# Presentation: 718139

## Using Non-human Sourced and Banked Human Specimen Material for Nucleic Acid Based Assay Development 

- David Norwood, Ph.D.
- Diagnostic Systems Division
- USAMRIID - Ft. Detrick

## Overview

- Assay design
- Assay optimization
- Purified viral RNAs
- Sensitivity
- Specificity
- Clinical samples
- Sensitivity
- Specificity

## Assay Design

- Determine Gene Target(s)
- 392 bp of sequence from the RNA-directed RNA polymerase (pol) gene region (New England Journal of Medicine, 348:20 1953-1966)
- Corresponds to region 15239 to 15630 of the Urbani strain.
- Design specific primers and probes (Taqman)
- Primer Express 2.0 for Windows
- Oligo 5.0

## Homology in pol Region 

## 100 % Homology in pol Region (SARS)

- BJ01
- BJ02
- BJ03
- BJ04
- CUHK-SU10
- CUHK-W1
- Frankfurt 1
- GZ01
- HKU-39849
- HSR1
- SIN2500

- SIN2677
- SIN2679
- SIN2748
- TAIWAN
- TAIWAN JC2003
- TOR2
- TW1
- TWC
- URBANI
- VIETNAM
- ZJ01

## Assay Optimization 

- Template was purified RNA from viral infected cell supernatants (Urbani strain).
- 56 primer pairs tested for amplification efficiency (SYBR Green).
- 13 primer pairs selected for probe testing.
- 38 primer-probe sets tested for robustness.
- 1 primer-probe set found superior.

## Sensitivity – Purified Viral RNA

**7.5 pfu**

**0.75 pfu**

**0.075 pfu**

**0.0075 pfu**

**0.00075 pfu**

| Reaction Input | Ct |
| --- | --- |
| 7.5 pfu | 21.6 |
| 0.75 pfu | 25.0 |
| 0.075 pfu | 28.7 |
| 0.0075 pfu | 31.2 |
| 0.00075 pfu | 35.1 |

## Specificity – Purified Viral RNA

- Additional strains of SARS
- Other related coronaviruses
- None of this work done to date

## Clinical Samples

- Positive clinical samples
- Did not have access to positive clinical samples
- Work was being done to determine if a non human primate model of disease was feasible
- Negative samples (specificity)
- Should be from individuals exhibiting similar clinical presentation
- Should be samples from a relevant clinical matrix
- Should be temporally separated from the SARS virus

## Non Human Primate Model

- Three cynomologus macaques
- NHP-1 and NHP-2 received an intra-nasal (IN) challenge
- NHP-3 received an intravenous challenge.
- Animals were monitored with telemetry implants
- Strain used for challenge was Urbani

## Sample Collection / Extraction

- Urine and Feces collected daily
- Nasal swabs, throat swabs, rectal swabs, and blood were collected every other day beginning with day 0.
- Animals were followed for 20 days post challenge
- RNA was extracted from all matrices using standard Trizol extraction methods
- LOD of 4x10-1 PFU/ml for Urine, Media, and Blood
- LOD of 4x10-1 PFU/ml for Feces

## NHP Results

- NHP-1 and NHP-2 (IN)
- No disease observed by blood chemistries
- NHP-2 appeared to have a mild pneumonia on day 6 which eventually resolved
- NHP-3 (IV)
- Developed a fever on days 5 and 6, died of a bacterial infection on day 16 (May have been from telemetry implant)
- Labored breathing observed, animal was clearly sick
- Left lower lobe pneumonia, resolved by the time the animal died
- No disease observed by blood chemistries
- PCR Results
- No virus detected in feces, blood, or rectal swabs
- Virus was detected in nasal swabs, throat swabs, and urine

## Throat Swabs

## Nasal Swabs

## Urine

## Negative samples

- CONUS military patients
- Exhibited signs of respiratory disease at time of examination
- Exhibited fever at time of examination
- 100 samples received, processed and tested
- Throat washes
- All samples were from patient examinations prior to the introduction of SARS into the US
- Would other coronaviruses/infectious agents cross-react with assay?
- All 100 samples were negative for by the SARS assay

## Additional work

- Plan to challenge 6 more NHPs in August
- 3 intra-nasal challenges and 3 intravenous challenges
- Plan is to use a different strain of SARS
- Obtain nearest neighbor panels for specificity testing
- Additional SARS strains
- Other coronaviruses

## Acknowledgements

- LTC David Kulesh
- Bonnie Loveless, MS
- Deanna Christensen, MS
- SPC Elizabeth Bode
- George Ludwig, PhD
- Tammy Clements
- Len Wasieloski, PhD
- Melanie Ulrich, PhD
- Philip Craw
- Alexandra Zalles-Ganley
- Susan Coyne

- COL Timothy Endy, MD
- Jason Paragas, PhD
- John Huggins, PhD
- Lisa Hensley, PhD
- Elizabeth Fritz, PhD
- Aura Garrison, MS
- LCDR James Lawler, MD