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 Cases in 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 YearTotal Cases# Neuro# DeathOnset Date Range 1999626162 Aug – 24 Sep 200021 220 Jul – 27 Sep 200166 913 Jul – 7 Dec 20024,1562,94228419 May – 14 Dec 20039,8622,86626411 April – 4 Dec 2004 Total 2,470 16,637 900 6,865 88 653 23 April – Dec Aug99 – Dec2004

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 SampleLab 1Lab 2Lab 3Lab 4Average FDA-Hu2002 10 -1 10 9 FDA-Hu2002 10 -4 10 6 ND10 6 FDA-Hu2002 10 -7 10 3 ND10 3 FDA-Hu2002 10 -1 60 o C/2hr 10 7 NY99-FDA 10 -1 10 9 NY99-FDA 10 -4 10 6 ND10 6 NY99-FDA 10 -7 10 3 ND10 3 NY99-FDA 10 -1 60 o C/2hr 10 7 10 8 10 6 10 4 10 6.5

8. Correlation Between Copies/mL and PFU/mL SampleAverage*PFU FDA-Hu200210 10 7.5 /mL FDA-Hu2002 60 o C/2 hr 10 7 0 NY99-FDA10 10 7.5 /mL NY99-FDA 60 o C/2 hr 10 6.5 0 * 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 10 10 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 Detection5 copies10 copies50 copies100 copies500 copies1000 copies In % Hu02NY99Hu02NY99Hu02NY99Hu02NY99Hu02NY99Hu02NY99 1002022555510 75020222112222 50003013350000 25213131310000 1124310000000 False Negative 770400000000 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 4 o C – 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