Intern Abstracts

Sequence-independent amplification of rotaviruses for high-throughput next-generation sequencing

Anthony K. Bennici1, Karla M. Stucker1, Asmik Akopov1, Nadia Fedorova1, Rebecca A. Halpin1, Timothy B. Stockwell2, David E. Wentworth
1Virology Group and 2Informatics Department, J. Craig Venter Institute, Rockville, MD 20850

Rotavirus is a segmented dsRNA virus that causes gastroenteritis and is the primary cause of severe pediatric diarrhea, which results in over half a million deaths per year globally. There are eight known species of rotaviruses (A-H) that infect humans and other animals, with Rotavirus A being the leading cause of human disease. Due to their great genetic diversity and broad public health implications, it is essential to increase existing genomic sequence data on rotaviruses. The goal of this project is to develop an in-house method for the sequence-independent amplification of viral dsRNA genomes that will be optimized for high-throughput viral genome sequencing using next generation sequencing platforms already in place at the J. Craig Venter Institute (JCVI). Sequence-independent amplification involves ligating a specialized adaptor to the dsRNA genome segments to allow for the synthesis of cDNA using an adaptor-specific primer. We will use a lab-adapted human strain of rotavirus with a known genome sequence to test and optimize this sequence-independent amplification method. Development of a high-throughput sequence-independent amplification strategy for dsRNA viruses will increase our ability to sequence more diverse rotavirus strains, including clinical isolates from both humans and animals, regardless of their nucleotide sequence diversity.

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.

Genomic and antigenic analysis of seasonal H3N2 influenza A virus from 2012-2013

Haley Hochstein1, Karla M. Stucker1, Seth Schobel2, Xudong Lin1, Randall J. Olsen3, Anju Subba1, Rebecca A. Halpin1, Asmik Akopov1, Nadia Fedorova1, Timothy B. Stockwell2, James M. Musser3, Suman R. Das1, David E. Wentworth1
1Virology Group and 2Informatics Department, J. Craig Venter Institute, Rockville, MD 20850
3Center for Molecular and Translational Human Infectious Diseases Research, The Methodist Hospital Research Institute, Houston, TX 77030

Influenza is an acute viral infection of the upper respiratory system that accounts for 3-5 million hospitalizations and 250,000-500,000 deaths annually worldwide (WHO). Influenza A virus is a segmented, negative-sense RNA virus that is classified into various subtypes based on its two main surface glycoproteins, hemagluttinin (HA) and neuraminidase (NA). The two influenza A subtypes that annually circulate in humans are seasonal H1N1 and seasonal H3N2. The 2012-2013 influenza season resulted in a severe epidemic of H3N2 viruses. We hypothesize that this epidemic resulted from genetic changes in the HA that resulted in significant drift and aim to prove this by analyzing 145 human nasopharyngeal isolates that were collected from The Methodist Hospital System in Houston, Texas from November, 2012 through February, 2013. Full influenza genomic sequencing was performed on all samples using the Ion Torrent next-generation sequencing platform to identify nucleotide substitutions important in the epidemic strain. The complete genome analysis suggested that the most important changes in this strain were in the HA gene. Using the HA sequence data from these samples, as well as from other publically available clinical and vaccine sequences, we performed an alignment and inferred a maximum-likelihood phylogenetic tree. Based on these analyses, 12 clinical isolates of interest were chosen for viral isolation and in vitro antigenic analyses. The combined genomic and antigenic analyses of these samples demonstrate biologically significant antigenic drift among H3N2 viruses from the 2012-2013 influenza season in Texas, which provides important information for the upcoming vaccine selection decisions.

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