PhD

The present investigation was undertaken to develop recombinant coat protein (rCP) of
Banana bunchy top virus (BBTV) and Banana bract mosaic virus (BBrMV) for
immunodetection of the viruses. The experiments were conducted at the Virology Lab,
Banana Research Station, Kannara; Department of Plant Pathology, College of Agriculture,
Vellanikkara, Kerala Agricultural University and Indian Institute of Science, Bengaluru
during the period of 2016-2020.
A roving survey in 10 districts of Kerala, divided into population subsets viz., North,
Central and Southern zones were conducted for sample collection. After a preliminary
DAC-ELISA, 17 and 12 representative samples respectively were selected and carried
forward for further evaluations. The CP gene of BBTV was amplified from the total DNA
isolated using reported primers by Polymerase Chain Reaction (PCR) and that of BBrMV
by Reverse Transcriptase-PCR (RT-PCR). The CP gene sequences of these isolates were
determined and submitted in the NCBI-GenBank Database. The 17 BBTV isolates were
designated as MT174314-MT174330 and the 12 BBrMV isolates as MT818176-
MT818187.
It was inevitable to evaluate the molecular diversity of the viruses prior to devising nucleic-
acid based and serological detection methods. The phylogeographic analysis depicted a
clear demarcation of BBTV Kerala isolates based on geography whereas no such clustering
was observed in the case of BBrMV isolates. Being an RNA virus, the molecular diversity
of BBrMV (ranging between 1-12 %) was higher than BBTV. However, the 5’ and 3’
terminal of BBrMV CP gene was hypervariable and found unsuitable to be targeted for
nucleic-acid based detection. Hence, forward primer was designed from the NIb region of
ssRNA genome of BBrMV and reverse primer from 3’ UTR region upstream and
downstream to the CP gene respectively. For nucleic-acid based detection of BBTV, highly
conserved non-coding region of DNA-S upstream and downstream to the CP ORF was
targeted. The primers were validated by detecting virus from the field samples collected
from various parts of the state.
The rCPs were chosen as a potential antigen for raising antibodies in order to develop
serodiagnostic assays for the early detection of the viruses. The BBTV CP gene was clonedin to three expression vectors viz., pRSET-C, pGEX-4T-2 and pET32a(+) and transformed
to expression hosts like BL21 (DE3) pLysS, Rosetta (DE3) pLysS and C41 strains of E.
coli after amplification in DH5α. The 20 kDa recombinant BBTV CP (rBBTV CP) cloned
in to pRSET-C, and overexpressed in various E. coli hosts had a hexa histidine (6X His)
tag at the N terminal.
Similarly, a 37 kDa fusion protein (pET/rBBTV CP) was
overexpressed from pET/BBTVCP clone had a thioredoxin (Trx) tag (17 kDa) along with
the 6X His tag. Whereas, a 45 kDa fusion protein (pGEX/rBBTV CP) with GST tag was
overexpressed from pGEX/BBTVCP clone. These affinity tags in the fusion rCP enabled
purification from other E. coli proteins. Although pRSET/rBBTV CP was soluble, the 20
kDa protein was highly unstable and partially degraded during purification at 4 °C.
Curiously, pGEX/rBBTV CP dissociated from its GST affinity tag and the rCP without the
tag degraded. On evaluating the protease cleavage sites in the fusion protein, trypsin
cleavage sites were present between the C terminal of GST and N terminal of BBTV CP
which might be the reason for cleavage of the ~20 kDa protein from its affinity tag. Thus,
it was impossible to purify the protein from the pool of E. coli proteins.
Restriction free (RF) cloning of BBTV CP to pGEX-4T-2 was attempted not only to replace
these trypsin cleavage sites but also the thrombin cleavage site present in the vector with
Tobacco etch virus (TEV) NIa protease site. Thrombin is a specific enzyme used to cleave
off the tag from the fusion protein after purification. However, its specificity is not
universal. Furthermore, the commercially available enzyme is costly. TEV protease on
other hand was produced in the laboratory and was highly specific. However, the cleavage
using TEV protease was unsuccessful apparently because of a steric hindrance contributed
by the two extremely ordered regions flanking the TEV cleavage site present in the
disordered region of the fusion protein. pET/rBBTV CP was highly soluble like
ΔpGEX/rBBTVCP.
Likewise, BBrMV CP gene was cloned into pRSET-C and pGEX-4T-2 to obtain
pRSET/rBBrMV CP and pGEX/rBBrMV CP of size 34 kDa and 60 kDa respectively. The
34 kDa pRSET/rBBrMV CP was insoluble. Overexpression and purification of the protein
was standardized in various conditions to increase solubility. On the contrary,
pGEX/rBBrMV CP was highly soluble and was purified by GSH Sepharose affinity
column chromatography. 360 μg/ml of untagged protein was obtained from 1 l culture.
However, like any other potyviral CP, the exposed N and C terminal of BBrMV CP was
also prone to proteolytic cleavage. It partially degraded when incubated with thrombin atroom temperature for GST tag cleavage. All these bands were detected by potyviral CP
specific antibody in Western blot. Further on storage complete degradation of the protein
was observed. Further standardisation of the protocol is necessary to either stabilise
monomeric CP or develop BBrMV VLPs in vitro for immunising animal in order to raise
the antiserum.
The immunogenicity of the antigens (rBBTV CP and rBBrMV CP) was confirmed by
Western blot using BBTV CP specific and potyvirus CP specific antibody procured from
NRC, Banana and IISc, Bangalore respectively. The rCPs were also characterized by
fluorescence spectroscopy, sucrose gradient ultra centrifugation and electron microscopy.
The fluorescent spectra of tagged and tag less rBBrMV CP deviated from 330 nm which is
typical for a partially disordered protein. However, the spectra of pET/rBBTV CP and
ΔpGEX/rBBTV CP were different. The former depicted the spectra of a mostly globular
protein. There were two λmax for the fluorescence spectra of ΔpGEX/rBBTV CP.
The epitope prediction of BBTV CP with Trx tag gave interesting insights. A single linear
epitope of 80 residues were detected in pET/rBBTV CP comprising of C terminal of the
affinity tag and the N terminal of BBTV CP. This was expected to increase the
immunogenicity of the antigen and administered for production of antiserum.
The titre value of polyclonal antiserum produced against the 37 kDa pET/rBBTV CP was
evaluated by DAC-ELISA and was found to be 1:128000. Titre value for serological assays
of field samples was standardized as 1:10000 to be more inclusive for detecting virus even
at early stages of infection. A total of 247 tissue culture samples and 10 field samples were
screened for the presence of the virus using the antiserum and was compared with the
procured antiserum. Seemingly, the latter non-specifically reacted with plant proteins
which gave a higher absorbance value in negative control and correspondingly high
absorbance in the infected samples. The polyclonal antiserum raised against rBBTV CP
was used to standardize serological detection assays like IC-PCR, DIBA and TAS-ELISA
apart from DAC-ELISA. DIBA and TAS-ELISA were the most sensitive assays which
could detect up to 1:80 dilution of the antigen.
In conclusion, due to the higher nucleotide variability of the CP gene, serological detection
is preferred over nucleic acid based assays. However, the quality of antigen used for raising
the antibody plays a major role in serodiagnostics. Hence, high quality rCPs of both BBTV
and BBrMV were developed in the laboratory in various vector/host systems. ThepET/rBBTV CP overexpressed in C41 strain of E.coli (1.1 mg/ ml obtained from 1 L
culture) was used for immunisation of the animal. A highly sensitive antiserum specific to
BBTV with a titre ten fold higher than that of the commercially available antiserum was
obtained. Using this antiserum raised against rBBTV CP, various serodiagnostic assays
were standardised in the laboratory. Among these, TAS-ELISA was the most sensitive,
detecting antigen even at higher dilution.