Complete L RNA segment of TSWV WA-US isolate
The complete L RNA of TSWV WA-USA isolate was 8914nts in length [GenBank Accession no. KP827649] and encodes an ORF of 8640 nts (from nt position 242 through 8793) in viral complementary sense. The deduced amino acid sequence of the ORF is 2879 in length and is predicted to code for a 330.9 kDa protein. The comparative sequence analysis of the ORF and amino acid sequence showed that this protein is an RNA dependent RNA polymerase (RdRp). The TSWV WA-USA isolate shared highest nt identity (98 %) with the known TSWV isolates reported from Japan (AB198742), Korea (HM581940; HM581937; AB190813) and Spain (KP008132). Further TSWV WA-USA isolate shared 97 % nt identity with another TSWV US isolate PA01 (KT160280). A 241 and 33 nt untranslated region (UTR) was identified at the 5 and 3′ ends of the L segment respectively. These UTRs shared the first 16 nt (agagcaatcaggtaac) that are conserved terminal sequences for Tospovirus genome segments [2].
Molecular phylogeny
The complete L RNA sequences of known Tospoviruses were analyzed to study its molecular phylogeny. The evolutionary history was inferred by using the maximum likelihood method based on the Tamura-Nei model (Fig. 2). The phylogenetic tree showed that all known L RNA sequences of Tospoviruses formed two distinct clades that consisted of sub-clades (Fig. 2). The L RNA sequence of the TSWV WA-USA isolate was within one major clade consisting of other American and Asiatic isolates [Peanut bud necrosis virus (PBNV) (AF025538), Watermelon bud necrosis virus (WBNV)(GU735408), Watermelon spotted wilt virus (WSWV) (AF133128), Calla lily chlorotic spot virus (CCSV) (FJ822962), Iris yellow spot virus (IYSV) (FJ623474), Tomato yellow ring spot virus (TYRV) (JN560178), Bean necrotic mosaic virus (BeNMV) (JF417980),and Tomato chlorotic spot virus (TCSV) (HQ700667)]. Within this clade, TSWV isolates formed a distinct sub-clade along with TCSV (HQ700667) and BeNMV (JF417980) suggesting its distinct lineage. The other major clade included Tospoviruses primarily of Euro-Asiatic origin (KJ541746 PolRSV Italy, X93218 INSV-The Netherlands, DQ256124 CaCV-Thailand, AB061774 MYSV-Japan, and EF552435, TZSV-China) along with an American Tospovirus that was a result of genetic re-assortment between GRSV and TCSV (HQ644142), SVNV isolate from the USA (HQ728385) and another TSWV isolate (PA01) characterized from pepper reported from USA [9].
Molecular phylogenetic relationships of RdRp amino acids sequences were inferred employing maximum-likelihood statistical method. Phylogeny reconstruction revealed that the RdRp encoded by TSWV WA-USA isolate was part of a group consisting of other TSWV isolates (Fig. 3). The phylogenetic study of RdRp sequences showed the presence of three distinct clades consistent with the distinct evolutionary lineages proposed for Tospoviruses, SVNV and BeNMV [18]. Phylogenetic analysis of the complete L RNA nucleotide sequences showed the presence of only two distinct lineages in which BeNMV clustered with TSWV isolates and SVNV clustered with Tospoviruses belonging to the Euro-Asiatic group (Fig. 2).
Conserved RdRp motifs
Sequence analysis of the core polymerase region of RdRp of the TSWV WA-USA isolate showed the presence of all the five conserved motifs (Fig. 4) characteristic of Tospovirus RdRps. These include motif A (DxxKWS), motif B (QGxxxYxSS), motif C (SDD), motif D (TxxxKK), and motif E (EFxSE) [13, 19]. These conserved regions were used in transgenic research to provide broad spectrum resistance against Tospoviruses [13].
Recombination detection
Analysis of the 23 complete L RNA sequences (including that of the TSWV WA-USA and TSWV PA01 isolates) revealed ten recombination events. Details of recombination detection study including the events, major and minor parents involved, beginning and end of break-points are provided in Additional file 1: Table S4. Among the ten recombination events, one event identified isolate TSWV WA-USA as a recombinant arising from the major parent (HM581937), a TSWV-Pepper isolate from South Korea and minor parent (KC261971) another South Korean TSWV isolate (TSWV-17). The event was localized at positions 4534 and 5536 in the alignment (Fig. 5). Further, seven among the nine algorithms used (RDP, Chimaera, BootScan, 3Seq, GENECONV, MaxChi, SiScan and LARD, PhylPro), detected this event except LARD and PhylPro. Moreover, the recently described Korean isolate KM076651, was found to be a recombinant evolved from nine different recombination events (Fig. 5). Among these nine recombination events, three events (Event numbers 1, 7 and 9) that resulted in two putative recombinants (TSWV WA-USA and KM076651) were found to be involved in conserved RdRp motifs (Fig. 4 and Additional file 1: Table S4). Despite recombination events within the conserved RdRp motifs, consensus amino acid sequences in the core polymerase region of RdRp are maintained among various TSWV isolates. This indicates the role of the functional significance of these conserved amino acid sequences in shaping viral genome evolution.