp) at the Genomics High-Throughput Sequencing Facility at the University of California, Irvine.
Data obtained from the ~40 million raw reads per composite ribosomal RNA-depleted total RNA were curated and assembled following the methods of Green et al. [2]. The resulting contigs were annotated by doing BLASTX searches of the NCBI virus sequence database. Annotated contigs revealed shared sequence identity with the previously characterized PMWaVs and secoviruses. Two contigs retrieved from the symptomatic composite sample had significant matches to PSV-A and other sadwaviruses but were sufficiently divergent to suggest they represented the two RNA components of a new species. Similar to PSV-A and the majority of secovirids, the potential new virus has a bipartite genome encoding two positive-sense RNA molecules.
To complete the genome sequence of the virus, 5’ and 3’ rapid amplification of cDNA ends (RACE) was performed. Both 5’ and 3’ ends were obtained using the Takara SMARTer RACE 5’/3’ kit according to the manufacturer’s instructions followed by PCR with a universal anchored primer and sequence specific primers (Table S1). Amplicons were cloned, and five to seven clones were Sanger sequenced. The complete genome comprised two RNA molecules; RNA-1 is 5,956 nt long (GenBank accession code OM777135) and RNA-2 is 3,808 nt long (GenBank accession code OM777136), each coding for large polyproteins referred as P1 and P2, respectively. The name pineapple secovirus B (PSV-B) is proposed for this putative new virus infecting pineapple.
The PSV-B P1 of 1875 aa is predicted to code for proteins involved in replication: protease cofactor (Pro-C), helicase (Hel), genome-linked viral protein (VPg), protease (Pro), and RNA-dependent RNA-polymerase (Pol). Likewise, PSV-B P2 of 1143 aa is predicted to code for the movement protein (MP) and one large coat protein (CP) (Fig. 1). Like other secovirids, both PSV-B RNAs are expected to possess a genome-linked viral protein termed “VPg” bound at the 5’ end and a poly(A) tail at the 3’ end, respectively [1, 7]. Analysis of conserved domains using the NCBI Conserved Domain Search Tool (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) revealed conserved motifs for the RdRp and Helicase for the P1; whereas a conserved MP motif was predicted for the P2. Parallel analysis using HMMER (https://www.ebi.ac.uk/Tools/hmmer/) and Pfam (http://pfam.xfam.org/) further predicted the presence of two CP domains corresponding to nepovirus and picornavirus CP domains for P2, although other members within the subgenus Cholivirus (Genus Sadwavirus) are predicted to encode only one large CP [4-6, 11]. Analysis of the predicted cleavage sites for P1 located four Q/S, and five E/G dipeptides were also found [3]. The cleavage sites recognized by the RNA-1-encoded 3C-like protease (3CL-Pro) likely cleave P1 at five sites, defining six domains; while 3CL-Pro likely cleaves P2 at one site, defining two domains (Fig. 1) [1, 6, 7].
The recently characterized PSV-A was found to be closely related to Dioscorea mosaic associated virus (DMaV) and to chocolate lily virus A (CLVA) [5]. In 2020, the proposed revision of the family Secoviridae classified DMaV and CLVA, previously denotated as unassigned secoviruses, as members of the Cholivirus subgenus within the Sadwavirus genus [7]. To study the taxonomic position PSV-B, and relatedness with PSV-A and other members of the family Secoviridae, phylogenetic analysis using the Maximum Likelihood method based on the aa sequence of the Pro-Pol region was carried out using LG+G as the best model of protein evolution. This analysis suggests that PSV-B is a new Sadwavirus member that is related to, but distinct from, the previously characterized Cholivirus member PSV-A (Fig. 2). It is placed on a branch distinct from PSV-A and basal to that containing DMaV and CLVA (Fig. 2). For members within the family Secoviridae, species demarcation criteria are <80% identity for the aa sequence of the Pro-Pol region, and <75% identity for the large and small CP together [10]. Identities of 45.1% and 53.5% were observed when comparing the Pro-Pol region of PSV-B to PSV-A and CLVA homolog regions, respectively, using pairwise comparisons. These results are consistent with the findings reported in Australia in 2002 where two isometric viruses were reported to infect pineapple and partial sequences showed similarities to strawberry mottle virus [8]. Combined results support the establishment of PSV-B as a member of a new species in the subgenus Cholivirus under the genus Sadwavirus in the family Secoviridae [7].
Specific primers were used for the detection of each of the two PSV-B RNAs from the 25 field samples collected in 2019. Primer pairs targeting the RdRp-coding region and CP-coding region for the detection of RNA1 and RNA2, respectively, were used in RT-PCR assays (Table S1). Total RNA was used for cDNA synthesis using the M-MLV reverse transcriptase from Promega. RT-PCR reactions were performed as follows: initial denaturation at 95ºC for 5 min, 35 cycles of 95ºC for 40 s, 57ºC for 30 s and 72ºC for 40 s, and the final extension at 72ºC for 10 minutes. Four out of the 12 MWP symptomatic samples revealed the expected band of 702 bp for RNA-1 and 380 bp for RNA-2, and their identity was confirmed by direct Sanger sequencing of the amplicons. The presence of PMWaVs and PSV-A was assessed as detailed previously (Green et al. 2019; Larrea-Sarmiento et al. 2020; Larrea-Sarmiento et al. 2021). Samples infected with PSV-B were also infected with PMWaV-2, PMWaV-3, PMWaV-6, and PSV-A. Nine out of the 13 healthy looking plant samples presented only single infections with PMWaV-3; while the remaining five did not test positive for any virus [5]. Further research is needed to determine the prevalence of PSV-B in other pineapple producing countries and to evaluate if PSV-B is involved in the etiology of MWP.