A Unique Multibasic Proteolytic Cleavage Site and Three Mutations in the HA2 Domain Confer High Virulence of H7N1 Avian Influenza Virus in Chickens

Abd el-Whab, El-Sayed Mohammed GND; Veits, Jutta GND; Tauscher, Kerstin GND; Ziller, Mario GND; Teifke, Jens Peter GND; Stech, Jürgen GND; Mettenleiter, Thomas C. GND

In 1999, after circulation for a few months in poultry in Italy low pathogenic (LP) avian influenza (AI) H7N1 virus mutated into a highly pathogenic (HP) form by acquisition of a unique multibasic cleavage site (mCS) PEIPKGSRVRR*GLF in the hemagglutinin (HA) and additional, alterations with hitherto unknown biological function. To elucidate these virulence-determining alterations, recombinant H7N1 viruses carrying specific mutations in the HA of LPAI A/chicken/Italy/473/1999 (Lp) and HPAI A/chicken/Italy/445/1999 (Hp) were generated. Hp with monobasic CS or carrying the HA of Lp induced only mild or no disease in chickens thus resembling Lp. Conversely, Lp with the HA of Hp was as virulent and transmissible as Hp. While Lp with a multibasic cleavage site (Lp_CS445) was less virulent than Hp, full virulence was exhibited when HA2 was substituted by that of Hp. In HA2, three amino acids differences consistently detected between LP and HP H7N1 viruses were successively introduced into Lp_CS445. Q450L in the HA2 stem domain increased virulence and transmission but was detrimental for replication in cell culture probably due to low pH-activation of HA. A436T and/or K536R restored viral replication in-vitro and in-vivo. Viruses possessing A436T and K536R were observed early in the HPAI outbreak but later superseded by viruses carrying all three mutations. Together, beside the mCS stepwise mutations in HA2 increased fitness of the Italian H7N1 virus in-vivo. The shift toward higher virulence in the field was most likely gradual with rapid optimization. IMPORTANCE In 1999, after 9 months of circulation of low pathogenic (LP) avian influenza virus a devastating highly pathogenic (HP) AIV H7N1 in poultry emerged marking the largest epidemic of AIV reported in a Western country. The HPAIV possessed a unique multibasic cleavage site (mCS) complying with the minimum motif for HPAIV. The main findings in this report are the identification of three mutations in the HA2 domain which are required for replication, stability as well as for virulence, transmission and tropism of H7N1 in chickens. In addition to the mCS, Q450L was required for full virulence and transmissibility of the virus. Nonetheless, it was detrimental for virus replication and required A436T and/or K536R to restore replication, systemic spread and stability. These results are important for better understanding the evolution of highly pathogenic avian influenza viruses from low pathogenic precursors.

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Abd el-Whab, El-Sayed Mohammed / Veits, Jutta / Tauscher, Kerstin / et al: A Unique Multibasic Proteolytic Cleavage Site and Three Mutations in the HA2 Domain Confer High Virulence of H7N1 Avian Influenza Virus in Chickens. 2015.

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