A nucleotide sequence rearrangement distinguishes two isolates of satellite tobacco ringspot virus RNA

Several strains of tobacco ringspot virus (TobRV) support the replication and encapsidation of satellite tobacco ringspot virus RNA (STobRV RNA). We have compared the nucleotide sequences of four STobRV RNAs, each initially associated with a different isolate of TobRV. A STobRV RNA from a geranium isolate of TobRV and STobRV RNA from the previously analyzed budblight isolate (J.M. Buzayan, W.L. Gerlach, G. Bruening, P. Keese, and A.R. Gould, 1986, Virology 151, 186-199) differed by a single nucleotide residue substitution. STobRV RNAs from TobRV isolates 62L and NC-87 have the same 360-residue nucleotide sequence. This sequence differs from that of the 359-nucleotide residue budblight STobRV RNA principally at locations 100 through 140. The differences between the two sequences in this region are consistent with a rearrangement of blocks of nucleotide residues. The two sequences can be folded with similar patterns of base pairing. All four STobRV RNAs share a sequence of eighty 5'-terminal and of twenty 3'-terminal residues, including the 5' hydroxyl group and 2':3'-cyclic phosphodiester group.     

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.     

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.     

Complete RNA1 sequences of two UK isolates of barley mild mosaic virus: a wild-type fungus-transmissible isolate and a non-fungus-transmissible derivative

The complete RNA1 sequences of two isolates (fungus transmissible and non-fungus transmissible) of barley mild mosaic virus (BaMMV) were obtained. The two isolates' RNA1 sequences had very high sequence identity (99.3%), and of the 15 amino acid differences (out of 2258) between the putative polyproteins, 11 were conservative and unlikely to affect the structure or function of the protein. The remaining amino acid differences were thought unlikely to affect fungus transmission because they occur in the CI- and NIb-coding regions. This strongly suggests that the P73 protein of RNA2 (which has a 364-aa deletion in the non-fungus-transmissible isolate) is involved in fungus transmission of BaMMV.     

Replication of tobacco ringspot virus. II. Differences in nucleotide sequences between the viral RNA components

Tobacco ringspot virus (TRSV)-specific double-stranded (ds)-RNA was used in competition hybridization experiments to compare the nucleotide sequences of the viral RNAs. The results indicate that the RNA of molecular weight 1.4 × 10⁶ (RNA₁) isolated from either middle or bottom component particles of TRSV have indistinguishable nucleotide sequences. However, the sequences of the RNA of molecular weight 2.3 × 10⁶ (RNA₂) are distinct from those of RNA₁ although these RNAs may have sequences of about 900 nucleotides in common.     

Multimeric forms of satellite of tobacco ringspot virus RNA

An approximately 350-nucleotide residue RNA replicates in association with tobacco ringspot virus (TobRV) and becomes encapsidated in TobRV coat protein. Here we show by electrophoretic analyses that this small satellite RNA, RNA S, is the most abundant and most rapidly migrating of a series of at least ten encapsidated RNAs with RNA S sequences. A largely double-stranded RNA fraction from infected tissue, when denatured, gave a similar series of up to 12 zones that contained both RNA S sequences and sequences that hybridized to RNA S. Analysis of the mobilities suggests a weight increment between each zone corresponding approximately to the size of RNA S. Thus the more slowly migrating zones appear to contain covalent multimers of RNA S or, for tissue RNA, both multimers of RNA S and multimers of the complement of RNA S sequences. Neither terminal structure of TobRV genomic RNAs was found in the satellite RNA. RNA S lacks detectable polyadenylate or oligoadenylate. Covalently linked protein was not detected in RNA S or its more slowly migrating forms, and satellite RNA biological activity, unlike that of the TobRV RNAs, was not protease sensitive. Polynucleotide kinase catalyzed the phosphorylation of satellite RNAs, indicating free 5'-hydroxyl groups.     

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.     

Phylogenetic and serological analysis of turnip ringspot virus and radish mosaic virus isolates

Turnip ringspot virus (TuRSV) has been proposed to be a member of a new species in the genus Comovirus. Its remarkable host-range similarity to radish mosaic virus (RaMV) may have led to its misrecognition in the past. Findings from both sequence analysis and serological tests support the assignment of TuRSV to a new comovirus species. In addition, phylogenetic analysis suggests that the two genome segments of some TuRSV isolates have a heterogeneous origin.     

Complete sequence of RNA1 of grapevine Anatolian ringspot virus

The nucleotide sequence of RNA1 of grapevine Anatolian ringspot virus (GARSV), a nepovirus of subgroup B, was determined from cDNA clones. It is 7,288 nucleotides in length excluding the 3′ terminal poly(A) tail and contains a large open reading frame (ORF), extending from nucleotides 272 to 7001, encoding a polypeptide of 2,243 amino acids with a predicted molecular mass of 250 kDa. The primary structure of the polyprotein, compared with that of other viral polyproteins, revealed the presence of all the characteristic domains of members of the order Picornavirales, i.e., the NTP-binding protein (1B^Hel), the viral genome-linked protein (1C^VPg), the proteinase (1D^Prot), the RNA-dependent RNA polymerase (1E^Pol), and of the protease cofactor (1A^Pro-cof) shared by members of the subfamily Comovirinae within the family Secoviridae. The cleavage sites predicted within the polyprotein were found to be in agreement with those previously reported for nepoviruses of subgroup B, processing from 1A to 1E proteins of 67, 64, 3, 23 and 92 kDa, respectively. The RNA1-encoded polyprotein (p1) shared the highest amino acid sequence identity (66 %) with tomato black ring virus (TBRV) and beet ringspot virus (BRSV). The 5′- and 3′-noncoding regions (NCRs) of GARSV-RNA1 shared 89 % and 95 % nucleotide sequence identity respectively with the corresponding regions in RNA2. Phylogenetic analysis confirmed the close relationship of GARSV to members of subgroup B of the genus Nepovirus.     

Unique variations of pbp2b sequences in penicillin-nonsusceptible Streptococcus pneumoniae isolates from Korea

pbp2b gene alterations were analyzed in 102 clinical isolates of Streptococcus pneumoniae (30 penicillin susceptible, 23 intermediate, and 49 resistant) from Korea. On the basis of PBP2B amino acid sequences, penicillin-nonsusceptible isolates of S. pneumoniae belonged to six groups, and 76% of the isolates in groups I to IV showed the same divergent block of amino acid alterations. Thirteen isolates (group II) also possessed a divergent block that was identical to that of Streptococcus oralis. The pbp2b genes of most Korean isolates showed novel mosaic mutations due to horizontal gene transfer. The Thr252 --> Ala substitution, previously thought to be associated only with penicillin-nonsusceptible strains, was also found in three penicillin-susceptible strains. On the basis of their pbp2b nucleotide sequences, all penicillin-nonsusceptible isolates can be detected by multiplex PCR, which can be used as a novel method for detection of antibiotic-resistant pneumococcal strains in clinical specimens.     

RNA2 of TRV SYM breaks the rules for tobravirus genome structure

Currently, all of the RNA2 molecules described for all of the more than thirty sequenced isolates of the three tobraviruses, Tobacco rattle virus (TRV), Pea early-browning virus (PEBV) and Pepper ringspot virus (PepRSV), have the virus coat protein (CP) gene located in the 5' proximal position. However, sequencing of the RNA2 of the SYM isolate of TRV revealed that this isolate has a unique genome structure in which the virus CP gene is located in the central region of RNA2 downstream of three completely novel open reading frames (ORFN1, ORFN2 and ORFN3). An infectious clone of SYM RNA2 was constructed and mutations were introduced separately into each of the novel genes to interrupt their translation. However, none of the mutations resulted in any noticeable change in the ability of TRV RNA1 or RNA2 to replicate and move systemically in the leaves or roots of infected plants. In addition, individual expression of the novel ORFs either from a Potato virus X (PVX) vector or from a binary plasmid in Agrobacterium tumefaciens did not reveal any potential function.     

Comparisons among isolates of Sweet potato feathery mottle virus using complete genomic RNA sequences

We determined the complete or partial nucleotide sequences of eight Sweet potato feathery mottle virus (SPFMV) isolates and compared them with 12 other partial SPFMV sequences. The genome organization of the isolate Bungo (strain group C) was very different from those of isolates in the russet crack, ordinary (O), and east Africa groups. 10-O appeared to be a recombinant of isolates S and O, with a recombination site within the P1 gene. This study will help to provide a better understanding of the taxonomy and biology of SPFMV and how these features relate to virulence.     

Phylogenetic analysis of two Chinese orf virus isolates based on sequences of B2L and VIR genes

Orf virus (ORFV) is an enveloped virus with a double-stranded DNA genome, causing a contagious pustular dermatitis, mainly in goats and sheep. In this study, two strains of ORFV were isolated from sheep and goat samples in Xinjiang and Shaanxi, China. The B2L and virus interferon resistance (VIR) genes of these two isolates were sequenced and analyzed after PCR amplification. Phylogenetic analysis showed that the two isolates clustered with other ORFV strains but were separated into different subgroups. The Xinjiang strain shared the highest homology with the Gansu strain, whereas the Shaanxi strain shared higher homology with the Taiwan and Hubei strains. This is the first report of the molecular characterization of ORFV in Northwest China, and it provides new information on the genotyping of the causative agents responsible for contagious ecthyma dermatitis outbreaks in China.     

The nucleotide sequences of the RNA 1 and RNA 2 of asparagus virus 2 show a close relationship to citrus variegation virus

Archives of Virology -     

Comparative genetic analysis of genomic DNA sequences of two human isolates of Tanapox virus

Members of the genus Yatapoxvirus, which include Tanapox virus (TPV) and Yaba monkey tumor virus, infect primates including humans. Two strains of TPV isolated 50 years apart from patients infected from the equatorial region of Africa have been sequenced. The original isolate from a human case in the Tana River Valley, Kenya, in 1957 (TPV-Kenya) and an isolate from an infected traveler in the Republic of Congo in 2004 (TPV-RoC). Although isolated 50 years apart the genomes were highly conserved. The genomes differed at only 35 of 144,565 nucleotide positions (99.98% identical). We predict that TPV-RoC encodes 155 ORFs, however a single transversion (at nucleotide 10241) in TPV-Kenya resulted in the coding capacity for two predicted ORFs (11.1L and 11.2L) in comparison to a single ORF (11L) in TPV-RoC. The genomes of TPV are A+T rich (73%) and 96% of the sequence encodes predicted ORFs. Comparative genomic analysis identified several features shared with other chordopoxviruses. A conserved sequence within the terminal inverted repeat region that is also present in the other members of the Yatapoxviruses as well as members of the Capripoxviruses, Swinepox virus and an unclassified Deerpox virus suggests the existence of a conserved near-terminal sequence secondary structure. Two previously unidentified gene families were annotated that are represented by ORF TPV28L, which matched homologues in certain other chordopoxviruses, and TPV42.5L, which is highly conserved among currently reported chordopoxvirus sequences.     

Update and comparison of the immediate-early gene DNA sequences of two pseudorabies virus isolates

Differences between the immediate-early gene DNA sequences of two pseudorabies virus isolates (Indiana-Funkhauser and Ka) were resolved and confirmed. The deduced amino acid sequences showed that regions 2 and 4 have fewer changes than the rest of the molecules. These two conserved regions may be functionally important.     

The first complete genome sequences of two distinct European tomato spotted wilt virus isolates

Tomato spotted wilt virus (TSWV) represents a major constraint to the production of important vegetable and ornamental crops in several countries around the world, including those in Europe. In spite of their economic importance, European TSWV isolates have only been partially characterized, and a complete genome sequence has not been determined yet. In this study, we completed the whole genome sequence of two distinct TSWV isolates from Italy, p105 and p202/3WT. The sequences of the L and M segments of p105 and of the L segment of p202/3WT were determined using a combined approach of RT-PCR and small RNA (sRNAs) contig assembly. Phylogenetic analysis based on RNA-dependent RNA polymerase and G_N/G_C protein sequences grouped the two isolates in two different clades, showing that different evolutive lineages are present among Italian TSWV isolates. Analysis of possible recombination/reassortment events among our isolates and other available full-length genome TSWV sequences showed a likely reassortment event involving the L segment.