Phylogenetic analysis of the Potyviridae with emphasis on legume-infecting potyviruses

Summary.  The 3′-terminal nucleotide sequences of thirteen authenticated strains of bean common mosaic virus (BCMV) and one strain of bean common mosaic necrosis virus (BCMNV) were obtained. The regions sequenced included the coat protein coding sequence and 3′-end non-coding region. These data, combined with sequence information from other legume-infecting potyviruses and the Potyviridae were used for phylogenetic analysis. Evidence is provided for delineation of BCMNV as distinct from BCMV and the inclusion of azuki mosaic, dendrobium mosaic, blackeye cowpea mosaic, and peanut stripe viruses as strains of BCMV. This relationship defines the members of the BCMV and BCMNV subgroups. These data also provide a basis upon which to define virus strains, in combination with biological data. Other aspects and implications of legume-infecting potyvirus phylogenetics are discussed. Received December 24, 1996 Accepted June 3, 1997     

Sequence of the 3′-terminal region of a Zimbabwe isolate of cowpea aphid-borne mosaic virus (CABMV)

Summary The 3-terminal 1221 nucleotides of a Zimbabwe isolate of cowpea aphid-borne mosaic potyvirus (CABMV) genome have been sequenced. The sequence comprises an open reading frame (ORF) of 990 nucleotides and a 3 non-coding-region of 231 nucleotides followed by a poly-A. The ORF has high similarity to NIb and coat proteins (CP) of potyviruses. A potential CP Q/S cleavage site was identified, yielding a CP of 30.5 kDa containing 275 amino acids. The CABMV sequence is closely related to that of South African passiflora virus (SAPV) which should therefore be regarded as a strain of CABMV.The sequence described in this paper has been deposited in the EMBL database under accession number X82873.     

The tryptic peptides and terminal sequences of the protein from the cowpea strain of tobacco mosaic virus

The use of ion exchange chromatography, gel filtration, and paper electrophoresis for the separation of the peptides obtained by tryptic digestion of the cowpea strain of tobacco mosaic virus protein is described. The amino acid compositions of the 13 tryptic peptides obtained were determined and accounted for 159 residues compared with the 158 found for type tobacco mosaic virus protein. The only tryptic peptide that cowpea tobacco mosaic virus protein and type tobacco mosaic virus protein have in common is asparaginyl-arginine despite the fact that the former is very similar to type tobacco mosaic virus protein in a number of its properties. The amino terminal sequence was found to be acetyl alanyl-tyrosine and the carboxyl terminal sequence was confirmed as alanine preceded by threonine.     

Sequences of the coat protein gene of five peanut stripe virus (PStV) strains from Thailand and their evolutionary relationship with other bean common mosaic virus sequences

Summary.  The coat protein gene and part of the 3′ non-coding region of five strains of peanut stripe virus (PStV) from Thailand have been cloned and sequenced. Phylogenetic comparisons of these strains, known as T1, T3, T5, T6 and T7, and related sequences showed that these strains are indeed strains of PStV. Further, PStV strains appear to be related to each other according to their geographic origin. That is, the Thai strains are more closely related to each other than they are to strains from the USA or Indonesia, despite the variety of symptoms caused by these strains and the overlap of symptom types between the strains from different locations. Like other PStV strains, PStV-Thai can be considered strains of bean common mosaic virus (BCMV) but can be distinguished from bean-infecting strains of BCMV and blackeye cowpea mosaic virus (BlCMV) through sequence and host range. No evidence was found that PStV-Thai strains, unlike PStV-Ib, are recombinants of PStV and BlCMV, although the T3 strain may be a recombinant of different PStV sequences. Phylogenetic analyses of viruses of the BCMV group suggest that acquisition of the ability to infect peanut may have occurred only once. Received January 19, 1998 Accepted May 5, 1998     

Molecular characterisation and evolutionary relationships of a potyvirus infecting Crotalaria in Brazil

Summary.  The 3′ terminal genomic region of a potyvirus causing mosaic disease in several Crotalaria species has been cloned and sequenced. Comparisons of the nucleotide and deduced amino acid (aa) sequences of the cloned cDNA with those from other potyviruses show that the Crotalaria-infecting virus (designated Crotalaria mosaic virus; CrMV) is closely related to Cowpea aphid-borne mosaic virus (CABMV). Maximum identity (95.4%) at the coat protein (CP) aa level was observed between CrMV and a Brazilian strain of CABMV. Phylogenetic analyses derived from the sequence alignments of the CP and 3′ untranslated region confirmed the identification of CrMV as a strain of CABMV and the name CABMV-Cr is suggested. Received April 24, 2001 Accepted October 5, 2001     

<Emphasis Type="BoldItalic">Bean common mosaic virus</Emphasis> isolates causing different symptoms in asparagus bean in China differ greatly in the 5′-parts of their genomes

Potyvirus isolates from asparagus bean ( Vigna sesquipedalis) plants in Zhejiang province, China, caused either rugose and vein banding mosaic symptoms (isolate R) or severe yellowing (isolate Y) in this host, but were otherwise similar in host range. Both isolates were completely sequenced and shown to be isolates of Bean common mosaic virus (BCMV). The complete sequences were 9992 (R) or 10062 (Y) nucleotides long and shared 91.7% identical nucleotides (93.2% identical amino acids) in their genomes and were more distantly related to the BCMV-Peanut stripe virus sequence (PStV). The isolates were much less similar to one another in the 5'-UTR and the N-terminal region of the P1 protein. In the P1, isolate Y was closer to PStV (76.1% identical amino acids) than to isolate R (64.8%). Phylogenetic analyses of the coat protein region showed that the new isolates grouped with other isolates from Vigna spp., forming the blackeye cowpea mosaic strain subgroup of BCMV with 94-98% nucleotides (96-99% amino acids) identical to one another and about 90% identity to other BCMV isolates. Other significant subgroupings amongst published BCMV isolates were detected.     

Cowpea aphid-borne mosaic virus (CABMV) is widespread in passionfruit in Brazil and causes passionfruit woodiness disease

Summary. Leaf samples of yellow passionfruit (Passiflora edulis f. flavicarpa) displaying fruit woodiness symptoms were collected in seven Brazilian states and the Federal District. Viral infection was confirmed by host range and ELISA, and fourteen viral isolates were obtained. All isolates were capable of infecting several leguminous host species, although differences in symptom severity were noticeable. Woodiness symptoms were reproduced in yellow passionfruit, and mosaic symptoms were induced in common bean. All isolates infected cowpea, reported as a non-host of passion fruit woodiness virus (PWV). Indirect ELISA demonstrated that all isolates were serologically related to each other and also to cowpea aphid-borne mosaic virus (CABMV). The complete sequence of the capsid protein was determined for all isolates. Comparison of these sequences with those of other potyviruses indicated the highest identity with CABMV isolates (85 to 94%). Identity with PWV isolates ranged from 54 to 70%. Phylogenetic analysis grouped all of the Brazilian isolates in a monophyletic cluster with the CABMV isolates, clearly distinct from the PWV isolates. Furthermore, this analysis demonstrated that a group of previously characterized isolates from Brazil that had been designated as PWV should be reclassified as CABMV. Together, these results provide unequivocal evidence that, in Brazil, passionfruit woodiness disease is primarily caused by CABMV. The presence of PWV in Brazil has yet to be confirmed. An erratum to this article is available at .     

Indigenous and introduced potyviruses of legumes and <Emphasis Type="Italic">Passiflora</Emphasis> spp. from Australia: biological properties and comparison of coat protein nucleotide sequences

Five Australian potyviruses, passion fruit woodiness virus (PWV), passiflora mosaic virus (PaMV), passiflora virus Y, clitoria chlorosis virus (ClCV) and hardenbergia mosaic virus (HarMV), and two introduced potyviruses, bean common mosaic virus (BCMV) and cowpea aphid-borne mosaic virus (CAbMV), were detected in nine wild or cultivated Passiflora and legume species growing in tropical, subtropical or Mediterranean climatic regions of Western Australia. When ClCV (1), PaMV (1), PaVY (8) and PWV (5) isolates were inoculated to 15 plant species, PWV and two PaVY P. foetida isolates infected P. edulis and P. caerulea readily but legumes only occasionally. Another PaVY P. foetida isolate resembled five PaVY legume isolates in infecting legumes readily but not infecting P. edulis. PaMV resembled PaVY legume isolates in legumes but also infected P. edulis. ClCV did not infect P. edulis or P. caerulea and behaved differently from PaVY legume isolates and PaMV when inoculated to two legume species. When complete coat protein (CP) nucleotide (nt) sequences of 33 new isolates were compared with 41 others, PWV (8), HarMV (4), PaMV (1) and ClCV (1) were within a large group of Australian isolates, while PaVY (14), CAbMV (1) and BCMV (3) isolates were in three other groups. Variation among PWV and PaVY isolates was sufficient for division into four clades each (I-IV). A variable block of 56 amino acid residues at the N-terminal region of the CPs of PaMV and ClCV distinguished them from PWV. Comparison of PWV, PaMV and ClCV CP sequences showed that nt identities were both above and below the 76-77% potyvirus species threshold level. This research gives insights into invasion of new hosts by potyviruses at the natural vegetation and cultivated area interface, and illustrates the potential of indigenous viruses to emerge to infect introduced plants.     

Genome sequence and phylogenetic analysis of a novel comovirus from tabasco pepper (Capsicum frutescens)

A virus isolate from tabasco pepper (Capsicum frutescens) has been reported as a strain of the comovirus Andean potato mottle virus (APMoV). Using the replicative intermediate viral dsRNA, the pepper virus strain was sequenced by Illumina MiSeq. The viral genome was de novo assembled resulting in two RNAs with lengths of 6028 and 3646 nt. Nucleotide sequence analysis indicated that they corresponded to the RNA-1 and RNA-2 of a novel comovirus which we tentatively named pepper mild mosaic virus (PepMMV). Predictions of the open reading frame (ORF) of RNA-1 resulted in a single ORF of 5871 nt with five cistrons typical of comoviruses, cofactor proteinase, helicase, viral protein genome-linked, 3C-like proteinase (Pro), and RNA-dependent RNA polymerase (RdRP). Similarly, sequence analysis of RNA-2 resulted in a single ORF of 3009 nt with two cistrons typical of comoviruses: movement protein and coat protein (large coat protein and small coat proteins). In pairwise amino acid sequence alignments using the Pro-Pol protein, PepMMV shared the closest identities with broad bean true mosaic virus and cowpea mosaic virus, 56% and 53.9% respectively. In contrast, in alignments of the amino acid sequence of the coat protein (small and large coat proteins) PepMMV shared the closest identities to APMoV and red clover mottle virus, 54% and 40.9% respectively. A phylogenetic tree constructed using the conserved domains for the Pro-Pol from all members of the family Secoviridae confirmed the comovirus nature of the virus. Phylogenetic and sequence analyses supports proposing PepMMV as a new species of the genus Comovirus.

     

Molecular characterization and interviral relationships of a flexuous filamentous virus causing mosaic disease of sugarcane (Saccharum officinarum L.) in India

Summary.  A virus isolate causing mosaic disease of commercial sugarcane was purified to homogeneity. Electron microscopy revealed flexuous filamentous virus particles of ca 890 × 15 nm. The virus isolate reacted positively with heterologous antiserum to narcissus latent virus form UK, but failed to react with potyvirus group specific antiserum. N-terminal sequencing of the intact coat protein (CP) and the tryptic peptides indicated that the virus was probably a potyvirus but distinct from several reported potyviruses. Comparison of the 3′-terminal 1084 nucleotide sequence of the RNA genome of this virus revealed 93.6% sequence identity in the coat protein coding region with the recently described sugarcane streak mosaic virus (Pakistani isolate). The molecular weight of the coat protein (40 kDa) was higher than that deduced from the amino acid sequence (34 kDa). The apparent increase in size was shown to be due to glycosylation of the coat protein which has not been reported thus far in the family, Potyviridae. This is the first report on the molecular characterization of a virus causing mosaic disease of sugarcane in India and the results demonstrate that the virus is a strain of sugarcane streak mosaic virus, a member of the Tritimovirus genus of the Potyviridae. We have named it sugarcane streak mosaic virus – Andhra Pradesh isolate (SCSMV-AP). Received October 14, 1997 Accepted August 7, 1998     

Improved diagnosis of cowpea aphid-borne mosaic virus in Africa: significance for cowpea seed-indexing,breeding programs and potyvirus taxonomy

Summary Large-scale surveys in Africa for blackeye cowpea mosaic (BlCMV) and cowpea aphid-borne mosaic (CABMV) showed that several CABMV isolates from Southern Africa were either not or poorly recognized by monoclonal antibodies prepared to isolates collected in West Africa. Selection of three new monoclonal antibodies prepared against the Maputo (Mozambique) isolate of CABMV, and their incorporation into a revised panel of monoclonal antibodies, resulted in the assignment of four of these new CABMV isolates to existing serotypes (II, IV, and V) and three others to a new serotype (VI). The South African isolate of passiflora mosaic virus was shown to be related to CABMV isolates in serotype IV. It is proposed that CABMV isolates be assembled into a distinct species in the legume-infecting, aphid-transmissible potyviruses.     

A mosaic of beach bean (Canavalia rosea) caused by an isolate of Cowpea aphid-borne mosaic virus (CABMV) in Brazil

Beach bean (Canavalia rosea) plants showing mosaic symptoms were found at Massaguaçú beach, Caraguatatuba, Brazil. A potyvirus was found to be responsible for the symptoms, based on transmission assays and electron microscopy. A positive reaction in ELISA was obtained against cowpea aphid-borne mosaic (CABMV) antisera. Viral identity was confirmed by RT-PCR using specific primers to amplify part of the NIb and the entire CP coding region of the genome and the 3′NTR. Comparison of the amplified sequences with that of CABMV showed a nucleotide sequence identity of 97% for the CP coding region. Thus, the potyvirus from beach bean should be considered a CABMV isolate, referred to as CABMV-Cr.     

Genome-associated proteins of comoviruses

Previously, a protein has been reported to be associated with the RNAs of cowpea mosaic virus. Here we present evidence for genome-associated proteins of other comoviruses: squash mosaic virus, Echtes Ackerbohnemosaik-Virus, and another strain of cowpea mosaic virus. Although the proteins and protein-oligonucleotide complexes derived from the RNAs of these viruses showed similar patterns of electrophoretic mobility, two-dimensional chromatograms of their tryptic peptides were distinct. Chromatograms of peptides from preparations of cowpea mosaic virus that were isolated from two hosts were very similar, indicating that the genome-associated protein may be virus specified. Upon digestion with ribonuclease T1, RNAs from the viruses gave rise to a distribution of large oligonucleotides that is consistent with the presence of polyriboadenylate.     

Watermelon mosaic virus-Morocco is a distinct potyvirus

Summary The relationship of the Morocco isolate of watermelon mosaic virus (WMV) to WMV 2, soybean mosaic virus (a virus closely related to WMV 2) and the W strain of papaya ringspot virus (PRSV-W), formerly WMV 1, was examined by comparing tryptic peptide profiles using high performance liquid chromatography. The profiles indicated that the coat protein sequence of WMV-Morocco differed substantially from those of the other potyviruses. This conclusion was supported by sequence data from five tryptic peptides from the coat protein of WMV-Morocco, which showed only 61–68% identity to equivalent sequences in PRSV-W, WMV 2 and zucchini yellow mosaic, another potyvirus infecting cucurbits. Based on the above data, and on known correlations between coat protein sequence similarities and potyvirus relationship, it is concluded that WMV-Morocco should be regarded as a distinct potyvirus.     

Towards a system for the identification and classification of potyviruses: I. Serology and amino acid composition of six distinct viruses

Antigenic relationships of six distinct potyviruses were studied by immunodiffusion tests using highly purified sonicated virus preparations and anti-intact virus sera devoid of detectable antibodies to host-plant antigens. Three variants of bean yellow mosaic virus (BYMV) including BYMV sensu stricto and two variants of pea mosaic virus (PMV and SPMV) were shown to be antigenically very similar and also relatively closely related to lettuce mosaic virus (LMV). Distant antigenic relationships were detected between the BYMV variants and bean common mosaic virus (BCMV); between BCMV and passionfruit woodiness virus (PWV); and between PWV and potato virus Y (PVY). No antigenic relationships were detected between any of these viruses and sugarcane mosaic virus (SCMV). Antibodies in anti-viral sera were very poor in recognizing coat proteins dissociated with LiCl from homologous viruses and failed altogether to recognise those dissociated with pyrrolidine. Attempts to prepare antisera in mice against isolated viral coat proteins dissociated with either LiCl or pyrrolidine were unsuccessful due to poor immunogenicity of the preparations.Electrophoretic mobilities of the viral coat proteins relative to marker proteins in the presence of sodium dodecyl sulphate suggest that the protein subunits of all the viruses studied have molecular weights of about 33,000. However, the coat proteins were prone to partial degradation. The amino acid compositions of the antigenically closely related viruses were very similar, but similarities of those distantly related were no greater than those of the apparently unrelated viruses.The problems in the use of serological and amino acid composition data obtainable with currently available techniques for the classification of potyviruses are discussed.     

A comparative study of the cowpea and bean strains of southern bean mosaic virus

Features of the primary structure and translation of the genomic RNAs of the cowpea and bean strains of southern bean mosaic virus have been investigated in order to assess the similarity of the two viruses. The sequence of 400 bases at their 3' termini have been determined. These include the 3' noncoding regions and extend well into the coat protein cistrons. The noncoding regions (136 bases for the cowpea strain RNA and 129 bases for the bean strain RNA) show no obvious sequence homology. However, extensive base as well as amino acid sequence homology exists in the coding region. RNAs from both strains have a small protein attached to their 5' terminus-the protein in the cowpea strain being the smaller of the two. In vitro studies show that there are similarities in the overall mode of translation of the genomes of the two viruses. Although corresponding proteins are synthesized they differ in size.     

Strains of BCMV and BCMNV characterized from lima bean plants affected by deforming mosaic disease in Peru

Summary Viruses of the species Bean common mosaic virus (BCMV) and Bean common mosaic necrosis virus (BCMNV) were simultaneously detected by the different size of PCR amplicons in lima bean plants (Phaseolus lunatus) displaying deforming mosaic symptoms in Peru. Phylogenetic analysis of partial deduced CP amino acid sequences indicated that the Peruvian BCMV isolates belong to new strains. One isolate differed from the other Peruvian isolates, and also from the ten previously described BCMV strains recognized by responses on differential bean varieties. The sequence of the 3′-proximal part (2547 nucleotides) of the genome confirmed that this isolate also belongs to BCMV.     

Identification and sequence analysis of potyviruses infecting crops in Vietnam

Summary Fifty-two virus isolates from 13 distinct potyvirus species infecting crops in Vietnam were identified and the 3′ region of each genome was sequenced. The viruses were: bean common mosaic virus (BCMV), potato virus Y (PVY), sugarcane mosaic virus (SCMV), sorghum mosaic virus (SrMV), chilli veinal mottle virus (ChiVMV), zucchini yellow mosaic virus (ZYMV), leek yellow stripe virus (LYMV), shallot yellow stripe virus (SYSV), onion yellow dwarf virus (OYDV), turnip mosaic virus (TuMV), dasheen mosaic virus (DsMV), sweet potato feathery mottle virus (SPFMV) and a novel potyvirus infecting chilli, tentatively named chilli ringspot virus (ChiRSV). With the exception of BCMV and PVY, this is first report of these viruses in Vietnam. Further, rabbit bell (Crotalaria anagyroides) and typhonia (Typhonium trilobatum) were identified as new natural hosts of the peanut stunt virus (PStV) strain of BCMV and of DsMV, respectively. Sequence and phylogenetic analyses of the entire CP-coding region revealed considerable variability in BCMV, SCMV, PVY, ZYMV and DsMV. Correspondence: A/Prof. Rob M. Harding, Tropical Crops and Biocommodities Domain, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane 4001, Australia     

Some tryptic peptides of bromovirus proteins

The action of trypsin on brome mosaic virus (BMV) at pH 8 resulted in the dissociation of the virus particles into an 8 S component which contained two proteins, I and F. Protein I was capable of reassembly at pH 8 or 5 into 16-nm spherical particles; protein F formed these particles only at pH 5. SDS-polyacrylamide-gel electrophoresis and amino acid analysis indicated that protein F was about 23 and protein I about 18 amino acid residues smaller than native BMV protein. Six peptides, a cleavage product of the acetylated N-terminal peptide, free arginine, and eight other peptides containing more than one basic amino acid were isolated and their amino acid compositions and N-terminal residues were determined. The BMV peptides were similar or identical to peptides isolated after the limited proteolysis of cowpea chlorotic mottle virus protein at the N-terminus. The sequences of these portions of both viral proteins are probably very similar and probably function as sites of RNA-protein interaction. Peptides isolated from a total tryptic digestion of broad bean mottle virus protein were unlike peptides cleaved in limited proteolysis of BMV and cowpea chlorotic mottle virus.