Generating a Library of Influenza a Virus Hemagglutanin and Neuraminidase Genes

Titas Bera, Anthony Bennici, Haley Hochstein, Karla M. Stucker, Suman R. Das, David E. Wentworth
Virology Group, J. Craig Venter Institute, Rockville, MD 20850

 


Every year, five to twenty percent of the United States population is infected with seasonal influenza, making the construction of a seasonal vaccine of high importance. However, due to antigenic drift among seasonal influenza viruses, the vaccine strains need frequent updating. Antigenic drift occurs as the genes encoding influenza’s major antigenic proteins, hemagglutinin (HA) and neuraminidase (NA), undergo mutations in response to selection pressures exerted by the host immune response. Therefore, careful surveillance of influenza viruses is required to monitor their evolution and determine when the vaccine needs to be updated. Currently, these monitoring efforts require both genomic sequencing and functional studies to fully understand how influenza antigenicity is changing. In order to facilitate these studies, a large library of historical and contemporary HA and NA genes is being constructed. This project is using previously synthesized linear HA and NA genes as templates for amplification, and the products are being cloned into a reverse genetics plasmid we designed to facilitate ligation independent cloning. We are using the In-Fusion cloning technique (Invitrogen) to insert each HA or NA gene segment into the linearized plasmid. The DNA is transformed into bacteria, and clones containing the insert are identified by colony PCR and sequencing. Having a library of cloned genes from the two seasonal Influenza A virus subtypes, H1N1 and H3N2, facilitates many functional studies of these genes and their viruses to understand seasonal influenza virus evolution and help in the development of new vaccines.

 

Generating a Library of Influenza a Virus Hemagglutanin and Neuraminidase Genes

Titas Bera, Anthony Bennici, Haley Hochstein, Karla M. Stucker, Suman R. Das, David E. Wentworth
Virology Group, J. Craig Venter Institute, Rockville, MD 20850

 


Every year, five to twenty percent of the United States population is infected with seasonal influenza, making the construction of a seasonal vaccine of high importance. However, due to antigenic drift among seasonal influenza viruses, the vaccine strains need frequent updating. Antigenic drift occurs as the genes encoding influenza’s major antigenic proteins, hemagglutinin (HA) and neuraminidase (NA), undergo mutations in response to selection pressures exerted by the host immune response. Therefore, careful surveillance of influenza viruses is required to monitor their evolution and determine when the vaccine needs to be updated. Currently, these monitoring efforts require both genomic sequencing and functional studies to fully understand how influenza antigenicity is changing. In order to facilitate these studies, a large library of historical and contemporary HA and NA genes is being constructed. This project is using previously synthesized linear HA and NA genes as templates for amplification, and the products are being cloned into a reverse genetics plasmid we designed to facilitate ligation independent cloning. We are using the In-Fusion cloning technique (Invitrogen) to insert each HA or NA gene segment into the linearized plasmid. The DNA is transformed into bacteria, and clones containing the insert are identified by colony PCR and sequencing. Having a library of cloned genes from the two seasonal Influenza A virus subtypes, H1N1 and H3N2, facilitates many functional studies of these genes and their viruses to understand seasonal influenza virus evolution and help in the development of new vaccines.

 

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