Molecular requirements for a pandemic influenza virus: An acid-stable hemagglutinin protein

Abstract

Influenza pandemics require that a virus containing a hemagglutinin (HA) surface antigen previously unseen by a majority of the population becomes airborne-transmissible between humans. Although the HA protein is central to the emergence of a pandemic influenza virus, its required molecular properties for sustained transmission between humans are poorly defined. During virus entry, the HA protein binds receptors and is triggered by low pH in the endosome to cause membrane fusion; during egress, HA contributes to virus assembly and morphology. In 2009, a swine influenza virus (pH1N1) jumped to humans and spread globally. Here we link the pandemic potential of pH1N1 to its HA acid stability, or the pH at which this one-time-use nanomachine is either triggered to cause fusion or becomes inactivated in the absence of a target membrane. In surveillance isolates, our data show HA activation pH values decreased during the evolution of H1N1 from precursors in swine (pH 5.5-6.0), to early 2009 human cases (pH 5.5), and then to later human isolates (pH 5.2-5.4). A loss-of-function pH1N1 virus with a destabilizing HA1-Y17H mutation (pH 6.0) was less pathogenic in mice and ferrets, less transmissible by contact, and no longer airborne-transmissible. A ferret-adapted revertant (HA1-H17Y/HA2-R106K) regained airborne transmissibility by stabilizing HA to an activation pH of 5.3, similar to that of human-adapted isolates from late 2009-2014. Overall, these studies reveal that a stable HA (activation pH ≤ 5.5) is necessary for pH1N1 influenza virus pathogenicity and airborne transmissibility in ferrets and is associated with pandemic potential in humans.

Keywords: fusion glycoprotein; influenza virus; membrane fusion; pandemic; transmission.

Fig. 1.

HA activation pH values for pH1N1 influenza viruses and potential H1 swine precursors. HA activation pH was determined by syncytia assays in virus-infected Vero cells. Each dot represents HA activation pH of an individual virus. Swine viruses are from classical (Csw), Eurasian avian-like (EAsw), and North American triple-reassortant (TRsw) lineages. Human isolates are pH1N1. Avian isolates are H1N1 duck and shorebird viruses. Mean (±SD) of two to three independent experiments with duplicates is shown. Virus abbreviations are described in SI Appendix, Fig. S1. **P < 0.01, ***P < 0.001, ****P < 0.0001, Student t test.

Fig. 2.

Pathogenicity in mice. DBA/2J mice were inoculated with 750 PFU of virus or with vehicle (PBS). Mean (±SD) percentage weight change (A) and survival (B) are reported at the indicated d.p.i. in groups of 15 mice. (C) Mean (±SD) virus titers in nasal turbinates, trachea, and lungs in groups of eight mice. Dashed line shows limit of detection. (D) Histology slides were stained with hematoxylin and eosin (H&E) or polyclonal anti-NP antisera. Representative lung sections at 3 d.p.i. Black arrows show lesions, including alveoli thickening, inflammatory cell infiltration of the airway, and epithelial necrosis of bronchi/bronchioles causing cell debris in the lumen. Numerous bronchiolar epithelial cells expressed viral NP proteins in WT virus-infected mice, but only a few in Y17H virus-infected mice. The control group showed no damage or NP staining. (Scale bar, 100 μm.) (E) Median (range) pathology scores for lung histology. (F) Mean (±SD) total number of cells (Top) and neutrophils (Bottom) in bronchoalveolar lavage fluid at the indicated d.p.i. in groups of five to six mice. For the Student test (C and E) and one-way ANOVA followed by Tukey post hoc test (F), significance is as follows: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 3.

Pathogenicity in ferrets. Ferrets were inoculated intranasally with 10⁶ PFU of virus or PBS. Nasal turbinates, trachea, and lung lobes were harvested from four ferrets at 3 and 6 d.p.i. (A) Virus titers in the trachea and cranial right (CrR), middle right (MR), caudal right (CaR), cranial left (CrL), and caudal left (CaL) lung lobes at 3 d.p.i. Each bar represents an individual animal. No virus was detected at 6 d.p.i. (B and C) Pathology scores. Median (range) pathology scores correspond to lesions in the front, middle, and anterior nasal cavity (B) and bronchi, bronchioles, alveoli, and perivascular areas (C). (D) Mean (±SD) fold change of cytokine and chemokine concentration in the lungs by RT-PCR at 3 and 6 d.p.i. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; Student test.

Fig. 4.

Influenza virus transmission in ferrets by contact and airborne routes. Four donor ferrets were inoculated intranasally with 10⁶ PFU of WT (black bars) or Y17H-mutant (red bars) virus and were caged separately. The next day, one naive contact ferret was introduced into each cage (Contact: WT, blue bars; Y17H, orange bars), and another was placed in an adjacent cage that permitted only airborne contact. (Airborne: WT, purple bars; Y17H, green bars). (A–C) Titers of infectious virus in nasal washes of donor ferrets (A), contact ferrets (B), and airborne-contact ferrets (C). Downward arrows indicate subpopulations in the Y17H contact and airborne-contact ferrets with stabilized HA proteins (pH < 5.6). Each bar shows an individual animal. ****P < 0.0001; Student test. ND, not determined because there was insufficient sample for phenotypic testing from the airborne-contact ferret on day 9 p.i.

Fig. 5.

Evolution of loss-of-function Y17H mutant after acquiring enhanced transmissibility in ferrets. Viruses from the Y17H group in the experiment described in Fig. 4 were isolated so that their HA genes could be sequenced and HA activation pH values measured. Bar charts for each of the four cages (5–8) show the proportion of mutations with each residue at positions HA1-17 and HA2-106 over the course of infection. Proportions were determined by next-generation sequencing. At HA1 position 17, red bars correspond to H17 (inoculated virus) and gray bars to Y17 (stabilized revertant). At HA2 position 106, gray bars correspond to R106 (inoculated virus) and blue bars to K106 (stabilized mutant). The isolated viruses were then propagated for measurement of the pH of HA activation by syncytia assay (mean of two independent assays, each performed in duplicate). Airborne transmission was only detected in cage 8. For the cage 5 and cage 6 donors on day 1 and the cage 5 contact recipient on day 5, sufficient sample was not available for next-generation sequencing, but Sanger sequencing showed no difference from the inoculated virus (HA1-H17 and HA2-R106).