scispace - formally typeset
Search or ask a question
Posted Content•DOI•

Altered Sub-Genomic RNA Expression in SARS-CoV-2 B.1.1.7 Infections

Matthew Parker1, Benjamin B Lindsey1, Dhruv R. Shah1, Sharon Hsu1, Alexander J Keeley1, David G Partridge, Shay Leary2, Alison Cope, Amy State, Katie Johnson, Nasar Ali, Rasha Raghei, Joe Heffer1, Nikki Smith1, Peijun Zhang1, Marta Gallis1, Stavroula F Louka1, Hailey Hornsby1, Max Whiteley1, Benjamin H Foulkes1, Stella Christou1, Paige Wolverson1, Manoj Baliram Pohare1, Sam E Hansford1, Luke R. Green1, Cariad Evans, Mohammad Raza, Dennis Wang, Silvana Gaudieri3, Silvana Gaudieri4, Silvana Gaudieri2, Simon Mallal3, Simon Mallal4, Thushan I de Silva1 •
University of Sheffield1, Murdoch University2, Vanderbilt University Medical Center3, University of Western Australia4
03 Mar 2021-bioRxiv (Cold Spring Harbor Laboratory)-
TL;DR: In this article, the authors applied periscope to ARTIC Network Oxford Nanopore genomic sequencing data from 4400 SARS-CoV-2 positive clinical samples, and showed that normalised sgRNA expression profiles are significantly increased in B.1.7 infections.
Abstract: SARS-CoV-2 lineage B.1.1.7 viruses are more transmissible, may lead to greater clinical severity, and result in modest reductions in antibody neutralization. subgenomic RNA (sgRNA) is produced by discontinuous transcription of the SARS-CoV-2 genome and is a crucial step in the SARS-CoV-2 life cycle. Applying our tool (periscope) to ARTIC Network Oxford Nanopore genomic sequencing data from 4400 SARS-CoV-2 positive clinical samples, we show that normalised sgRNA expression profiles are significantly increased in B.1.1.7 infections (n=879). This increase is seen over the previous dominant circulating lineage in the UK, B.1.177 (n=943), which is independent of genomic reads, E gene cycle threshold and days since symptom onset at sampling. A noncanonical sgRNA which could represent ORF9b is found in 98.4% of B.1.1.7 SARS-CoV-2 infections compared with only 13.8% of other lineages, with a 16-fold increase in median expression. We hypothesise that this is a direct consequence of a triple nucleotide mutation in nucleocapsid (28280:GAT>CAT, D3L) creating a transcription regulatory-like sequence complementary to a region 3’ of the genomic leader. These findings provide a unique insight into the biology of B.1.1.7 and support monitoring of sgRNA profiles in sequence data to evaluate emerging potential variants of concern. One Sentence Summary The recently emerged and more transmissible SARS-CoV-2 lineage B.1.1.7 shows greater subgenomic RNA expression in clinical infections and enhanced expression of a noncanonical subgenomic RNA near ORF9b.

Summary (1 min read)

Jump to: [Main Text] – [Methods] – [Subgenomic Read Classification and Normalisation] and [Phylogenetic Tree Generation]

Main Text

  • Reasons for the potential viral load increase and enhanced mortality are currently unclear ( 9 ) .
  • It is possible that the increased GAT>CTA, resulting in an amino acid substitution, D3L, in the nucleocapsid protein.
  • The authors propose that this represents the ORF9b sgRNA, which was detected at low levels in 13 3A&C ).
  • Finally the authors believe that sgRNA expression analysis should be carried out on all compatible genomic surveillance platforms to give an instant readout of altered expression profiles in emerging SARS-CoV-2 variants.

Methods

  • Testing SARS-CoV-2 positivity was determined from nose/throat swabs diagnostically by Sheffield Teaching Hospitals NHS Foundation trust either using the Hologic Panther to generate Relative Light Units (RLU) ( 26 ) or an in house dual E/RdRp real time PCR assay to generate a cycle threshold (ECT or RCT respectively) ( 16) .
  • Sample Preparation, ARTIC Network PCR and Nanopore Sequencing RNA was extracted from viral transport medium (VTM) with Qiagen QIAamp MinElute Virus Spin Kit (50).
  • Resultant RNA was subject to the ARTIC network ( 10) tiled amplicon protocol and subsequently sequenced on an Oxford Nanopore GridION X5.
  • Bases were called with the default basecaller in MinKNOW (currently guppy v4) with --require-both-ends set for de-multiplexing.

Subgenomic Read Classification and Normalisation

  • Briefly, reads containing the leader sequence at their start are identified by a local alignment, the quality of this alignment determines which quality "bin" sgRNA reads are placed (HQ, LQ or LLQ).
  • The amplicon from which the sgRNA evidence was generated is determined and a count of genomic reads for this amplicon used to normalise the raw sgRNA read counts.
  • Samples were excluded from the subsequent analysis if: Their consensus coverage was < 0.9 .
  • They had less than 50,000 mapped reads (the authors have previously shown that fewer reads produce a less robust analysis).

Phylogenetic Tree Generation

  • The grapevine pipeline ( https://github.com/COG-UK/grapevine ) was used for generating the phylogeny based on all data available on GISAID and COG-UK up until 16th February 2021.
  • First by randomly selecting one sequence per country per epi week followed by random sampling of the remaining sequences to generate a sample of 4000 sequences.
  • The global tree was then pruned using code adapted from the tree-manip package ( https://github.com/josephhughes/tree-manip ).
  • The authors then identified samples with D3L mutations and colour coded these tips according to their lineages.
  • The visualisation was produced using R/ape, R/ggplot2, R/ggtree, R/treeio, R/phangorn, R/stringr, R/dplyr, R/aplot.

Did you find this useful? Give us your feedback

Figures (3)

Content maybe subject to copyright    Report

Title
Altered Subgenomic RNA Expression
in SARS-CoV-2 B.1.1.7 Infections
Authors
Matthew D Parker
1,2,3
, Benjamin B. Lindsey
4,5
, Dhruv R Shah
5
, Sharon Hsu
2,5
,
Alexander J
Keeley
4,5
, David G Partridge
4
, Shay Leary
6
, Alison Cope
4
, Amy State
4
, Katie Johnson
4
, Nasar
Ali
4
, Rasha Raghei
4
, Joe Heffer
7
, Nikki Smith
5
, Peijun Zhang
5
, Marta Gallis
5
, Stavroula F
Louka
5
, Hailey R Hornsby
5
, Max Whiteley
5
, Benjamin H Foulkes
5
, Stella Christou
5
, Paige
Wolverson
5
, Manoj Pohare
5
, Samantha E Hansford
5
, Luke R Green
5
, Cariad Evans
4
,
Mohammad Raza
4
, Dennis Wang
1,2,3,8
, Silvana Gaudieri
6,9,10
, Simon Mallal
9,10
, The COVID-19
Genomics UK (COG-UK) consortium
11十
, Thushan I. de Silva
4,5
*
十
Full list of consortium names and affiliations located in the supplementary material
*Corresponding Author
Email: t.desilva@sheffield.ac.uk
Affiliations
1
Sheffield Biomedical Research Centre, The University of Sheffield
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted March 4, 2021. ; https://doi.org/10.1101/2021.03.02.433156doi: bioRxiv preprint
2
Sheffield Bioinformatics Core, The University of Sheffield
3
The Department of Neuroscience/Neuroscience Institute, The University of Sheffield
4
Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield, UK.
5
The Florey Institute for Host-Pathogen Interactions & Department of Infection, Immunity
and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK.
6
Institute for Immunology and Infectious Diseases, Murdoch University, Murdoch, Western
Australia, Australia
7
IT Services, The University of Sheffield, Sheffield, UK
8
Department of Computer Science, The University of Sheffield, Sheffield, UK
9
Division of Infectious Diseases, Department of Medicine, Vanderbilt University Medical
Center, Nashville, Tennessee, USA
10
School of Human Sciences, University of Western Australia, Crawley, Western Australia,
Australia
11
https://www.cogconsortium.uk
One Sentence Summary
The recently emerged and more transmissible SARS-CoV-2 lineage B.1.1.7 shows greater
subgenomic RNA expression in clinical infections and enhanced expression of a
noncanonical subgenomic RNA near ORF9b.
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted March 4, 2021. ; https://doi.org/10.1101/2021.03.02.433156doi: bioRxiv preprint
Abstract
SARS-CoV-2 lineage B.1.1.7 viruses are more transmissible, may lead to greater clinical
severity, and result in modest reductions in antibody neutralization. subgenomic RNA
(sgRNA) is produced by discontinuous transcription of the SARS-CoV-2 genome and is a
crucial step in the SARS-CoV-2 life cycle. Applying our tool (periscope) to ARTIC Network
Oxford Nanopore genomic sequencing data from 4400 SARS-CoV-2 positive clinical
samples, we show that normalised sgRNA expression profiles are significantly increased in
B.1.1.7 infections (n=879). This increase is seen over the previous dominant circulating
lineage in the UK, B.1.177 (n=943), which is independent of genomic reads, E gene cycle
threshold and days since symptom onset at sampling. A noncanonical sgRNA which could
represent ORF9b is found in 98.4% of B.1.1.7 SARS-CoV-2 infections compared with only
13.8% of other lineages, with a 16-fold increase in median expression. We hypothesise that
this is a direct consequence of a triple nucleotide mutation in nucleocapsid
(28280:GAT>CAT, D3L) creating a transcription regulatory-like sequence complementary to
a region 3’ of the genomic leader. These findings provide a unique insight into the biology of
B.1.1.7 and support monitoring of sgRNA profiles in sequence data to evaluate emerging
potential variants of concern.
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted March 4, 2021. ; https://doi.org/10.1101/2021.03.02.433156doi: bioRxiv preprint
Main Text
The recently emerged SARS-CoV-2 lineages B.1.1.7 (20I/501Y.V1 or VOC-202012/01),
B.1.351 (20H/501Y.V2 or VOC-202012/02) and P.1 (20J/501Y.V3 or VOC-202101/02) (1
)
have been classified as variants of concern by public health agencies. An increasing body of
evidence suggests B.1.1.7 is more transmissible (2
, 3
) and rapidly became the dominant
circulating virus in the United Kingdom (UK) during October 2020 to February 2021 (Figure
1A). To date, B.1.1.7 has been reported in 93 countries (https://cov-lineages.org/, 22nd
February 2021), with increasing prevalence. Recent data suggest a similar pattern for P.1 in
Manaus (4
), Brazil (5
), and now 19 other countries (https://cov-lineages.org/, 22nd February
2021), where it appears to be more transmissible than it’s ancestral lineage B.1.1.28 (4
).
Along with B.1.351, these variants of concern harbour functionally important mutations in the
SARS-CoV-2 Spike protein, some of which demonstrate evidence of convergent evolution
across all three lineages (Supplementary Table S1). Preliminary data (6
, 7
) shows that
B.1.1.7 positive diagnostic respiratory samples may have lower cycle threshold (Ct) values,
therefore higher viral loads, compared to other lineages. These findings suggest a potential
reason for enhanced transmissibility, though they did not account for potential confounders
such as days since symptom onset at sampling. Many of these studies also use S gene
target failure (SGTF) as a surrogate for the presence of B.1.1.7 (6
), which might misclassify
samples, depending on the prevalence of B.1.1.7 (8
). Several analyses from community
tested cases also suggest increased mortality associated with B.1.1.7 (8
). Reasons for the
potential viral load increase and enhanced mortality are currently unclear (9
).
Genomic surveillance has been critical in rapidly identifying these variants and Nanopore
sequencing of ARTIC Network (10
) prepared SARS-CoV-2 amplicons is used by many
laboratories to generate this data. We have recently reported an approach to quantify
subgenomic RNA (sgRNA) expression profiles from genomic sequence data, which is
produced as a result of a critical step in the SARS-CoV-2 replication cycle (11
). sgRNA is
produced from the genomically encoded SARS-CoV-2 RNA Dependent RNA polymerase
(RdRp) using discontinuous transcription of the positive, single-stranded SARS-CoV-2
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted March 4, 2021. ; https://doi.org/10.1101/2021.03.02.433156doi: bioRxiv preprint
genome from the 3’ end. Negatively stranded RNAs are produced, which are shorter than
the genome, owing to a template switch from the ORF to the leader sequence at the 5’ end
of the genome when RdRp encounters a transcription regulatory sequence in the genome
body (TRS-B) to a complementary TRS 3’ of the leader sequence (TRS-L). All sgRNAs
therefore contain a leader sequence at their 3’ end which can be used computationally for
their identification. There are thought to be nine such canonical sgRNAs; Spike:S, E:
Envelope, M: Membrane, N: Nucleocapsid, ORF3a, ORF6, ORF7a, ORF8 and ORF10,
although multiple studies have found negligible ORF10 expression (12
, 13
).
As part of COVID-19 Genomics Consortium UK (COG-UK) (14
) we are sequencing
SARS-CoV-2 positive nose-throat swabs from healthcare workers and patients at Sheffield
Teaching Hospitals NHS Foundation Trust, Sheffield, UK (‘Pillar 1’ testing). Additionally, to
relieve pressure on centralised sequencing services, we also sequence a selection from
‘Pillar 2’ testing, which represent SARS-CoV-2 positive samples from the community, tested
at the UK’s Lighthouse Laboratories (Figure S1). We hypothesised that we would see
differences in sgRNA expression profiles in distinct lineages of SARS-CoV-2, in particular,
increased sgRNA in B.1.1.7 that may relate to its altered phenotype.
We stratified sgRNA expression by lineage in 4400 SARS-CoV-2 sequences that reached
our previously defined quality control thresholds (>90% genome coverage, >50K mapped
reads, Table S2), normalized for the genomic coverage from the corresponding amplicon for
the five most abundantly expressed open reading frames (S, E, M, N and ORF6). We
focused our analyses primarily on comparing B.1.1.7 with B.1.177, the dominant lineage in
the UK, which was displaced by B.1.1.7. These two lineages are also the most represented
sequences from both sampling pillars in our dataset (Figure 1A&B, B.1.1.7; Pillar 1: 729,
Pillar 2: 150, B.1.177; Pillar 1: 764 Pillar 2: 179). A significant increase in normalized sgRNA
expression of S, N, and to a lesser extent E, ORF6 and M, was apparent in B.1.1.7
SARS-CoV-2 infections (Figure 1C&D, Wilcoxon effect sizes; S: 0.475, E: 0.191, M 0.0700,
N: 0.469, ORF6: 0.105). Negligible differences were seen in other ORF sgRNA (Figure S2).
Consistent with previous findings (6
, 15
), we also found significantly decreased E gene Ct
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted March 4, 2021. ; https://doi.org/10.1101/2021.03.02.433156doi: bioRxiv preprint
Citations
More filters
Journal Article•DOI•
The biological and clinical significance of emerging SARS-CoV-2 variants.

[...]

Kaiming Tao1, Philip L Tzou1, Janin Nouhin1, Ravindra K. Gupta2, Tulio de Oliveira3, Sergei L Kosakovsky Pond4, Daniela Fera5, Robert W. Shafer1 •
Stanford University1, University of Cambridge2, University of KwaZulu-Natal3, Temple University4, Swarthmore College5
17 Sep 2021-Nature Reviews Genetics
TL;DR: The emergence of SARS-CoV-2 variants with novel spike protein mutations that are influencing the epidemiological and clinical aspects of the COVID-19 pandemic has been witnessed as mentioned in this paper.
Abstract: The past several months have witnessed the emergence of SARS-CoV-2 variants with novel spike protein mutations that are influencing the epidemiological and clinical aspects of the COVID-19 pandemic. These variants can increase rates of virus transmission and/or increase the risk of reinfection and reduce the protection afforded by neutralizing monoclonal antibodies and vaccination. These variants can therefore enable SARS-CoV-2 to continue its spread in the face of rising population immunity while maintaining or increasing its replication fitness. The identification of four rapidly expanding virus lineages since December 2020, designated variants of concern, has ushered in a new stage of the pandemic. The four variants of concern, the Alpha variant (originally identified in the UK), the Beta variant (originally identified in South Africa), the Gamma variant (originally identified in Brazil) and the Delta variant (originally identified in India), share several mutations with one another as well as with an increasing number of other recently identified SARS-CoV-2 variants. Collectively, these SARS-CoV-2 variants complicate the COVID-19 research agenda and necessitate additional avenues of laboratory, epidemiological and clinical research.

593 citations

Posted Content•DOI•
Evolution of enhanced innate immune evasion by the SARS-CoV-2 B.1.1.7 UK variant

[...]

Lucy Thorne1, Mehdi Bouhaddou, Ann-Kathrin Reuschl2, Lorena Zuliani-Alvarez, Benjamin J. Polacco, Adrian Pelin, Jyoti Batra, Matthew Whelan2, Manisha Ummadi, Ajda Roic, Jane Turner2, Kirsten Obernier, Hannes Braberg, Margaret Soucheray, Alicia L. Richards, Kuei-Ho Chen, Bhavya Harjai, Danish Memon3, Myra Hosmillo4, Joseph Hiatt, Aminu S Jahun4, Ian Goodfellow4, Mahdad Noursadeghi2, Jacqueline M. Fabius, Kevan M. Shokat, Natalia Jura, Kliment A. Verba5, Kliment A. Verba1, Pedro Beltrao3, Danielle L. Swaney, Adolfo García-Sastre, Clare Jolly2, Greg J. Towers2, Nevan J. Krogan •
University of California, San Francisco1, University College London2, European Bioinformatics Institute3, University of Cambridge4, University of California, Berkeley5
07 Jun 2021-bioRxiv
TL;DR: In this article, the authors used unbiased abundance proteomics, phosphoproteomics, mRNA sequencing and viral replication assays to show that B.1.7 isolates more effectively suppress host innate immune responses in airway epithelial cells.
Abstract: Emergence of SARS-CoV-2 variants, including the globally successful B.1.1.7 lineage, suggests viral adaptations to host selective pressures resulting in more efficient transmission. Although much effort has focused on Spike adaptation for viral entry and adaptive immune escape, B.1.1.7 mutations outside Spike likely contribute to enhance transmission. Here we used unbiased abundance proteomics, phosphoproteomics, mRNA sequencing and viral replication assays to show that B.1.1.7 isolates more effectively suppress host innate immune responses in airway epithelial cells. We found that B.1.1.7 isolates have dramatically increased subgenomic RNA and protein levels of Orf9b and Orf6, both known innate immune antagonists. Expression of Orf9b alone suppressed the innate immune response through interaction with TOM70, a mitochondrial protein required for RNA sensing adaptor MAVS activation, and Orf9b binding and activity was regulated via phosphorylation. We conclude that B.1.1.7 has evolved beyond the Spike coding region to more effectively antagonise host innate immune responses through upregulation of specific subgenomic RNA synthesis and increased protein expression of key innate immune antagonists. We propose that more effective innate immune antagonism increases the likelihood of successful B.1.1.7 transmission, and may increase in vivo replication and duration of infection.

85 citations

Journal Article•DOI•
Infection and transmission of ancestral SARS-CoV-2 and its alpha variant in pregnant white-tailed deer.

[...]

Konner Cool1, Natasha N. Gaudreault1, Igor Morozov1, Jessie D. Trujillo1, David A. Meekins1, Chester McDowell1, Mariano Carossino2, Dashzeveg Bold1, Dana N Mitzel3, Taeyong Kwon1, Velmurugan Balaraman1, Daniel W. Madden1, Bianca Libanori Artiaga1, Roman Pogranichniy1, Gleyder Roman Sosa1, Jamie Henningson1, William C. Wilson3, Udeni B. R. Balasuriya2, Adolfo García-Sastre, Juergen A. Richt1 •
Kansas State University1, Louisiana State University2, United States Department of Agriculture3
29 Nov 2021-Emerging microbes & infections
TL;DR: In this paper, the authors investigated the susceptibility and transmission of SARS-CoV-2 in adult white-tailed deer (WTD) and found that adult WTD are highly susceptible to SARS infection and can transmit the virus through direct contact as well as vertically from doe to fetus.
Abstract: SARS-CoV-2 was first reported circulating in human populations in December 2019 and has since become a global pandemic. Recent history involving SARS-like coronavirus outbreaks have demonstrated the significant role of intermediate hosts in viral maintenance and transmission. Evidence of SARS-CoV-2 natural infection and experimental infections of a wide variety of animal species has been demonstrated, and in silico and in vitro studies have indicated that deer are susceptible to SARS-CoV-2 infection. White-tailed deer (WTD) are amongst the most abundant and geographically widespread wild ruminant species in the US. Recently, WTD fawns were shown to be susceptible to SARS-CoV-2. In the present study, we investigated the susceptibility and transmission of SARS-CoV-2 in adult WTD. In addition, we examined the competition of two SARS-CoV-2 isolates, representatives of the ancestral lineage A and the alpha variant of concern (VOC) B.1.1.7 through co-infection of WTD. Next-generation sequencing was used to determine the presence and transmission of each strain in the co-infected and contact sentinel animals. Our results demonstrate that adult WTD are highly susceptible to SARS-CoV-2 infection and can transmit the virus through direct contact as well as vertically from doe to fetus. Additionally, we determined that the alpha VOC B.1.1.7 isolate of SARS-CoV-2 outcompetes the ancestral lineage A isolate in WTD, as demonstrated by the genome of the virus shed from nasal and oral cavities from principal infected and contact animals, and from virus present in tissues of principal infected deer, fetuses and contact animals.

56 citations

Posted Content•DOI•
Infection and transmission of SARS-CoV-2 and its alpha variant in pregnant white-tailed deer

[...]

Konner Cool1, Natasha N. Gaudreault1, Igor Morozov1, Jessie D. Trujillo1, David A. Meekins1, Chester McDowell1, Mariano Carossino2, Dashzeveg Bold1, Taeyong Kwon1, Velmurugan Balaraman1, Daniel W. Madden1, Bianca Libanori Artiaga1, Roman Pogranichniy1, Gleyder Roman Sosa1, Jaimie Henningson1, William C. Wilson3, Udeni B. R. Balasuriya2, Adolfo García-Sastre, Juergen A. Richt1 •
Kansas State University1, Louisiana State University2, United States Department of Agriculture3
16 Aug 2021-bioRxiv
TL;DR: In this article, the authors investigated the susceptibility and transmission of SARS-CoV-2 in adult white-tailed deer and examined the competition of two SARS CoV2 isolates, representatives of the ancestral lineage A and the alpha variant of concern (VOC) B.1.7 through co-infection of white-tail deer.
Abstract: SARS-CoV-2, a novel Betacoronavirus, was first reported circulating in human populations in December 2019 and has since become a global pandemic. Recent history involving SARS-like coronavirus outbreaks (SARS-CoV and MERS-CoV) have demonstrated the significant role of intermediate and reservoir hosts in viral maintenance and transmission cycles. Evidence of SARS-CoV-2 natural infection and experimental infections of a wide variety of animal species has been demonstrated, and in silico and in vitro studies have indicated that deer are susceptible to SARS-CoV-2 infection. White-tailed deer (Odocoileus virginianus) are amongst the most abundant, densely populated, and geographically widespread wild ruminant species in the United States. Human interaction with white-tailed deer has resulted in the occurrence of disease in human populations in the past. Recently, white-tailed deer fawns were shown to be susceptible to SARS-CoV-2. In the present study, we investigated the susceptibility and transmission of SARS-CoV-2 in adult white-tailed deer. In addition, we examined the competition of two SARS-CoV-2 isolates, representatives of the ancestral lineage A (SARS-CoV-2/human/USA/WA1/2020) and the alpha variant of concern (VOC) B.1.1.7 (SARS-CoV-2/human/USA/CA_CDC_5574/2020), through co-infection of white-tailed deer. Next-generation sequencing was used to determine the presence and transmission of each strain in the co-infected and contact sentinel animals. Our results demonstrate that adult white-tailed deer are highly susceptible to SARS-CoV-2 infection and can transmit the virus through direct contact as well as vertically from doe to fetus. Additionally, we determined that the alpha VOC B.1.1.7 isolate of SARS-CoV-2 outcompetes the ancestral lineage A isolate in white-tailed deer, as demonstrated by the genome of the virus shed from nasal and oral cavities from principal infected and contact animals, and from virus present in tissues of principal infected deer, fetuses and contact animals.

31 citations

Posted Content•DOI•
Quantitative measurement of infectious virus in SARS-CoV-2 Alpha, Delta and Epsilon variants reveals higher infectivity (viral titer:RNA ratio) in clinical samples containing the Delta and Epsilon variants.

[...]

Hannah W Despres1, Margaret G. Mills2, David J. Shirley, Madaline M. Schmidt1, Meei-Li Huang2, Keith R. Jerome2, Keith R. Jerome3, Alexander L. Greninger3, Alexander L. Greninger2, Emily A. Bruce1 •
University of Vermont1, University of Washington2, Fred Hutchinson Cancer Research Center3
20 Sep 2021-medRxiv
TL;DR: In this article, the authors measured infectious viral titer (using a micro-focus forming assay) as well as total and subgenomic viral RNA levels (using RT-PCR) in a set of 165 clinical samples containing SARS-CoV-2 Alpha, Delta and Epsilon variants that were processed within two days of collection from the patient.
Abstract: Background Novel SARS-CoV-2 Variants of Concern (VoC) pose a challenge to controlling the COVID-19 pandemic. Previous studies indicate that clinical samples collected from individuals infected with the Delta variant may contain higher levels of RNA than previous variants, but the relationship between viral RNA and infectious virus for individual variants is unknown. Methods We measured infectious viral titer (using a micro-focus forming assay) as well as total and subgenomic viral RNA levels (using RT-PCR) in a set of 165 clinical samples containing SARS-CoV-2 Alpha, Delta and Epsilon variants that were processed within two days of collection from the patient. Results We observed a high degree of variation in the relationship between viral titers and RNA levels. Despite the variability we observed for individual samples the overall infectivity differed among the three variants. Both Delta and Epsilon had significantly higher infectivity than Alpha, as measured by the number of infectious units per quantity of viral E gene RNA (6 and 4 times as much, p=0.0002 and 0.009 respectively) or subgenomic E RNA (11 and 7 times as much, p<0.0001 and 0.006 respectively). Conclusion In addition to higher viral RNA levels reported for the Delta variant, the infectivity (amount of replication competent virus per viral genome copy) may also be increased compared to Alpha. Measuring the relationship between live virus and viral RNA is an important step in assessing the infectivity of novel SARS-CoV-2 variants. An increase in the infectivity of the Delta variant may further explain increased spread and suggests a need for increased measures to prevent viral transmission. SIGNIFICANCE STATEMENT Current and future SARS-CoV-2 variants threaten our ability to control the COVID-19 pandemic. Variants with increased transmission, higher viral loads, or greater immune evasion are of particular concern. Viral loads are currently measured by the amount of viral RNA in a clinical sample rather than the amount of infectious virus. We measured both RNA and infectious virus levels directly in a set of 165 clinical specimens from Alpha, Epsilon or Delta variants. Our data shows that Delta is more infectious compared to Alpha, with ∼ six times as much infectious virus for the same amount of RNA. This increase in infectivity suggests increased measures (vaccination, masking, distancing, ventilation) are needed to control Delta compared to Alpha.

29 citations

References
More filters
Journal Article•DOI•
Integrative genomics viewer

[...]

James T. Robinson1, Helga Thorvaldsdottir1, Wendy Winckler1, Mitchell Guttman1, Eric S. Lander2, Eric S. Lander1, Gad Getz1, Jill P. Mesirov1 •
Massachusetts Institute of Technology1, Harvard University2
10 Jan 2011-Nature Biotechnology
TL;DR: In this article, the authors present an approach for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data.
Abstract: Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.

10,798 citations

Journal Article•DOI•
Welcome to the Tidyverse

[...]

Hadley Wickham, Mara Averick, Jennifer Bryan, Winston Chang, Lucy D'Agostino McGowan, Romain François, Garrett Grolemund, Alex Hayes, Lionel Henry, Jim Hester, Max Kuhn, Thomas Lin Pedersen, Evan Miller, Stephan Milton Bache, Kirill Müller, Jeroen Ooms, David Robinson, Dana P. Seidel, Vitalie Spinu, Kohske Takahashi, Davis Vaughan, Claus O. Wilke, Kara H. Woo, Hiroaki Yutani 
21 Nov 2019-The Journal of Open Source Software
TL;DR: This is a list of winners and nominees for the 2016 Paralympic Games in Rio de Janeiro, Brazil.
Abstract: Hadley Wickham1, Mara Averick1, Jennifer Bryan1, Winston Chang1, Lucy D’Agostino McGowan8, Romain François1, Garrett Grolemund1, Alex Hayes12, Lionel Henry1, Jim Hester1, Max Kuhn1, Thomas Lin Pedersen1, Evan Miller13, Stephan Milton Bache3, Kirill Müller2, Jeroen Ooms14, David Robinson5, Dana Paige Seidel10, Vitalie Spinu4, Kohske Takahashi9, Davis Vaughan1, Claus Wilke6, Kara Woo7, and Hiroaki Yutani11

7,298 citations

Journal Article•DOI•
A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology.

[...]

Andrew Rambaut1, Edward C. Holmes2, Áine O'Toole1, Verity Hill1, John T. McCrone1, Christopher Ruis3, Louis du Plessis4, Oliver G. Pybus4 •
University of Edinburgh1, University of Sydney2, University of Cambridge3, University of Oxford4
15 Jul 2020-Nature microbiology
TL;DR: A rational and dynamic virus nomenclature that uses a phylogenetic framework to identify those lineages that contribute most to active spread and is designed to provide a real-time bird’s-eye view of the diversity of the hundreds of thousands of genome sequences collected worldwide.
Abstract: The ongoing pandemic spread of a new human coronavirus, SARS-CoV-2, which is associated with severe pneumonia/disease (COVID-19), has resulted in the generation of tens of thousands of virus genome sequences. The rate of genome generation is unprecedented, yet there is currently no coherent nor accepted scheme for naming the expanding phylogenetic diversity of SARS-CoV-2. Here, we present a rational and dynamic virus nomenclature that uses a phylogenetic framework to identify those lineages that contribute most to active spread. Our system is made tractable by constraining the number and depth of hierarchical lineage labels and by flagging and delabelling virus lineages that become unobserved and hence are probably inactive. By focusing on active virus lineages and those spreading to new locations, this nomenclature will assist in tracking and understanding the patterns and determinants of the global spread of SARS-CoV-2.

2,093 citations

Journal Article•DOI•
The Architecture of SARS-CoV-2 Transcriptome.

[...]

Dong Wan Kim1, Joo Yeon Lee2, Jeong Sun Yang2, Jun Won Kim2, V. Narry Kim1, Hyeshik Chang1 •
Seoul National University1, Centers for Disease Control and Prevention2
14 May 2020-Cell
TL;DR: Functional investigation of the unknown transcripts and RNA modifications discovered in this study will open new directions to the understanding of the life cycle and pathogenicity of SARS-CoV-2.

1,626 citations

Journal Article•DOI•
Early transmissibility assessment of the N501Y mutant strains of SARS-CoV-2 in the United Kingdom, October to November 2020.

[...]

Kathy Leung1, Marcus Ho-Hin Shum, Gabriel M. Leung1, Tommy Tsan-Yuk Lam, Joseph T. Wu1 •
University of Hong Kong1
07 Jan 2021-Eurosurveillance
TL;DR: In this article, two new SARS-CoV-2 lineages with the N501Y mutation in the receptor-binding domain of the spike protein spread rapidly in the United Kingdom.
Abstract: Two new SARS-CoV-2 lineages with the N501Y mutation in the receptor-binding domain of the spike protein spread rapidly in the United Kingdom. We estimated that the earlier 501Y lineage without amino acid deletion Δ69/Δ70, circulating mainly between early September and mid-November, was 10% (6-13%) more transmissible than the 501N lineage, and the 501Y lineage with amino acid deletion Δ69/Δ70, circulating since late September, was 75% (70-80%) more transmissible than the 501N lineage.

593 citations

Related Papers (5)
Estimated transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England.

[...]

09 Apr 2021-Science
Nicholas G Davies, Sam Abbott, Rosanna C. Barnard, Christopher I Jarvis, Adam J. Kucharski, James D Munday, Carl A. B. Pearson, Timothy W Russell, Damien C. Tully, Alex D. Washburne, Tom Wenseleers, Amy Gimma, William Waites, Kerry L. M. Wong, Kevin van Zandvoort, Justin D. Silverman, Karla Diaz-Ordaz, Ruth H. Keogh, Rosalind M Eggo, Sebastian Funk, Mark Jit, Katherine E. Atkins, Katherine E. Atkins, W. John Edmunds 
Evolution of enhanced innate immune evasion by the SARS-CoV-2 B.1.1.7 UK variant

[...]

07 Jun 2021-bioRxiv
Lucy Thorne, Mehdi Bouhaddou, Ann-Kathrin Reuschl, Lorena Zuliani-Alvarez, Benjamin J. Polacco, Adrian Pelin, Jyoti Batra, Matthew Whelan, Manisha Ummadi, Ajda Roic, Jane Turner, Kirsten Obernier, Hannes Braberg, Margaret Soucheray, Alicia L. Richards, Kuei-Ho Chen, Bhavya Harjai, Danish Memon, Myra Hosmillo, Joseph Hiatt, Aminu S Jahun, Ian Goodfellow, Mahdad Noursadeghi, Jacqueline M. Fabius, Kevan M. Shokat, Natalia Jura, Kliment A. Verba, Kliment A. Verba, Pedro Beltrao, Danielle L. Swaney, Adolfo García-Sastre, Clare Jolly, Greg J. Towers, Nevan J. Krogan 
Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage.

[...]

29 Aug 2003-Journal of Molecular Biology
Eric J. Snijder, Peter J. Bredenbeek, Jessika C. Dobbe, Volker Thiel, John Ziebuhr, Leo L.M. Poon, Yi Guan, Mikhail Rozanov, Willy J. M. Spaan, Alexander E. Gorbalenya 
Assessing transmissibility of SARS-CoV-2 lineage B.1.1.7 in England.

[...]

25 Mar 2021-Nature
Erik M. Volz, Swapnil Mishra, Meera Chand, Jeffrey C. Barrett, Robert Johnson, Lily Geidelberg, Wes Hinsley, Daniel J. Laydon, Gavin Dabrera, Áine O'Toole, Roberto Amato, Manon Ragonnet-Cronin, Ian Harrison, Ben Jackson, Cristina V. Ariani, Olivia Boyd, Nicholas J. Loman, Nicholas J. Loman, John T. McCrone, Sónia Gonçalves, David Jorgensen, Richard M. Myers, Verity Hill, David K. Jackson, Katy A. M. Gaythorpe, Natalie Groves, John Sillitoe, Dominic P. Kwiatkowski, Seth Flaxman, Oliver Ratmann, Samir Bhatt, Samir Bhatt, Susan Hopkins, Axel Gandy, Andrew Rambaut, Neil M. Ferguson 
Increased mortality in community-tested cases of SARS-CoV-2 lineage B.1.1.7.

[...]

15 Mar 2021-Nature
Nicholas G Davies, Christopher I Jarvis, W. John Edmunds, Nicholas P. Jewell, Karla Diaz-Ordaz, Ruth H. Keogh 
Frequently Asked Questions (6)
Q1. What contributions have the authors mentioned in the paper "Altered subgenomic rna expression in sars-cov-2 b.1.1.7 infections" ?

Applying their tool ( periscope ) to ARTIC Network Oxford Nanopore genomic sequencing data from 4400 SARS-CoV-2 positive clinical samples, the authors show that normalised sgRNA expression profiles are significantly increased in B. 1. 1. 7 infections ( n=879 ). 4. 0 International license made available under a ( which was not certified by peer review ) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. These findings provide a unique insight into the biology of B. 1. 1. 7 and support monitoring of sgRNA profiles in sequence data to evaluate emerging potential variants of concern. 

These noncanonical sgRNA tend to be enriched around canonical sites, presumably due to the frequency of RdRp template switching that occurs in close proximity. 

This could have led to a transcriptionally driven recombination event resulting in the GAT > CTA mutation in the genomic sequence (Figure 3E panel 2&3). 

4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 

This demonstrates the importance of stratifying sequences by lineage when studying sgRNA..CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 

Of note, this site is upstream of the ORF9b ATG and this noncanonical sgRNA retains the full coding region of the ORF9b, but lacks the canonical start codon of N.