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1.
Viruses ; 14(2)2022 02 05.
Article in English | MEDLINE | ID: covidwho-1674824

ABSTRACT

SARS-CoV-2 can efficiently infect both children and adults, albeit with morbidity and mortality positively associated with increasing host age and presence of co-morbidities. SARS-CoV-2 continues to adapt to the human population, resulting in several variants of concern (VOC) with novel properties, such as Alpha and Delta. However, factors driving SARS-CoV-2 fitness and evolution in paediatric cohorts remain poorly explored. Here, we provide evidence that both viral and host factors co-operate to shape SARS-CoV-2 genotypic and phenotypic change in primary airway cell cultures derived from children. Through viral whole-genome sequencing, we explored changes in genetic diversity over time of two pre-VOC clinical isolates of SARS-CoV-2 during passage in paediatric well-differentiated primary nasal epithelial cell (WD-PNEC) cultures and in parallel, in unmodified Vero-derived cell lines. We identified a consistent, rich genetic diversity arising in vitro, variants of which could rapidly rise to near fixation within two passages. Within isolates, SARS-CoV-2 evolution was dependent on host cells, with paediatric WD-PNECs showing a reduced diversity compared to Vero (E6) cells. However, mutations were not shared between strains. Furthermore, comparison of both Vero-grown isolates on WD-PNECs disclosed marked growth attenuation mapping to the loss of the polybasic cleavage site (PBCS) in Spike, while the strain with mutations in Nsp12 (T293I), Spike (P812R) and a truncation of Orf7a remained viable in WD-PNECs. Altogether, our work demonstrates that pre-VOC SARS-CoV-2 efficiently infects paediatric respiratory epithelial cells, and its evolution is restrained compared to Vero (E6) cells, similar to the case of adult cells. We highlight the significant genetic plasticity of SARS-CoV-2 while uncovering an influential role for collaboration between viral and host cell factors in shaping viral evolution and ultimately fitness in human respiratory epithelium.


Subject(s)
Evolution, Molecular , Respiratory Mucosa/virology , SARS-CoV-2/genetics , Animals , Cells, Cultured , Child , Chlorocebus aethiops , Genotype , Humans , Mutation , Nose/cytology , Nose/virology , Phenotype , SARS-CoV-2/classification , SARS-CoV-2/growth & development , Vero Cells , Whole Genome Sequencing
4.
The FASEB Journal ; 35(S1), 2021.
Article in English | Wiley | ID: covidwho-1233952

ABSTRACT

Introduction Interferon (IFN) alpha (IFNα) and lambda3 (IFN?3) constitute the first line of immunity against viral infection by increasing expression of interferon-stimulated genes (ISGs). Both are produced in SARS-CoV-2 infection and have been considered therapeutically in COVID-19. In epithelial cells IFNs have been shown to influence the expression of angiotensin-converting enzyme 2 (ACE2), the receptor for S-protein (S1P) of SARS-CoV-2. ACE2 is part of the vascular renin angiotensin system and may be important in COVID-19-associated endotheliitis. Whether SP1 influences immune responses in endothelial cells unknown. We investigated effects of SP1 on ACE2 expression and immune responses in human endothelial cells. Methods Culturedhuman Microvascular, Lymphatic, Aortic and Pulmonary Endothelial Cells (MEC, LEC, AEC, and PEC respectively) were stimulated with SP1 of SARS-CoV-2 (1µg/106 cells), IFNα (100ng/mL) or IFN?3 (100IU/mL). Because ACE2, metalloproteinase domain 17 (ADAM17) and type II transmembrane serine protease (TMPRSS2) are important for SARS-CoV-2 infection, cells were treated with inhibitors of ADAM17 (marimastat, 3.8nM and TAPI-1, 100nM), ACE2 (MLN4760, 440pM), and TMPRSS2 (camostat, 50µM). Expression of ISGs (ISG15, IFIT1, and MX1) was investigated by real-time PCR (5h), cytokine by ELISA and protein expression by immunoblotting (24h). Results AEC, LEC, MEC, and PEC stimulated with SP1 exhibited increased expression of ISGs: ISG15 (1.9-2.3-fold), IFIT1 (2.0-7.9-fold), MX1 (3.1-5.8-fold), indicating activation of the IFN pathway. MEC and LEC exhibited higher responses to IFNα activation. MEC (ISG15: 16-fold, IFIT1: 21-fold, MX1: 31-fold) and LEC (ISG15: 48-fold, IFIT1: 105-fold, MX1: 134-fold). Only MEC exhibited ISG expression induced by IFN?3 (ISG15: 1.7-fold, IFIT1: 1.9-fold, MX1: 1.7-fold) (P<0.05). IL-6 production was increased in MEC stimulated with IFNα (1230pg/mL) and IFN?3 (1124pg/mL) vs control (591pg/mL). Marimastat, but not camostat or MLN4760 inhibited SP1 effects. IFNα induced IL-6 production in AEC and PEC (178pg/mL vs control 109pg/mL). No changes in IL-6 were observed in LEC. IFNα increased expression of the short form of ACE2 (75kDa) (63%), ADAM17 (36%), and TMPRSS2 (65%) in MECs. This was associated with increased activation of pro-inflammatory pathways with increased phosphorylation of Stat1 (134%), Stat2 (102%), ERK1/2 (42%). IFN?3 also increased phosphorylation of Stat2 (60%) and ERK1/2 (75%). In MEC, phosphorylation of the eNOS activation motif Ser1177 was reduced by IFNLα and (40%) and IFN?3 (40%). Conclusions In human endothelial cells from multiple vascular beds, SP1, IFNα and IFN?3 induced an immune response characterised by increased ISG expression and IL-6 production, processes that involve ADAM17. These effects were associated with increased expression of the short form of ACE2 and activation of pro-inflammatory pathways. Our novel findings demonstrate that SP-1 induces an endothelial immune and inflammatory response that may be important in endotheliitis associated with COVID-19.

5.
J Infect ; 83(1): 96-103, 2021 07.
Article in English | MEDLINE | ID: covidwho-1198895

ABSTRACT

OBJECTIVES: Patients requiring haemodialysis are at increased risk of serious illness with SARS-CoV-2 infection. To improve the understanding of transmission risks in six Scottish renal dialysis units, we utilised the rapid whole-genome sequencing data generated by the COG-UK consortium. METHODS: We combined geographical, temporal and genomic sequence data from the community and hospital to estimate the probability of infection originating from within the dialysis unit, the hospital or the community using Bayesian statistical modelling and compared these results to the details of epidemiological investigations. RESULTS: Of 671 patients, 60 (8.9%) became infected with SARS-CoV-2, of whom 16 (27%) died. Within-unit and community transmission were both evident and an instance of transmission from the wider hospital setting was also demonstrated. CONCLUSIONS: Near-real-time SARS-CoV-2 sequencing data can facilitate tailored infection prevention and control measures, which can be targeted at reducing risk in these settings.


Subject(s)
COVID-19 , SARS-CoV-2 , Bayes Theorem , Hospitals , Humans , Molecular Epidemiology , Renal Dialysis/adverse effects
6.
Genome Res ; 31(4): 645-658, 2021 04.
Article in English | MEDLINE | ID: covidwho-1135943

ABSTRACT

We have developed periscope, a tool for the detection and quantification of subgenomic RNA (sgRNA) in SARS-CoV-2 genomic sequence data. The translation of the SARS-CoV-2 RNA genome for most open reading frames (ORFs) occurs via RNA intermediates termed "subgenomic RNAs." sgRNAs are produced through discontinuous transcription, which relies on homology between transcription regulatory sequences (TRS-B) upstream of the ORF start codons and that of the TRS-L, which is located in the 5' UTR. TRS-L is immediately preceded by a leader sequence. This leader sequence is therefore found at the 5' end of all sgRNA. We applied periscope to 1155 SARS-CoV-2 genomes from Sheffield, United Kingdom, and validated our findings using orthogonal data sets and in vitro cell systems. By using a simple local alignment to detect reads that contain the leader sequence, we were able to identify and quantify reads arising from canonical and noncanonical sgRNA. We were able to detect all canonical sgRNAs at the expected abundances, with the exception of ORF10. A number of recurrent noncanonical sgRNAs are detected. We show that the results are reproducible using technical replicates and determine the optimum number of reads for sgRNA analysis. In VeroE6 ACE2+/- cell lines, periscope can detect the changes in the kinetics of sgRNA in orthogonal sequencing data sets. Finally, variants found in genomic RNA are transmitted to sgRNAs with high fidelity in most cases. This tool can be applied to all sequenced COVID-19 samples worldwide to provide comprehensive analysis of SARS-CoV-2 sgRNA.


Subject(s)
Genome, Viral , RNA, Viral/genetics , SARS-CoV-2/genetics , Sequence Analysis, RNA/methods , Animals , Base Sequence , Chlorocebus aethiops , Humans , Limit of Detection , Vero Cells
9.
Nat Microbiol ; 6(1): 112-122, 2021 01.
Article in English | MEDLINE | ID: covidwho-989837

ABSTRACT

Coronavirus disease 2019 (COVID-19) was first diagnosed in Scotland on 1 March 2020. During the first month of the outbreak, 2,641 cases of COVID-19 led to 1,832 hospital admissions, 207 intensive care admissions and 126 deaths. We aimed to identify the source and number of introductions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into Scotland using a combined phylogenetic and epidemiological approach. Sequencing of 1,314 SARS-CoV-2 viral genomes from available patient samples enabled us to estimate that SARS-CoV-2 was introduced to Scotland on at least 283 occasions during February and March 2020. Epidemiological analysis confirmed that early introductions of SARS-CoV-2 originated from mainland Europe (the majority from Italy and Spain). We identified subsequent early outbreaks in the community, within healthcare facilities and at an international conference. Community transmission occurred after 2 March, 3 weeks before control measures were introduced. Earlier travel restrictions or quarantine measures, both locally and internationally, would have reduced the number of COVID-19 cases in Scotland. The risk of multiple reintroduction events in future waves of infection remains high in the absence of population immunity.


Subject(s)
COVID-19/epidemiology , COVID-19/virology , SARS-CoV-2/genetics , Adult , Aged , Europe/epidemiology , Genome, Viral , High-Throughput Nucleotide Sequencing , Humans , Male , Middle Aged , Molecular Epidemiology , Phylogeny , SARS-CoV-2/isolation & purification , Spain/epidemiology , Travel/statistics & numerical data
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