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Characterization of a Neuraminidase-Deficient Influenza A Virus as a Potential Gene Delivery Vector and a Live Vaccine

Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639,¹ Department of Virology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551,² Department of Veterinary Public Health, Faculty of Agriculture, Tottori University, Tottori 680-8553,³ CREST, Japan Science and Technology Corp., Saitama 332-0012, Japan,⁴ Department of Pathobiological Sciences, University of Wisconsin, Madison, Wisconsin 53706⁵

Received 28 July 2003/ Accepted 25 November 2003

We recently identified a packaging signal in the neuraminidase (NA) viral RNA (vRNA) segment of an influenza A virus, allowing us to produce a mutant virus [GFP(NA)-Flu] that lacks most of the NA open reading frame but contains instead the gene encoding green fluorescent protein (GFP). To exploit the expanding knowledge of vRNA packaging signals to establish influenza virus vectors for the expression of foreign genes, we studied the replicative properties of this virus in cell culture and mice. Compared to wild-type virus, GFP(NA)-Flu was highly attenuated in normal cultured cells but was able to grow to a titer of >10⁶ PFU/ml in a mutant cell line expressing reduced levels of sialic acid on the cell surface. GFP expression from this virus was stable even after five passages in the latter cells. In intranasally infected mice, GFP was detected in the epithelial cells of nasal mucosa, bronchioles, and alveoli for up to 4 days postinfection. We attribute the attenuated growth of GFP(NA)-Flu to virion aggregation at the surface of bronchiolar epithelia. In studies to test the potential of this mutant as a live attenuated influenza vaccine, all mice vaccinated with 10⁵ PFU of GFP(NA)-Flu survived when challenged with lethal doses of the parent virus. These results suggest that influenza virus could be a useful vector for expressing foreign genes and that a sialidase-deficient virus may offer an alternative to the live influenza vaccines recently approved for human use.

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