Mapping the lipid envelope composition of SARS-CoV2 for reducing transmission, thrombosis and inflammation

Coronaviruses are enveloped viruses, like influenza, Herpex Simplex and HIV. Thus, they are surrounded by a lipid bilayer derived from the host cell, in this case oral/lung epithelia. Surprisingly little is known about coronavirus membranes. Old studies suggest that virions bud from the phospholipid (PL)-rich ERGIC membrane, unlike many other enveloped viruses which bud from the plasma membrane, this a high level of PLs is likely. Importantly, the envelope of the SARS-CoV2 virus has not been mapped. We successfully purified SARS-CoV2 virus from Vero cell cultures on sucrose gradients, validated purity by nanosight, and are analysing its lipid composition using established mass spectrometry protocols (Stanton/O'Donnell laboratories). We can detect common PL and the full repertoire is currently being mapped and quantified. However, this now needs to be extended using human patient virus isolates since the composition is predicted to depend on the host cell and its metabolism. Notably, how virus membranes vary dependent on the host is unknown. Virus will be isolated from saliva, sputum and BAL of hospitalised patients with COVID 19. We have already obtained BAL, and subglottic aspirates, from patients intubated on ITU, and isolated live virus with titres >104 PFU/ml. Saliva will be obtained from patients using infrastructure set up as part of our ongoing clinical trial (ISRCTN25647404). We have isolated virus from saliva (104 - 105 PFU/ml) from patients on our ward. Virally-infected epithelial cells can also be harvested from the nasal mucosa using direct brushing. We plan to obtain virus from asymptomatic students donating to our university testing centre (if samples are available at that time). Virus will be purified using sucrose gradient centrifugation (already validated using Vero-derived virus). Lipids will be extracted using a chloroform/methanol method before lipidomics as described below. As this is a pilot study, we will aim for 20 BAL and 50-100 saliva samples as first step. Ethical approval processes for observational studies are well established and we anticipate no problems. A second approach will be to analyse virus cultured in A549 (human alveolar epithelial cell line transfected to overexpress ACE2 and TMPRSS2). This can be manipulated through exposure to cytokines consistent with the inflammatory status observed in COVID19 (PMID:32302401,32353870, 32678432). This model will allow scale up of virus isolation, enabling in-depth mechanistic studies to be undertaken. Virus will be cultured in A549 (+ACE2/TMPRSS2). The impact of inflammation will be tested using cytokines known to be elevated in COVID19, to mimic inflamed airway cytokine storm. These will include: IL-1b, IL-2, IL-6, IL-7, IL-8/CXCL8, IL-10, IL-17, IFNg, IFNg-inducible protein 10, monocyte chemoattractant protein 1 (MCP1), G-CSF, macrophage inflammatory protein 1a, and TNFa (PMID:32302401,32353870, 32678432). Two other cytokines of significant interest are IL-4/IL-13, known to play a key role in allergic lung diseases like asthma. IL-4/IL-13 will be tested +/- dexamethasone. Importantly, IL-4/IL-13 are inducers of procoagulant eoxPL generation in airway epithelia (PMCID:PMC5765418). It is therefore critical that we determine the impact these cytokines have on envelope composition, to determine how this may impact dissemination of bioactive lipids via the virion envelope. This will be conducted on in vitro and in vivo isolated virus and to complement this we will also analyse the subcellular membranes of A549 cells. Third, we will conduct a transcriptomic analysis of A549 cells during infection focusing on lipid metabolism. This will determine which lipid signalling pathways are utilised/hijacked by the virus to support replication (particularly PL synthesis), aiming to identify druggable targets for intervention based on interfering with the lipid