# Presentation: 042318

## 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 |  |

## Medically Important Arboviruses in the United States

| Pathogen | Vector | Host | Cases 
(1964-2002) | Human Disease |
| --- | --- | --- | --- | --- |
| EEE | Mosquito | Avian
 | 191 | Encephalitis;
CF-50% (children) |
| WEE | Mosquito | Avian | 640 | Encephalitis;
CF-< 5% (children) |
| SLE | Mosquito | Avian | 4561 | Encephalitis;
CF-10% (elderly) |
| WN | Mosquito | Avian | 2870
(>4000) | Encephalitis;
CF-10% (elderly) |
| LAC | Mosquito | Small mammals | 2910 | Encephalitis;
CF < 1% (children) |

**Medically Important Arboviruses in the United States**

## 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 / Estimated Number of St. Louis**
**Encephalitis Cases, U.S., 1932-2002**

## Spread of WN Virus in the US

**1999**

**2000**

**2001**

**2002**

**Spread of WN Virus in the US**

## CDC Tests for WN Virus

| Specimen | 1st Choice | Other | Comments |
| --- | --- | --- | --- |
| Human serum/CSF | IgM ELISA
Plaque Reduction Neutralization | TaqMan/NASBA
Virus Isolation | TaqMan (57%) for acute CSF; <10% serum |
| Human tissue | TaqMan/NASBA
 | Virus Isolation | Fatal WN cases: TaqMan/NASBA positive ~ 100% |
| Donated blood products | TaqMan/NASBA |  |  |
| Non-Human |  |  |  |
| 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

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

**Interpretation of Results**

## Slide 13

## Slide 14

|  | IgM | IgG |
| --- | --- | --- |
| EEE | 0.92 | 1.88 |
| SLE | 4.42 | 2.86 |
| LAC | 1.26 | 1.12 |

| WN | 9.97 | 4.62 |
| --- | --- | --- |

| PRNT |
| --- |
| NEG |
| 1:20 |
| NEG |
| 1:160 |

## 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) |

## 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
Low IgM; high PRNT to several flaviviruses

- 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
- Low IgM; high PRNT to several flaviviruses

**WN Human Serological Data**

***Lessons Learned 1999-2002***

## CDC IgM ELISA Assay

_**Good Points**_
-  Sensitive
-  Specific (WN & SLE P/N ratio)
-  IgM Positive in csf & serum at onset

_**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

***Future Directions***

## 1.

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

**1.**

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

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

**NASBA**

**Agarose gel**

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

**3.**

**2.**

***RNA Extraction***

***Amplification***

***Detection***

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

**melting curve**
**analysis**

**TaqMan probe**

**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

- 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)**

## 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

- 1,000 copies/ml
**5 copies**
_**DS DNA**_
**2,400 copies/ml**
**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**

## Slide 30

## 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**

## 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**

## 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***

## 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**

## 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**

## Diagnostic & Reference SectionTaqMan & NASBA Assays 

| Viral Target | Sensitivity | Specificity/Comments |
| --- | --- | --- |
| WN | < 0.1 pfu | Lineage 1 WN |
| SLE | < 0.15 pfu | All NA & SA SLE  |
| EEE | < 0.10 pfu | NA EEE only |
| WEE | < 0.35 pfu | All NA & SA WEE; TaqMan > sensitivity |
| LAC | < 1 pfu | 15 LAC strains; no other CAL serogroup |
| In Progress |  |  |
| DEN | < 0.1 pfu | Multiplex with serotype probes |
| SYBR Green | < 1 pfu | Consensus assays for DEN, alphavirus, flavivirus, CAL serogroup bunyavirus. |
| VEE |  |  |