scholarly journals HiSpike: A high-throughput cost effective sequencing method for the SARS-CoV-2 spike gene

2021 ◽  
Author(s):  
Ephraim Fass ◽  
Gal Zizelski Valenci ◽  
Mor Rubinstein ◽  
Paul J Freidlin ◽  
Shira Rosencwaig ◽  
...  
Keyword(s):  
Real Time ◽  
South African ◽  
High Throughput ◽  
Spike Protein ◽  
Medical Community ◽  
Effective Vaccine ◽  
Gene Variants ◽  
Spike Gene ◽  
Field Samples ◽  
The Uk

ABSTRACTThe changing nature of the corona virus of the SARS-CoV-2 pandemic poses unprecedented challenges to the world’s health systems. New and virulent emerging spike gene variants, such as the UK 20I/501Y.V1 and South African 20H/501Y.V2, could jeopardize global efforts to produce immunity and reduce mortality. These challenges require effective real-time genomic surveillance solutions that the medical community can quickly adopt. The SARS-CoV-2 spike protein mediates host receptor recognition and entry into the cell and therefore, it is most susceptible to generation of variants with increased transmissibility and pathogenicity. The spike protein is also the primary target of neutralizing antibodies in COVID-19 patients and the most common antigen for induction of effective vaccine immunity. Therefore, tight monitoring of the spike protein gene variants is key to mitigating COVID-19 spread and vaccine escape mutants. Currently, the ARTIC method for SARS-CoV-2 whole genome sequencing is applied worldwide. However, this method commonly requires more than 96 hours (4-5 days) from start to finish and at present high sample sequence demands, sequencing resources are quickly exhausted. In this work, we present HiSpike, a method for high-throughput targeted next generation sequencing (NGS) of the spike gene. This simple three-step method can be completed in less than 30 hours and can sequence 10-fold more samples compared to the conventional ARTIC method and at a fraction of the cost. HiSpike was proven valid, and has identified, at high quality, multiple spike variants from real-time field samples, such as the UK and the South African variants. This method will certainly be effective in discovering future spike mutations. Therefore, running HiSpike for full sequencing of the spike gene of all positive SARS-CoV-2 samples could be considered for near real-time detection of known and emerging spike mutations as they evolve. HiSpike provides affordable sequencing options to help laboratories conserve resources, hence it provides a tool for widespread monitoring, that can support critical knowledge-based decisions.

scholarly journals HiSpike: A high-throughput cost effective sequencing method for the SARS-CoV-2 spike gene

2021 ◽  
Author(s):  
Ephraim Fass ◽  
Gal Valenci Zizelski ◽  
Mor Rubinstein ◽  
Paul Freidlin ◽  
Shira Rosencwaig ◽  
...  
Keyword(s):  
Real Time ◽  
South African ◽  
High Throughput ◽  
Spike Protein ◽  
Medical Community ◽  
Effective Vaccine ◽  
Gene Variants ◽  
Spike Gene ◽  
Field Samples ◽  
The Uk

Abstract The changing nature of the corona virus of the SARS-CoV-2 pandemic poses unprecedented challenges to the world’s health systems. New and virulent emerging spike gene variants, such as the UK 20I/501Y.V1 and South African 20H/501Y.V2, could jeopardize global efforts to produce immunity and reduce mortality. These challenges require effective real-time genomic surveillance solutions that the medical community can quickly adopt. The SARS-CoV-2 spike protein mediates host receptor recognition and entry into the cell and therefore, it is most susceptible to generation of variants with increased transmissibility and pathogenicity. The spike protein is also the primary target of neutralizing antibodies in COVID-19 patients and the most common antigen for induction of effective vaccine immunity. Therefore, tight monitoring of the spike protein gene variants is key to mitigating COVID-19 spread and vaccine escape mutants. Currently, the ARTIC method for SARS-CoV-2 whole genome sequencing is applied worldwide. However, this method commonly requires more than 96 hours (4–5 days) from start to finish and at present high sample sequence demands, sequencing resources are quickly exhausted. In this work, we present HiSpike, a method for high-throughput targeted next generation sequencing (NGS) of the spike gene. This simple three-step method can be completed in less than 30 hours and can sequence 10-fold more samples compared to the conventional ARTIC method and at a fraction of the cost. HiSpike was proven valid, and has identified, at high quality, multiple spike variants from real-time field samples, such as the UK and the South African variants. This method will certainly be effective in discovering future spike mutations. Therefore, running HiSpike for full sequencing of the spike gene of all positive SARS-CoV-2 samples could be considered for near real-time detection of known and emerging spike mutations as they evolve. HiSpike provides affordable sequencing options to help laboratories conserve resources, hence it provides a tool for widespread monitoring, that can support critical knowledge-based decisions.

scholarly journals HiSpike Method for High-Throughput Cost Effective Sequencing of the SARS-CoV-2 Spike Gene

2022 ◽  
Vol 8 ◽  
Author(s):  
Ephraim Fass ◽  
Gal Zizelski Valenci ◽  
Mor Rubinstein ◽  
Paul J. Freidlin ◽  
Shira Rosencwaig ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Cost Effective ◽  
Developed Countries ◽  
Spike Protein ◽  
Medical Community ◽  
Effective Vaccine ◽  
Gene Variants ◽  
Spike Gene ◽  
Field Samples

The changing nature of the SARS-CoV-2 pandemic poses unprecedented challenges to the world's health systems. Emerging spike gene variants jeopardize global efforts to produce immunity and reduce morbidity and mortality. These challenges require effective real-time genomic surveillance solutions that the medical community can quickly adopt. The SARS-CoV-2 spike protein mediates host receptor recognition and entry into the cell and is susceptible to generation of variants with increased transmissibility and pathogenicity. The spike protein is the primary target of neutralizing antibodies in COVID-19 patients and the most common antigen for induction of effective vaccine immunity. Tight monitoring of spike protein gene variants is key to mitigating COVID-19 spread and generation of vaccine escape mutants. Currently, SARS-CoV-2 sequencing methods are labor intensive and expensive. When sequence demands are high sequencing resources are quickly exhausted. Consequently, most SARS-CoV-2 strains are sequenced in only a few developed countries and rarely in developing regions. This poses the risk that undetected, dangerous variants will emerge. In this work, we present HiSpike, a method for high-throughput cost effective targeted next generation sequencing of the spike gene. This simple three-step method can be completed in < 30 h, can sequence 10-fold more samples compared to conventional methods and at a fraction of their cost. HiSpike has been validated in Israel, and has identified multiple spike variants from real-time field samples including Alpha, Beta, Delta and the emerging Omicron variants. HiSpike provides affordable sequencing options to help laboratories conserve resources for widespread high-throughput, near real-time monitoring of spike gene variants.

scholarly journals In Silico Investigation of the New UK (B.1.1.7) and South African (501Y.V2) SARS-CoV-2 Variants with a Focus at the ACE2–Spike RBD Interface

2021 ◽  
Vol 22 (4) ◽  
pp. 1695
Author(s):  
Bruno O. Villoutreix ◽  
Vincent Calvez ◽  
Anne-Geneviève Marcelin ◽  
Abdel-Majid Khatib
Keyword(s):  
Amino Acid ◽  
South African ◽  
In Silico ◽  
Neutralizing Antibodies ◽  
Viral Escape ◽  
Spike Protein ◽  
Amino Acid Substitutions ◽  
The South ◽  
African Strain ◽  
The Uk

SARS-CoV-2 exploits angiotensin-converting enzyme 2 (ACE2) as a receptor to invade cells. It has been reported that the UK and South African strains may have higher transmission capabilities, eventually in part due to amino acid substitutions on the SARS-CoV-2 Spike protein. The pathogenicity seems modified but is still under investigation. Here we used the experimental structure of the Spike RBD domain co-crystallized with part of the ACE2 receptor, several in silico methods and numerous experimental data reported recently to analyze the possible impacts of three amino acid replacements (Spike K417N, E484K, N501Y) with regard to ACE2 binding. We found that the N501Y replacement in this region of the interface (present in both the UK and South African strains) should be favorable for the interaction with ACE2, while the K417N and E484K substitutions (South African strain) would seem neutral or even unfavorable. It is unclear if the N501Y substitution in the South African strain could counterbalance the K417N and E484K Spike replacements with regard to ACE2 binding. Our finding suggests that the UK strain should have higher affinity toward ACE2 and therefore likely increased transmissibility and possibly pathogenicity. If indeed the South African strain has a high transmission level, this could be due to the N501Y replacement and/or to substitutions in regions located outside the direct Spike–ACE2 interface but not so much to the K417N and E484K replacements. Yet, it should be noted that amino acid changes at Spike position 484 can lead to viral escape from neutralizing antibodies. Further, these amino acid substitutions do not seem to induce major structural changes in this region of the Spike protein. This structure–function study allows us to rationalize some observations made for the UK strain but raises questions for the South African strain.

scholarly journals Reliability of Spike Gene Target Failure for ascertaining SARS-CoV-2 lineage B.1.1.7 prevalence in a hospital setting

2021 ◽  
Author(s):  
José Afonso Guerra-Assunção ◽  
Paul A. Randell ◽  
Florencia A. T. Boshier ◽  
Michael A. Crone ◽  
Juanita Pang ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Lower Cost ◽  
Hospital Setting ◽  
Qpcr Assay ◽  
Spike Protein ◽  
Whole Genome ◽  
Gene Target ◽  
Spike Gene ◽  
The Uk

AbstractThe appearance of the SARS-CoV-2 lineage B.1.1.7 in the UK in late 2020, associated with faster transmission, sparked the need to find effective ways to monitor its spread. The set of mutations that characterise this lineage include a deletion in position 69 and 70 of the spike protein, which is known to be associated with Spike Gene Target Failure (SGTF) in a commonly used three gene diagnostic qPCR assay. The lower cost and faster turnaround times compared to whole genome sequencing make the use of qPCR for monitoring of the variant spread an attractive proposition. However, there are several potential issues with this approach. Here we use 826 SARS-CoV-2 samples collected in a hospital setting as part of the Hospital Onset COVID Infection (HOCI) study where qPCR was used for viral detection, followed by whole genome sequencing (WGS), to identify the factors to consider when using SGTF to infer lineage B.1.1.7 prevalence in a hospital setting, with potential implications for locations where this variant has recently been introduced.

scholarly journals Structural Analysis of Receptor Binding Domain Mutations in SARS-CoV-2 Variants of Concern that Modulate ACE2 and Antibody Binding

2021 ◽  
Author(s):  
Dhiraj Mannar ◽  
James W Saville ◽  
Xing Zhu ◽  
Shanti S. Srivastava ◽  
Alison M. Berezuk ◽  
...  
Keyword(s):  
Structural Analysis ◽  
South African ◽  
Receptor Binding ◽  
Antibody Binding ◽  
Binding Domain ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Biochemical Assays ◽  
The Uk ◽  
Spike Proteins

SummaryThe recently emerged SARS-CoV-2 South African (B. 1.351) and Brazil/Japan (P.1) variants of concern (VoCs) include a key mutation (N501Y) found in the UK variant that enhances affinity of the spike protein for its receptor, ACE2. Additional mutations are found in these variants at residues 417 and 484 that appear to promote antibody evasion. In contrast, the Californian VoCs (B.1.427/429) lack the N501Y mutation, yet exhibit antibody evasion. We engineered spike proteins to express these RBD VoC mutations either in isolation, or in different combinations, and analyzed the effects using biochemical assays and cryo-EM structural analyses. Overall, our findings suggest that the emergence of new SARS-CoV-2 variant spikes can be rationalized as the result of mutations that confer either increased ACE2 affinity, increased antibody evasion, or both, providing a framework to dissect the molecular factors that drive VoC evolution.

scholarly journals Transformations, Comparisons, and Analysis of Down to Up Protomer States of Variants of the SARS-CoV-2 Prefusion Spike Protein Including the UK Variant B.1.1.7

2021 ◽  
Author(s):  
Michael H. Peters ◽  
Oscar Bastidas ◽  
Daniel S. Kokron ◽  
Christopher E. Henze
Keyword(s):  
South African ◽  
Mitigation Strategies ◽  
Spike Protein ◽  
Triple Mutant ◽  
Cell Receptors ◽  
Potential Impact ◽  
The Uk ◽  
Protein Variants ◽  
Hinge Angle ◽  
Structure Breaking

AbstractMonitoring and strategic response to variants in SARS-CoV-2 represents a considerable challenge in the current pandemic, as well as potentially future viral outbreaks of similar magnitude. In particular mutations and deletions involving the virion’s prefusion Spike protein has significant potential impact on vaccines and therapeutics that utilize this key structural viral protein in their mitigation strategies. In this study, we have demonstrated how dominant energetic landscape mappings (“glue points”) coupled with sequence alignment information can potentially identify or flag key residue mutations and deletions associated with variants. Surprisingly, we also found excellent homology of stabilizing residue glue points across the lineage of β coronavirus Spike proteins, and we have termed this as “sequence homologous glue points”. In general, these flagged residue mutations and/or deletions are then computationally studied in detail using all-atom biocomputational molecular dynamics over approximately one microsecond in order to ascertain structural and energetic changes in the Spike protein associated variants. Specifically, we examined both a theoretically-based triple mutant and the so called UK or B.1.1.7 variant. For the theoretical triple mutant, we demonstrated through Alanine mutations, which help “unglue” key residue-residue interactions, that these three key stabilizing residues could cause the transition of Down to Up protomer states, where the Up protomer state allows binding of the prefusion Spike protein to hACE2 host cell receptors, whereas the Down state is believed inaccessible. For the B.1.1.7 variant, we demonstrated the critical importance of D614G and N5017 on the structure and binding of the Spike protein associated variant. In particular, we had previously identified D614 as a key glue point in the inter-protomer stabilization of the Spike protein. Other mutations and deletions associated with this variant did not appear to play a pivotal role in structure or binding changes. The mutant D614G is a structure breaking Glycine mutation demonstrating a relatively large hinge angle and highly stable Up conformation in agreement with previous studies. In addition, we demonstrate that the mutation N501Y may significantly increase the Spike protein binding to hACE2 cell receptors through its interaction with Y41 of hACE2 forming a potentially strong hydrophobic residue binding pair. We note that these two key mutations, D614G and N501Y, are also found in the so-called South African (SA; B.1.351) variant of SARS-CoV-2. Future studies along these lines are therefore aimed at mapping glue points to residue mutations and deletions of associated prefusion Spike protein variants in order to help direct and optimize efforts aimed at the mitigation of this deadly virion.

The Invention of the ‘Tropical Worker’: Medical Research and the Quest for Central African Labor on the South African Gold Mines, 1903–36

1993 ◽  
Vol 34 (2) ◽  
pp. 271-292 ◽  
Author(s):  
Randall M. Packard
Keyword(s):  
South African ◽  
Central Africa ◽  
Mining Industry ◽  
Primary Source ◽  
Cultural Construction ◽  
Medical Community ◽  
Effective Vaccine ◽  
The South ◽  
Gold Mines ◽  
Political Economic

In 1903 the South African mining industry began recruiting African labor from Central Africa in order to shore up their labor supplies. From the outset, Central African recruitment was problematic, for Central African mine workers died at very high rates. The primary source of Central African mortality was pneumonia. In response to this high mortality the Union government threatened to close down Central African recruitment, a threat which they carried out in 1913. From 1911 to 1933, the mining industry fought to maintain, and then after 1913 to regain access to Central African labor. Of central importance in this struggle were efforts to develop a vaccine against pneumonia. While the mine medical community failed to produce an effective vaccine against pneumonia, the Chamber of Mines successfully employed the promise of a vaccine eventually to regain access to Central African Labor in 1934. The mines achieved this goal by controlling the terrain of discourse on the health of Central African workers, directing attention away from the unhealthy conditions of mine labor and toward the imagined cultural and biological peculiarities of these workers. In doing so the mines constructed a new social category, ‘tropical workers’ or ‘tropicals’. The paper explores the political, economic and intellectual environment within which this cultural construction was created and employed.

scholarly journals Rapid System to Detect the Main Variants of SARS-CoV-2 in Biological Specimens

2021 ◽  
Author(s):  
Marco Favaro ◽  
Paola Zampini ◽  
Enrico S. Pistoia ◽  
Roberta Gaziano ◽  
Sandro Grelli ◽  
...  
Keyword(s):  
South African ◽  
Real Time Pcr ◽  
Biological Samples ◽  
Rna Viruses ◽  
Rapid Identification ◽  
Replacement Rate ◽  
Spike Gene ◽  
Containment Measures ◽  
Selection For ◽  
The Uk

Abstract Since its appearance in late 2019, SARS-CoV-2 has been reported to acquire substitutions more slowly than other RNA viruses, but its tendency to manifest recurrent deletions/mutations in the spike glycoprotein exceeds this slow replacement rate. To date, variants have been identified in many countries, some of which are transmitted efficiently and also present several lineages. The rapid identification of such variants is paramount to quickly implement containment measures. We developed a novel assay using traditional real-time PCR to detect the main reported variants of the spike gene of SARS CoV-2. Primers and probes were designed to detect the following deletions and mutations as well as to cover all lineages known to date (B.1.617, B.1.617.1, B.1.617.2, B.1.617.3 and B.1.618): delta 69:70 and delta 144:145 deletions, which denote the UK variant (VOC 202012/01, now called Alpha); delta 242:244 deletion, which identifies the South African variant (now named Beta); delta 3675:3677 deletion in the ORF1a gene, which denotes the Brazilian variant (now called Gamma); and P681R mutation as well as delta 145:146 and delta 157:158 deletions, which identify the Indian variant (also known as Delta). Our assay will help clinical microbiologists and clinicians to rapidly recognize the presence of variants in biological samples (particularly nasopharyngeal swabs), and it may also be useful for epidemiological purposes in the early selection for successive tracing of patients harbouring virus variants that may be more diffusive and/or not responsive to vaccines.

scholarly journals Design of a High-Throughput Real-Time PCR System for Detection of Bovine Respiratory and Enteric Pathogens

2021 ◽  
Vol 8 ◽  
Author(s):  
Nicole B. Goecke ◽  
Bodil H. Nielsen ◽  
Mette B. Petersen ◽  
Lars E. Larsen
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Negative Impact ◽  
Animal Health ◽  
Simultaneous Detection ◽  
Individual Sample ◽  
Vast Number ◽  
Positive Individual ◽  
Field Samples

Bovine respiratory and enteric diseases have a profound negative impact on animal, health, welfare, and productivity. A vast number of viruses and bacteria are associated with the diseases. Pathogen detection using real-time PCR (rtPCR) assays performed on traditional rtPCR platforms are costly and time consuming and by that limit the use of diagnostics in bovine medicine. To diminish these limitations, we have developed a high-throughput rtPCR system (BioMark HD; Fluidigm) for simultaneous detection of the 11 most important respiratory and enteric viral and bacterial pathogens. The sensitivity and specificity of the rtPCR assays on the high-throughput platform was comparable with that of the traditional rtPCR platform. Pools consisting of positive and negative individual field samples were tested in the high-throughput rtPCR system in order to investigate the effect of an individual sample in a pool. The pool tests showed that irrespective of the size of the pool, a high-range positive individual sample had a high influence on the cycle quantification value of the pool compared with the influence of a low-range positive individual sample. To validate the test on field samples, 2,393 nasal swab and 2,379 fecal samples were tested on the high-throughput rtPCR system as pools in order to determine the occurrence of the 11 pathogens in 100 Danish herds (83 dairy and 17 veal herds). In the dairy calves, Pasteurella multocida (38.4%), rotavirus A (27.4%), Mycoplasma spp. (26.2%), and Trueperella pyogenes (25.5%) were the most prevalent pathogens, while P. multocida (71.4%), Mycoplasma spp. (58.9%), Mannheimia haemolytica (53.6%), and Mycoplasma bovis (42.9%) were the most often detected pathogens in the veal calves. The established high-throughput system provides new possibilities for analysis of bovine samples, since the system enables testing of multiple samples for the presence of different pathogens in the same analysis test even with reduced costs and turnover time.

scholarly journals Structural Analysis of Spike Protein Mutations in an Emergent SARS-CoV-2 Variant from the Philippines

2021 ◽  
Author(s):  
Neil Andrew D. Bascos ◽  
Denise Mirano-Bascos ◽  
Cynthia P. Saloma
Keyword(s):  
South African ◽  
Neutralizing Antibodies ◽  
Clinical Investigation ◽  
The Philippines ◽  
Molecular Surface ◽  
Spike Protein ◽  
Structure Stability ◽  
Whole Genome ◽  
Surface Character ◽  
The Uk

AbstractA SARS-CoV-2 emergent lineage with multiple signature mutations in the Spike protein region was recently reported with cases centered in Cebu Island, Philippines. Whole genome sequencing revealed that the 33 samples with the Ph-B.1.1.28 emergent variant merit further investigation as they all contain the E484K, N501Y, and P681H Spike mutations previously found in other variants of concern such as the South African B.1.351, the Brazil P.1 and the UK B.1.1.7 variants. This is the first known report of these mutations co-occurring in the same virus. The possible implications of the mutations found in the Spike protein were analyzed for their potential effects on structure, stability, and molecular surface character. The analysis suggests that these mutations could significantly impact the possible interactions of the Spike protein monomer with the ACE2 receptor and neutralizing antibodies and warrants further clinical investigation. Some of the mutations affecting the N and C terminal domains may have effects on Spike monomer and trimer stability. This report provides insights on relevant targets for the design of future diagnostics, therapeutics and vaccines against the evolving SARS-CoV-2 variants in the Philippines.

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