Relationship between hemagglutinin stability and influenza virus persistence after exposure to low pH or supraphysiological heating.

Guohua Yang,Charles J Russell,Chet R Ojha,Anice C Lowen

doi:10.1371/journal.ppat.1009910

Guohua Yang, Charles J Russell + Show 2 more

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https://doi.org/10.1371/journal.ppat.1009910

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Abstract

The hemagglutinin (HA) surface glycoprotein is triggered by endosomal low pH to cause membrane fusion during influenza A virus (IAV) entry yet must remain sufficiently stable to avoid premature activation during virion transit between cells and hosts. HA activation pH and/or virion inactivation pH values less than pH 5.6 are thought to be required for IAV airborne transmissibility and human pandemic potential. To enable higher-throughput screening of emerging IAV strains for “humanized” stability, we developed a luciferase reporter assay that measures the threshold pH at which IAVs are inactivated. The reporter assay yielded results similar to TCID50 assay yet required one-fourth the time and one-tenth the virus. For four A/TN/09 (H1N1) HA mutants and 73 IAVs of varying subtype, virion inactivation pH was compared to HA activation pH and the rate of inactivation during 55°C heating. HA stability values correlated highly with virion acid and thermal stability values for isogenic viruses containing HA point mutations. HA stability also correlated with virion acid stability for human isolates but did not correlate with thermal stability at 55°C, raising doubt in the use of supraphysiological heating assays. Some animal isolates had virion inactivation pH values lower than HA activation pH, suggesting factors beyond HA stability can modulate virion stability. The coupling of HA activation pH and virion inactivation pH, and at a value below 5.6, was associated with human adaptation. This suggests that both virologic properties should be considered in risk assessment algorithms for pandemic potential.

Highlights

IntroductionDiverse influenza A viruses (IAVs) circulate in wild aquatic birds, wild animals (e.g., turkeys, whales, seals, mink), domestic animals (e.g., poultry, horses, canines, swine), humans, and most likely bats [1]
Diverse influenza A viruses (IAVs) circulate in wild aquatic birds, wild animals, domestic animals, humans, and most likely bats [1]
To determine the minimum multiplicity of infection (MOI) required for luciferase reporter gene expression above background, Luc9.1 cells in 96-well plates were inoculated with 10-fold dilutions of wildtype (WT) virus or the mutant HA1-Y17H, which contains a destabilizing mutation in the HA2 stalk [33, 34, 73]

Summary

Introduction

Diverse influenza A viruses (IAVs) circulate in wild aquatic birds, wild animals (e.g., turkeys, whales, seals, mink), domestic animals (e.g., poultry, horses, canines, swine), humans, and most likely bats [1]. Avian IAVs preferentially bind glycans with SA α-2,3-linked to its subterminal galactose, while those adapted to humans and ferrets contain mutations in the receptor-binding pocket that allow engagement of glycans with α-2,6-linked SA [6,7,8]. IAV virions and IAV-infected cells contain terminal glycans, and the neuraminidase (NA) surface glycoprotein cleaves terminal SA moieties from these glycans to prevent virus aggregation and superinfection [9]. Airborne transmission between humans has been correlated with a functional balance between HA affinity for SA-containing receptors and NA enzymatic activity to cleave SA [10,11,12]. In addition to the HA and NA surface glycoproteins, viral internal genes and their interactions with host cell proteins regulate interspecies adaptation of influenza viruses

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