Development of S tandard Reagents for WNV NAT

1. Development of Standard Reagents for WNV NAT M. Rios, A. Grinev, K. Sirnivasan, O. Wood, S. Daniel, I. Hewlett CBER/FDA

2. WNV Human Disease Casesin Six Outbreaks 1999-2004 in the U.S. • 2002 : documented Human to Human transmission • transfusion, transplantation, breast feeding & transplacental • Public health concern: DHHS, State Dept Public Health, Blood community, and test kit manufacturers - screening assays

3. Chronology of Assays • Biological assays: • In vivo: intracerebral injections in mice • In vitro infectivity: Cytopathic effect (CPE) Plaque assays (PFU) • Serological assays: • ELISA; MAC-ELISA, PRNT • Short asymptomatic acute phase need to be detected by nucleic acid assays: PCR, TMA, etc

4. Need for Standards • Lack of consensus for viral titer • Viral titer has been defined in plaque forming units (PFU) • Number of viral particles per PFU has broad range (1 – 1000 virions) • Need for correlation of RNA copies with PFU • Non-infectious particles (defective) may be detected by NAT but not by infectivity assays

5. Reagent Standardization • Consensus in viral titers: • Determination of copy number is necessary to define analytical sensitivity and fulfill regulatory requirements • Animal isolates were available • Needed to include both animal and human strains • Potential for genetic variation • Genetic characterization of isolates to be used in the panel

6. FDA WNV NAT Panels • Genetic characterization • NY99 (flamingo) isolate from CDC – available during assay development • FDA-Hu2002 isolated at the FDA • Characterization of viral stocks • PFU – at both FDA and NY Dept. of Health Lab. and by cytopathic assays at FDA (TCID50) • Measurement of RNA copy number • Using intermediate dilutions sent to 4 laboratories who had quantitative assay • The final panel specification were to be defined in collaborative studies on prototype panel formulated with heat inactivated stocks

7. Viral Titer Determination Copies/mL

8. Correlation Between Copies/mL and PFU/mL * Assay were performed in two independent laboratories

9. Genetic Characterization of FDA-Hu2002 Isolate (AY646354) Comparing to NY99 flamingo isolate (AF 196835) Previously observed mutations In red: Hu 2001 (AF533540) In green: Hu 2001 (AF533540) and Hu 2002 TX (AY289214)

10. Reagent Characterization Summary • Both NY99-FDA and FDA-Hu2002 stocks have a viral titer of 1010 copies/mL • The PFU titers at both NY State Dept of Health Laboratory and at the FDA were 2.5 logs lower then the RNA copy numbers • Heat treatment of the virus results in loss of infectivity by PFU and 2 to 3 log reduction of copy number as determined by TaqMan

11. WNV Panel Formulation and Evaluation in Collaborative Studies • Panel formulated using both NY99-FDA and FDA-Hu2002 strains • composed of 14 coded members (1000, 500, 100, 50, 10, 5 and 0 viral copies/mL, one from each isolate) • Distributed to 7 independent laboratories • Results reported to FDA

12. Panel Evaluation: Results The seven participant laboratories performed 12 different assays on the panel Results of the 12 different assays on the panel Only one assay had 2 false positive results in the 0 copies panel member

13. Panel Evaluation in Collaborative Studies • Great variability of results with low copy number panel members • Qualitative assays performed better than quantitative assays • A second round of testing was needed for evaluation of viral quantification - statistical analysis ongoing • Stability studies: panel stable for at least 17 months at 4oC – further studies ongoing

14. Analytical sensitivity • FDA’s current standard for WNV NAT assays is 100 copies/ml for the individual donation • Standard may be revised as assay sensitivity improves and additional data on viremia and infectivity become available in future studies