Genome sequence of a Japanese isolate of Radish mosaic virus: the first complete nucleotide sequence of a crucifer-infecting comovirus

Summary The complete nucleotide sequences of RNA1 and RNA2 of a Japanese isolate of Radish mosaic virus (RaMV-J), a crucifer-infecting comovirus, were determined. RNA1 is 6064 nucleotides long and encodes a 210-kDa polyprotein containing conserved motifs that are required for replication. RNA2 is 4020 nucleotides long and encodes a 123-kDa polyprotein containing the putative movement protein and two coat proteins. Comparisons of the encoded proteins confirmed that RaMV-J and a Czech RaMV isolate are isolates of the same species in the genus Comovirus. A phylogenetic analysis of RaMV-J and other comoviruses revealed that legume-infecting comoviruses constitute a single branch to which RaMV is distantly related.     

Molecular characterization of two prunus necrotic ringspot virus isolates from Canada

We determined the entire RNA1, 2 and 3 sequences of two prunus necrotic ringspot virus (PNRSV) isolates, Chr3 from cherry and Pch12 from peach, obtained from an orchard in the Niagara Fruit Belt, Canada. The RNA1, 2 and 3 of the two isolates share nucleotide sequence identities of 98.6%, 98.4% and 94.5%, respectively. Their RNA1- and 2-encoded amino acid sequences are about 98% identical to the corresponding sequences of a cherry isolate, CH57, the only other PNRSV isolate with complete RNA1 and 2 sequences available. Phylogenetic analysis of the coat protein and movement protein encoded by RNA3 of Pch12 and Chr3 and published PNRSV isolates indicated that Chr3 belongs to the PV96 group and Pch12 belongs to the PV32 group.     

Coat protein gene sequence of an Austrian isolate of grapevine fanleaf virus

Summary The nucleotide sequence of the coat protein cistron of an Austrian isolate of grapevine fanleaf virus (GFLV-FC) fromVitis vinifera cv. French Colombard was determined. It shows small differences at the RNA level as well as at the protein level compared to the sequences of already published grapevine fanleaf virus strains. The differences may be a result of the natural variation among virus populations or a consequence of selection in a special host plant. As the virus RNA sequence reported here was isolated directly from its natural woody host by an immunocapture-PCR technique, passage of the virus through a herbaceous host could be avoided and a possible bias introduced by a different host environment was excluded. The sequence similarity of known GFLV coat protein cistrons to the sequence described is as high as among the other strains.     

Complete nucleotide sequence of an isolate of the nepovirus raspberry ringspot virus from grapevine

The complete nucleotide sequence of the RNAs 1 and 2 of the nepovirus Raspberry ringspot virus cherry isolate (RpRSV-ch) from grapevine was determined. The RNA 1 is 7935 nucleotides (nt) long excluding the poly(A) tail, and contains one long open reading frame (ORF) encoding a polypeptide of 2367 amino acids. This ORF is preceeded by a 136nt 5' non-coding region, and followed by a 695nt 3' non-coding region. Conserved amino acid motifs, characteristic of the viral protease cofactor, the NTP-binding protein, proteinase and polymerase, were found in the sequence of the RNA 1-encoded polyprotein. The RNA 2 is 3915nt long excluding the poly(A) tail, and contains one long ORF encoding a polypeptide of 1106 amino acids. This ORF is preceeded by a 203nt 5' non-coding region, and followed by a 390nt 3' non-coding region. When compared to the corresponding sequences of other nepoviruses, a maximum level of 34% identity was found between the RNA 1-encoded polypetides of RpRSV-ch and other nepoviruses. For the RNA 2-encoded polypeptide, 88% identity was found between RpRSV-ch and RpRSV-S, a Scottish isolate of RpRSV from raspberry, and a maximum 29% identity between RpRSV-ch and other nepoviruses.     

Identification and characterization of a new vitivirus from grapevine

New virus-like sequences, TvAQ7 and TvP15, were found in a Japanese grapevine accession of OKY-AQ7 (cv. Aki Queen) and of OKY-P15 (cv. Pione) by nested RT-PCR to simultaneously amplify a segment of the RNA-dependent RNA polymerase (RdRp) gene from members of the genera Vitivirus and Foveavirus. The TvAQ7 genome, except for an exact 5′ terminus, is composed of about 7.6 kb containing five potential open reading frames. The genomic organization resembles those of grapevine virus A and other known vitiviruses. The 199-amino-acid sequence deduced from the ORF4 of TvAQ7 has 38.5–54.0% identity with the coat protein (CP) of known grapevine vitiviruses and 86.9% identity with TvP15. Phylogenetic analysis based on amino acid sequences of the CP showed that TvAQ7 and TvP15 were closely related to the vitiviruses. In addition, we confirmed that TvAQ7 and TvP15 were transmitted from infected grapevine to healthy seedlings by the mealybug Pseudococcus comstocki Kuwanae. On the basis of our findings, TvAQ7 and TvP15 should be considered isolates of a new species of the genus Vitivirus, and both isolates are probably genetic variants of the new species. We propose to name this virus grapevine virus E (GVE).     

Complete genomic sequence of a Tobacco rattle virus isolate from Michigan-grown potatoes

Tobacco rattle virus (TRV) causes stem mottle on potato leaves and necrotic arcs and rings in potato tubers, known as corky ringspot disease. Recently, TRV was reported in Michigan potato tubers cv. FL1879 exhibiting corky ringspot disease. Sequence analysis of the RNA-1-encoded 16-kDa gene of the Michigan isolate, designated MI-1, revealed homology to TRV isolates from Florida and Washington. Here, we report the complete genomic sequence of RNA-1 (6,791 nt) and RNA-2 (3,685 nt) of TRV MI-1. RNA-1 is predicted to contain four open reading frames, and the genome structure and phylogenetic analyses of the RNA-1 nucleotide sequence revealed significant homologies to the known sequences of other TRV-1 isolates. The relationships based on the full-length nucleotide sequence were different from than those based on the 16-kDa gene encoded on genomic RNA-1 and reflect sequence variation within a 20–25-aa residue region of the 16-kDa protein. MI-1 RNA-2 is predicted to contain three ORFs, encoding the coat protein (CP), a 37.6-kDa protein (ORF 2b), and a 33.6-kDa protein (ORF 2c). In addition, it contains a region of similarity to the 3′ terminus of RNA-1, including a truncated portion of the 16-kDa cistron. Phylogenetic analysis of RNA-2, based on a comparison of nucleotide sequences with other members of the genus Tobravirus, indicates that TRV MI-1 and other North American isolates cluster as a distinct group. TRV M1-1 is only the second North American isolate for which there is a complete sequence of the genome, and it is distinct from the North American isolate TRV ORY. The relationship of the TRV MI-1 isolate to other tobravirus isolates is discussed.     

Complete genomic characterization of a potato mop-top virus isolate from the United States

Potato mop-top virus (PMTV; family Virgaviridae) was reported recently in the Pacific Northwestern USA. To better understand the genetic diversity of this virus, the complete genome of an isolate from Washington State (WA), USA, was characterized. Sequence comparisons of the WA isolate with other known sequences revealed that the RNA-Rep-encoded RdRp protein and the RNA-CP-encoded coat protein displayed >99 % amino acid sequence identity to those of two Nordic (RdRp) and several European and North American isolates (CP), respectively. The RNA-TGB-encoded TGB 1 and TGB 3 protein sequences had >99 % amino acid sequence identity to the corresponding proteins of Czech and Danish isolates, whereas the TGB 2 protein is identical to those of Colombian isolates. Phylogenetic analysis of the viral genes of the WA isolate reflected the close relationship between WA and European isolates. RFLP analysis of corresponding DNA of RNA TGB and RNA CP revealed that the WA isolate has the RNA TGB-II and RNA CP-B types, which are prevalent in Europe and other parts of world. This is the first report of the complete genome characterization of PMTV from the Americas.     

Comparison of the coat protein, movement protein and RNA polymerase gene sequences of Australian, Chinese, and American isolates of barley yellow dwarf virus transmitted by Rhopalosiphum padi

Summary.  Barley yellow dwarf luteovirus-GPV (BYDV-GPV) is a common problem in Chinese wheat crops but is unrecorded elsewhere. A defining characteristic of GPV is its capacity to be transmitted efficiently by both Schizaphis graminum and Rhopaloshiphum padi. This dual aphid species transmission contrasts with those of BYDV-RPV and BYDV-SGV, globally distributed viruses, which are efficiently transmitted only by Rhopaloshiphum padi and Schizaphis graminum respectively. The viral RNA sequences encoding the coat protein (22K) gene, the movement protein (17K) gene, the region surrounding the conserved GDD motif of the polymerase gene and the intergenic sequences between these genes were determined for GPV and an Australian isolate of BYDV-RPV (RPVa). In all three genes, the sequences of GPV and RPVa were more similar to those of an American isolate of BYDV-RPV (RPVu) than to any other luteovirus for which there is data available. RPVa and RPVu were very similar, especially their coat proteins which had 97% identity at the amino acid level. The coat protein of GPV had 76% and 78% amino acid identity with RPVa and RPVu respectively. The data suggest that RPVu and RPVa are correctly named as strains of the same serotype and that GPV is sufficiently different from either RPV strain to be considered a distinct BYDV type. The coat protein and movement protein genes of GPV are very dissimilar to SGV. The polymerase sequences of RPVu, RPVa and GPV show close affinities with those of the sobemo-like luteoviruses and little similarity with those of the carmo-like luteoviruses. The sequences of the coat proteins, movement proteins and the polymerase segments of BYDV serotypes, other than RPV and GPV, form a cluster that is separate from their counterpart sequences from dicot-infecting luteoviruses. The RPV and GPV isolates consistently fall within a dicot-infecting cluster. This suggests that RPV and GPV evolved from within this group of viruses. Since these other viruses all infect dicots it seems likely that their common ancestor infected a dicot and that RPV and GPV evolved from a virus that switched hosts from a dicot to a monocot. Accepted December 19, 1997 Received September 9, 1997     

The complete nucleotide sequence of a Spanish isolate of <Emphasis Type="Italic">Citrus psorosis virus</Emphasis>: comparative analysis with other ophioviruses

Summary. The complete genomic sequence (11278nt) of Citrus psorosis virus (CPsV), isolate P-121 from Spain, was determined and compared with those from isolate CPV-4 and from other ophioviruses. The three RNAs of P-121 had similar size and identical organization as those of CPV-4. The 24K and the RdRp proteins were potentially encoded in the viral complementary (vc) strand of RNA 1, the 54K protein potentially encoded in vcRNA 2 and the coat protein encoded in vcRNA 3. These four proteins from P-121 and CPV-4 had 87, 92, 93 and 94% amino acid identity, respectively, but only 22, 38, 25 and 33% identity with their homologous proteins from Mirafiori lettuce big vein virus (MLBVV), the only other ophiovirus completely sequenced. Biological and genetic differences between CPsV and MLBVV (and the other ophioviruses), would support their future allocation in different genera within a tentative family Ophioviridae.     

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.     

The nucleotide sequence of Indian peanut clump virus RNA 2: sequence comparisons among pecluviruses

Summary.  The RNA-2 molecule of an isolate of the L serotype of Indian peanut clump virus (IPCV) was shown to consist of 4290 nucleotides with five open reading frames (ORF). The arrangement of the ORFs resembled that in RNA-2 of Peanut clump virus (PCV) from West Africa. The proteins encoded by the ORFs in IPCV-L RNA are between 32% and 93% identical to those encoded by PCV RNA. Partial sequence data for the RNA-2 of isolates of the H and T serotypes of IPCV show that the coat and P40 proteins encoded by the 5′-most ORFs of RNA-2 of IPCV-L, IPCV-H and IPCV-T are as similar to each other as any is to the corresponding proteins of PCV. A conserved motif ‘F-E-x₆-W’ is present near the C-termini of the coat proteins of all three IPCV serotypes and of PCV, as it is in the coat proteins of other viruses that have rod-shaped particles, such as Tobacco mosaic virus and Tobacco rattle virus. The results support the distinction of IPCV and PCV as separate virus species, but also raise the question of how the serotypes of IPCV should be classified. Received December 17, 1999/Accepted March 15, 2000     

A RT/PCR-partial restriction enzymatic mapping (PREM) method for the molecular characterisation of the large satellite RNAs of Arabis mosaic virus isolates

The satellite RNA of the grapevine isolate NW of Arabis mosaic virus (ArMV) was cloned and sequenced, and showed 75% identity at the nucleotide level to the satellite RNA of the lilac isolate of ArMV. In order to survey ArMV isolates from various geographical origins and natural hosts for the presence of large satellite RNAs and analyse their degree of variability, a RT/PCR-partial restriction enzymatic mapping (PREM) method was developed. The method is based on the incorporation of 5-methyl-dCTP in the RT/PCR reaction, and the subsequent digestion of the RT/PCR products by methyl-sensitive restriction enzymes. Satellites RNAs were detected by RT/PCR in eight isolates out of 47, six of them originating from grapevine, one from hop and one from lilac. The partial restriction digestion patterns allowed to distinguish six different types of satellites. Cloning and sequencing of the different satellites confirmed these results, the PREM proving able to discriminate sequences with 96% identity. The sizes of the different satellites varied between 1092 and 1139 nucleotides, their encoded proteins between 338 and 360 amino acids. Conserved domains were found in the amino and carboxy-termini between the sequences of the proteins encoded by the satellites of the different isolates of ArMV.     

Complete nucleotide sequence of the Japanese isolate S of beet necrotic yellow vein virus RNA and comparison with European isolates

Summary The complete nucleotide sequences of beet necrotic yellow vein virus RNA-1 to RNA-4 of the Japanese isolate S (BNYVV-S) were determined and compared with those of French isolate (BNYVV-F2). The nucleotide sequences of the two isolates were very similar, differing by only 1.7% (RNA-1), 4.1% (RNA-2), 2.9% (RNA-3) and 3.6% (RNA-4), respectively. The differences of the amino acid sequences of the two isolates depended upon the open reading frames (ORF) as follows: P237, 1.4%; P22 (coat protein), 2.1%; 54k ORF, 3.4%; P42, 0.5%; P13, 1.7%; P15, 3.0%; P14, 7.0% P25, 6.4%; P31, 3.5%. Comparison of the coat protein and triple gene block (P42, P13 and P15) regions of RNA-2 with other isolates revealed that BNYVV-S was much more similar to the Yugoslavian isolate (BNYVV-Yu2) than to BNYVV-F2. The nucleotide differences between BNYVV-S and BNYVV-Yu2 were less than 1%. Based upon the grouping of BNYVV variants reported by Kruse et al. [10], BNYVV-S is thus considered to belong to the A type along with BNYVV-Yu2, whereas BNYVV-F2 is classified in the B type. Our data suggest that the Japanese isolate S may have been derived from European countries other than France or Germany.The BNYVV-S nucleotide sequences have been assigned the accession numbers D84410, D84411, D84412 and D84413 in the DDBJ.     

Cydonia japonica, Pyrus calleryana and P. amygdaliformis: three new ornamental or wild hosts of Apple stem pitting virus

Japanese quince, ornamental and wild pear symptomless samples were infected with Apple stem pitting virus (ASPV). Identification of ASPV was achieved by different PCR assays that amplified either the RNA polymerase or coat protein gene regions. For further confirmation, 312 bp amplicons within the polymerase gene were sequenced and compared with previously published ASPV sequences and additional sequences of isolates from ancient Italian cultivars. Comparison of the partial sequences isolated from wild/ornamental hosts and from cultivated species revealed significant divergence levels. Among the wild/ornamental isolates, the PCT88 isolate from Pyrus calleryana was the most divergent, having an amino acid deletion and incorporating a unique stretch of amino acids not present in any other isolate. Further to this preliminary partial sequence data, statistical analysis demonstrated that the isolates from wild or ornamental hosts were not more closely related to each other than to isolates from cultivated hosts. These results represent the first report of natural ASPV infection in these novel ornamental and wild Rosaceae hosts.     

The partial sequencing of the genomic RNA of a UK isolate of Pepino mosaic virus and the comparison of the coat protein sequence with other isolates from Europe and Peru

Summary.  A 3599 nucleotide portion of the genomic RNA of a UK isolate of Pepino mosaic virus (PepMV), isolated from tomato, has been sequenced (Accession No. AF340024). The region sequenced includes the 3′-end of the RNA polymerase, the triple gene block (TGB), the coat protein (CP) and 3′ untranslated region (UTR). In addition, the CP sequences of another 15 PepMV isolates, including 14 European tomato isolates and a Peruvian pepino isolate, have been determined and compared. This analysis shows that all the tomato isolates share over 99% identity, but only between 96–97% identity with the Peruvian pepino isolate. Received June 1, 2001 Accepted July 17, 2001     

Complete nucleotide sequence of Alfalfa mosaic virus isolated from alfalfa (Medicago sativa L.) in Argentina

The complete nucleotide sequence of an Alfalfa mosaic virus (AMV) isolate infecting alfalfa (Medicago sativa L.) in Argentina, AMV-Arg, was determined. The virus genome has the typical organization described for AMV, and comprises 3,643, 2,593, and 2,038 nucleotides for RNA1, 2 and 3, respectively. The whole genome sequence and each encoding region were compared with those of other four isolates that have been completely sequenced from China, Italy, Spain and USA. The nucleotide identity percentages ranged from 95.9 to 99.1 % for the three RNAs and from 93.7 to 99 % for the protein 1 (P1), protein 2 (P2), movement protein and coat protein (CP) encoding regions, whereas the amino acid identity percentages of these proteins ranged from 93.4 to 99.5 %, the lowest value corresponding to P2. CP sequences of AMV-Arg were compared with those of other 25 available isolates, and the phylogenetic analysis based on the CP gene was carried out. The highest percentage of nucleotide sequence identity of the CP gene was 98.3 % with a Chinese isolate and 98.6 % at the amino acid level with four isolates, two from Italy, one from Brazil and the remaining one from China. The phylogenetic analysis showed that AMV-Arg is closely related to subgroup I of AMV isolates. To our knowledge, this is the first report of a complete nucleotide sequence of AMV from South America and the first worldwide report of complete nucleotide sequence of AMV isolated from alfalfa as natural host.     

Molecular characterization of divergent grapevine Pinot gris virus isolates and their detection in Slovak and Czech grapevines

Analysis of complete genome sequences of three Slovak isolates of grapevine Pinot gris virus (GPGV) showed their low heterogeneity (reaching 1.7 %) and a close relationship to the Italian NC_015782 isolate (4.2-4.5 % divergence). Comparison of Slovak and Italian isolates revealed an unusual accumulation of 21 indel mutations in ORF1, resulting in a localized high divergence in the encoded amino acid sequences. An elevated divergence in the 5′ extremity of the GPGV genomes is suggestive of a recombination between Slovak isolates and grapevine berry inner necrosis virus. RT-PCR allowed the frequent detection of closely related GPGV isolates in grapevines from Slovakia and the Czech Republic.     

Sequence analysis of RNA-2 of different isolates of broad bean wilt virus confirms the existence of two distinct species

Summary.  The complete nucleotide sequence of RNA-2 from a Japanese isolate IP of broad bean wilt virus (BBWV) was determined. The sequence encodes a single large polyprotein, which contains a putative movement protein and two coat proteins (CPs). The 3′-terminal sequences of RNA-2 were also determined for three other Japanese isolates and two ATCC isolates (PV132 and PV176) of BBWV. The CPs of the four Japanese isolates share 86.8–98.0% amino acid sequences homology with one another and 88.3–96.5% with those reported for the isolate PV131 (BBWV-2). However, they have only 57.9–66.2% homology with those of PV132 and PV176 (BBWV-1). Received December 7, 1998 Accepted February 23, 1999