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Inactivation of Influenza A virus under expiratory aerosol conditions is mediated by structural protein changes, as determined by whole virus mass-spectrometry.

ABSTRACT: Multiple respiratory viruses including Influenza A virus (IAV) can be transmitted via expiratory aerosol particles, and many studies have established that environmental conditions can affect viral infectivity during airborne transmission. Low aerosol pH was recently identified as a major factor influencing the infectivity of aerosol-borne IAV and SARS-CoV-2, however, there is a fundamental lack of understanding as to the mechanisms leading to viral inactivation within the acidic aerosol micro-environment. Here, we identified that the early stages of the IAV infection cycle were impacted by transient exposure to acidic aerosol conditions (pH below 5.5), which was primarily attributed to loss of binding function of the viral protein haemagglutinin. Viral capsid integrity was also somewhat affected by transient acidic exposure. We then characterised the structural changes associated with loss of viral infectivity using whole-virus hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS), and observed discrete regions of unfolding in the external viral protein haemagglutinin and in the internal matrix protein 1. Viral nucleoprotein structure appeared to be unaffected by exposure to acidic aerosol conditions, and no changes to viral genome integrity or to lipids within the viral envelope were detected using our whole-virus methods. Collectively, these data indicate that viral inactivation observed under indoor aerosol conditions is mediated by specific protein conformational changes, particularly to haemagglutinin. This study additionally provides a proof-of-concept that HDX-MS is a highly effective method for characterisation of internal and external proteins of whole enveloped viruses such as IAV. Overall, improved understanding of the fate of respiratory viruses within exhaled aerosols will aid the development of novel strategies and therapeutics to control the severity of seasonal and/or pandemic influenza, and constitutes a global public health priority.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Influenza A Virus (a/wsn/1933(h1n1))

DISEASE(S): Influenza

SUBMITTER: Oscar Vadas

LAB HEAD: Oscar Vadas

PROVIDER: PXD037176 | Pride | 2023-08-10

REPOSITORIES: Pride

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Project description:Influenza A virus (IAV) infection is a global respiratory disease, which annually leads to 3-5 million cases of severe illness, resulting in 290,000-650,000 deaths. Additionally, during the past century, four global IAV pandemics have claimed millions of human lives. The epithelial lining of the trachea plays a vital role during IAV infection, both as point of viral entry and replication as well as in the antiviral immune response. Tracheal tissue is generally inaccessible from human patients, which makes animal models crucial for the study of the tracheal host immune response. In this study, pigs were inoculated with swine- or human-adapted H1N1 IAV to gain insight into how host adaptation of IAV shapes the innate immune response during infection. In-depth multi-omics analysis (global proteomics and RNA sequencing) of the host response in upper and lower tracheal tissue was conducted, and results were validated by microfluidic qPCR. Additionally, a subset of samples was selected for histopathological examination. A classical innate antiviral immune response was induced in both upper and lower trachea after infection with either swine- or human-adapted IAV with upregulation of genes and higher abundance of proteins associated with viral infection and recognition, accompanied by a significant induction of interferon stimulated genes with corresponding higher proteins concentrations. Infection with the swine-adapted virus induced a much stronger immune response compared to infection with a human-adapted IAV strain in the lower trachea, which could be a consequence of a higher viral load and a higher degree of inflammation. Central components of the JAK-STAT pathway, apoptosis, pyrimidine metabolism, and the cytoskeleton were significantly altered depending on infection with swine- or human-adapted virus and might be relevant mechanisms in relation to antiviral immunity against putative zoonotic IAV. Based on our findings, we hypothesize that during host adaptation, IAV evolve to modulate important host cell elements to favor viral infectivity and replication.

Project description:The amino acid sequences of immunodominant domains of hemagglutinin (HA) on the surface of influenza A virus (IAV) evolve rapidly, producing viral variants. HA mediates receptor recognition, binding and cell entry, and serves as the target for IAV vaccines. Glycosylation, a post-translational modification that places large, branched polysaccharide molecules on proteins, can modulate the function of HA, and can shield antigenic regions allowing for viral evasion from immune responses. Our previous work showed that subtle changes in the HA protein sequence can have a measurable change in glycosylation. Thus, being able to measure quantitatively how glycosylation changes in viral variants is critical for understanding how HA function may change throughout viral evolution. Moreover, understanding quantitatively how the choice of viral expression systems affects glycosylation can help in the process of vaccine design and manufacture. Although IAV vaccines are most commonly expressed in chicken eggs, cell-based vaccines have many advantages and the adoption of more cell-based vaccines would be an important step in mitigating seasonal influenza and protecting against future pandemics. Here, we have investigated the use of data-independent acquisition (DIA) mass spectrometry for quantitative glycoproteomics. We found that DIA improved the sensitivity of glycopeptide detection for four variants of A/Switzerland/9715293/2013 IAV: WT and mutant, each expressed in chicken eggs and MDCK cells. We used the Tanimoto similarity metric to quantify changes in glycosylation between WT and mutant, and between egg-expressed and cell-expressed virus. Our DIA site-specific glycosylation similarity comparison of WT and mutant expressed in eggs confirmed our previous analysis while achieving greater depth of coverage. We found that sequence changes resulting from different viral expression systems affected distinct glycosylation sites of HA. Our methods can be applied to track glycosylation changes in circulating IAV variants to bolster the genomic surveillance already being done, for a more complete understanding of IAV evolution.

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	20211011_OT_HDXMS_42.raw	Raw
	20211011_OT_HDXMS_73.raw	Raw
	20211011_OT_HDXMS_74.raw	Raw
	20211011_OT_HDXMS_76.raw	Raw
	20220207_OT_HDXMS03.raw	Raw

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Inactivation of Influenza A virus under expiratory aerosol conditions is mediated by structural protein changes, as determined by whole virus mass-spectrometry.

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