Complete nucleotide sequence and molecular probing of potato virus S genome.

Complete genomes of three isolates of Potato virus S (PVS) were cloned and sequenced. The PVS ORF-1 was characterized for the first time. It encodes a putative replication protein (RPT) that shares the highest homology (about 52%) with that of Blueberry scorch virus (BlScV). ORF-1 motifs, characteristic for carlaviruses were found for methyltransferase (MTR), helicase (HEL) and RNA-dependent RNA polymerase (RdRp). The complete sequence of PVS genome enabled to develop an immunocapture RT-PCR probing of the PVS genome. Using this system, the sequence variability of 11 genome zones was examined for 34 PVS isolates including 15 PVS-CS variants that caused a systemic infection in Chenopodium quinoa. A broad variability between PVS isolates and diverse sequence variants was found. cDNA fragments covering the coat protein (CP) leader and CP-coding region (approx. 420 bp) were pooled for PVS-O and Chenopodium-systemic PVS isolates (PVS-CS) and corresponding cDNA libraries were screened for sequence variants. Both cDNA pools differred mainly in the 5'-end of the CP gene. Methionine at the position 17 in combination with serine at the position 34 were frequently associated with the CS character of PVS. In general, hydrophobic and polar amino acids were characteristic for the positions 17 and 34, respectively in PVS-CS isolates. Genome probing and evolutionary distances suggested that the PVS-CS isolates analyzed were close to the ordinary European isolates of ordinary strain of PVS (PVS-O) but distant to the original Andean strain of PVS (PVS-A).

Complete nucleotide sequence of the genomic RNA of poplar mosaic virus (Genus Carlavirus).

The complete nucleotide sequence of the Poplar mosaic virus (PopMV) genome was determined. The genomic RNA of PopMV is 8,742 nucleotides in length. Comparative sequence analysis supports earlier research suggesting that this virus is a member of the genus Carlavirus. For example, as is the case for all carlaviruses, there are 6 predicted ORFs in the PopMV genome. The first ORF, ORF1, encodes a predicted helicase/replicase, which corresponds to ORF1 from other carlaviruses, while ORF2, ORF3, ORF4, ORF5 and ORF6 encode the three triple block proteins, the coat protein, and a putative nucleic acid-binding protein respectively.

The complete nucleotide sequence of the genome RNA of Lily symptomless virus and its comparison with that of other carlaviruses.

The complete genomic nucleotide sequence and genome structure of Lily symptomless virus (LSV), a lily-infecting carlavirus, have been obtained. The genome of the Korean strain of LSV, LSV-Kr, was 8394 nucleotides long and contained six open reading frames (ORFs) coding for proteins of Mr 220 kDa (1948 aa), 25 kDa (228 aa), 12 kDa (106 aa), 7 kDa (64 aa), 32 kDa (291 aa) and 16 kDa (140 aa) from the 5' to 3' end, respectively, which is typical of carlaviruses. Genetic heterogeneity was observed in the ORF1 gene. A total of 221 of 5,847 nucleotides (nt) were heterologous in the ORF1 of replicase; 162 nt portions were silent and 59 nt resulted in amino acid changes. This heterogeneity indicates that the LSV-infecting lily plants contained a genetically heterogeneous population of LSV (quasispecies). Overall similarities to those of other carlaviruses for the six ORFs of LSV were from 76.1% to 31.6% and from 87.3% to 13.7%, at nucleotide and amino acid levels, respectively. The ORF1 replicase gene of LSV shares 40.9% to 56.8% and 48.9% and 58.6% identities with that of 5 other carlaviruses at the amino acid and nucleotide levels, respectively. LSV was closest to Blueberry scorch virus (BlScV) in this ORF, among the carlaviruses for which sequence information is available. The three triple gene blocks (ORF2-4), ORF5 (coat protein) and 3'-proximal 16 kDa ORF6 genes were further analyzed, and phylogenetic trees for the coding regions indicate that the LSV was the most closely related to Kalanchoe latent virus and BlScV. This is the first report of the complete nucleotide sequence and genome structure of LSV.

Characterization of a distinct carlavirus isolated from Verbena.

Avirus was isolated from Verbena plants that bore virus-like symptoms. The virus, for which the name Verbena latent virus (VeLV) is proposed, was consistently isolated from these plants, both with and without disease symptoms. Electron microscopy studies of ultrathin sections of infected Verbena tissues revealed the presence of elongated flexuous virus particles, ca. 650 nm in length. Its experimental host range was limited to Verbena spp. and Nicotiana clevelandii. No inclusion bodies or specific cytopathological effects, were observed. Electrophoresis of dissociated purified virus preparation in sodium dodecyl sulfate-polyacrylamide gel revealed a major protein component with a molecular mass of 38.9 kDa. Polyclonal antibodies which could specifically bind to virus particles were produced. A portion of the viral RNA was cloned and sequenced; it comprised 2503 nucleotides and contained part of three open reading frames (ORFs) which from the 5' to the 3'-ends, potentially encode for 489 amino acids (ORF1), a 25.8-kDa protein (ORF2) and a 12-kDa protein (ORF3). Comparison of the predicted amino acid sequence with those of other plant viruses revealed 40-60% identity with several carlaviruses. In the light of particle morphology, absence of specific cytopathological effects in ultrathin sections, and genomic and serological properties, it is suggested that this virus belongs to the genus Carlavirus.

Complete genome sequence of garlic latent virus, a member of the carlavirus family.

The complete genome sequence of the garlic latent virus (GLV) has been determined. The whole GLV genome consists of 8,353 nucleotides, excluding the 3'-end poly(A)+ tail, and contains six open-reading frames (ORFs). Putative proteins that were encoded by the reading frames contain the motifs that were conserved in carlavirus-specific RNA replicases, NTP-dependent DNA helicases, two viral membrane-bound proteins, a viral coat protein, and a zinc-finger. Overall, the GLV genome shows structural features that are common in carlaviruses. An in vitro translation analysis revealed that the zinc-finger protein is not produced as a transframe protein with the coat protein by ribosomal frameshifting. A Northern blot analysis showed that GLV-specific probes hybridized to garlic leaf RNA fragments of about 2.6 and 1.5 kb long, in addition to the 8.5 kb whole genome. The two subgenomic RNAs might be encapsidated into smaller viral particles. In garlic plants, 700 nm long flexuous rod-shaped virus particles were observed in the immunoelectron microscopy using polyclonal antibodies against the GLV coat proteins.

Complete nucleotide sequence of the genomic RNA of Aconitum latent virus (genus Carlavirus) isolated from Delphinium sp..

We determined the complete nucleotide sequence of Aconitum latent virus (AcLV-D) isolated from Delphinium sp. The genomic RNA of AcLV-D is 8,657 nucleotides in length, excluding the poly (A) tail. Based on computer analysis, six open reading frames (ORFs) were identified as for other carlaviruses. Although each ORF differs from those of previously reported carlaviruses, the coat protein of AcLV and Potato virus M (PVM) shows a high level of identity. The results suggest that AcLV is a distinct carlavirus species but closely related to PVM.

Differentiation of Allium carlaviruses isolated from different parts of the world based on the viral coat protein sequence.

Common primers which amplify the 3' terminal genomic RNAs of Allium carlaviruses were designed based on the nucleotide sequence of shallot latent virus (SLV), garlic latent virus (GLV) and garlic common latent virus (GCLV). A total of fifteen cDNAs encoding the coat protein (CP) of the carlaviruses, including the biologically identified isolates SLV, GLV and GCLV as well as viruses from infected Allium plants cultivated in different parts of the world, were amplified by RT-PCR with the common primers. The cDNAs were then cloned and sequenced. The predicted viral CP amino acid sequence as well as the nucleotide sequence revealed that SLV and GLV, previously considered as separate viruses on the basis of their biological and physical properties, belong to the same species of the genus Carlavirus. Both viruses are clearly differentiated from GCLV. In addition, every SLV and GLV isolate from the Allium plants in Taiwan showed characteristic and common variations in their CP sequences, suggesting the possible presence of geographical variants. However, no apparent sequence variations of SLV and GLV related to their host plant species, including A. sativum, A. wakegi, A. chinense, A. fistulosum, A. cepa and A. ampeloprasum, were observed. These findings suggested that the sequence variations observed in the respective virus isolates do not correlate with the specificity of their infectivities for Allium species.