Molecular characterization and detection of plum bark necrosis stem pitting-associated virus.

The complete RNA genome of plum bark necrosis stem pitting-associated virus (PBNSPaV) was cloned and sequenced and was determined to be 14, 214 nts long. The genome structure revealed seven major open reading frames (ORFs), and nontranslated regions at the 5' and 3' ends. PBNSPaV represents the simplest genome organization in the genus Ampelovirus, family Closteroviridae. The ORFs 1a and 1b encode, respectively, a large polyprotein with a molecular mass (Mr) of 259.6 kDa containing conserved domains characteristic of a papain-like protease, methyltransferase and helicase (ORF1a) and a 64.1-kDa protein of eight conserved motifs characteristic of viral RNA-dependent RNA polymerase (RdRp) (ORF1b). ORF1b is presumably expressed via a +1 ribosomal frameshift mechanism. ORF2 encodes a small 6.3-kDa hydrophobic protein of unknown function. ORF3 encodes a 57.4-kDa protein, a homologue of the HSP70 family of heat shock proteins. ORF4 encodes a 61.6-kDa protein with unknown function. ORF5 encodes a 35.9-kDa capsid protein (CP). Lastly, ORF6 encodes a 25.2-kDa minor capsid protein (CPm). Phylogenetic analyses performed on sequences of the HSP70h RdRp and CP support classification of the virus in the genus Ampelovirus. A real-time TaqMan RT-PCR assay and a one-step RT-PCR were developed for PBNSPaV detection and compared using three different sample preparation methods.

Nucleotide sequence and rt-PCR detection of a virus associated with grapevine rupestris stem-pitting disease.

ABSTRACT Grapevine rupestris stem pitting (RSP) is a graft-transmissible disease of unknown etiology. We have characterized a virus associated with this disease. The entire genomic sequence (GenBank accession number AF026278) consisted of 8,725 nucleotides excluding a poly(A) tail. Six open reading frames (ORF) were found. ORF1 potentially encodes a polypeptide with a methyltransferase domain, a papain-like proteinase domain, a helicase domain, and a RNA-dependent RNA polymerase domain; ORF2, ORF3, and ORF4 compose a triple-gene block; ORF5 encodes a coat protein; and ORF6 is located near the 3' end with unknown function. Sequence analysis indicated that the virus is most similar to apple stem-pitting virus and may be allied with the carla- and potexviruses and grouped with other viruses that infect woody hosts. A specific reverse-transcription polymerase chain reaction (RT-PCR)-based detection method was developed. Among 62 grapevine sources known to be infected with rupestris stem-pitting disease, 60 sources tested positive by RT-PCR. Among 43 healthy vines tested, all were negative. The name grapevine rupestris stem-pitting-associated virus is proposed.

Long, nearly identical untranslated sequences at the 3' terminal regions of the genomic RNAs of cherry leafroll virus (walnut strain).

Hybridization analyses of cDNA clones derived from the two genomic RNAs, RNA1 and RNA2, of the walnut strain of the nepovirus cherry leafroll nepovirus (wCLRV) demonstrated a long region of high homology between the two viral RNAs. Subsequent mapping and nucleotide sequencing revealed a long, noncoding, presumably untranslated, region (3' UTR) immediately 5' of the terminal polyadenylate, a region that is almost identical in the two RNAs. This 3' UTR is 1567 nucleotide residues long in RNA1. Homologies of about 80% were found with corresponding regions of genomic RNAs from other strains of CLRV, but not with the corresponding regions of other nepovirus genomic RNAs.