Development of new diagnostic reagents against Banana streak Mysore virus Jenny Vo PhD student
biosecurity built on science Cooperative Research Centre for National Plant Biosecurity
Banana streak virus (BSV)
BSV is a plant-infecting pararetrovirus (dsDNA genome). BSV is a cryptic species complex, comprising >10 viral species. can only be reliably distinguished using molecular methods. Field spread of BSV is slow. BSV is best controlled by using clean planting material.
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Endogenous and exogenous forms of BSV exist Two forms of BSV DNA
Integrated in plant chromosomes
© Vo.J 2009
Encapsidated in virus particles
Replication-competent DNA (‘alive’) Replication-defective DNA (‘dead’) biosecurity built on science
Detection of BSV
PCR incapable of distinguishing endogenous from exogenous forms of viral DNA.
Reliable detection of BSV is based on viral protein markers.
Proteins are most easily detected using antibodies.
But…worldwide shortage of BSV antibodies.
Objective 1: to characterise the BSV coat protein Objective 2: to generate novel immunodiagnostic reagents using recombinant antibody technology and phage display biosecurity built on science
Characterisation of the BSV coat protein via proteomic approaches • Coat protein released from polyprotein through action of viral aspartic protease. • Two coat proteins of c. 37-39 kDa observed by SDS PAGE. • Amino acid sequencing results suggest alternative aspartic protease cleavage sites.
C-terminus
N-terminus Coat protein
Mass analysis
N terminal sequencing Internal peptide sequencing
Zinc finger Peptide 1 P2 P3 P4 P5 P6 SQYRPADIDMAGPTGYAPATSQQGLLGSTAPDRLGKGSFKWKSPTEYFNLPSAQQ domain QAGAMFVMPANFDPKVFERWESITLNHMADKVFSTAEDKLIYLENLLGEAEKIMF CXCX2CX4HX4CX2 QSWRMTYANEYEEMKGQALGNNGTQNLLSQIRRIFYLEDPKTGTTVSQDAAYKAI CKCYACGEEGHYASECRN SQYRP STAPD KSLVCHEMTGTAIKRYMADYWTLAAKTGRIWQGPELSDEFFTKLPSGLGDRVAKA FKEKYPGNTVGVPARITFTQLYLEELCREAAYQRSLKNLDFCREFPIPGYYKKPGR KFGVRKSTSYKGKPHKTHIKIDKRKYLRNKKCKCYACGEEGHYASECRN biosecurity built on science
Mapping the distribution of linear epitopes of the coat protein ď ą designed a library of 16-mer overlapping peptides spanning the entire CP ď ą tested with rabbit antibody raised against BSMYV ď ą used pre-immunised serum as negative control CP1-N terminus SQYRP
CP 2- N terminus STAPD
1.2
Pre-Immune Immune
Identified linear antigenic peptides of coat protein
1.0
A405 nm
0.8 0.6
use to produce antibodies
0.4 0.2
coat protein
P82
P79
P76
P73
P70
P67
P64
P61
P58
P55
P52
P49
P46
P43
P40
P37
P34
P31
P28
P25
P22
P19
P16
P13
P7
P10
Nterminus
P4
P1
0.0
Cterminus biosecurity built on science
Chicken recombinant antibodies Antigen binding site
VH
Variable domains
CH1 CH1 CL CH2
VL
CH3
VH
CH1
CL Fc region
linker
VH
Constant domains
VL VL
CL
Fab
ScFv
Recombinant antibodies
immunoglobulin scFvs: the smallest functional VH–VL domains of an antibody can be cloned directly from lymphocytes of animals can be re-engineered for improved stability and specificity advantage of using chicken as the animal model for immunization biosecurity built on science
Antibody Phage Display displaying peptides or proteins on the surface bacteriophage via physical linkage between the displayed protein (phenotype) and the DNA encoding it (genotype). allows screening of large number protein variants. VH
VL
scFv phage display library
each phage receives a different gene
exposed to target antigen
Biopanning: in vitro affinity selection Resulting in stepwise enrichment of the phage pool in favour of the best binders.
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Construction and selection of BSV-scFv Virus purification
Amplification of VH and VL
Transformation
E. coli
Phagemid rescue phage displayed scFv library Immunisation
Cloning VH
VL
Panning RNA extraction scFv phagemid library
Selective pressure on phenotype biosecurity built on science
Results Polyclonal phage ELISA showing the enrichment of BSV specific binders
Screening of individual clones by monoclonal phage ELISA
1.2
Absorbance 405 nm
1 0.8 0.6 0.4
PBS Healthy Milk Infected
0.2 0
Phage library
•Total 288 clones were screened •Majority of clones was positive for BSV binding
DNA analysis •Many identical sequences after panning selective binders were amplified biosecurity built on science
Characterisation of monoclonal scFv Specificity test 3.0
BSMYV
A405 nm
2.5
Healthy
2.0
1.5
1.0
0.5
0.0 3A10
3F3
3G12
B9
B17
M13
chicken
Phage displayed scFvs
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Binding properties of some scFvs against different BSV strains Cross-reactivity test ďƒ˜ identified 2 patterns of binding, (Mys+, OL+, GF+) and (Mys+, OL-, GF+)
scFv amino acid sequences 2.0
Strains
A405 nm
1.5
Mys 1.0
OL 0.5
GF 0.0
B9
B17
3F3
3G12
3A10
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Conclusion 1st study to identify/ predict BSMYV-CP isolated a panel of scFv capable to detect BSV using phage display selection and a functional screening approach
Direction for future research Express scFvs as soluble proteins. Develop new detection test using scFvs. Develop scFvs against every banana virus for use in an antibody array. biosecurity built on science
Acknowledgements •Principal advisor: Dr Andrew Geering (QAAFI-UQ) •Associate advisors: Professor Ross Barnard (SCMB/QAAFI-UQ) Dr Hans Heine (CSIRO) Dr Paul Campbell (DEEDI) •Professor Ben Lockhart (University of Minnesota) •Dr Katleen Braet (Medical Research, University of Ghent, Belgium)
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