# Presentation: 060796

## 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

| Family/Genus | Pathogens |
| --- | --- |
| Togaviridae/Alphavirus | Eastern equine encephalitis |
| ss + RNA +; 70 nm particle | Western equine encephalitis |
|  | Venezuelan equine encephalitis |
|  |  |
| Flaviviridae/Flavivirus | St. Louis encephalitis |
| ss + RNA; 40-60 nm particle | Powassan |
|  | West Nile  |
|  | Dengue |
|  |  |
| Bunyaviridae/Bunyavirus |  |
|            California serogroup | California encephalitis |
| ss -RNA; 3 segment genome | La Crosse encephalitis |
|  | Jamestown Canyon |
|  | Snowshoe hare |
|  | Cache Valley (bunyamwera) |
|  |  |
| Reoviridae/Coltivirus | Colorado tick fever |
| ds RNA |  |

## Eastern Equine Encephalitis Human cases: 1964-2002

**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

## La Crosse Virus Encephalitis Human cases: 1964-2002

**La Crosse Virus Encephalitis Human cases: 1964-2002**

** ****2910 cases**
** ****76 cases/year **
** ****children < 16**
** ****other CAL viruses**

## 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

| Year | # Cases | # States | # Counties | Onset Date Range |
| --- | --- | --- | --- | --- |
| 1999 | 62 | 1 | 6 | 2 AUG – 24 SEP |
| 2000 | 21 | 3 | 10 | 20 JUL – 27 SEP |
| 2001 | 66 | 10 | 39 | 13 JUL – 7 DEC |
| 2002 | 4,156 |     39* | 740 | 19 MAY – 19 DEC |
| 2003 | 8,977 | 46 | 1048 | 28 MAR – 30 NOV |

*** Plus D.C.**

**Notes:**
The first 3 years of WN in the US there were a limited number of human cases.
2002; 4156 total cases, about 2700 ME
2003; 8393 total cases; about 2400 ME
Last 2 years human cases 2/3 of the year

## WNV Human Infection “Iceberg” in 2002 

**~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

**Notes:**
The crows remain an important part of the avian mortality surveillance

## 1999

**1999**

## 2000

**2000**

## 2001

**2001**

## 2002

**2002**

## 2003

**2003**

**Notes:**
50% OF ALL HUMAN CASES IN 2003 WERE IN COLORADO & NEBRASKA

## 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

## Slide 14

**Notes:**
AS A RESULT OF WN TRANSMISSION DOCUMENTED BY BLOOD TRANSFUSION SCREENING OF BLOOD PRODUCTS BEGAN IN JUNE 2003.

740 CONFIRMED DONORS-WILL LIKELY EXCEED 1000
6 “BREAKTHROUGH” CASES

## 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)

**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

|  | Bird Surveillance | Mosquito Surveillance | Veterinary Diagnostic | Human Diagnostic |
| --- | --- | --- | --- | --- |
| Test Target  | Virus | Virus | Antibody | Antibody |
| Sample Type | Tissues, oral swabs | Mosquito pools | serum | Serum, plasma, csf
tissues |
| Available
Tests | TaqMan RT-PCR
NASBA
RT-PCR
Isolation in Vero
VecTest
 | TaqMan RT-PCR
NASBA
RT-PCR
Isolation in Vero
VecTest
 | IgM ELISA
Plaque Reduction
Neutralization | IgM ELISA
IgG ELISA
Plaque Reduction
Neutralization
IgA ELISA
IFA |
| Comments | Birds have high viremia; 106 - 109 | Mosquito pool titers vary; VecTest will detect approx. 65% | Tissues from fatal equine cases tested by RT-PCR | Tissues from fatal human cases tested by RT-PCR.
Plasma/serum/csf can be tested by NAT. |

## IgM

**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**

**Notes:**
Viremia precedes clinical illness.
There is some crossover period where people are clinically ill are viremic, and are IgM positive; but this is a rare event.,
The typical patient presents with clinical illness and is NAT negative, IgM positive.

## 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***

## 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

- 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**

## 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

- P/N > 3 = positive
- P/N < 2 = negative
- P/N 2-3 = equivocal

**Interpretation of Results**

## Slide 22

## 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. **

**Notes:**
We have also observed that using IgM ELISA data alone we can predict with a high degree of accuracy a true WN case.

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

| WN/SLE ratio | % WN Cases | % SLE Cases | % Unresolved | Total # specimens |
| --- | --- | --- | --- | --- |
| < 1.00
SLE>WN | 32% | 68% | 0% | 34 |
| 1.00-1.99 | 85.8% | 6.7% | 7.5% | 120 |
| 2.00-2.99 | 93.5% | 3.6% | 2.9% | 139 |
| 3.00-3.99 | 93.1% | 1.9% | 5% | 159 |
| 4.00-4.99 | 97.1% | 0.7% | 2.2% | 139 |
| >5.00 | 98.8% | 0% | 1.2% | 745 |

## 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) |

## WN Serological Data

***2002 WN Case Tested in 2003***

|  | WNV
IgM | SLE
IgM | WNV
IgG | SLE
IgG | WNV
PRNT | SLE
PRNT |
| --- | --- | --- | --- | --- | --- | --- |
| DAY 7 | 5.2 | NEG | 12.0 | 3.4 | 1:160 | 1:10 |
| DAY 25 | 5.0 | NEG | 11.2 | 3.2 | 1:160 | 1:10 |

_**West Nile Virus IgA Assay  **_
-   95% WN IgM positive serum samples are IgA positive days 11 – 40
-   No IgA positives after day 51

## WN Serological Data

***Secondary Flavivirus Infection***

|  | WNV IgM | SLE IgM | WNV PRNT | SLE PRNT | JE PRNT | YF PRNT |
| --- | --- | --- | --- | --- | --- | --- |
| CASE 1 | 7.1 | 5.8 | 1:2560 | 1:2560 | 1:5120 | 1:640 |
|  |  |  |  |  |  |  |
|  | WNV IgM | DEN IgM | WNV PRNT | SLE PRNT | DEN PRNT | YF PRNT |
| CASE 2 | 33.2 | 2.4 | 1:2560 | 1:1280 | 1:640 | 1:640 |

**Notes:**
Secondary flavivirus infections are difficult to interpret.
The typical result is high PRNT titer to several flaviviruses with no clear 4-fold “winner.”  
In fact the PRNT titer is often highest to the 1st infecting flavivirus

## 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.”

- 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***

## 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 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**

## 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

***Future Directions***

## Microsphere-based assay to detect IgM to WN and SLE viruses in human serum

**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

**Notes:**
Anti-human IgM Red-phycoerythrin

Microsphere immunofluorescence intensity

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

| Serum
Sample | MFI (32)
WN | MFI (57)
SLE | MAC-ELISA P/N WN | MAC-ELISA P/N SLE |
| --- | --- | --- | --- | --- |
| 1 (WN) | 7464 | 408 | 37.3 | 4.3 |
| 2 (WN) | 430 | 38 | 5.8 | 0.9 |
| 3 (SLE) | 46 | 2296 | 2.9 | 7.5 |
| 4 (SLE) | 152 | 836 | 3.3 | 6.6 |
| 5 (NEG) | 17 | 41 | 0.8 | 0.9 |
| 6 (NEG) | 36 | 56 | 1.2 | 0.8 |

## 1.

**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**

## 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

**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;***** 4****th**** Ed.**

## Sample Preparation for Molecular Amplification Assays

**Mosquito pool (n=50)**
**Tissues**

**Homogenized **
**Lysate**

**RNA in H****2****O**

**Disruption**
**In QIAGEN**
**Mixer Mill**

**RNA extraction &**
**purification in QIAGEN**
**BioRobot or kits**

- Molecular Amplification Test

**Serum/plasma**
**CSF/breast milk**

**RNA in H****2****O**

## 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

- 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)**

## WNV TaqMan

**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**

## WN Virus TaqMan Assay With 

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

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

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

## Slide 41

## NASBA Detection Formats

- NASBA ECL Reader

- NASBA Molecular Beacon

**NASBA Detection Formats**

- SS RNA

**FAM**

**Q**

## Molecular Beacon Probe for WN Virus NASBA Assay

**Molecular Beacon Probe for WN Virus NASBA Assay**

**WN-specific sequence**

**WN-specific sequence**

**Stem**

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

## Ancestor

**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**

**All US WN strains  >99.7% identical (nucleotide)**
-  **<3 amino acid differences between any 2 isolates**

- 1999 & 2000

- 2001 & 2002

**WN TX HUMAN  2003**

## 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

**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**

## 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

- 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**

## 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

- 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***

## Flavivirus primers (Chang & Kuno)
California & Bunyamwera serogroup (Kuno)
Dengue
Alphavirus

- California & Bunyamwera serogroup (*Kuno)*
- Dengue
- Alphavirus

**Diagnostic & Reference Section**_***SYBR Green Consensus Assays***_*** ***

**primer-dimer**

**DEN-1**

**DEN-2**

**DEN-3**

**DEN-4**

## 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**