Laboratory Testing for West Nile Virus Infections Testing Human & Non-Human Tissues

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1 Laboratory Testing for West Nile Virus Infections Testing Human & Non-Human Tissues Robert S Lanciotti Chief; Diagnostic & Reference Laboratory Arbovirus Diseases Branch Fort Collins, Colorado

2 Presentation Summary Standard Laboratory Diagnostic Protocol CDC Experience With Human & Non- Human Tissue Testing

3 Laboratory Testing for WNV Environmental Surveillance (NAAT) Detection of virus and/or viral RNA in mosquito vectors. Detection of virus and/or viral RNA in amplifying hosts. **Detection of antibody or virus in sentinel animals.

4 WNV Human Viremia & Immune Response Virus Assays #pfu/ml 500 IgM Serology Assays ELISA P/N 20 WN viremia IgG 2 infection 3 to 10 days CNS illness DAYS POST ONSET

5 WNV RT-PCR Testing of Human Serum/CSF Tissue Specimens Donor Screening WN Fever Cases Too few tested at CDC Tilley & Fox reported 35% POS Neuroinvasive Disease Cases Serum/Plasma 30% to 45% POS CSF 60% POS

6 Laboratory Testing for WNV Human Diagnosis (primarily serology) Acute Antibody (IgM) in serum/csf Seroconversion IgG and/or neutralizing antibody in acute/convalescent paired specimens Virus or viral RNA in serum, csf, or other tissues can enhance serological diagnosis.

7 Laboratory Tests Used for WNV IgM Antibody Testing FDA cleared: PanBio, FOCUS, InBios, Spectral Published: CDC IgM ELISA, CDC MIA, PRNT

8 IgM Capture ELISA 1. Coat With Goat anti-human IgM 4 Overnight 2. Add Patient 1: Hour 3. Add Viral (Recombinant) Antigen 4 Overnight HRP HRP 4. Add HRP anti- McAb 37 1 Hour

9 Microsphere-based assay to detect IgM to WN and SLE viruses in human serum Beadsets are coupled to 6B6C-1 One beadset is reacted with WNV antigen and the other with SLEV antigen Add reacted beadsets to IgGdepleted serum and anti-igm R- PE. The assay gives concurrent WN and SLE virus IgM values All samples reacted on viral and control antigens Time of reaction 1.5 hours Assay for multiplex WN, SLE, DEN 1-4, LAC, JE, POW, MVE, YF, RR, MAY, VEE, EEE, WEE, BF, CHIK, in progress.

10 Neutralization Assay for West Nile Virus Plaque Reduction Neutralization Test (PRNT) Patient serum dilutions pfu of virus; incubate with cells. 100 plaques = no virus antibody present 90% reduction of virus plaques = virus antibody present neutralization No neutralization

11 WNV serological testing is complicated by antibody cross-reactivity with other flaviviruses & original antigenic sin phenomenon

12 > 2 is positive

13 WN Serological Data Typical Human WN Case Days IgM P/N IgG P/N PRNT Sample post-onset WN SLE WN SLE WN SLE Typical WN Case acute serum :80 1:20 conv. serum :1280 1:80 In primary flavivirus infections ; Martin et al 2002: IgM P/N to WN is 2-5X greater than SLE.

14 Original Antigenic Sin Phenomenon Case 1 Case 2 Case 3 Days post WNV RNA detected PRNT WNV PRNT SLE ,560 10, ,280 20, ,280 20, , ,560 20,480

15 Sensitivity of WN Virological Assays TEST DETECTION DETECTION ASSAY TIME VecTest 100,000 pfu/ml NT 15 min CDC Ag-cap EIA 10,000 pfu/ml NT 24 hour RAMP 1,500 pfu/ml NT 90 min Isolation in SMB 100 pfu/ml NT 5-10 days Isolation in Vero 100 pfu/ml NT 5-10 days RT-PCR-standard 5 pfu/ml 2,500 copies/ml 8 hours NASBA 0.1 pfu/ml 50 copies/ml 4 hours RT-PCR-probe 0.1 pfu/ml 50 copies/ml 4 hours GenProbe TMA 0.02 pfu/ml 10 copies/ml 4 hours

16 NAAT Assay Platform Options Standard RT-PCR Agarose gels, nested Real time RT-PCR Single\multiplex format SYBR Green NASBA (nucleic acid sequence based amplification) ECL, molecular beacons TMA (transcription-mediated amplification) LAMP (Loop-mediated isothermal amplification)

17 Laboratory Tests Used for WNV Testing Viral RNA FDA cleared: GenProbe & Roche (plasma) Published: CDC Real-time RT-PCR, NASBA (mosquitoes, serum, plasma, csf, solid tissue specimens.)

18 WNV RT-PCR Testing of Non- Human Tissue Specimens Tested > 1000 avian specimens. In experimentally infected or naturally infected competent avian hosts, all major organ tissues demonstrate high levels of WNV. Brain, Heart, Liver, Kidney, Spleen, Lung. Cloacal swab & feathers

19 WNV RT-PCR Testing of Human Organ Tissue Specimens Tissue Approx. # tested Result Brain 100 POS Fetal Cord 100 NEG Placenta 80 NEG (1 low pos) Liver 20 NEG Kidney 20 NEG (Urine) 100 NEG Spleen 10 NEG Heart 10 NEG Lungs 10 NEG Bone Marrow 1 NEG

20 Summary: WNV RT-PCR Testing of Human Specimens Plasma/Serum or csf testing of symptomatic patients can augment diagnosis. csf (60%) Plasma/serum (30% to 45%) Brain tissue obtained from confirmed WNV fatal cases is typically positive. Limited testing of solid tissue autopsy specimens from liver, heart, kidney, spleen, lungs, have not yielded positive results.

21 Special Thanks To CDC Arbovirus Diagnostic & Reference Section Roselyn Hochbien Janeen Laven Mara Miller Jaimie Robinson Brandy Russell Jason Velez Rob Lanciotti Barbara Johnson Amy Lambert Olga Kosoy Jane Johnson Amanda Panella