Qualification Panels for West Nile Virus RNA Tests Mark Manak, Ph.D. Boston Biomedica, Inc. West Bridgewater, MA SoGAT Langen, Germany July 3, 2003

WNV Controls/Panels Panel/Controls Design Designed to closely mimic a true serum sample Cultured Virus from an infected individual Heat Treated Diluted in Defibrinated Plasma (Basematrix ) Panel/Controls Uses Method optimization and comparison Standardization of WNV RNA concentration Useful for quantitation Monitoring assay performance

West Nile Virus Qualification Panels QWN702 - WNV Lineage 1, NY 1999 (flamingo CDC) QWN701 - WNV Lineage 2, Uganda Member Panel 2 10,000 copies/ml copies/ml 2 1,000 copies/ml 3 30 copies/ml copies/ml 3 Negative Randomly Assorted Semi blinded

WNV Run Control: Accurun 365 Accurun 365 WNV Positive Run Control Manufactured from cultured West Nile Virus* Target level of 300 copies/ml Ideally suited to monitor assay precision Validation of analytical runs Run Control for Blood Screening Assays Accurun 865 WNV Negative Run Control * NY99 isolate, Lineage 2

RT-PCR Quantitative WNV Assay Specific Primers for WNV 5’UTR: Detects all known isolates of WNV Env: Lineage 1 only, more sensitive Closed-tube, Real-time fluorescent assay Quantified with TaqMan probe Broad linear dynamic range Between 300 – 10,000,000 copies/ml Ultrasensitive assay Sensitivity to 30 copies/ml

WNV RNA Standards-RNA Transcripts

WNV RNA Quantitation Standard Ultra 1, ,000 1, Copies/ml 30 Neg.

Reproducibility of Ultrasensitive Assay Ave. Ct STD %CV (n = 12)

QWN702 Panel Quantitation Panel MemberTarget 5’-LTR 140uL Env Primer 140uL Env CDC BBI Ultra Assay Ct QWN , QWN , QWN , QWN , QWN QWN QWN QWN QWN QWN QWN QWN QWN QWN QWN

Comparison of Assays

Negative Specimens Material Source of materials No. of samples CtResults Serum American Red Cross 12850Negative Plasma BBI Diagnostics 10050Negative Basematrix BBI Diagnostics 9250Negative

WNV Positive Samples Target Average (Copy/mL) 1.1x x x x x x 10 3 STDEV 2.1x x x x x x 10 3 CV 19.0%28.5%31.3%18.6%78%76.2% #Samples

WNV Titers in Avian and Mosquito Tissues Avian Brains Mosquito Pools ID copies/g ID copies/ml ZL SU ZL SL ZL SL ,360 ZL EM ZL NF ,000 ZL ,000,000 NF ,000 ZL ,800,000 NF ,200,000 ZL ,200,000 SU ZL ,400,000 SU ZL ,000,000 SU ,000

BBI WNV Panel and Controls BBI Panels and Controls –ACCURUN WNV RNA Positive Control A365 (NY99 Strain) –ACCURUN WNV RNA Negative Control A865 –QWN702 WNV Qualification Panel (Lineage 1) (NY99 Strain) –QWN701 WNV Qualification Panel (Lineage 2) (Uganda Strain) –WNV Characterized Samples (US Source ) Panels and controls Uses –Assay Development –Comparison/standardization of Assays –Training of Laboratory Personnel –Assay Validation –Monitoring Assay Performance

Acknowledgements Boston Biomedica Inc. Alan Doty Barbara Weiblen Harmesh Sharma American Red Cross Chyang Fang, Ph.D. MA State Health Depart. Barbara Werner, Ph.D. FDA Maria Rios, Ph.D. CDC Robert Lanciotti, Ph.D. BBI Biotech Xiuli Chen, Ph.D. Jienli Wu Ron Meixell