Universal SOP for Generation, Barcoding and Amplification of cDNA from Genomic RNA of BSL-3/4 Viruses
Foreign Animal Disease Research
Project Number: 8064-32000-057-61
Nonfunded Cooperative Agreement
Start Date: Feb 27, 2014
End Date: Feb 26, 2015
The J. Craig Venter Institute has been awarded at grant for whole genome approach to microbial forensics in order to achieve the development of a procedure to support the transfer of viral cDNA generated in a BSL-3/4 laboratory to BSL-2 laboratory for genomic analysis. ARS, PIADC is a co-investigator of this research project. Funding for ARS, PIADC is provided through an Interagency Agreement with US DHS. This cooperative research agreement details the scope of work between JCVI and ARS, PIADC.
Positive strand RNA viruses (ssRNA+), such as Foot-and-Mouth Disease Virus (FMDV) must be converted to cDNA prior to DNA sequencing in a bio-safety level (BSL)2 laboratories. However, the genomic RNA derived from those viruses remains infectious, and in the case of FMDV, remains a Select Agent itself. Therefore, the cDNA must be treated to remove all remaining RNA and tested to ensure the absence of infectious RNA prior to transfer to a BSL-2 laboratory. An additional concern is that full-length cDNAs from ssRNA+ viruses can be recovered.
The specific objectives of this collaborative research project are:
1. Establish a standard operating procedure (SOP) for generating high quality cDNAs from FMDV that can be removed from BSL-3 laboratory for subsequent genomic sequencing in a BSL-2 laboratory.
2. Demonstrate that the SOP completely inactivates FMDV by removing the infectious genomic RNAs and amplifying barcoded cDNAs that cannot be used to rescue and/or recover the virus.
1. A standard operating procedure (SOP) for generating high quality cDNAs from FMDV that can be removed from BSL-3 laboratory for subsequent genomic sequencing in a BSL-2 will be jointly developed by JCVI and ARS and validated at PAIDC.
2. Foot-and-Mouth Disease (FMD) viral RNA will be isolated by ARS, PIADC using a JCVI protocol which will result in inactivation of the virus particles. The purified viral RNA will be used as a template for cDNA production and amplification using the enhanced sequence independent single primer amplification (SISPA) strategy developed by JCVI. The RNA genome will be destroyed by treatment with RNases.
3. The remaining DNA composed of the SISPA products will be purified and tested for viral infectivity and the presence of infectious full-length viral genomes.
4. Safety testing will be carried by APHIS under this protocol and the modified DNA will be sent to JCVI, where it will be sequenced, analyzed and viral and host senquence will be send back to PIADC for further analysis.
5. Based on the SOP and safety test, a new protocol for bringing FMDV cDNA out of the BSL3Ag for high throughput sequencing will be established and published.