Characterization of a flowering cherry strain of <Emphasis Type="Italic">Cherry necrotic rusty mottle virus</Emphasis>

The host range and complete nucleotide sequences of two Cherry necrotic rusty mottle virus (CNRMV) isolates (FC4 and FC5) infecting flowering cherry accessions imported from Japan are described. Of the plants tested, cherry, peach, apricot and almond became infected, but only sweet cherry cv. ‘Canindex’, Nanking cherry and apricot cv. ‘Tilton’ showed a mild foliar mottle. The genomic sequences of CNRMV-FC4 and CNRMV-FC5 are 8,430 and 8,429 nt in length, excluding the 3′ poly (A) tail. They contain seven open reading frames encoding for a putative virus replicase, “triple gene block” proteins, a coat protein and two proteins with unknown functions. The two CNRMV-FC isolates share 96% identity in the genomic sequences, and their genome organizations are virtually identical to that of a German CNRMV isolate (CNRMV-GER). However, they differ from CNRMV-GER by 14% in the overall nucleotide sequence and 2% (ORF2) to 30% (ORF5a) in the derived amino acid sequences of individual gene products.     

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     

Genome characterization of a flexuous rod-shaped mycovirus, Botrytis virus X, reveals high amino acid identity to genes from plant ‘potex-like’ viruses

Summary. This study reports the molecular characterization of a flexuous rod-shaped mycovirus, Botrytis virus X (BVX), infecting the plant-pathogenic fungus, Botrytis cinerea. BVX contains a ssRNA genome of 6966 nucleotides, and a poly(A) tract at or very near the 3′ terminus. Computer analysis of the genomic cDNA sequence of BVX revealed five potential open reading frames (ORFs). ORF1 showed significant amino acid sequence identity to the replicase proteins of plant ‘potex-like’ viruses, including 73% identity to the RNA-dependent RNA polymerase (RdRp) region of the allexivirus, garlic virus A (GarV-A). The C-terminal region of ORF3 shared amino acid homology with plant ‘potex-like’ coat proteins. The remaining ORFs did not reveal significant homology with known protein sequences. BVX differs substantially from Botrytis virus F (BVF), another flexuous rod-shaped mycovirus characterized from the same B. Cinerea isolate. It is proposed that the mycovirus BVX belongs to a new, as yet unassigned genus in the plant ‘potex-like’ virus group, distinct from BVF.     

Complete genomic RNA sequence of the Polish <Emphasis Type="Italic">Pepino mosaic virus</Emphasis> isolate belonging to the US2 strain

We have determined the complete nucleotide and amino acid sequences of the Polish Pepino mosaic virus (PepMV) isolate marked as PepMV-PK. The PepMV-PK genome consists of a single positive-sense RNA strand of 6412-nucleotide-long that contains five open reading frames (ORFs). ORF1 encodes the putative viral polymerase (RdRp), ORFs 2–4 the triple gene block (TGB 1–3), and ORF5-coat protein CP. Two short untranslated regions flank the coding ones and there is a poly (A) tail at the 3′ end of the genomic RNA. Thus, the genome organization of PepMV-PK is that of a typical member of the genus Potexvirus. Phylogenetic analysis based on full-length genomes of PepMV sequences showed that PepMV-PK was most closely related to the Ch2 isolate from Chile. Comparison of PepMV-PK and Ch2 showed the following nucleotide identities: 98% for the RdRp, 99% for the CP genes, and 98, 99, and 98% for the TGB1, TGB2, and TBG3, respectively. This high level of nucleotide sequence identity between the Chilean and Polish PepMV-PK isolates suggest their common origin.     

Complete nucleotide sequence of Sesbania mosaic virus: a new virus species of the genus Sobemovirus

Summary.  The complete nucleotide sequence of the Sesbania mosaic virus (SeMV) genomic RNA was determined by sequencing overlapping cDNA clones. The SeMV genome is 4149 nucleotides in length and encodes four potential overlapping open reading frames (ORFs). Comparison of the nucleotide sequence and the deduced amino acid sequence of the four ORFs of SeMV with that of other sobemoviruses revealed that SeMV was closest to southern bean mosaic virus Arkansas isolate (SBMV-Ark, 73% identity). The 5′ non-coding regions of SeMV, SBMV and southern cowpea mosaic virus (SCPMV) are nearly identical. However ORF1 of SeMV which encodes for a putative movement protein of M_r 18370 has only 34% identity with SBMV-Ark. ORF 2 encodes a polyprotein containing the serine protease, genome linked viral protein (VPg) and RNA dependent RNA polymerase domains and shows 78% identity with SBMV-Ark. The N-terminal amino acid sequence of VPg was found to be TLPPELSIIEIP, which mapped to the region 326–337 of ORF2 product and the cleavage site between the protease domain and VPg was identified to be E³²⁵-T³²⁶. The cleavage site between VPg and RNA dependent RNA polymerase was predicted to be E⁴⁴⁵-T⁴⁴⁶ based on the amino acid sequence analysis of the polyprotein from different sobemoviruses. ORF3 is nested within ORF2 in a − 1 reading frame. The potential ribosomal frame shift signal and the downstream stem-loop structure found in other sobemoviruses are also conserved in SeMV RNA sequence, indicating that ORF3 might be expressed via − 1 frame shifting mechanism. ORF4 encodes the coat protein of SeMV, which shows 76 and 66% identity with SBMV-Ark and SCPMV, respectively. Thus the comparison of the non-coding regions and the ORFs of SeMV with other sobemoviruses clearly revealed that it is not a strain of SBMV. Phylogenetic analysis of six different sobemoviruses, including SeMV, suggests that recombination event is not frequent in this group and that SeMV is a distinct member of the genus sobemovirus. The analysis also shows sobemoviruses infecting monocotyledons and dicotyledons fall into two distinct clusters. Received April 20, 2000 Accepted August 28, 2000     

Complete genome sequence of an Argentinean isolate of <Emphasis Type="Italic">Solenopsis invicta</Emphasis> virus 3

Solenopsis invicta virus 3 (SINV-3) is a recently described positive-strand RNA virus that infects the red imported fire ant, S. invicta. The genome of an Argentinean isolate of Solenopsis invicta virus 3 (SINV-3^ArgSF ) obtained from the Santa Fe region of Argentina was sequenced in entirety. Assembly of nine overlapping fragments yielded a consensus genome sequence 10,386 nucleotides long, excluding the poly(A) tail present on the 3′ end (Genbank accession number GU017972). With the exception of the poly(A) tail, the genome length of SINV-3^ArgSF was identical to the North American isolate (SINV-3^USDM ). The SINV-3^ArgSF genome possessed three major open reading frames (ORFs) (comprised of ≥100 codons) in the sense orientation; SINV-3^USDM possessed only two. ORFs 1 and 2 had identical start and stop genome positions for both isolates. Blastp analysis of the translated ORF 1 of SINV-3^ArgSF recognized conserved domains for helicase, protease, and RNA-dependent RNA polymerase. These domains and their corresponding positions were identical to those reported for SINV-3^USDM . ORF 2a, unique to the SINV-3^ArgSF genome, was also found in frame 2 and had a canonical start codon located at nucleotide position 8,351 and a stop codon ending at position 8,827. Blastp analysis of the translated amino acid sequence of ORF 2a revealed no significant similarity in the Genbank database. The two SINV-3 isolates exhibited 96.2% nucleotide sequence identity across the entire genome. The amino acid sequences of ORFs 1 and 2 exhibited higher identities (99.0 and 98.2%, respectively) than the corresponding nucleotide regions within the genome. These data indicated that the nucleotide differences between the SINV-3 isolates were largely synonymous. This observation was corroborated by codon substitution rate analysis. Thus, the majority of the SINV-3 codon changes were silent in the two polyproteins, indicating purifying selection pressure on the viral genome.     

The molecular diversity and evolution of Rice tungro bacilliform virus from Indian perspective

Rice tungro disease is caused by a combination of two viruses: Rice tungro spherical virus and Rice tungro bacilliform virus (RTBV). This study was performed with the objective to decipher the molecular variability and evolution of RTBV isolates present in the tungro-affected states of Indian subcontinent. Phylogenetic analysis based on ORF-I, ORF-II, and ORF-IV sequences showed distinct divergence of Indian RTBV isolates into two groups; one consisted isolates from Hyderabad (Andhra Pradesh), Cuttack (Orissa), and Puducherry and another from West Bengal, Chinsura West Bengal, and Kanyakumari (Tamil Nadu). The results obtained from phylogenetic analysis were further supported with the single nucleotide polymorphisms (SNPs), insertion and deletion (INDELs) and evolutionary distance analysis. In addition, sequence difference count matrix revealed a maximum of 56 (ORF-I), 13 (ORF-II) and 73 (ORF-IV) nucleotides differences among all the Indian RTBV isolates taken in this study. However, at the protein level these differences were not significant as revealed by K (a)/K (s) ratio calculation. Sequence identity at nucleotide and amino acid level was 92-100 % (ORF-I), 96-100 % (ORF-II), 94-100 % (ORF-IV) and 86-100 % (ORF-I), 98-100 % (ORF-II) and 95-100 % (ORF-IV), respectively, among Indian isolates of RTBV. The divergence of RTBV isolates into two independent clusters of Indian and non-Indian was shown with the help of the data obtained from phylogeny, SNPs, and INDELs, evolutionary distance analysis, and conserved motifs analysis. The important role of ORF-I and ORF-IV in RTBV diversification and adaptation to different rice growing regions is also discussed.     

Molecular characterization of Hop latent virus and phylogenetic relationships among viruses closely related to carlaviruses

Summary.  The complete nucleotide sequence of the hop latent virus (HpLV) genome was determined. The viral RNA genome is 8,612 nucleotides long, excluding the poly(A) tail, and contains six open reading frames (ORFs), which encode putative proteins of 224-kDa (ORF 1), 25-kDa (ORF 2), 11-kDa (ORF 3), 7-kDa (ORF 4), 34-kDa (ORF 5), and 12-kDa (ORF 6). ORF 5 encodes the coat protein as demonstrated by N-terminal sequencing of three proteolytic peptides derived from the virus particle. The genome organization of HpLV is similar to that of other species in the genus Carlavirus, and the overall sequence of HpLV is more similar to that of Potato virus M than to sequences of other carlaviruses reported to date. The amino acid sequences of the putative methyltransferase, RNA helicase, and RNA-dependent RNA polymerase encoded in ORF 1 and an ‘accessory’ helicase encoded in ORF 2 of the HpLV genome were compared with those of viruses in the ‘tymo’ lineage: the genera Carlavirus, Potexvirus, Allexivirus, Foveavirus, Trichovirus, Capillovirus, Vitivirus, and Tymovirus. The phylogenetic relationships among the viruses in these genera are discussed. This is the first molecular characterization of a carlavirus infecting hop plants. Accepted June 15, 2000 Received January 19, 2000     

A near-complete genome sequence of a distinct isolate of <Emphasis Type="Italic">Sugarcane yellow leaf virus</Emphasis> from China,representing a sixth new genotype

The 5803 nt genomic sequence of a Sugarcane yellow leaf virus (SCYLV) isolate (SCYLV-chn1) from China was determined. It covered more than 98% of the complete viral genome and contained all the six ORFs and the entire intergenic untranslated region. This isolate was most closely related to SCYLV genotype CUB (isolates CUB-YL1 and CB86010) with identities of 95.2–97.4% (nt) (93.2–97.2% aa) in ORF0, ORF1, and ORF2. Sequence comparison and phylogenetic analyses supported the view that this isolate represents a new genotype; SCYLV CHN1 was suggested as the name for this new genotype.     

Analysis of the complete DNA sequence of rice tungro bacilliform virus from southern India indicates it to be a product of recombination

The complete nucleotide sequence of an isolate of rice tungro bacilliform virus (RTBV), collected from Kanyakumari, India, where RTBV was reported recently for the first time, has been analyzed. Sequence comparison revealed that the RTBV isolate from Kanyakumari (RTBV-KK) has a high degree of identity to the two previously reported RTBV sequences from India, RTBV-AP and RTBV-WB, which had been collected from field locations about 10 years ago and 1000–2000 km away from the collection site of RTBV-KK. Most of the sequence domains reported previously in other RTBV isolates were found to be conserved in RTBV-KK. Closer inspection revealed RTBV-KK to be a possible recombinant between RTBV-AP and RTBV-WB in the genomic region encompassing the coat protein gene.     

Short communication: Molecular analysis of Potato leafroll virus isolates from the Czech Republic

The complete genomes of three Czech isolates VIRUBRA 1/045, VIRUBRA 1/046, and VIRUBRA 1/047 of Potato leafroll virus (PLRV) were sequenced and compared with 13 complete sequences of PLRV isolates available in GenBank. Among the Czech isolates, VIRUBRA 1/046 and 1/047 showed the highest nucleotide (nt) identity (98.7%). PLRV was the most conserved virus in both open reading frames (ORFs) 3 and 4. The most variable regions were ORFs 0 and Rap1. Interestingly, isolate VIRUBRA 1/045 significantly differed from the other two Czech isolates in ORFs 0 and 1. Moreover, we identified mutations in the amino acid (aa) sequences, which were specific for the Czech isolates. Phylogenetic analysis based on ORF0 showed that the Czech isolates could be classified in two of the three groupings of the phylogenetic tree obtained. This is the first report on sequence analysis of the genome sequences of PLRV isolates from the Czech Republic. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Sequence analysis of open reading frames (ORFs) 2 to 4 of a U.S. isolate of porcine reproductive and respiratory syndrome virus

Summary The sequence of ORFs 2 to 4 of a U.S. isolate of porcine reproductive and respiratory syndrome virus (PRRSV), ATCC VR2385, was determined by analysis of a cDNA library. The cDNA clones containing PRRSV specific sequences were selected using a VR2385 ORF 4 specific PCR probe and sequenced. The ORFs 2, 3 and 4 overlapped each other and encoded polypeptides with predicted M _r of 29.5 kDa (ORF 2), 28.7 kDa (ORF 3) and 19.5 kDa (ORF 4), respectively. No overlap was found between ORFs 4 and 5, and instead there was a 10 bp sequence which separated these two ORFs. The nucleic acid homology with corresponding ORFs of the European PRRSV isolate Lelystad virus (LV) was 65% for ORF 2, 64% for ORF 3 and 66% for ORF 4. Comparison of the ORF 4 sequences of VR2385 with that of another U.S. isolate MN-1b revealed only 86% amino acid sequence homology and the presence of deletions in the ORF 4 of MN-1b. Our results further strengthen the observation that there is sequence variation between US and European PRRSV isolates.     

Genetic diversity of beak and feather disease virus detected in psittacine species in Australia

The complete nucleotide (nt) sequence of eight isolates of beak and feather disease virus (BFDV) obtained from a range of psittacine species with psittacine beak and feather disease (PBFD) from throughout Australia were compared with the sequences of two BFDV isolates previously reported from Australia (BFDV-AUS) and America (BFDV-USA), respectively. All isolates had the same basic structure including the position of the open reading frames, the hairpin structure between ORF1 and ORF2, the nonanucleotide motif (TAGTATTAC) therein, the three motifs of Rep protein encoded from ORF1 and involved in rolling circle replication, and the P-loop motif previously described, but the genome size of the eight isolates ranged from 1992 to 2018 nt. Overall nt identity of the isolates compared to BFDV-AUS ranged from 84 to 97%; the variation was due to a combination of point mutations and a number of deletions and insertions ranging from 1 to 17 nt in size detected in both coding and noncoding regions. The identity of the nt sequence of ORF2 compared to BFDV-AUS varied from 80 to 99%, while the identity of the deduced amino acid sequences varied from 73 to 99%. Phylogenetic analysis grouped the isolates into four clusters but there were no apparent regional differences or differences related to the psittacine species of origin. While seven ORFs with the potential to encode proteins greater than 8.7 kDa were detected in the BFDV-AUS isolate described previously, only three of these ORFs were detected in all 10 BFDV isolates for which sequence data were available. The three ORFs were ORF1 that presumably encodes the Rep protein, ORF2 presumably the major capsid protein, and the ORF previously designated ORF5. The ORF5 was of two size classes in different isolates, 303 and 474 nt, and only the first 303 nt of the viruses with an ORF of 474 nt were common to the other isolates.     

Comparison of the structural protein coding sequences of the VR-2332 and Lelystad virus strains of the PRRS virus

Summary The 3-portion of the genome of a U.S. isolate of the porcine reproductive and respiratory syndrome (PRRS) virus, ATCC VR-2332, was cloned and sequenced. The resultant 3358 nucleotides contain 6 open reading frames (ORFs) with homologies to ORFs 2 through 7 of the European strain of the PRRS virus and other members of the free-standing genus of arteriviruses. Both VR-2332 and the European isolate (called the Lelystad virus) have been identified as infectious agents responsible for the swine disease called PRRS. Comparative sequence analysis indicates that there are degrees of amino acid identity to the Lelystad virus open reading frames ranging from 55% in ORF 5 to 79% in ORF 6. Hydropathy profiles indicate that the ORFs of VR-2332 and Lelystad virus correspond structurally despite significant sequence differences. These results are consistent with the biological similarities but distinct serological properties of North American and European isolates of the virus.     

Complete nucleotide sequences of the genomes of two isolates of apple chlorotic leaf spot virus from peach (Prunus persica) in China

The complete nucleotide sequences of two isolates of apple chlorotic leaf spot virus (Z1 and Z3) collected from peach in Henan Province, China, were determined. The genomes of both Z1 and Z3 were found to contain three open reading frames (ORFs). Sequence analysis showed that genomic sequences of Z1 and Z3 isolates shared 67.4%-82.9% and 67.2%-82.6% identity, respectively, with the other eight isolates of ACLSV that have been reported previously. Based on the putative amino acid sequences of the products of the three ORFs, Z1 and Z3 isolates showed the greatest identity to isolate PBM1 (GenBank accession number AJ243438) from plum and the least identity with isolate Ta Tao5 (GenBank Accession Number: EU223295) from peach. Considering the low level of sequence identity between Z1/Z3 isolate and Ta Tao5 isolate, two types of ACLSV may exist in peach.     

Segment-2 sequence analysis and cross-neutralization studies on some Indian bluetongue viruses suggest isolates are VP2-variants of serotype 23

Sequence analysis of segment 2 (seg-2) of three Indian bluetongue virus (BTV) isolates, Dehradun, Rahuri and Bangalore revealed 99% nucleotide identity amongst them and 96% with the reference BTV 23. Phylogenetic analysis grouped the isolates in ‘nucleotype D’. The deduced amino acid (aa) sequence of the Bangalore isolate showed a high variability in a few places compared to other isolates. B-cell epitope analyses predicted an epitope that is present exclusively in the Bangalore isolate. Two-way cross serum neutralization confirmed that Bangalore isolate is antigenically different from the other two isolates. The results of this study suggest that these three isolates are VP2 variants of BTV 23. This signifies that non-cross-neutralizing variants of the same BTV serotype should be included in vaccine preparation.