Universal Sop for Generation, Barcoding and Amplification of Cdna from Genomic Rna of Bls-3/4 Viruses
Foreign Animal Disease Research
Project Number: 1940-32000-057-65
Interagency Reimbursable Agreement
Start Date: Feb 27, 2014
End Date: Feb 26, 2016
This collaborative research proposal seeks to develop and standardize the process to convert BSL 3 and 4 level viruses to cDNAs for use the purpose of genomic sequencing in BSL-2 laboratories inclusive of Foot-and-Mouth Disease Virus (FMDV. A technique will be applied, which creates overlapping cDNA fragments that are barcoded by the addition of nucleotides to the termini which has many advantages including; the barcoded DNAs can’t be used to recover or rescue viruses, it allows the PCR amplification of the genomic material and it is scalable for high throughput sequencing of hundreds of samples simultaneously. If successful, the process will be applied to African Swine Fever Virus and Classical Swine Fever Virus.
Specific objectives include:
1. Establish a standard operating procedure (SOP) for generating high quality cDNAs from high consequence viruses that can be safely and securely removed from BSL-3/4 containment for subsequent genomic sequencing in BSL-2 laboratories.
2. Demonstrate that the SOP completely inactivates virues from diverse virus families that are considered high consequence, removes the infectious genomic RNAs and amplifies barcoded cDNAs that cannot be used to rescue or recover viruses.
3. Prove that the SOP generates high quality templates spanning the entire genome for multiple next-generation sequencing platforms.
4. Perform validation on a reagent kit provided by JCVI to generate high quality cDNAs for deep sequencing of FMDV and FMDV infected tissues.
5. Conduct viral RNA isolation and generation of cDNA using JCVI SISPA protocol and reagent kit.
6. Conduct additional purification of DNA containing SISPA products.
7. A new protocol for brining FMDV cDNA out of BSL3Ag for high throughput sequencing will be established and submitted for publication based on the SOP and safety testing results.
Viruses will be inactivated and nucleic acid manipulated to obtain barcoded cDNA. This material will be safety tested and shown to be inadequate to infect cells or derive infectious virus. Foot-and-Mouth Disease Virus (FMDV) will be tested at ARS, PIADC, and if successful, in African Swine Fever Virus (ASFV) and Classical Swine Fever Virus (CSFV). Additional viruses will be manipulated and tested by collaborators from the J.Craig Venter Institute, the University of Wisconsin, the University of Maryland, the University of North Carolina and the Centers for Disease Control and Prevention in Altanta GA to develop the standard operating procedure for viral inactivation.
The specific methodology includes the isolation of the viral RNA, conversion to cDNA using reverese transcriptase and a barcoded SISPA primer and the removal of infectious genomic RNAs and purification and subsequent demonstration that the SOP completely inactivates viruses and removes the genomic RNA. Data will be submitted to USDA, APHIS for review. If requested safety tests will be run by USDA-APHIS at PIADC BSL3Ag laboratory.
Objective 4: USDA, ARS will use the locked-down SOP and test the reagent kit developed by JCVI for inactivation of FMDV.
Objective 5: Inactivation of FMDV will be confirmed in-vitro using infection/transfection protocols previously developed. CDNA will be submitted for safety testing in animals.
Objective 6: SISPA cDNA products will be submitted for protocol approval for plasmid removal from BSL3 and determine its viability for deep sequencing. The modified DNA will be transferred to JCVI to conduct high throughput sequencing and analysis.
Objective 7: A SOP for removing FMDV cDNA out of a BSL3Ag facility for deep sequencing will be developed based on safety test results.