Synergism of the polybasic proteolytic cleavage site and mutations in the HA2 protein for exhibition of full virulence and transmissibility of H7N1 virus in chickens

In 1999, low pathogenic (LP) avian influenza (AI) H7N1 virus in Italy caused widespread outbreaks in poultry. After few months the virus transformed into highly pathogenic (HP) form causing severe economic losses. Comparison of full genome sequences of LP and HP viruses indicated a considerable number of mutations in all gene segments. The hemagglutinin (HA) protein of the HPAI virus has a peculiar polybasic proteolytic cleavage site motif (pPCS). To date, the genetic markers responsible for increased virulence and transmissibility of this virus has not been fully understood. In this study, recombinant LPAI and HPAI H7N1 viruses of chicken-origin were generated using reverse genetics. A panel of mutants carrying specific mutations in the HA of LP and HP viruses were used for experimental infection of chickens as well as replication kinetics in cell culture. HPAI H7N1 virus with monobasic PCS or carrying the whole HA of LPAI H7N1 virus induced very limited clinical signs in chickens resembling the LP virus. On the contrary, LPAI virus with the whole HA of HPAI virus was as virulent and transmissible as the HPAI strain where all infected and contact chickens died. Meanwhile, recombinant LPAI virus with pPCS alone was significantly less virulent than the HPAI virus. One out of three mutations in the HA2, in the vicinity of the cleavage site, was required for the exhibition of full virulence in chickens although it reduced the replication efficiency of the HPAI virus on cell culture.