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     

Apricot latent virus: a new species in the genus Foveavirus

Summary.  Extraction of viral double-stranded RNA from peach leaves infected with Apricot latent virus (ALV) followed by molecular cloning of synthesized cDNA and its sequencing, suggested that ALV is a new virus, whose coat protein (CP) coding region contains Apple stem pitting virus (ASPV)-related sequences. The sequenced portion of the ALV genome (1444 nt) includes the putative CP gene and the 3′ non-translated region. The 5′ portion of this fragment (1-651 nt) is highly distinct whereas the 3′ portion is 77% identical to the corresponding region of ASPV. Molecular hybridization experiments using a cRNA probe to ASPV with ALV-infected leaf tissue extracts also revealed that the genome of ALV contains nucleotide sequences related to that of ASPV. Western blots of tissue extracts indicated that ALV coat protein reacted with polyclonal antiserum against ASPV; however, the ALV CP differs in size from that of ASPV. ALV was graft-transmitted to several Prunus rootstocks. Based on the available sequence data, serological observations and bioassays we propose that ALV is a new species in the genus Foveavirus, typified by ASPV. ALV-specific PCR-primers and viral-specific cRNA probes developed in this investigation may be useful for detecting the virus and for studying its epidemiology and geographical distribution. Received February 10, 2000/Accepted April 3, 2000     

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     

Sequence variability within the 3′-proximal part of the <Emphasis Type="Italic">Sweet potato mild mottle virus</Emphasis> genome

Summary.  Sweet potato mild mottle virus (SPMMV) is the type member of the genus Ipomovirus (family Potyviridae) and is only known to occur in East Africa. In Uganda, SPMMV is the third most prevalent virus infecting sweet potato. The sequence variability of SPMMV was studied by cloning and sequencing a 1.8-kb fragment representing the 3′-end of the genome of eight SPMMV isolates collected from different districts of Uganda. Sequence comparisons indicated 85.9–99.9% nucleotide sequence identity and 92.8–100% amino acid sequence similarity for the coat protein (CP) encoding region. The nucleotide sequence identity within the 3′-untranslated region (3′ UTR) was 84.7–100%, and the region was variable in length (303–308 nucleotides) due to some deletions within the 5′-proximal part of the 3′ UTR. Phylogenetic analysis of the CP amino acid sequences revealed significant clustering, indicating the existence of distinguishable sequence variants or strains. The low CP amino acid sequence similarity of SPMMV isolates with other characterised viruses of the family Potyviridae and the unusual putative proteolytic cleavage site at the NIb/CP junction further demonstrate SPMMV as a very distinct virus in the family Potyviridae. Received July 9, 2002; accepted October 1, 2002     

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.     

Genetic analysis of a novel Alaska barley yellow dwarf virus in the family <Emphasis Type="Italic">Luteoviridae</Emphasis>

Summary. A new plant virus belonging to the family Luteoviridae and isolated from diseased oat (Avena sativa L.) plants was discovered in Alaska in 2003. Even though plants with red/orange leaves were indicative of barley yellow dwarf disease, they were not reactive to specific antibodies corresponding to barley yellow dwarf virus (BYDV)-MAV, -PAV, -SGV, and cereal yellow dwarf virus-RPV from enzyme-linked immunosorbent assays (ELISA). An alternative RT-PCR assay that incorporated Shu-F/Yan-R primers for detection of BYDV-MAV, -PAS, -PAV, and SGV was effective in producing ∼830-nt fragments that contained genomic sequences to the 3′-terminus of the polymerase gene (ORF 2), the intergenic region (∼113 nt), the coat protein gene (ORF 3), and the putative movement gene (ORF 4). The Alaskan isolates were most similar to BYDV-MAV with only about 77 and 80% amino acid identity in the CP and ORF 4, respectively. The Alaska isolates coat protein gene sequences differed in several regions that otherwise are conserved among BYDV-MAV isolates, and may be important in serological variations, accounting for the negative ELISA results. Based upon sequence and serological differences, we concluded that the Alaskan BYDV-MAV-like isolates formed a novel species tentatively in the genus Luteovirus, and propose the name BYDV-ORV (oat red-leaf virus).     

Molecular characterization of Hop mosaic virus: its serological and molecular relationships to Hop latent virus

Summary.  The 3′-terminal sequence of hop mosaic virus (HpMV) genomic RNA was determined. A cDNA of approximately 1.8 kbp was amplified from the HpMV genome by 3′ RACE using a degenerate primer, which was designed to anneal to the overlapping region of open reading frames (ORFs) 2 and 3 of eight carlavirus genomes. The sequence contained three ORFs, encoding proteins of 7-, 34-, and 11-kDa, which corresponded to ORFs 4, 5, and 6 of the carlavirus genome, respectively. The amino acid sequence of ORF 5, encoding the coat protein (CP) of HpMV, shows the highest identity (67%) to that of Hop latent virus (HpLV). The HpMV CP N-terminal sequence differs from that of HpLV, but the central and C-terminal sequences of the CP of both viruses are similar. The sequence similarity possibly causes the cross-reaction of heterologous antibodies of HpMV and HpLV. Phylogenetic analyses based on the CP amino acid and 3′ non-coding region sequences indicate close relationships among HpMV, HpLV, and Potato virus M. We report here the first molecular characterization of HpMV genomic RNA. Received November 30, 2000 Accepted April 11, 2001     

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.     

Vanilla mosaic virus isolates from French Polynesia and the Cook Islands are Dasheen mosaic virus strains that exclusively infect vanilla

Summary. Sequence was determined for the coat protein (CP) gene and 3′ non-translated region (3′NTR) of two vanilla mosaic virus (VanMV) isolates from Vanilla tahitensis, respectively from the Cook Islands (VanMV-CI) and French Polynesia (VanMV-FP). Both viruses displayed distinctive features in the N-terminal region of their CPs; for VanMV-CI, a 16-amino-acid deletion including the aphid transmission-related DAG motif, and for VanMV-FP, a stretch of GTN repeats that putatively belongs to the class of natively unfolded proteins. VanMV-FP CP also has a novel DVG motif in place of the DAG motif, and an uncommon Q//V protease cleavage site. The sequences were compared to a range of Dasheen mosaic virus (DsMV) strains and to potyviruses infecting orchids. Identity was low to DsMV strains across the entire CP coding region and across the 3′NTR, but high across the CP core and the CI-6K2-NIa region. In accordance with current ICTV criteria for species demarcation within the family Potyviridae, VanMV-CI and VanMV-FP are strains of DsMV that exclusively infect vanilla.     

Analysis of the complete genome of peach chlorotic mottle virus: identification of non-AUG start codons, <Emphasis Type="Italic">in vitro</Emphasis> coat protein expression,and elucidation of serological cross-reactions

Summary The entire genome of peach chlorotic mottle virus (PCMV), originally identified as Prunus persica cv. Agua virus (4N6), was sequenced and analysed. PCMV cross-reacts with antisera to diverse viruses, such as plum pox virus (PPV), genus Potyvirus, family Potyviridae; and apple stem pitting virus (ASPV), genus Foveavirus, family Flexiviridae. The PCMV genome consists of 9005 nucleotides (nts), excluding a poly(A) tail at the 3′ end of the genome. Five open reading frames (ORFs) were identified with four untranslated regions (UTR) including a 5′, a 3′, and two intergenic UTRs. The genome organisation of PCMV is similar to that of ASPV and the two genomes share a nucleotide (nt) sequence identity of 58%. PCMV ORF1 encodes the replication-associated protein complex (M_r 241,503), ORF2 – ORF4 code for the triple gene block proteins (TGBp; M_r 24,802, 12,370, and 7320, respectively), and ORF5 encodes the coat protein (CP) (M_r 42,505). Two non-AUG start codons participate in the initiation of translation: ³⁵AUC and ⁷⁶⁷⁶AUA initiate translation of ORF1 and ORF5. In vitro expression with subsequent Western blot analysis confirmed ORF5 as the CP-encoding gene and confirmed that the codon AUA is able to initiate translation of the CP. Expression of a truncated CP fragment (M_r 39, 689) was demonstrated, and both proteins are expressed in vivo, since both were observed in Western blot analysis of PCMV-infected peach and Nicotiana occidentalis. The expressed proteins cross-reacted with an antiserum against ASPV. The amino acid sequences of the CPs of PCMV and ASPV CP share only 37% identity, but there are 11 shared peptides 4–8 aa residues long. These may constitute linear epitopes responsible for ASPV antiserum cross reactions. No significant common linear epitopes were associated with PPV. Extensive phylogenetic analysis indicates that PCMV is closely related to ASPV and is a new and distinct member of the genus Foveavirus.     

Barley yellow dwarf virus-PAV RNA does not have a VPg

Summary.  The genus Luteovirus is divided into two subgroups. It has already been shown that a genome-linked protein (VPg) is covalently attached to the 5′-end of the genomic RNA of subgroup II luteoviruses, but whether a VPg is present or absent on the genomic RNA of subgroup I luteoviruses is unknown. In the present work, a simple and sensitive method was developed for the detection of the VPg on virion genomic RNA. This method was used to show that a VPg is not attached to the 5′-end of the RNA genome of barley yellow dwarf virus-PAV which is a member of subgroup I luteoviruses. Accepted August 1, 1997 Received April 6, 1997     

Nucleotide sequence analysis of RNA-2 of a flat apple isolate of Cherry rasp leaf virus with regions showing greater identity to animal picornaviruses than to related plant viruses

Summary.  RNA-2 of a flat apple isolate of Cherry rasp leaf virus (CRLV-FA) appears to consist of 3274 nucleotides, excluding a 3′ poly (A) tail. The data supports re-classification of CRLV in a new genus in the family Comoviridae. A single open reading frame (ORF) encoding a putative 108 kDa polyprotein was identified. Potential protease cleavage sites were identified which would result in the production of a putative movement protein (41 kDa), and 3 capsid protein subunits (24, 20, and 22 kDa, respectively). A 5′-UTR and 3′-UTR were identified, 248 nt and 146 nt long, respectively. The genome organisation of CRLV-FA RNA-2 is similar to that of Apple latent spherical virus (ALSV) RNA-2, a new member of the family Comoviridae. The Vp25 amino acid sequences were unique to CRLV-FA and ALSV (54% identity), with no relationship identified to any other virus. CRLV-FA Vp20 and Vp24 amino acid sequences were closely related to ALSV (59 and 65%, respectively) but the only other relationships identified were with a range of animal ssRNA positive-strand viruses. Received December 7, 2001; accepted April 12, 2002 Published online June 21, 2002     

Complete sequence of a viral nervous necrosis virus (NNV) isolated from red-spotted grouper (Epinephelus akaara) in China

A nodavirus isolated from red-spotted grouper (Epinephelus akaara) larvae in China has been subjected to genome analysis. The full-length genome sequences of RNA1 and RNA2 were determined, and the 5′-non-coding region (NCR) and 3′NCR sequences were determined by 5′ rapid amplification of cDNA ends (RACE) and 3′RACE. RNA1 is 3,103 nt in length and contains a 982-amino-acid open reading frame (ORF) encoding protein A with a calculated molecular mass of 110.74 kDa. RNA2 is 1,433 nt long and contains a 338-amino-acid major ORF encoding coat protein with a calculated molecular mass of 37.059 kDa. Multiple alignment and phylogenetic analysis clearly supported including this virus in the species Redspotted grouper nervous necrosis virus, genus Betanodavirus, family Nodaviridae.     

Molecular characterization of a new potyvirus infecting sunflower

Summary.  We have sequenced 1873 nucleotides from the 3′-end region of a sunflower potyvirus genome including the 3′-NIb protein coding region (813 nucleotides), the entire coat protein coding region (807 nucleotides) and 3′-NCR (253 nucleotides), excluding the poly (A) tail. Amino acids identity of the whole CP between the sunflower virus and Potyvirus members ranged from 49.5% (SCMV) to 81.5% (PVY-NsNr), and the core ranged from 55% (TVMV) to 87% (PVY-NsNr; PepMoV). The 3′-NCR nucleotides showed 38.7% homology to PeSMV and 61% to PepMoV-C. The sequence of 3′end region and analysis of phylogenetic relationships suggest this sunflower virus could belong to PVY subgroup and the name of “sunflower chlorotic mottle virus” (SuCMoV) is proposed. This is the first report on the partial nucleotide sequence of a potyvirus infecting sunflower. Received May 19, 2000/Accepted June 9, 2000     

Nucleotide sequence and genome organization of Dweet mottle virus and its relationship to members of the family Betaflexiviridae

The nucleotide sequence of Dweet mottle virus (DMV) was determined and compared to sequences of members of the families Alphaflexiviridae and Betaflexiviridae. The DMV genome has 8,747 nucleotides (nt) excluding the 3′ poly-(A) tail. DMV genomic RNA contains three putative open reading frames (ORFs) and untranslated regions of 73 nt at the 5′ and 541 nt at 3′ termini. ORF1 potentially encoding a 227.48-kDa polyprotein, which has methyltransferase, oxygenase, endopeptidase, helicase, and RNA-dependent RNA polymerase (RdRP) domains. ORF2 encodes a movement protein of 40.25 kDa, while ORF3 encodes a coat protein of 40.69 kDa. Protein database searches showed 98–99% matches of DMV ORFs with citrus leaf blotch virus (CLBV) sequences. Phylogenetic analysis based on the RdRP core domain revealed that DMV is closely related to CLBV as a member of the genus Citrivirus. DMV did not satisfy the molecular criteria for demarcation of an independent species within the genus Citrivirus, family Betaflexiviridae, and hence, DMV can be considered a CLBV isolate.     

Nucleotide sequence and genome organisation of cherry mottle leaf virus and its relationship to members of the Trichovirus genus

Summary.  The nucleotide sequence of cherry mottle leaf virus (CMLV) was determined and compared to sequences of a number of plant viruses including the type member of the Trichovirus genus (apple chlorotic leafspot virus, ACLSV), and members of the Vitivirus genus including grapevine virus B, (GVB). The CMLV genome was determined to consist of 8003 nt excluding the poly(A) tail at the 3′ end of the genome. The overall A+U content of CMLV genomic RNA was 59. 1%, which is similar to ACLSV, but significantly different from GVB. Four putative open reading frames were identified (ORFs 1, 2, 3, and 4) encoding proteins of M_r 215. 8 kDa, 47 kDa, 21.6 kDa, and 15. 3 kDa, respectively. This differs from ACLSV which has 3 ORFS, and GVB which has 5 ORFs. Protein database searches showed no matches of CMLV ORF4 with ACLSV sequences, but found similarities between ORF4 of CMLV and ORF5 of GVB, suggesting that this may be a nucleic acid-binding protein. CMLV and ACLSV formed a common virus clade in phylogenetic analysis of the coat protein amino acid sequence and except for CMLV’s ORF4, these viruses show high levels of similarity throughout the genome. CMLV appears to be a member of the Trichovirus genus. Accepted November 19, 1999/Received August 12, 1999     

The complete genome sequence of pepper severe mosaic virus and comparison with other potyviruses

Summary. The complete nucleotide sequence of pepper severe mosaic virus (PepSMV) was determined. The viral genome consisted of 9890 nucleotides, excluding a poly (A) tract at the 3′ end of the genome. The PepSMV RNA genome encoded a single polyprotein of 3085 amino acid residues, resulting in ten functionally distinct potyviral proteins. The lengths of the 5′ nontranslated region (NTR) and the 3′ NTR were 164 and 468 nucleotides, respectively. The genome organization of the virus was typical for members of the genus Potyvirus in the family Potyviridae. The coat protein amino acid sequence identity between PepSMV and the other 45 potyviruses ranged from 53.4 to 79.7%. Sequence alignments and phylogenetic analyses of the potyviral polyprotein sequences revealed that PepSMV was the closest to potato virus Y (PVY) and closely related to members of the PVY subgroup. Our genome sequence data clearly confirmed that PepSMV belongs to a separate species in the genus Potyvirus.     

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     

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     

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