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Abstract #165393

Bin Zhou, Wadsworth Center, NYSDOH, Griffin Laboratory, Bldg 1, Room 117, 5668 State Farm Road, Slingerlands, NY 12159, 518-862-4335, bxz03@health.state.ny.us

Epidemics of influenza A virus affect 15-60 million people in the US annually, and these infections cause significant morbidity and mortality. Many subtypes of influenza A, which are differentiated on the basis of their hemagglutinin (H1-H16) and neuraminidase (N1-N9) surface glycoproteins, circulate in other species, such as pigs, horses, dogs, seals and most importantly birds. Infrequently, novel influenza A viruses emerge from such reservoirs and give rise to pandemics, in which morbidity and mortality are dramatically increased. The development of plasmid-based reverse-genetic technology, to engineer a negative-sense segmented RNA genome and recover infectious influenza A virus, has revolutionized basic research and shows great promise for vaccine production. Current reverse-genetic systems use restriction digestion and ligation to create clones for each gene segment. To accelerate the rescue of new influenza A viruses for research and vaccine production, we modified the reverse-genetic system (Neumann et al. 1999), for recombination-based cloning of the entire influenza A genome, amplified using a multi-segment RT-PCR (M-RTPCR) strategy to simultaneously amplify all eight genomic segments. We used M-RTPCR to amplify the genome of a recent human H3N2 virus (A/NY/238/2005) directly from a clinical swab specimen, and then cloned the amplicons into our recombination-based reverse-genetic plasmids. Clones containing each A/NY/238/2005 gene segment were identified, and used to generate a genetically engineered virus (rA/NY/238/2005). The data show that the combination of M-RTPCR and recombination-based cloning confers sensitivity, speed, fidelity, and flexibility to the rescue of any subtype of influenza A virus, without the need for in vitro propagation.

Learning Objectives:

• At the conclusion of the presentation, the participant should be able to understand the significance of Influenza A virus as a human and animal pathogen, describe the current methods of genetic engineering and vaccine development for Influenza A virus, and explain how this new technique benefits public health.

Presenting author's disclosure statement:

Any relevant financial relationships? No
Any institutionally-contracted trials related to this submission?

I agree to comply with the American Public Health Association Conflict of Interest and Commercial Support Guidelines, and to disclose to the participants any off-label or experimental uses of a commercial product or service discussed in my presentation.