Role of the B Allele of Influenza A Virus Segment 8 in Setting Mammalian Host Range and Pathogenicity

Abstract

Two alleles of segment 8 (NS) circulate in nonchiropteran influenza A viruses. The A allele is found in avian and mammalian viruses, but the B allele is viewed as being almost exclusively found in avian viruses. This might reflect the fact that one or both of its encoded proteins (NS1 and NEP) are maladapted for replication in mammalian hosts. To test this, a number of clade A and B avian virus-derived NS segments were introduced into human H1N1 and H3N2 viruses. In no case was the peak virus titer substantially reduced following infection of various mammalian cell types. Exemplar reassortant viruses also replicated to similar titers in mice, although mice infected with viruses with the avian virus-derived segment 8s had reduced weight loss compared to that achieved in mice infected with the A/Puerto Rico/8/1934 (H1N1) parent. In vitro, the viruses coped similarly with type I interferons. Temporal proteomics analysis of cellular responses to infection showed that the avian virus-derived NS segments provoked lower levels of expression of interferon-stimulated genes in cells than wild type-derived NS segments. Thus, neither the A nor the B allele of avian virus-derived NS segments necessarily attenuates virus replication in a mammalian host, although the alleles can attenuate disease. Phylogenetic analyses identified 32 independent incursions of an avian virus-derived A allele into mammals, whereas 6 introductions of a B allele were identified. However, A-allele isolates from birds outnumbered B-allele isolates, and the relative rates of Aves-to-Mammalia transmission were not significantly different. We conclude that while the introduction of an avian virus segment 8 into mammals is a relatively rare event, the dogma of the B allele being especially restricted is misleading, with implications in the assessment of the pandemic potential of avian influenza viruses. IMPORTANCE Influenza A virus (IAV) can adapt to poultry and mammalian species, inflicting a great socioeconomic burden on farming and health care sectors. Host adaptation likely involves multiple viral factors. Here, we investigated the role of IAV segment 8. Segment 8 has evolved into two distinct clades: the A and B alleles. The B-allele genes have previously been suggested to be restricted to avian virus species. We introduced a selection of avian virus A- and B-allele segment 8s into human H1N1 and H3N2 virus backgrounds and found that these reassortant viruses were fully competent in mammalian host systems. We also analyzed the currently available public data on the segment 8 gene distribution and found surprisingly little evidence for specific avian host restriction of the B-clade segment. We conclude that B-allele segment 8 genes are, in fact, capable of supporting infection in mammals and that they should be considered during the assessment of the pandemic risk of zoonotic influenza A viruses.

Mutation of influenza A virus PA-X decreases pathogenicity in chicken embryos and can increase the yield of reassortant candidate vaccine viruses

“…Cells were incubated at 37°C, 5% CO 2 . Reverse genetics plasmids were kindly provided by Professor Ron Fouchier (A/Puerto Rico/8/34; (59)), Professor Wendy Barclay (A/England/195/2009 (60) and A/turkey/England/50-92/91 (61)), Dr John McCauley (A/California/07/2009; (62)), Dr Laurence Tiley (A/mallard/Netherlands/10/1999 (63)), Professor Robert Lamb (A/Udorn/307/72 (64)), Professor Earl Brown (A/Hong Kong/1/68 (65)) and Professor Munir Iqbal (A/chicken/Pakistan/UDL-01/2008 (66)). RG plasmids for A/mallard/Netherlands/12/2000 (NIBRG-60) and A/turkey/Turkey/1/2005 (NIBRG-23; with the multi-basic cleavage site removed (67)) were made by amplifying HA and NA genes by PCR from cDNA clones available within NIBSC and cloning into pHW2000 vector using BsmB1 restriction sites.…”

“…Tissue sections were cut and mounted onto slides and stained with haematoxylin and eosin (H&E) by the Easter Bush Pathology Service. Further sections were examined by immunohistofluorescence performed for influenza NP (62). Sections were deparaffinised and rehydrated and heat-induced antigen retrieval was performed using sodium citrate buffer (10 mM sodium citrate, 0.05% Tween20, pH 6.0).…”

Constitutive TRIM22 expression within the respiratory tract identifies tissue-specific and cell-type dependent intrinsic immune barriers to influenza A virus infection

“…Polyclonal antibodies were used to detect TRIM22 (Sigma-Aldrich; HPA003575), Mx1 (Santa Cruz; sc-50509), GBP1 (Proteintech; 15303-1-AP), IFIH1 (Proteintech; 21775-1-AP), TLR3 (Proteintech; 17766-1-AP), histone H3K27ac (AbCam; ab4729), and actin (Sigma-Aldrich; A5060). IAV hybridoma antisera were used to detect NP, M1, and NS1, as previously described [89]. Monoclonal antibodies were used to detect UBA7 (AbCam; ab133499), Actin (DSHB; 224-236-1), IFITM1 (Proteinech; 60074-1g), and IAV NP (AbCam; ab20343).…”

Comparison of the efficacy of a commercial inactivated influenza A/H1N1/pdm09 virus (pH1N1) vaccine and two experimental M2e-based vaccines against pH1N1 challenge in the growing pig model

“…Serum samples collected at the day of challenge were also characterized by western blot utilizing lysates from MDCK cells that had been infected with either PR8 virus or a PR8 7:1 reassortant with segment 7 from the pH1N1 strain A/California/ 07/2009 at an MOI of 5 as targets [ 21 ]. Cell lysates were harvested 24 hours post infection and run on SDS-PAGE gradient gels (4–20%) for separation of proteins before transferring to nitrocellulose membranes.…”