Molecular characterisation of a novel recombinant Ribgrass mosaic virus strain FSHS

The viruses of the genus Tobamovirus (http://​ictvonline.​org/​virusTaxonomy.​asp) that exhibit co-divergence and host-switching have a widespread geographical distribution, an extensive host range and prevail even in abiotic natural ecosystems [8, 15, 21, 24]. Subgroup 1 tobamoviruses mostly infect solanaceous species, with the exception of the orchid infecting Odontoglossum ringspot virus (ORSV). The subgroup 2 viruses are highly diverse infecting members of Cactaceae, Cucurbitaceae, Fabaceae, Malvaceae and Passifloraceae, showing host range specificity and serological relatedness that may prelude an increased number of subgroups [14]. Subgroup 3 includes crucifer and plantain infecting viruses. In subgroup 1 the origin-of-assembly (OA) is located in the movement protein (ORF3), while in subgroup 2 it is in the coat protein (ORF4) and in subgroup 3 it’s located in the overlap between ORF3 and ORF4 [8, 15]. The subgroup 3 tobamoviruses are further classified into Ribgrass mosaic virus (RMV), Turnip vein clearing virus (TVCV) and Youcai mosaic virus (YoMV) clusters, based on coat protein phylogeny and host range [12]. Although they were historically considered as Brassicaceae infecting viruses they have a wide host range, with RMV isolates alone infecting taxonomically diverse Rosid and Asterid groups [1], comprising Actinidiaceae, Balsaminaceae, Brassicaceae, Caryophyllaceae, Liliaceae, Plantaginaceae and Scrophulariaceae [4, 12].

RMV was first reported by Holmes from Plantago lanceolata L. (Plantaginaceae) and has been described with various acronyms and synonyms over the years [8], with isolates designated as RMV being reported from species of 15 dicot and monocot families [4, 5]. The reference genome of RMV strain: Kons. 1105 isolate R14 (HQ667979) was sequenced from infected Plantago sp., followed by isolation of two strains of RMV viz. Kons. 1105-V2 (HQ667980) and NZ-439 (HQ667978) from the same genus [4]. RMV strains Actinidia-AC (GQ401365.1) and Actinidia-AD (GQ401366.1) were isolated from infected leaves of Actinidia chinensis and A. deliciosa respectively (Actinidiaceae) [4]. These genomes helped to resolve the nomenclature ambiguity and phylogenetic separation of RMV (sensu stricto), confirming three distinct clusters within subgroup 3 tobamoviruses [12] and placing several isolates previously regarded as RMV into the YoMV cluster. None of the previously published RMV genomes were identified as recombinants.

In the family Virgaviridae, recombinants have been recorded in Furovirus [10], Hordeivirus [6], Tobamovirus [11, 15] and Tobravirus [17] groups. The immediate ancestors of orchid-associated tobamovirus ORSV was hypothesized to be a recombinant between 5′ and 3′ genome fragments from subgroup 3 and 1, respectively [8, 15]. Within subgroup 1 recombination in the replicase read through component in Tobacco mosaic virus (TMV) and Tobacco mild green mosaic virus (TMGMV) appeared to have resulted in a novel TMGMV isolate H7 [7]. Natural recombinations have also been shown between infectious isolates of TMV and Tomato mosaic virus (ToMV), including RdRp, movement and coat protein genes [11]. In subgroup 2 tobamoviruses, different ORFs of the genomes of Cactus mild mottle virus (CMMoV) and Frangipani mosaic virus (FrMV) were shown to be related to viruses infecting diverse host groups, indicating possible recombinations [16, 20]. However, unlike the diverse subgroups 1 and 2, natural recombinations have not previously been reported between subgroup 3 tobamoviruses.

The RMV isolate FSHS was collected from leaf tissues of P. major L., showing mild interveinal mottling, from the Waitakere ranges, Auckland region, New Zealand. The presence of tobamoviruses in symptomatic leaves was confirmed using a TMV immunostrip (Agdia #ISK 37800/0024) and also by indirect ELISA using both TMV and RMV specific antisera [3, 22]. RNA extraction and genome amplification by RT-PCR, cloning, the assembly of consensus sequences and gene translations were conducted as described previously [3, 4]. The genome was amplified as 10 overlapping fragments (using the primer sets listed in Additional file 1: Table S1). Two separate amplicons spanned the individual recombination points and two others amplicons both spanned the whole of the integrated fragment from RMV Actinidia-AC plus adjacent sequence from NZ-439, demonstrating that the complete genome sequence was not an artifact from the assembly of sequence fragments from a mixed infection of two strains.

The complete genome, nucleotide (nt) and amino acid (aa) sequences of all the ORFs and the 5′ and 3′UTR sequences of the new strain (JQ319720.1) were compared with GenBank (http://​blast.​ncbi.​nlm.​nih.​gov/​Blast.​cgi), all other complete RMV genome sequences [4] and representative genomes from the TVCV and YoMV clusters [12]. Phylogenetic and molecular evolutionary analyses of the FSHS genome and the complete genomes of 33 representative tobamoviruses, and the relationship of the aa sequences of the four ORFs to selected tobamovirus species were elucidated by the neighbour joining method using MEGA 6.0 [25].

The whole genome and nucleotide sequence of all the ORFs of the strains FSHS (JQ319720.1), Kons. 1105 (HQ667979), NZ-439 (HQ667978) and Actinidia-AC (GQ401365.1), representing the RMV cluster, were analysed using seven different recombination detection algorithms viz. RDP, GeneConv, Bootscan, MaxChi, Chimaera, SiScan and 3Seq (http://​web.​cbio.​uct.​ac.​za/​~darren/​rdp) for the possible recombination events with RDP4 [19]. Each of the RMV genomes was individually used as the reference sequence for analysis of the potential recombinant genome, the major and minor parents. The recombination breakpoint locations were determined based on highest consensus recombination scores (<0.5) and the least average P values obtained for the various algorithms used. Additionally, phylogenetic trees were constructed exclusively for RMV sequences ignoring recombinations, using NJ, UPGMA, FastNJ, ML and Bayesian tree programs built in RDP 4; also ML (GTRCAT) tree incorporating the recombinations [19]. In addition recombination was examined using SplitsTree4 analysis [13].

The genome of RMV strain FSHS (JQ319720.1), comprising 6311nts, showed the typical organisation of a subgroup 3 tobamovirus with four open reading frames (ORFs), with untranslated regions at 5′ and 3′ ends (Fig. 1a). ORF1 and 2 encode replicase proteins of 125.23 kDa and 181.9 kDa, respectively. ORF1, the putative replicase component, has viral-methyltransferase and viral-helicase domains and codes for 1107 amino acids. ORF2 codes for a replicase read-through component that is associated with RNA-dependent RNA polymerase activity, comprising 1601 amino acids. ORF3 codes for the movement protein, consisting of 267 amino acids (30.1 kDa). ORF4 overlaps ORF3 by 77 nucleotides, spanning nucleotides 5603 to 6076, and codes for 157 amino acids (17.5 kDa) of the coat protein. The 5′ and 3′ UTRs are 67 and 235 nucleotides long, respectively [4].

The complete FSHS genome differs from the reference sequence RMV strain Kons. 1105 isolate R14 (HQ667979) and the Actinidia strains (GQ401365.1 and GQ401366.1) by 8 %, from RMV NZ-439 (HQ667978) by 3 %, and from representative members of TVCV and YoMV clusters by 13 % and 19 %, respectively. The 5′UTR sequence of FSHS was identical to all other sequences in the RMV cluster and differed by 1-9 % from TVCV and YoMV respectively. The 3′UTR differed by <3 % to other sequences in the RMV cluster and 6-8 % from representative members of TVCV and YoMV clusters respectively. Comparisons between the nucleotide (nt) and amino acid (aa) sequences of the four ORFs of strain FSHS (JQ319720.1) and representative species from three clades within subgroup 3 are summarised in Table 1. Sequences of the individual ORFs of FSHS differed by 1-9 % (nt) and 0-3 % (aa) to other sequences within the RMV cluster, the most similar being NZ-439 with 1-3 % (nt) and 0-2 % (aa) difference, and by 12-15 % (nt) and 6-13 % (aa) from TVCV and 17-21 % (nt) and 11-20 % (aa) from YoMV. The new genome exhibited the phylogenetically conserved nucleotide motif identified in the RNA polymerase gene of the tobamovirus [9], between nucleotides 4303-4349 of the replicase read-through component as observed in previously published strains of RMV [4], with an exception at position 4312 where adenine (A) is replaced by thymine (T). In addition the recombinant genome showed two other polymorphisms (position 4332, T instead of A/C/G; position 4345, A instead of G) that are specific to RMV [4].

The phylogenetic analyses of the whole genome of the RMV strain FSHS (JQ319720.1) with 33 different tobamoviruses (Fig. 2a) placed FSHS in the RMV cluster of subgroup 3 tobamoviruses [4, 12], as did the nt and aa sequences of the four individual ORFs (results not shown). Within the RMV cluster strains FSHS and NZ-439 (HQ667978) formed a clade, while strains Kons. 1105 (HQ667979) and Actinidia-AC (GQ401365.1) clustered separately (Fig. 2a). Similar clustering was observed in the phylogenetic trees constructed using exclusively RMV sequences ignoring recombinations, identified by RDP 4 (not shown) [19].

Recombination detection analyses indicated the occurrence of recombination events within the RMV cluster, as summarised in Table 2. The analyses showed that isolate FSHS (JQ319720.1) is a recombinant composed of a 659 nt fragment of the replicase component, between viral-methyltransferase and viral-helicase of the minor parent Actinidia-AC (GQ401365.1) incorporated into the major parent RMV NZ-439 (HQ667978) (Fig. 1b, c, d). The exact position of the beginning and ending break points varied, depending on the reference sequence and algorithm used in the analyses (Table 2). The phylogenetic association of the fragments of the recombinant strain and the major and minor parents are shown in Fig. 2b. The large fragments of strain FSHS (nts 1-1921 and nts 2582-6311) clustered with major parent RMV strain NZ-439 (HQ667978) and the small fragment (nts 1906-2564) with the minor parent RMV Actinidia-AC, at positions between nts 1921-2581 (Fig. 2b). The segments from 5′UTR to the beginning breaking point (1-1905 nt) and the ending break point to 3′UTR (2565-6311 nt) of FSHS showed 99 % (nt) sequence identity with the major parent RMV NZ-439 (HQ667978). The inserted sequence of the recombinant strain revealed 92 % (nt) and 97 % (aa) identity with RMV strain Actinidia-AC (minor parent). SplitsTree4 analysis [13] of the published RMV sequences also confirmed the results of recombination analyses (results not shown).

The fact that RMV is readily mechanically transmissible provides an opportunity for cross-species transmission, possibly resulting in mixed infections and subsequent recombination. Such recombination events might prelude the ability of the new strain to infect both herbaceous and woody asterids expanding the host range, enabling the recombinant to adapt to new micro-replicative niches producing a new range of symptoms, as has been observed in artificially derived tobamovirus recombinants [8, 18, 23, 24]. Recombinations in different ORFs of the genome may variously impact the fitness of recombinants to adapt to environmental change. Capsid genes involved in recombinations are considered to be important as they have multiple roles in the viral life-cycle [2]. Recombinations in the error prone RNA dependent RNA polymerase gene have been reported as a mechanism to restore the levels of precision of viral replication proteins in plant RNA viruses [11] and in tobamoviruses the polymerase domain (54 k) of the gene encoding RdRP has been linked to pathogenicity, based on investigations of artificial chimeras [18]. However the recombination observed in FSHS strain located between viral-methyltransferase and viral-helicase of the replicase component appears to be unique.