The authors have declared that no competing interests exist.
Studies of the genetic diversity and evolutionary relationships of plant viruses are important for understanding plant virus epidemiology and for planning control measures [
TuMV is a member of a clearly defined lineage of potyviruses, the TuMV phylogenetic group, all of which, except TuMV, have been isolated from monocotyledonous plants [
Here we report a further study of TuMV group viruses from
Our interest in narcissus viruses was aroused as
Wild and domestic
The viruses were directly identified from the collected
Similarly, three or four fragments covering the full genomic regions of NYSV-like viruses, including their NIb-3’ region, were amplified by RT-PCR (
The alignment for Neighbour-net (NN), recombination and maximum likelihood (ML) phylogenetic analyses was made from the deduced amino acid sequences of the complete CP coding and polyprotein regions of NYSV-like viruses together with those of the same viruses from the public nucleotide sequence databases using outgroup sequences of NLSYV, JYMV, ScaMV, TuMV and WoSV. The alignments were made using CLUSTAL_X2 [
Putative recombination sites were identified using the RDP [
The phylogenetic relationships of the aligned sequences of complete CP coding and polyprotein regions of NYSV-like viruses were inferred using the NN method in SPLITSTREE version 4.11.3 [
The nucleotide sequence identities were estimated using EMBOSS Needle [
One hundred and eighty-eight symptomatic
Numbers of plants | |||
---|---|---|---|
Examined | detected | ||
District | Virus in the family |
NYSV-like virus (%) | |
Hokkaido | 24 | 9 (37.5) | 0 (0.0) |
Tohoku | 25 | 7 (28.0) | 4 (16.0) |
Kanto | 30 | 23 (76.7) | 10 (33.3) |
Chubu | 19 | 15 (78.9) | 4 (21.1) |
Kinki | 20 | 12 (60.0) | 8 (40.0) |
Chugoku | 9 | 8 (88.9) | 4 (44.4) |
Shikoku | 15 | 10 (66.7) | 5 (33.3) |
Kyushu and Okinawa | 46 | 35 (76.1) | 22 (47.8) |
Total | 188 | 119 (63.3) | 57 (0.3) |
a This includes narcissus late season yellows virus (NLSYV), cyrtanthus elatus virus A (CyEVA), narcissus latent virus (NLV), narcissus degeneration virus (NDV) and/or ornithogalum mosaic virus (OrMV).
We chose 91 clones of the NYSV-like viruses using the sequences obtained by the POTYNIB5P primer, and then analysed the remaining region of their NIb-3’ sequences (
Red dots show the narcissus plants infected with NYSV-like viruses. The major area of
The 91 CP sequences determined in this study, together with 46 sequences obtained from the public nucleotide sequence databases, were analysed. The length of aligned CP sequences was 798 nucleotides after gaps were removed. SPLITSTREE analyses using both nucleotide and amino acid sequences showed similar topology and found a reticulated phylogenetic network linking the CP genes.
The sequences of complete coat protein coding region of narcissus yellow stripe virus (NYSV)-like viruses obtained in this study with those of outgroup sequences of Japanese yam mosaic virus (JYMV), narcissus late season yellows virus (NLSYV), scallion mosaic virus (ScaMV), turnip mosaic virus (TuMV) and wild onion symptomless virus (WoSV). Isolates used in this study and isolates with accession numbers obtained from the public nucleotide sequence databases were listed. Black dots indicate the isolates used for full genomic sequencing. For details of lineages, see text and
We compared the complete genomes of the nine NYSV-like viruses that we sequenced together with seven NYSV and NLSYV sequences from the public nucleotide sequence databases (JQ326210, JQ395042, JQ911732, JX156421, KU516386, NC_011541, NC_023628). The genomes of Japanese isolates of NYSV-like viruses were 9626–9639 nucleotides in length excluding the 5’ end 24 nucleotide primer sequences (
SPLITSTREE was used to analyse the polyprotein-encoding regions of the 13 NYSV-like viruses together with NLSYV and outgroup sequences (
One unequivocal recombination site was found in the middle of the CI coding region (near nt 4600) of the NYSV Zhangzhou sequence (NC_011541) as previously reported [
The International Committee on Taxonomy of Viruses (ICTV) discriminates members of different potyvirus species using pairwise sequence comparisons. The polyprotein ORFs of isolates of different species in a large sample of potyviruses were found to have less than 76% pairwise nucleotide identity and less than 82% pairwise amino acid identity [
NY-HG16 (%) | NY-HR38 (%) | |
---|---|---|
wild onion symptomless virus (WoMV) | ||
TUR256-1 (NC_030391) | 64.3 |
64.7 (68.2) |
TUR256-2 (LC159495) | 64.3 (68.1) | 64.7 (68.2) |
scallion mosaic virus (ScaMV) | ||
China (NC_003399) | 62.4 (62.1) | 62.3 (62.1) |
turnip mosaic virus (TuMV) | ||
OM (AB701690) | 61.6 (61.0) | 61.8 (60.9) |
ASP (AB701697) | 62.1 (61.5) | 61.8 (61.3) |
Japanese yam mosaic virus (JYMV) | ||
China (KJ701427) | 61.7 (59.8) | 62.6 (59.9) |
JY1 (AB016500) | 61.7 (59.3) | 61.7 (59.2) |
narcissus late season yellows virus (NLSYV) | ||
Marijiniup8 (NC_023628) | 69.9 (75.8) | 69.6 (75.7) |
Marijiniup9 (JX156421) | 69.7 (76.0) | 69.4 (75.9) |
Zhangzhou (JQ326210) | 70.1 (76.0) | 70.3 (76.0) |
narcissus yellow stripe virus-like virus (NYSV-like virus) | ||
Marijiniup3 (JQ395042) | 70.4 (77.2) | 70.5 (77.5) |
NY-CB5 | 69.4 (76.7) | 69.2 (76.5) |
NY-EH173 | 69.1 (76.7) | 69.1 (76.4) |
NY-HG19 | 70.4 (77.4) | 70.5 (77.4) |
NY-HG27 | 70.4 (77.6) | 70.9 (77.3) |
NY-KM1O | 70.6 (77.0) | 70.6 (77.4) |
NY-KM1P | 70.2 (77.4) | 70.1 (77.6) |
NY-OI1 | 70.9 (77.6) | 70.7 (77.6) |
ZZ-2 (JQ911732) | 69.8 (76.7) | 69.6 (76.6) |
a Narcissus virus 1 isolates were basal isolates of narcissus viruses in turnip mosaic virus phylogenetic group (see
b Nucleotide identity. The identities were calculated using EMBOSS Needle [
c Amino acid identity
Virus and isolate | narcissus virus 1 | narcissus late season yellows virus | narcissus yellow stripe virus-like virus | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
NV-1 | NYSV-1 | NYSV-2 | NYSV-3 | |||||||||||
NY-HG16 | NY-HR38 | Marijiniup8 | Marijiniup9 | Zhangzhou | Marijiniup3 | NY-KM1O | NY-KM1P | NY-CB5 | NY-EH173 | ZZ-2 | NY-HG19 | NY-HG27 | NY-OI1 | |
NY-HG16 | - | 69.9 | 69.7 | 70.1 | 70.4 | 70.6 | 70.2 | 69.4 | 69.4 | 69.8 | 70.4 | 70.4 | 70.9 | |
NY-HR38 | - | 69.6 | 69.4 | 70.3 | 70.5 | 70.6 | 70.1 | 69.2 | 69.1 | 69.6 | 70.5 | 70.9 | 70.7 | |
Marijiniup8 | 75.8 | 75.7 | - | 70.7 | 70.5 | 70.8 | 70.2 | 70.2 | 69.7 | 70.6 | 70.7 | 71.0 | ||
Marijiniup9 | 76.0 | 75.9 | - | 71.0 | 70.6 | 71.0 | 70.3 | 70.4 | 70.1 | 70.9 | 71.2 | 70.8 | ||
Zhangzhou | 76.0 | 76.0 | - | 71.3 | 71.5 | 71.6 | 71.5 | 71.2 | 71.7 | 75.6 | 75.8 | 71.7 | ||
Marijiniup3 | 77.2 | 77.5 | 79.5 | 79.3 | 79.5 | - | 72.3 | 72.5 | 72.3 | 72.5 | 72.6 | 72.8 | ||
NY-KM1O | 77.0 | 77.4 | 79.3 | 78.9 | 79.3 | - | 72.4 | 72.3 | 72.5 | 72.7 | 72.6 | 73.4 | ||
NY-KM1P | 77.4 | 77.6 | 79.3 | 78.9 | 79.3 | - | 72.8 | 72.5 | 72.5 | 72.4 | 72.6 | 73.6 | ||
NY-CB5 | 76.7 | 76.5 | 76.9 | 77.4 | 77.2 | 81.3 | 81.4 | 81.1 | - | 73.9 | 74.1 | 73.1 | ||
NY-EH173 | 76.7 | 76.4 | 76.9 | 77.5 | 77.2 | 81.3 | 81.3 | 81.0 | - | 73.7 | 73.5 | 73.3 | ||
ZZ-2 | 76.7 | 76.6 | 77.0 | 77.5 | 77.2 | 81.2 | 81.2 | 80.8 | - | 73.7 | 73.7 | 73.9 | ||
NY-HG19 | 77.4 | 77.4 | 78.0 | 78.4 | 78.4 | - | ||||||||
NY-HG27 | 77.6 | 77.3 | 78.1 | 78.5 | 78.5 | - | ||||||||
NY-OI1 | 77.6 | 70.7 | 78.4 | 78.6 | 78.9 | - |
Percent identities of the polyprotein coding regions were calculated by EMBOSS Needle [
The similarities and differences we observed were uniformly distributed through the sequences as was shown by the SIMPLOT method.
The relationships among the narcissus potyviruses and their relatives was most clearly shown by phylogenetic analyses.
The amino acid sequences of polyprotein (major open reading frame) regions of Japanese yam mosaic virus (JYMV), scallion mosaic virus (ScaMV), turnip mosaic virus (TuMV) and wild onion symptomless virus (WoSV) related to TuMV phylogenetic group were used. Sweet potato latent virus (SPLV), sweet potato virus 2 (SPV2), sweet potato virus C (SPVC), sweet potato virus G (SPVG) and sweet potato feathery mottle virus (SPFMV) were used as outgroup sequences. Circles at each node indicate the statistical support using the SH method, green circle; SH 1.0 (full support), yellow circle; 0.8 < SH <1.0. (between SH 0.8 and SH 0.9999). Horizontal branch lengths are drawn to scale with the bar indicating 0.1 nt replacements per site. The calculated tree was displayed by TREEVIEW [
Although these clusters were detected using a small number of genomic sequences, they are monophyletic and have strong statistical support in ML phylogenetic analyses. Their nucleotide sequences and the encoded amino acid sequences gave closely similar trees;
Our phylogenetic analyses show that the narcissus viruses fall into five statistically robust clusters, but these do not completely coincide with the groupings indicated by the pairwise comparisons used by the ICTV, probably because pairwise sequence identities computed from multiple independent pairwise alignments are not the same as those computed from multiple sequence alignments (i.e. phylogenetic analyses), and are less informative [
Cross-protection, which was independently discovered by several researchers [
Genetic recombination is perhaps also emerging as an alternative biologically-based test of relatedness. Intra-species recombination is common in most potyvirus populations [
In the early 20th century, plant virus species were distinguished from one another by biological characters, especially host range and symptoms, and many groupings made that way were subsequently found to be of immunologically and genetically related viruses. However recently, the process has been reversed, first the genetics is studied and then the biology, and although this is the only option for viral gene sequences obtained by metagenomic studies [
The biological question highlighted by our studies is to ask what evolutionary process has caused the component populations of narcissus viruses to form a series of five phylogenetically distinct clusters. It could be a result of chance divergences, or it could be the result of adaptation to, or co-evolution with, an ancient, diverse though small, host population [
Arrow indicates primers used to amplify cDNA. Isolates are shown in parenthesis. The primer sequences are listed in
(TIF)
The sequences of polyproteins of narcissus yellow stripe virus (NYSV)-like viruses obtained in this study with those of outgroup sequences of Japanese yam mosaic virus (JYMV), narcissus late season yellows virus (NLSYV), scallion mosaic virus (ScaMV), turnip mosaic virus (TuMV) and wild onion symptomless virus (WoSV). Isolates with accession numbers were obtained from the public nucleotide sequence databases. Isolates NYSV Zhangzhou (NC_011541) and NYSV NAR-2 (KU516386) were added (A) or removed (B), and the trees were constructed.
(TIF)
The sequences of NY-HR38 and NY-HG19 isolates represent the likely parental sequences of NYSV NAR-2 isolate. Note the support (i.e.
(TIF)
Isolates NY-HG16 (A) and NY-KM1P (B) were used as the query isolate. The similarities were estimated using SIMPLOT version 3.5.1 with a window size of 200 nt.
(TIF)
(TIF)
a Rows in brown show that
b Number of clone sequenced for approximately 600–700 bp by POTYNIB5P primer. c Not detected. d Number of clone for NYSV-like virus sequence.
(PDF)
a Correspond to the genome of Chinese isolate [
(PDF)
a NYSV; narcissus yellow stripe virus, NLSYV; narcissus late season yellows virus. b P1; protein 1, HC-Pro; helper component-proteinase protein, P3; protein 3, 6K1; 6kda 1 protein, CI; cylindrical inclusion protein, 6K2; 6kda 2 protein, VPg; genome-linked viral protein, NIa-Pro; nuclear inclusion a-proteinase protein, NIb; nuclear inclusion b protein, CP; coat protein.
c Numbers colored in red show different amino acid residues at cleavage sites.
(PDF)
Percent identities of protein 1 and cytoplasmic inclusion coding regions were calculated by EMBOSS Needle [
(PDF)
We thank Rei Nomiyama, Kenta Soejima, Yuichiro Hirosue, Miho Fujita, Kaoru Tominaga, Hironori Satomoto, Ryosuke Yasaka (Laboratory of Plant Virology, Saga University) and Yukio Nagano (Analytical Research Center for Experimental Sciences, Saga University) for their careful technical assistance.