Arch Virol (2009) 154:1125–1128 DOI 10.1007/s00705-009-0415-8
ORIGINAL ARTICLE
The complete nucleotide sequence of the barley yellow dwarf GPV isolate from China shows that it is a new member of the genus Polerovirus Wenwei Zhang Æ Zhuomin Cheng Æ Lei Xu Æ Maosen Wu Æ Peter Waterhouse Æ Guanghe Zhou Æ Shifang Li
Received: 11 November 2008 / Accepted: 27 May 2009 / Published online: 24 June 2009 Ó Springer-Verlag 2009
Abstract The complete nucleotide sequence of the ssRNA genome of a Chinese GPV isolate of barley yellow dwarf virus (BYDV) was determined. It comprised 5673 nucleotides, and the deduced genome organization resembled that of members of the genus Polerovirus. It was most closely related to cereal yellow dwarf virus-RPV (77% nt identity over the entire genome; coat protein amino acid identity 79%). The GPV isolate also differs in vector specificity from other BYDV strains. Biological properties, phylogenetic analyses and detailed sequence comparisons suggest that GPV should be considered a member of a new species within the genus, and the name Wheat yellow dwarf virus-GPV is proposed.
Barley yellow dwarf disease was first described from California USA in 1950 [6] and has since been reported on wheat, barley, oats and many grass species worldwide [5, 12]. In China, the disease is most serious on wheat and occurs throughout the northern and northwestern provinces. The
W. Zhang and Z. Cheng contributed equally to this work. W. Zhang Z. Cheng (&) L. Xu M. Wu G. Zhou S. Li State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, 100193 Beijing, People’s Republic of China e-mail:
[email protected] W. Zhang e-mail:
[email protected] P. Waterhouse CSIRO Plant Industry, Clunies Ross St., Canberra, ACT 2601, Australia
causal agent was first described as barley yellow dwarf virus (BYDV), a phloem-limited virus transmitted by aphids in a persistent, circulative manner [8]. Although at least 25 aphid species have been reported as vectors of BYDV, each virus isolate is transmitted by only one or a few aphid species. Based on the predominant aphid vector and serological relationships, Rochow described five virus strains from the USA, separated into two groups. Group I included the strains MAV, (transmitted by Macrosiphon [=Sitobion] avenae), PAV (transmitted by Rhopalosiphum padi and S. avenae) and SGV (transmitted by Schizaphis graminum). Group II included RPV (transmitted by R. padi) and RMV (transmitted by Rhopalosiphum maidis) [9, 10]. In China, four BYDV strains were reported by Zhou et al. [17]. The GAV and PAV strains showed strong serological cross-reactivity with the MAV and PAV strains from the US, and an RMV isolate similar to that from the US was also found. The most common strain in China, however, was of a fourth type, named GPV. It was transmitted by R. padi and S. graminum and had no serological relationship with the US isolates [16]. In recent years, the nucleotide sequences and genome organization of several BYDV isolates have been determined, and this has led to a major reorganization of their taxonomy. The PAV and MAV strains are similar in genome organization [4, 14] but sufficiently different from one another that they are now recognized as members of different species within the genus Luteovirus, with Barley yellow dwarf virus—PAV being the type species. The RPV strain proved to have a different genomic organization [15] and belongs to a species named Cereal yellow dwarf virusRPV (CYDV-RPV) within the genus Polerovirus (type species, Potato leafroll virus, PLRV). Both genera are classified within the family Luteoviridae [3]. We now report the complete sequence of the Chinese GPV strain, showing that it should be classified with the group II
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isolates in the genus Polerovirus, probably as a member of a new species. A Chinese field isolate of GPV was isolated, purified [1, 2] and stored at -70°C. It was inoculated to oats (cultivar Huabei no. 2) using Schizaphis graminum and the membrane feeding method. Antisera were obtained from rabbits after a series of 6 injections with about 2.77 mg of purified virus preparations. First-strand cDNA was synthesized with GPV RNA as the template and bovine thymus DNA as the random primer. The second strand was synthesized by the RNaseH method. ds-cDNA was inserted into pUC8 using the BamHI linker. The cDNA fragment was cut out from the pGP plasmid using BamHI (Promega) and subcloned into BamHI -digested M13mp19RF. E. coli JPA101 was then transformed with this construct using a Gene Pulser (Bio-Rad). Transformants were identified from 2X YT ? 0.7% agar mini cultures containing X-gal and IPTG, and ssDNA was prepared as recommended by ABI. DNA was sequenced in both directions by the dideoxynucleotide chain termination method [11]. This was done either using dye-labelled primer and Taq DNA polymerase (ABI) followed by autosequencing or using T7 DNA polymerase (Promega), isotope labeling and high-voltage electrophoresis. Terminal sequences were determined using the 30 RACE and 50 RACE kits (Invitrogen) according to the manufacturer’s instructions. The complete nucleotide sequence was assembled and analyzed
using the software NTI Suite 8.0 (InforMax Inc). Phylogenetic analysis was done using MEGA4 [13]. Particles of GPV were polyhedral and about 26 nm in diameter, similar to those of other BYDV strains [7]. The polyclonal antibody raised to purified GPV virions was tested by immunosorbent electron microscopy (ISEM) and shown to decorate particles of GPV but not those of MAV, PAV, SGV, RPV, RMV or GAV strains (results not shown). The complete GPV genomic sequence was assembled from a total of 23 overlapping clones together with the terminal sequences obtained by 50 RACE and 30 RACE. It was 5673 nucleotides (nts) long and had six predicted open reading frames (ORFs) that were similar to those in the genus Polerovirus. The chief features of the sequences and the sizes of the ORFs are shown in Fig. 1. ORF0 is particularly characteristic of members of the genus Polerovirus, but its function remains unknown. As in all other members of the family, ORF2, which encodes the RdRp, lacks a stop codon and is expressed as a fusion protein with ORF1, entered by a -1 frameshift. By comparison with other members of the genus, the frameshift site is predicted to be the heptanucleotide GGGAAAC at nt position 1595– 1601. ORF1 and the fusion protein provide the genomereplication components. In common with other members of the genus, the N-terminus of the coat protein is argininerich, and ORF4, the cell-to-cell movement protein, is
Fig. 1 Organization of the genome of the GPV sequence. The predicted frameshift heptanucleotide is marked with an asterisk, and the ‘‘leaky’’ amber stop codon with a filled diamond
Table 1 Nucleotide sequence identity (%) between the GPV isolate and some members of the family Luteoviridae Virus
Polerovirus
Accession no.
CYDV-RPV L25299
50 -UTR
35
Luteovirus CYDV-RPS AF235168 54
PLRV D00530 36
BWYV AF473561 56
BYDV-PAV D85783
BYDV-MAV D11028
BYDV-GAV AY220739
BYDV-SGV U06865
47
43
44
N/A
ORF0
54 (41.4)
66 (54.1)
44 (26.4)
38 (16.1)
–
–
–
–
ORF1
68 (55.6)
66 (63.1)
49 (25.2)
46 (31.0)
22
23
23
N/A
ORF2
81 (81.5)
80 (80.4)
62 (63.6)
57 (60.0)
33
33
33
N/A
UTR CP
91 84 (78.6)
80 82 (73.6)
66 68 (61.2)
48 62 (58.1)
39 53
42 51
42 51
N/A 51
ORF4
90
88
71
51
52
50
51
N/A
CP-RT
71
70
40
54
49
50
49
46
3 -UTR
83
85
82
78
7
68
10
N/A
Genome
77
75
57
57
32
38
32
N/A
0
For selected comparisons, the percentage amino acid identity is also shown in parentheses
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variable, and the ORF4-coat protein region was the most conserved, with nucleotide identities exceeding 80% for CYDV-RPV and CYDV-RPS (amino acid identities of about 79 and 74%). Phylogenetic analysis of the 50 -part of the genome and the coat protein confirmed the relationships between the viruses and placed GPV with RPV and RPS within the genus Polerovirus (Fig. 2). Members of different species are expected to have CP amino acid sequence identities below 80%, and their entire genomes are expected to be less than 85% identical [3], suggesting that GPV should be considered a member of a new species within the genus. This is supported by its different vector specificity. Because GPV mainly infects wheat in China, we suggest that the name Wheat yellow dwarf virus-GPV would be the most appropriate. The sequence reported here was deposited in the EMBL/ Genbank/DDBJ databases with the accession number FM865413. Acknowledgments We thank Dr. Michael J. Adams for helpful suggestions and critical reading of this manuscript.
References
Fig. 2 Phylogenetic trees based on the codon-aligned nucleotide sequences of (a) concatenated ORFs 0, 1 and 2 of members of the genus Polerovirus and (b) the coat proteins of members of the entire family Luteoviridae. Distance trees were constructed in MEGA4 [13] using the maximum composite likelihood algorithm. The values at the forks indicate the percentage of trees in which this grouping occurred after bootstrapping (10,000 replicates; shown only when [60%). The scale bar shows the number of substitutions per base. Both trees are rooted with the corresponding sequences of pea enation mosaic virus1 (genus Enamovirus) and highlight the position of the new GPV sequence
located within it but in a different reading frame. The coat protein terminates at an amber stop codon and is followed by a readthrough domain generating a minor coat protein that is expected to be involved in aphid transmission. Preliminary comparisons showed that the GPV isolate was most closely related to the other cereal-infecting members of the genus Polerovirus, CYDV-RPV and CYDV-RPS (77 and 75% nt identity, respectively, over the entire genome). Selected comparisons between different regions of the genomes of the GPV isolate, other poleroviruses and BYDV viruses in the genus Luteovirus are shown in Table 1. In most regions of the genome, GPV was most closely related to CYDV-RPV. ORF0 was the most
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