# Presentation: 514455

## Serological and Molecular Amplification Assays for West Nile Virus

- Arbovirus Diseases Branch
- Diagnostic & Reference Laboratory
- Fort Collins, Colorado

## CDC Tests for WN Virus

| Specimen | 1st Choice | Other | Comments |
| --- | --- | --- | --- |
| Human serum/CSF | ELISA
Plaque Reduction Neutralization | TaqMan/NASBA
Virus Isolation | TaqMan (57%) for acute CSF; <10% serum |
| Human tissue | TaqMan/NASBA
 | Virus Isolation
IHC | Fatal WN cases: TaqMan/NASBA positive ~ 100% |
| Non-Human | 1st Choice | 2nd Choice |  |
| Avian tissue | TaqMan/NASBA
Virus isolation | VecTest/ Ag. Cap. ELISA/RT-PCR | Ag.-based tests require
1000 pfu |
| Mosquito pool | TaqMan/NASBA
Virus isolation | VecTest/Ag. Cap. ELISA/RT-PCR |  |

## 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***

## IgM Capture ELISA

- 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**

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

ELISA Assay must be standardized in each lab

- P/N > 3 = positive
- P/N < 2 = negative
- P/N 2-3 = equivocal
*ELISA Assay must be standardized in each lab*

**Interpretation of Results**

## Slide 6

## Slide 7

## WN Serological Data

***Typical Human WN Case***

**In primary flavivirus infections ;**
*Martin et al 2002***: IgM P/N to WN is 3-5X greater than SLE.  **
**2002 data: Use 2X criteria WN to SLE ratio: only 1 exception in 417 WN confirmed cases. **

## WN Serological Data

**WN Serological Data**

## Longevity of Human WN Virus-Reactive IgM in Serum

| Days P.I. | N | Positive MAC-ELISA |  | Total (%) | Ave. P/N (Range) |
| --- | --- | --- | --- | --- | --- |
|  |  | Positive (%) | Equivocal |  |  |
| 200 | 22 | 13 (60) | 4 | 17 (77) | 6.0    (3.0-10.8) |
| 300-400 | 21 | 9 (43) | 2 | 11 (52) | 4.0    (31.-6.5) |
| 500 | 12 | 5 (42) | 2 | 6 (60) | 5.0    (3.1-6.9) |

## Early Season: Before WN Cases in a Geographic Region.
IgM & IgG ELISA with WN, SLE, other arboviruses
PRNT with WN, SLE, & others?

After WN Confirmed Cases in a Geographic Region:
IgM ELISA with WN & SLE (probable)
PRNT, IgG ?
SLE ?
Travel?
P/N WN < 2X SLE

- IgM & IgG ELISA with WN, SLE, other arboviruses
- PRNT with WN, SLE, & others?
- After WN Confirmed Cases in a Geographic Region:
- IgM ELISA with WN & SLE (***probable***)
- PRNT, IgG ?
- SLE ?
- Travel?
- P/N WN < 2X SLE

**WN Human Serological Testing Algorithm**

## IgM Detectable in serum & csf by onset (99%)
6 exceptions serum of 800 – 1999 - 2002 cases
10 exceptions csf of 800 - 1999 - 2002 cases

IgG Positive by day 7 Post-Onset

P/N 3-5X Higher to WN than SLE

IgM Persistence > 1 Year

Secondary Flavivirus Infections are Problematic

- 6 exceptions serum of 800 – 1999 - 2002 cases
- 10 exceptions csf of 800 - 1999 - 2002 cases
- IgG Positive by day 7 Post-Onset
- P/N 3-5X Higher to WN than SLE
- IgM Persistence > 1 Year
- Secondary Flavivirus Infections are Problematic

**WN Human Serological Data**

***Lessons Learned 1999-2002***

## CDC IgM ELISA Assay

_**Good Points**_
-  Sensitive
-  Relatively Specific (WN & SLE P/N ratio)
-  Technology Transferable

_**Bad Points**_
-  Cross-reactivity among flaviviruses
-  Limited utility in secondary infections
-  Two day test
-  IgM persistence

## CDC Training Course
Trained > 60 Public Health Laboratories

Proficiency Panel
100% agreement IgM ELISA
92%   agreement IgG ELISA (false neg’s)

- 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

- Automation of IgM & IgG ELISA
- Reagent Stability
- Incubation Times
- Luminex Assay
- Commercial Assays
-     (flavivirus)

***Future Directions***

## 1.

**serum, csf, tissue, & mosquito pools**

**1.**

**Standard**
**RT-PCR**

**TaqMan**
**RT-PCR**

**NASBA**

**Agarose gel**

**NucliSens****TM**
**Reader/ECL**
**Molecular**
**beacons**

**NA sequencing;**
**S. blot**

**3.**

**2.**

***RNA Extraction***

***Amplification***

***Detection***

**SYBR Green**
**RT-PCR**

**melting curve**
**analysis**

**TaqMan probe**
**PE7700/5700/7000**
**iCycler**
**Smart Cycler**
**LightCycler**
**OPTICAN**

**Molecular Amplification Assays**

## CDC TaqMan Testing Algorithm

-  Extract RNA (100 ul to 500 ul)
-  TaqMan with ENV primer set + internal control
-  Ct < 37 positive; Ct 37 – 45 equivocal
-  All positives & equivocal are repeated with a
-     second primer set; using newly extracted RNA

## RNA Extraction & Purification

**Chemical/Phase Separation**
- guanidine isothiocyanate, phenol/chloroform, ethanol precipitation. (Home-made;TRIzol)
- 40 samples per day
**Silica-gel Kits**
- column (QIAGEN)
- beads (Nuclisens, Bio-101)
- 80 samples per day
**Robotics QIAGEN 9604**
- 300 samples per day

**Notes:**
Our current protocol is similar to what we utilize in IgM ELISA testing

## TaqMan RT-PCR of West Nile Virus Dilutions

**TaqMan RT-PCR of West Nile Virus Dilutions**

**15 pfu/ml**

**15.000 pfu/ml**

**threshold**

**Ct = 37**

## Detection Limit

**0.80 pfu/ml (100 ul)**
**0.10 pfu/ml (500 ul)**

**Detection Limit**
**Plaque forming units (pfu)**
**       ****Ct = 37**

- _3’NC set_
**3.2 pfu/ml**

- _NS5 set (Lipken)_
**1.2 pfu/ml**

## WN Virus TaqMan Assay Detection Limit

**5 copies**
_**DS DNA**_
**12 copies**
_**RNA (Kramer)**_
**37 copies**

**WN Virus TaqMan Assay Detection Limit**

## WN Virus TaqMan Assay With 

**WN Virus TaqMan Assay With **
**JOE-Labeled Internal Positive Control**

**WN virus**
**primer/probe set**

**HEX internal control**
**primer/probe set**

**Sensitivity of WN Virus NASBA & TaqMan  Assays**

## Specificity of WNV NASBA & TaqMan  Assays

**Specificity of WNV NASBA & TaqMan  Assays**

## Molecular Evolution of WN Virus Strains in the U.S.

**Molecular Evolution of WN Virus Strains in the U.S.**

- All US WN strains  >99.8% identical (nucleotide)
-  <3 amino acid differences between any 2 isolates
-  WNV NY1999 & WNV FLA 2002:
- 25 nucleotide differences & 1 amino acid substitution

## 1999: No WNV isolated
2000: No WNV isolated
2001: 1 virus isolated csf (NY State Lab)
2002: 13 WNV isolated CDC + 1 from MD Dept. Health
 5 serum/plasma
 3 csf
 4 brain tissue
 1 liver 

**2000: No WNV isolated**
**2001: 1 virus isolated csf (NY State Lab)**
**2002: 13 WNV isolated CDC + 1 from MD Dept. Health**
** ****5 serum/plasma**
** ****3 csf**
** ****4 brain tissue**
** ****1 liver **

**WNV Isolates From Humans:****1999 - 2002**

***Virus Isolates are Important!***

## Human viremia is low:
Transfusion studies: 1-130 pfu/ml
 Average 24 pfu/ml
 Virus isolation is rare (asymptomatic IgM neg donors)

Human viremia is short-lived 
Rarely 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

- Transfusion studies: 1-130 pfu/ml
-  Average 24 pfu/ml
-  Virus isolation is rare (asymptomatic IgM neg donors)
**Human viremia is short-lived**
- Rarely 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***

## IgM

**DAYS POST ONSET**

**1    2    3    4    5    6    7    8    9    10**

**-5   -4   -3   -2  -1    0**

**IgM**

**IgG**

**ELISA**
**P/N**

**#pfu/ml**

**WN**
**viremia**

- 150

***illness***

## CDC Training Course
Trained > 60 Public Health Laboratories

Proficiency Panel
76% Complete Agreement
False positives
Failure to detect the lowest positive

- Trained > 60 Public Health Laboratories
- Proficiency Panel
- 76% Complete Agreement
- False positives
- Failure to detect the lowest positive

**TaqMan Technology Transfer**

## West Nile is a BSL3 virus
ELISA: Biosafety Cabinet (BSC) until serum is washed, then BSL2
PRNT: BSL3
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

**ELISA:** Biosafety Cabinet (BSC) until serum is washed, then BSL2
**PRNT:** BSL3
**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;***** 4****th**** Ed.**

## CDC Reagent Production
1995 - 1999:  100 – 150 Reagent Requests/year
2002 - 560 reagent requests
No change in personnel or policy
Commercial Partners – patent license agreements for WN antigen  production.
Abbott Laboratories; Focus Technologies; GenBio; Hennessey Research Associates; Immucor; InBIOS; RMZ; Biotech Corporation; Rapid Medical Diagnostic Corp.
USDA Permit for WN RNA lysate

Select Agent Issues

Vaccine Strains (Acambis) for WN & SLE PRNT

- 1995 - 1999:  100 – 150 Reagent Requests/year
- 2002 - 560 reagent requests
- No change in personnel or policy
**Commercial Partners** – patent license agreements for WN antigen  production.
- Abbott Laboratories; Focus Technologies; GenBio; Hennessey Research Associates; Immucor; InBIOS; RMZ; Biotech Corporation; Rapid Medical Diagnostic Corp.
**USDA Permit** for WN RNA lysate
**Select Agent Issues**
**Vaccine Strains** (Acambis) for WN & SLE PRNT

**Reagent Production & Shipping**

## Diagnostic & Reference SectionPersonnel 

**Diagnostic & Reference Section**_***Personnel***_*** ***

**Denise Martin**

**Amanda Noga**

**Olga Kosoy**

**Jane Johnson**

**Amy Lambert**

**Rob Lanciotti**

**Jason Velez **

**Kathy Wolff**

**Barbara Johnson**

**Trudy Chambers**