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Based on the observation that an internally located 3' promoter sequence can be functional (R. Flick and G. Hobom, Virology, 1999, 262(1), 93-103), we generated transfectant influenza A viruses harboring a dicistronic segment containing the CAT gene (660 nt) or a fragment of the Mengo virus VP0 capsid gene (306 nt) under the control of a duplicated 3' promoter sequence. Despite slightly reduced NA expression, the transfectant viruses replicated efficiently and proved to be stable upon both serial passage in vitro in MDCK cells and in vivo replication in the pulmonary tissue of infected mice. Internal initiation of replication and transcription from the second, internal, 3' promoter directed the synthesis of subgenomic vRNA and mRNA and therefore permitted expression of the foreign gene product, e.g., the CAT enzyme. The design of this vector may prove particularly appropriate for the utilization of influenza virus for the expression of heterologous proteins in their native form.

4-Guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (4-guanidino-Neu5Ac2en) specifically inhibits the influenza virus neuraminidase (NA) through interaction of the guanidino group with conserved Glu 119 and Glu 227 residues in the substrate binding pocket of the enzyme. To understand the mechanism by which influenza viruses become resistant to 4-guanidino-Neu5Ac2en, we investigated mutations at amino acid residues 119 and 227 in the influenza virus NA for their effects on this compound and on NA activity. The NA gene was cloned from the NWS-G70c virus, and mutations were introduced at the codon for amino acid residue 119 or 227. All of the 13 mutants containing a change at residue 119 were transported to the cell surface, although their expression levels ranged from 68.2 to 91.3% of wild type. Mutant NAs that retained at least 20% of the wild-type enzymatic activity were tested for their sensitivity to 4-guanidino-Neu5Ac2en and found to be sevenfold less sensitive to this compound than was the wild-type NA. By contrast, only 6 of 13 mutants defined by modifications at residue 227 were transported to the cell surface, and those NAs lacked substantial enzymatic activity (9% of wild type, at most). These results suggest that only a limited number of resistant viruses arise through mutations at Glu 119 and Glu 227 under selective pressure from 4-guanidino-Neu5Ac2en and that the development of compounds which interact with 227 Glu more strongly than does 4-guanidino-Neu5Ac2en may reduce the likelihood of drug-resistant viruses still further.

The hemagglutinin-neuraminidase (HN) gene of the Hitchner B1 strain of Newcastle disease virus (NDV) was cloned as a cDNA and inserted into a baculovirus expression vector. The recombinant HN (recHN) expressed in Spodoptera frugiperda cells had both hemagglutinating and neuraminidase activities both of which were inhibited by polyclonal anti-NDV sera or a monoclonal antibody (MAb) against HN. Infected insect cells could hemadsorb chicken red blood cells suggesting that the recHN is properly glycosylated and transported to the cell surface. A 67-kDa recHN precursor and a 74-kDa, presumably mature, recHN from infected cells were detected by Western blot analysis and were found to comigrate with similar proteins from NDV-infected chick embryo fibroblast cells. The kinetics of synthesis of recHN was similar to that for polyhedrin and some HN appeared in the extracellular medium. HN was copurified with extracellular virus (ECV) from the extracellular medium and was used to immunize chickens. The anti recHN serum was specific to NDV in both ELISA and Western blot analysis.