Codon usage bias and recombination events for neuraminidase and hemagglutinin genes in Chinese isolates of influenza A virus subtype H9N2

The representative complete neuraminidase sequences (NA) of 138 Chinese isolates and hemagglutinin sequences (HA) of 128 Chinese isolates of influenza A virus subtype H9N2 were analyzed to reveal codon usage bias and recombination events. A plot of the effective number of codons (ENC) against G + C context at the third position of the codons (ENC-plot) and correlation analysis demonstrated that mutational pressure is the main factor determining the codon usage bias in the H9N2 subtype. The high ENC values (51.31 for NA and 48.83 for HA) indicated that the codon usage bias in Chinese H9N2 isolates was very low. Plots of correspondence analysis and an ENC plot indicated that the HA and NA genes had different codon usage bias. Recombination analysis revealed two possible recombination events for HA and NA gene sequences in the Chinese isolates. Subsequent phylogenetic analysis further supported the validity of these recombination events. In addition, one false-positive recombinant for HA gene was found, which may have been derived from contamination during PCR amplification.