Distinct Patterns of Emergence of SARS-CoV-2 Spike Variants including N501Y in Clinical Samples in Columbus Ohio

AbstractFollowing the worldwide emergence of the p.Asp614Gly shift in the Spike (S) gene of SARS-CoV-2, there have been few recurring pathogenic shifts occurring during 2020, as assessed by genomic sequencing. This situation has evolved in the last several months with the emergence of several distinct variants (first identified in the United Kingdom and South Africa) that manifest multiple changes in the S gene, particularly p.Asn501Tyr (N501Y), that likely have clinical impact. We report here the emergence in Columbus, Ohio in December 2020 of two novel SARS-CoV-2 clade 20G variants. One variant, that has become the predominant virus found in nasopharyngeal swabs in the December 2020-January 2021 period, harbors S p.Gln677His (Q677H), affecting a consensus QTQTN domain near the S1/S2 furin cleavage site, nucleocapsid (N) p.Asp377Tyr (D377Y) and membrane glycoprotein (M) p.Ala85Ser (A85S) mutations, with additional S mutations in subsets. The other variant present in two samples, contains S N501Y, which is a marker of the UK-B.1.1.7 (clade 20I/501Y.V1) strain, but lacks all other mutations from that virus. The Ohio variant is from a different clade and shares multiple mutations with the clade 20G viruses circulating in the area prior to December 2020. These two SARS-CoV-2 viruses, which we show are also present and evolving currently in several other parts of North America, add to the diversity of S gene shifts occurring worldwide. These and other shifts in this period of the pandemic support multiple independent acquisition of functionally significant and potentially complementing mutations affecting the S QTQTN site (Q675H or Q677H) and certain receptor binding domain mutations (e.g., E484K and N501Y).

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Viral and Host Attributes Underlying the Origins of Zoonotic Coronaviruses in Bats

Comparative Medicine ◽

10.30802/aalas-cm-21-000027 ◽

2021 ◽

Author(s):

Alison E Stout ◽

Qinghua Guo ◽

Jean K Millet ◽

Gary R Whittaker

Keyword(s):

Viral Pathogenesis ◽

Spike Protein ◽

Future Research ◽

Receptor Binding Domain ◽

Viral Reservoirs ◽

Furin Cleavage ◽

Proteolytic Activation ◽

Furin Cleavage Site ◽

Unique Aspect ◽

Genome Level

With a presumed origin in bats, the COVID-19 pandemic has been a major source of morbidity and mortality in the humanpopulation, and the causative agent, SARS-CoV-2, aligns most closely at the genome level with the bat coronavirusesRaBtCoV4991/RaTG13 and RmYN02. The ability of bats to provide reservoirs of numerous viruses in addition to coronavirusesremains an active area of research. Unique aspects of the physiology of the chiropteran immune system may contributeto the ability of bats to serve as viral reservoirs. The coronavirus spike protein plays important roles in viral pathogenesis and the immune response. Although much attention has focused on the spike receptor-binding domain, a unique aspect of SARS-CoV-2 as compared with its closest relatives is the presence of a furin cleavage site in the S1–S2 region of the spike protein. Proteolytic activation is likely an important feature that allows SARS-CoV-2—and other coronaviruses—to overcome the species barriers and thus cause human disease. The diversity of bat species limits the ability to draw broad conclusions about viral pathogenesis, but comparisons across species and with reference to humans and other susceptible mammals may guide future research in this regard.

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2016 Laboratory guidelines for postvasectomy semen analysis: Association of Biomedical Andrologists, the British Andrology Society and the British Association of Urological Surgeons

Journal of Clinical Pathology ◽

10.1136/jclinpath-2016-203731 ◽

2016 ◽

Vol 69 (7) ◽

pp. 655-660 ◽

Cited By ~ 7

Author(s):

P Hancock ◽

B J Woodward ◽

A Muneer ◽

J C Kirkman-Brown

Keyword(s):

United Kingdom ◽

General Practitioner ◽

Semen Analysis ◽

Single Sample ◽

British Association ◽

Motile Sperm ◽

Iso 15189 ◽

The United Kingdom ◽

Two Samples ◽

The Uk

Post-vasectomy semen analysis (PVSA) is the procedure used to establish whether sperm are present in the semen following a vasectomy. PVSA is presently carried out by a wide variety of individuals, ranging from doctors and nurses in general practitioner (GP) surgeries to specialist scientists in andrology laboratories, with highly variable results.Key recommendations are that: (1) PVSA should take place a minimum of 12 weeks after surgery and after a minimum of 20 ejaculations. (2) Laboratories should routinely examine samples within 4 h of production if assessing for the presence of sperm. If non-motile sperm are observed, further samples must be examined within 1 h of production. (3) Assessment of a single sample is acceptable to confirm vasectomy success if all recommendations and laboratory methodology are met and no sperm are observed. Clearance can then be given. (4) The level for special clearance should be <100 000/mL non-motile sperm. Special clearance cannot be provided if any motile sperm are observed and should only be given after assessment of two samples in full accordance with the methods contained within these guidelines. Surgeons are responsible both preoperatively and postoperatively for the counselling of patients and their partners regarding complications and the possibility of late recanalisation after clearance. These 2016 guidelines replace the 2002 British Andrology Society (BAS) laboratory guidelines and should be regarded as definitive for the UK in the provision of a quality PVSA service, accredited to ISO 15189:2012, as overseen by the United Kingdom Accreditation Service (UKAS).

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Detection of Enterovirus D68 in Wastewater Samples from the UK between July and November 2021

Viruses ◽

10.3390/v14010143 ◽

2022 ◽

Vol 14 (1) ◽

pp. 143

Author(s):

Alison Tedcastle ◽

Thomas Wilton ◽

Elaine Pegg ◽

Dimitra Klapsa ◽

Erika Bujaki ◽

...

Keyword(s):

Clinical Samples ◽

Acute Flaccid Myelitis ◽

The United Kingdom ◽

Virus Surveillance ◽

Enterovirus D68 ◽

Wastewater Samples ◽

The Uk ◽

Human Viruses ◽

Nonpharmaceutical Interventions ◽

Next Generation Sequencing Ngs

Infection with enterovirus D68 (EV-D68) has been linked with severe neurological disease such as acute flaccid myelitis (AFM) in recent years. However, active surveillance for EV-D68 is lacking, which makes full assessment of this association difficult. Although a high number of EV-D68 infections were expected in 2020 based on the EV-D68′s known biannual circulation patterns, no apparent increase in EV-D68 detections or AFM cases was observed during 2020. We describe an upsurge of EV-D68 detections in wastewater samples from the United Kingdom between July and November 2021 mirroring the recently reported rise in EV-D68 detections in clinical samples from various European countries. We provide the first publicly available 2021 EV-D68 sequences showing co-circulation of EV-D68 strains from genetic clade D and sub-clade B3 as in previous years. Our results show the value of environmental surveillance (ES) for the early detection of circulating and clinically relevant human viruses. The use of a next-generation sequencing (NGS) approach helped us to estimate the prevalence of EV-D68 viruses among EV strains from other EV serotypes and to detect EV-D68 minor variants. The utility of ES at reducing gaps in virus surveillance for EV-D68 and the possible impact of nonpharmaceutical interventions introduced to control the COVID-19 pandemic on EV-D68 transmission dynamics are discussed.

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The SARS-CoV-2 transcriptome and the dynamics of the S gene furin cleavage site in primary human airway epithelia

10.1101/2021.02.03.429670 ◽

2021 ◽

Author(s):

Wei Zou ◽

Min Xiong ◽

Siyuan Hao ◽

Elizabeth Yan Zhang-Chen ◽

Nathalie Baumlin ◽

...

Keyword(s):

Amino Acids ◽

Viral Genome ◽

Cleavage Site ◽

Vero Cells ◽

Furin Cleavage ◽

Airway Epithelia ◽

Human Airway ◽

S Gene ◽

Furin Cleavage Site ◽

Human Airway Epithelia

AbstractThe novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) caused the devastating ongoing coronavirus disease-2019 (COVID-19) pandemic which poses a great threat to global public health. The spike (S) polypeptide of SARS-CoV-2 consists of the S1 and S2 subunits and is processed by cellular proteases at the S1/S2 boundary. The inclusion of the 4 amino acids (PRRA) at the S1/S2 boundary forms a furin cleavage site (FCS), 682RRAR↓S686, distinguishing SARS-CoV-2 from its closest relative, the SARS-CoV. Various deletions surrounding the FCS have been identified in patients. When SARS-CoV-2 propagated in Vero cells, the virus acquired various deletions surrounding the FCS. In the present study, we studied the viral transcriptome in SARS-CoV-2 infected primary human airway epithelia (HAE) cultured at an air-liquid interface (ALI) with an emphasis on the viral genome stability at the S1/S2 boundary using RNA-seq. While we found overall the viral transcriptome is similar to that generated from infected Vero cells, we identified a high percentage of mutated viral genome and transcripts in HAE-ALI. Two highly frequent deletions were found at the S1/S2 boundary of the S gene: one is a deletion of 12 amino acids, 678TNSPRRAR↓SVAS689, which contains the FCS, another is a deletion of 5 amino acids, 675QTQTN679, which is two amino acids upstream of the FCS. Further studies on the dynamics of the FCS deletions in apically released virions revealed that the selective pressure for the FCS maintains the S gene stability in HAE-ALI but with exceptions, in which the FCS deletions are remained at a high rate. Thus, our study presents evidence for the role of unique properties of human airway epithelia in the dynamics of the FCS region during infection of human airways, which is donor-dependent.

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Characterisation of the transcriptome and proteome of SARS-CoV-2 using direct RNA sequencing and tandem mass spectrometry reveals evidence for a cell passage induced in-frame deletion in the spike glycoprotein that removes the furin-like cleavage site

10.1101/2020.03.22.002204 ◽

2020 ◽

Cited By ~ 29

Author(s):

Andrew D. Davidson ◽

Maia Kavanagh Williamson ◽

Sebastian Lewis ◽

Deborah Shoemark ◽

Miles W. Carroll ◽

...

Keyword(s):

Mass Spectrometry ◽

Tandem Mass Spectrometry ◽

Rna Sequencing ◽

Cleavage Site ◽

Clinical Samples ◽

Tandem Mass ◽

Furin Cleavage ◽

Furin Cleavage Site ◽

Novel Coronavirus ◽

Almost All

ABSTRACTDirect RNA sequencing using an Oxford Nanopore MinION characterised the transcriptome of SARS-CoV-2 grown in Vero E6 cells. This cell line is being widely used to propagate the novel coronavirus. The viral transcriptome was analysed using a recently developed ORF-centric pipeline. This revealed the pattern of viral transcripts, (i.e. subgenomic mRNAs), generally fitted the predicted replication and transcription model for coronaviruses. A 24 nt in-frame deletion was detected in subgenomic mRNAs encoding the spike (S) glycoprotein. This feature was identified in over half of the mapped transcripts and was predicted to remove a proposed furin cleavage site from the S glycoprotein. This motif directs cleavage of the S glycoprotein into functional subunits during virus entry or exit. Cleavage of the S glycoprotein can be a barrier to zoonotic coronavirus transmission and affect viral pathogenicity. Allied to this transcriptome analysis, tandem mass spectrometry was used to identify over 500 viral peptides and 44 phosphopeptides, covering almost all of the proteins predicted to be encoded by the SARS-CoV-2 genome, including peptides unique to the deleted variant of the S glycoprotein. Detection of an apparently viable deletion in the furin cleavage site of the S glycoprotein reinforces the point that this and other regions of SARS-CoV-2 proteins may readily mutate. This is of clear significance given the interest in the S glycoprotein as a potential vaccine target and the observation that the furin cleavage site likely contributes strongly to the pathogenesis and zoonosis of this virus. The viral genome sequence should be carefully monitored during the growth of viral stocks for research, animal challenge models and, potentially, in clinical samples. Such variations may result in different levels of virulence, morbidity and mortality.

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SARS-CoV-2 bearing a mutation at the S1/S2 cleavage site exhibits attenuated virulence and confers protective immunity

10.1101/2021.04.29.442060 ◽

2021 ◽

Author(s):

Michihito Sasaki ◽

Shinsuke Toba ◽

Yukari Itakura ◽

Herman M. Chambaro ◽

Mai Kishimoto ◽

...

Keyword(s):

Cleavage Site ◽

Neutralizing Antibodies ◽

Body Weight Loss ◽

Airway Epithelial Cells ◽

Spike Protein ◽

Furin Cleavage ◽

Growth Property ◽

S Gene ◽

Furin Cleavage Site ◽

The Impact

AbstractSevere Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) possesses a discriminative polybasic cleavage motif in its spike protein that is recognized by host furin protease. Proteolytic cleavage activates the spike protein and influences both the cellular entry pathway and cell tropism of SARS-CoV-2. Here, we investigated the impact of the furin cleavage site on viral growth and pathogensis using a hamster animal model infected with SARS-CoV-2 variants bearing mutations at the furin cleavage site (S gene mutants). In the airway tissues of hamsters, the S gene mutants exhibited a low growth property. In contrast to parental pathogenic SARS-CoV-2, hamsters infected with the S gene mutants showed no body weight loss and only a mild inflammatory response, indicating the attenuated variant nature of S gene mutants. We reproduced the attenuated growth of S gene mutants in primary differenciated human airway epithelial cells. This transient infection was enough to induce protective neutralizing antibodies crossreacting with different SARS-CoV-2 lineages. Consequently, hamsters inoculated with S gene mutants showed resistance to subsequent infection with both the parental strain and the currently emerging SARS-CoV-2 variants belonging to lineages B.1.1.7 and P.1. Together, our findings revealed that the loss of the furin cleavage site causes attenuation in the airway tissues of SARS-CoV-2 and highlights the potential benefits of S gene mutants as potential immunogens.

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Coronaviruses with a SARS-CoV-2-like receptor-binding domain allowing ACE2-mediated entry into human cells isolated from bats of Indochinese peninsula

10.21203/rs.3.rs-871965/v1 ◽

2021 ◽

Author(s):

Sarah Temmam ◽

Khamsing Vongphayloth ◽

Eduard Baquero Salazar ◽

Sandie Munier ◽

Max Bonomi ◽

...

Keyword(s):

Receptor Binding ◽

Human Cells ◽

Binding Domain ◽

Receptor Binding Domain ◽

Furin Cleavage ◽

Animal Reservoir ◽

Angiotensin Converting Enzyme 2 ◽

Furin Cleavage Site ◽

Spike Sequence ◽

Early Human

Abstract The animal reservoir of SARS-CoV-2 is unknown despite reports of various SARS-CoV-2-related viruses in Asian Rhinolophus bats, including the closest virus from R. affinis, RaTG13. Several studies have suggested the involvement of pangolin coronaviruses in SARS-CoV-2 emergence. SARS-CoV-2 presents a mosaic genome, to which different progenitors contribute. The spike sequence determines the binding affinity and accessibility of its receptor-binding domain (RBD) to the cellular angiotensin-converting enzyme 2 (ACE2) receptor and is responsible for host range. SARS-CoV-2 progenitor bat viruses genetically close to SARS-CoV-2 and able to enter human cells through a human ACE2 pathway have not yet been identified, though they would be key in understanding the origin of the epidemics. Here we show that such viruses indeed circulate in cave bats living in the limestone karstic terrain in North Laos, within the Indochinese peninsula. We found that the RBDs of these viruses differ from that of SARS-CoV-2 by only one or two residues, bind as efficiently to the hACE2 protein as the SARS-CoV-2 Wuhan strain isolated in early human cases, and mediate hACE2-dependent entry into human cells, which is inhibited by antibodies neutralizing SARS-CoV-2. None of these bat viruses harbors a furin cleavage site in the spike. Our findings therefore indicate that bat-borne SARS-CoV-2-like viruses potentially infectious for humans circulate in Rhinolophus spp. in the Indochinese peninsula.

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S-variant SARS-CoV-2 is associated with significantly higher viral loads in samples tested by ThermoFisher TaqPath RT-QPCR

10.1101/2020.12.24.20248834 ◽

2020 ◽

Cited By ~ 2

Author(s):

Michael Kidd ◽

Alex Richter ◽

Angus Best ◽

Jeremy Mirza ◽

Benita Percival ◽

...

Keyword(s):

Viral Load ◽

N Gene ◽

Clinical Samples ◽

Published Data ◽

List Type ◽

Recent Information ◽

Viral Loads ◽

S Gene ◽

Viral Genes ◽

The Uk

AbstractBirmingham University Turnkey laboratory is part of the Lighthouse network responsible for testing clinical samples under the UK government ‘Test & Trace’ scheme. Samples are analysed for the presence of SARS-CoV-2 in respiratory samples using the Thermofisher TaqPath RT-QPCR test, which is designed to co-amplify sections of three SARS-CoV-2 viral genes.Since more recent information became available regarding the presence of SARS-CoV-2 variants of concern (S-VoC), which can show a suboptimal profile in RT-QPCR tests such as the ThermoFisher TaqPath used at the majority of Lighthouse laboratories, we analysed recently published data for trends and significance of the S-gene ‘dropout’ variant.Results showed that:the population of S-gene dropout samples had significantly lower median Ct values of ORF and N-gene targets compared to samples where S-gene was detectedon a population basis, S-gene dropout samples clustered around very low Ct values for ORF and N targetslinked Ct values for individual samples showed that a low Ct for ORF and N were clearly associated with an S-dropout characteristicwhen conservatively inferring relative viral load from Ct values, approximately 35% of S-dropout samples had high viral loads between 10 and 10,000-fold greater than 1 × 106, compared to 10% of S-positive samples.This analysis suggests that patients whose samples exhibit the S-dropout profile in the TaqPath test are more likely to have high viral loads at the time of sampling. The relevance of this to epidemiological reports of fast spread of the SARS-CoV-2 in regions of the UK is discussed.

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Detection of Enterovirus D68 in wastewater samples from the United Kingdom during outbreaks reported globally between 2015 and 2018

10.1101/738948 ◽

2019 ◽

Author(s):

Manasi Majumdar ◽

Thomas Wilton ◽

Yara Hajarha ◽

Dimitra Klapsa ◽

Javier Martin

Keyword(s):

United Kingdom ◽

Temporal Distribution ◽

Clinical Samples ◽

Acute Flaccid Myelitis ◽

The United Kingdom ◽

The Usa ◽

Stool Samples ◽

Enterovirus D68 ◽

Wastewater Samples ◽

The Uk

ABSTRACTDetection of enterovirus D68 (EV-D68) in wastewater samples from the UK between December 2014 and December 2018 showed a marked seasonal distribution with a high proportion of samples containing EV-D68 during periods when identification of this virus in clinical samples was most common. This includes a recent upsurge of EV-D68 detection in respiratory samples from the United Kingdom between August and December 2018 associated with cases of acute flaccid myelitis, following similar reports in the USA. Phylogenetic analysis of EV-D68 sewage strains demonstrated that strains belonging to distinct genetic clades followed the same temporal distribution as that observed for EV-D68 clinical strains in the UK and that they showed very close genetic relationship with EV-D68 strains circulating elsewhere in the world during the same periods. The results demonstrated a clear association between detecting EV-D68 in wastewater and finding it in clinical samples which was somehow unexpected given that EV-D68 is rarely detected in stool samples. We conclude that the use of environmental surveillance is a valuable tool to detect and monitor outbreaks due to EV-68 infection.

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Rapid Rise of S-Gene Target Failure and the UK variant B.1.1.7 among COVID-19 isolates in the Greater Toronto Area, Canada

10.1101/2021.02.09.21251225 ◽

2021 ◽

Cited By ~ 1

Author(s):

Kevin Antoine Brown ◽

Jonathan Gubbay ◽

Jessica Hopkins ◽

Samir Patel ◽

Sarah A Buchan ◽

...

Keyword(s):

Reproduction Number ◽

Early Warning Systems ◽

Warning Systems ◽

Gene Target ◽

Health Measures ◽

S Gene ◽

Greater Toronto Area ◽

The United Kingdom ◽

Novel Variant ◽

The Uk

A novel variant of SARS-CoV-2, B.1.1.7, originally discovered in the United Kingdom (UK), is rapidly overtaking other strains around the globe. In certain assays, absence of detection of the S-gene target, also known as S-gene target failure (SGTF) can be a sensitive surrogate of B.1.1.7. We analyzed daily counts of SGTF among samples from Dynacare Laboratory Ontario (which draws samples from the Greater Toronto Area) and resulted between December 16, 2020 and February 3, 2021. We identified 11,485 positive COVID-19 tests, of which 448 had SGTF (3.9%). The estimated prevalence of SGTF rose from 2.0% on December 16 to 15.2% on February 3 (1.8-fold weekly increase, 95%CI: 1.5, 2.2). The estimated reproduction number for SGTF cases was 1.17 (95%CI: 0.94 to 1.46), while the reproduction number for non-SGTF cases was 0.82 (95%CI: 0.65 to 1.01); the relative reproduction number was 1.44 (95%CI: 1.03, 1.99). 59 samples were sent for confirmatory testing, of which 53 (90%) were identified as B.1.1.7 using whole genome sequencing or found to have the N501Y mutation. In order to pre-emptively plan and implement public health measures to control COVID-19 now and through the spring, accurate and up-to-date early warning systems for new variants, including B.1.1.7, are essential across North America and the globe.

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