4d.Progress report.
This report serves to document research conducted under a specific cooperative agreement between ARS and The University of California-Davis. Additional details can be found in the report for the parent CRIS 1940-32000-041 00X, "Development of Rapid Real Time PCR-Based Assays for Selected OIE Class A Diseases." This report serves to document the accomplishments for Fiscal Year 2006.

This project was initiated in May 2003 with the following objectives:.
1)Perform initial analytic validation of a real-time PCR to detect VSV-New Jersey and VSV-Indiana serotypes using a single tube multiplex format and.
2)Design and provide an armored RNA containing target sequences for VSV NJ and IN, and modified for use as an assay internal control and.
3)Participate in validation of real-time RT-PCR test VSV by testing negative cohort samples. During the last year, this assay has been adapted to a multiplex format capable of identifying both VSV New Jersey and VSV Indiana serotypes in a single-tube assay. Through this agreement UC-Davis will design and provide an armored RNA to be used as a positive control in the multiplex assay and participate in negative cohort sample processing.

Accomplishments Fiscal Year 2006: California Animal Health and Food Safety Laboratory (CAHFS) at UC-Davis personnel completed processing of all samples from the negative cohort as part of the validation of a VSV-real time RT-PCR. Negative cohort samples were tested both by single tube extraction method and also by 96-well magnetic bead extraction method which allows rapid sample processing. We have now completed all samples and processed them with version 1.0 of the ARS-VSV –rRT-PCR test. As part of the VSV rRT-PCR validation, a total of 1534 head of cattle (1371 dairy and 163 beef) from Colorado were tested by the dry v1.0 test.

The CAHFS completed serum-virus neutralization testing for VSV-New Jersey and VSV-Indiana on all samples from 30 previously selected herds, and completed realtime RT PCR testing on oral and/or hoof lesions from the same animals using version1.0 of the VSV primer/probe set (dried format).

A sub-set of approximately five-hundred samples were selected from the 30 premises, which included 18 premises with antibody responses ranging between titers 1:4 and 1:256, and 12 premises with no serologic evidence of vesicular stomatitis virus exposure. The sub-set of specimens was evaluated by realtime RT PCR in a 96-well format for side-by-side comparison of version 1.0 (dry format) and version 1.2 (wet format). A 100 sample pre-validation was performed to demonstrate equivalency between the 96-well PCR and the prototype single tube PCR format. Positive extraction controls for VSV-New Jersey and VSV-Indiana yielded similar results in both formats, and all field samples tested negative in both formats. Reagents were available to complete testing on 468 of the 500 samples.

No realtime RT PCR positive reactions were detected by either format v1.0 or v1.2. PCR positive controls demonstrated excellent assay reproducibility over the study period for both formats. The V1.2 (wet format) had a slightly better reproducibility between runs (Indiana mean Ct = 28.98, SD = 0.35, New Jersey mean Ct = 30.01, SD = 0.66) compared to V1.0 with an average 1.13 Ct variability between runs. Version v1.0 had 4 assay failures tracked to failure of the desiccant or missing probe in the dried format, version 1.2 had no assay failures.

The data have been incorporated into the VSV realtime PCR validation packet.

The armored RNA construct containing the target for VSV-Indiana and VSV-New Jersey realtime RT PCR was evaluated by spiking into clinical material (oral swab fluid), and testing time and temperature stability. The armored RNA was reproducible (between run standard deviation 0.06 for VSV-NJ, 1.0 for VSV-Ind) at a mimic concentration of 10-5. The armored RNA spiked into clinical material was stable under storage conditions selected to approximate the extremes of time and temperature expected for shipping and handling samples in a diagnostic laboratory setting, including 24 hours at 37C, 2 weeks at ambient room temperature, -20C, and -70C. The analytic performance mimicked the linearity and dynamic range in the realtime RT PCR assay using reference VSV-Indiana virus, indicating that the armored RNA can be used as a non-infectious, quantifiable, and stabile PCR reagent for an assay positive control, for training panels, and for proficiency test purposes.

This research falls within component 1: Biodefense Research of the NP-103 National Program.