SynTura: A New Ribonuclease Resistant Viral RNA Control Material

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SynTura A New Ribonuclease Resistant Viral RNA
Control Material

• Ralf Schönbrunner, Ph.D.
• SoGAT XXI
• Brussels, Belgium
• May 28, 2009

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SynTura Project

• The SynTura project was created to provide RNA
  Internal Controls and Quantification Standards
  which behave like the target they intend to
  control
• Synthetic Natural SynTura

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SynTura Project Objectives

• Develop technology platform which can provide
  control material close to real analyte
• HCV was used as model system
• Also functional as QS and IC

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The Best Theoretical Alternative for Real HCV

• Similar virus to HCV
• Easy to culture
• Grows to high titers
• Non infectious to humans
• Detectable by HCV assays

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Viruses Related to HCV
Flaviviridae Flavivirus Yellow fever virus, West Nile virus Dengue virus St. Louis encephalitis virus Tick-borne encephalitis virus Japanese encephalitis virus
Flaviviridae Pestivirus Classical swine fever virus (CSFV) Border disease virus (BDV) Bovine Viral Diarrhea Virus 1 2 (BVDV)
Flaviviridae Hepacivirus HCV
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BVDV, HCV and MS2
BVDV HCV MS2
Mammalian virus Mammalian virus E. Coli Bacteriophage
Flaviviridae Flaviviridae Leviviridae
Genome 12 kb Genome 9.5 kb Genome 3.57 kB
Enveloped (lipid bilayer) Enveloped (lipid bilayer) Protein coat
Detergent sensitive Detergent sensitive Detergent resistant
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HCV BVDV Genome Comparison
Target Region of NAT HCV assays
BVDV genome is similar to HCV but can be easily
cultured and manipulated
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Flaviviridae TranslationHCV BVDV
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5UTR Secondary Structure

• Critical 5UTR secondary structure
• Transcripts often do not have correct secondary
  structure
• Might give misleading results for certain HCV
  genotypes
• Is the Flaviviridae 5-3 Ring formation
  important?

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5 3 NTR Strategies
?
SynTura
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Position of HCV 5NTR in BVDV
5HCV NTR
NFAR
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Generation of a BVDV-Hybrid
Make RNA Transcripts
BVDV Plasmid (BVDV in blue)
Insert desired Sequence (red)
DNA
RNA
Viral Particles
Transfect Cell Line
Grow transfected Cell in culture
Infected Cells produce Virus
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Development Timeline
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SynTura HCV Growth Curve
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Inactivation of BVDV

• Inactivated with ß-Propiolactone
• Protein-modifying agent
• reacts with amides of Lys or Arg
• Commonly used for BVDV vaccines
• Alternative Heat 90 loss

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Initial Titer Determination
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Effect of Inactivation on TMA

• Results with ProCleix Ultrio TMA Assay
• No impact of ß-PL inactivation on TMA

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Sequence Stability

• 3 passages 5 clones partially sequenced
• No mutations found in 5 clones
• 7 passages 5 clones partially sequenced
• 1 clone no mutation
• 2 clones had 1 point mutation in BVDV sequence
• 2 clones had 1 point mutation in HCV insert
• Rate of mutation in insert similar to virus

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SynTura Thermal Stability
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37ºC Accelerated Stability
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SynTura HCV as Calibrator
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OptiQuant-S HCV RNA Quantification Panel

• Based on SynTura Technology
• 100, 500, 5e3, 5e4, 5e5, 5e6 2.5e7 IU/mL
• Plasma Matrix

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Multicenter Study
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Roche CAP/CTM HCV IVD
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Comparison of two Abbott realTime HCV ASR Assays
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Key Results

• SynTura HCV titer 2.0E08 IU/mL
• Concentrated to 1.5eE10 IU/mL
• Stable over 7 passages
• Insert remains intact
• No mutation after 3 passages
• Only 1 point mutation after 7 passages

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Summary

• SynTura has properties very similar to HCV and
  probably other enveloped mammalian viruses
• SynTura technology allows integration of defined
  RNA sequences
• SynTura technology can be used for RNA assays as
• QS and IC
• Positive Control
• Calibrator

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Thank You

• Martin Luther Universität Halle/Saale
• Sven Behrens
• Martina Behrens
• Acrometrix
• Mona Shahbazian
• Jerry Boonyaratanakornkit
• Reina Karunaratne

• Steve Young, Jessie Kilgore Tricore
• Hanna Rennert, John Sipley, - NYPH, Cornell
  Medical Center
• Melody Hung-Fan, Kara Lee, -Contra Costa Public
  Health Lab
• Linda Sabatini, Lech Mazur, -ACL Central Lab
• Maura Pieretti, Carolyn Dowell, -BayCare Lab
• Ted Schutzbank- Covance