Virus Genes DOI 10.1007/s11262-014-1073-9
Genome sequence of two isolates of Yellow oatgrass mosaic virus, a new grass-infecting Tritimovirus Mohamed Hassan
Received: 13 December 2013 / Accepted: 29 March 2014 Ó Springer Science+Business Media New York 2014
Abstract Complete genome sequences of two Yellow oatgrass mosaic virus (YOgMV) isolates have been determined to be 9,292 nucleotides excluding the 30 polyadenylated tail. The viral RNA encodes a large putative open reading frame (ORF) of a single polyprotein consisting of 3,002 amino acids with typical genome organization of monopartite potyvirids. A small overlapping ORF encoding a pretty interesting Potyviridae ORF of 122 amino acids is found in the P3 cistron of both YOgMV isolates. The nucleotide and amino acid identities between the two YOgMV isolates are 90 and 97 %, respectively. Pairwise comparison of YOgMV putative mature proteins and proteinase cleavage sites with those of representative members of the family Potyviridae indicated that YOgMV is more closely related to members of the genus Tritimovirus. In phylogenetic trees constructed with sequences of putative polyprotein, YOgMV consistently groups with members of the genus Tritimovirus. These results suggest that YOgMV should be classified as a distinct species in the genus Tritimovirus in the family Potyviridae. Keywords Trisetum flavescens Potyviridae Tritimovirus Poaceae Virus taxonomy Electronic supplementary material The online version of this article (doi:10.1007/s11262-014-1073-9) contains supplementary material, which is available to authorized users. M. Hassan (&) Department of Agricultural Botany, Faculty of Agriculture, Fayoum University, Fayoum, Egypt e-mail: [email protected] M. Hassan Department of Plant Pathology, University of Arkansas, Fayetteville, AR 72701, USA e-mail: [email protected]
Introduction Trisetum flavescens L., commonly known as yellow oatgrass or golden oatgrass, is a perennial species of Poaceae family. It is native to Europe, Asia, and North Africa and grows in seeded pastures, roadsides, and as a weed in croplands. Recently, there is an increased interest to study plant virus ecology in nature for several economic and scientific perspectives [1–5]. One of these reasons is that wild grasses may serve as reservoirs of viruses that can cause epidemics in important cultivated crops [5]. Thus, it is important to extend our knowledge of viruses infecting wild plants to better predict the potential of native viruses to infect cultivated crops. Potyviridae is the largest family of positive-sense RNA viruses infecting plants. The International Committee on Taxonomy of Viruses (ICTV) assigned eight genera within the family Potyviridae based on their genetic relatedness, vector transmission, and genome organization [6]. One of these genera, Tritimovirus, consists of six species: Wheat streak mosaic virus (WSMV) as the type member, Brome streak mosaic virus (BrSMV), Oat necrotic mottle virus (ONMV), Wheat eqild mosaic virus (WEqMV), Tall oatgrass mosaic virus (TOgMV), and Yellow oatgrass mosaic virus (YOgMV; for which only 30 end of the viral genome has been reported) [4, 7–12]. In addition, Cocksfoot streak mosaic virus (CfSMV), a new proposed Tritimovirus, was isolated from a diseased cocksfoot plant in Germany [13]. According to Rabenstein et al. [14], CfSMV is a distinctive member of the genus Tritimovirus based on sequence comparisons and phylogenetic analysis. However, the sequence data for CfSMV are not available. The availability of full genome sequences has significantly clarified the taxonomic status of several potyvirids
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Virus Genes
and has led to the establishment of criteria that can be used to distinguish closely related virus species [7]. In this study we report the complete genome sequence of two isolates of YOgMV, a new Tritimovirus infecting wild yellow oatgrass and confirmed their presence in multiple locations in the Czech Republic. In addition, the genome organization and identities of the putative gene products of the new virus are discussed in relation to previously described Tritimoviruses.
Materials and methods Mechanical inoculation Yellow oatgrass plants with mild mosaic symptoms were collected from Stare´ Brˇ´ısˇteˇ (Sb) and Radeˇtice (Ra) counties, Czech Republic. The two YOgMV isolates (Sb and Ra) were transmitted by sap inoculation to seedlings of the oat (cv. Abel), the propagation host of YOgMV [12]. Symptomatic leaves infected with YOgMV isolates at 2–3 weeks post-inoculation were tested for the presence of the virus by RT-PCR.
A
RNA extraction, full-length amplification, and genome sequencing Total RNA was extracted using the Qiagen RNeasy Kit according to manufacturer’s recommendations (Qiagen). Total RNA was used as a template for RT-PCR and 30 end of YOgMV isolates were amplified using primers published by Gibbs and Mackenzie [15]. To amplify the rest of the viral genome, a genome walking strategy was employed as described previously by Hassan et al. [4] using specific reverse primers from known sequences and upstream degenerate primers (Supplementary Table 1). All RT-PCR reactions were carried out using a one-step RT-PCR kit (Takara BIO Inc.), according to the manufacturer’s recommendations. Amplicons of expected size were gel purified using QIAquick gel extraction kit (Qiagen), ligated into pGEM-T Easy plasmid (Promega), and transformed into Escherichia coli DH5a. The 50 terminus was amplified using a 50 RACE kit (Invitrogen), according to the manufacturer’s recommendations. At least 39 coverage of the virus genome was obtained. Nucleotide sequences were determined using a fluorescent DNA sequencer CEQ2000XL, with dye terminator reagents (Beckman Coulter). Sequence analysis and phylogenetic relatedness The complete nucleotide consensus was assembled from overlapping cDNA clones using ProSeq program [16].
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B Fig. 1 a Potted plant of yellow oatgrass transplanted from Stare´ Brˇ´ısˇteˇ, Czech Republic. The plant shows mild mosaic symptoms on the new emerging leaf, faint mosaic streaks pointed by arrows. b Locations in Czech Republic where yellow oatgrass diseased plants were observed and collected for characterization of YOgMV isolates. The towns’ names, where Sb and Ra YOgMV isolates were collected are underlined
Nucleotides and amino acid sequences were analyzed using CLUSTALW in BioEdit package [17]. BlastX and BlastP at the website of NCBI were used to search for related proteins from the Protein Information Resources (PIR) database [18]. Protein function analysis and transmembrane helices in Tritimovirus proteins were predicted using TMHMM [19]. Similarity plot depicting the identity among the aligned nucleotide sequences were generated with SimPlot, Version 3.5.1 [20]. Similarity was calculated within a sliding
Virus Genes Fig. 2 a, b Systemic infection symptoms induced by YOgMV isolates Sb and Ra on oat cv. Abel, respectively. Infected plant leaves presented are from the same physiological age. c–e Chlorosis and necrotic lesions symptoms observed on different oat plants parts after 21 days of inoculation with Ra isolate. f Pronounced dwarfing on an oat plant inoculated with Ra isolate compared to a healthy oat plant on the right
Healthy
Infected
A
C
window of 200 nt, with a step of 20 residues between points. Positions containing gaps were excluded from the analysis. Phylogenetic analysis was done using the Neighbor-Joining (NJ) and Maximum Likelihood (ML) methods of phylogenetic inference with 1,000 bootstrap replicates using MEGA 5.0 [21]. RT-PCR Specific primer pairs were designed to amplify a 200-nt fragment of YOgMV coat protein gene (Supplementary
Infected
Healthy
B
D
E
F
Table 1). Reverse transcription was carried out at 50 °C for 30 min., activation of the hot start Taq polymerase at 95 °C for 15 min, followed by 35 cycles of 94 °C at 30 s., 55 °C at 30 s., 72 °C at 30 s., and a final extension step at 72 °C for 10 min according to the manufacturer’s instructions of QiagenÒOne-step RT-PCR kit (Qiagen). Amplification products were electrophoresed on 2 % agarose gels, stained with ethidium bromide, and visualized under UV light. PCR products of expected size were extracted from 2 % agarose gel using QIAquick gel extraction kit (Qiagen) and RT-PCR products were cloned
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P3
HC-Pro
P1
2685 E/A
NIa NIa VPg Pro
3006
2186
Q/A E/G E/G
CI
Q/S
1957
Q/G Q/D
1709 1760
G/G
Amino acids
6k2
1013 1064
Y/G
6k1
5’UTR
735
1
A
CP
NIb
3’UTR
(A)n 9292
9149
8229
6735
6045
Nucleotides
5299 5454
AAG(A6)
2751-2760
3213 3366
2379
1230
129
1
PIPO
B
Similarity (%)
Fig. 3 a Schematic diagram of Yellow oatgrass mosaic virus (YOgMV) genome organization RNA. The map of the YOgMV genome depicts the locations of polyprotein open reading frame start and stop codons; the amino acid (above) and the nucleotide (below) coordinates of predicated mature viral proteins (P1, HC-Pro, P3, 6K1, CI, 6K2, VPg-NIa, NIb, and CP). The specific dipeptides of a cleavage site and their location in the genome are shown above the map. b Similarity plots of YOgMV isolates and TOgMV nucleotide sequence, calculated and plotted by SimPlot 3.5.1 (Lole et al. [20]). The entire genetic map of YOgMV is shown at the top, approximately to scale. YOgMV isolate Sb used as a reference (the query sequence), YOgMV isolate Ra curve (blue) and TOgMV curve (green)
352
Virus Genes
Position (nt)
into the pGEM-T vector (Promega); five clones were sequenced.
symptom induction by Ra isolate is more rapid than that of symptom production by Sb isolate (Fig. 2a). Genome organization of YOgMV
Results and discussion Symptoms induced by YOgMV isolates and RT-PCR detection Twelve yellow oatgrass samples, exhibiting mild mosaic symptoms, were collected from different districts in Czech Republic (Fig. 1a, b). YOgMV was detected from all samples collected from geographically different districts by RT-PCR analysis with primers specific to the coat protein gene of YOgMV. The virus causes similar symptoms when mechanically inoculated into yellow oatgrass seedlings [12]. However, when YOgMV isolates are inoculated into oat (cv. Abel), they cause severe mosaic symptoms, necrosis, and dwarfing (Fig. 2a–f). This observation suggests that YOgMV coevolved with wild yellow oatgrass. Roossinck [22] proposed that recent plant–virus association induces more severe symptoms compared to older associations. Ra isolate showed more severe symptoms than Sb isolate in oat plants (cv. Abel), though both YOgMV isolates induced leaf mosaic and necrotic blotch symptoms, as progress of infection (Fig. 2a). In the oat plants, timing of
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The universal potyvirid primers [15], designed to amplify the 30 end portion of viruses belonging to Potyviridae, were used to generate amplicons of the expected size (1.7 Kbp), which were cloned and sequenced. BlastN search of the NCBI databases revealed that WSMV, the type member of genus Tritimovirus [8], was the most closely related viral species to YOgMV in Potyviridae. The complete nucleotide sequence of YOgMV was determined from multiple overlapping cDNA clones, generated by genome walking and 50 RACE. The sequence of YOgMV isolates (Sb and Ra) consists of 9,292 nucleotides (nt) excluding the 30 -poly (A) tail, and have been submitted to GenBank under the accession numbers KF984546 and KF984547 for Sb and Ra isolates, respectively. The genome of YOgMV has the typical features of the monopartite of Potyviridae members, consisting of a single large ORF. The putative ORF starts with AUG codon at (nt 129–131) and ends with the stop codon UAG at (nt 9,147–9,149). The putative ORF consist of 9,090 nucleotides and encodes a polyprotein of 3,006 amino acid (aa) with a predicted molecular mass 341.5 kDa (Fig. 3a).
Virus Genes Table 1 Predicted protease cleavage sites of YOgMV isolates by comparison to other Tritimoviruses Virus
P1/HC-Pro
HC-Pro/P3
P3/6K1
6K1/CI
CI/6K2
6k2/VPg
VPg/NIa
NIa/NIb
NIb/CP
YOgMV(Sb)
HGLRWY/G
RDYAIG/G
KLVKYQ/G
VNAQYQ/D
AHVSYQ/A
ASVKYE/G
NASTFE/G
GLVHWQ/S
QYCVFE/A
YOgMV(Ra)
HGLRWY/G
RDYAIG/G
KLVKYQ/G
VNAQYQ/D
AHVSYQ/A
ASVKYE/G
NASTFE/G
GLVHWQ/S
QYCVFE/A
TOgMV
KGLKWC/G
RDYEIG/G
NEVEYQ/G
VNCEYQ/G
AHVEYQ/A
AKSSYE/G
NKSLYE/S
DLANWQ/S
RCCMYE/Y
ONMV
HGLRWY/S
KDYKIG/G
ELVEYQ/S
VNCEYQ/S
SHVSYQ/A
HRVKYE/G
NKSLYE/G
ELVNWQ/S
KYCVYE/S
WSMV
HGLRWY/G
KDYKIG/G
ELVEYQ/G
FNCEYQ/S
SHVSYQ/A
RSVKFE/G
NKSTFE/G
DLVSWQ/S
QYCVYE/S
BrSMV
ERIEYY/S
KEYEIG/G
EVVVFQ/S
VGSIYQ/S
AHVMYQ/K
HEAKFE/G
PYAVFE/S
KLVGFQ/N
DVCKFE/S
WEqMV
EGIQLY/G
KDYKIG/G
QIVEYQ/A
VNCEFQ/A
SHVMYQ/S
KRVRYE/G
NAAQFE/S
SRVQWQ/S
QFCEWE/S
Dipeptide of protease cleavage sites are indicated in bold
The 50 terminus of the genomic RNA sequence is AAAUUAAAC, similar to that of other members of the genus Tritimovirus [8, 10]. The 50 UTR is 128 nt long (21 % A and 33 % U), a common feature in other Tritimoviruses [4]. The 30 UTR of the virus is 143 nt long excluding the polyadenosine tail, which is similar to the lengths of other Tritimoviruses (WSMV, ONMV, TOgMV, and WEqMV). Pairwise comparison of the YOgMV polyprotein sequence with proteinase recognition motifs of WSMV, ONMV, and TOgMV (genus Tritimovirus) was perfectly consistent with the viral encoded proteinase cleavage sites in polyprotein (Table 1). Thus, like other potyvirids, the polyprotein is cleaved into ten putative proteins (P1, HCpro, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb, and CP) [7]. Cleavage was mediated by the three viral encoded proteinases P1, Helper Component-Protease (HC-Pro), and Nuclear Inclusion a (NIa). All proteinase cleavage sites for YOgMV were located at the same genomic coordinates and similar to that of other Tritimoviruses; however, substitutions at each cleavage site were observed (Table 1). NCBI BlastP searches revealed potential potyviral conserved motifs in the YOgMV polyprotein. YOgMV P1, a serine proteinase, has a conserved peptidase S30 potyvirus P1 proteinase present in C-terminal region. The conserved catalytic triad was predicted to be at H258, D272, and S304, similar to that of WSMV and TOgMV [4, 23]. WSMV P1 was found to mediate suppression of RNA silencing [24]; therefore, it may play a significant role in virus replication and symptom expression. HC-Pro contains a cysteine proteinase domain in its C-terminal region. The conserved catalytic triad was predicted at C272 and H343 flanked by a highly conserved region found in all other members of Tritimovirus. The zinc-finger-like motif H13–(X2)–C16–(X29)–C46–(X2)–C49 was present in YOgMV HC-Pro. This motif is essential for transmission by eriophyid mites [25]. NIa-Pro is the third proteinase and likely is responsible for the cleavages in the rest of YOgMV polyprotein. The four catalytic triads were predicted to be H36, D75, C147, and H163. Using the NCBI ORF-Finder, a putative P3N-Pretty Interesting Potyviridae ORF (PIPO) was found in the ?2
reading frame embedded in the YOgMV-P3 [26]. YOgMVPIPO ORF found downstream of a conserved A2G1–2A6–7 motif (nucleotides 2,751–2,760) could code for a protein of 122 amino acids. The slippery motif (A2G1–2A6–7) found to be highly conserved at the 50 end of P3 cistron of all other Tritimoviruses [4, 11, 23, 27], indicating that the YOgMVPIPO ORF might be expressed as a P3-PIPO fusion product via ribosomal frameshift or transcriptional slippage at conserved A2G1–2A6–7 motif [23]. The two small proteins, 6K1 and 6K2, share the highest identities with orthologs of Tritimovirus. A 51-aa 6K1 peptide was located upstream of CI cistron and contained the conserved motif (A–X22–K), present in all orthologs of Tritimoviruses; thus, it may contribute to a yet unknown biological function. YOgMV-6K2 was located downstream of CI cistron and is believed to anchor the virus replication complex to the membrane of endoplasmic reticulum [28]. In silico protein analysis, using TMHMM program revealed that 6K1 and 6K2 proteins contain a signature transmembrane domain, which suggests an association with membrane structures. In 6K1 and 6K2, TM helixes with high probability were found between residues 13–35 amino acids, similar to TOgMV, WSMV, and ONMV (data not shown). The highly conserved RNA helicase motifs were present in YOgMV-CI, which contain an NTP-binding motif G–C– G–K–S–X3–P (aa 1,157–1,165), and helicase-C domains. At C-terminus of CI mature protein, the conserved Potyviridae polyprotein (PP-poty) domain was present. A tyrosine residue (Y82), in the motif F–Y–G–F–D (aa 1,841–1,845), was found in YOgMV-VPg peptide and matched exactly the context of the tyrosine, in TEV VPg reported to be linked to the 50 end of the viral RNA [29]. Pairwise alignment of YOgMV-CP and HC-Pro cistrons with aphid-transmissible potyviruses showed missing motifs associated with aphid transmission [30, 31]. Sequence comparison and phylogenetic relationships within Potyviridae family Pairwise comparisons of amino acid identities of 10 putative proteins of YOgMV with other five Tritimoviruses
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Virus Genes Table 2 Percent identities of YOgMV (Sb isolate) genes compared with representative members of other genera in the family Potyviridae Virus
50 UTR (nt)
Polyprotein (nt)
Polyprotein (aa)
P1 (aa)
HC-Pro (aa)
P3 (aa)
6K1 (aa)
CI (aa)
6K2 (aa)
NIa-Pro (aa)
NIb (aa)
VPg (aa)
CP (aa)
30 UTR (nt)
WSMV
77
63
62
52
57
55
67
67
56
71
75
55
50
45
ONMV
69
63
61
52
55
53
69
65
56
70
75
60
52
49
WEqMV
59
53
61
41
39
35
56
50
35
50
66
49
40
34
BrSMV
43
51
45
21
39
38
49
51
37
53
63
44
40
34
TOgMV
47
61
61
54
54
55
73
63
58
68
73
60
52
54
The highest scores are indicated in bold
YOgMV-Ra
100
A
B
YOgMV-Sb
88
TOgMV 99
100
98 WSMV-R
73
100 WSMV-CZ
100
100
ONMV-Type
Tritimovirus
100
100 67
WEqMV 100
BrSMV
BrSMV-11-Cal
SCsMV
BrSMV-A
100
100
BrSMV-Hm 100
TriMV 100
ScSMV
100
100
Potyvirus
RgMV-AV
Rymovirus
AgMV
100 100
NLV MacMV BaYMV 100
100
100
Potyvirus
PVY
Brambyvirus
BlVY
99
Rymovirus
RyMV
BlVY
RGMV
100
AgMV
100
JGMV
HoMV
65
HoMV
100
JGMV
99
CVYV 100
PVY
Ipomovirus
CBSV
100
Ipomovirus
SpMMV
WYMV
Poacevirus
TriMV SPMMV
Poacevirus
CVYV
86
Tritimovirus
WSMV
100
WEqMV 96
ONMV
100
100 ONMV-Pp
100
82
TOgMV
WSMV-Type
58
YOgMV-Sb YOgMV-Ra
CYNMV OMV
100
Maculovirus
Brambyvirus Maculovirus
BaYMV
100 100
Bymovirus
WYMV
Bymovirus 0.2
0.1
Fig. 4 a Phylogenetic tree generated with the CP amino acid sequences of YOgMV isolates and other members of Potyviridae. The trees were generated using Maximum Likelihood algorithm; Bootstrap values out of 100 replicas are indicated at branches node and the scale bar represents 0.1 amino acid substitution per site. Genera with representative members are indicated on the right side of phylogenetic tree. Genbank accession numbers of potyvirids species are Agropyron mosaic virus (AgMV, NC_005903), Barley yellow mosaic virus (BaYMV, NC_002990), Blackberry virus Y (BlVY, NC_008558), Brome streak mosaic virus (BrSMV-11-Cal, Z48506), Brome streak mosaic virus (BrSMV-Hm, X78485), Brome streak mosaic virus (BrSMV-A, AJ271086), Cucumber vein yellowing virus (CVYV, AY578085), Johnsongrass mosaic virus (JGMV, z26920), Hordeum mosaic virus (HoMV, NC_005904), Maclura mosaic virus AAB02823.), Narcissus latent virus (NLV, DQ450199), Potato virus Y (PVY, NC_001616), Oat necrotic mottle virus (ONMV-Pp, AF454461), Oat necrotic mottle virus (ONMV-Type, AY377938), Sugarcane mosaic virus (SCMV, NC_003398), Sweet potato mild mottle virus (SPMMV, NC_003797), Tall oatgrass mosaic virus (TOgMV, KF260962), Triticum mosaic virus (TriMV, FJ669487), Wheat eqlid mosaic virus (WEqMV, NC_009805), Wheat streak mosaic virus (WSMV-El Batan3, AF285170), Wheat streak mosaic virus (WSMV-Type, AF285169), Wheat streak mosaic virus (WSMV-Cz, AF454454), and Wheat yellow mosaic virus (WYMV,
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NC_002350). b Phylogenetic relationships inferred with NeighborJoining tree were obtained with the complete amino acid sequences of a polyprotein of Yellow oatgrass mosaic virus (YOgMV) and with representative species of various genera of the family Potyviridae. The values at the forks indicate the number of times out of 100 trees that this grouping occurred after bootstrapping the data. The scale bar represents a genetic distance of 0.2. Bootstrap values above 60 % are indicated for each node. Genera with representative members are indicated on the right side of phylogenetic tree. The viruses used for alignment and its Genbank accession numbers were as follows: Agropyron mosaic virus (AgMV, NC_005903), Barley yellow mosaic virus (BaYMV, NC_002990), Blackberry virus Y (BlVY, NC_008558), Brome streak mosaic virus (BrSMV, NC_003501), Chinese yam necrotic mosaic virus (CYNMV, AB710145), Cucumber vein yellowing virus (CVYV, AY578085), Hordeum mosaic virus (HoMV, NC_005904), Johnsongrass mosaic virus (JGMV, z26920), Oat mosaic virus (OMV, AJ306718), Oat necrotic mottle virus (ONMV, NC_005136), Potato virus Y (PVY, NC_001616), Ryegrass mosaic virus (RGMV, NC_001814), Sugarcane streak mosaic virus (SCsMV, Y17738), Sweet potato mild mottle virus (SPMMV, NC_003797), Tall oatgrass mosaic virus (TOgMV, KF260962), Triticum mosaic virus (TriMV, FJ669487), Wheat eqlid mosaic virus (WEqMV, NC_009805), Wheat streak mosaic virus (WSMV, NC_001886), and Wheat yellow mosaic virus (WYMV, NC_002350)
Virus Genes
(WSMV, ONMV, WEqMV, BrSMV, and TOgMV) revealed that YOgMV is a distinct species in the genus Tritimovirus (Table 2). Overall, nucleotide and amino acid identities of YOgMV individual gene products were the highest for those of TOgMV and WSMV (Table 2). NIb of YOgMV was the most conserved gene and shared the highest amino acid identity (63–75 %) with orthologs of other Tritimoviruses. P1 of YOgMV was the least conserved, sharing 21–54 % identity (Table 2). Interestingly, YOgMV-CP was highly variable (40–52 % amino acid identity) when compared to other Tritimoviruses. In addition, the 30 UTR shared the highest nucleotide identity with TOgMV (54 %) (Table 2). The similarity plot performed for the whole genome sequences of YOgMV isolates (Sb and Ra) and TOgMV (Fig. 3b) showed that all genes contribute to the differences between these two viruses. The highest similarities were observed for the CI and NIb genes and the lowest for P1 and CP genes. According to Adams et al. [7], the values that discriminate between species in family Potyviridae are 76–77 % nucleotide identity and 81–82 % amino acid identity in the whole genome. Pairwise comparisons between the sequences of the complete ORF of two YOgMV isolates exhibited relatively high genetic diversity (Table 2). The two YOgMV isolates shared 90 % nucleotide sequence identity (97 % amino acid identity). High diversity is expected in indigenous viruses naturally infecting wild plants collected from multiple geographical locations [2, 3, 32]. Given that these two isolates of YOgMV were collected from different areas, it is likely that the observed polymorphisms occur as a result of two distinct viral populations and do not represent a ‘‘mutant cloud’’ within a single population [33]. Phylogenetic relationships were inferred using two different methods (NJ and ML) with complete polyprotein and CP data sets representative of virus species from eight genera assigned within the family Potyviridae [6]. Different phylogenetic trees clearly clustered YOgMV within Tritimovirus genus with high bootstrap support (Fig. 4a, b). Methods applied to infer the phylogenetic analysis (NJ and ML) using different data sets gave a unique consensus tree topology (data not shown). The sequence of YOgMV isolates clustered most closely with TOgMV, a grass-infecting Tritimovirus in Czech Republic, and it appears that they share a most recent common ancestor. Recently, several viruses were identified in wild grasses from central European countries such as Germany, Austria, and Czech Republic [4, 12, 14, 34]. In most of the cases, the infected wild grasses were found along field edges of cereal crops. The incidence of viral infections in wild grasses is probably much higher than expected [14, 35].
This highlights the need for more in-depth studies on the threat of viruses in native vegetation to agricultural production, and the threat of viruses of agricultural crop systems to native vegetation. Acknowledgments The author would like to thank Mrs Lenka Sˇirlova´ for providing YOgMV isolates used in this study and Mrs Terri L. Phelan for critically reviewing the manuscript.
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34. F. Rabenstein, H. Herbert, in Resist. Gegen Abiotischen Stress Pflanzenzu¨cht. (Lehr- und Forschungszentrum fu¨r Landwirtschaft ¨ sterreichs, 2012), pp. 11–13 Raumberg-Gumpenstein, O 35. L.L. Ingwell, S.D. Eigenbrode, N.A. Bosque-Pe´rez, Sci. Rep. 2, 578 (2012)
Genome sequence of two isolates of Yellow oatgrass mosaic virus, a new grass-infecting Tritimovirus Mohamed Hassan
Received: 13 December 2013 / Accepted: 29 March 2014 Ó Springer Science+Business Media New York 2014
Abstract Complete genome sequences of two Yellow oatgrass mosaic virus (YOgMV) isolates have been determined to be 9,292 nucleotides excluding the 30 polyadenylated tail. The viral RNA encodes a large putative open reading frame (ORF) of a single polyprotein consisting of 3,002 amino acids with typical genome organization of monopartite potyvirids. A small overlapping ORF encoding a pretty interesting Potyviridae ORF of 122 amino acids is found in the P3 cistron of both YOgMV isolates. The nucleotide and amino acid identities between the two YOgMV isolates are 90 and 97 %, respectively. Pairwise comparison of YOgMV putative mature proteins and proteinase cleavage sites with those of representative members of the family Potyviridae indicated that YOgMV is more closely related to members of the genus Tritimovirus. In phylogenetic trees constructed with sequences of putative polyprotein, YOgMV consistently groups with members of the genus Tritimovirus. These results suggest that YOgMV should be classified as a distinct species in the genus Tritimovirus in the family Potyviridae. Keywords Trisetum flavescens Potyviridae Tritimovirus Poaceae Virus taxonomy Electronic supplementary material The online version of this article (doi:10.1007/s11262-014-1073-9) contains supplementary material, which is available to authorized users. M. Hassan (&) Department of Agricultural Botany, Faculty of Agriculture, Fayoum University, Fayoum, Egypt e-mail: [email protected] M. Hassan Department of Plant Pathology, University of Arkansas, Fayetteville, AR 72701, USA e-mail: [email protected]
Introduction Trisetum flavescens L., commonly known as yellow oatgrass or golden oatgrass, is a perennial species of Poaceae family. It is native to Europe, Asia, and North Africa and grows in seeded pastures, roadsides, and as a weed in croplands. Recently, there is an increased interest to study plant virus ecology in nature for several economic and scientific perspectives [1–5]. One of these reasons is that wild grasses may serve as reservoirs of viruses that can cause epidemics in important cultivated crops [5]. Thus, it is important to extend our knowledge of viruses infecting wild plants to better predict the potential of native viruses to infect cultivated crops. Potyviridae is the largest family of positive-sense RNA viruses infecting plants. The International Committee on Taxonomy of Viruses (ICTV) assigned eight genera within the family Potyviridae based on their genetic relatedness, vector transmission, and genome organization [6]. One of these genera, Tritimovirus, consists of six species: Wheat streak mosaic virus (WSMV) as the type member, Brome streak mosaic virus (BrSMV), Oat necrotic mottle virus (ONMV), Wheat eqild mosaic virus (WEqMV), Tall oatgrass mosaic virus (TOgMV), and Yellow oatgrass mosaic virus (YOgMV; for which only 30 end of the viral genome has been reported) [4, 7–12]. In addition, Cocksfoot streak mosaic virus (CfSMV), a new proposed Tritimovirus, was isolated from a diseased cocksfoot plant in Germany [13]. According to Rabenstein et al. [14], CfSMV is a distinctive member of the genus Tritimovirus based on sequence comparisons and phylogenetic analysis. However, the sequence data for CfSMV are not available. The availability of full genome sequences has significantly clarified the taxonomic status of several potyvirids
123
Virus Genes
and has led to the establishment of criteria that can be used to distinguish closely related virus species [7]. In this study we report the complete genome sequence of two isolates of YOgMV, a new Tritimovirus infecting wild yellow oatgrass and confirmed their presence in multiple locations in the Czech Republic. In addition, the genome organization and identities of the putative gene products of the new virus are discussed in relation to previously described Tritimoviruses.
Materials and methods Mechanical inoculation Yellow oatgrass plants with mild mosaic symptoms were collected from Stare´ Brˇ´ısˇteˇ (Sb) and Radeˇtice (Ra) counties, Czech Republic. The two YOgMV isolates (Sb and Ra) were transmitted by sap inoculation to seedlings of the oat (cv. Abel), the propagation host of YOgMV [12]. Symptomatic leaves infected with YOgMV isolates at 2–3 weeks post-inoculation were tested for the presence of the virus by RT-PCR.
A
RNA extraction, full-length amplification, and genome sequencing Total RNA was extracted using the Qiagen RNeasy Kit according to manufacturer’s recommendations (Qiagen). Total RNA was used as a template for RT-PCR and 30 end of YOgMV isolates were amplified using primers published by Gibbs and Mackenzie [15]. To amplify the rest of the viral genome, a genome walking strategy was employed as described previously by Hassan et al. [4] using specific reverse primers from known sequences and upstream degenerate primers (Supplementary Table 1). All RT-PCR reactions were carried out using a one-step RT-PCR kit (Takara BIO Inc.), according to the manufacturer’s recommendations. Amplicons of expected size were gel purified using QIAquick gel extraction kit (Qiagen), ligated into pGEM-T Easy plasmid (Promega), and transformed into Escherichia coli DH5a. The 50 terminus was amplified using a 50 RACE kit (Invitrogen), according to the manufacturer’s recommendations. At least 39 coverage of the virus genome was obtained. Nucleotide sequences were determined using a fluorescent DNA sequencer CEQ2000XL, with dye terminator reagents (Beckman Coulter). Sequence analysis and phylogenetic relatedness The complete nucleotide consensus was assembled from overlapping cDNA clones using ProSeq program [16].
123
B Fig. 1 a Potted plant of yellow oatgrass transplanted from Stare´ Brˇ´ısˇteˇ, Czech Republic. The plant shows mild mosaic symptoms on the new emerging leaf, faint mosaic streaks pointed by arrows. b Locations in Czech Republic where yellow oatgrass diseased plants were observed and collected for characterization of YOgMV isolates. The towns’ names, where Sb and Ra YOgMV isolates were collected are underlined
Nucleotides and amino acid sequences were analyzed using CLUSTALW in BioEdit package [17]. BlastX and BlastP at the website of NCBI were used to search for related proteins from the Protein Information Resources (PIR) database [18]. Protein function analysis and transmembrane helices in Tritimovirus proteins were predicted using TMHMM [19]. Similarity plot depicting the identity among the aligned nucleotide sequences were generated with SimPlot, Version 3.5.1 [20]. Similarity was calculated within a sliding
Virus Genes Fig. 2 a, b Systemic infection symptoms induced by YOgMV isolates Sb and Ra on oat cv. Abel, respectively. Infected plant leaves presented are from the same physiological age. c–e Chlorosis and necrotic lesions symptoms observed on different oat plants parts after 21 days of inoculation with Ra isolate. f Pronounced dwarfing on an oat plant inoculated with Ra isolate compared to a healthy oat plant on the right
Healthy
Infected
A
C
window of 200 nt, with a step of 20 residues between points. Positions containing gaps were excluded from the analysis. Phylogenetic analysis was done using the Neighbor-Joining (NJ) and Maximum Likelihood (ML) methods of phylogenetic inference with 1,000 bootstrap replicates using MEGA 5.0 [21]. RT-PCR Specific primer pairs were designed to amplify a 200-nt fragment of YOgMV coat protein gene (Supplementary
Infected
Healthy
B
D
E
F
Table 1). Reverse transcription was carried out at 50 °C for 30 min., activation of the hot start Taq polymerase at 95 °C for 15 min, followed by 35 cycles of 94 °C at 30 s., 55 °C at 30 s., 72 °C at 30 s., and a final extension step at 72 °C for 10 min according to the manufacturer’s instructions of QiagenÒOne-step RT-PCR kit (Qiagen). Amplification products were electrophoresed on 2 % agarose gels, stained with ethidium bromide, and visualized under UV light. PCR products of expected size were extracted from 2 % agarose gel using QIAquick gel extraction kit (Qiagen) and RT-PCR products were cloned
123
P3
HC-Pro
P1
2685 E/A
NIa NIa VPg Pro
3006
2186
Q/A E/G E/G
CI
Q/S
1957
Q/G Q/D
1709 1760
G/G
Amino acids
6k2
1013 1064
Y/G
6k1
5’UTR
735
1
A
CP
NIb
3’UTR
(A)n 9292
9149
8229
6735
6045
Nucleotides
5299 5454
AAG(A6)
2751-2760
3213 3366
2379
1230
129
1
PIPO
B
Similarity (%)
Fig. 3 a Schematic diagram of Yellow oatgrass mosaic virus (YOgMV) genome organization RNA. The map of the YOgMV genome depicts the locations of polyprotein open reading frame start and stop codons; the amino acid (above) and the nucleotide (below) coordinates of predicated mature viral proteins (P1, HC-Pro, P3, 6K1, CI, 6K2, VPg-NIa, NIb, and CP). The specific dipeptides of a cleavage site and their location in the genome are shown above the map. b Similarity plots of YOgMV isolates and TOgMV nucleotide sequence, calculated and plotted by SimPlot 3.5.1 (Lole et al. [20]). The entire genetic map of YOgMV is shown at the top, approximately to scale. YOgMV isolate Sb used as a reference (the query sequence), YOgMV isolate Ra curve (blue) and TOgMV curve (green)
352
Virus Genes
Position (nt)
into the pGEM-T vector (Promega); five clones were sequenced.
symptom induction by Ra isolate is more rapid than that of symptom production by Sb isolate (Fig. 2a). Genome organization of YOgMV
Results and discussion Symptoms induced by YOgMV isolates and RT-PCR detection Twelve yellow oatgrass samples, exhibiting mild mosaic symptoms, were collected from different districts in Czech Republic (Fig. 1a, b). YOgMV was detected from all samples collected from geographically different districts by RT-PCR analysis with primers specific to the coat protein gene of YOgMV. The virus causes similar symptoms when mechanically inoculated into yellow oatgrass seedlings [12]. However, when YOgMV isolates are inoculated into oat (cv. Abel), they cause severe mosaic symptoms, necrosis, and dwarfing (Fig. 2a–f). This observation suggests that YOgMV coevolved with wild yellow oatgrass. Roossinck [22] proposed that recent plant–virus association induces more severe symptoms compared to older associations. Ra isolate showed more severe symptoms than Sb isolate in oat plants (cv. Abel), though both YOgMV isolates induced leaf mosaic and necrotic blotch symptoms, as progress of infection (Fig. 2a). In the oat plants, timing of
123
The universal potyvirid primers [15], designed to amplify the 30 end portion of viruses belonging to Potyviridae, were used to generate amplicons of the expected size (1.7 Kbp), which were cloned and sequenced. BlastN search of the NCBI databases revealed that WSMV, the type member of genus Tritimovirus [8], was the most closely related viral species to YOgMV in Potyviridae. The complete nucleotide sequence of YOgMV was determined from multiple overlapping cDNA clones, generated by genome walking and 50 RACE. The sequence of YOgMV isolates (Sb and Ra) consists of 9,292 nucleotides (nt) excluding the 30 -poly (A) tail, and have been submitted to GenBank under the accession numbers KF984546 and KF984547 for Sb and Ra isolates, respectively. The genome of YOgMV has the typical features of the monopartite of Potyviridae members, consisting of a single large ORF. The putative ORF starts with AUG codon at (nt 129–131) and ends with the stop codon UAG at (nt 9,147–9,149). The putative ORF consist of 9,090 nucleotides and encodes a polyprotein of 3,006 amino acid (aa) with a predicted molecular mass 341.5 kDa (Fig. 3a).
Virus Genes Table 1 Predicted protease cleavage sites of YOgMV isolates by comparison to other Tritimoviruses Virus
P1/HC-Pro
HC-Pro/P3
P3/6K1
6K1/CI
CI/6K2
6k2/VPg
VPg/NIa
NIa/NIb
NIb/CP
YOgMV(Sb)
HGLRWY/G
RDYAIG/G
KLVKYQ/G
VNAQYQ/D
AHVSYQ/A
ASVKYE/G
NASTFE/G
GLVHWQ/S
QYCVFE/A
YOgMV(Ra)
HGLRWY/G
RDYAIG/G
KLVKYQ/G
VNAQYQ/D
AHVSYQ/A
ASVKYE/G
NASTFE/G
GLVHWQ/S
QYCVFE/A
TOgMV
KGLKWC/G
RDYEIG/G
NEVEYQ/G
VNCEYQ/G
AHVEYQ/A
AKSSYE/G
NKSLYE/S
DLANWQ/S
RCCMYE/Y
ONMV
HGLRWY/S
KDYKIG/G
ELVEYQ/S
VNCEYQ/S
SHVSYQ/A
HRVKYE/G
NKSLYE/G
ELVNWQ/S
KYCVYE/S
WSMV
HGLRWY/G
KDYKIG/G
ELVEYQ/G
FNCEYQ/S
SHVSYQ/A
RSVKFE/G
NKSTFE/G
DLVSWQ/S
QYCVYE/S
BrSMV
ERIEYY/S
KEYEIG/G
EVVVFQ/S
VGSIYQ/S
AHVMYQ/K
HEAKFE/G
PYAVFE/S
KLVGFQ/N
DVCKFE/S
WEqMV
EGIQLY/G
KDYKIG/G
QIVEYQ/A
VNCEFQ/A
SHVMYQ/S
KRVRYE/G
NAAQFE/S
SRVQWQ/S
QFCEWE/S
Dipeptide of protease cleavage sites are indicated in bold
The 50 terminus of the genomic RNA sequence is AAAUUAAAC, similar to that of other members of the genus Tritimovirus [8, 10]. The 50 UTR is 128 nt long (21 % A and 33 % U), a common feature in other Tritimoviruses [4]. The 30 UTR of the virus is 143 nt long excluding the polyadenosine tail, which is similar to the lengths of other Tritimoviruses (WSMV, ONMV, TOgMV, and WEqMV). Pairwise comparison of the YOgMV polyprotein sequence with proteinase recognition motifs of WSMV, ONMV, and TOgMV (genus Tritimovirus) was perfectly consistent with the viral encoded proteinase cleavage sites in polyprotein (Table 1). Thus, like other potyvirids, the polyprotein is cleaved into ten putative proteins (P1, HCpro, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb, and CP) [7]. Cleavage was mediated by the three viral encoded proteinases P1, Helper Component-Protease (HC-Pro), and Nuclear Inclusion a (NIa). All proteinase cleavage sites for YOgMV were located at the same genomic coordinates and similar to that of other Tritimoviruses; however, substitutions at each cleavage site were observed (Table 1). NCBI BlastP searches revealed potential potyviral conserved motifs in the YOgMV polyprotein. YOgMV P1, a serine proteinase, has a conserved peptidase S30 potyvirus P1 proteinase present in C-terminal region. The conserved catalytic triad was predicted to be at H258, D272, and S304, similar to that of WSMV and TOgMV [4, 23]. WSMV P1 was found to mediate suppression of RNA silencing [24]; therefore, it may play a significant role in virus replication and symptom expression. HC-Pro contains a cysteine proteinase domain in its C-terminal region. The conserved catalytic triad was predicted at C272 and H343 flanked by a highly conserved region found in all other members of Tritimovirus. The zinc-finger-like motif H13–(X2)–C16–(X29)–C46–(X2)–C49 was present in YOgMV HC-Pro. This motif is essential for transmission by eriophyid mites [25]. NIa-Pro is the third proteinase and likely is responsible for the cleavages in the rest of YOgMV polyprotein. The four catalytic triads were predicted to be H36, D75, C147, and H163. Using the NCBI ORF-Finder, a putative P3N-Pretty Interesting Potyviridae ORF (PIPO) was found in the ?2
reading frame embedded in the YOgMV-P3 [26]. YOgMVPIPO ORF found downstream of a conserved A2G1–2A6–7 motif (nucleotides 2,751–2,760) could code for a protein of 122 amino acids. The slippery motif (A2G1–2A6–7) found to be highly conserved at the 50 end of P3 cistron of all other Tritimoviruses [4, 11, 23, 27], indicating that the YOgMVPIPO ORF might be expressed as a P3-PIPO fusion product via ribosomal frameshift or transcriptional slippage at conserved A2G1–2A6–7 motif [23]. The two small proteins, 6K1 and 6K2, share the highest identities with orthologs of Tritimovirus. A 51-aa 6K1 peptide was located upstream of CI cistron and contained the conserved motif (A–X22–K), present in all orthologs of Tritimoviruses; thus, it may contribute to a yet unknown biological function. YOgMV-6K2 was located downstream of CI cistron and is believed to anchor the virus replication complex to the membrane of endoplasmic reticulum [28]. In silico protein analysis, using TMHMM program revealed that 6K1 and 6K2 proteins contain a signature transmembrane domain, which suggests an association with membrane structures. In 6K1 and 6K2, TM helixes with high probability were found between residues 13–35 amino acids, similar to TOgMV, WSMV, and ONMV (data not shown). The highly conserved RNA helicase motifs were present in YOgMV-CI, which contain an NTP-binding motif G–C– G–K–S–X3–P (aa 1,157–1,165), and helicase-C domains. At C-terminus of CI mature protein, the conserved Potyviridae polyprotein (PP-poty) domain was present. A tyrosine residue (Y82), in the motif F–Y–G–F–D (aa 1,841–1,845), was found in YOgMV-VPg peptide and matched exactly the context of the tyrosine, in TEV VPg reported to be linked to the 50 end of the viral RNA [29]. Pairwise alignment of YOgMV-CP and HC-Pro cistrons with aphid-transmissible potyviruses showed missing motifs associated with aphid transmission [30, 31]. Sequence comparison and phylogenetic relationships within Potyviridae family Pairwise comparisons of amino acid identities of 10 putative proteins of YOgMV with other five Tritimoviruses
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Virus Genes Table 2 Percent identities of YOgMV (Sb isolate) genes compared with representative members of other genera in the family Potyviridae Virus
50 UTR (nt)
Polyprotein (nt)
Polyprotein (aa)
P1 (aa)
HC-Pro (aa)
P3 (aa)
6K1 (aa)
CI (aa)
6K2 (aa)
NIa-Pro (aa)
NIb (aa)
VPg (aa)
CP (aa)
30 UTR (nt)
WSMV
77
63
62
52
57
55
67
67
56
71
75
55
50
45
ONMV
69
63
61
52
55
53
69
65
56
70
75
60
52
49
WEqMV
59
53
61
41
39
35
56
50
35
50
66
49
40
34
BrSMV
43
51
45
21
39
38
49
51
37
53
63
44
40
34
TOgMV
47
61
61
54
54
55
73
63
58
68
73
60
52
54
The highest scores are indicated in bold
YOgMV-Ra
100
A
B
YOgMV-Sb
88
TOgMV 99
100
98 WSMV-R
73
100 WSMV-CZ
100
100
ONMV-Type
Tritimovirus
100
100 67
WEqMV 100
BrSMV
BrSMV-11-Cal
SCsMV
BrSMV-A
100
100
BrSMV-Hm 100
TriMV 100
ScSMV
100
100
Potyvirus
RgMV-AV
Rymovirus
AgMV
100 100
NLV MacMV BaYMV 100
100
100
Potyvirus
PVY
Brambyvirus
BlVY
99
Rymovirus
RyMV
BlVY
RGMV
100
AgMV
100
JGMV
HoMV
65
HoMV
100
JGMV
99
CVYV 100
PVY
Ipomovirus
CBSV
100
Ipomovirus
SpMMV
WYMV
Poacevirus
TriMV SPMMV
Poacevirus
CVYV
86
Tritimovirus
WSMV
100
WEqMV 96
ONMV
100
100 ONMV-Pp
100
82
TOgMV
WSMV-Type
58
YOgMV-Sb YOgMV-Ra
CYNMV OMV
100
Maculovirus
Brambyvirus Maculovirus
BaYMV
100 100
Bymovirus
WYMV
Bymovirus 0.2
0.1
Fig. 4 a Phylogenetic tree generated with the CP amino acid sequences of YOgMV isolates and other members of Potyviridae. The trees were generated using Maximum Likelihood algorithm; Bootstrap values out of 100 replicas are indicated at branches node and the scale bar represents 0.1 amino acid substitution per site. Genera with representative members are indicated on the right side of phylogenetic tree. Genbank accession numbers of potyvirids species are Agropyron mosaic virus (AgMV, NC_005903), Barley yellow mosaic virus (BaYMV, NC_002990), Blackberry virus Y (BlVY, NC_008558), Brome streak mosaic virus (BrSMV-11-Cal, Z48506), Brome streak mosaic virus (BrSMV-Hm, X78485), Brome streak mosaic virus (BrSMV-A, AJ271086), Cucumber vein yellowing virus (CVYV, AY578085), Johnsongrass mosaic virus (JGMV, z26920), Hordeum mosaic virus (HoMV, NC_005904), Maclura mosaic virus AAB02823.), Narcissus latent virus (NLV, DQ450199), Potato virus Y (PVY, NC_001616), Oat necrotic mottle virus (ONMV-Pp, AF454461), Oat necrotic mottle virus (ONMV-Type, AY377938), Sugarcane mosaic virus (SCMV, NC_003398), Sweet potato mild mottle virus (SPMMV, NC_003797), Tall oatgrass mosaic virus (TOgMV, KF260962), Triticum mosaic virus (TriMV, FJ669487), Wheat eqlid mosaic virus (WEqMV, NC_009805), Wheat streak mosaic virus (WSMV-El Batan3, AF285170), Wheat streak mosaic virus (WSMV-Type, AF285169), Wheat streak mosaic virus (WSMV-Cz, AF454454), and Wheat yellow mosaic virus (WYMV,
123
NC_002350). b Phylogenetic relationships inferred with NeighborJoining tree were obtained with the complete amino acid sequences of a polyprotein of Yellow oatgrass mosaic virus (YOgMV) and with representative species of various genera of the family Potyviridae. The values at the forks indicate the number of times out of 100 trees that this grouping occurred after bootstrapping the data. The scale bar represents a genetic distance of 0.2. Bootstrap values above 60 % are indicated for each node. Genera with representative members are indicated on the right side of phylogenetic tree. The viruses used for alignment and its Genbank accession numbers were as follows: Agropyron mosaic virus (AgMV, NC_005903), Barley yellow mosaic virus (BaYMV, NC_002990), Blackberry virus Y (BlVY, NC_008558), Brome streak mosaic virus (BrSMV, NC_003501), Chinese yam necrotic mosaic virus (CYNMV, AB710145), Cucumber vein yellowing virus (CVYV, AY578085), Hordeum mosaic virus (HoMV, NC_005904), Johnsongrass mosaic virus (JGMV, z26920), Oat mosaic virus (OMV, AJ306718), Oat necrotic mottle virus (ONMV, NC_005136), Potato virus Y (PVY, NC_001616), Ryegrass mosaic virus (RGMV, NC_001814), Sugarcane streak mosaic virus (SCsMV, Y17738), Sweet potato mild mottle virus (SPMMV, NC_003797), Tall oatgrass mosaic virus (TOgMV, KF260962), Triticum mosaic virus (TriMV, FJ669487), Wheat eqlid mosaic virus (WEqMV, NC_009805), Wheat streak mosaic virus (WSMV, NC_001886), and Wheat yellow mosaic virus (WYMV, NC_002350)
Virus Genes
(WSMV, ONMV, WEqMV, BrSMV, and TOgMV) revealed that YOgMV is a distinct species in the genus Tritimovirus (Table 2). Overall, nucleotide and amino acid identities of YOgMV individual gene products were the highest for those of TOgMV and WSMV (Table 2). NIb of YOgMV was the most conserved gene and shared the highest amino acid identity (63–75 %) with orthologs of other Tritimoviruses. P1 of YOgMV was the least conserved, sharing 21–54 % identity (Table 2). Interestingly, YOgMV-CP was highly variable (40–52 % amino acid identity) when compared to other Tritimoviruses. In addition, the 30 UTR shared the highest nucleotide identity with TOgMV (54 %) (Table 2). The similarity plot performed for the whole genome sequences of YOgMV isolates (Sb and Ra) and TOgMV (Fig. 3b) showed that all genes contribute to the differences between these two viruses. The highest similarities were observed for the CI and NIb genes and the lowest for P1 and CP genes. According to Adams et al. [7], the values that discriminate between species in family Potyviridae are 76–77 % nucleotide identity and 81–82 % amino acid identity in the whole genome. Pairwise comparisons between the sequences of the complete ORF of two YOgMV isolates exhibited relatively high genetic diversity (Table 2). The two YOgMV isolates shared 90 % nucleotide sequence identity (97 % amino acid identity). High diversity is expected in indigenous viruses naturally infecting wild plants collected from multiple geographical locations [2, 3, 32]. Given that these two isolates of YOgMV were collected from different areas, it is likely that the observed polymorphisms occur as a result of two distinct viral populations and do not represent a ‘‘mutant cloud’’ within a single population [33]. Phylogenetic relationships were inferred using two different methods (NJ and ML) with complete polyprotein and CP data sets representative of virus species from eight genera assigned within the family Potyviridae [6]. Different phylogenetic trees clearly clustered YOgMV within Tritimovirus genus with high bootstrap support (Fig. 4a, b). Methods applied to infer the phylogenetic analysis (NJ and ML) using different data sets gave a unique consensus tree topology (data not shown). The sequence of YOgMV isolates clustered most closely with TOgMV, a grass-infecting Tritimovirus in Czech Republic, and it appears that they share a most recent common ancestor. Recently, several viruses were identified in wild grasses from central European countries such as Germany, Austria, and Czech Republic [4, 12, 14, 34]. In most of the cases, the infected wild grasses were found along field edges of cereal crops. The incidence of viral infections in wild grasses is probably much higher than expected [14, 35].
This highlights the need for more in-depth studies on the threat of viruses in native vegetation to agricultural production, and the threat of viruses of agricultural crop systems to native vegetation. Acknowledgments The author would like to thank Mrs Lenka Sˇirlova´ for providing YOgMV isolates used in this study and Mrs Terri L. Phelan for critically reviewing the manuscript.
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