Digital transcriptomics to capture SARS-CoV-2 persistence in blood

Project Summary:

A project to further validate findings from a long COVID study that identified SARS-CoV-2 RNA transcripts in long COVID blood via digital transcriptomics. Upregulated transcripts included antisense ORF1ab RNA which suggests ongoing viral replication. The same methods will be applied to long COVID samples obtained via LongCovid Research Consortium teams in both France and the UK. Sample analysis is performed on whole blood which contains platelets, monocytes, and epithelial cells that are reduced in blood plasma preparations. This should allow persistent viral RNA inside patient host cells to be captured by the digital transcriptomics method. Certain samples will also be analyzed with a metabolomic analysis with the goal of developing a comprehensive multi-omic signature (digital transcriptomics, proteomics and metabolomics) panel that might eventually be developed into a long COVID blood test.

Project Background:

Persistence of the SARS-CoV-2 virus in tissue (viral reservoir) is increasingly being studied as a driver of long COVID. However, identifying persistent viral RNA in samples obtained from long COVID patients is a challenge because SARS-CoV-2 acts within host cells, meaning that reservoirs are often localized to tissue. While biopsy procedures that collect tissue samples – for example from the gut or lymph nodes – can be performed in some settings to identify persistent viral RNA, the procedures are expensive and invasive. There is consequently a very important need to develop and iterate testing methods that can identify SARS-CoV-2 RNA in blood – a fluid that can be collected non-invasively at at the average doctor’s office.

To address this gap, the project team recently used blood digital transcriptomics (nCounter platform) in a small study where they compared 48 long COVID patients with 12 controls carefully matched for age, sex, vaccine status, comorbidities and time since acute COVID. They found that several viral RNAs were upregulated: nucleocapsid, ORF7a, ORF3a, Mpro (a Paxlovid target), and antisense ORF1ab RNA. Specifically, the upregulation of antisense ORF1ab RNA suggests ongoing viral replication. SARS-CoV-2-related host RNAs (ACE2/TMPRSS2 receptors, DPP4/FURIN proteases) and RNAs prototypical for memory B-cells and platelets were also upregulated. Since both patients and controls were sampled for a median of 2 years after acute COVID, the team was able to show viral persistence at the RNA level in 60% of patients and 8% of controls, for 2 years or more after acute COVID.

The current project will determine if these early but encouraging findings can be replicated in whole blood samples obtained from a larger set of long COVID patients. Specially, the project team will use digital transcriptomcis to validate viral persistence and immune biomarkers in samples obtained from the PERSICOT cohort in Paris France (led by LCRC team member Dominique Salmon) and samples obtained by LCRC team member David Price and team at the University of Cardiff. They will also be able to further confirm and possibly extend their clinical findings of a link between persistent viral RNA and patient-reported outcomes in their own Belgium long COVID patient group.

Key to the digital transcriptomics analysis is that viral RNA is not necessarily free floating in long COVID blood. Instead, it may be located inside host cells. For example, the PERISCOT team is identifying SARS-CoV-2 RNA in platelets and megakaryokytes. Digital transcriptomics of whole blood is able to capture all relevant fractions: plasma carrying free virus, monocytes having phagocytosed infected cells, and other cells that may contain virus or viral genetic material such as platelets as well as rare circulating endothelial/epithelial cells or cell fragments.