Differential Contribution of PB1-F2 to the Virulence of Highly Pathogenic H5N1 Influenza A Virus in Mammalian and Avian Species

Abstract

Highly pathogenic avian influenza A viruses (HPAIV) of the H5N1 subtype occasionally transmit from birds to humans and can cause severe systemic infections in both hosts. PB1-F2 is an alternative translation product of the viral PB1 segment that was initially characterized as a pro-apoptotic mitochondrial viral pathogenicity factor. A full-length PB1-F2 has been present in all human influenza pandemic virus isolates of the 20 ^th century, but appears to be lost evolutionarily over time as the new virus establishes itself and circulates in the human host. In contrast, the open reading frame (ORF) for PB1-F2 is exceptionally well-conserved in avian influenza virus isolates. Here we perform a comparative study to show for the first time that PB1-F2 is a pathogenicity determinant for HPAIV (A/Viet Nam/1203/2004, VN1203 (H5N1)) in both mammals and birds. In a mammalian host, the rare N66S polymorphism in PB1-F2 that was previously described to be associated with high lethality of the 1918 influenza A virus showed increased replication and virulence of a recombinant VN1203 H5N1 virus, while deletion of the entire PB1-F2 ORF had negligible effects. Interestingly, the N66S substituted virus efficiently invades the CNS and replicates in the brain of Mx+/+ mice. In ducks deletion of PB1-F2 clearly resulted in delayed onset of clinical symptoms and systemic spreading of virus, while variations at position 66 played only a minor role in pathogenesis. These data implicate PB1-F2 as an important pathogenicity factor in ducks independent of sequence variations at position 66. Our data could explain why PB1-F2 is conserved in avian influenza virus isolates and only impacts pathogenicity in mammals when containing certain amino acid motifs such as the rare N66S polymorphism.

Author Summary

Influenza A viruses can infect avian and mammalian hosts. Human infections with seasonal influenza virus strains are usually confined to the respiratory tract and are cleared within days by the immune system. In contrast, highly pathogenic avian influenza viruses can spread throughout the whole body, usually resulting in multi-organ failure and even death in immune competent hosts. Here, we investigated the species-specific function of an influenza A virus protein, PB1-F2, that is highly conserved in avian influenza virus strains but which is lost in many isolates from mammalian hosts. Our data indicate that PB1-F2 allows successful spreading of the virus throughout the body in experimentally infected ducks. In contrast, PB1-F2 does not contribute to the severity of HPAIV infections in mice. Nevertheless, a polymorphism at position 66 of PB1-F2 (N66S) that was found in the devastating 1918 pandemic virus and in several early H5N1 HPAIV isolates clearly increased pathogenicity of a HPAIV influenza virus in mice. Our findings might explain why the whole PB1-F2 ORF is conserved in avian influenza viruses, since it contributes to viral dissemination and pathogenicity, but can be lost in mammalian hosts as it has minimal effects on virulence.