Emergence of highly pathogenic avian influenza A(H5N1) virus PB1-F2 variants and their virulence in BALB/c mice.

Emergence of highly pathogenic avian influenza A(H5N1) virus PB1-F2 variants and their virulence in BALB/c mice.

1. Ram P. Kamal1,2, 
2. Amrita Kumar1,2, 
3. Charles T. Davis1, 
4. Wen-Pin Tzeng1,
5. Tung Nguyen3, 
6. Ruben O. Donis1, 
7. Jacqueline M. Katz1 and 
8. Ian A. York1*

+Author Affiliations

1. 1Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia; United States of America
2. 2Battelle Memorial Institute, Atlanta, Georgia; United States of America
3. 3National Centre for Veterinary Diagnostics, Department of Animal Health, Hanoi, Viet Nam

ABSTRACT

Influenza A viruses (IAV) 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 of mammalian IAV frequently have premature stop codons that are expected to cause truncations of the protein, whereas avian IAV usually express a full-length 90 amino acid PB1-F2. However, in contrast to other avian IAV, 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 1000-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, truncated (25 amino acids), 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 co-localized 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.

IMPORTANCE Although 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.

FOOTNOTES

• ↵*Corresponding author. Please direct correspondence to: Ian A York, 1600 Clifton Rd NE, MS-G16, Atlanta, GA 30329, United States of America. Phone:+1 404-639-7286, Email: ITE1@cdc.gov

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