UBC Theses and Dissertations
Molecular cloning of blueberry shock ilarvirus (BSIV) RNA 3 containing the coat protein gene and identification of a virus associated with BSIV-infected blueberry blossoms Bremsak, Irene Jane
Blueberry shock ilarvirus (BSIV) was cloned using random primers. Complementary DNA (cDNA) was ligated into Bluescript II and used to transform into DH5a Escherichia coil. The plasmids were screened for BSIV RNA 3 cDNA by Northern, Southern and dot blots. Seven clones were restriction mapped. A 412 base pair region of three clones was sequenced and compared to tobacco streak, citrus variegation, apple mosaic and prune dwarf ilarvirus coat proteins (cp) at the nucleotide and amino acid levels. The BSIV cp amino acid sequence was 61% homologous with that of apple mosaic virus cp. BSIV virions were localized in ‘Bluetta’ and ‘Rancocas’ blueberry pollen by immunoelectron microscopy. The identity of a virus co-isolated with BSIV from blueberry blossoms was determined. It’s host range and symptomatology differed significantly from BSIV. Purified virions were isometric, Ca. 32 nm in diameter, with electron dense centres. The cp subunit had a molecular weight (Mw) of 30 kD. The genome was single-stranded and tripartite with M’s of 1.19, 1.01, 0.74 and 0.37 x 106 D and when the double-stranded RNA profile was compared to that of cucumber mosaic virus (CMV) isolated from primula, they were identical. Polyclonal antiserum and monoclonal antibodies were produced. The polyclonal antiserum reacted with CMV serotypes I and II; all the monoclonal antibodies reacted with CMV-II, although some monoclonal antibodies reacted with CMV-I. The virus, called CMV-B since it was isolated from blueberry blossoms, was an isolated of CMV-II. A relationship between BSIV and CMV in blueberry plants was investigated. None of the 650 BSIV infected blueberry tissue samples tested in 1993 were infected with CMV. Vegetation within and near the BSIV-infected field, was surveyed and seven of nineteen species tested were infected with CI\4V. It was concluded that CMV-infected pollen deposited by pollinators on the blueberry blossoms was the source and that blueberry was probably not a host of CMV.
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