Nucleotide sequence of both genomic RNAs of a North American tobacco rattle virus isolate

Summary.  The complete sequence of a North American tobacco rattle virus (TRV) isolate, ‘Oregon yellow’ (ORY), was determined from cDNA and RT-PCR clones derived from the two genomic RNAs of this isolate. The RNA-1 is 6790 bases and RNA-2 is 3261 bases. The sequence of TRV-ORY RNA-1 was similar to RNA-1 of TRV isolate SYM, and differs in 48 nucleotides. TRV-ORY RNA-1 was one base shorter than -SYM, and had 47 base substitutions resulting in 12 amino acid substitutions of which 4 were conservative. The RNA-2 of TRV-ORY was distinct from RNA-2 of other characterized TRV isolates and contained three open reading frames (ORFs) that could potentially code for proteins of MW 22.4 kDa, 37.6 kDa and 17.9 kDa. Based on the homology of the predicted amino acid sequence with those of other tobraviruses, ORF1 of RNA-2 encodes the coat protein (CP). The protein sequence of ORF2 had regions of limited similarity with those of ORF2 of two other TRV isolates and pea early browning tobravirus. The ORF3 was unique to TRV-ORY. Phylogenetic analysis of tobravirus CPs indicated that TRV-ORY was most closely related to pepper ringspot tobravirus and TRV-TCM. The relationship of tobravirus CPs to other rod-shaped tubular plant viruses vis also discussed. Accepted March 21, 1998     

Genetic variability of genomic RNA 2 of four tobacco rattle tobravirus isolates from potato fields in the Northwestern United States

Sequence analysis of RNA 2 of four Tobacco rattle virus (TRV) isolates collected from potato fields in Oregon (OR2, Umt1), Washington (BM), and Colorado (Cot2) revealed significant homologies to the ORY isolate from North America. Phylogenetic analysis based on a comparison of nucleotide (nt) and amino acid (aa) sequences with other members of the genus Tobravirus indicates that the North American isolates cluster as a distinct group. All of the RNAs are predicted to contain open reading frames (ORFs) potentially encoding the coat protein (CP, ORF 2a) and 37.6 kDa (ORF 2b) ORFs. In addition, they all contain a region of similarity to the 3' terminus of RNA 1 of ORY, including a truncated portion of the 16 kDa cistron from the 3' end of RNA 1. Three of the isolates, which are nematode transmissible, OR2, BM, and Cot2, also contain a third putative ORF (ORF 2c) which encodes a protein of 33.6 kDa. The fourth isolate, Umt1, which is not nematode transmissible, is the most divergent of the isolates as it encodes a truncated version of ORF 2c. The ORF 2c deletion in Umt1 may contribute to its inability to be transmitted by the vector. The results reported in this article indicate again that the TRV genome is flexible. Interestingly, although both isolates Umt1 and Cot2 were mechanically transmitted to tobacco from potato, only Umt1 exhibits the deletion in RNA 2. TRV Isolate Umt1, therefore, appears to be another example of rapid adaptation of the TRV genome to non-field conditions.     

Genetic structure of a population of Potato virus Y inducing potato tuber necrotic ringspot disease in Japan; comparison with North American and European populations

The structure of Potato virus Y (PVY) populations causing potato tuber necrotic ringspot disease (PTNRD) was analysed. The full-length sequences of the genomic RNAs of five geographically distinct isolates from Japan were determined. Recombination and phylogenetic analyses of European, North American and Japanese isolates of PVY showed that the world PVY population has three major lineages and two sublineages. Most recombinants were interlineage, and one isolate from Europe was identified as an intralineage recombinant. No recombinants were found among Japanese PTNRD isolates, which were most closely related to PTNRD isolates previously found in North America. Comparison of the within- and between population nucleotide diversities in the N lineage sequences from Japan, Europe and North America showed that Japanese population was distinct from the European and North American populations. The nucleotide sequences of the protein 1 and coat protein genes of a further 18 isolates were determined. One Japanese clade had radiated in a star burst as shown by its deviation from the neutral equilibrium model and its small nucleotide diversity. Our results suggest that PVY PTNRD was recently introduced into Japan more than once, and has expanded throughout Japan from founder populations.     

Genome structure of tobacco rattle virus strain PLB: further evidence on the occurrence of RNA recombination among tobraviruses

RNA-2 (2196 nucleotides) of tobacco rattle virus (TRV) strain PLB was found to consist of a 5'-terminal sequence of 1376 nucleotides identical to the 5'-sequence of RNA-2 of TRV strain PSG and a 3'-terminal sequence of 820 nucleotides that is identical to the 3'-sequence of RNA-1 of strain PLB. Thus, in strains PLB and PSG the same coat protein gene is fused to different RNA-1 derived 3'-termini. By combining RNA-1 of TRV strain TCM with RNA-2 of strain PLB, a viable pseudo-recombinant was formed with genome segments that have nonidentical 3'-sequences. After 25 passages in tobacco each RNA retained its strain-specific 3'-sequences. This indicates that the perfect 3'-homology that occurs between the two genome segments of all natural TRV isolates analyzed so far is not a prerequisite for a stable genotype.     

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     

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.     

Single-nucleotide polymorphisms and reading frame shifts in RNA2 recombinant regions of tobacco rattle virus isolates Slu24 and Deb57

Two previously sequenced tobacco rattle virus (TRV) isolates, Slu24 and Deb57, from Polish potato fields have recombinant RNA2 species. The 3’-proximal region of the Slu24 RNA2 is derived from the 3’ terminus of its supporting RNA1, while that of the Deb57 RNA2 is derived from the 3’ terminus of the unrelated RNA1 from the isolate SYM or PpK20. Gene structure annotation revealed open reading frames encoding truncated 16-kDa proteins in the recombinant regions of the RNA2 of Deb57 and Slu24. Reading frame shifts, single nucleotide substitutions and deletions occurred during recombination, including shifts from a stop codon or replacements of an internal stop codon. In the recombinant region of the Deb57 RNA2, the first reading frameshift event starts from the AUG start codon of the truncated 16-kDa protein. The second frameshift event, caused by a single nucleotide deletion upstream of the stop codon, leads to the splitting of the stop codon into two amino acid codons and the continuation of translation. In addition, a U-to-A substitution changes a potential internal stop codon UAA, which is caused by recombination-related frame shifts, into the codon AAA, encoding lysine. The replacement of this internal stop codon with an amino acid codon prevented the premature translation termination of the truncated 16-kDa protein. These recombination-related reading frame shifts are the driving force underlying the major differences in the translated amino acids, consequently leading to their translation into distinct polypeptides. Conversely, single nucleotide substitutions in the recombinant regions of the RNA2 of Deb57 or Slu24 resulted in only minor changes in the translated amino acids.     

The nucleotide sequence of the coat protein gene and 3′ untranslated region of papaya ringspot virus type W (Aust)

Summary The nucleotide sequence of the 3 terminal region of the Australian isolate of papaya ringspot virus type W [PRSV-W (Aust)] was determined. An open reading frame (864 bp), encoding the putative coat protein gene, occurs upstream of the putative 3 untranslated region (206 bp) and poly(A) tail. A 23 amino acid sequence was obtained from N-terminal analysis of the coat protein from purified virions. This sequence has 100% homology with a region of the amino acid sequence inferred from the nucleic acid sequence of the coat protein gene. However, this region is 13 amino acids downstream from the N terminus predicted for two American isolates of PRSV. The coat protein gene of PRSV-W (Aust) was shown to have 96.8% and 96.4% nucleotide sequence similarity with American isolates of PRSV-W and PRSV-P, respectively.     

Nucleic acid sequence of the VP1 region of attenuated MS-1 hepatitis A virus

The nucleotide sequence of the VP1 region of marmoset-attenuated hepatitis A virus (HAV), MS-1, was determined by incorporative dideoxynucleotide sequencing of the RNA obtained from purified, liver-derived virus. Comparison of this nucleotide sequence to those of four previously published isolates revealed that one of the isolates, HM-175, which was obtained from Australia and passed three times in marmosets, had a 8.5-11% nucleotide variability compared to the remaining four isolates which were isolated from North American sources. This nucleotide variability does not result in amino acid differences with the exception of two of the North American isolates, which were derived from tissue culture passage. These isolates have been shown to contain regions of variability generated by nucleotide insertions and/or deletions, while the remaining three isolates, including the Australian isolate, demonstrate limited amino acid differences within the VP1 molecule.     

Cloning and sequencing of the coat protein gene of tobacco ringspot virus isolates from UK and Iran

The coat protein (CP) gene and the 3' untranslated region (UTR) of genomic RNA 2 of Tobacco ringspot virus (TRSV, the genus Nepovirus, subgroup a) isolates from the UK and Iran were cloned from total viral RNA and sequenced. Comparison of these isolates with an isolate from the USA revealed a high degree of nucleotide and amino acid identity which extends the knowledge of molecular relationships between these three TRSV isolates. The UK isolate shared the highest nucleotide identity (95%) with the US isolate as compared to a lower identity with the Iranian isolate (92%). The highest identity (98%) was found between the UK and US isolates at amino acid level. Comparative analysis of the Iranian, UK and US isolates revealed some differences concerning some members of other subgroups of nepoviruses. For example, the N-terminal FDAYXR and the C-terminal FYGRXS motifs conserved among some nepoviruses, which occur adjacent to each other in folded CP molecules, were not detected in the Iranian, UK or US TSRV isolates. These isolates shared similarity only with Tomato ringspot virus (TomRSV) belonging to the subgroup c of nepoviruses. Another similarity of these isolates with TomRSV and Raspberry ringspot virus (RRSV) was the presence of a 34 nucleotide (nt) long sequence within the 3'-UTR.     

The complete genome sequences of two isolates of potato black ringspot virus and their relationship to other isolates and nepoviruses

The complete nucleotide sequences of RNA 1 and RNA 2 of the nepovirus potato black ringspot virus (PBRSV) from two different isolates were determined, as well as partial sequences from two additional isolates. RNA1 is 7,579-7,598 nucleotides long and contains one single open reading frame (ORF), which is translated into a large polyprotein with 2,325 amino acids and a molecular weight of 257 kDa. The complete sequence of RNA2 ranges from 3857 to 3918 nt between the different isolates. It encodes a polyprotein of 1079-1082 amino acids with a molecular weight of 120 kDa. Sequence comparison using the Pro-Pol region and CP showed that all four isolates formed two distinct groups, corresponding to potato and arracacha, that were closely related to each other and also to tobacco ringspot virus (TRSV). Comparing our data to those obtained with other nepoviruses, our results confirm that PBRSV belongs to a distinct species and is a member of subgroup A in the genus Nepovirus based on its RNA2 size, genome organization, and nucleotide sequence.     

Nucleotide sequence of a Japanese isolate of Squash mosaic virus. Brief report

Summary.  The complete nucleotide sequence of the RNA-1 of Squash mosaic virus (SqMV) was determined using a Japanese isolate (Y-SqMV). The sequence consisted of 5865 nucleotides excluding the poly (A) at the 3′ terminus and contained a single long open reading frame with a coding capacity for a protein of M_r209971. Analysis of the deduced amino acid sequence suggested a genomic organization typical of comoviruses. The nucleotide sequence of the RNA-2 of Y-SqMV was also determined and compared with the SqMV isolates from the United States. The larger and smaller capsid protein (CP) coding region was compared to those of K-SqMV and Z-SqMV, which represent two subgroups of SqMV. The larger CP gene of Y-SqMV showed 93.0% and 88.0% identities with those of K-SqMV and Z-SqMV, respectively at the nucleotide level. The smaller CP gene of Y-SqMV was 94.1% and 88.4% identical with those of K-SqMV and Z-SqMV. The results suggested that the Japanese SqMV isolate (Y-SqMV) is distinct from those in the United States, and might represent a third subgroup. Received May 7, 2001 Accepted August 1, 2001     

Heterogeneity in Nsp2 of European-like porcine reproductive and respiratory syndrome viruses isolated in the United States

Recently, isolates of porcine reproductive and respiratory syndrome virus (PRRSV) that possess nucleotide sequences similar to European isolates have been reported in United States herds. The origin, diversity and prevalence of European-like North American PRRSV isolates in the U.S. remain unknown. Nucleotide sequence analysis of the 12 kb ORF1 of a North American isolate, SDPRRS 01-08 (01-08), showed 93.7% identity with Lelystad virus (LV), the prototypic European isolate, but only 58% identity with VR-2332, the prototypic North American isolate. Comparisons between LV and 01-08 at the peptide sequence level of the predicted non-structural proteins (Nsp) showed that Nsp9 (98.9% amino acid identity) was the most conserved and the least conserved was Nsp2 at 90.6% identity. For the purpose of comparison, GP5, the principal envelope structural protein, showed a 93.5% identity between 01-08 and LV. The most dramatic differences between the Nsp2 proteins of LV and 01-08 were a single 17 amino acid deletion between residues 734 and 750, as well as several amino acid differences. The same deletion was identified in the Nsp2 in five of seven other EuroPRRSV isolates submitted to the South Dakota Animal Disease Research and Diagnostic Laboratory. The remaining two isolates contained small deletions, but in other regions of Nsp2. Peptide sequence diversity in the form of hypervariability and deletions in Nsp2 demonstrate that European-like PRRSV isolates in the USA represent a heterogeneous group. Furthermore, areas in Nsp2 with deletions and amino acid hypervariability localize to regions that are predicted to be immunologically important.     

An onion isolate of tobacco rattle virus: reactivity with anantiserum to Hypochoeris mosaic virus, a putative furovirus, and molecular analysis of its RNA 2

Summary.  A tubular virus from onion was found to react with an antiserum to Hypochoeris mosaic virus (HyMV), a putative furovirus. Sequence analysis of its genomic RNAs and further serological tests, however, indicated it to be a tobravirus rather than a furovirus. The reactivity of the HyMV antiserum with several isolates of tobacco rattle virus (TRV) suggests that HyMV itself may be a tobravirus. The deduced amino acid sequences of the putative proteins encoded on RNA 2 of the onion virus isolate (ON) suggest close evolutionary relationships to the TRV isolate TCM from tulip. However, RNA 2 of the ON isolate contains a shorter RNA 1-like sequence on its 3′-end and an additional small ORF upstream of its RNA 1-like part. The sequence of its 315 5′-terminal nucleotides is more similar to that of RNA 2 of the PLB isolate from potato than to that of TCM RNA 2 Received December 9, 1997 Accepted February 5, 1998     

Probable geographical grouping of PVY(N) and PVY(NTN) based on sequence variation in P1 and 5'-UTR of PVY genome and methods for differentiating North American PVY(NTN)

An increasing number of countries in recent years have reported the occurrence of potato tuber necrotic ringspot disease (PTNRD), caused by tobacco veinal necrosis strain of Potato virus Y (PVY(N)), belonging to the sub-group tuber necrosis (PVY(NTN)). Methods for the differentiation of PVY(NTN), based on primer sequences often detect isolates of European (EU) type but not the North American (NA) type. To resolve this problem, the nucleotide sequence of 5'-untranslated region (5'-UTR) and the P1 gene of 11 isolates of PVY(N) and PVY(NTN) from the European Union and North America was determined. Sequence comparison and phylogenetic analysis of 5'-UTR and P1 region indicated that PVY(N) isolates from the European Union and North America formed their own separate groups. Intra-group sequence identity for all except one was over 98%, as opposed to the inter-group identity of 90%. Additionally, the PVY(NTN) isolates from the European Union and North America clustered with their respective PVY(N) isolates. This indicates a possible evolution of PVY(NTN) isolates from the PVY(N) isolates of a geographical region. With this information of regional relationships of PVY(NTN) and PVY(N) isolates, two approaches were developed based on a competitive RT-PCR and a restriction pattern, for the differentiation of NA-PVY(NTN) from the local PVY(N) and from EU-PVY(NTN). Thus sequencing of the P1 gene and use of competitive RT-PCR approach could be applicable for determining the possible origin of new occurrences of PVY(NTN) from other geographical regions.     

Sequence analysis of grapevine isolates of Raspberry ringspot nepovirus

Summary. The nucleotide sequences of RNAs 1 and 2 of a German isolate of Raspberry ringspot virus (RpRSV) infecting grapevine (RpRSV-Grapevine), as well as partial sequences of another grapevine isolate from Switzerland (RAC815) were determined. The sequences of the protease-polymerase region encoded by RNA1, and the movement protein and coat protein genes encoded by RNA 2, of these isolates were compared with those of other isolates available in databases. The coat proteins of the grapevine isolates formed a sister group to all those from other RpRSV isolates, but whether this resulted from divergence or recombination was uncertain.     

Complete Nucleotide Sequence of the RNA-2 of Grapevine Deformation and Grapevine Anatolian Ringspot Viruses

The nucleotide sequence of RNA-2 of Grapevine Anatolian ringspot virus (GARSV) and Grapevine deformation virus (GDefV), two recently described nepoviruses, has been determined. These RNAs are 3753 nt (GDefV) and 4607 nt (GARSV) in size and contain a single open reading frame encoding a polyprotein of 122 kDa (GDefV) and 150 kDa (GARSV). Full-length nucleotide sequence comparison disclosed 71–73% homology between GDefV RNA-2 and that of Grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV), and 62–64% homology between GARSV RNA-2 and that of Grapevine chrome mosaic virus (GCMV) and Tomato black ring virus (TBRV). As previously observed in other nepoviruses, the 5 non-coding regions of both RNAs are capable of forming stem-loop structures. Phylogenetic analysis of the three proteins encoded by RNA-2 (i.e. protein 2A, movement protein and coat protein) confirmed that GDefV and GARSV are distinct viruses which can be assigned as definitive species in subgroup A and subgroup B of the genus Nepovirus, respectively. The nucleotide sequences data reported in this paper were submitted to the GenBank database and given the accession numbers AY291207 for the Grapevine Anatolian ringspot virus and AY291208 for the Grapevine deformation virus.     

Genome segment RNA-1 of a flat apple isolate of <Emphasis Type="Italic">Cherry rasp leaf virus</Emphasis>: nucleotide sequence analysis and RT-PCR detection

Summary. The sequence of the RNA-1 of a flat apple isolate of Cherry rasp leaf virus (CRLV-FA) was determined using overlapping cDNA fragments. CRLV-FA RNA-1 consists of 6992 nucleotides (nt), excluding a 3′ poly (A) tail. A single open reading frame (ORF) consisting of 6705 nt was identified. This ORF encodes a putative polyprotein consisting of 2235 amino acid (aa) residues, approximately 249.6 kDa. When compared to CRLV-pot (potato isolate) RNA-1 ORF, 2 deletions of 5 aa and 10 aa (total 15 aa) were observed at the variable N-terminus of the protease cofactor of CRLV-FA. Non-coding regions were identified at the 5′-(142 nt) and 3′-end (145 nt). CRLV-FA and CRLV-pot are isolates of the same virus with identity levels for the RNA-1 associated nt and deduced aa of 94% and 95%, respectively. RT-PCR targeting CRLV-FA RNA-1 appear to be of similar sensitivity and just as reliable as RT-PCR targeting RNA-2.