Molecular characterization and coat protein serology of watermelon leaf mottle virus (Potyvirus)

Summary.  A cDNA library was generated from purified RNA of watermelon leaf mottle virus (WLMV) (Genus Potyvirus). Two overlapping clones totaling 2,316 nucleotides at the 3′terminus of the virus were identified by immunoscreening with coat protein antiserum. The sequence analyses of the clones indicated an open reading frame (ORF) of 2,050 nucleotides which encoded part of the replicase and the coat protein, a 243-nucleotide non-coding region (3′UTR), and 23 adenine residues of the poly (A) tail. The taxonomic status of WLMV was determined by comparisons of the sequence of the cloned coat protein gene and 3′UTR with potyvirus sequences obtained from GenBank. The nucleotide sequence identities of WLMV compared with 17 other potyviruses ranged from 55.6 to 63.5% for the coat protein, and from 37.2 to 48.3% for the 3′UTR. Phylogenetic analyses of the coat protein region and the 3′UTR indicated that WLMV did not cluster with other potyviruses in a clade with high bootstrap support. The coat protein gene was expressed in Escherichia coli and a polyclonal antiserum was prepared to the expressed coat protein. In immunodiffusion tests, WLMV was found to be serologically distinct from papaya ringspot virus type W, watermelon mosaic virus 2, zucchini yellow mosaic virus, and Moroccan watermelon mosaic virus. In Western blots and ELISA, serological cross-reactivity with other cucurbit potyviruses was observed. Serological and sequence comparisons indicated that watermelon leaf mottle virus is a distinct member of the Potyvirus genus. Accepted September 23, 1999     

Sequencing and characterization of the coat protein and 3′ non-coding region of a newsweet potato potyvirus

Summary.  A cDNA library was constructed from viral genomic RNA purified from sweet potato plants affected by “Sweet Potato Chlorotic Dwarf disease” in an attempt to clarify the etiology of this viral complex in Argentina. By sequence analysis, some of the obtained clones were found to belong to sweet potato feathery mottle potyvirus (SPFMV), to a closterovirus and to a new potyvirus. A cDNA clone of 1,103 bp representing the coat protein cistron and 3^′ non-coding region of the newly identified potyvirus was further characterized. The sequence contained an ORF of 855 nucleotides with a coding capacity of 285 amino acids, followed by a 3^′ untranslated tail of 248 nucleotides. The core and C-terminal regions have sequences well conserved among potyviruses. Furthermore, amino acid sequence comparisons of the capsid protein with those of other described potyviruses showed 63% homology with SPFMV, 68 to 70% with two different isolates of sweet potato latent potyvirus (SPLV), 57% with sweet potato G potyvirus (SPGV) and 73% with potato virus Y (PVY). These data allowed us to propose the inclusion of this virus as a new member of the family Potyviridae, genus Potyvirus with the designation sweet potato mild speckling potyvirus (SPMSV). Received January 16, 1997 Accepted March 4, 1997     

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     

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     

Molecular characterization of a distinct potyvirus from whitegrass in China

Summary.  A potyvirus isolated from perennial whitegrass (Pennisetum centrasiaticum Tzvel.) in North China was characterized at the molecular level. The 3′ terminal nucleotide (nt) sequence of 1669 nt of the viral RNA genome has been determined, which covered the coding region of the C-terminal part of the large nuclear inclusion protein (NIb, RNA polymerase), capsid protein (CP) gene and the 3′ nontranslated region (NTR). The CP gene consisted of 909 nt (including the stop codon) encoding 302 amino acid residues, and the 3′ NTR was 241 nt in length excluding the polyadenylated tract. Sequence comparison of the amino acids of CPs showed that this virus was most closely related to Sorghum mosaic virus and Maize dwarf mosaic virus with percent identities of 77% to 78% while that of the 3′ NTRs suggested that it was most closely related to Zea mosaic virus with identity of 72%. This virus isolate was to some extent closely related to other members of the Sugarcane mosaic virus subgroup of potyviruses for the CP amino acid sequences. Phylogenetic analyses of the sequences indicated that this virus isolate represented a distinct potyvirus, and the name Pennisetum mosaic virus (PenMV) is proposed. Received November 22, 2002; accepted January 8, 2003 Published online March 21, 2003     

The nucleotide sequence of the 3′-terminal region of dasheen mosaic virus (Caladium isolate) and expression of its coat protein in Escherichia coli for antiserum production

Summary.  A caladium isolate of dasheen mosaic virus (DsMV-Ch) was cloned as cDNA from genomic RNA. The sequence of the 3′-terminal 3 158 nucleotides, which consisted of the 3′-terminus of the NIa gene, the NIb gene, the coat protein (CP) gene, and a 246-nucleotide non-coding region, was between 57–68% similar at the nucleotide level and 72–82% similar at the amino acid level when compared with other potyviruses. Phylogenetic analysis of aligned, selected potyviral CP sequences indicate that DsMV-Ch is similar to DsMV isolates infecting taro and closely related to the bean common mosaic virus subgroup in the genus Potyvirus. A recombinant DsMV-Ch CP (∼39 kDa) expressed in E. coli was used as an immunogen and the resulting antiserum reacted with DsMV and several other potyviruses in Western blots and indirect ELISA. Received March 19 Accepted June 26, 1998     

Sequence comparison of the 3′-terminal parts of the RNA of four German isolates of sugarcane mosaic potyvirus (SCMV)

Summary.  The 3′-termini of the genome of four German isolates of sugarcane mosaic potyvirus (SCMV) were cloned and sequenced. The sequence data covered the 3′ non-coding region (3′NCR), coat protein and part of the nuclear inclusion b (NIb) genes of the isolates. Comparisons of the sequences revealed that the investigated isolates are very closely related. An alignment of the predicted coat protein amino acid sequences of the German isolates with sequence data for other members of the SCMV subgroup, in particular the two SCMV strains, SCMV-SC and SCMV-MDB, showed a limited degree of homology indicating that the German isolates may represent a distinct virus. However, this is mainly due to the previously reported unexpected sequence diversity in the surface exposed N-terminal region of coat protein of SCMV isolates. Comparisons of the amino acid sequences of the core region of the coat proteins and the nucleotide sequences of the 3′ NCR clearly show that the German isolates are strains of SCMV. Received June 7, 1996 Accepted October 24, 1996     

Molecular taxonomy of a new potyvirus isolated from chilli pepper in Thailand

Summary.  A virus isolated from chilli pepper plants in Kamphaengsaen, Nakorn Pathom, showing vein banding mottle symptoms was classified using sequence analysis and phylogeny of the coat protein gene and 3′ noncoding region (3′ NCR). This virus was found to be a typical potyvirus on the basis of particle morphology, biological properties and cytopathology. The 3′ terminus region of the genome of 1,309 nucleotides, representing the viral coat protein gene and 3′ NCR was cloned and sequenced. Nucleotide sequence analysis indicated that the 3′ region of the viral genome had a polyA tail of at least 12 nucleotides, a noncoding region of 272 nucleotides, a coat protein gene of 864 nucleotides and 161 nucleotides representing the 3′ terminus of the polymerase gene. The amino acid sequence of the coat protein was compared with those of 23 distinct potyviruses, and 63.1% shown to be the highest homology. However the 3′ NCR had, at most, 29.7% random homology, thus indicating that this virus is a distinct species in the genus Potyvirus in the family Potyviridae. The result is well supported by previous studies on the biology and biochemical properties of this virus. Received February 13, 1998 Accepted June 26, 1998     

Pepper yellow mosaic virus,a new potyvirus in sweetpepper,Capsicum annuum

Summary.  A potyvirus was found causing yellow mosaic and veinal banding in sweetpepper in Central and Southeast Brazil. The sequence analysis of the 3′ terminal region of the viral RNA revealed a coat protein of 278 amino acids, followed by 275 nucleotides in the 3′-untranslated region preceding a polyadenylated tail. The virus shared 77.4% coat protein amino acid identity with Pepper severe mosaic virus, the closest Potyvirus species. The 3′-untranslated region was highly divergent from other potyviruses. Based on these results, the virus found in sweetpepper plants could be considered as a new potyvirus. The name Pepper yellow mosaic virus (PepYMV) is suggested. Received March 12, 2001 Accepted August 28, 2001     

Identification and molecular characterization of a new potyvirus from Panax notoginseng

A potyvirus causing distortion and mosaic symptoms in the herbal plant Sanqi (Panax notoginseng) was isolated from Yunnan province, China, and the complete nucleotide sequence of one isolate and the partial sequences of two other isolates were determined. The viral RNA genome comprised 9,750 nt excluding the 3′-terminal poly(A) tail, with the capacity to encode a single polyprotein of 3,089 amino acids. Phylogenetic analysis with other completely sequenced potyviruses revealed that the virus in this study was most closely related to plum pox virus, with 56.3% nt identity in the genomic RNA sequence and 53.3% aa identity in the polyprotein. However, the most closely related 3′-terminal sequences were from four partially sequenced potyviruses infecting plants of the family Apiaceae (67.7–75.3% nt identity and 73.8–76.7% aa identity in their coat protein cistrons), especially Angelica virus Y. These results suggest that this virus isolate should be designated a member of a new species in the genus Potyvirus, which is tentatively named Panax virus Y (PanVY).     

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     

Identification and molecular characterization of a new potyvirus infecting Triteleia species

Plants of Triteleia hyacinthina, Triteleia ixioides Starlight, and Triteleia laxa Corina with severe mosaic and yellow vein-banding were found to be infected with a potyvirus. The 3′-terminal region of the virus was amplified by RT-PCR from total RNA using a potyvirus-specific degenerate primer (poty5P: 5′ GGN AAY AAY AGY GGN CAR CC 3′) and an oligo-dTprimer. The sequence generated included the 3′-NIb protein coding region (680 nucleotides), the entire coat protein coding region (840 nucleotides), and 3’-untranslated region (UTR) (253 nucleotides). Amino acid identity of the whole CP between the triteleia virus and potyvirus member ranged from 54% Apium virus Y (ApVY) to 67% Auraujia mosaic virus (ArjMV) and Twisted-stalk chlorotic streak virus (TSCSV) and the core ranged from 59% (ApVY) to 75% (ArjMV). The 3-UTR showed no significant homology with other known potyviruses. Phylogenetic relationships suggest this triteleia virus is a new member of the Potyvirus genus and the name of “Triteleia mosaic virus” (TriMV) is proposed. This is the first report of a potyvirus infecting triteleia.     

Comparison of the complete sequences of five different isolates of Potato virus A (PVA), genus Potyvirus

Summary.  The complete nucleotide (nt) and deduced amino acid (aa) sequences of isolates Ali, U, Her (from potato, Solanum tuberosum) and TamMV (from tamarillo, Solanum betacea) of Potato virus A (PVA, genus Potyvirus) were determined and compared with the previously reported sequence of PVA isolate B11. Most parts (proteins) of the polyprotein showed over 95% aa sequence similarity. The cylindrical inclusion (CI) protein and the 6K 1 protein were the most conserved proteins among the five isolates. TamMV was the most different isolate. Sequence similarity between TamMV and the other isolates was the lowest in regions close to the 5′-end [5′-non-translated region (NTR) and P1 region] and 3′-end (N-terminus of coat protein) of the genome. However, the termini of the genome (the first 60 nt of the 5′-NTR and the entire 3′-NTR) were highly similar in all five isolates. A frameshift region in the replicase (NIb) was identified the PVA isolates Ali, B11, Her and U, as compared to TamMV and other potyviruses. Received May 25, 1999/Accepted July 23, 1999     

Konjak mosaic virus: the complete nucleotide sequence of the genomic RNA and its comparison with other potyviruses

Summary. Konjak mosaic virus (KoMV) belongs to the genus Potyvirus, family Potyviridae. The complete nucleotide sequence of KoMV F isolate (KoMV F) was determined. The genome is 9,544 nucleotides long excluding the 3′ terminal poly A tail and encodes a typical potyviral 350-kDa polyprotein of 3,087 amino acids. Phylogenetic analysis using known potyvirus polyproteins shows that KoMV constitutes a branch with yam mosaic virus, close to another branch including Japanese yam mosaic virus, turnip mosaic virus, scallion mosaic virus and lettuce mosaic virus. The 3′ terminal 1,842 nucleotides of a different isolate of KoMV, K-2, was also determined, covering the C-terminal 292 amino acids of the nuclear inclusion protein b (NIb), coat protein (CP), and the 3′ untranslated region. The amino acid sequences of the KoMV F CP and the nucleotide sequences of the KoMV F 3′ untranslated region showed 92.5 and 90.5% identity to the corresponding genes of K-2, 88.7–96.8 and 92.7–94.4% to those of Zantedeschia mosaic virus (ZaMV) isolates, 87.5–89.7% and 85.5–90.3% to those of Japanese hornwort mosaic virus (JHMV) isolates. These results showed that KoMV is a distinct potyvirus and that KoMV, ZaMV, and JHMV are members of the same potyvirus species. Considering that KoMV was the first of these to be described, ZaMV and JHMV may be considered isolates of KoMV.     

Complete nucleotide sequence and genome organization of sweet potato feathery mottle virus (S strain) genomic RNA:the large coding region of the P1 gene

Summary. The complete nucleotide sequence of a sweet potato feathery mottle virus severe strain (SPFMV-S) genomic RNA was determined from overlapping cDNA clones and by directly sequencing viral RNA. The viral RNA genome is 10 820 nucleotides long, excluding the poly(A) tail and contains one open reading frame (ORF) starting at nucleotide 118 and ending at 10 599, potentially encoding a polyprotein of 3 493 amino acids (Mr 393 800). The ORF was followed by a 3 untranslated region of 221 nucleotides. The deduced polyprotein includes P1 (74K), HC-Pro (52K), P3 (46K), 6K1, CI (72K), 6K2, NIa-VPg (22K), NIa-Pro (28K), NIb (60K) and coat (35K) proteins, after an analysis of protein cleavage sites analogous to other potyvirus polyproteins. The polyprotein had a high level of amino acid identity with those of other potyviruses, except in the regions of P1 and P3. The P1 of SPFMV-S RNA has 664 amino acid residues, and is the largest and least similar to those of other potyviruses. HC-Pro and CI show high identity with those of other potyviruses. P3 has relatively low identity, however, the length of P3 was within the range of variability among other potyviruses. The 6K1 protein between P3 and C1 is also highly similar to those of other potyviruses. This is the first report on the complete nucleotide sequence of the sweet potato-infecting virus. Received October 28, 1996 Accepted April 3, 1997     

Characterisation of an isolate of <Emphasis Type="Italic">Narcissus degeneration virus</Emphasis> from Chinese narcissus (<Emphasis Type="Italic">Narcissus tazetta</Emphasis> var. <Emphasis Type="Italic">chinensis</Emphasis>)

Summary. A potyvirus from Chinese narcissus was transmitted mechanically to three species of Narcissus and to Lycoris radiata but not to 22 other test species. In western blot, the coat protein reacted strongly with Narcissus degeneration virus (UK isolate) antiserum. Antiserum raised to the Chinese virus did not react with eighteen other potyviruses. The complete nucleotide sequence (9816 nt) had the typical genome organisation for a member of the genus Potyvirus. Sequence comparisons and phylogenetic analysis showed that the Chinese virus was different from all previously sequenced potyviruses but distantly related to onion yellow dwarf and shallot yellow stripe viruses.     

Nucleotide sequence of coat protein gene of yam mild mosaic virus, isolated in Papua New Guinea

Summary.  We determined the 3′-termimus 1353 nucleotides (nts) in length excluding the poly (A) tail of yam mild mosaic potyvirus (YMMV) RNA. The sequence starts within a long open reading frame (ORF) 1209 nts and is followed by untranslated region (3′-UTR) of 144 nts. The coat protein (CP) contains 266 amino acids (aa) with molecular ratio (Mr) of approximately 30 kDa. The CP of YMMV differs substantially from yam mosaic virus (YMV), Japanese yam mosaic virus (JYMV) (57 and 61% of amino acid sequence identity) and other potyvirus species. This result suggests that YMMV should be classified as a new yam potyvirus. Received January 29, 1999 Accepted February 26, 1999     

Determination of 3′-terminal nucleotide sequence of pepper\break vein banding virus RNA and expression of its coat protein in Escherichia coli

Summary.  Pepper vein banding virus (PVBV) is an important virus infecting chilli pepper in south India. Earlier reports suggested it to be a distinct potyvirus. The nucleotide sequence of PVBV RNA from the 3′-end (3862 nt) was determined. Analysis of the nucleotide and deduced amino acid sequence revealed that it encompasses a partial open reading frame encoding the partial sequence of VPg, NIa-protease, NIb, coat protein (CP) and 3′-untranslated region (UTR). Comparison of the amino acid sequence of CP and the nucleotide sequence of 3′-UTR with those of other potyviruses confirmed an earlier observation that PVBV is a distinct member of the Potyvirus sub-group and it had significant similarity to a recently characterized virus infecting chilli pepper, chilli vein-banding mottle virus (CVbMV), from Thailand. The analysis showed that both PVBV and CVbMV might represent strains of the same virus. Further, the PVBV CP gene was overexpressed in E. coli, which assembled into potyvirus-like particles (PVLPs). The assembled particles were shown to encapsidate the CP mRNA. Received March 17, 1999 Accepted April 28, 1999     

Bean yellow mosaic,clover yellow vein,and pea mosaic are distinct potyviruses: evidence from coat protein gene sequences and molecular hybridization involving the 3′ non-coding regions

Summary The sequences of the 3 1019 nucleotides of the genome of an atypical strain of bean yellow mosaic virus (BYMV-S) and of the 3 1018 nucleotides of the clover yellow vein virus (CYVV-B) genome have been determined. These sequences contain the complete coding region of the viral coat protein followed by a 3 non-coding region of 173 and 178 nucleotides for BYMV-S and CYVV-B, respectively. When the deduced amino acid sequences of the coat protein coding regions were compared, a sequence identity of 77% was found between the two viruses, and optimal alignment of the 3 untranslated regions of BYMV-S and CYVV-B gave a 65% identity. However, the degree of homology of the amino acid sequences of coat proteins of BYMV-S with the published sequences for three other strains of BYMV ranged from 88% to 94%, while the sequence homology of the 3 untranslated regions between the four strains of BYMV ranged between 86% and 95%. Amplified DNA probes corresponding to the 3 non-coding regions of BYMV-S and CYVV-B showed strong hybridization only with the strains of their respective viruses and not with strains of other potyviruses, including pea mosaic virus (PMV). The relatively low sequence identities between the BYMV-S and CYVV-B coat proteins and their 3 non-coding regions, together with the hybridization results, indicate that BYMV, CYVV, and PMV are distinct potyviruses.     

Ceratobium mosaic potyvirus: another virus from orchids

Summary.  A potyvirus, which we call ceratobium mosaic virus, has been detected in about one third of more than 100 plants representing c. 33 orchid genera in two collections in Australia. It was detected using RT-PCR with redundant primers that are Potyviridae-specific and have additional sequences corresponding to either the SP6 or T7 bacteriophage promoters at their 5′-termini. Thus the nucleotide sequence of the resulting PCR fragments, consisting of about 1.7 kb of the 3′ portion of the viral genome, could be determined directly. Viral sequences obtained from five infected orchids indicate that they contained different isolates of a single potyvirus species most closely related to the bean common mosaic group of potyviruses, but clearly distinct from all whose virion protein genes have been reported to the international gene sequence databases. Accepted December 18, 1997 Received September 9, 1997