Arch Virol DOI 10.1007/s00705-014-2231-z
ANNOTATED SEQUENCE RECORD
Complete genome sequence of a divergent strain of Japanese yam mosaic virus from China Pingxiu Lan • Fan Li • Mingqiang Wang Ruhui Li
•
Received: 17 April 2014 / Accepted: 8 September 2014 Ó Springer-Verlag Wien (outside the USA) 2014
Abstract A novel strain of Japanese yam mosaic virus (JYMV-CN) was identified in a yam plant with foliar mottle symptoms in China. The complete genomic sequence of JYMV-CN was determined. Its genomic sequence of 9701 nucleotides encodes a polyprotein of 3247 amino acids. Its organization is virtually identical to that of two JYMV isolates from Japan. With the latter, it shares nucleotide sequence identities of only 74.7–74.8 %, indicating it might be a member of a new species. However, sequence analysis of the polyprotein and individual proteins suggested that the Chinese isolate is a divergent JYMV strain in the process of speciation.
Yams (Dioscorea spp.) are a multi-species group of tuber crops grown in many tropical and sub-tropical countries, especially in West Africa [1, 2]. Yams are economically important for their food and medicinal value. Since yams are propagated vegetatively, viral diseases constitute a major production threat [3]. Approximately 10 viruses infect yam [3–6]. One of them, Japanese yam mosaic virus (JYMV), was first reported in Japanese yam (D. japonica) as a strain of the potyvirus yam mosaic virus (YMV) [7]. The virus induced mosaic and green-banding symptoms on leaves and caused significant tuber yield losses. JYMV has flexuous, filamentous particles of approximately 680–780 nm in length and induces typical cylindrical inclusions in P. Lan M. Wang R. Li (&) USDA-ARS, National Germplasm Resources Laboratory, Beltsville, MD 20705, USA e-mail: [email protected] P. Lan F. Li Yunnan Agricultural University, Kunming 650201, Yunnan, People’s Republic of China
the cytoplasm of infected cells. It infects several members of the genus Dioscorea and is transmitted by aphids in a nonpersistent manner. The virus was later renamed JYMV based on its genomic sequences [3, 8]. During a survey for yam viruses, a total of 86 leaf samples were collected from both symptomatic and symptomless yam plants (D. polystachya Turcz.) in seven counties (Binchuan, Chengjiang, Chuxiong, Fumin, Jianshui, Yanshan and Yongsheng) of Yunnan Province, China. The samples were initially tested by RT-PCR using degenerate potyvirus primers CIFor/CIRev [9], and then virus-specific primers [10, 11]. Total nucleotide acids were extracted from leaf tissue using a CTAB-based method [12]. RT-PCR was carried out using the SuperScriptTM III One-Step RT-PCR System (Invitrogen, Carlsbad, CA, USA). The amplicons of selected samples were cloned into pGEM-T Easy Vector (Promega, Madison, WI, USA). Plasmid DNA was isolated from overnight cultures and sequenced (MacrogenUSA, Rockville, MD, USA). Only a single one of the sequenced samples, from Yanshan, was found to have a mixed infection of yam mild mosaic virus (YMMV) with JYMV. The infected plant showed mild mottle symptoms. This JYMV isolate (JYMV-CN) shared nucleotide sequence identity of only 78 % with two Japanese isolates, M (NC_000947) and J1 (AB016500) [3, 8], within the CI region, indicating that it is possibly distinct from them. To obtain the complete genomic sequence of JYMVCN, 10 pairs of JYMV-specific primers were initially designed according to an alignment of the genomic sequences of two Japanese JYMV isolates and used in RTPCR to amplify overlapping fragments covering the whole genome. Only one fragment covering the 30 -terminus was obtained. The genome-walking strategy described by Xu et al. [13] was then used to obtain an 8.5-kb portion of the
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30 end of the genome of in a series of five sequential RTPCR cloning steps. To obtain the sequence at the 50 end, a 50 RACE reaction was performed using a GeneRacerÒ Kit (Invitrogen). Contig assembly was performed using the DNASTAR 5.01 package (DNASTAR Inc., Madison, WI, USA). Pairwise comparisons of nucleotide and protein sequences were performed using the EMBOSS Needle Pairwise Sequence Alignment at http://www.ebi.ac.uk/ Tools/psa/emboss_needle/nucleotide.html. Phylogenetic analysis was done using MEGA6 [14], and neighbor-joining and maximum-likelihood trees were constructed using 1000 bootstrap replicates. The complete nucleotide sequence of JYMV-CN was determined to be 9701 nt, excluding the poly (A) tail, and it has been submitted to the GenBank database under accession code KJ701427. The genomic organization and structure of JYMV-CN is virtually identical to that of the two Japanese JYMV isolates [3, 8]. The putative ORF starts at the first in-frame AUG (nt 91–93), which is in the appropriate context of GCCAATGGC (underlined bases are different) and ends at the termination codon UAA at nt 9493–9495 in the sequence. The 50 -untranslated region (50 -UTR) of 90 nt of JYMV-CN is 63–66 nt shorter than that of the Japanese isolates due to three deletions. Sequence comparison showed that the first 38-nt stretch was conserved in the 50 UTR sequences of the Chinese isolate and seven Japanese isolates (NC_000947, AB016500, AB079771-AB079775). The 50 half of this stretch is also conserved in almost all potyviruses. This stretch also contained two slightly changed potyboxes ‘a’ (UCAACAM) at position 14–20 (nt 15–21 for gp1) and ‘b’ (HYAACGA) at position 24–30 (nt 25–31 for the M isolate). Similar deletions also occurred in two Japanese isolates, J3 and J4, resulting in a shorter 50 -UTR. The results indicated that the 50 -UTR sequence of JYMV-CN was complete. The genome of JYMV-CN encodes a large polyprotein of 3134 amino acid (aa) residues with a calculated molecular mass of 357 kDa. All nine putative cleavage sites identified in the polyprotein of JYMV-CN are very similar to those of the Japanese isolates, resulting in ten mature proteins (P1, HCPro, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb and CP) [3, 8]. Only minor changes occurred at two cleavage sites of P3/6K1 (VSHQ/A, the underlined amino acid residue is different) and NIa-Pro/NIb (VHPQ/M), respectively. The putative P3N-PIPO ORF was also identified by the presence of 2940GGAAAAAA2947 [15]. With the exception of the P1 and P3N-PIPO, the size of all other mature proteins of JYMV-CN is the same as those of the Japanese isolates. The P1 protein of JYMV-CN is 3 aa longer than that of the J1 isolate and 4 aa longer than that of the M isolate, mainly due to the addition of 16TEL18 at its N-terminus. The P3N-PIPO of 229 aa of JYMV-CN is 5 aa longer than that of the two Japanese isolates, and the addition occurred at the
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C-terminus of the protein. All conserved potyviral motifs are present in the individual proteins of JYMV-CN and the two Japanese isolates. Comparison of the Chinese isolate with the two Japanese isolates revealed nucleotide sequence identities of 74.7–74.8 % at the whole-genome level, which is much lower than the 83.5 % between the two Japanese isolates. These values are lower than the potyvirus species demarcation threshold of 76 %, suggesting that JYMV-CN might be a member of a distinct species [16]. Further sequence comparisons of individual regions were conducted, and the results are presented in Table 1. Comparisons of JYMV-CN with YMV, YMMV, narcissus yellow stripe virus (NYSV), turnip mosaic virus (TuMV) and scallion mosaic virus (ScaMV) were also included in the analysis to show the sequence identities between distinct potyviruses. The results showed that P1 is the most divergent region. The P1 amino acid sequence identities are 39.7–43.5 % between JYMV-CN and the Japanese isolates (Table 1) and 62.2 % between the Japanese isolates. The identities of the other mature proteins between JYMV-CN and the Japanese isolates ranged from 73.6 to 79.2 % in 6K2 to 89.3 to 89.8 % in CI (Table 1), which are lower than those between the two Japanese isolates (89.2 % in HC-Pro, 84.0 % in P3, 91.1 % in P3N-PIPO, 96.2 % in 6K1, 96.1 % in CI, 86.8 % in 6K2, 97.4 in VPg, 97.1 % in NIa-Pro, 93.4 in NIb and 93.8 % in CP). The sequence identities of most of the mature proteins, including CP (79.5–80.8 %), were higher than the species thresholds (76 % in the nt sequence and 80 % in the aa sequence). Thus, we propose that this Chinese isolate is, in fact, a divergent strain belonging to the species Japanese yam mosaic virus. The sequence data suggest that it is in process of speciation. A phylogenetic analysis was initially conducted using the deduced polyprotein sequences of the three JYMV isolates and 87 other members of the genus Potyvirus. Both neighbor-joining and maximum-likelihood trees clearly placed the three isolates together in the TuMV subgroup (subgroup 1) of the PVY group [17]. The tree was then simplified to include members of subgroup 1 and potato virus Y (Fig. 1A). With the exception of proteins P1 and 6K2, a similar clustering of JYMV within the TuMV subgroup is maintained in phylogenetic trees based on sequences of the other proteins and the complete genome of all available potyviruses (data not shown). Relationships among more available JYMV isolates were further analyzed. A neighbor-joining tree based on the sequence of the 1110 50 -terminal nucleotides showed that the Chinese isolate was separated from the seven Japanese isolates in a distinct branch (Fig. 1B). JYMV-CN shared nucleotide sequence identities of 54.7–56.6 % with the Japanese isolates, supporting this distinction. This analysis also revealed the presence of four distinct JYMV clusters in
A divergent strain of Japanese yam mosaic virus from China Table 1 Nucleotide and amino acid sequence identities between JYMV1-CN and other JYMV strains or selected potyviruses Region
Genome
JYMV-M (NC 000947)
JYMV-J1 (AB016500)
YMV2 (NC 004752)
YMMV3 (NC 019412)
NYSV4 (NC 011541)
TuMV5 (NC 002509)
ScaMV6 (NC 003399)
nt7
nt
nt
nt
nt
nt
nt
aa8
74.7
74.8
Polyprotein
81.2
50 -UTR9 30 -UTR
47.7 81.1
P1
55.9
aa
aa
55.4
53.9
81.2 44.9 82.1
39.7
58.1
46.4 44.3 45.7
48.6
20.4
aa
63.1
49.7 47.0 49.5
43.5
aa
62.1 60.0
56.6 52.2
50.1
21.4
aa
50.2
59.7 59.4
54.6 55.2 23.4
aa
49.0
37.0 49.3 50.7
23.4
38.4
19.9
HC-Pro
75.2
84.7
76.6
86.6
59.1
50.4
57.1
49.3
65.4
64.4
62.0
62.9
64.3
65.9
P3
76.8
82.3
74.6
80.4
51.0
29.4
52.1
24.2
46.4
42.8
55.2
33.7
57.0
44.5
P3N-PIPO
79.0
84.3
77.1
83.4
51.1
33.2
51.7
21.1
61.0
48.5
55.6
38.4
57.2
46.4
6K1
76.9
92.3
73.7
84.6
61.9
53.8
61.5
63.5
62.8
65.4
60.9
63.5
60.9
59.6
CI
78.2
89.8
78.3
89.3
60.2
58.1
58.7
55.9
66.3
69.6
66.8
70.0
56.6
70.0
6K2
73.0
79.2
69.2
73.6
56.2
52.8
54.3
31.2
55.3
45.3
54.1
52.8
61.2
47.2
VPg
79.5
88.5
77.6
89.1
62.1
58.4
57.9
51.8
65.2
66.1
62.5
64.6
65.2
65.6
NIa-Pro
76.3
83.5
76.5
82.7
55.7
52.3
56.9
44.9
63.4
63.0
64.9
68.3
63.1
63.4
NIb
75.6
84.9
76.8
84.9
65.2
64.0
64.6
61.1
67.8
72.8
67.8
74.5
68.0
70.8
CP
80.8
88.0
79.5
86.9
60.8
56.8
61.6
57.7
67.0
66.6
68.7
69.3
68.5
1
2
3
4
Japanese yam mosaic virus, yam mosaic virus, yam mild mosaic virus, narcissus yellow stripe virus, mosaic virus, 7 nucleotide sequence, 8 amino acid sequence, 9 untranslated region
A
5
turnip mosaic virus,
scallion
Japanese mosaic virus-gp1 (NC 00947)
100 100
Japanese mosaic virus-J1 (AB016500) Japanese yam mosaic virus-CN
100
Turnip mosaic virus (NC 002509) Narcissus yellow stripe virus (NC 011541)
98
90
69.3 6
84
1 PVY group
Scallion mosaic virus (NC 003399) Yam mosaic virus (NC 004752) Potato virus Y (NC 001616) Yam mild mosaic virus-Brazil (NC 019412) Sugarcane streak mosaic virus (NC 014037) 94
B
Japanese yam mosaic virus-M (NC 000947) Japanese yam mosaic virus-J2 (AB079771)
98
Japanese yam mosaic virus-O1 (AB079774) Japanese yam mosaic virus-J1 (AB016500)
98 91
Japanese yam mosaic virus-O2 (AB079775) Japanese yam mosaic virus-J4 (AB079773)
84
Japanese yam mosaic virus-J3 (AB079772) Japanese yam mosaic virus-CN Turnip mosaic virus (NC 002509)
Fig. 1 Neighbor-joining trees based on polyprotein sequences of three Japanese yam mosaic virus (JYMV) isolates and other viruses of subgroup 1 in the PVY group (A) and 5-terminal 1110-nucleotide
sequences of known JYMV isolates (B). Bootstrap analysis was applied using 1000 replicates. Solid triangles indicate the JYMV isolate characterized in this study
Japan. The nucleotide sequence identities ranged from 55.1 to 99.7 %, supporting a relatively distant relationship among them.
Recombination analysis of the full-length genomes of the potyviruses infecting yams (JYMV, YMV and YMMV) and three potyviruses (NYSV, ScaMV, and TuMV) that are
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closely related to JYMV in subgroup 1 using the RDP4 software package [18] detected a major interspecies recombination event in the lineage leading to JYMV. The analysis revealed that the three JYMV isolates are recombinants between the major parent, an unknown virus (likely TuMV), and the minor parent, NYSV. This event is found within the P3 region at nt 2773–3027 of JYMV-CN, nt 2790–3081 of JYMV-M, and nt 2842–3078 of JYMV-J1. It was supported by clear P-values (\0.05) in the BOOTSCAN, MAXCHI, CHIMAERA programs of the RDP4 software. Several other putative recombination sites were also found, in all three isolates, only in the Chinese isolate, or only in the Japanese isolates, but these were detected by only one program of the RDP4 package. The results show that interspecies recombination events may have been involved in the evolution of JYMV and its isolates. This is the first report of JYMV in a new geographic region (China). The virus was associated with mild symptoms and does not appear to be common in the region. Sequence analysis showed that this Chinese isolate is a distinct strain of JYMV and that JYMV isolates are highly divergent. Although the whole-genome sequence identities of JYMV-CN to the two Japanese isolates fall below the currently accepted potyvirus species demarcation threshold of 76 % [16], the evidence above suggests that all of the JYMV isolates originated by recombination. Sequence identity is not the only criterion for potyvirus species demarcation [16], and JYMV-CN has, to the best of our knowledge, a host range restricted to yam, like the Japanese isolates; therefore, the biology of JYMV-CN is equivalent to that of other JYMV isolates, with the sequence divergence possibly resulting from adaptation to a different yam species (D. polystachya rather than D. japonica) or from additional recombination events not clearly identified by our analysis. Our results also show that interspecies recombination events might play an important role in the evolution of JYMV. The genomic data presented here will facilitate future investigations of the origins and evolution of JYMV. Acknowledgments The authors thank Gaili Bao and Lijuan Li for sample collections, and Drs. Gary Kinard, Dimitre Mollov and John Hartung for critical review of the manuscript.
References 1. Asiedu P, Sartie A (2010) Crops that feed the World 1. YamsYams for income and food security. Food Sec 2:305–315
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2. FAO (2011) Food and Agricultural Organisation of the United Nations. FAO Statistical Yearbook 2010, Rome 3. Fuji S, Nakamae H (1999) Complete nucleotide sequence of the genomic RNA of a Japanese yam mosaic virus, a new potyvirus in Japan. Arch Virol 144:231–240 4. Kenyon L, Shoyinka SA, Hughes J, Odu BO (2001) An overview of viruses infecting Dioscorea yams in sub-Saharan Africa. Plant Virology in sub-Saharan Africa. In: Proceeding of a conference organized by IITA, International institute of tropical agricultural, Ibadan, pp 432–493 5. Eni AO, Hughes J, Rey MEC (2008) Survey of the incidence and distribution of five viruses infecting yams in the major-producing zones in Benin. Ann Appl Biol 153:223–232 6. Eni AO, Hughes J, Asiedu R, Rey MEC (2010) Survey of the incidence and distribution of viruses infecting yam (Dioscorea spp.) in Ghana and Togo. Ann Appl Biol 156:243–251 7. Okuyama S, Saka H (1978) Yam mosaic virus. Science Reports of the Faculty of Agriculture Ibaraki University 26:29–34 8. Fuji S, Nakamae H (2000) Complete nucleotide sequence of the genomic RNA of a mild strain of Japanese yam mosaic virus. Arch Virol 145:635–640 9. Ha C, Coombs S, Revill PA, Harding RM, Vu M, Dale JL (2008) Design and application of two novel degenerate primer pairs for the detection and complete genomic characterization of potyviruses. Arch Virol 153:25–36 10. Mumford RA, Seal SE (1997) Rapid single-tube immunocapture RT-PCR for the detection of two yam potyviruses. J Virol Methods 69:73–79 11. Kajihara H, Muramoto K, Fuji S, Tanaka S, Ito S (2009) Simultaneous detection of Japanese yam mosaic virus and yam mild mosaic virus from yam leaves using a tube capture reverse transcription-polymerase china reaction assay. J Gen Plant Pathol 75:72–75 12. Li R, Mock R, Huang Q, Abad J, Hartung J, Kinard G (2008) A reliable and inexpensive method of nucleic acid extraction for the PCR-based detection of diverse plant pathogens. J Virol Methods 154:48–55 13. Xu DL, Liu HY, Koike ST, Li F, Li R (2010) Biological characterization and complete genomic sequence of Apium virus Y infecting celery. Virus Res 156(917):920 14. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol Biol Evol 30:2725–2729 15. Chung BYW, Miller WA, Atkins JF, Firth AE (2008) An overlapping essential gene in the Potyviridae. Proc Natl Acad Sci USA 105:5897–5902 16. Adams MJ, Antoniw JF, Fauquet CM (2005) Molecular criteria for genus and species discrimination within the family Potyviridae. Arch Virol 150:459–479 17. Gibbs A, Ohshima K (2010) Potyviruses and the digital revolution. Annu Rev Phytopathol 48:205–223 18. Martin DP, Lemey P, Lott M, Moulton V, Posada D, Lefeuvre P (2010) RDP3: a flexible and fast computer program for analyzing recombination. Comput Appl Biosci 26:2462–2463
ANNOTATED SEQUENCE RECORD
Complete genome sequence of a divergent strain of Japanese yam mosaic virus from China Pingxiu Lan • Fan Li • Mingqiang Wang Ruhui Li
•
Received: 17 April 2014 / Accepted: 8 September 2014 Ó Springer-Verlag Wien (outside the USA) 2014
Abstract A novel strain of Japanese yam mosaic virus (JYMV-CN) was identified in a yam plant with foliar mottle symptoms in China. The complete genomic sequence of JYMV-CN was determined. Its genomic sequence of 9701 nucleotides encodes a polyprotein of 3247 amino acids. Its organization is virtually identical to that of two JYMV isolates from Japan. With the latter, it shares nucleotide sequence identities of only 74.7–74.8 %, indicating it might be a member of a new species. However, sequence analysis of the polyprotein and individual proteins suggested that the Chinese isolate is a divergent JYMV strain in the process of speciation.
Yams (Dioscorea spp.) are a multi-species group of tuber crops grown in many tropical and sub-tropical countries, especially in West Africa [1, 2]. Yams are economically important for their food and medicinal value. Since yams are propagated vegetatively, viral diseases constitute a major production threat [3]. Approximately 10 viruses infect yam [3–6]. One of them, Japanese yam mosaic virus (JYMV), was first reported in Japanese yam (D. japonica) as a strain of the potyvirus yam mosaic virus (YMV) [7]. The virus induced mosaic and green-banding symptoms on leaves and caused significant tuber yield losses. JYMV has flexuous, filamentous particles of approximately 680–780 nm in length and induces typical cylindrical inclusions in P. Lan M. Wang R. Li (&) USDA-ARS, National Germplasm Resources Laboratory, Beltsville, MD 20705, USA e-mail: [email protected] P. Lan F. Li Yunnan Agricultural University, Kunming 650201, Yunnan, People’s Republic of China
the cytoplasm of infected cells. It infects several members of the genus Dioscorea and is transmitted by aphids in a nonpersistent manner. The virus was later renamed JYMV based on its genomic sequences [3, 8]. During a survey for yam viruses, a total of 86 leaf samples were collected from both symptomatic and symptomless yam plants (D. polystachya Turcz.) in seven counties (Binchuan, Chengjiang, Chuxiong, Fumin, Jianshui, Yanshan and Yongsheng) of Yunnan Province, China. The samples were initially tested by RT-PCR using degenerate potyvirus primers CIFor/CIRev [9], and then virus-specific primers [10, 11]. Total nucleotide acids were extracted from leaf tissue using a CTAB-based method [12]. RT-PCR was carried out using the SuperScriptTM III One-Step RT-PCR System (Invitrogen, Carlsbad, CA, USA). The amplicons of selected samples were cloned into pGEM-T Easy Vector (Promega, Madison, WI, USA). Plasmid DNA was isolated from overnight cultures and sequenced (MacrogenUSA, Rockville, MD, USA). Only a single one of the sequenced samples, from Yanshan, was found to have a mixed infection of yam mild mosaic virus (YMMV) with JYMV. The infected plant showed mild mottle symptoms. This JYMV isolate (JYMV-CN) shared nucleotide sequence identity of only 78 % with two Japanese isolates, M (NC_000947) and J1 (AB016500) [3, 8], within the CI region, indicating that it is possibly distinct from them. To obtain the complete genomic sequence of JYMVCN, 10 pairs of JYMV-specific primers were initially designed according to an alignment of the genomic sequences of two Japanese JYMV isolates and used in RTPCR to amplify overlapping fragments covering the whole genome. Only one fragment covering the 30 -terminus was obtained. The genome-walking strategy described by Xu et al. [13] was then used to obtain an 8.5-kb portion of the
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30 end of the genome of in a series of five sequential RTPCR cloning steps. To obtain the sequence at the 50 end, a 50 RACE reaction was performed using a GeneRacerÒ Kit (Invitrogen). Contig assembly was performed using the DNASTAR 5.01 package (DNASTAR Inc., Madison, WI, USA). Pairwise comparisons of nucleotide and protein sequences were performed using the EMBOSS Needle Pairwise Sequence Alignment at http://www.ebi.ac.uk/ Tools/psa/emboss_needle/nucleotide.html. Phylogenetic analysis was done using MEGA6 [14], and neighbor-joining and maximum-likelihood trees were constructed using 1000 bootstrap replicates. The complete nucleotide sequence of JYMV-CN was determined to be 9701 nt, excluding the poly (A) tail, and it has been submitted to the GenBank database under accession code KJ701427. The genomic organization and structure of JYMV-CN is virtually identical to that of the two Japanese JYMV isolates [3, 8]. The putative ORF starts at the first in-frame AUG (nt 91–93), which is in the appropriate context of GCCAATGGC (underlined bases are different) and ends at the termination codon UAA at nt 9493–9495 in the sequence. The 50 -untranslated region (50 -UTR) of 90 nt of JYMV-CN is 63–66 nt shorter than that of the Japanese isolates due to three deletions. Sequence comparison showed that the first 38-nt stretch was conserved in the 50 UTR sequences of the Chinese isolate and seven Japanese isolates (NC_000947, AB016500, AB079771-AB079775). The 50 half of this stretch is also conserved in almost all potyviruses. This stretch also contained two slightly changed potyboxes ‘a’ (UCAACAM) at position 14–20 (nt 15–21 for gp1) and ‘b’ (HYAACGA) at position 24–30 (nt 25–31 for the M isolate). Similar deletions also occurred in two Japanese isolates, J3 and J4, resulting in a shorter 50 -UTR. The results indicated that the 50 -UTR sequence of JYMV-CN was complete. The genome of JYMV-CN encodes a large polyprotein of 3134 amino acid (aa) residues with a calculated molecular mass of 357 kDa. All nine putative cleavage sites identified in the polyprotein of JYMV-CN are very similar to those of the Japanese isolates, resulting in ten mature proteins (P1, HCPro, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb and CP) [3, 8]. Only minor changes occurred at two cleavage sites of P3/6K1 (VSHQ/A, the underlined amino acid residue is different) and NIa-Pro/NIb (VHPQ/M), respectively. The putative P3N-PIPO ORF was also identified by the presence of 2940GGAAAAAA2947 [15]. With the exception of the P1 and P3N-PIPO, the size of all other mature proteins of JYMV-CN is the same as those of the Japanese isolates. The P1 protein of JYMV-CN is 3 aa longer than that of the J1 isolate and 4 aa longer than that of the M isolate, mainly due to the addition of 16TEL18 at its N-terminus. The P3N-PIPO of 229 aa of JYMV-CN is 5 aa longer than that of the two Japanese isolates, and the addition occurred at the
123
C-terminus of the protein. All conserved potyviral motifs are present in the individual proteins of JYMV-CN and the two Japanese isolates. Comparison of the Chinese isolate with the two Japanese isolates revealed nucleotide sequence identities of 74.7–74.8 % at the whole-genome level, which is much lower than the 83.5 % between the two Japanese isolates. These values are lower than the potyvirus species demarcation threshold of 76 %, suggesting that JYMV-CN might be a member of a distinct species [16]. Further sequence comparisons of individual regions were conducted, and the results are presented in Table 1. Comparisons of JYMV-CN with YMV, YMMV, narcissus yellow stripe virus (NYSV), turnip mosaic virus (TuMV) and scallion mosaic virus (ScaMV) were also included in the analysis to show the sequence identities between distinct potyviruses. The results showed that P1 is the most divergent region. The P1 amino acid sequence identities are 39.7–43.5 % between JYMV-CN and the Japanese isolates (Table 1) and 62.2 % between the Japanese isolates. The identities of the other mature proteins between JYMV-CN and the Japanese isolates ranged from 73.6 to 79.2 % in 6K2 to 89.3 to 89.8 % in CI (Table 1), which are lower than those between the two Japanese isolates (89.2 % in HC-Pro, 84.0 % in P3, 91.1 % in P3N-PIPO, 96.2 % in 6K1, 96.1 % in CI, 86.8 % in 6K2, 97.4 in VPg, 97.1 % in NIa-Pro, 93.4 in NIb and 93.8 % in CP). The sequence identities of most of the mature proteins, including CP (79.5–80.8 %), were higher than the species thresholds (76 % in the nt sequence and 80 % in the aa sequence). Thus, we propose that this Chinese isolate is, in fact, a divergent strain belonging to the species Japanese yam mosaic virus. The sequence data suggest that it is in process of speciation. A phylogenetic analysis was initially conducted using the deduced polyprotein sequences of the three JYMV isolates and 87 other members of the genus Potyvirus. Both neighbor-joining and maximum-likelihood trees clearly placed the three isolates together in the TuMV subgroup (subgroup 1) of the PVY group [17]. The tree was then simplified to include members of subgroup 1 and potato virus Y (Fig. 1A). With the exception of proteins P1 and 6K2, a similar clustering of JYMV within the TuMV subgroup is maintained in phylogenetic trees based on sequences of the other proteins and the complete genome of all available potyviruses (data not shown). Relationships among more available JYMV isolates were further analyzed. A neighbor-joining tree based on the sequence of the 1110 50 -terminal nucleotides showed that the Chinese isolate was separated from the seven Japanese isolates in a distinct branch (Fig. 1B). JYMV-CN shared nucleotide sequence identities of 54.7–56.6 % with the Japanese isolates, supporting this distinction. This analysis also revealed the presence of four distinct JYMV clusters in
A divergent strain of Japanese yam mosaic virus from China Table 1 Nucleotide and amino acid sequence identities between JYMV1-CN and other JYMV strains or selected potyviruses Region
Genome
JYMV-M (NC 000947)
JYMV-J1 (AB016500)
YMV2 (NC 004752)
YMMV3 (NC 019412)
NYSV4 (NC 011541)
TuMV5 (NC 002509)
ScaMV6 (NC 003399)
nt7
nt
nt
nt
nt
nt
nt
aa8
74.7
74.8
Polyprotein
81.2
50 -UTR9 30 -UTR
47.7 81.1
P1
55.9
aa
aa
55.4
53.9
81.2 44.9 82.1
39.7
58.1
46.4 44.3 45.7
48.6
20.4
aa
63.1
49.7 47.0 49.5
43.5
aa
62.1 60.0
56.6 52.2
50.1
21.4
aa
50.2
59.7 59.4
54.6 55.2 23.4
aa
49.0
37.0 49.3 50.7
23.4
38.4
19.9
HC-Pro
75.2
84.7
76.6
86.6
59.1
50.4
57.1
49.3
65.4
64.4
62.0
62.9
64.3
65.9
P3
76.8
82.3
74.6
80.4
51.0
29.4
52.1
24.2
46.4
42.8
55.2
33.7
57.0
44.5
P3N-PIPO
79.0
84.3
77.1
83.4
51.1
33.2
51.7
21.1
61.0
48.5
55.6
38.4
57.2
46.4
6K1
76.9
92.3
73.7
84.6
61.9
53.8
61.5
63.5
62.8
65.4
60.9
63.5
60.9
59.6
CI
78.2
89.8
78.3
89.3
60.2
58.1
58.7
55.9
66.3
69.6
66.8
70.0
56.6
70.0
6K2
73.0
79.2
69.2
73.6
56.2
52.8
54.3
31.2
55.3
45.3
54.1
52.8
61.2
47.2
VPg
79.5
88.5
77.6
89.1
62.1
58.4
57.9
51.8
65.2
66.1
62.5
64.6
65.2
65.6
NIa-Pro
76.3
83.5
76.5
82.7
55.7
52.3
56.9
44.9
63.4
63.0
64.9
68.3
63.1
63.4
NIb
75.6
84.9
76.8
84.9
65.2
64.0
64.6
61.1
67.8
72.8
67.8
74.5
68.0
70.8
CP
80.8
88.0
79.5
86.9
60.8
56.8
61.6
57.7
67.0
66.6
68.7
69.3
68.5
1
2
3
4
Japanese yam mosaic virus, yam mosaic virus, yam mild mosaic virus, narcissus yellow stripe virus, mosaic virus, 7 nucleotide sequence, 8 amino acid sequence, 9 untranslated region
A
5
turnip mosaic virus,
scallion
Japanese mosaic virus-gp1 (NC 00947)
100 100
Japanese mosaic virus-J1 (AB016500) Japanese yam mosaic virus-CN
100
Turnip mosaic virus (NC 002509) Narcissus yellow stripe virus (NC 011541)
98
90
69.3 6
84
1 PVY group
Scallion mosaic virus (NC 003399) Yam mosaic virus (NC 004752) Potato virus Y (NC 001616) Yam mild mosaic virus-Brazil (NC 019412) Sugarcane streak mosaic virus (NC 014037) 94
B
Japanese yam mosaic virus-M (NC 000947) Japanese yam mosaic virus-J2 (AB079771)
98
Japanese yam mosaic virus-O1 (AB079774) Japanese yam mosaic virus-J1 (AB016500)
98 91
Japanese yam mosaic virus-O2 (AB079775) Japanese yam mosaic virus-J4 (AB079773)
84
Japanese yam mosaic virus-J3 (AB079772) Japanese yam mosaic virus-CN Turnip mosaic virus (NC 002509)
Fig. 1 Neighbor-joining trees based on polyprotein sequences of three Japanese yam mosaic virus (JYMV) isolates and other viruses of subgroup 1 in the PVY group (A) and 5-terminal 1110-nucleotide
sequences of known JYMV isolates (B). Bootstrap analysis was applied using 1000 replicates. Solid triangles indicate the JYMV isolate characterized in this study
Japan. The nucleotide sequence identities ranged from 55.1 to 99.7 %, supporting a relatively distant relationship among them.
Recombination analysis of the full-length genomes of the potyviruses infecting yams (JYMV, YMV and YMMV) and three potyviruses (NYSV, ScaMV, and TuMV) that are
123
P. Lan et al.
closely related to JYMV in subgroup 1 using the RDP4 software package [18] detected a major interspecies recombination event in the lineage leading to JYMV. The analysis revealed that the three JYMV isolates are recombinants between the major parent, an unknown virus (likely TuMV), and the minor parent, NYSV. This event is found within the P3 region at nt 2773–3027 of JYMV-CN, nt 2790–3081 of JYMV-M, and nt 2842–3078 of JYMV-J1. It was supported by clear P-values (\0.05) in the BOOTSCAN, MAXCHI, CHIMAERA programs of the RDP4 software. Several other putative recombination sites were also found, in all three isolates, only in the Chinese isolate, or only in the Japanese isolates, but these were detected by only one program of the RDP4 package. The results show that interspecies recombination events may have been involved in the evolution of JYMV and its isolates. This is the first report of JYMV in a new geographic region (China). The virus was associated with mild symptoms and does not appear to be common in the region. Sequence analysis showed that this Chinese isolate is a distinct strain of JYMV and that JYMV isolates are highly divergent. Although the whole-genome sequence identities of JYMV-CN to the two Japanese isolates fall below the currently accepted potyvirus species demarcation threshold of 76 % [16], the evidence above suggests that all of the JYMV isolates originated by recombination. Sequence identity is not the only criterion for potyvirus species demarcation [16], and JYMV-CN has, to the best of our knowledge, a host range restricted to yam, like the Japanese isolates; therefore, the biology of JYMV-CN is equivalent to that of other JYMV isolates, with the sequence divergence possibly resulting from adaptation to a different yam species (D. polystachya rather than D. japonica) or from additional recombination events not clearly identified by our analysis. Our results also show that interspecies recombination events might play an important role in the evolution of JYMV. The genomic data presented here will facilitate future investigations of the origins and evolution of JYMV. Acknowledgments The authors thank Gaili Bao and Lijuan Li for sample collections, and Drs. Gary Kinard, Dimitre Mollov and John Hartung for critical review of the manuscript.
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