Genome characterization of the mild strain of Banana bunchy top babuvirus
Date Issued
2005
Date
2005
Author(s)
Lin, Chia-Hua
DOI
zh-TW
Abstract
The causal agent of Banana bunchy top disease is Banana bunchy top babuvirus
(BBTV). The BBTV-infected banana plants show symptoms of dwarf, bunchy top,
leaf atrophy, vein clearing, dark-green streak on pseudostem, and were unable to
produce fruits in severe cases. BBTV is a complex circular single-stranded DNA
virus consist of six genomic integral components, DNA 1 ~ 6. DNA 1 encodes
master replication initiation protein (master Rep) for viral genomic replication.
Besides master Rep, components encode additional replication initiation protein
(additional Rep) with the self-only-replication function could also be isolated from
some BBTV-infected banana plants. BBTV strains can be classified into five types,
type I ~ V, defined by symptoms, and could be differentiated by polymerase chain
reaction (PCR)-based assay using C1, S and SR primer pairs. Type V is a mild strain,
and its complete genome organization has yet been characterized. In this study,
primer pairs based on conserved and specific regions of BBTV components were
designed. The designed primer pairs and total nucleic acids extracted from each type
V isolate was used in PCR reaction. The PCR products were cloned to construct
PCR-libraries. Clones from each PCR-library were analyzed by restriction fragment
length polymorphisms (RFLP). Randomly selected clones from each RFLP group
were sequenced. Based on result of analysis, five integral components, DNA 1 ~ 5,
were identified from isolates of type V, V-1 and V-2. However we were unable to
obtain DNA 6 from both isolates even two primer pairs designed from DNA 6
conserved region. No signal could be detected from total nucleic acids extracted of
V-1 or V-2 isolates by Southern blot analysis using DNA 6 open reading frame as
probes. However we were also unable to detect DNAs in the nucleic acids by
Southern blot analysis. It indicates that the amounts of BBTV genomic DNA are
much lower in the mild strains. Real-time quantitative PCR were performed to
quantify the amounts of DNA 1 and DNA 3. The result indicated the amount of
DNA 1 and DNA 3 in mild strain isolates were fewer than in severe strain isolates by
more than 1000 folds. These data suggest that DNA 6 was absent or had some
sequence changes in mild strain. Besides, mild strain had a ca. 0.5 kb PCR product
amplified by S primer pairs. After cloning and sequencing of these fragments, we
found that they are defective forms of master Reps, and they could be classified into
five subgroups by sequence homologies and genome organizations. The result of
immunocapture PCR suggests these defective Reps are incapsidated. In addition to
the 0.5 kb defective master Reps, another 0.7 kb defective master Rep could also be
identified from V-2 isolate. Further studies are needed to resolve if the absence of
ordinary DNA 6 and/or the presence of defective Rep could account for symptoms
amelioration of mild strain virus infection.
Subjects
香蕉萎縮病毒
輕微病徵型系統
基因體
Banana bunchy top virus
mild strain
genome
Type
other
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