Phylogenetic relationships of closely related potyviruses infectingsweet potato determined by genomic characterizationof Sweet potato virus G and Sweet potato virus 2

Fan Li • Donglin Xu • Jorge Abad •

Ruhui Li

Received: 8 February 2012 / Accepted: 10 April 2012 / Published online: 5 May 2012

� Springer Science+Business Media, LLC (Outside the USA) 2012

Abstract Complete nucleotide sequences of Sweet potato

virus G (SPVG) and Sweet potato virus 2 (SPV2) were

determined to be 10,800 and 10,731 nucleotides, respec-

tively, excluding the 30-poly(A) tail. Their genomic orga-

nizations are typical of potyviruses, encoding a polyprotein

which is likely cleaved into 10 mature proteins by three

viral proteinases. Conserved motifs of orthologous proteins

of viruses in the genus Potyvirus are found in corre-

sponding positions of both viruses. Pairwise comparisons

of individual protein sequences of the two viruses with

those of 78 other potyviruses show that P1 protein and coat

protein (CP) of both viruses are significantly large, with the

SPVG CP as the largest among the all the known species of

the genus Potyvirus. The extended N-terminal region of the

P1 protein is conserved in the potyviruses and ipomovirus

infecting sweet potato. A novel ORF, PISPO, is identified

within the P1 region of SPVG, SPV2, Sweet potato feath-

ery mottle virus (SPFMV), and Sweet potato virus C

(SPVC). The C-terminal half of CP is highly conserved

among SPFMV, SPVC, SPVG, SPV2, and Sweet potato

virus-Zimbabwe. Phylogenetic analysis based on the

deduced CP amino acid sequences supports the view that

these five viruses are grouped together in a SPFMV line-

age. The analysis also reveals that Sweet potato virus Y

and Ipomoea vein mosaic virus are grouped with SPV2 as

one species, and these two viruses should be consolidated

with SPV2.

Keywords Sweet potato virus G � Sweet potato virus 2 �Potyvirus � Genomic sequence � PISPO � Phylogeny

Introduction

Among *30 different viruses infecting sweet potato, six of

them are members of the genus Potyvirus in the family Poty-

viridae [1, 2]. Some of these potyviruses, such as Sweet potato

feathery mottle virus (SPFMV) and Sweet potato virus G

(SPVG), have been approved to be components of sweet potato

viral disease (SPVD), the most devastating viral disease of

sweet potato worldwide [1, 3, 4]. Among six potyviruses,

SPFMV is the best characterized and found in almost all growth

regions. The Common strain of SPFMV is now considered to

be a different potyvirus—Sweet potato virus C (SPVC) [5],

whereas Sweet potato latent virus (SPLV), Sweet potato mild

speckling virus (SPMSV), SPVG, and Sweet potato virus 2

(SPV2) have been recognized as distinct species of the genus

Potyvirus based on partial genomic sequences and/or serolog-

ical reactions [6–9].

SPVG was first described as a potyvirus distinct from

SPFMV in a sweet potato clone from China [8] and has since

been reported in countries of Africa, Asia, America, and

Oceania [10–15]. Sequence analysis of the coat protein (CP)

nucleotide (nt) sequences of 78 SPVG isolates available in the

GenBank databases showed that the virus was 67–82 %

identical to SPFMV, SPV2, and a sweet potato potyvirus

isolate from Zimbabwe (SPV-Zw) [16]. However, the C-ter-

minal half of the CP region of these viruses was more than

90 % identical, indicating a close relationship among them [8,

10, 14, 16]. Phylogenetic analysis revealed a limited genetic

diversity among the SPVG isolates [10].

SPV2 was the second potyvirus described from diseased

sweet potato collected from Taiwan based on its biological

F. Li � D. Xu � R. Li (&)

National Germplasm Resources Laboratory, USDA-ARS,

Beltsville, MD 20705, USA

e-mail: Ruhui.Li@ars.usda.gov

J. Abad

Plant Germplasm Quarantine Program,

USDA-APHIS-PPQ-PHP, Beltsville, MD 20705, USA

123

Virus Genes (2012) 45:118–125

DOI 10.1007/s11262-012-0749-2

and serological distinctions from SPFMV [17]. Sequence

analysis of the 30 partial genome (2,006 bp) of SPV2

confirmed that it was a distinct potyvirus closely related to

SPFMV, SPVG, and SPV-Zw [18]. Together with SPV-Zw

and two isolates (LSU-2, LSU-5) of Ipomoea vein mosaic

virus (IVMV) from the USA, SPV2 was renamed as Sweet

potato virus Y (SPVY). SPVY has since been used in some

recent literature. SPV2 occurs in most sweet potato pro-

duction areas [8, 12, 18, 19]. Sequence analyses of the CP

sequences revealed identities of 81–99 % among the SPV2

isolates as well as partial correlation of the geographic

origin of the SPV2 isolates and their phylogenetic clus-

tering [19].

Several other potyviruses have been reported to infect

sweet potato based on their biological and serological

reactions [6, 7, 12]. Some of these viruses such as SPV-Zw

and IVMV were later recognized as isolates of SPV2 based

on their 30 partial genome [9]. Others including Sweet

potato vein mosaic virus (SPVMV) [20] are not available

for direct comparison. SPVMV is also used to describe a

sweet potato potyvirus from China [15], but it is not clear

how the Chinese SPVMV is related to the Argentinean one.

Sequence information has been confirmed as a more reli-

able scheme for the classification of potyviruses than bio-

logical and serological properties [7]. In this study, the

complete nucleotide sequences of SPVG and SPV2 were

obtained for comparative and phylogenetic analyses of the

potyviruses infecting sweet potato for the first time. The

results confirm the previous reports of SPVG and SPV2 as

distinct species in the genus Potyvirus. A novel ORF (PI-

SPO) nested within the P1 region is identified only in

SPVG, SPV2, SPFMV, and SPVC among all the species of

the genus Potyvirus. Together with SPFMV, SPVC, and

SPV-Zw, the five sweet potato potyviruses form a phylo-

genetic lineage.

Materials and methods

Virus resource

A Korean sweet potato accession (Q21990) infected

with SPVG was used to obtain genomic sequence of

SPVG. The accession was also infected with SPLCV

but did not show obvious symptoms. A diseased sweet

potato plant infected with Sweet potato chlorotic stunt

virus (SPCSV), SPFMV, SPV2, SPVG, and Sweet

potato leaf curl virus (SPLCV) from North Carolina

(NC), USA was used for cloning the SPV2 genome.

The plant showed severe symptoms including rugosity,

leaf distortion, mosaic, and blistering. The infected

plants were maintained in an insect-proof greenhouse by

vegetative cuttings.

RT-PCR, cloning, and sequencing

Total nucleic acids were extracted from infected sweet potato

plants by a CTAB method and used as templates for RT-PCR

amplifications. RT-PCR was carried out using the Super-

Script� III One-Step RT-PCR System (Invitrogen, Carlsbad,

CA, USA) following the manufacturer’s instructions in a

C1000 thermal cycler (Bio-Rad, Hercules, CA, USA).

Approximately 1.8 kb of NIb–30 terminus sequences were

amplified using virus-specific forward primers designed

according to the alignments of the available SPVG or SPV2

sequences, and oligo(dT) primer, respectively. To amplify HC-

Pro–NIb region, a fragment of *0.7 kb was first obtained

within the CI and HC-Pro, respectively, from each virus as

described by Ha et al. (2008) [21]. For the central region of the

SPVG genome, virus-specific primers were then designed

based on the sequences of the three regions and used in sub-

sequent RT-PCR amplifications. For cloning the same region of

SPV2 from the NC plant, the CI or HC-Pro fragment sequences

different from SPFMV, SPVC, and SPVG were used to design

forward primers to be paired with SPV2-specific reverse

primers designed based on the 50-terminal region of the NIb–30

terminus or CI–30terminus contigs obtained during this study.

For the 50-partial genome of *2.6 kb, a semi-nested RT-PCR

using a forward primer of FMV5EF (50-AAAAATAACA-

CAACWCAATACAACA-30) based on the alignment of the

available 50 end of SPFMV and two virus-specific reverse

primers was conducted for each virus. The 50 terminal

sequences were obtained by 50 RACE using the GeneRacer 50

primers and two virus-specific reverse primers using the 50

RACE System (Invitrogen) according to the manufacturer’s

instructions. To insure that the genomic sequences of both

viruses obtained from above process represented the genome

from the same virus, five pairs of virus-specific primers were

designed to amplify DNA fragments with *200–300 bp

overlapping region at each end. For SPV2, the complete

genomic sequence was also obtained from 10 RT-PCR

amplicons with longer ([300 bp) overlapping regions.

All the amplified PCR products were purified using

QIAquick PCR Purification Kit (Qiagen, Valencia, CA,

USA), and subsequently cloned into pCR�2.1-TOPO�

Vector (Invitrogen) following the manufacturer’s instruc-

tions. Plasmid DNA was isolated from overnight culture

using the FastPlasmidTM

Mini Kit (5 Prime, Inc., Gai-

thersburg, MD, USA). At least five clones from each

ligation were sequenced on both strands using M13 for-

ward/reverse primers and specific sequencing primers if

necessary (MacrogenUSA, Rockville, MD, USA).

Sequence analysis and phylogeny

The complete genomic sequence of each virus was

assembled from 5 to 10 overlapping RT-PCR clones by

Virus Genes (2012) 45:118–125 119

123

sequence assembly in the DNAStar 5.01 package (DNA-

Star Inc., Madison, WI, USA). Sequences of other viruses

were obtained from the GenBank databases. The putative

cleavage sites of the polyprotein of each virus were

determined by comparison of the sequences with other

potyviruses. The conserved domains of the putative gene

products were searched by MyHits at http://myhits.isb-sib.

ch/cgi-bin/motif_scan. Multiple sequence alignments were

performed with ClustalW2 at http://www.ebi.ac.uk/Tools/

msa/clustalw2/. Pairwise comparisons of nucleotide

sequences, their deduced polyprotein sequences, and indi-

vidual protein sequences were performed using EMBOSS

Pairwise Alignment Algorithms at http://www.ebi.ac.uk/

Tools/emboss/align/. Phylogenetic relationships were ana-

lyzed by MEGA 5.05 with aligned nucleotide or amino acid

(aa) sequences of SPVG, SPV2 (IVMV, SPVMV, and

SPVY), and other species in the family Potyviridae. Both

neighbor-joining and maximum likelihood trees were con-

structed using 1,000 bootstrap replicates. Putative recombi-

nation breakpoints were identified using RDP4 Beta 4.9

program [22].

Results

Genomic organization of SPV2 and SPVG

Sequence analysis of the clones from RT-PCR products

using the potyvirus degenerate primers within the CI and

HC-Pro regions not only confirmed the presence of

SPFMV, SPVG, and SPV2 in the NC plant and SPVG in

the Korean accession, respectively, but also revealed the

existence of SPVC and at least one other distinct potyvirus/

variant in the NC plant. The presence of SPVC in the NC

plant was confirmed by RT-PCR using the virus-specific

primers (data not shown).

The genomic sequence of the Korean isolate of SPVG

(SPVG-Kr) was first determined from seven overlapping

clones from the accession Q21990 infected only with one

potyvirus (SPVG). The complete genomic sequences of

both SPVG-Kr and SPVG-NC isolates were then obtained

from five overlapping RT-PCR clones of 2.0–2.5 kb

amplified by the SPVG-specific primers. The mixed

infection of SPV2 with other three potyviruses in the NC

plant made the cloning process more complicated. To

confirm the genomic sequence of SPV2-NC obtained in

initial process, the sequence was determined again from 10

overlapping DNA fragments. These sequences were

deposited into GenbBank under accession numbers

JN613805 (SPVG-NC), JN613806 (SPVG-Kr), and

JN613807 (SPV2-NC). The nucleotide sequences of two

SPVG isolates are the same in length, and they share

identities of 98.9 % (110 nt difference), indicating that the

genomic sequence of SPVG obtained from the NC plant

represents the true assembly of the virus. The genomic

sequence of SPVG-NC, therefore, was used in subsequent

sequence analyses. The genomes of SPVG and SPV2

comprise 10,800 nt and 10,731 nt, respectively, excluding

the poly(A) tail at the 30-end, and contain a single large

open reading frame (ORF). The predicted ORF starts at the

first inframe AUG (nt 116–118 for SPVG or nt 118–200 for

SPV2), and ends at the termination codon UAA (nt

10,580–10,582 for SPVG or nt 10,516–10,518 for SPV2),

thereby encoding a single large polyprotein of 3,488 aa for

SPVG and 3,466 aa for SPV2. The 50-untranslated region

(50-UTR) is 115 nt long for SPVG and 117 nt long for

SPV2, and contains a highly conserved block, potybox ‘a’,

as 29AAAGAACAT37 (underlined bases are different) for

SPVG or 29AAAAGACAT37 for SPV2, and several scat-

tered CAA motifs [10].

Nine putative protease cleavage sites were identified in

the polyprotein of both viruses by comparison with the

consensus protease recognition motifs of selected potyvi-

ruses, resulting in 10 putative mature proteins (P1, HC-Pro,

P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb, and CP) (Fig. 1)

[23]. Almost all functional motifs that are highly conserved

among members of the genus Potyvirus were identified in

the 10 mature proteins of SPVG and SPV2 (Table 1). A

small putative P3N-PIPO ORF is identified with presence

of GGAAAAAA (nt 3,808–3,815 for SPVG and nt

3,810–3,817 for SPV2), the highly conserved motif in

potyviruses within the P3 protein [24].

A third ORF encoding a protein of significant size (224

aa for SPVG and 206 aa for SPV2) is identified at nt

1,246–1,920 for SPVG and nt 1,204–1,922 for SPV2 within

the P1 region, respectively. It is interesting that the same

conserved motif, GGAAAAAA, is also found at nt

1,228–1,235 for SPVG and 1,230–1,237 for SPV2. This

indicates that the ribosomal frameshifting used for the

P3N-PIPO expression may also be involved in expression a

protein of 601 aa within the P1 protein by both SPVG and

SPV2. Unlike that of the P3N-PIPO, the size of this protein

is very similar to that of the P1 protein. The same structure

is also found in both SPFMV and SPVC, but not in Sweet

potato mild mottle virus (SPMMV) and 78 other viruses in

the genus Potyvirus. Therefore, this putative protein named

PIPSO is unique to the four closely related potyviruses

infecting sweet potato [1].

With the exception of the 6K1 of SPVG (53 aa) and VPg

of SPV2 (194 aa), the sizes of the other eight deduced

proteins (HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa-Pro, and

NIb) are identical among SPFMV, SPV2, SPVC, and

SPVG (Fig. 1). Except for their large P1 and CP, the sizes

of the eight other proteins of these viruses are similar to

those of other potyviruses (data not shown). The P1 protein

of both SPVG and SPV2 is 618 aa, slightly smaller than

120 Virus Genes (2012) 45:118–125

123

those of SPFMV (664–724 aa) and SPVC (654 aa), but

significantly larger than those of other potyviruses. The

SPVG CP is 355 aa long with a calculated Mr of 39.4 kDa,

the largest of all known species of the genus Potyvirirus.

The SPV2 CP of 332 aa (37.2 kDa) is the third largest,

which is three aa shorter than that of SPV-Zw. The large

sizes of both P1 and CP result from additional amino acids

at the N-terminal regions.

Phylogenetic relationships of SPVG and SPV2 to other

potyviruses

Pairwise comparisons of the complete genomic sequence

of SPVG or SPV2 with those of other potyviruses available

in the GenBank show that the two viruses share identities

that ranged from *48.3 % (Freesia mosaic virus, FreMV)

to 64.6 % (SPFMV) with members of the genus Potyvirus

and ranged from 40.9 % (Cassava brown streak virus) to

49.6 % (Agropyron mosaic virus) with species of other

genera in the family Potyviridae, respectively (data not

shown). The two viruses are 69.1 % identical to each other,

and they share nucleotide sequence identities of

63.5–64.6 % with SPFMV and 62.6–64.1 % with SPVC,

respectively, indicating that these four sweet potato pot-

yviruses are closely related. These values show clearly that

SPVG and SPV2 are two closely related but distinct spe-

cies in the genus Potyvirus [2]. A phylogenetic analysis

was conducted initially using the complete genomic and

polyprotein sequences of SPVG, SPV2, and 78 other spe-

cies in the genus Potyvirus. The topology of both the

neighbor-joining and maximum likelihood trees was very

similar. The phylogenetic trees placed SPVG and SPV2 in

a lineage including SPFMV and SPVC within a group

containing Turnip mosaic virus (TuMV) and 10 other

potyviruses in the PVY supergroup of the genus Potyvirus

(data not shown) [25]. The tree was then simplified to

include most viruses in the TuMV group and representative

species in other groups (Fig. 2). The results also showed

PISPOP3N-PIPO

2166K153 aa 6K1

P1618 aa

601 aa

HC-Pro458 aa

P3352 aa

CI643 aa

CP353 aa352 aa

NIb521 aa

P3192 aa194 aa

NIa-Pro243 aa

216 aa

5’ UTR 3’ UTR VPg Poly(A)

115 nt117 nt

218 nt213 nt

53 aa52 aa

6K153 aa

SPVG MEQY(D)/S YLVG/G VQHQ/A VYHQ/S VQHQ/S VQHQ/A VEHE/S VYAQ/S VHHQ/SSPVG MEQY(D)/S YLVG/G VQHQ/A VYHQ/S VQHQ/S VQHQ/A VEHE/S VYAQ/S VHHQ/SSPV2 MEQY/S YLVG/G VQHQ/A VYHQ/S VQHQ/S VQHQ/G VEHE/S VYAQ/A VHHQ/S

Fig. 1 Schematic representation of the genomic organization and

putative protein cleavage sites of SPVG and SPV2. Proteolytic

cleavage sites from P1 to CP are indicated by solid arrows, conserved

His residues is underlined. Each cleavage site is indicated by a slash,

and different amino acid in the cleavage site between P1 and HC-Pro

is indicated by a parenthesis. Lengths of the untranslated regions

(UTR) at both ends, and the number of aa residues of mature proteins

of each virus are indicated

Table 1 Several conserved protein motifs with proposed functions identified in SPVG and SPV2

Protein Motifa, b Locationc Proposed functions Reference

SPVG SPV2

P1 H-8X-D-31X-G-X-S-G 526–570 526–570 Proteolytic activity [23]

HC-Pro G-Y-C-Y-71X-H 960–1,035 960–1,035 Proteolytic activity [23]

C–C–C 910–912 910–912 Long-distance movement [32]

F-R-N–K 799–802 799–802 Symptom expression [33, 34]

P–T-K 928–930 928–930 Aphid transmission [32, 35]

CI G-A-V-G-S-G-K–S-T 1,566–1,574 1,566–1,574 NTP-binding [36, 37]

V-L-L-V-E-P–T-R-P-L 1,586–1,595 1,586–1,595 Helicase [37, 38]

D-E-X–H 1,655–1,658 1,654–1,657 Helicase [37, 38]

NIa-Pro H-34X-D-67X-G-X-C-G-X14-H 2,415–2,536 2,416–2,537 Proteolytic activity [23]

NIb C-D-A-D-G-S 2,857–2,862 2,858–2,863 RdRp [39]

S-G-X3-T-X3-N-T-X30-G-D-D 2,922–2,965 2,923–2,966 RdRp [39]

CP D-A-G 3,140–3,142 3,142–3,144 Aphid transmission [32, 40]

a All motifs are identical for both SPVG and SPV2b The different amino acids are underlinedc The location of each motif on the polyprotein sequence

Virus Genes (2012) 45:118–125 121

123

that SPVG and SPV2 were more closely related to each

other than to SPFMV and SPVC. A similar clustering of

the SPFMV lineage with the TuMV-related species is

maintained in phylogenetic trees based on other individual

mature proteins (data not shown).

Genetic relationships among the potyviruses infecting

sweet potato

Sequence identities of individual proteins among SPVG,

SPV2, SPFMV, and SPVC were found to be 38.1–65.9 % for

P1, 71.0–83.8 % for HC-Pro, 48.9–68.2 % for P3,

66.0–82.7 % for 6K1, 75.7–85.7 % for CI, 56.6–75.5 % for

6K2, 67.0–83.3 % for VPg, 76.5–92.2 % for NIa-Pro,

72.2–86.8 % for NIb, and 66.1–82.7 % for CP, respectively.

The P1 protein of these viruses is the most divergent of all

proteins. The variations of the sequences and insertions/

deletions occur mainly in the central hypervariable region (aa

212–332 for SPVG and SPV2) of these viruses, whereas their

N-terminal (aa 1–208) (Fig. 3) and the C-terminal (aa

334–618) regions are conserved (data not shown). The P1

protein of the above four viruses is also similar to that of

SPMMV at the N-terminus (Fig. 3). Two of three conserved

W-G/G-W motifs of SPMMV required for the P1-mediated

silencing suppression [26] were also found as 25W-G and137G-W/Y in SPVG and SPV2. A conserved Cys-rich domain

that resembles a zinc finger-like structure was identified at the

same region of these viruses [27]. Unlike the P1 protein, the

C-terminus of PISPO (207–239 aa residues) is not conserved

among the four viruses. The identities of aa sequences at this

region are only 26.1 % between SPVG and SPV2, and range

from 25.5 % (SPVC-C1 vs. SPV2) to 41.6 % (SPVC-Bungo

vs. SPFMV-Piu3) among the four viruses. Only two motifs,456E-W-X-D-X-L/I-X-A-F-X-E-X-E-E/K469 and 591R-G-T/

A/S–W-G/S–F-X-R–R/S-X-I601 (location in both SPVG and

SPV2), are found at this region of PISPO of the four viruses.

This region, however, are conserved within a species, with

identities of 68.1–98.7 % among SPFMV strains/isolates,

89.0 % between SPVC-Bungo and SPVC-C1 and 91.2 %

between SPVG-Kr and SPVG-NC.

A neighbor-joining tree based on the CP nucleotide

sequences of strains/isolates of the sweet potato potyviruses

SPFMV lineage

SPFMV-10-O (AB439206)

SPFMV-O (AB465608)

SPFMV-Piu3 (FJ155666)

SPFMV-S (D86371)

SPVC-Bungo (AB509453)

SPVC-C1 (GU207957)

SPV2-NC

SPVG-NC

SPVG-Kr

ScaMV (NC_003399)

TuMV (NC_002509)

PPV (NC_001445)

ApVY (NC_014905)

KoMV (NC_009713)

TEV (NC_001555)

BCMV (NC_003397)

FreMV (NC_014064)

JGMV (NC_003606)

SCMV (NC_003398)

NDV (NC_008824)

SYSV (NC_007433)

SPMMV (NC_003797)

100

100

100100

100

100

100

100

100

99

100

93

94

9899

100

100

55

63

0.1

Fig. 2 Phylogenetic tree based on deduced polyprotein sequences of

SPVG, SPV2, and representative potyviruses in the genus Potyvirus.

The values at the forks indicate the percentage of 1,000 bootstrap

replicates, and the scale bar represents a genetic distance of 0.1. Solidtriangles indicate the viruses characterized in this study. The viruses

selected from the GenBank for analysis were as follows: Apium virusY (ApVY), Bean common mosaic virus (BCMV), Freesia mosaicvirus (FreMV), Johnsongrass mosaic virus (JGMV), Konjac mosaic

virus (KoMV), Narcissus degeneration virus (NDV), Plum pox virus(PPV), Sugarcane mosaic virus (SCMV), Scallion mosaic virus(ScaMV, NC_003399), Sweet potato feathery mottle virus (SPFMV,

RC strain of isolate S; O strain of isolates 10-O and O; EA strain of

isolate Piu3), Sweet potato virus C (SPVC, isolates C1, and Bungo),

Shallot yellow stripe virus (SYSV), Tobacco etch virus (TEV), Turnipmosaic virus (TuMV). Sweet potato mild mottle virus (SPMMV) was

used as an outgroup

122 Virus Genes (2012) 45:118–125

123

shows that SPV-Zw also groups with the above four viruses

in the SPFMV lineage (Fig. 4) [10]. Despite being more

closely related to SPV2, SPV-Zw is clearly separated from

the other four viruses on a distinct branch at species level.

SPLV and SPMSV are separated from the SPFMV lineage

as distinct viruses, but SPLV is more closely related to the

lineage. Pairwise comparisons of the CP sequences show

that SPV-Zw ranges from 66.0 % (SPFMV-10-O) to

73.5 % (SPV2) identical to those of other viruses in the

lineage. These values are lower than the species thresholds

(76 % nt identity) for the potyviruses [2]. These five viruses

have CP sequence identities ranging from 57.9 % to 60.1 %

with SPLV and 51.9 % to 54.0 % with SPMSV, respec-

tively, confirming that SPLV and SPMSV are only distantly

related to the viruses in the SPFMV lineage [6, 7].

Relationships among the viruses in the SPFMV lineage

were refined by a closer analysis of the CP region. The CP

sizes vary from 313 aa (SPVC) to 355 aa (SPVG-NC)

among these viruses because of insertions/deletions at the

N-terminal half region (CP-N). The CP-N regions differ

not only in the sizes (196 aa for SPVG, 176 aa for SPV-Zw,

173 aa for SPV2, 156 aa for SPFMV, and 154 aa for SPVC)

but also in the sequence identities (43.4–59.0 %) among

the viruses. The CP-N regions are only conserved within

isolates of the same virus, with identities of 89.8–100 %

among 72 SPVG isolates and 86.7–100 % among 38 SPV2

isolates. However, the C-terminal regions (CP-C) of 159 aa

are highly conserved among the viruses in the SPFMV

lineage, and they share identities of 90.1 % (SPVC vs

SPVG) to 99.4 % (SPVG vs. SPV2) between the viruses.

The CP-C identities among the viruses in the SPFMV

lineage and SPLV or SPMSV ranging from 64.8 % to

78.6 %, similar to those among most species of Potyvirus.

Analysis of the complete genomic sequences of the

potyviruses and ipomovirus infecting sweet potato using

the RDP4 program did not reveal any positive

recombination breakpoints identification in SPVG and

SPV2 (data not shown).

Discussion

Several potyviruses have been identified to cause syner-

gistic diseases when co-infection with SPCSV in sweet

potato [4, 28], and mixed infections of these viruses are

common [10, 14]. As a well-studied and widely distributed

potyvirus, SPFMV has often masked the presence of clo-

sely related potyviruses such as SPVG and SPV2 in sweet

potato, making these viruses less studied. SPFMV, SPVG,

and SPV2 alone usually do not cause visual symptoms on

sweet potato, and they are detected by grafting onto I.

setosa or SPCSV-infected sweet potato clone such as TIB8,

by serological assays and/or by RT-PCR. The three viruses

induce similar symptoms on the biological indicator plants

[10, 19] and react to some SPFMV antisera [9, 10, 12]. The

C-terminal half of CP is highly conserved among SPFMV,

SPVC, SPVG, SPV2, and SPV-Zw in the SPFMV lineage.

Therefore, an antiserum made from one virus could contain

antibodies against common epitopes of these viruses,

resulting in cross reactions in serological study. The high

nucleotide sequence identities at the 30-partial sequences of

SPFMV, SPVC, SPVG, and SPV2 could result in ampli-

fication of the non-target virus or co-amplification of the

viruses from mixed infections, especially when degenerate

primers were used in RT-PCR. Therefore, the detection

methods used in earlier studies might erroneously identify

SPVG and SPV2 as SPFMV. The SPVG-Kr isolate, indeed,

was initially identified as SPFMV based on biological

assay and nitrocellulose membrane-ELISA in our study

(data not shown). A virus survey of 279 field samples in

southwestern China (Yunnan Province) showed that

infections of potyviruses were common in fields, and

Fig. 3 Alignment of available N-terminal sequences of the P1

protein of potyviruses infecting sweet potato. Identical (asterisk)

and conserved (colon) aa residues among these viruses are indicated.

The WG/GW(Y) motifs involved in silencing suppression are bolded.

The Cys residues of a zinc finger-like structure are indicated by solid

rumbas (filled diamond). Numbers of the starting and ending aa

residues are indicated before and after each sequence, respectively.

The numbers of residues (gaps) between blocks are indicated. The

type isolates of SPFMV (NC_001841), SPVC-C1 (GU207957), and

SPMMV (NC_003797) were used in the comparison

Virus Genes (2012) 45:118–125 123

123

SPVG, not SPFMV, was the most prevalent virus in the

region. A recent GenBank list of the CP sequences of 52

SPVG isolates from different areas of China further con-

firmed that the virus was common in the country. Similar

result was obtained from field samples in New Zealand

[13]. Infection rates of SPV2 were not high, but the virus

was always detected in mixed infections with other virus/

viruses in sweet potato [10, 13, 18, 19]. SPVG and SPV2

may play a more important role in SPVD than previously

thought.

Phylogenetic analyses of the complete genomic

sequences show that SPVG and SPV2 are grouped with

SPFMV and SPVC in a cluster, and SPVG is the most

closely related to SPV2 (Fig. 2), confirming both the dis-

tinctions and similarities among the four viruses [10–12].

Further analysis of the CP aa sequences suggests that these

four viruses and SPV-Zw cluster together in the SPFMV

lineage (Fig. 4) [10], suggesting that the five viruses might

share a common ancestor. The viruses in this lineage have

large genomes that range from 10,731 (SPV2) to 10,996 nt

(SPFMV-Piu3), due to an N-terminal extension of the P1

and CP regions (Figs. 1, 3) [10]. The P1 protein of these

viruses consists of the conserved N-terminal (P1-N),

hypervariable central and conserved C-terminal poty-like

protease (P1-Pro) regions. The P1-N domains are unique to

the sweet potato viruses including SPMMV in the family

Potyviridae, indicating they might have a common ances-

tor and similar functions in host fitness (Fig. 3). The P1

protein of some viruses, indeed, has been shown to be

involved in host selection [29]. The presence of W-G/G-W/

Y motifs in the P1 protein supports that it might be a RNA

silencing suppressor in the SPFMV lineage [26], but fur-

ther investigations are necessary. The conservation of the

P1-N domain among these viruses also suggests that it

might be important for fitness in sweet potato [27]. The

four viruses in the SPFMV lineage also contain a novel

ORF, PISPO, within the P1 protein region, and it is unique

to these viruses [1]. Further study is needed to understand

the expression and function of PISPO. The CP variations

occur mainly at the N-terminal half region among different

species or different strains of these viruses. The high level

of homology at the C-terminal half regions of the lineage

indicates that this region has an important function in these

viruses.

Previous studies indicate that recombination between dif-

ferent strains of a potyvirus or closely related potyvirus spe-

cies may be common [30]. Evidence of recombination events

was found in the P1 N-terminal region involving intraspecies

of SPFMV O and RC strains [5, 31], and intergeneric of

SPMMV and an unknown potyvirus resulting in the SPFMV

P1 [27]. RDP3 analysis did not predict a history of recombi-

nation in respect of SPVG or SPV2 with SPFMV, SPVC, and

SPMMV using either the complete genomic sequence or the

nucleotide sequences of the P1 region. Further analysis of the

complete sequence data of different strains of SPVG and

SPV2 is needed to provide better understanding of the evo-

lution among the isolates of each virus and with other viruses

in the SPFMV lineage.

SPV2

SPVG

SPFMV

SPVC

SPV-Zw

SPV2-NC

SPV2-VTSB-Tschilombo(AY459609)

SPV2-Hua3(EU218529)

SPV2-Zimbia(AY459610)

SPV-Zw(AF016366)

SPVG-Hua2(EU218528)

SPVG-NC

SPVG-Yunnan(HQ844203)

SPVC-Bungo(AB509453)

SPVC-C1(GU207957)

SPFMV-S(D86371)

SPFMV-Piu3(FJ155666)

SPFMV-10-O(AB439206)

SPFMV-O(AB465608)

SPLV(EF492050)

SPMSV(U61228)

SPMMV(NC 003797)

100

100

55

100

10099

99

100

78100

87

74

82

87

0.1

Fig. 4 Neighbor-joining tree

based on the CP nt sequences of

five potyviruses infecting sweet

potato. Bootstrap analysis was

applied using 1,000 bootstrap

replicates, and the scale barrepresents a genetic distance of

0.1. Selected strains/isolates of

SPFMV, SPVC, SPVG, and

SPV2 were used for analysis.

Only one CP sequence of Sweetpotato mild speckling virus(SPMSV) or Sweet potato virus-

Zimbabwe (SPV-Zw) was

available for analysis. SPMMV

was used as an outgroup

124 Virus Genes (2012) 45:118–125

123

Acknowledgments The authors thank Whitney Hymes, Crindi

Loschinkohl, and Sam Grinstead for their providing the excellent

technical assistance; Drs. Joseph Foster, John Hartung, Hsing-Yeh

Liu, and Gary Kinard are also thanked for their critical review of the

manuscript.

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