# Presentation: 699445

## TESTING OF FIELD SPECIMENS FOR WEST NILE AND OTHER ARBOVIRUSES 

***Laura D. Kramer******1******, Elizabeth B. Kauffman******1******, Roger Nasci et al.***
***and the Arbovirus Laboratory******1***
**1****Wadsworth Center, NYSDOH**
**and**
**2****CDC, Arbovirus Diseases Branch**

***2***

## What is the question being asked? Issues to consider in choice of assay

**Sensitivity**
**Specificity**
**Speed**
**Staffing**
**Safety**
**$$**

## Specimens tested to monitoractivity of West Nile virus -1

**SERA**** for ****antibody**** detection**** **
**Wild-caught avian species**
**Captive birds**
**Sentinels**
**Cases - equine, human**

**Notes:**

We monitored viral activity by testing sera for antibody and tissues for virus. Human and other mammal sera were also tested by the state. ELISAs were performed followed by IFA and PRNT.

## Diagnostic Assays for field and case specimens - 1

**Serological**
**Assays**

**ELISA - indirect; competitive**
**    ****IgM**
**    ****IgG**
**PRNT**
**HI**
**CF**
**IFA**
**Dipsticks**
**Luminex**

***Diagnostic Assays for field and case specimens - 1***

## Serologic Assays

**Indirect**** ****ELISA**** ****using wild bird conjugate (Chiles and Reisen, 1998; Ebel, 2002)**
**Passeriformes (white-crowned sparrows)**
**Columbiformes (ringed turtle doves)**
**Galliformes (domestic chickens)**
**Anseriformes (muscovy ducks)**

**  ****Competitive**** ****ELISA**** ****(Hall, 1994)**

**  ****Confirmatory PRNT***********

## Indirect enzyme-linked immunosorbent assay

***Indirect****** enzyme-linked immunosorbent assay***

## Competitive enzyme-linked immunosorbent assay

***Competitive****** enzyme-linked immunosorbent assay***

## Suspension Array Technology Sequence of Events

*Suspension Array Technology Sequence of Events*

- Antigen - White
- Patient’s Antibody - Blue
- PE Labeled Anti Antibody - Red

- Fluorescent Bead -
- Yellow

**Notes:**
Luminex : highly sensitive homogeneous assay system based on fluorescent microbeads and reporter molecules that can detect multiple proteins in the same assay well. 
The luminex technology is based on the recognition of fluorescently colour-coded beads by a flow cytometry device. The reader identifies the beads by the use of two fluorophores inserted in the beads. It also reads a third fluorescence at the surface of the beads which allows to detect modification at the surface of these beads. For example, the phosphorylation of a peptide coupled to the beads can be detected by adding a fluorescently labelled (PE or equivalent fluorophore) antibody. The reader picks samples from tubes or from a 96-well plate. 

## Flow Cell

- Quantitative
- Measure of
- Extrinsic
- Fluorescence

- Bead
- Identification

- Laser 1

- Laser 2

**Notes:**
Using microfluidics to align the beads in a single file, the Luminex 100 instrument employs two lasers: one for the detection of the fluorescent bead itself and the other for the fluorescent reporter. The color signatures are captured optically and processed digitally into binding data. 
AdvantagesNon-Radioactive 
Highly Sensitive 
Homogeneous 
Economical 
Low Variability 

## TISSUES for virus isolation or detection

**TISSUES**** ****for virus isolation or detection**
** ****Mosquito  pools**
** ****Dead vertebrate tissues**
**  ****Oral/cloacal swabs**
** ****Living vertebrate - sera**

***Specimens tested to monitor******viral****** activity***

- (50)

## Diagnostic Assays for Arboviruses - 2

**Virus Detection**
**Assays**

**Virus isolation (cells, mice)**
**IFA, IHC**
**TaqMan RT-PCR**
**Ang-capture ELISA**
**RT-PCR**
**Dipsticks**
**NASBA**

***Diagnostic Assays for Arboviruses - 2***

## s

- e

**Antiviral MAb to well**

**Unknown virus sample**

**Antiviral MAb conjugate**

**Substrate addition**

***Antigen Capture Assay***

- Hunt et al.

## Antigen-capture ELISA

Mosquito homogenate
Avian tissue
Oral / cloacle swabs of avian carcasses
Crows & blue jays
100% agreement with assay of brain tissues

**Mosquito homogenate**
**Avian tissue**
**Oral / cloacle swabs of avian carcasses**
**Crows & blue jays**
**100% agreement with assay of brain tissues**

***Rapid assay for virus/antigen detection ***
***VecTest******TM***** *****dip sticks***

**15 min**

- R Nasci 2002

## Procedure

**VecTest****TM**** assays for arbovirus antigen**

**Control Zone:  captures unbound Ab-gold complex, confirms migration of sample through test zone.**

**Test Zone:  virus-specific antibodies immobilized on membrane, SLE (top) and WN (bottom) in this assay.**

**Reagent Zone: virus-specific antibodies conjugated to gold particle.  **

**Specificity depends on antibodies**

## Results

***Results***

**Specificity:  No evidence of cross reaction (false positives) in TaqMan and VecTest  even at highest virus titer.  **

- R Nasci

## Results – Mosquito pools

**TaqMan as standard**

**Plaque**

**TaqMan**

**VecTest**

**100%**

**60%**

**74%**

**34 TaqMan WNV-positive pools from Staten Island, NY 2000**

**(No VecTest false positives) **

- R Nasci

**67%**

**= EIA**

## Results – Oral swabs

_**Ontario, Canada (Barker et al.)**_
**	****Crows / oropharyngeal swabs**
**	****	**** ****83% sensitivity**
**	****	**** ****95% specificity**
**	**

- _Oral swabs (Komar et al.)_

- Sensitivi

**Sensitivity**:

- 85% RT-PCR +
- 79% VecTest +

## Flow chart for virologic testing of vertebrate and mosquito specimens

## RT-PCR assays on avian tissue, 2002

**Number of tests**

***RT-PCR assays on ******avian****** tissue, 2002***

**Tests done, Total 8,477**

**Birds tested, Total 4,297**

**Number**

## RT-PCR assays on mosquito pools, 2002

***RT-PCR assays on ******mosquito pools******, 2002***

**Tests done, Total 10,289**

**Mosq pools tested, Total 7,096**

**Month of year**

**Number**

**N**

**N**

**Number**

##  Disrupts

-  **Disrupts**
** ****2 x 96 samples**** ****(1.2 ml)**** **
** ****or ****           **
** ****2 x 24 samples**** ****(2.0 ml)**
** ****in 2-4 min**

**              ****TRITURATION: Qiagen Mixer Mill MM 300**

## WNV Taqman primer / probe sets

***WNV Taqman primer / probe sets***

**1,160**

**3,111**

**10,668**

**Lanciotti 2000**

## High Throughput Testing

**Automated Nucleic Acid Workstation**
- Automates sample and reaction preparation for nucleic acid analysis
**increase in productivity **
**cost efficient**
**high quality of product**
**decreased cross-contamination**
**consistency and reproducibility**

## 	

Class II biosafety cabinet enclosure (HEPA filtered)
Sets up dilutions and replicate samples in up to four 96-well output trays for TaqMan analysis
Automatically seals output trays with a full cover optical blanket and holds them at 4C
Completely compatible with TaqMan sequence detection system without additional manipulation
Software synergy 
One-step process from RNA purification  to assay plate

**Class II biosafety cabinet enclosure (****HEPA filtered****)**
**Sets up ****dilutions**** and ****replicate**** samples in up to four 96-well output trays for TaqMan analysis**
**Automatically seals**** output trays with a full cover optical blanket and holds them at 4C**
**Completely ****compatible**** ****with TaqMan sequence detection system without additional manipulation**
**Software synergy **
**One-step process**** from RNA purification  to assay plate**

***ABI Prism 6700***

## Slide 25

## Slide 26

## Slide 27

## Slide 28

##   Comparison of TaqMan Ct values on RNA samples from naturally infected bird kidneys

**  ****Comparison of TaqMan Ct values on RNA samples from naturally infected bird kidneys**

## Comparative assays on infected mosquito parts*

**Comparative assays on infected mosquito parts***

- * Added to pool of 50 uninfected mosquitoes

## Summary of High Throughput  Techniques

- Submission of sample data to laboratory on Excel spreadsheets
- High capacity mixer mill
- Robotic workstation for RNA extraction and real time RT-PCR setup
- Real time RT-PCR

## Sensitivity: Log 10 PFU by real time RT-PCR assay 

***Sensitivity: ******Log ******10 ******PFU****** by real time RT-PCR assay ***
***              ******(primer  - probe set to E gene)***

**  **_**SENSITIVITY **_
**0.08 PFU in 5 ul**

**Notes:**
the point at which the fluorescence rises appreciably above background, CT values

## Sensitivity: Copy number by real time RT-PCR assay 

**  **_**SENSITIVITY **_
**40-60 copies in 5 ul**

***Sensitivity: ******Copy number****** by real time RT-PCR assay ***
***                   ******(primer  - probe set to E gene)***

**Notes:**
The method described in the last slide quantitated samples in terms of the number of infectious viral particles. It gave no direct quantitation of the copy numbers of viral RNA because not all viral particles are infectious in a virus stock. In order to determine the detection limit in RNA copy number, the envelope gene of the WNV was cloned, in vitro transcribed by T7 polymerase, and quantitated. This is a plot of the CT values versus the log of titrated copy numbers of the in vitro transcribed RNA using primer/probe set 1160. 

## Sensitivity of multiplex real time RT-PCRusing 2 sets of primer-probes for WNV

**Sensitivity of single assay RT-PCR, 0.08 PFU**

## West Nile Virus Positive Kidney 107A

- Frozen Section, 200x, Evan’s Blue counterstain
- First antibody = rabies glycoprotein-specific Mab (3D7)
- Second antibody = FITC labeled anti-mouse IgM

## West Nile Virus Positive Crow Kidney 107A

- Frozen Section, 200x, Evan’s Blue counterstain
- First antibody = West Nile Virus E protein-specific Mab (H5.46 )
- Second antibody = FITC labeled anti-mouse IgM

## Cell culture inoculation

**Vero cell cuture**

**Cytopathology**

***Cell culture inoculation***

**Notes:**
If after inoculation of primary CC, no CPE is observed, the media is harvested and passed into 2nd CC to amplify virus if any present. Can also go back to original material and inoculate different types of CC, e.g., mosquito CC - C636, or CER -hamster cells

## WNV infected Vero cells stained with WNV specific monoclonal 

**WNV infected Vero cells stained with WNV specific monoclonal **
**         ****antibody directed against the envelope glycoprotein**
**     **

## Arbovirus Laboratory staff

**Arbovirus Laboratory staff**

**2002**