A viral dsRNA element of the chestnut blight fungus with a distinct genetic organization.

We have sequenced overlapping complementary DNA clones representing the viral double-stranded (ds) RNA from hypovirulent strain NB58 of the chestnut blight fungus Cryphonectria parasitica. Cryphonectria hypovirus 2-NB58 (CHV2-NB58) dsRNA contains 12,507 base pairs, excluding the poly(A) tail at the 3' end of the plus strand, and is organizationally similar to the largest dsRNA from the virus of strain EP713 (CHV1-713; identical to HAV; Shapira et al., (1991), EMBO J. 10, 731-739). CHV2-NB58 and CHV1-713 dsRNAs share approximately 60% nucleotide sequence identity. On the poly(A)-containing strand of CHV2-NB58, a 487-residue nontranslated region precedes two open reading frames, designated ORF A (438 codons) and ORF B (3291 codons). The connecting pentanucleotide sequence UAAUG (1802-1806) terminates ORF A and initiates ORF B. In contrast to the 69-kDa ORF A product of CHV1-713, the 50-kDa CHV2-NB58 ORF A product did not undergo autoproteolysis under the conditions tested, nor were motifs associated with cysteine proteases present in the CHV2-NB58 ORF A sequence. CHV2-NB58 ORF B products appear to be homologous with CHV1-713 ORF B products, and the motifs involved in autoproteolysis of the N-terminal 48 kDa of CHV1-713 ORF B were identified in the CHV2-NB58 ORF B product. Motifs associated with RNA polymerase and helicase activities were highly conserved between CHV2-NB58 and CHV1-713 and were found at similar genomic positions in the C-terminal half of ORF B.

Nucleotide sequence of the carlavirus associated with blueberry scorch and similar diseases.

We have synthesized and mapped a library of cDNA clones representing the RNA genome of a strain of blueberry scorch carlavirus (BBScV) associated with a disease known locally, in New Jersey, U.S.A., as Sheep Pen Hill disease. The nucleotide sequence of that strain was determined to be 8514 residues, excluding the poly(A) tail. In addition, cDNA clones representing the 3' terminus of another strain of the virus from the same field were synthesized, mapped and sequenced. The overall identity between sequences of these two strains was approximately 90% spanning the 1634 residue overlap, confirming their identity as distinct strains and not simply different isolates of a single strain. Finally, the coat protein gene of a distinct strain of the virus, isolated from plants with blueberry scorch disease in the Puyallup Valley in Washington State, U.S.A., was cloned from total cDNA by PCR. Sequence analysis revealed that the strain from Washington was more divergent from the two New Jersey strains than they were from each other. Comparisons of these sequences with other carlavirus sequences indicated that BBScV is more closely related to lily symptomless virus and potato virus S than to potato virus M, Helenium virus S, carnation latent virus or poplar mosaic virus. BBScV and potato virus M shared approximately 54% nucleotide sequence identity overall.

First field isolation of wound tumor virus from a plant host: minimal sequence divergence from the type strain isolated from an insect vector.

A new strain of wound tumor virus (WTV) has been isolated from a periwinkle plant (Catharanthus roseus) that was among several used as bait plants in a blueberry field. The 12 segments of double-stranded RNA of the viral genome were isolated directly from infected tissue and found to have mobilities through agarose gels that were identical to those of the type strain WTV. Coupled complementary DNA (cDNA) and polymerase chain reactions (PCR) primed with oligonucleotides complementary to the termini of segments 4-12 of the type strain of WTV successfully amplified those segments. Amplification products of the 9 segments were of the size expected for the full-length segment, with no shorter than full-length products representing defective RNAs detected. PCR products representing segments 7, 11, and 12 were cloned and sequenced in their entirety. The sequence of each segment varied only slightly from the homologous segment of the type strain. Variation ranged from less than 1% for segment 12 to approximately 3% for segment 7, but even these low levels of variation were much greater than the variation found in WTV isolates maintained in the laboratory. Most of the variation in each of the three segments was confined to the coding regions, and most of the differences were third position transitions. The new WTV strain has been designated WTVNJ.