Author response: HA stabilization promotes replication and transmission of swine H1N1 gamma influenza viruses in ferrets (2020)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Pandemic influenza A viruses can emerge from swine, an intermediate host that supports adaptation of human-preferred receptor-binding specificity by the hemagglutinin (HA) surface antigen. Other HA traits necessary for pandemic potential are poorly understood. For swine influenza viruses isolated in 2009–2016, gamma-clade viruses had less stable HA proteins (activation pH 5.5–5.9) than pandemic clade (pH 5.0–5.5). Gamma-clade viruses replicated to higher levels in mammalian cells than pandemic clade. In ferrets, a model for human adaptation, a relatively stable HA protein (pH 5.5–5.6) was necessary for efficient replication and airborne transmission. The overall airborne transmission frequency in ferrets for four isolates tested was 42%, and isolate G15 airborne transmitted 100% after selection of a variant with a stabilized HA. The results suggest swine influenza viruses containing both a stabilized HA and alpha-2,6 receptor binding in tandem pose greater pandemic risk. Increasing evidence supports adding HA stability to pre-pandemic risk assessment algorithms. Introduction Aside from recent bat isolates (Tong et al., 2012; Tong et al., 2013; Kandeil et al., 2019), influenza A viruses (IAVs) originate from avian species and transmit to humans directly or via other animals (e.g. swine) (Long et al., 2019b; Russell et al., 2018). Genetically and antigenically diverse IAV strains circulate in wild and domestic birds, aquatic mammals, canine, swine, and humans (Long et al., 2019b). IAVs contain eight RNA gene segments (Shaw and Palese, 2013; Noda et al., 2018), and three previous human pandemic viruses (1957, 1968, and 2009) underwent gene reassortment before spreading to humans (Smith et al., 2009; Garten et al., 2009). Swine can serve as an intermediate host between avian species and humans in part by expressing both avian IAV-preferred (α2,3-linked) and human IAV-preferred (α2,6-linked) sialic-acid-terminated receptors (Rajao et al., 2018; de Graaf and Fouchier, 2014). Currently, H1N1, H1N2, and H3N2 subtypes are endemic in swine (Gao et al., 2017; Walia et al., 2019), and it is challenging to triage these genetically diverse viruses for pre-pandemic countermeasures. In addition to pandemics, swine IAVs cause sporadic spillovers from swine to humans. More than 400 cases of swine-IAV infections in humans have been documented between 2005 and 2018 (World Health Organization (WHO), 2018). H1N1-subtype IAVs are currently enzootic in swine, constituting a pool of genetically diverse and continuously evolving strains. Swine H1N1 strains are divided into three major lineages: classical swine (1A), human seasonal (1B), and Eurasian avian (1C) (Anderson et al., 2016). The classical swine lineage branches into three clades based on early regional considerations: alpha (α; 1A.1), beta (β; 1A.2), and gamma (γ; 1A.3) (Anderson et al., 2016). In approximately 1999, gamma viruses split into two branches: swine gamma (1A.3.3.3) and swine viruses that later contributed the hemagglutinin (HA) gene to the 2009 human pandemic virus (1A.3.3.2). (Gao et al., 2017). Frequently after 2009, HA and other gene segments from H1N1pdm viruses transmitted from humans to swine, generating diverse reassortant viruses. Swine gamma viruses isolated after 2009 contain varying numbers of internal genes originating from human H1N1pdm viruses (Ducatez et al., 2011). Here, we studied H1N1pdm and gamma clade viruses because these two clades contain HA genes that are closely related to the 2009 pandemic and many gamma clade viruses have one or more H1N1pdm internal genes that are already human adapted. During IAV entry, HA trimers bind to sialic-acid-terminating moieties, triggering endocytosis of virions. Avian IAVs have HA proteins evolved to bind glycoprotein and glycolipid receptors having terminal sialic acids with an α−2,3 linkage, and human-adapted IAVs have HA proteins containing receptor-binding pocket mutations that allow higher affinity binding to α−2,6-linked sialic acid (de Graaf and Fouchier, 2014; Xiong et al., 2014; Gambaryan and Matrosovich, 2015). Within minutes after endocytosis, virions are exposed to increasing acidification (Mellman et al., 1986; Cain et al., 1989). Upon reaching a threshold pH that varies by virus and typically ranges from pH 4.8 to 6.2 (Galloway et al., 2013; Russell et al., 2018), spring-loaded HA nanomachines are triggered to irreversibly change their structures and fuse the viral envelope with the endosomal membrane (Skehel and Wiley, 2000). Thus, IAV entry is regulated by HA receptor binding specificity and HA stability. Avian IAVs typically have HA proteins triggered at pH 5.5–6.2 while H1N1pdm isolates (2010–2011) have been shown to trend lower at pH 5.3–5.5 (Russell et al., 2018). This suggests that avian-to-human adaptation of the HA protein may include a decrease in activation pH. Multiple mechanisms may be operational. HA proteins with a lower activation pH have increased resistance to extracellular inactivation and have increased environmental stability (Poulson et al., 2016; Labadie et al., 2018; Russier et al., 2020). This may be important because the human upper respiratory tract is mildly acidic (England et al., 1999; Man et al., 2017). Intracellularly, opposing intracellular forces may select for intermediate HA stability (Singanayagam et al., 2019), which may help minimize interferon responses both in dendritic cells triggered by high-pH HA proteins (Russier et al., 2020) and in late endosomes triggered by low-pH HA proteins via interactions with IFITM2 and IFITM3 proteins (Gerlach et al., 2017). Consequently, an optimal HA stability is essential for virus replication. A pandemic-capable IAV requires its HA protein has been unseen immunologically by the majority of the population and is able to support efficient replication and transmission between humans. Pandemic risk assessment tools developed by the Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO) consider virologic, population, and ecological/epidemiological factors (Centers for Disease Control and Prevention (CDC), 2019 and World Health Organization (WHO), 2016). These are called IRAT and TIPRA, respectively. The algorithms explicitly consider HA receptor-binding specificity; however, other virologic properties that may be important are not included or are only considered indirectly by virtue of transmission experiments (reviewed in Russell et al., 2018; Lipsitch et al., 2016; Long et al., 2019b). Presently, HA stability is not considered in risk assessment tools but has been linked to IAV transmission and host adaptation. HA stabilization occurred after H1N1pdm circulation in humans (Maurer-Stroh et al., 2010; Russier et al., 2016) and has been shown to be necessary for airborne transmission of avian H5N1 and 2009 H1N1 pandemic (H1N1pdm) viruses in ferrets (Russier et al., 2016; Imai et al., 2012; Herfst et al., 2012). As swine provide an established pathway for the emergence of pandemic influenza and HA stability is an intrinsic property of the key gene involved in pandemics, we considered it important to determine the impact of HA stability on swine IAV replication and transmissibility. In this study, we hypothesized that contemporary swine viruses vary in HA stability and strains having more stable HA proteins are more likely to transmit by the airborne route in ferrets. To test this hypothesis, we measured HA activation pH for a panel of H1N1 swine gamma viruses and infected ferrets with genetically related pairs of H1N1 swine gamma viruses that differed in HA stability. Results H1N1 gamma isolates from swine had higher HA activation pH values than pandemic isolates from swine and humans To survey HA stability of contemporary H1N1 influenza A viruses (IAVs), we collected 34 swine and 21 human H1N1 IAVs (2009–2016) (Supplementary file 1). Phylogenetic analyses of HA segments showed that 22 swine isolates were gamma clade while 21 human and 12 swine isolates were pandemic (pdm) clade (Figure 1—figure supplement 1). All isolates were widely distributed within gamma and pdm clades and thus could be taken as genetically diverse representatives of the two targeted genetic clades (Figure 1—figure supplement 1). We measured HA stability by syncytia assay, recording the highest pH at which virus-expressed HA proteins were induced to cause membrane fusion. A/Tennessee/1-560/2009 (H1N1) (A/TN/2009) had an HA activation pH of 5.5, a moderately stable value previously reported for human pandemic isolates at the start of the 2009 pandemic (Russier et al., 2016; Galloway et al., 2013). The pdm clade viruses isolated from humans and swine after 2009 had HA activation pH values ranging from 5.0 to 5.5 with an average of 5.3 (Figure 1A, Supplementary file 1). In contrast, gamma clade swine viruses had significantly higher HA activation pH values that ranged from 5.5 to 5.9 and averaged 5.7 (Figure 1A, p<0.0001 compared to the pdm clade). Overall, pdm-origin HA proteins were more stable than swine gamma, and HA stability for the two clades overlapped at pH 5.5. Figure 1 with 2 supplements see all Download asset Open asset HA activation pH values and replication capacities of H1N1 pandemic (pdm) and gamma virus isolates from humans and swine. (A) HA activation pH values. Vero cells were infected with IAVs (human pdm n = 21; swine pdm n = 12; swine gamma n = 22) at a multiplicity of infection (MOI) of 3 PFU/cell. At 16 hr post-infection, HA activation pH values were measured by syncytia assay. Each symbol represents the mean HA activation pH value of an individual isolate. A dashed line is shown at pH 5.5. (B) Replication capacities in MDCK cells. MDCK cells were infected by pandemic and gamma viruses at an MOI of 0.01 PFU/cell. Cell culture supernatants were harvested at 12, 24, and 36 hr, and were titrated by TCID50 on MDCK cells. Each symbol represents the mean peak titer of an individual isolate. The white square in the human pdm group is the reference virus A/TN/09. All experiments were independently performed at least twice. P values were determined according to one-way ANOVA followed by a Tukey’s multiple comparisons test. Swine gamma viruses had higher average peak titers in MDCK cells To evaluate replication capacities of the viruses in vitro, we inoculated viruses into MDCK cells at a multiplicity of infection (MOI) of 0.01 PFU/cell and measured virus titers at 12, 24, and 36 hr post infection (hpi). A/TN/2009, a reference isolate, had a peak titer of 3.63 × 106 TCID50/ml (Figure 1B, open square). Peak titers of human pdm viruses (2.86 × 105 to 3.29 × 107 TCID50/ml) were similar to those from swine pdm isolates (7.87 × 106 to 6.76 × 107 TCID50/ml; p=0.937). However, peak titers of swine gamma viruses (2.80 × 106 to 6.56 × 108 TCID50/ml) averaged 10-fold higher than pdm clade (p<0.001, Figure 1B, Figure 1—figure supplement 2). Two similar gamma virus pairs differed in HA stability For further work, we selected two pairs of gamma viruses that within each pair contained (a) substantial differences in HA stability, (b) only one amino-acid substitution in HA, and (c) limited amino-acid variations in other genes. Pair 1 consisted of A/swine/Illinois/2A-1213-G15/2013 (G15) and A/swine/Illinois/2B-0314-P4/2014 (P4), which contain NP and M genes from the pdm clade and the six other genes (including HA) from swine gamma (Figure 2A). Consensus sequences of these viruses differed at amino-acid positions HA1-210, PA-271, and PB2-648. HA1-210 is located at the HA receptor-binding domain near the trimer interface, where it could potentially modulate HA head dissociation during acid-induced activation (Figure 2B). Next-generation sequencing (completed after ferret studies) revealed isolate P4 contained approximately 100% HA1-S210 while isolate G15 contained approximately 85% HA1-N210 and 15% HA1-S210 (Figure 2C). Virus isolates P4 and G15 induced syncytia formation at pH 5.5 and 5.8, respectively, whether the HA and NA genes were expressed from the background of wild-type viruses or 6+2 reassortant viruses containing internal genes from A/TN/2009 (Figure 3A). Thus, the PA and PB2 variations did not appear to contribute to differences in HA stability. Acid inactivation titrations showed G15 virus was susceptible to inactivation at 0.2 pH units higher than P4 (Figure 3B). Viruses G15 and P4 grew to similar titers that were nearly 100-fold higher than A/TN/2009 in swine testicular (ST) and MDCK cells (Figure 3C). The Pair 1 viruses bound to both α2,6- and α2,3-linked receptors (Figure 3D). P4 had higher binding to α2,6 and less binding to α2,3 than G15. In summary, Pair 1 gamma swine viruses were capable of efficient replication in cultured cells and binding to both α2,6- and α2,3-linked receptors. However, P4 had an HA activation pH of 5.5 and receptor-binding skewed toward an α2,6 preference, whereas G15 had a substantially higher activation pH of 5.8 and receptor-binding skewed toward an α2,3 preference. Figure 2 Download asset Open asset Genetic, structural, and sequence analyses of swine gamma Pair 1 and Pair 2 viruses. (A) Lineages of Pair 1 and 2 viruses. Virus gene segments were analyzed by using CLC Genomics Workbench version 11.0.1. A/Ohio/09/2015 and A/Iowa/39/2015 H1N1 viruses are gamma viruses from human infections (Pulit-Penaloza et al., 2018). Gene segments from pandemic and swine endemic are marked in black and white, respectively. (B) HA protein structure and locations of HA residue variations. HA1 residue 210 (yellow) is located in the HA receptor-binding domain head (green) in contact with an adjacent protomer (gray). HA2 residue 117 (yellow) is located in the stalk region (red) in contact with the fusion peptide (black) of an adjacent protomer. HA1 residues in the stalk are colored blue, and two protomers of the trimer are colored gray. The structure was generated by Mac PyMOL using A/California/04/2009 (H1N1) (PDB entry 3UBE). (C) Sequence variations between Pair 1 isolates G15 and P4. (D) Sequence variations between Pair 2 isolates P19 and P24. Whole-genome analyses were performed by next-generation sequencing. Amino-acid variations with frequencies greater than 5% are shown in colored pie charts. H3 numbering was used for the HA segment. Figure 3 Download asset Open asset Pair 1 (G15 and P4) virus characterization in vitro. (A) HA activation pH measured by syncytia assay. Viruses were inoculated into Vero cells at an MOI of 3 PFU/cell. Recombinant viruses with HA and NA segments from G15 and P4 were rescued in the background of the six internal genes from A/TN/09 using reverse genetics. Representative images of three independent experiment results are shown. (B) HA inactivation pH measured by loss of infectivity as a function of acid exposure. Virus aliquots were treated with pH-adjusted PBS, re-neutralized, and subjected to measurement of residual virus infectivity by TCID50. (C) Virus growth capacity in swine testicular (ST) and MDCK cells. Viruses were inoculated into ST and MDCK cells at an MOI of 0.01 PFU/cell. Cell culture supernatants were harvested at the indicated time points and quantified by TCID50. (D) Binding specificities toward α(2, 6)- or α(2, 3)-linked sialic acid receptors. Viruses were inoculated onto fetuin-coated plates. Virus binding affinities toward α2,6- or α2,3-linked sialylglycopolymers were measured by solid-phase receptor binding assay. Recombinant A/Puerto Rico/9/1934 (H1N1) with the HA segment from A/Mallard/Alberta/383/2009 (H5N1) was used as a control. All data were means ± SD of at least two independent experiments. Pair 2 consisted of A/swine/Illinois/2E-0113-P19/2013 (P19) and A/swine/Illinois/2E-0113-P24/2013 (P24), which contain PB2, PB1, PA, and M genes from the pdm clade and the four other genes (including HA) from swine gamma (Figure 2A). Consensus sequences of these viruses had only one major amino-acid variation in the genome, residue HA2-117. HA2-117 is located in helix D of the central triple-stranded coiled coil in the stalk, where it forms part of a pocket engaged by the fusion peptide of an adjacent protomer in the prefusion structure (Figure 2B). Next-generation sequencing (completed after ferret studies) showed that isolate P24 contained approximately 100% HA2-N117 while isolate P19 contained approximately 83% HA2-T117 and 17% HA2-N117 (Figure 2D). Virus isolates P24 and P19 caused syncytia formation at pH 5.6 and 5.9, respectively (Figure 4A). In the background of 6 + 2 reassortment viruses containing internal genes from A/TN/2009, P24 caused membrane fusion at pH 5.6 while P19 was shifted up to 6.0 (Figure 4A). P19 virus was susceptible to acid inactivation at 0.15 pH units higher than P24 (Figure 4B). Viruses P24 and P19 grew to similar titers and approximately 10 to 100-fold higher than A/TN/2009 in ST and MDCK cells (Figure 4C). The Pair 2 viruses bound to both α2,6- and α2,3-linked receptors similarly (Figure 4D). In summary, the Pair 2 viruses replicated efficiently in cell culture and bound similarly to α2,6- and α2,3-linked receptors. However, the Pair 2 viruses differed at stalk residue HA2-117, which resulted in P24 becoming activated at pH 5.6 for membrane fusion while P19 was activated at pH 5.9. Figure 4 Download asset Open asset Pair 2 (P19 and P24) virus characterization in vitro. (A) Syncytia assay results for wild-type and 6+2 reassortant viruses. (B) HA inactivation pH values quantified by TCID50. (C) Virus replication in ST and MDCK cells inoculated at an MOI of 0.01 PFU/cell and quantified by TCID50. (D) Receptor binding specificities to α2,6- or α2, 3-linked sialic acid receptors. All data were mean ± SD of at least two independent experiments. Pair 1 viruses showed consistently efficient transmissibility by contact but varying transmissibility through airborne exposures in ferrets To investigate the impact of HA stability on virus pathogenicity and transmission, three donor ferrets were intranasally inoculated with 106 PFU of isolates G15 and P4, which had a low HA stability (activation pH 5.8) and a moderate HA stability (activation pH 5.5), respectively. Each group was in an isolated cubicle, and the G15 cubicle was processed before decontamination and handling the P4 group. Additionally, each group had three cages containing one donor ferret each. One day following inoculation, three naive contact ferrets were cohoused with the donor ferrets, and three airborne ferrets were introduced individually into adjacent non-contact compartments separated by perforated dividers that allowed air flow. All ferrets were monitored for body weight and temperature daily, and nasal washes were collected every other day until day 14. Ferret sera were collected on day 21. The results showed that temperature and body weight changes of ferrets infected with G15 and P4 were limited and similar to each other (Figure 5—figure supplement 1), consistent with swine viruses typically causing only mild disease in ferrets (Russier et al., 2017; Pulit-Penaloza et al., 2018). On day 1, ferrets inoculated with G15 had nasal wash titer of 1.5 × 105 TCID50/ml, whereas those from P4-infected ferrets were 10-fold higher (2.2 × 106 TCID50/ml, p=0.23) (Figure 5A). Peak days (day 2.3 and 3.0 for G15 and P4, respectively, p=0.21) and peak titers of infection (6.4 and 6.7 log10 TCID50/ml for G15 and P4, respectively) were similar (Table 1). Moreover, both groups displayed similar contact transmission contact virus and contact peak nasal titers and log10 TCID50/ml for G15 and P4, respectively, (Figure to airborne transmission, only ferrets in the P4 group (activation pH and had nasal wash which at × 105 TCID50/ml (Figure The G15 (pH 5.8) airborne group had and nasal virus titers of × 106 TCID50/ml and In summary, both virus isolates replicated efficiently in transmitted efficiently to and replicated to titers in inoculated with G15 efficient airborne transmission while those inoculated with P4 had only Figure with 1 supplement see all Download asset Open asset Pair 1 (G15 and P4) replication and transmission in ferrets. inoculated ferrets were On the 3 naive ferrets each were cohoused with the and 3 naive ferrets were into perforated cages transmission through the Virus TCID50 titers of nasal washes from and (C) ferrets. Each represents the virus titer of an individual and the dashed the of (D) titers of sera of ferrets infected or exposed to the tested viruses. The of was not 1 H1N1 swine gamma virus characterization before and after infection and transmission in ferrets. Virus 1 2 activation inactivation in binding of peak peak titer titers activation pH activation pH activation activation pH measured by syncytia assay. 6+2 viruses contained HA and NA genes from swine gamma isolates and the six internal genes from inactivation pH measured by acid-induced inactivation pH with TCID50 binding specificity measured by solid-phase receptor binding assay. activation pH of virus on day of variant HA1-S210 in the and transmitted ferrets airborne transmission by the G15 group was as HA1-N210 population resulted in a higher HA activation pH and a binding of α2,6- α2,3-linked receptors compared to P4. viruses collected from ferret nasal we measured HA stability by syncytia assay. For the group inoculated with P4 isolate (pH 5.5), HA activation pH values in and airborne animals at an intermediate stability of pH (Figure The HA activation pH of the G15 was donor and exposed contact ferrets nasal viruses with HA activation pH ranging from 5.6 to 5.9. three airborne ferrets and viruses were from only one airborne ferret virus for which revealed a value of 5.6 on the day collected (day Figure Thus, P4 with a moderately stable HA) its after transmission while G15 with an HA) had its HA protein stabilized during airborne transmission. Figure 6 with 1 supplement see all Download asset Open asset and of Pair 1 (G15 and P4) viruses after infection and transmission in ferrets. The transmission was performed as in Figure (A) Virus HA activation pH values ± measured by at least two independent syncytia assay experiments. (B) HA variant in G15 and P4 viruses after infection and contact transmission in ferrets by using mutations and with a frequency than 5% during the transmission in to the sequences of the P values were determined by test. (C) of HA1-N210 and HA1-S210 in G15 before and after infection and transmission in ferrets. We ferret nasal washes by next-generation sequencing. The of HA and in G15 donor and contact ferrets were significantly higher than those of P4 (Figure G15 was less genetically stable in ferrets. P4 had mutations in and airborne however, of these increased time or (Figure supplement G15 had a of that did not or substantially from (Figure supplement As in Figure the of G15 contained 85% HA1-N210 and 15% HA1-S210 HA1-N210 within one day in two inoculated ferrets and by 3 days in the ferret (Figure Figure supplement In contact and airborne G15 consisted of by P4 contained was as and residues that were not in P4. airborne transmission for both Pair 1 groups occurred with a frequency of virus transmitted virus contained and the HA activation pH of virus was In the G15 virus containing the HA1-N210 variation (pH 5.8) was and did not airborne transmit in the HA1-S210 (pH was airborne transmitted with its and variations. HA stabilization was during infection and transmission with Pair 2 viruses to the HA1-S210 that in ferrets had increased HA stability and increased binding to α2,6- α2,3-linked receptors (Figure 3D). these changes have been shown to IAV replication and transmission in ferrets (Russell et al., 2018). we performed a ferret transmission using Pair 2 isolates P19 and P24 that had only one variation at HA2-117 that HA stability but not receptor-binding specificity (Figure The results for Pair 2 those for Pair 1, with a lower frequency of airborne transmission. one day of infection in inoculated the average nasal wash titer of P24 was higher than that of P19 × 107 TCID50/ml and × 105 TCID50/ml, respectively) (Figure This was not and both groups had similar titers on day Peak titers were approximately days in the P24 group but were similar in for both groups and log10 TCID50 for P19 and respectively, 1). P24 donor ferrets had higher body on days and in weight loss compared to P24 (Figure supplement 1), but these differences were relatively and both groups had only mild of P19 and P24 transmitted with 100% by contact (Figure and virus titers were between groups (Figure transmission was less efficient for these viruses than for Pair 1, as Pair 2 virus and were for only ferrets in the P19 group and in the P24 group (Figure Figure with 1 supplement see all Download asset Open asset Pair 2 (P19 and P24) replication and transmission in ferrets. The transmission was performed as in Figure Virus TCID50 titers of nasal washes from and (C) ferrets. Each represents the virus titer of an individual and the dashed the of (D) titers of sera of ferrets infected or exposed to the tested viruses. The of was not HA activation pH of viruses from donor and contact ferrets in the P24 group were at pH (Figure In the P19 group inoculated with virus having an HA activation pH of 5.9, HA stability in one donor ferret was to pH 5.7 at 3 and was approximately 5.7 in all three contact ferrets (Figure as with Pair 1 P19 virus had an HA activation pH of 5.6 (Figure HA stabilization had Figure with 1 supplement see all Download asset Open asset and of Pair 2 (P19 and P24) viruses after infection and transmission in ferrets. Virus and were determined as in Figure (A) Virus HA activation pH values ± measured by at least two independent syncytia assay experiments. P values were determined by one-way ANOVA followed by a