BPAC Meeting

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BPAC Meeting
Washington D. C. March 13 14, 2003
Development of a Supplemental WNV Taqman Assay
and Other Critical Reagents in Support of the
Procleix WNV Assay
Bruce Phelps Blood Testing RD Chiron Corporation
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Objectives of the Chiron WNV Program

• WNV Taqman Assay Development
• Propagation of West Nile Virus
• Development of Assay Standards
• Reagents for Serological Assay Development

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WNV Taqman Assay Development

• A qualitative WNV Taqman assay is being developed
  at Chiron in support of the Procleix WNV assay
  development program.
• The assay will be fully validated and may be used
  as a supplemental alternative NAT assay during
  Phase 2 trials to be initiated at the advent of
  the 2003 mosquito season.

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WNV Taqman Assay Development

• The capsid region of the WNV genome is targeted
  for amplification.
• A 965 nucleotide RNA transcript from the capsid
  region is included as an Internal Control.
• The Taqman assay detects 10-50 copies of purified
  RNA transcript.

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Detection of WNV RNA purified from CDC viral
lysate
WNV Taqman Assay Development

• RNA Dilution Target Ct IC Ct
• 4 x 10-2 24.5 Neg
• 4 x 10-3 28.2 Neg
• 4 x 10-4 31.6 40
• 4 x 10-5 35.6 36.7
• 4 x 10-6 39.5 36.5
• 4 x 10-7 43.9 (2/3) 36.6
• 4 x 10-8 Neg 36.6
• No RNA Neg 36.6
• Cycles to exceed threshold, tested as
  triplicate,
• with 100 cps of IC/reaction.
• CDC Taqman assay detection limit 10-6 dilution.

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WNV Taqman Assay Development

• Two methods are being evaluated for target
  isolation
• Magnetic silica adsorption
• Magnetic beads for oligonucleotide capture

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Propagation of West Nile Virus

• West Nile virus strain 385-99, originally
  isolated from a snowy owl in the Bronx Zoo, New
  York, 1999, is being propagated in Vero cells in
  our BL3 facility.
• Viral titers of 107 PFU/mL have been obtained
  from several viral cultures.

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Propagation of West Nile Virus

• A quantitative TaqMan assay (developed in-house)
  is being used to estimate viral genomic
  equivalents/mL using supernatants of viral
  infected cells.
• Efficient WNV inactivation, using a validated HCV
  heat-inactivation protocol, has been confirmed by
  Vero cells infectivity studies.

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Development of Assay Standards

• Viral suspensions of heat-inactivated virus
  (total of 109 PFU of pre-inactivated virus) have
  been prepared.
• Three RNA transcripts of different genomic
  regions have been prepared
• 5'NC/C/Pre M/M (900 nt)
• E/NS1/NS2a (1500 nt)
• NS5/3'NC (1000 nt)

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Development of Assay Standards

• A genetically engineered non-infectious
  full-length West Nile Virus genome is being
  cloned in vectors suitable for RNA production.
• Once fully characterized, this full length West
  Nile virus transcript could be used as a standard
  for nucleic acid test evaluation.

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Reagents for Serological Assay Development

• Cloning and yeast expression of West Nile Virus
  recombinant proteins
• PrM/E region
• Envelope protein
• Capsid protein
• NS1 region
• New York Strain, 385-99

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Reagents for Serological Assay Development

• These recombinant West Nile virus proteins will
  facilitate development of supplemental or
  diagnostic tests to detect IgM and/or IgG
  antibodies in the serum/blood of infected
  individuals.
• These recombinant proteins can also be used to
  generate MAbs for antigen test development.

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Acknowledgements

• We acknowledge Dr. Laura Kramer and Alan P.
  Dupuis of the Arbovirus Laboratories, Wadsworth
  Center, New York State Dept. of Health, for
  providing protocols and valuable assistance to
  culture and propagate West Nile virus.