1. Development of a Panel for Dengue Virus Maria Rios, PhD CBER/FDA Blood Products Advisory Committee Meeting December 14, 2010

2. The Need for Standard Reagents for Dengue virus (DENV) There are four known serotypes of DENV Reference reagents are needed to evaluate test performance Reference reagents require large volumes of the 4 DENV serotypes; clinical specimens are limited in volume and not suitable Laboratory strains for all 4 serotypes are available for the development of standard reagents

3. Detection of Active DENV Infection Two types of assays can detect active early infection : Nucleic acid testing (NAT) - for the detection of viral RNA which is produced soon after initiation of viral infection much earlier than the antibody production Antigen assays - Non-structural protein (NS1antigen) known to be produced and secreted in the early stages of infection after appearance of viral RNA and before antibody production

4. Blood Screening for Viruses NAT platform for viral RNA detection has been extensively used to screen blood donations HIV, HCV and WNV are examples of RNA viruses where detection of genetic material is the most sensitive approach to prevent transmission by transfusion Epidemiological studies have used NAT assays for the detection of DENV among blood donors FDA is proactively involved in the production of RNA standard reagent for DENV to facilitate development and evaluation of performance of new assays, and future need for lot release DENV antigen panel will be developed, if needed

5. Efforts Towards Standards Development CDC has provided viral isolates from each of the DENV serotypes to be used as standards: –DENV-1, Hawaii –DENV-2, New Guinea C –DENV-3, H-87 –DENV-4, H-241 These are laboratory strains that have undergone multiple propagations after virus isolation from human specimens from Philippines

6. DENV RNA Titer Standardization Lack of consensus for viral titer –Viral titer has been defined by plaque assay (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 Copy number assignment is necessary to define analytical sensitivity

7. Steps of DENV RNA Standard Preparation 1.240 mL of virus stocks from each of the 4 serotypes were produced in C6/36 mosquito cells 2.Preliminary characterization of stocks in house Genetic sequencing Infectivity titer by plaque assay (PFU and FFU) Viral RNA concentration determined in PDU (PCR Detectable Units) by limiting dilution TaqMan 3.A portion of the Stocks were heat inactivated (HI) by incubation at 62°C for 1 hour 4.Dilutions of both live and HI stocks were prepared and frozen at -80°C

8. Standardization of DENV RNA stocks: Preliminary characterization 1. Two concentrations of stocks from each serotype were retested at: FDA NIAID/NIH (Drs Eagle and Goncalves) CDC (Dr Lanciotti) NYSDoH (Dr Kramer) Gen-Probe (Dr Linnen) 2. HI was independently confirmed by cultivation at FDA, NIAID/NIH; NYSDoH and CDC laboratories

9. Standardization of DENV RNA stocks: Preliminary characterization 3. The Stocks concentrations were defined by average of preliminary characterization performed by 5 different laboratories Viral Load (PDU/ml) Titer (PFU/ml) DENV-1 10 11.7 10 5 DENV-2 10 11.6 10 5 DENV-3 10 10.6 10 4 DENV-4 10 10.5 10 4 4. Stability studies in pre-characterized stocks are ongoing

10. Ongoing Stability Studies For evaluation of the candidate preparation stocks were diluted in: –VP-SFM – tissue culture medium –BaseMatrix – defibrinated / delipidated human plasma Stored at various temperatures: +4 ° C, –20 ° C and –80 ° C Testing time points: 0, 15, 30, 90, 180, 365 days

11. DENV-2 preparation (concentration in PCR Detectable units = PDU) DENV-1 preparation (concentration in PCR Detectable units = PDU)

12. DENV-3 preparation (concentration in PCR Detectable units = PDU) 12 DENV-3 preparation (concentration in PCR Detectable units = PDU) DENV-4 preparation (concentration in PCR Detectable units = PDU)

13. Next Steps and Future Plans 1.Second round of NAT testing will be performed using stocks diluted in BaseMatrix 2.Perform data analysis on NAT results from second round of testing to further define PDU 3.Initiate contact with WHO collaborating centers to proceed with international evaluation 4.Panels will be made available when adequately standardized and needed