To gain insight into the intertypic incompatibility between typeThe genomes of influenza A and B viruses each consist of eight single-stranded RNA segments of negative polarity. Both types of viruses have two envelope glycoproteins, hemagglutinin (HA) and neuraminidase (NA). HA is responsible for viral binding to cellular sialic acid-containing receptors, followed by fusion between envelope and cell endosomal membranes, while NA is responsible for viral release from infected cells through removal of sialic acids from cellular glycoconjugates and viral glycoproteins (11). Type A viruses are further divided into subtypes based on HA (H1 to H15) and NA (N1 to N9) antigenicities. In cells infected with two different type A viruses, intratypic reassortants possessing various combinations of gene segments are produced (24). However, intertypic reassortants between type A and B viruses have not been detected in nature, although both viruses are cocirculating in human populations.Attempts to generate reassortants between type A and B viruses have been unsuccessful (9, 15, 21). With reverse genetics, Muster et al. (17) produced a mutant type A virus whose NA noncoding regions were replaced with those of the nonstructural (NS) gene of type B virus. This result suggests that at least the noncoding regions of the type B NS segment are compatible with influenza A viral components at the level of RNA transcription and replication. However, this mutant virus replicated more slowly than wild-type WSN virus in cell culture and was attenuated in mice (17). Animals infected with the chimeric virus were resistant to challenge with the wild-type virus, suggesting that such manipulation of the influenza virus genome might be useful in producing live attenuated vaccine strains.Recently, we established a plasmid-based reverse genetics system (18) that has provided a powerful tool for generating recombinant influenza viruses. To gain insight into the intertypic incompatibility between type A and B viruses, we used this technology to study the functionality of chimeric (A/B) HAs and then evaluated the potential of the chimeric (A/B) HA viruses as live vaccines.
MATERIALS AND METHODSCells. 293T human embryonic kidney cells and COS-7 cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum and antibiotics. Madin-Darby canine kidney (MDCK) cells were grown in minimal essential medium (MEM) with 5% newborn calf serum and antibiotics. Cells were maintained at 37°C in 5% CO 2 .Construction of plasmids. The generation of plasmid constructs for viral RNA production (pPolI), containing the HA genes of wild-type A/WSN/33 (H1N1, A/WSN) (pPolI-WSN-HA) and wild-type B/Lee/40 (B/Lee) (pPolI-B-HA) flanked by the human RNA polymerase I promoter and the mouse RNA polymerase I terminator, was described in a previous publication (18). A series of chimeric (A/B) HA pPolI constructs (Fig. 1) were produced with these wild-type HA constructs, PCR amplification with ProofStart polymerase (Qiagen), and ligation. Primer sequences will...