Project Number: 6040-32000-081-009-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Mar 1, 2021
End Date: Feb 28, 2023
1. Review the repositories of the collaborating laboratories and will identify contemporary influenza A viruses (IAV) and viruses that are similar to those posing a threat to the U.S. poultry industry. Use high-quality short-read sequencing to obtain the complete genomes of these representative IAV isolates from different continents. The data will be used as references during the development of the nanopore next generation secquencing (NGS) protocol. Design universal custom-barcoded primers that allow identification of all influenza A subtypes. Develop a rapid targeted influenza A viruses nanopore NGS protocol that will allow sequencing of IAV within one day of sample receiving. Develop a custom bioinformatics pipeline for analysis of NGS data. The pipeline will utilize publically available tools, will be optimized for use on a single personal computer, and will be made available to the National Animal Health Laboratory Network (NAHLN) laboratories for use with nanopore NGS analyses. 2. Utilize host depletion and RNA capture approaches to efficiently decrease the amount of host nucleic acid and increase the relative amount of pathogen RNA in the tested samples, respectively. Arguably, this will increase the efficiency of the downstream steps and will improve the specificity and sensitivity of the developed protocol. Further measure limit of detection, specificity and diagnostic sensitivity of the developed protocol and will compare them to the NAHLN validated real-time RT-PCR IAV M-gene assay and estimate their positive and negative predictive values. 3. Use clinical samples to validate the developed protocol. Use diverse sets of field samples, experimental samples, as well as field collected and spiked Finders Technology Associates (FTA) cards. All validations will be performed in parallel with the NAHLN validated real-time RT-PCR IAV M-gene assay and statistical comparisons will be done. All results will be summarized and analyzed. A Standard Operating Procedure (SOP) for targeted nanopore NGS of IAV from clinical samples will be developed. The finalized bioinformatics pipeline will be published and made available to NAHLN laboratories.
For objective 1, Eight to ten IAV isolates will be selected from the Southeast Poultry Research Laboratory repositories. The isolates will be selected to represent both North American and Eurasian lineages of influenza A viruses (IAV). At least one H5 and H7 virus will be selected from each lineage as highly pathogenic influenza A viruses historically belonging to one of these two subtypes. The complete genomes of the selected viruses will be sequenced using the short-read Illumina MiSeq sequencing platform. This sequencing will be performed with the aim to obtain high quality genome sequences of the selected isolates to be used as a reference during the development and validation of the nanopore next generation sequencing (NGS) assay. Custom barcoded pan-influenza A primers will be designed for targeted sequencing of influenza A viruses. This will allow the elimination of one step during the library preparation and will expedite the process. A targeted nanopore NGS protocol will be developed and tested at all laboratories. For objective 2, at least 15 influenza A positive clinical samples will be tested with the developed NGS protocol side-by-side with and without using the Southeast Poultry Research Laboratories' custom-developed host-depletion approach. This approach utilizes custom-designed host nucleic acid probes to deplete the host nucleic acid and increases the efficiency of the downstream sequencing steps. This approach has been successfully applied to other RNA viruses (e.g. virulent Newcastle disease virus). Once the host-depletion and RNA capture protocols are optimized, they will be used to test 10-fold serial dilutions of four selected viruses. The protocol with better analytical sensitivity (limit of detection) will be selected for further validation. The serial dilutions will also be tested by the currently validated real-time RT-PCR IAV M-gene assay to compare the limit of detection of both assays. At least 30 known positive and negative samples will be tested by the selected nanopore NGS protocol. The specificity of the NSG protocol will be estimated. For objective 3, at least 40 field samples will be tested by the developed nanopore NGS protocol. All positive samples will be confirmed by the currently validated NAHLN real-time RT-PCR M-gene assay. At least 40 experimental samples will be tested by the developed nanopore NGS protocol. All positive samples will be confirmed by the currently validated NAHLN real-time RT-PCR M-gene assay. At least 40 Finders Technology Associates (FTA) card samples will be tested by the developed nanopore NGS protocol. All positive samples will be confirmed by the currently validated NAHLN real-time RT-PCR M-gene assay. The validated targeted nanopore NGS protocol will be used for drafting a Standard Operating Procedure and shared with NAHLN laboratories. The developed bioinformatics pipeline for NGS data analysis will be summarized and made available to all NAHLN laboratories.