Analysis of SARS-CoV-2 in Nasopharyngeal Samples from Patients with COVID-19 Illustrates Population Variation and Diverse Phenotypes, Placing the Growth Properties of Variants of Concern in Context with Other Lineages

Prince, Tessa; Dong, Xiaofeng; Penrice-Randal, Rebekah; Randle, Nadine; Hartley, Catherine; Goldswain, Hannah; Jones, Benjamin; Semple, Malcolm G; Baillie, J Kenneth; Openshaw, Peter; Turtle, Lance; Hughes, Grant L.; Anderson, Enyia R; Patterson, Edward I; Druce, Julian; Screaton, Gavin; Carroll, Miles W.; Stewart, James; Hiscox, Julian A.

doi:10.1128/msphere.00913-21

Cited by 12 publications

(5 citation statements)

References 57 publications

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“…SARS-CoV-2 variants display various degrees of transmissibility and confer differing levels of disease severity in infected patients (Harvey et al, 2021; 10.3389/fmicb.2022.997539 10.3389/fmicb.2022.997539 -Pastrana et al, 2022). Moreover, studies have shown that SARS-CoV-2 variants have different replication rates and produce distinct viral titers from infected Vero cells (Jeong et al, 2021;Prince et al, 2022). Because inhibition of stress granule formation is related to viral replication (McCormick and Khaperskyy, 2017), we hypothesized that SARS-CoV-2 variants might also show differing degrees of stress granule inhibition.…”

Section: Discussionmentioning

confidence: 99%

Differential effect of SARS-CoV-2 infection on stress granule formation in Vero and Calu-3 cells

et al. 2022

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Stress granule formation is induced by numerous environmental stressors, including sodium arsenite treatment and viral infection. Accordingly, stress granules can inhibit viral propagation and function as part of the antiviral host response to numerous viral infections. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antagonizes stress granule formation, in part, via interaction between SARS-CoV-2 nucleocapsid (N) protein and Ras-GTPase-activating SH3-domain-binding protein 1 (G3BP1). However, it is unclear whether there are differential effects in different cell types. In this study, we assessed interaction between the N protein of SARS-CoV-2 S clade and G3BP1/2 in Vero and Calu-3 cells and investigated the effect of various SARS-CoV-2 strains on sodium arsenite-induced stress granule formation. Our data show that SARS-CoV-2 S clade N protein interacts with both G3BP1 and G3BP2 more strongly in Calu-3 vs. Vero cells. Consistent with this observation, infection with SARS-CoV-2 S clade induces stress granule formation in Vero but not in Calu-3 cells. However, infection with SARS-CoV-2 S clade, as well as other SARS-CoV-2 variants, inhibits sodium arsenite-induced stress granule formation in both cell lines. Taken together, our results show differential effects of SARS-CoV-2 infection on stress granule formation that is dependent on host cell type, rather than virus strain type.

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Section: Discussionmentioning

confidence: 99%

Differential effect of SARS-CoV-2 infection on stress granule formation in Vero and Calu-3 cells

et al. 2022

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“…The second method, DiversiTools (code available at https://github.com/josephhughes/DiversiTools ), uses the primer-trimmed alignment file (named as [sampleID]_L001.final.bam) and its associated index file (produced in the EasySeq pipeline) along with the reference genome and a coding region file to analyse the minor genomic variation and predict the amino acid sequence based on the genomic data. DiversiTools allows for an in-depth analysis of viral diversity in each sample, rather than just the consensus/dominant genomic information, as previously described 17 . Participants were included in the minor variant analysis if all three of their samples met the following criteria: 1) the dominant genome sequence had a minimum 90% consensus called and 2) 90% of genome positions had a minimum coverage of 200X.…”

Section: Methodsmentioning

confidence: 99%

Characterisation of SARS-CoV-2 genomic variation in response to molnupiravir treatment in the AGILE Phase IIa clinical trial

et al. 2022

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Molnupiravir is an antiviral, currently approved by the UK Medicines and Healthcare products Regulatory Agency (MHRA) for treating at-risk COVID-19 patients, that induces lethal error catastrophe in SARS-CoV-2. How this drug-induced mechanism of action might impact the emergence of resistance mutations is unclear. To investigate this, we used samples from the AGILE Candidate Specific Trial (CST)−2 (clinical trial number NCT04746183). The primary outcomes of AGILE CST-2 were to measure the drug safety and antiviral efficacy of molnupiravir in humans (180 participants randomised 1:1 with placebo). Here, we describe the pre-specified exploratory virological endpoint of CST-2, which was to determine the possible genomic changes in SARS-CoV-2 induced by molnupiravir treatment. We use high-throughput amplicon sequencing and minor variant analysis to characterise viral genomics in each participant whose longitudinal samples (days 1, 3 and 5 post-randomisation) pass the viral genomic quality criteria (n = 59 for molnupiravir and n = 65 for placebo). Over the course of treatment, no specific mutations were associated with molnupiravir treatment. We find that molnupiravir significantly increased the transition:transversion mutation ratio in SARS-CoV-2, consistent with the model of lethal error catastrophe. This study highlights the utility of examining intra-host virus populations to strengthen the prediction, and surveillance, of potential treatment-emergent adaptations.

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“…Vero hSLAM cells that express the human signalling lymphocytic activation molecule (SLAM) 14 , and VAT cells -Vero E6 expressing both human angiotensin-converting enzyme 2 (ACE2), the major receptor of SARS-CoV-2, and transmembrane serine protease 2 (TMPRSS2), which cleaves the viral S protein priming it for cellular infection 15 . Both viral growth kinetics and changes to cell morphology (i.e., cytopathic effects; CPE) can vary between these different cell lines thereby impacting the outcome of tests used to assess culture positivity 16 . For example, a 2020 study found that cell culture supernatants from Vero E6 cells expressing TMPRSS2 had more than 100 times more viral RNA copies than Vero E6 cells not expressing this protein 17 .…”

Section: Introductionmentioning

confidence: 99%

Optimisation of SARS-CoV-2 culture from clinical samples for clinical trial applications

Wooding,

Buist,

Romero-Ramirez

et al. 2024

Preprint

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Clinical trials of SARS-CoV-2 therapeutics often include virological secondary endpoints to compare viral clearance and viral load reduction between treatment and placebo arms. This is typically achieved using RT-qPCR, which cannot differentiate replicant competent virus from non-viable virus or free RNA, limiting its utility as an endpoint. Culture based methods for SARS-CoV-2 exist; however, these are often insensitive and poorly standardised for use as clinical trial endpoints. We report optimisation of a culture-based approach evaluating three cell lines, three detection methods, and key culture parameters. We show that Vero-ACE2-TMPRSS2 (VAT) cells in combination with RT-qPCR of culture supernatants from the first passage provides the greatest overall detection of Delta viral replication (22/32, 68.8%), being able to identify viable virus in 83.3% (20/24) of clinical samples with initial Ct values <30. Likewise, we demonstrate that RT-qPCR using culture supernatants from the first passage of Vero hSLAM cells provides the highest overall detection of Omicron viral replication (9/31, 29%), detecting live virus in 39.1% (9/23) of clinical samples with initial Ct values < 25. This assessment demonstrates that combining RT-qPCR with virological end point analysis has utility in clinical trials of therapeutics for SARS-CoV-2; however, techniques may require optimising based on dominant circulating strain.

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Analysis of SARS-CoV-2 in Nasopharyngeal Samples from Patients with COVID-19 Illustrates Population Variation and Diverse Phenotypes, Placing the Growth Properties of Variants of Concern in Context with Other Lineages

Abstract: SARS-CoV-2 is the causative agent of COVID-19. The virus has spread across the planet, causing a global pandemic.

Cited by 12 publications

References 57 publications

Differential effect of SARS-CoV-2 infection on stress granule formation in Vero and Calu-3 cells

Differential effect of SARS-CoV-2 infection on stress granule formation in Vero and Calu-3 cells

Characterisation of SARS-CoV-2 genomic variation in response to molnupiravir treatment in the AGILE Phase IIa clinical trial

Optimisation of SARS-CoV-2 culture from clinical samples for clinical trial applications

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