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

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 degrees 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 degrees 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. Author summary Many influenza viruses circulate among wild aquatic birds, sporadically infecting domestic animals and humans. Avian influenza viruses need to humanize several properties before efficiently replicating in mammals and transmitting between humans. To triage emerging zoonotic viruses for pandemic likelihood and pathogenic impact in humans, the Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO) use risk assessment algorithms that consider viral properties, human population attributes, and non-human virus ecology. While receptor-binding specificity of the HA protein is the only molecular property explicitly considered, recent studies have shown the HA protein needs to become stabilized to enable human adaptation and airborne transmission in ferret experimental models. In this work, we developed a high-throughput assay to measure the HA stability of surveillance isolates by monitoring the pH at which these viruses become inactivated. Our results showed HA activation pH and virion inactivation pH are similar for human viruses but differ in some animal viruses. Knowing that these two values, previously considered equivalent, can differ substantially will spur researchers to determine the relative importance of each for humanizing emerging influenza viruses so that pre-pandemic preparations can better target viruses of greatest risk to humans.

Automated summary

The stability of HA correlates with the acid and thermal stability of the virus, but does not correlate with thermal stability at 55 degrees Celsius. Some animal isolates have lower virion inactivation pH values than HA activation pH, indicating factors beyond HA stability can affect virion stability. The coupling of HA activation pH and virion inactivation pH below 5.6 is associated with human adaptation, and both virologic properties should be considered in risk assessment algorithms for pandemic potential.