A Combination of HA and PA Mutations Enhances Virulence in a Mouse-Adapted H6N6 Influenza A Virus

H6N6 viruses are commonly isolated from domestic ducks, and avian-to-swine transmissions of H6N6 viruses have been detected in China. Whether subsequent adaptation of H6N6 viruses in mammals would increase their pathogenicity toward humans is not known. To address this, we generated a mouse-adapted (MA) swine influenza H6N6 virus (A/swine/Guangdong/K6/ 2010 [GDK6-MA]) which exhibited greater virulence than the wild-type virus (GDK6). Amino acid substitutions in PB2 (E627K), PA (I38M), and hemagglutinin ([HA] L111F, H156N, and S263R) occurred in GDK6-MA. HA with the H156N mutation [HA(H156N)] resulted in enlarged plaque sizes on MDCK cells and enhanced early-stage viral replication in mammalian cells. PA(I38M) raised polymerase activity in vitro but did not change virus replication in either mammalian cells or mice. These single substitutions had only limited effects on virulence; however, a combination of HA(H156N S263R) with PA(I38M) in the GDK6 backbone led to a significantly more virulent variant. This suggests that these substitutions can compensate for the lack of PB2(627K) and modulate virulence, revealing a new determinant of pathogenicity for H6N6 viruses in mice, which might also pose a threat to human health. IMPORTANCE Avian H6N6 influenza viruses are enzootic in domestic ducks and have been detected in swine in China. Infections of mammals by H6N6 viruses raise the possibility of viral adaptation and increasing pathogenicity in the new hosts. To examine the molecular mechanisms of adaptation, a mouse-adapted avian-origin swine influenza H6N6 virus (GDK6-MA), which had higher virulence than its parental virus, was generated. Specific mutations were found in PB2 (E627K), PA (I38M), and HA (L111F, H156N, and S263R) and were assessed for their virulence in mice. The combination of HA(H156N S263R) and PA(I38M) compensated for the lack of PB2(627K) and showed increased pathogenicity in mice, revealing a novel mechanism that can affect the virulence of influenza viruses. H6N6 viruses should be monitored in the field for more virulent forms that could threaten human health.