Variability and transmission by <Emphasis Type="Italic">Aphis glycines</Emphasis> of North American and Asian <Emphasis Type="Italic">Soybean mosaic virus</Emphasis> isolates

Summary. The variability of North American and Asian strains and isolates of Soybean mosaic virus was investigated. First, polymerase chain reaction (PCR) products representing the coat protein (CP)-coding regions of 38 SMVs were analyzed for restriction fragment length polymorphisms (RFLP). Second, the nucleotide and predicted amino acid sequence variability of the P1-coding region of 18 SMVs and the helper component/protease (HC/Pro) and CP-coding regions of 25 SMVs were assessed. The CP nucleotide and predicted amino acid sequences were the most similar and predicted phylogenetic relationships similar to those obtained from RFLP analysis. Neither RFLP nor sequence analyses of the CP-coding regions grouped the SMVs by geographical origin. The P1 and HC/Pro sequences were more variable and separated the North American and Asian SMV isolates into two groups similar to previously reported differences in pathogenic diversity of the two sets of SMV isolates. The P1 region was the most informative of the three regions analyzed. To assess the biological relevance of the sequence differences in the HC/Pro and CP coding regions, the transmissibility of 14 SMV isolates by Aphis glycines was tested. All field isolates of SMV were transmitted efficiently by A. glycines, but the laboratory isolates analyzed were transmitted poorly. The amino acid sequences from most, but not all, of the poorly transmitted isolates contained mutations in the aphid transmission-associated DAG and/or KLSC amino acid sequence motifs of CP and HC/Pro, respectively.Received March 20, 2002; accepted May 14, 2003     

High sequence conservation among <Emphasis Type="Italic">Odotoglossum ringspot virus</Emphasis> isolates from orchids

The variability in the nucleotide (nt) and amino acid (aa) sequences of the coat protein (CP) of Odontoglossum ringspot virus (ORSV), which naturally infects orchids worldwide, was investigated. The CP genes of 48 ORSV isolates originating from different locations in Korea were amplified using RT-PCR and sequenced. The encoded CP consists of 158 aa. The CP sequences of the Korean isolates were compared at the nt and aa levels with those of the previously published ORSV isolates originating from different countries. The Korean isolates share 94.8–100% and 92.4–100% CP identity to ORSV isolates from other countries at the nt and aa levels, respectively. No particular region of variability could be found in either sequence of the viruses. In the deduced aa sequence, the N-terminal region was more conserved than the C-terminal region in ORSV. The phylogenetic tree analysis and recombination analysis revealed that there was no distinct grouping between geographic locations and sequence identity, and nor distinct intra-specific recombination events among ORSV isolates.     

Coat protein gene diversity among <Emphasis Type="Italic">Chrysanthemum virus B</Emphasis> isolates from India

Summary. The complete coat protein (CP) sequences from 29 Indian isolates of Chrysanthemum virus B (CVB) were determined and analysed in relation to other previously characterized carlaviruses. The CP genes of the Indian CVB isolates were highly heterogeneous, sharing nucleotide sequence identities of 74–98%. Based on phylogenetic analyses, the isolates formed three groups potentially representing either two or three major CVB strain groupings. Recombination analysis revealed at least one definite recombination event involving the exchange of sequences between members of different groups. To our knowledge this is the first reported evidence of homologous recombination in carlaviruses.     

Comparison of the complete sequences of three different isolates of <Emphasis Type="Italic">Pepino mosaic virus</Emphasis>: Size variability of the TGBp3 protein between tomato and <Emphasis Type="Italic">L. peruvianum</Emphasis> isolates

Summary. The complete nucleotide sequence of the genomes of two Spanish isolates (LE-2000 and LE-2002) from tomato and one Peruvian isolate (LP-2001) from Lycopersicon peruvianum of the Pepino mosaic virus (PepMV) were determined. The tomato isolates share identities higher than 99%, while the genome of LP-2001 had mean nucleotide identities of 95.6% to 96.0% with tomato isolates. The predicted amino acid sequences showed similarities ranging between 95.2% and 100% with TGBp3 and TGBp2 and CP proteins, respectively. In LP-2001 two main differences were found with respect to the tomato isolates; (i) the 5 untranslated region (UTR) was 2nt shorter by deletion at position 12–13 and it had some polymorphims at the putative promoter sequence reported for PepMV tomato isolates and other potexviruses, which could be functionally significant for RNA replication, and (ii) the TGBp3 protein had two extra amino acids in the C-terminal region.     

Molecular criteria for genus and species discrimination within the family <Emphasis Type="Italic">Potyviridae</Emphasis>

Summary. A phylogenetic analysis of the optimised nucleotide (nt) alignment of the entire ORFs of a representative of each fully-sequenced species in the family Potyviridae provided strong support for several subgroups within the genus Potyvirus. A complete set of two-way comparisons was done between the sequences for the entire ORF and for each gene amongst all the 187 complete sequences from the family. Most species had 50–55% nt identity to other members of their genus in their ORFs but there were significant groups of more closely related species and species demarcation criteria were <76% nt identity and <82% amino acid identity. The corresponding thresholds for species demaracation using nt identity values for the individual genes ranged from 58% (P1 gene) to 74–78% (other genes) although a few comparisons between different species exceeded these values. For the entire ORF, genus demarcation criteria were <46% nt identity but this did not separate rymoviruses from potyviruses. Comparisons in the CI gene most accurately reflected those for the complete ORF and this region would therefore be the best for diagnostic and taxonomic studies if only a sub-portion of the genome is to be sequenced. Further comparisons were then made using all the 1220 complete capsid protein (CP) genes. These studies suggest that 76–77% nt identity is the optimal species demarcation criterion for the CP. The study has also helped to allocate the correct virus name to some sequences from the international databases that currently have incorrect or redundant names. The taxonomic status of the current genus Rymovirus and of three unassigned species in the family is discussed. Significant discontinuities in the distributions within and between the currently defined species suggest that the continuum of variation that is theoretically available is constrained or disrupted by molecular barriers that must have some biological significance.     

Molecular characterization and evolutionary analysis of <Emphasis Type="Italic">soybean mosaic virus</Emphasis> infecting <Emphasis Type="Italic">Pinellia ternata</Emphasis> in China

Twenty-nine Pinellia ternata specimens were collected from representative areas in China, including the major production provinces of Zhejiang, Henan, Shanxi, Hunan, Shandong and Hubei. Seven isolates related to soybean mosaic virus (SMV), which could be pathogenic on P. ternata and some soybean [Glycine max (L.) Merr.] cultivars, were detected using double antibody sandwich immunosorbent assay (DAS-ELISA) and RT-PCR amplification performed with degenerate primer of potyviruses. It is revealed that the common potyvirus infecting P. ternata is, indeed, only SMVs rather than Dasheen mosaic virus (DsMV) as previously reported. Further molecular phylogenetic analysis of the coat protein (CP) genes of these SMV isolates from P. ternata and G. max, along with some other potyvirus members, such as DsMV and Watermelon mosaic virus (WMV) reconstructed the evolutionary route on both nucleotide and amino acid levels. Similarity and homology of nucleotide sequences for SMV CP genes demonstrated high host correlation and low partial habitat correlation, while those of amino acid sequences also showed that the host correlation was more notable than the habitat correlation. The amino acid sequence of conserved region within CP determines the main function, which shows high homology between species. This study outspreaded from the viruses themselves and their relationship to the infected hosts and revealed the evolutionary strategies, especially the rapid variation or recombination of SMV of P. ternata, in order to adapt itself naturally to the special host. The GenBank Accession numbers of the sequences reported in this article are DQ360817-DQ360823.     

Low genetic diversity among <Emphasis Type="Italic">Cucumber vein yellowing virus</Emphasis> isolates from Spain

The population structure and genetic diversity of Cucumber vein yellowing virus (CVYV) from Spain were estimated by analyses of partial nucleotide sequences of the P1-proteinase (P1-Pro), P3 protein (P3), and the coat protein (CP) coding regions. Analysis of 56 CVYV Spanish field isolates collected from 2001 to 2005 showed low genetic diversity (0.0026, 0.0013, and 0.0012 for the P1-Pro, P3, and CP regions, respectively). The ratio between nonsynonymous and synonymous substitutions was among the lowest found in a plant virus, indicating a strong negative selective pressure in the regions analyzed. Nonsynonymous nucleotide substitutions were only found within the P1-Pro regions, although these do not appear to have been selected with time. The results support the hypothesis that the Spanish CVYV population could derive from a single origin of recent introduction.     

Complete genomic RNA sequence of the Polish <Emphasis Type="Italic">Pepino mosaic virus</Emphasis> isolate belonging to the US2 strain

We have determined the complete nucleotide and amino acid sequences of the Polish Pepino mosaic virus (PepMV) isolate marked as PepMV-PK. The PepMV-PK genome consists of a single positive-sense RNA strand of 6412-nucleotide-long that contains five open reading frames (ORFs). ORF1 encodes the putative viral polymerase (RdRp), ORFs 2–4 the triple gene block (TGB 1–3), and ORF5-coat protein CP. Two short untranslated regions flank the coding ones and there is a poly (A) tail at the 3′ end of the genomic RNA. Thus, the genome organization of PepMV-PK is that of a typical member of the genus Potexvirus. Phylogenetic analysis based on full-length genomes of PepMV sequences showed that PepMV-PK was most closely related to the Ch2 isolate from Chile. Comparison of PepMV-PK and Ch2 showed the following nucleotide identities: 98% for the RdRp, 99% for the CP genes, and 98, 99, and 98% for the TGB1, TGB2, and TBG3, respectively. This high level of nucleotide sequence identity between the Chilean and Polish PepMV-PK isolates suggest their common origin.     

Molecular characterization of two Chinese isolates of <Emphasis Type="Italic">Beet mosaic virus</Emphasis>

The complete genomic sequences of Beet mosaic virus Xinjiang (BtMV-XJ) and Inner Mongolia (BtMV-IM) isolates from China were determined and compared with US and German isolates, reported previously. Results showed that viral genome of the two isolates both comprise 9,591 nucleotides, and contain the large single open reading frame (ORF) encoding a single polyprotein of 3,085 amino acid residues, from which ten putative functional proteins may be produced by autolytic cleavage processing as the US (BtMV-Wa) and German (BtMV-G) isolates. Sequence comparisons showed that BtMV-XJ shared 89.8% and 98.3% overall nucleotide identity with BtMV-Wa and BtMV-G isolates, and BtMV-IM exhibited the overall identities of 91.6% and 93.8% with BtMV-Wa and BtMV-G, respectively. Further, analyses revealed that BtMV-XJ shared higher identities in almost every region to BtMV-G than to BtMV-Wa both at the nucleotide and the amino acid levels. While BtMV-IM in the regions (6,666–7,671 and 7,672–9,591) showed highest homology with BtMV-XJ and BtMV-G, especially, after nt 7,672 with similarity up to 99.2% with BtMV-G; the region (2,331–4,083) showed highest identity (98.0% nt identity) with BtMV-Wa. That suggested BtMV-XJ had a more close relationship to BtMV-G, while BtMV-IM was more likely to be a natural recombination virus. In addition, phylogenetic analysis of the available BtMV CP sequences showed that BtMV isolates fell into two distinct groups: Euroasia group (Europe and China) and America group (USA). To the best of our knowledge, this study reported the complete sequences of two BtMV isolates from Asia for the first time. H. Xiang and Y.-H. Han contributed equally to this paper.     

Phylogenetic analysis of <Emphasis Type="Italic">Beet necrotic yellow vein virus</Emphasis> isolates from China

A survey detected Beet necrotic yellow vein virus (BNYVV) infection in six Chinese sugar-beet-growing regions. To study the diversity of virus isolates among the regions, nucleotide sequences of four proteins, namely CP, p25, p31, and p26, were determined and the amino acid sequences thus deduced were analyzed using sequence alignments and the phylogenetic method, respectively. Amino acid sequence analysis of CP revealed A-type isolates in Harbin, Hohhot, Baotou, Wuwei, and Jiuquan and B-type isolates in Hohhot and Changji; no Chinese isolate was found to cluster with European P-type isolates. Chinese p25 proteins clustered into three groups with seven tetrad motifs (positions 67–70). Of the seven, the tetrad ASHG has not been reported previously. Most Chinese p31 proteins clustered in p31-2 group, diverging from European p31 proteins. Isolates containing RNA 5 occurred in most of the investigated regions and were associated with both A-and B-types. Phylogenetic analyses of these four proteins showed complex patterns of genetic diversity among these Chinese isolates and indicated that the isolates of China and Japan were more closely related and may have a common origin. The nucleotide sequence data reported in this article have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers EF473089–EF473126.     

Strawberry necrotic shock virus is a distinct virus and not a strain of <Emphasis Type="Italic">Tobacco streak virus</Emphasis>

Summary. Fragaria (strawberry) and Rubus species (blackberry, wild blackberry, red raspberry and black raspberry) were thought to be infected with distinct isolates of Tobacco streak virus (TSV). Employing serology and nucleic acid hybridization it has been shown that these isolates form a cluster distinct from other strains of TSV. In this study we have cloned and sequenced the complete RNA 3 of an isolate of TSV from strawberry (Fragaria) as well as the coat protein (CP) gene of 14 additional isolates of TSV originating from Fragaria and Rubus species. Our data suggest that the isolates of TSV that infect Fragaria and Rubus belong to a distinct virus for which we propose the name Strawberry necrotic shock virus (SNSV). The RNA 3 of SNSV contains 2248 nucleotides, 43 more than the type isolate of TSV from white clover (TSV-WC), with a CP gene that is 669 nucleotides long, in contrast to the 714–7 nucleotides of the TSV CP sequences found in the database. The movement protein gene of SNSV is 897 nucleotides in length, 27 more than that of the TSV-WC isolate of TSV. The CP genes of the 15 Fragaria and Rubus isolates that we studied form two distinct phylogenetic clusters that share about 95% amino acid sequence identity, while they only share 60–65% amino acid sequence identity with TSV-WC.     

Identification of a Naturally Occurring Recombinant Isolate of <Emphasis Type="Italic">Sugarcane Mosaic Virus</Emphasis> Causing Maize Dwarf Mosaic Disease

The complete nucleotide sequence of a potyvirus causing severe maize dwarf mosaic disease in Shaanxi province, northwestern China was determined (GenBank accession No. AY569692). The full genome is 9596 nucleotides in length excluding the 3 -terminal poly (A) sequence. It contains a large open reading frame (ORF) flanked by a 149 nt 5-untranslated region (UTR) and a 255 nt 3-UTR. The putative polyprotein encoded by this large ORF comprises of 3063 amino acid residues. Sequence comparisons and phylogenetic analyses showed that this potyvirus is an isolate of Sugarcane mosaic virus (SCMV). The entire sequences shared identities of 89.6–97.6 % and 79.3–93.3% with 9 sequenced SCMV isolates at the nucleotide and deduced amino acid levels, respectively. But it showed much lower identities with Maize dwarf mosaic virus (MDMV), Sorghum mosaic virus (SrMV) and Johnsongrass mosaic virus (JGMV) isolates. The putative coat protein sequence is identical to that of a Chinese maize isolate SCMV-HZ. However, partition comparisons and phylogenetic profile analyses of the viral nucleotide sequences indicated that it is a recombinant isolate of SCMV. The recombination sites are located within the 6K1 and CI coding regions.     

A virus related to <Emphasis Type="Italic">Soybean mosaic virus</Emphasis> from <Emphasis Type="Italic">Pinellia ternata</Emphasis> in China and its comparison with local soybean SMV isolates

Summary. A potyvirus isolated from Pinellia ternata in China was characterised and shown to be related to Soybean mosaic virus (SMV). The virus was pathogenic on P. ternata and some soybean cultivars, whereas the local soybean SMV isolate HH5 did not infect P. ternata. Western blot experiments demonstrated a serological relationship between the virus from Pinellia, SMV and Watermelon mosaic virus (WMV). The complete nucleotide sequences of the Pinellia virus (isolate P-1, 9735nt) and of the Chinese soybean SMV isolates HH5 (9585nt) and HZ (9588nt) were determined. A 1733nt sequence at the 3-terminus of a second isolate from Pinellia (isolate P-2) was also determined. The predicted polyprotein of isolate P-1 has 83% amino acid (aa) identity with those of published SMV sequences. In many parts of the genome, aa identity was about 90% but it was much lower in the P1 protein region (24–29%), where it more closely resembled Dasheen mosaic virus (62%). The partial sequence of isolate P-2 had 91%nt identity to P-1 and both isolates resembled a recent sequence in the public databases (AF469171) wrongly named Zantedeschia mosaic virus. The two complete SMV soybean sequences had 93–95%nt identity with those of the previously sequenced isolates and >97% amino acid identity. Phylogenetic analysis and comparisons of coat proteins suggest that the Pinellia, WMV and SMV potyviruses should probably be treated as strains of the same species.     

The complete sequence of <Emphasis Type="Italic">Cymbidium mosaic virus</Emphasis> from <Emphasis Type="Italic">Vanilla fragrans</Emphasis> in Hainan,China

The complete nucleotide sequence of Cymbidium mosaic virus (CymMV) isolated from vanilla in Hainan province, China was determined for the first time. It comprised 6,224 nucleotides; sequence analysis suggested that the isolate we obtained was a member of the genus Potexvirus, and its sequence shared 86.67–96.61% identities with previously reported sequences. Phylogenetic analysis suggested that CymMV from vanilla fragrans was clustered into subgroup A and the isolates in this subgroup displayed little regional difference.     

Complete nucleotide sequence and experimental host range of <Emphasis Type="Italic">Okra mosaic virus</Emphasis>

Okra mosaic virus (OkMV) is a tymovirus infecting members of the family Malvaceae. Early infections in okra (Abelmoschus esculentus) lead to yield losses of 12–19.5%. Besides intensive biological characterizations of OkMV only minor molecular data were available. Therefore, we determined the complete nucleotide sequence of a Nigerian isolate of OkMV. The complete genomic RNA (gRNA) comprises 6,223 nt and its genome organization showed three major ORFs coding for a putative movement protein (MP) of M_r 73.1 kDa, a large replication-associated protein (RP) of M_r 202.4 kDa and a coat protein (CP) of M_r 19.6 kDa. Prediction of secondary RNA structures showed three hairpin structures with internal loops in the 5′-untranslated region (UTR) and a 3′-terminal tRNA-like structure (TLS) which comprises the anticodon for valine, typical for a member of the genus Tymovirus. Phylogenetic comparisons based on the RP, MP and CP amino acid sequences showed the close relationship of OkMV not only to other completely sequenced tymoviruses like Kennedya yellow mosaic virus (KYMV), Turnip yellow mosaic virus (TYMV) and Erysimum latent virus (ErLV), but also to Calopogonium yellow vein virus (CalYVV), Clitoria yellow vein virus (CYVV) and Desmodium yellow mottle virus (DYMoV). This is the first report of a complete OkMV genome sequence from one of the various OkMV isolates originating from West Africa described so far. Additionally, the experimental host range of OkMV including several Nicotiana species was determined. The nucleotide sequence data reported in this article have been submitted to the Genbank nucleotide sequence database and have been assigned the accession number EF554577.     

Analysis of Iranian <Emphasis Type="Italic">Potato virus S</Emphasis> isolates

Two hundred forty potato samples with one or more symptoms of leaf mosaic, distortion, mottling and yellowing were collected between 2005 and 2008 from seven Iranian provinces. Forty-four of these samples tested positive with double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) using a Potato virus S (PVS) polyclonal antibody. Of these 12 isolates of PVS were selected based on the geographical location for biological and molecular characterization. The full coat protein (CP) and 11K genes from 12 PVS isolates were PCR amplified, cloned and sequenced. All 12 PVS isolates showed mosaic symptoms on Nicotiana debneyii and N. tabacum cv. Whiteburly and local lesion on Chenopodium amaranticolor, C. quinoa and C. album. The Iranian isolates share between 93 and 100% pairwise nucleotide identity with other PVS^O isolates. Based on maximum likelihood phylogenetic analysis coupled with pairwise identity analysis, we propose 15 genotypes for the PVS^O strain and 3 genotypes for the PVS^A strain.     

Two unique US isolates of <Emphasis Type="Italic">Pepino mosaic virus</Emphasis> from a limited source of pooled tomato tissue are distinct from a third (European-like) US isolate

Summary. Three strains of Pepino mosaic virus (PepMV) found in the US have been cloned and sequenced by RT-PCR using total RNA from infected tissue as template, and degenerate potexvirus- and PepMV species- and isolate-specific primers. Despite limited source material, the complete nucleotide sequences (6413 and 6410nts, respectively) of two isolates, PepMV-US1 and PepMV-US2, were obtained and analyzed using total RNA from less than 0.2g of a pooled infected tomato leaf sample from Arizona. Sequence of the 3-end of the third isolate from infected fresh tomato fruits from Maryland (PepMV-US3) was also determined. The genome organizations of PepMV-US1 and US2 were typical of the genus Potexvirus, with the following reading frame order: ORF 1, encoding a putative replicase; ORFs 2–4, triple gene block proteins (TGBp) 1–3; and ORF 5, coat protein (CP). Gene-for-gene comparison between PepMV-US1 and US2 revealed the following amino acid identities: 91% in replicase, 89% in TGBp1, 92% in TGBp2, 85% in TGBp3, and 93% in the CP; with an overall nucleotide identity of 86%. Nucleotide sequence comparisons between US1 and US2 and the European isolates showed only 79–82% identity, whereas the identity among the European isolates was over 99%. Sequence comparisons and phylogenetic analysis indicate that PepMV-US1 and US2 are distinctly different from the European isolates, while the CP of PepMV-US3 is nearly identical to the European isolates. The results presented also suggest that TGBp1 and TGBp3 are more suitable than either the replicase or coat protein gene products for discriminating PepMV isolates.     

The complete sequence of a Spanish isolate of <Emphasis Type="Italic">Broad bean wilt virus</Emphasis> 1 (BBWV-1) reveals a high variability and conserved motifs in the genus <Emphasis Type="Italic">Fabavirus</Emphasis>

Summary. The genome of a Spanish isolate of Broad bean wilt virus-1 (BBWV-1) was completely sequenced and compared with available sequences of other isolates of the genus Fabavirus (BBWV-1 and BBWV-2). This consisted of two RNAs of 5814 and 3431 nucleotides, respectively, and their organization was similar to that of other members of the family Comoviridae. Its mean nucleotide identity with a BBWV-1 American isolate was 81.5%, and between 59.8 and 63.5% with seven BBWV-2 isolates. Our analysis showed sequence stretches in the 5′ non-coding regions which are conserved in both genomic RNAs and in BBWV-1 and BBWV-2 isolates.     

Polymorphism of a specific region in gene <Emphasis Type="Italic">p23</Emphasis> of <Emphasis Type="Italic">Citrus tristeza virus</Emphasis> allows discrimination between mild and severe isolates

Summary. The pathogenicity determinants of Citrus tristeza virus (CTV) are presently unknown, although transgenic Mexican limes over-expressing CTV p23, an RNA-binding protein involved in regulating the asymmetrical accumulation of viral RNA strands, display typical CTV symptoms. Here we compared the predominant sequence variants of gene p23 from 18 CTV isolates of different geographic origin and pathogenicity characteristics. Phylogenetic analysis of these sequences revealed three groups of isolates: i) mild, inducing only symptoms in lime and/or decline of citrus species grafted on sour orange rootstock, ii) severe, causing additionally stem pitting on sweet orange and/or grapefruit, and iii) an atypical group of isolates inciting variable symptoms. The sequences of the isolates located at the periphery of each group were recombinants. Pairwise comparisons of the predicted amino acid sequences showed that residues at positions 78–80 were characteristic of each group of isolates. Group-specific primers based on these differences allowed RT-PCR detection of each sequence type in dsRNA-rich preparations from infected tissues. While mild isolates contained only the sequence characteristic of this group, most severe isolates contained the sequences characteristic of their group, and additionally, sequences characteristic of the mild and/or the atypical groups, suggesting that the severe phenotype is associated with the presence of the severe and/or the atypical sequence types. This association can be exploited for quick detection of potentially damaging sequence variants and for monitoring cross protection.