GENOME CHARACTERIZATION, INFECTIVITY AND DEVELOPMENT OF IMMUNODIAGNOSTIC FOR A BADNAVIRUS ASSOCIATED WITH BANANA
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Date
2015
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DIVISION OF PLANT PATHOLOGY INDIAN AGRICULTURAL RESEARCH INSTITUTE NEW DELHI
Abstract
A cryptic Badnavirus species complex, known as banana streak viruses (BSV) poses a
serious threat to banana production and genetic improvement worldwide. The
presence of endogenous BSV (eBSV) sequences in the banana genome and their
potential to express as episomal viruses in natural genetic hybrids of banana
complicates the diagnosis, characterization and management of BSV. Complete
genome sequences of three episomal Banana streak MY virus (BSMYV) isolates
sampled from triploid banana hybrids (Chini Champa: AAB; Malbhog: AAB and
Monthan: ABB), grown in North-East and South India was achieved by sequence
independent improved rolling circle amplification (RCA). Episomal origin of two
shorter Banana streak OL virus (BSOLV) variants containing deletions at C-terminal
of ORF3 and intergenic region was confirmed by RCA. RCA coupled with restriction
fragment length polymorphism (RFLP) revealed diverse restriction profiles in five
BSMYV isolates including above three isolates. Episomal BSMYV isolates
characterized in present study shared an identity of 45-50% with other BSV species
and 43-44% with sugarcane bacilliform viruses (SCBV) and clustered in major cluster
1 along with other badnaviruses infecting banana, together with sugarcane infecting
badnaviruses from Guadeloupe (SCBGAV and SCBGDV). Nucleotide substitution
rates of BSMYV subpopulation and BSOLV subpopulation was 7.13 x 10-3 to 1.59 x
10-2
and 2.65 x 10-3
to 5.49 x 10-3
respectively, for the different coding regions,
indicating low subpopulation variation. The evolutionary analysis indicated that
banana and sugarcane badnaviruses share similar patterns for different coding regions
with both the virus populations being under negative or purifying selection. In
addition to the existence of extensive recombination within the banana streak viruses
and sugarcane bacilliform viruses (intra-BSV and intra-SCBV recombination events),
a total of 32 unique recombination events within banana and sugarcane badnaviruses
(inter BSV-SCBV) were detected. Many unique fragments were shown to contain
similar ruminant sequence fragments which indicated that banana and sugarcane
badnaviruses or their ancestors might have colonized the same host before making the
host shift. The patterns of distribution of recombination events, hot-spots (intergenic
region and C-terminal of ORF3) as well as cold-spots (distributed in ORF3) displayed
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the mirroring of recombination traces in both the group of badnaviruses. The putative
coat protein (CP) coding region (p37) of BSMYV was identified in silico by
comparison with caulimoviruses, retroviruses and Rice tungro bacilliform virus. The
p37 was in vitro expressed as recombinant protein in pMAL system and used as
antigen for raising polyclonal antiserum. The antiserum specifically reacted with
BSMYV virions in immunosorbent electron microscopy (ISEM) and antigen coated
plate-enzyme linked immunosorbent assay (ACP-ELISA). The anti MBP-p37
antiserum (1:2000) was successfully used in ACP-ELISA for specific detection of
BSMYV infection in field and tissue culture raised banana plants. To further simplify
the methodology of antigen preparation, synthetic peptides representing antigenic
epitopes were successfully used for production of polyclonal antibodies to BSMYV.
Two immunodominant linear epitopes were identified at N and C-terminal of putative
CP of BSMYV (pep-I and pep-II), synthesized and used for polyclonal antiserum
production. Only anti pep-I antiserum strongly reacted with BSMYV virions in ISEM
and ACP-ELISA (1:2000 and 1:4000) in crude sap exhibiting >3 folds differences in
optical density (OD) values of infected and healthy samples. Globulin (IgG) fraction
of the anti pep-I antiserum was conjugated with alkaline phosphatase (ALP) and used
successfully as secondary antibodies in double antibody sandwich-ELISA (DASELISA)
with good serological differentiation among healthy and infected samples
although showing slow reaction. Employing the immunoreagents developed in present
study a sensitive duplex-immunocapture-PCR (D-IC-PCR) was standardized for the
sensitive, reliable and accurate routine indexing of episomal BSV infection in tissue
cultured and field banana samples. In a survey for banana streak disease in North,
North-East, East, West and South India, 46% of the collected samples were indexed
positive for BSV infection indicating its widespread occurrence. A partial tandem
dimer containing 1.8-mer of BSMYV-IN1 was constructed in binary vector. The
agroinoculated banana plants were not positive for BSMYV DNA in RCA three
months post-inoculation which might be due to the homology dependent silencing
because of the presence of integrant eBSV sequences in inoculated banana plants.
Banana plants agroinoculated with a combination of 1.8-mer construct
(pC1.8BSMYV-IN1) and viral suppressor constructs (HC-Pro and p19) (2:1) were
detected positive for BSMYV DNA in D-IC-PCR, which is a step towards proving the
Koch’s postulates.
Description
T-9115
Keywords
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