Molecular Cloning, Sequence Analysis, and Detection of Banana Bunchy Top Virus in Hawaii

Abstract

The Hawaiian isolate of banana bunchy top virus (BBTV) was purified from infected banana cultivar Williams. Three single-stranded DNA (ssDNA) components were cloned and sequenced; they were named component 1, 3, and 4, respectively. Component 1 is 1,110 nucleotides in length and shares 98% nucleotide sequence identity with the BBTV DNA component 1 of the Australian isolate. This component contains two open reading frames (ORF) capable of encoding a protein of 33.5 kDa, which may function as a replease, and a protein about 15.2 kDa, with unknown functions. Component 3 is 1,057 nucleotides in length and does not contain any ORFs larger than 10 kDa. Component 4 is 1,017 nucleotides in length and potentially encodes a protein of 18.9 kDa. All three ssDNA components share the same stem-loop sequence and have a conserved noncoding region. The sequence of each of these three components is different from that of BBTV DNA components of two Taiwanese isolates. BBTV-specific clones were used in dot-blot hybridization assays for detection of BBTV in plants using radioactive and nonradioactive probes. A polymerase chain reaction (PCR) assay was developed for detection of BBTV in banana samples and single aphids. Dot-blot hybridization assays were as sensitive as enzyme-linked immunosorbent assay (ELISA), while PCR was 1,000 times more sensitive than dot-blot and ELISA assays for detection of BBTV in banana