Nanoparticles for Detection of Viruses and Estimation of Viral Surface Protein Expression

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Nanoparticles for Detection of Viruses and
Estimation of Viral Surface Protein Expression

• Amit Agrawal

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Overview

• Respiratory Syncytial Virusbackground
• Nanoparticle probes and detection technology
• Intact viral particle detection scheme
• Results and Discussion
• Conclusion

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Respiratory Syncytial Virus

• Discovered in 1956 ve sense RNA, enveloped
  virus
• 120-300 nm in diameter
• Major cause of respiratory problems in children
• More than 125,000 hospitalizations/year
• No vaccine yet
• Member of the Paramyxoviridae family of viruses

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Paramyxoviridae
Morbillivirus Measles Canine Dis- -temper Virus
Rublavirus Mumps, ParaInfluenza (PIV) 2,4
Paramyxovirus PIV 1,3
Pneumovirinae
Paramyxovirinae
Pneumovirus RSV A, Along, B1, B18537
Metapneumovirus APV hMPV
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Paramyxoviruses Structure

• Lipid bilayer membrane
• HN/H/G glycoprotein for attachment with host cell
• F glycoprotein for fusion with the host cell
• Estimation of F and G protein on the particle
  surface
• No technology available
• Viruses too small for flow cytometry

Taken from University of South Carolina Medical
School Website
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Unlabeled, Single Target Detection

• No target labeling needed
• Two antibody binding ensures greater specificity
• Sample purification and processing not needed
• A simple fixed-point confocal set up
• Up to femtomolar target detection possible
• Need to excite two fluorophores simultaneously

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Quantum Dots

• ZnS capped CdSe Nanocrystals 2-8 nm in size
• Broad absorption and narrow, symmetric emission
  Multiplexing and optical advantages
• Size and composition tunable emission
• Highly photostable over several hours
• High photoluminescence lifetime
• Can be conjugated to several biomolecules

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FRET Nanobeads

• 40 nm to 1 micron in size
• Polystyrene bead filled with one (or more) FRET
  pair
• Same absorption, different emission
• Highly Photostable
• Very Bright
• Chemically Activated
• Commercially available (Invitrogen)

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Detection Set Up

• Microfluidic channel increases sample throughput,
  helps reduce assay time
• High detection efficiency due to single
  excitation source
• No offline data processing needed Direct
  detection of coincidence

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Intact Viral Particles Detection and Surface
Protein Expression Estimation
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Results RSV-A vs. PIV3
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Results RSV-A2 vs. RSV- ? G

• 103-105 pfu /ml sample (Vero cell lysate)
• RSV- ? G doesnt have G protein on surface
• RSV G and F protein specific monoclonal
  antibodies were conjugated with the nanobeads in
  11 ratio
• A representative run of 8 second is shown
• Notice the difference is y-axis scale (photon
  counts) between RSV-?G and RSV-A2 virus plots
• Low signal corresponding to F-protein for RSV- ?G!

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Counting viral particles

• Relative concentration of viral particles can be
  estimated
• PIV3 serves as a control
• 30 -60 min needed for very low concentration of
  viral particles

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Intact Viral Particles Detection and Surface
Protein Expression Estimation
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RSV surface F protein estimation
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RSV particle F protein expression levels

• RSV-A2 expresses greater amount of F protein than
  RSV-?G
• RSV-A2 has greater particle to particle
  variability in F protein levels
• G-protein gene precedes F-protein gene in RSV
  genome
• Deletions in G protein gene may alter gene
  termination signal affecting F-protein expression
  (Moudy et. al, 2004)

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Severity of RSV infection is related to the
strain type

• RSV-A causes more severe infections than RSV-B

The Journal of Infectious Diseases 199717581420
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Extent of HPIV3 or SV5 fusion is related to
HN-protein density and F-HN ratio
JOURNAL OF VIROLOGY, Oct. 1998, p. 77457753

• CV-1 cells were infected with viruses expressing
  variable amount of HN protein
• Fluorophore labeled HN protein antibody was used
  to label the protein in the cells
• Flow cytometry was performed on the 10,000 cells

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Summary and Conclusion

• Luminescent nanoparticle probes used for rapid
  detection and quantification of viral particles
• Methodology to probe surface protein expression
  directly on the viral particles developed
• It should be possible to ask questions about
  relationship of protein expression levels and
  severity of infection for closely related viral
  strains
• More studies needed for broad / practical
  applications in diagnostics and research focus
  of ongoing work

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Acknowledgements

• Prof. Shuming Nie
• PI and dissertation advisor
• Biomedical Engineering Dept., Georgia Institute
  of Technology and Emory University
• Prof. Ralph Tripp
• University of Georgia and Centers for Disease
  Control and Prevention
• Dr. Larry Anderson
• Centers for Disease Control and Prevention
• Rene Alvarez
• University of Georgia, Athens
• Funding National Institutes of Health and
  Georgia Cancer Coalition Award to SN

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Thanks

• Questions?