Emergence of Highly Pathogenic Avian Influenza A(H5N1) Virus PB1-F2 Variants and Their Virulence in BALB/c Mice

Abstract

Influenza A viruses (IAVs) express the PB1-F2 protein from an alternate reading frame within the PB1 gene segment. The roles of PB1-F2 are not well understood but appear to involve modulation of host cell responses. As shown in previous studies, we find that PB1-F2 proteins of mammalian IAVs frequently have premature stop codons that are expected to cause truncations of the protein, whereas avian IAVs usually express a full-length 90-amino-acid PB1-F2. However, in contrast to other avian IAVs, recent isolates of highly pathogenic H5N1 influenza viruses had a high proportion of PB1-F2 truncations (15% since 2010; 61% of isolates in 2013) due to several independent mutations that have persisted and expanded in circulating viruses. One natural H5N1 IAV containing a mutated PB1-F2 start codon (i.e., lacking ATG) was 1,000-fold more virulent for BALB/c mice than a closely related H5N1 containing intact PB1-F2. In vitro, we detected expression of an in-frame protein (C-terminal PB1-F2) from downstream ATGs in PB1-F2 plasmids lacking the well-conserved ATG start codon. Transient expression of full-length PB1-F2, truncated (24-amino-acid) PB1-F2, and PB1-F2 lacking the initiating ATG in mammalian and avian cells had no effect on cell apoptosis or interferon expression in human lung epithelial cells. Full-length and C-terminal PB1-F2 mutants colocalized with mitochondria in A549 cells. Close monitoring of alterations of PB1-F2 and their frequency in contemporary avian H5N1 viruses should continue, as such changes may be markers for mammalian virulence. IMPORTANCEAlthough most avian influenza viruses are harmless for humans, some (such as highly pathogenic H5N1 avian influenza viruses) are capable of infecting humans and causing severe disease with a high mortality rate. A number of risk factors potentially associated with adaptation to mammalian infection have been noted. Here we demonstrate that the protein PB1-F2 is frequently truncated in recent isolates of highly pathogenic H5N1 viruses. Truncation of PB1-F2 has been proposed to act as an adaptation to mammalian infection. We show that some forms of truncation of PB1-F2 may be associated with increased virulence in mammals. Our data support the assessment of PB1-F2 truncations for genomic surveillance of influenza viruses.

Improved detection of influenza A virus from blue‐winged teals by sequencing directly from swab material

“…This sample also contained the PB1-F2 S66 marker, which has been associated with increased pathogenicity in mammals (Conenello et al, 2011). In chickens, PB1-F2 has been implicated in increasing the duration of virus shedding upon infection and lowering pathogenicity (Kamal et al, 2015;Krumbholz et al, 2011;Zell et al, 2007). We also found N and T at position 66; interestingly, PB1-F2 T66 was only found in samples (n = 3 samples) sequenced through swab-NGS.…”

“…The presence of PB1-F2 T66 in databases is low, suggesting either low circulation of this marker in avian species and/or detection bias. In chickens, PB1-F2 has been implicated in increasing the duration of virus shedding upon infection and lowering pathogenicity (Kamal et al, 2015;Krumbholz et al, 2011;Zell et al, 2007). The consequence of the total length and residue preference at position 66 in PB1-F2 for fitness in natural hosts remains to be determined.…”

Influenza A virus PB1-F2 protein prolongs viral shedding in chickens lengthening the transmission window

“…Recently it has been reported that approximately 61 % of HPAI H5N1 isolates from 2013 had a truncated PB1-F2 (Kamal et al , 2015); therefore we also examined if avian virus pathogenicity as defined by a multiple basic cleavage site (two or more basic aa), in the HA protein, was associated with PB1-F2 length. We found little variation in the frequency of full-length PB1-F2 in HPAI or LPAI viruses and a marginal reduction in HPAI (87.7 % HPAI vs 91.0 % LPAI) was entirely due to the H5 subtype (Table S3).…”

“…More than half of all human and swine isolate sequences deposited in the public sequence databases encode a PB1-F2 less than 90 aa in length. In contrast, more than 90 % of sequences from isolates sequenced from avian hosts retain the coding capacity for a full-length PB1-F2 protein (Kamal et al , 2015; Zell et al , 2007). This disparity of PB1-F2 full-length conservation between virus isolates from mammalian and avian hosts suggests a unique pressure to maintain a full-length PB1-F2 protein with evolutionary significance in avian hosts.…”

Genetically Different Highly Pathogenic Avian Influenza A(H5N1) Viruses in West Africa, 2015

“…Moreover, the alternative reading frame of the polymerase basic protein 1 of the WA2 viruses is truncated (57 aa long), as it is in the Asian and European progenitors. This truncation is common among influenza A viruses of mammals and in HPAI A(H5N1) viruses, and it has been associated with increased virulence in mammals ( 12 ). …”