Characterization of recombinant chimeric influenza B/Yamagata/88 viruses. Western blot analysis of the rIBV-NS110-PR8HA (A) and rIBV-NS110-HK68HA (B). Extracts from MDCK cells mock infected or infected with the indicated viruses (15 hpi) were probed with specific antibodies against PR8 HA (PY102) (α-PR8 HA), HK68 HA (12D1) (α-HK68 HA), B/NS1 (polyclonal antibody) (α-B/NS1), and B/NP (B017) (α-B/NP) to monitor viral infection, and β-actin (α-Actin) was used as an internal loading control. (C) The stable expression of the chimeric hemagglutinin during infection was further assessed by immunofluorescence analysis using either a PR8 HA-specific or an HK68 HA-specific monoclonal antibody. Nuclei were stained with DAPI. IFN-α/β induction by B viruses was evaluated using the MDCK pIFNβ cell line, which expresses the firefly luciferase (FF-Lucif) gene under the control of the IFN-β promoter (9). (D) The cells were infected with the recombinant influenza B viruses. Fifteen hours postinfection, the activation of the IFN-β promoter was determined by assessing firefly luciferase activity. (E) Multicycle growth curves of the recombinant viruses in MDCK cells infected at an MOI of 0.05 and titrated by plaque assay on MDCK cells. (F) Plaque size phenotypes of the chimeric B NS1-110 viruses in MDCK cells at 3 days p.i.