Molecular cloning and nucleotide sequence of the coat protein gene of a Cuban isolate of potato leafroll virus and its expression inEscherichia coli

Total RNA from infectedPhysalis floridana was isolated to generate complementary DNA corresponding to the coat protein (CP) gene of a Cuban isolate of potato leaf roll virus (PLRV). This cDNA was amplified by the polymerase chain reaction (PCR) and cloned into the bacterial expression vectors pEX(1–3) for fusion protein expression inE. coli. The product was detected by antibodies specific for the PLRV CP. The coding sequence of the CP gene was determined, and the predicted length of the CP was 208 amino acids (23 kD). The nucleotide sequences and deduced amino acid sequences were compared with the other PLRV isolates and found to be 97–99.5% identical at both the nucleotide and amino acid sequence level of other isolates. Comparison of the deduced amino acid sequences of the PLRV_cub CP revealed considerable homology to other luteoviruses. We believe that the protocol described could be applicable to other plant viruses of low abundance or of cumbersome isolation, since this method is less time consuming than the traditional methods of cloning coat protein genes of plant viruses with known sequences.     

Molecular analysis of coat protein coding region of tobacco stunt virus shows that it is a strain of <Emphasis Type="Italic">Lettuce big-vein virus</Emphasis> in the genus <Emphasis Type="Italic">Varicosavirus</Emphasis>

Summary. To evaluate the relationship between tobacco stunt virus (TStV) and Lettuce big-vein virus (LBVV), we determined nucleotide sequences of the coat protein (CP) coding region of five TStV and three LBVV isolates and compared them with those of one Japanese and four Spanish isolates of LBVV. CP coding regions were identical in size and the nucleotide and amino acid sequence identities between TStV and LBVV were 95.6–96.5% and 97.2–98.7%, respectively. Phylogenetic analysis of nucleotide sequences indicated that TStV was very closely related to LBVV and a strain of LBVV rather than a distinct species.     

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.     

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.     

Genetic variability in the coat protein genes of Cymbidium mosaic virus isolates from orchids

The variability in the nucleotide (nt) and amino acid (aa) sequences of the coat protein (CP) of Cymbidium mosaic virus (CymMV), which naturally infects orchids worldwide, was investigated. The CP genes of 55 CymMV isolates originating from different locations in Korea were amplified using RT-PCR and sequenced. The encoded CP consists of 223 aa. The CP sequences of the Korean isolates were compared with those of previously published CymMV isolates originating from different countries at both nt and aa levels. The Korean isolates shared 74.9–98.3 and 52.7–100% CP homology with CymMV isolates from other countries at the nt and aa levels, respectively. No particular region of variability could be found in either grouping of viruses. In the deduced CymMV CP aa sequence, the C-terminal region was more divergent than the N-terminal. The phylogenetic tree analysis based on nt sequence diversity of CP genes of CymMV isolates supported the hypothesis that CymMV isolates were divided into two subgroups. However, these subgroups were not formed by phylogenetic tree analysis of CP aa sequences. There was no distinct correlation between geographical locations and specific sequence identity, while recombination analysis revealed that there were no intra-specific recombination events among CymMV isolates.     

Biological and molecular variability among geographically diverse isolates of sweet potato virus 2

Summary. Sweet potato virus 2 (SPV2) is a tentative member of the genus Potyvirus, family Potyviridae. In addition to the type isolate of SPV2 recently characterised in greater detail, twelve additional isolates of this virus were obtained from sweet potato clones originating from China, Portugal, South Africa and Zambia. Sequences of the coat protein (CP) gene and 3′ non-translated region (NTR) were determined. Comparisons of the CP gene sequences of these isolates revealed nucleotide and amino acid sequence identities ranging from 81 to 99% and from 86 to 99%, respectively. Phylogenetic analysis of sequences distinguished several groups, which partially correlated with the geographic origin of the isolates, and indicated that some isolates from South Africa and a Zambian isolate are most distinct both in CP and 3′NTR sequences. Host range studies of a selected number of isolates revealed some differences in test plant reactions, which appeared to correlate to some extent with the geographic origin and molecular distinctness of the SPV2 isolates. The results strongly suggest the occurrence of biologically and genetically diverse strains of SPV2.     

Zucchini tigré mosaic virus is a distinct potyvirus in the papaya ringspot virus cluster: molecular and biological insights

In recent years, three new potyviruses have been described in the papaya ringspot virus (PRSV) cluster. In addition, two types of PRSV are recognized, type W, infecting cucurbit plants, and type P, infecting papaya and also cucurbits. A third type, PRSV-T, was also partially described in Guadeloupe. Complete genome sequencing of four PRSV-T isolates showed that this virus is a related virus that is distinct from PRSV, and the name zucchini tigré mosaic virus (ZTMV) is proposed, in reference to the typical symptoms observed in zucchini squash. Eleven other viral isolates from different geographic origins were confirmed as ZTMV isolates using the complete sequence of the cylindrical inclusion (CI) coding region, whereas pairwise sequence similarities in the coat protein (CP) coding region did not unambiguously distinguish ZTMV isolates from PRSV isolates. The use of the CI coding region for species demarcation appears more suitable than the CP coding region for closely related viruses. Principal coordinates analysis based on the biological behavior of the viral isolates studied clustered PRSV-P, PRSV-W and ZTMV isolates into three different groups. Therefore, ZTMV is different from PRSV in its molecular and biological properties.     

Variation in coat protein genes among five geographicallydifferent isolates of rice tungro spherical virus

Summary.  The variation in the sequence of the coat protein genes of four isolates of rice tungro spherical virus from different countries, Malaysia, Thailand, India and Bangladesh, was compared with an isolate from the Philippines. The evidence from RT-PCR, Southern blot hybridization and sequences of the coat protein genes indicated that the isolates appeared to fall into two groups. One comprised the Philippine and Malaysian isolates (about 95% sequence similarity) and the other the Bangladeshi and Indian isolates, the sequences of which differed by about 15% from that of the Philippine isolate. The Thai isolate seemed to be a mixture of these two subgroups. Received March 4, 1997 Accepted April 23, 1997     

Association of a satellite DNA β molecule with mesta yellow vein mosaic disease

The yellow vein mosaic disease infected mesta samples exhibited positive amplification with different primers specific for coat protein (CP) gene of DNA-A molecule of begomoviruses and full-length DNA β molecule. The amplified product of a full-length DNA β and the CP gene of two different isolates were cloned and sequenced. The DNA β molecule was 1,354 nt in length having highest sequence identity (86.1%) with two reported DNA β molecules of Indian isolates of begomovirus infecting cotton (accession number DQ191161 and AJ316038). Highest sequence identity (85.5%) of βC1 gene product was found with that encoded by DNA β associated with begomovirus infecting tomato (AJ316035), originating from Pakistan. The predicted βC1 protein consisted of 118 amino acids. The nucleotide sequences of the CP genes from both was 771 nt in length and showed sequence identity with CP genes of begomoviruses infecting tomato (82.2–92.4%), tobacco (AY007616, 94.2%) and Croton (AJ507777, 93.9%). The highest percentage sequence identity (97.6%) of the CP gene product was found with that encoded by DNA-A of two isolates of begomovirus infecting tomato (AJ810364 and AJ810357). The predicted CP consisted of 256 amino acids. The results indicate for the first time that the begomovirus associated with mesta yellow vein mosaic disease contains DNA β molecule along with DNA-A in its genome. The phylogenetic tree also indicated that the DNA β molecule reported here is distinct from other known geminiviruses or nanovirus components.     

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.     

Sequence Divergence of Four Soilborne Sugarbeet-Infecting Viruses

Soilborne viruses are among the most harmful pathogens of sugarbeet (Beta vulgaris L.ssp. vulgaris) but most of them lack information on genetic variability due to paucity of sequence data. Only one isolate of Beet soil borne virus (BSBV; genus Pomovirus), Beet virus Q (BVQ; genus Pomovirus) and Beet soil borne mosaic virus (BSBMV; genus Benyvirus) has been characterised for the coat protein (CP) gene. In this study, the CP gene sequences of three isolates each of BSBV and Beet necrotic yellow vein virus (BNYVV; genus Benyvirus) (France, Germany and USA), two isolates of BVQ (France and Germany), and one isolate of BSBMV (USA) were determined. Phylogenetic analyses including sequences from databanks indicated that the French BNYVV isolate of this study belongs to so-called P-type, the American isolate to A-type and the German isolate to B-type. The CP genes of the three BSBV isolates characterised in this study and the one available from databank were highly identical (98.4–99.0% at nucleotide level; one variable amino acid). The BSBMV isolate studied here differed from the previously characterised isolate for five nucleotides and four amino acids in the CP region. The two BVQ isolates characterised in this study contained three additional nucleotides resulting in an additional amino acid residue (arginine) at CP position 86, as compared to the only isolate available in databank.     

Genetic variability and phylogenetic analysis of hosta virus X

Triple gene block 1 (TGB1) and coat protein (CP) sequences of 30 hosta virus X (HVX) isolates from Tennessee (TN), USA, were determined and compared with available sequences in GenBank. The CPs of all known HVX isolates, including those from TN, shared 98.3–100% and 98.2–100% nucleotide and amino acid sequence identity, respectively, whereas TGB1 shared 97.4–100% nucleotide and 97–100% amino acid sequence identity. TGB1 of TN isolates were all longer by one codon from that of a Korean isolate, which is the only sequence publicly available. Phylogenetic analysis of nucleotide and amino acid sequences of TGB1 and CP of all known HVX isolates, separately or combined, revealed a close relationship, suggesting that all of them are derived from a common ancestor. Phylogenetic analysis with the type member of each genus of the family Flexiviridae confirmed that HVX is a member of a distinct species of the genus Potexvirus.     

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.     

Evidence that <Emphasis Type="Italic">Cymbidium mosaic virus</Emphasis> (CymMV) isolates divide into two subgroups based on nucleotide diversity of coat protein and replicase genes

Summary The genetic diversity of Cymbidium mosaic virus (CymMV, family Flexiviridae) was assessed by analysing the nucleotide sequences of coat protein (CP) and partial RNA-dependent RNA polymerase (RdRp) genes. Thirty CymMV sequences from vanilla isolates, obtained in this work by direct sequencing of RT-PCR products, were compared to the sequences from ornamental orchid isolates available in GenBank. The CymMV population exhibited overall low genetic diversity (π = 0.054 and π = 0.053 for CP and RdRp genes, respectively). Phylogenetic analyses of the 85 CP and 37 RdRp sequences revealed the segregation of the isolates into two congruent monophyletic clusters; however these two subgroups did not cluster in amino sequence analysis because most of the nt mutations were synonymous. Nevertheless, the subgrouping was confirmed by highly significant Kst tests for the CP and RdRp genes. Analysis of population genetic parameters and distribution of synonymous and nonsynonymous mutations revealed that both genes were under negative selection with no recombination events. These results suggested that the CymMV isolates found in cultivated orchids worldwide have a dual origin and are expanding as if following bottlenecks.     

Identification of new isolates of <Emphasis Type="Italic">Turnip mosaic virus</Emphasis> that cluster with less common viral strains

Summary Turnip mosaic virus (TuMV) was found infecting cultivated brassicas and wild and cultivated ornamental Brassicaceae plants in different regions of Spain. Five new TuMV isolates, originating from different host plant species (Brassica cretica, Brassica juncea, Brassica napus, Eruca vesicaria subsp. sativa and Sisymbrium orientale), have been identified. The nucleotide sequences of the coat protein (CP) genes of the five isolates were determined. Phylogenetic analysis of the CP sequences showed that the five isolates grouped into two different clusters. The three isolates from the central region of Spain clustered with a previously reported Pisum sativum isolate from southeastern Spain, whereas the other two isolates from the eastern region clustered with two Italian and two Greek isolates. Both clusters were genetically distinct and belonged to the multi-lineage group OBR. The OBR group contains mainly TuMV isolates from hosts other than Brassica spp. and Raphanus sativus and mostly originating from Mediterranean countries. These new sequences provide further phylogenetic resolution of the OBR group. Although new TuMV isolates have been found in Spain, they were not associated with any serious disease outbreaks.     

Complete nucleotide sequences of attenuated and severe isolates of <Emphasis Type="Italic">Leek yellow stripe virus</Emphasis> from garlic in northern Japan: Identification of three distinct virus types in garlic and leek world-wide

Summary. We have determined the complete nucleotide sequence of three isolates (two severe and one attenuated) of the potyvirus Leek yellow stripe virus (LYSV), the main causal agent of the garlic mosaic disease that is prevalent in Aomori prefecture, northern Japan. They contained 10,296–10,297 nucleotides and encoded a deduced polyprotein of 3,215 amino acids. Sequence variation among the three isolates was 1.5% at both the nucleotide and amino acid levels. Surprisingly, the sequences of all three isolates were quite different from that of recently described garlic isolate of LYSV from China. Sequence similarities among the 5-UTR and P1 regions were only 56–57% and 52–53%, respectively, raising the possibility that these isolates may actually be distinct virus species. Phylogenetic analyses using 5-UTR, P1 protein and coat protein revealed that all LYSV isolates described to date can be divided into three groups; i.e., those from leek, those from garlic in northern Japan, and those from garlic in southern Japan, China, and other parts of the world. The garlic isolates from northern Japan were more closely related to LYSV isolates from leek than to garlic isolates from southern Japan and China, suggesting a different origin for the garlic cultivars now cultivated in northern Japan.     

Genetic variability in the coat protein genes of lettuce big-vein associated virus and Mirafiori lettuce big-vein virus

Summary. Available data suggests that lettuce big-vein disease is caused by the ophiovirus Mirafiori lettuce big-vein virus (MLBVV) but not by the varicosavirus Lettuce big-vein-associated virus (LBVaV), although the latter is frequently associated with the disease. Since the disease occurs worldwide, the putative coat protein (CP) open reading frames of geographically distinct isolates of MLBVV and LBVaV were sequenced. Comparison of both nucleotide and amino acid sequences showed a high level of sequence similarity among LBVaV isolates. Phylogenetic analysis of LBVaV CP nucleotide sequences showed that most of the Spanish isolates clustered in a phylogenetic group whereas English isolates were more similar to the USA isolate. An Australian isolate was closely related to the Dutch isolate. Genetic diversity among MLBVV CP nucleotide sequences was higher ranging from 0.2% to 12%. Phylogenetic analysis of MLBVV CP nucleotide sequences revealed two distinct subgroups. However, this grouping was not correlated with symptom development on lettuce or the geographic origin of the MLBVV isolates. Finally, a quick method based on RFLP analysis of RT-PCR amplicons was developed for assigning MLBVV isolates to the two subgroups.     

Serological and coat protein sequence studies suggest that necrosis disease on sunflower in India is caused by a strain of Tobacco streak ilarvirus

Summary.  Serological and coat protein sequence studies were conducted to identify an ilarvirus associated with necrosis disease on sunflower in India. In electroblot immunoassay, sunflower ilarvirus reacted strongly only with antiserum to Tobacco streak virus (TSV). The coat protein gene of sunflower ilarvirus was cloned and sequenced. The sequence analyses also showed that the CP gene was most closely related to TSV, the member of subgroup I of Ilarvirus. The sunflower ilarvirus CP shared 90% amino acid sequence identity with TSV. On the basis of serological relatedness and sequence identity, it is proposed that the sunflower ilarvirus from India should be considered a strain of TSV belonging to subgroup I and designated as TSV-SF. This is the first report of the molecular characterization of TSV on sunflower from the Indian subcontinent. Received August 9, 2001 Accepted October 11, 2001