Evolutionary sites drive avian influenza virus adaptation

A mutant library for the A/chicken/Beijing/16/2013 (designated as BJ16) HA gene was created using a PCR-based mutagenesis approach with two rounds of mutagenesis to introduce random mutations into the plasmid-encoded HA. The gel-purified PCR amplicons and the pHW2000 plasmid were digested with BsmBI. The digested insert was gel-purified and ligated into the linearized plasmid using T4 DNA ligase. The ligation products were electroporated into E. coli electrocompetent cells. A separate culture of the wild-type HA proviral plasmid was prepared as a control.DMS virus library was generated by transfecting HEK-293T cells with the reverse genetics plasmids. At 6 hours post-transfection, the inoculum was aspirated and replaced with 8 ml of virus infection media. Supernatants were collected at 48 hpt. To amplify the rescue viruses while preserving diversity, the library was passaged once in MDCK cells grown in T175 flasks, infecting at a low multiplicity of infection of ~0.001 for 60 hours. This once-passaged DMS virus library was used for subsequent animal experiments. Samples were analyzed by the next generation sequencing.