Center for Biologics Evaluation and Research

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.
Year Total Cases Neuro Death Onset Date Range
1999 62 61 6 2 Aug 24 Sep
2000 21 21 2 20 Jul 27 Sep
2001 66 66 9 13 Jul 7 Dec
2002 4,156 2,942 284 19 May 14 Dec
2003 9,862 2,866 264 11 April 4 Dec
2004 Total 2,470 16,637 900 6,865 88 653 23 April Dec Aug99 Dec2004

• 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
Sample Lab 1 Lab 2 Lab 3 Lab 4 Average
FDA-Hu2002 10-1 109 109 109 109 109
FDA-Hu2002 10-4 106 ND 106 106 106
FDA-Hu2002 10-7 103 ND 103 103 103
FDA-Hu2002 10-1 60 oC/2hr 107 107 107 107 107
NY99-FDA 10-1 109 109 109 109 109
NY99-FDA 10-4 106 ND 106 106 106
NY99-FDA 10-7 103 ND 103 103 103
NY99-FDA 10-1 60 oC/2hr 107 108 106 104 106.5
8
Correlation Between Copies/mL and PFU/mL
Sample Average PFU
FDA-Hu2002 1010 107.5/mL
FDA-Hu2002 60 oC/2 hr 107 0
NY99-FDA 1010 107.5/mL
NY99-FDA 60 oC/2 hr 106.5 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 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
Detection 5 copies 5 copies 10 copies 10 copies 50 copies 50 copies 100 copies 100 copies 500 copies 500 copies 1000 copies 1000 copies
In Hu02 NY99 Hu02 NY99 Hu02 NY99 Hu02 NY99 Hu02 NY99 Hu02 NY99
100 2 0 2 2 5 5 5 5 10 10 10 10
lt100 gt 75 0 2 0 2 2 2 1 1 2 2 2 2
lt 75 gt 50 0 0 3 0 1 3 3 5 0 0 0 0
lt 50 gt 25 2 1 3 1 3 1 3 1 0 0 0 0
lt 25 gt 1 1 2 4 3 1 0 0 0 0 0 0 0
False Negative 7 7 0 4 0 0 0 0 0 0 0 0
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

• FDAs 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