WN NLTN March 9 2004

Serological and Molecular Amplification Assays for West Nile & Other Arboviruses: 

Serological and Molecular Amplification Assays for West Nile & Other Arboviruses Arbovirus Diseases Branch Diagnostic & Reference Laboratory Fort Collins, Colorado

Medically Important Arboviruses in the United States: 

Medically Important Arboviruses in the United States

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Eastern Equine Encephalitis Human cases: 1964-2002 Western Equine Encephalitis Human cases: 1964-2002 191 cases 5 cases/year no epidemic years 40% FL & GA 640 cases some epidemic years 65% cases 1964-66 & 1975 8 cases/year non epidemic 4 cases since 1990

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La Crosse Virus Encephalitis Human cases: 1964-2002 2910 cases 76 cases/year children < 16 other CAL viruses

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St. Louis Encephalitis Human cases: 1964-2002 4561 cases Epidemic cycles 50% 1975 & 1976 70% TX, IL, OH, IN, FL, MS

Reported WNV Disease Cases in Humans,United States, 1999-2003: 

Reported WNV Disease Cases in Humans, United States, 1999-2003 * Plus D.C.

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~80% Asymptomatic ~20% “West Nile Fever” <1% CNS disease WNV Human Infection “Iceberg” in 2002 284 fatalities ~ 3300 severe disease ~400,000 asymptomatic ~100,000 mild illness

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1999

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2000

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2001

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2002

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2003

Novel Modes of West Nile Virus Transmission, 2002: 

Novel Modes of West Nile Virus Transmission, 2002 Transplanted organs One donor to four recipients Transfused blood 23 confirmed cases in 2002, many more likely WNV NAT screening in Blood Banks began in July; >700 positive Breast milk One case, infant asymptomatic Transplacental transmission One case, severe outcome to infant Occupational exposure

West Nile Virus Diagnostic Assays: 

Serological Assays for WN Virus Acute & convalescent serum, csf. IgM ELISA (CDC, FOCUS, PanBio, Abbott) IgG ELISA (CDC, FOCUS) Blocking ELISA (avian & mammals) Plaque Reduction Neutralization (PRNT) IFA IgA ELISA Microsphere Immunoassay (CDC & NYSDH) Virus Detection Assays Acute csf, tissues, donated blood, environmental surveillance. Real Time Fluorescent RT-PCR (CDC, Roche, & Reference Labs) TMA (GenProbe) NASBA (BioMerieux) Virus Isolation Antigen Detection (ELISA & Dipstick) West Nile Virus Diagnostic Assays

Testing for West Nile Virus: 

Testing for West Nile Virus

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DAYS POST ONSET 1 2 3 4 5 6 7 8 9 10 -14 to -2 0 IgM IgG ELISA P/N #pfu/ml WN viremia 250 CNS illness Theoretical Depiction of WNV Human Viremia & Immune Response 2 20 Serology Assays Virus Assays Neutralizing Ab

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Serological Testing Algorithm for West Nile Virus human serum/csf IgM ELISA WN & SLE IgG ELISA WN & SLE POS NEG Plaque reduction Neutralization test (PRNT) with: SLE, WN, (other flaviviruses) STOP National Case Definition Confirmed: IgM pos csf IgM pos serum + PRNT >4-fold increase PRNT titer

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Secondary flavivirus infections Old versus recent infections IgG POS & IgM NEG indicates a previous flavivirus infection Additional Confirmation of IgM assay Seroconversion in paired specimens Why Run the IgG ELISA? IgG for early season testing and/or special cases; not during a confirmed WN epidemic

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Coat With Goat anti-Human IgM 4° Overnight Add Patient Serum @ 1:400 37° 1 Hour Add West Nile Recombinant Antigen 4° Overnight Add HRP anti-Flavivirus McAb 37° 1 Hour IgM Capture ELISA

Interpretation of Results: 

P/N: O.D. patient serum/O.D. negative control serum. P/N > 3 = positive P/N < 2 = negative P/N 2-3 = equivocal Interpretation of Results

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WN Serological Data Typical Human WN Case In primary flavivirus infections ; Martin et al 2002: IgM P/N to WN is 2-5X greater than SLE.

Analysis of 1,336 IgM Positive Serum Specimens for WN to SLE Ratio: 

Analysis of 1,336 IgM Positive Serum Specimens for WN to SLE Ratio

Longevity of Human WN Virus-Reactive IgM in Serum: 

Longevity of Human WN Virus-Reactive IgM in Serum

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WN Serological Data 2002 WN Case Tested in 2003 West Nile Virus IgA Assay 95% WN IgM positive serum samples are IgA positive days 11 – 40 No IgA positives after day 51

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WN Serological Data Secondary Flavivirus Infection

WN Human Serological Data: 

IgM remains the front-line screening assay IgM detectable in serum & csf by CNS illness onset (99%); not WN fever; IgG Positive by day 7 Post-Onset In primary WN cases: ELISA reactivity is 2-5X higher to WN than to SLE PRNT may not be necessary to confirm all WN IgM positives IgM Persistence > 1 Year in 50% cases in 1999 study WNV IgM positives detected in endemic areas could be previous years cases; additional laboratory testing is necessary IgA can be an additional marker for recent infection along with IgM Secondary flavivirus infections are problematic High PRNT to several flaviviruses; no clear “winner.” WN Human Serological Data Lessons Learned 1999-2003

WN EIA Serological Reagents: 

IgM & IgG EIA Kits from FOCUS & PanBio (FDA approved) WN antigen from FOCUS for Public Health Labs; 2004; not likely for 2005 & beyond Hennessey Research Associates SLE antigen from CDC Hoping for commercial partners HRP conjugate & IgG coating antibody from CDC Commercial sources possible WN EIA Serological Reagents http://www2a.cdc.gov/ncidod/dvbid/misc/index.asp

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CDC Training Course Trained > 60 Public Health Laboratories Proficiency Panel 100% agreement IgM ELISA 92% agreement IgG ELISA (false neg’s) IgM & IgG ELISA Technology Transfer

WN Serological Assays: 

WN Serological Assays Automation of IgM & IgG ELISA Reagent Stability Incubation Times Luminex Assay Commercial Assays Future Directions

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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 IgG-depleted 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 Cut-off determination and validation in progress

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Detection of WNV and SLEV IgM in microsphere-based duplexed immunoassay

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RNA extraction from: serum, csf, tissues, & mosquito pools 1. Standard RT-PCR TaqMan RT-PCR Agarose gel 3. 2. RNA Extraction Amplification Detection TaqMan probe CDC Molecular Amplification Assays

Laboratory Safety Issues: 

West Nile is a BSL3 virus ELISA: Biosafety Cabinet (BSC) until serum is washed, then BSL2 PRNT: BSL3 YF/WN chimera virus attenuated available from CDC Virus Isolation: BSL3 PCR: BSC until viral lysis buffer is added, then BSL2 Antigen (Dipstick) Assays: BSC until detergent lysis buffer is added, then BSL2 Animal Necropsy: BSL3 Laboratory Safety Issues CDC Implementation of Biosafety in Microbiological & Biomedical Laboratories; 4th Ed.

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Sample Preparation for Molecular Amplification Assays Mosquito pool (n=50) Tissues Homogenized Lysate RNA in H2O Disruption In QIAGEN Mixer Mill RNA extraction & purification in QIAGEN BioRobot or kits Molecular Amplification Test Serum/plasma CSF/breast milk RNA in H2O

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CDC TaqMan Testing Algorithm Extract RNA (100 ul to 1 ml or >) TaqMan with ENV primer set + internal control Ct < 38 positive; Ct 38 – 45 equivocal All positives & equivocal are repeated with a second primer set; using newly extracted RNA

TaqMan RT-PCR: 

TaqMan RT-PCR Primer 1 (position 500) Primer extension by reverse transcriptase cDNA – RNA hybrid Primer 2 (position 400) Primer extension by Taq polymerase DS DNA product (100 BP) Denature, primer & probe anneal, primer extension 40X Probe displacement & cleavage Dye release & fluorescence Primer 2 VS-probe (No fluorescence 535 nm) (fluorescence 535 nm)

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ENV set 0.10 pfu/ml or 40 copies/ml WNV TaqMan Detection Limit Plaque forming units (pfu) 3’NC set 0.4 pfu/ml or 160 copies/ml NS5 set (Lipken) 0.2 pfu/ml or 80 copies/ml

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WN Virus TaqMan Assay With HEX-Labeled Internal Positive Control WN virus primer/probe set HEX internal control primer/probe set

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NASBA ECL Reader NASBA Molecular Beacon NASBA Detection Formats SS RNA FAM Q

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Molecular Beacon Probe for WN Virus NASBA Assay WN-specific sequence WN-specific sequence Stem

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  Sensitivity of WN Virus NASBA & TaqMan Assays

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WN MD CROW 2000 WN NJ MOSQUITO 2000 WN NY GROUSE 2000 WN FL CROW 2001 WN OH HUMAN 2002 WN TX HUMAN 2002 WN GA HUMAN (donor) 2002 WN GA HUMAN (recipient) 2002 WN IN HUMAN 2002 WN NY CROW 2000 WN NY EQUINE 1999 WN CN MOSQUITO 1999 WN NY HUMAN 1999 WN NY FLAMINGO 1999 WN Israel STORK 1998 WN EGYPT 1951 Ancestor All US WN strains >99.7% identical (nucleotide) <3 amino acid differences between any 2 isolates 1999 & 2000 2001 & 2002 WN TX HUMAN 2003

WNV Isolates From Humans:1999 - 2002: 

1999: No WNV isolated 2000: No WNV isolated 2001: 1 virus isolated csf (NY State Lab) 2002: 16 WNV isolated CDC + 1 from MD Dept. Health 5 serum/plasma 3 csf 4 brain tissue 1 liver 2003: Numerous isolates from donated blood WNV Isolates From Humans: 1999 - 2002

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Most sensitive virus detection tests are TaqMan (quantitative) & NASBA (TMA) Use of internal, negative, & copy number controls is critical to validating the assay Copy number WN virus controls in human plasma available from Boston Biomedica. WN virus strains in the U.S. are highly conserved; 99.7% identical. Only 1 mutation in 9 primers commonly used Virus isolation West Nile Virus Testing Summary

WN Human Viremia: 

Human viremia is low: Transfusion studies: 1-130 pfu/ml Average 24 pfu/ml Virus isolation is rare Human viremia is short-lived Not detectable by Day 1 of onset 2 TaqMan Positives/ 100 Acute IgM positives Viremia is absent when IgM is detectable 2 IgM & TaqMan positives in transfusion studies Israel study 2002 LA Fever Study WN Human Viremia Data Summary

Diagnostic & Reference SectionSYBR Green Consensus Assays : 

Flavivirus primers (Chang & Kuno) California & Bunyamwera serogroup (Kuno) Dengue Alphavirus Diagnostic & Reference Section SYBR Green Consensus Assays primer-dimer DEN-1 DEN-2 DEN-3 DEN-4

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Denise Martin Jane Johnson Amanda Noga Olga Kosoy Amy Lambert Jason Velez Kathy Wolff Barbara Johnson Trudy Chambers Special Thanks to the CDC Arbovirus Diagnostic Lab Staff Brandy Russell Janeen Laven Roseyln Hochbein