Role of the Untranslated Regions of the Influenza a Virus Replication and Vaccines
This project will explore determine how sequence variation in the UTR’s of the influenza vRNA’s (particularly, HA and NA) impact the replication/fitness of influenza A viruses by focusing our analysis on the UTR’s of high yield reassortants used as vaccine seed stocks and selected naturally circulating strains.
Genome sequencing and analysis:
The sequence of the complete genomes including the HA and NA UTRs of low and high yield reassortant vaccine candidate, and seed stocks and their parental viruses (wild type). This will be done by combining the M-RTPCR process currently used in the JCVI influenza sequencing pipeline with RNA ligation RT-PCR that we have developed. Table of strains to be analyzed is included below.
A. RNA ligation is used to link the 5’ and 3’ termini, which circularizes the genomic RNAs, then we use RT-PCR to amplify the region across the junction that contains the UTR.
B. These will then be sequenced using our SISSPA and next generation pipeline that employs Roche 454, and Illumina HiSeq technologies. As an alternative we will use Ion torrent to sequence these small RNA-ligation RT-PCR amplicons.
C. Some of the viruses to be studied have been sequenced or are in process via the MRTPCR based pipeline. For these samples we will only need to add RNA ligation RT-PCR data to complete the UTRs.
We will also investigate changes in the HA and NA UTRs of a subset of ~6-10 reassortants vaccine seeds which have been serially passaged in mammalian cell culture or eggs to determine if the substrate/species used to propagate the viruses selects for changes in the UTRs that enhance growth under specific conditions.
White Paper Access
The initial white paper submitted can be downloaded here. Since white papers are not always approved exactly as submitted, this document may not exactly describe the final form of the project. Please contact firstname.lastname@example.org if you have any questions.
This project has been funded in whole or part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services under contract numbers N01-AI30071 and/or HHSN272200900007C.