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    A rapid, cost efficient and simple method to identify current SARS-CoV-2 variants of concern by Sanger sequencing part of the spike protein PROTEIN gene

    Authors: Tue Sparholt Joergensen; Kai Blin; Franziska Kuntke; Henrik K. Salling; Thomas Yssing Michaelsen; Mads Albertsen; Helene Larsen

    doi:10.1101/2021.03.27.21252266 Date: 2021-03-29 Source: medRxiv

    In 2020, the novel coronavirus, SARS-CoV-2, caused a pandemic, which is still raging at the time of writing this. Many countries have set up high throughput RT-qPCR based diagnostics for people with COVID-19 MESHD symptoms and for the wider population. In addition, with the use of whole genome sequencing ( WGS MESHD) new lineages of SARS-CoV-2 have been identified that have been associated with increased transmissibility or altered vaccine efficacy, so-called Variants of Concern (VoC). WGS is generally too labor intensive and expensive to be applied to all positive samples from the diagnostic tests, and often has a turnaround time too long to enable VoC focused contact tracing. Here, we propose to use Sanger sequencing for the detection of common variants of concern and key mutations in early 2021, using a single set of the recognized ARTIC Network primers. The proposed setup relies entirely on materials and methods already in use in diagnostic RT-qPCR labs and on existing infrastructure from companies that have specialized in cheap and rapid turnaround Sanger sequencing. In addition, we provide an automated mutation calling software (https://github.com/kblin/covid-spike-classification). We have validated the setup on 195 SARS-CoV-2 positive samples, and we were able to profile >85% of RT-qPCR positive samples, where the last 15% largely stem from samples with low viral count. At approximately 4 {euro} per sample in material cost, with minimal hands-on time, little data handling, and a turnaround time of less than 30 hours, the setup is simple enough to be implemented in any SARS-CoV-2 RT-qPCR diagnostic lab. Our protocol provides results that can be used to focus contact-tracing efforts and it is cheap enough for the tracking and surveillance of all positive samples for emerging variants such as B.1.1.7, B.1.351 and P.1 as of January 2021.

    A Sanger-based approach for scaling up screening of SARS-CoV-2 variants of interest and concern

    Authors: Matheus Filgueira Bezerra; Lais Ceschini Machado; Viviane Carvalho; Cassia Docena; Sinval Brandao-Filho; Constancia Flavia Junqueira Ayres; Marcelo Henrique Santos Paiva; Gabriel Luz Wallau

    doi:10.1101/2021.03.20.21253956 Date: 2021-03-25 Source: medRxiv

    The global spread of new SARS-CoV-2 variants of concern underscore an urgent need of simple deployed molecular tools that can differentiate these lineages. Several tools and protocols have been shared since the beginning of the COVID-19 pandemic MESHD, but they need to be timely adapted to cope with SARS-CoV-2 evolution. Although whole-genome sequencing (WGS) of the virus genetic material have been widely used, it still presents practical difficulties such as high cost, shortage of available reagents in the global market, need of a specialized laboratorial infrastructure and well-trained staff. These limitations result in genomic surveillance blackouts across several countries. Here we propose a rapid and accessible protocol based on Sanger sequencing of a single PCR fragment that is able to identify and discriminate all SARS-CoV-2 variants of concern (VOCs) identified so far, according to each characteristic mutational profile at the Spike-RBD region (K417N/T, E484K, N501Y, A570D). Twelve COVID-19 MESHD samples from Brazilian patients were evaluated for both WGS MESHD and Sanger sequencing: three from P.2, two from P.1 and seven from B.1.1 lineage. All results from the Sanger sequencing method perfectly matched the mutational profile of VOCs and non-VOCs described by WGS. In summary, this approach allows a much broader network of laboratories to perform molecular surveillance of SARS-CoV-2 VOCs MESHD and report results within a shorter time frame, which is of utmost importance in the context of rapid public health decisions in a fast evolving worldwide pandemic.

    Association of CXCR6 HGNC with COVID-19 MESHD severity: Delineating the host genetic factors in transcriptomic regulation

    Authors: Yulin Dai; Junke Wang; Hyun-Hwan Jeong; Wenhao Chen; Peilin Jia; Zhongming Zhao

    doi:10.1101/2021.02.17.431554 Date: 2021-02-17 Source: bioRxiv

    Background: The coronavirus disease 2019 MESHD ( COVID-19 MESHD) is an infectious disease MESHD that mainly affects the host respiratory system with ~80% asymptomatic or mild cases and ~5% severe cases. Recent genome-wide association studies ( GWAS MESHD) have identified several genetic loci associated with the severe COVID-19 MESHD symptoms. Delineating the genetic variants and genes is important for better understanding its biological mechanisms. Methods: We implemented integrative approaches, including transcriptome-wide association studies (TWAS) and colocalization analysis, to interpret the genetic risks using two independent GWAS datasets in lung and immune cells. We further performed single cell transcriptomic analysis on a bronchoalveolar lavage fluid (BALF) dataset from moderate and severe COVID-19 MESHD patients. Results: We discovered and replicated the genetically regulated expression of CXCR6 HGNC and CCR9 HGNC genes. These two genes have a protective effect on the lung and a risk effect on whole blood, respectively. The colocalization analysis of GWAS and cis-expression quantitative trait loci highlighted the regulatory effect on CXCR6 HGNC expression in lung and immune cells. In the lung resident memory CD8+ T (TRM) cells, we found a 3.32-fold decrease of cell proportion and lower expression of CXCR6 HGNC in the severe than moderate patients using the BALF transcriptomic dataset. Conclusion: CXCR6 HGNC from the 3p21.31 locus is associated with severe COVID-19 MESHD. CXCR6 HGNC tends to have a lower expression in lung TRM cells of severe patients, which aligns with the protective effect of CXCR6 HGNC from TWAS analysis. We illustrate one potential mechanism of host genetic factor impacting the severity of COVID-19 MESHD through regulating the expression of CXCR6 HGNC and TRM cell proportion. Our results shed light on potential therapeutic targets for severe COVID-19 MESHD.

    Novel COVID-19 MESHD phenotype definitions reveal phenotypically distinct patterns of genetic association and protective effects

    Authors: Genevieve H.L. Roberts; Raghavendran Partha; Brooke Rhead; Spencer C. Knight; Danny S. Park; Marie V. Coignet; Miao Zhang; Nathan Berkowitz; David A. Turrisini; Michael Gaddis; Shannon R. McCurdy; Milos Pavlovic; Luong Ruiz; Yambazi Banda; Ke Bi; Robert Burton; Marjan Champine; Ross Curtis; Karen Delgado; Abby Drokhlyansky; Ashley Elrick; Cat Foo; Jialiang Gu; Heather Harris; Shea King; Christine Maldonado; Evan McCartney-Melstad; Patty Miller; Keith Noto; Jingwen Pei; Jenna Petersen; Chodon Sass; Alisa Sedghifar; Andrey Smelter; Sarah South; Barry Starr; Cecily Vaughn; Yong Wang; Asher K. Haug Baltzell; Harendra Guturu; Ahna R. Girshick; Kristin A. Rand; Eurie L. Hong; Catherine A. Ball

    doi:10.1101/2021.01.24.21250324 Date: 2021-01-26 Source: medRxiv

    Multiple large COVID-19 MESHD genome-wide association studies ( GWAS MESHD) have identified reproducible genetic associations indicating that some infection susceptibility and severity risk is heritable. Most of these studies ascertained COVID-19 MESHD cases in medical clinics and hospitals, which can lead to an overrepresentation of cases with severe outcomes, such as hospitalization, intensive care unit admission, or ventilation. Here, we demonstrate the utility and validity of deep phenotyping with self-reported outcomes in a population with a large proportion of mild and subclinical cases. Using these data, we defined eight different phenotypes related to COVID-19 MESHD outcomes: four that align with previously studied COVID-19 MESHD definitions and four novel definitions that focus on susceptibility given exposure, mild clinical manifestations, and an aggregate score of symptom severity. We assessed replication of 13 previously identified COVID-19 MESHD genetic associations with all eight phenotypes and found distinct patterns of association, most notably related to the chr3/ SLC6A20 HGNC/ LZTFL1 HGNC and chr9/ABO regions. We then performed a discovery GWAS, which suggested some novel phenotypes may better capture protective associations and also identified a novel association in chr11/ GALNT18 HGNC that reproduced in two fully independent populations.

    Utilization of Whole Genome Sequencing to Understand SARS-CoV-2 Transmission Dynamics in Long-Term Care Facilities, Correctional Facilities and Meat Processing Plants in Minnesota, March - June 2020

    Authors: Nicholas B Lehnertz; Xiong Wang; Jacob Garfin; Joanne Taylor; Jennifer Zipprich; Brittany VonBank; Karen Martin; Dana Eikmeier; Carlota Medus; Brooke Wiedinmyer; Carmen Bernu; Matthew Plumb; Kelly Pung; Margaret A Honein; Rosalind Carter; Duncan MacCannell; Kirk E. Smith; Kathryn Como-Sabetti; Kris Ehresmann; Richard Danila; Ruth Lynfield

    doi:10.1101/2020.12.30.20248277 Date: 2021-01-08 Source: medRxiv

    Congregate settings and high-density workplaces have endured a disproportionate impact from COVID-19 MESHD. In order to provide further understanding of the transmission patterns of SARS-CoV-2 in these settings, whole genome sequencing (WGS) was performed on samples obtained from 8 selected outbreaks in Minnesota from March -June, 2020. WGS MESHD and phylogenetic analysis was conducted on 319 samples, constituting 14.4% of the 2,222 total SARS-CoV-2-positive individuals associated with these outbreaks. Among the sequenced specimens, three LTCFs and both correctional facilities had spread associated with a single genetic sequence. A fourth LTCF had outbreak cases associated with two distinct sequences. In contrast, cases associated with outbreaks in the two meat processing plants represented multiple SARS-CoV-2 sequences. These results suggest that a single introduction of SARS-CoV-2 into a facility can result in a widespread outbreak, and early identification and cohorting of cases, along with continued vigilance with infection prevention and control measures is imperative.

    Genomic epidemiology of the SARS-CoV-2 epidemic in Zimbabwe: Role of international travel and regional migration in spread

    Authors: Tapfumanei Mashe; Faustinos Tatenda Takawira; Leonardo de Oliveira Martins; Muchaneta Gudza-Mugabe; Joconiah Chirenda; Manes Munyanyi; Hlanai Gumbo; Blessmore V Chaibva; Charles Nyagupe; Andrew Tarupiwa; Agnes Juru; Isaac Phiri; Portia Manangazira; Alexander Goredema; Sydney Danda; Israel Chabata; Janet Jonga; Rutendo Munharira; Kudzai Masunda; Innocent Mukeredzi; Douglas Mangwanya; - The COVID-19 Genomics UK (COG-UK) Consortium; - SARS-CoV-2 Research Group; Alex Trotter; Thanh Le Viet; Gemma Kay; David Baker; Gaetan Thilliez; Ana-Victoria Gutierrez; Justin O Grady; Maxwell Hove; Sekesai Mutapuri-Zinyowera; Andrew J. Page; Robert A. Kingsley; Gibson Mhlanga

    doi:10.1101/2021.01.04.20232520 Date: 2021-01-06 Source: medRxiv

    Zimbabwe reported its first case of SARS-Cov-2 infection MESHD in March 2020, and case numbers increased to more than 8,099 to 16th October 2020. An understanding of the SARS-Cov-2 outbreak in Zimbabwe will assist in the implementation of effective public health interventions to control transmission. Nasopharyngeal samples from 92,299 suspected and confirmed COVID-19 MESHD cases reported in Zimbabwe between 20 March and 16 October 2020 were obtained. Available demographic data associated with those cases identified as positive (8,099) were analysed to describe the national breakdown of positive cases over time in more detail (geographical location, sex, age and travel history). The whole genome sequence ( WGS MESHD) of one hundred SARS-CoV-2-positive samples from the first 120 days of the epidemic in Zimbabwe was determined to identify their relationship to one another and WGS from global samples. Overall, a greater proportion of infections were in males (55.5%) than females (44.85%), although in older age groups more females were affected than males. Most COVID-19 MESHD cases (57 %) were in the 20-40 age group. Eight lineages, from at least 25 separate introductions into the region were found using comparative genomics. Of these, 95% had the D614G mutation on the spike protein PROTEIN which was associated with higher transmissibility than the ancestral strain. Early introductions and spread of SARS-CoV-2 were predominantly associated with genomes common in Europe and the United States of America (USA), and few common in Asia at this time. As the pandemic evolved, travel-associated cases from South Africa and other neighbouring countries were also recorded. Transmission within quarantine centres occurred when travelling nationals returning to Zimbabwe. International and regional migration followed by local transmission were identified as accounting for the development of the SARS-CoV-2 epidemic in Zimbabwe. Based on this, rapid implementation of public health interventions are critical to reduce local transmission of SARS-CoV-2. Impact of the predominant G614 strain on severity of symptoms in COVID-19 MESHD cases needs further investigation.

    Detection of the Novel SARS-CoV-2 European Lineage B.1.177 in Ontario, Canada

    Authors: Jennifer L. Guthrie; Sarah Teatero; Sandra Zittermann; Yao Chen; Ashleigh Sullivan; Heather Rilkoff; Esha Joshi; Karthikeyan Sivaraman; Richard de Borja; Yogi Sundaravadanam; Michael Laszloffy; Lawrence Heisler; Vanessa G. Allen; Jared T. Simpson; Nahuel Fittipaldi

    doi:10.1101/2020.11.30.20241265 Date: 2020-12-02 Source: medRxiv

    Background: Travel-related dissemination of SARS-CoV-2 continues to contribute to the global pandemic. A novel SARS-CoV-2 lineage (B.1.177) reportedly arose in Spain in the summer of 2020, with subsequent spread across Europe linked to travel by infected individuals. Surveillance and monitoring through the use of whole genome sequencing ( WGS MESHD) offers insights into the global and local movement of pathogens such as SARS-CoV-2 and can detect introductions of novel variants. Methods: We analyzed the genomes of SARS-CoV-2 sequenced for surveillance purposes from specimens received by Public Health Ontario ( Sept 6 HGNC - Oct HGNC 10, 2020), collected from individuals in eastern Ontario. Taxonomic lineages were identified using pangolin (v2.08) and phylogenetic analysis incorporated publicly available genomes covering the same time period as the study sample. Epidemiological data collected from laboratory requisitions and standard reportable disease case investigation was integrated into the analysis. Results: Genomic surveillance identified a COVID-19 MESHD case with SARS-CoV-2 lineage B.1.177 from an individual in eastern Ontario in late September, 2020. The individual had recently returned from Europe. Genomic analysis with publicly available data indicate the most closely related genomes to this specimen were from Southern Europe. Genomic surveillance did not identify further cases with this lineage. Conclusions: Genomic surveillance allowed for early detection of a novel SARS-CoV-2 lineage in Ontario which was deemed to be travel related. This type of genomic-based surveillance is a key tool to measure the effectiveness of public health measures such as mandatory self-isolation for returned travellers, aimed at preventing onward transmission of newly introduced lineages of SARS-CoV-2.

    Mutations in SARS-CoV-2 spike PROTEIN protein and RNA polymerase complex are associated with COVID-19 MESHD mortality risk

    Authors: Georg Hahn; Chloe M. Wu; Sanghun Lee; Julian Hecker; Sharon M. Lutz; Sebastien Haneuse; Nan M. Laird; Katharina Ribbeck; Christoph Lange; Ivet Bahar; Jinan Suliman; Elias Tayar; Hasan Ali Kasem; Meynard J. A. Agsalog; Bassam K. Akkarathodiyil; Ayat A. Alkhalaf; Mohamed Morhaf M. H. Alakshar; Abdulsalam Ali A. H. Al-Qahtani; Monther H. A. Al-Shedifat; Anas Ansari; Ahmad Ali Ataalla; Sandeep Chougule; Abhilash K. K. V. Gopinathan; Feroz J. Poolakundan; Sanjay U. Ranbhise; Saed M. A. Saefan; Mohamed M. Thaivalappil; Abubacker S. Thoyalil; Inayath M. Umar; Zaina Al Kanaani; Abdullatif Al Khal; Einas Al Kuwari; Adeel A. Butt; Peter Coyle; Andrew Jeremijenko; Anvar Hassan Kaleeckal; Ali Nizar Latif; Riyazuddin Mohammad Shaik; Hanan F. Abdul Rahim; Hadi M. Yassine; Gheyath K. Nasrallah; Mohamed G. Al Kuwari; Odette Chaghoury; Hiam Chemaitelly; Laith J Abu-Raddad

    doi:10.1101/2020.11.17.386714 Date: 2020-11-24 Source: bioRxiv

    SARS-CoV-2 mortality has been extensively studied in relationship to a patient's predisposition to the disease. However, how sequence variations in the SARS-CoV-2 genome affect mortality is not understood. To address this issue, we used a whole-genome sequencing ( WGS MESHD) association study to directly link death of SARS-CoV-2 patients with sequence variation in the viral genome. Specifically, we analyzed 3,626 single stranded RNA-genomes of SARS-CoV-2 patients in the GISAID database (Elbe and Buckland-Merrett, 2017; Shu and McCauley, 2017) with reported patient's health status from COVID-19 MESHD, i.e. deceased versus non-deceased. In total, evaluating 28,492 loci of the viral genome for association with patient/host mortality, two loci, 12,053bp and 25,088bp, achieved genome-wide significance (p-values of 1.24e-12, and 1.24e-26, respectively). Mutations at 25,088bp occur in the S2 subunit of the SARS-CoV-2 spike PROTEIN protein, which plays a key role in viral entry of target host cells. Additionally, mutations at 12,053bp are within the ORF1ab PROTEIN gene, in a region encoding for the protein nsp7, which is necessary to form the RNA polymerase complex responsible for viral replication and transcription. Both mutations altered amino acid coding sequences, potentially imposing structural changes that could enhance viral infectivity and symptom severity, and may be important to consider as targets for therapeutic development.

    The Pathogen Genomics in Public HeAlth Surveillance Evaluation (PG-PHASE) Framework: An Implementation Science Approach to Evaluating Pathogen Whole Genome Sequencing in Public Health

    Authors: Angeline S Ferdinand; Margaret Kelaher; Courtney R Lane; Anders Gonçalves da Silva; Norelle Sherry; Susan A Ballard; Patiyan Andersson; Tuyet Hoang; Justin T Denholm; Marion Easton; Benjamin P Howden; Deborah A Williamson

    doi:10.21203/rs.3.rs-104489/v1 Date: 2020-11-07 Source: ResearchSquare

    Background Pathogen whole genome sequencing ( WGS MESHD) is being incorporated into public health surveillance and disease control systems worldwide and has the potential to make significant contributions to infectious disease MESHD surveillance, outbreak investigation and infection prevention and control. However, to date, there are limited data regarding: (i) the optimal models for integration of genomic data into epidemiological investigations, and (ii) how to quantify and evaluate public health impacts resulting from genomic epidemiological investigations. Methods We developed the Pathogen Genomics in Public HeAlth Surveillance Evaluation (PG-PHASE) Framework to guide examination of the use of WGS in public health surveillance and disease control. We illustrate the use of this framework with three pathogens as case studies: Listeria monocytogenes, Mycobacterium tuberculosis MESHD and SARS-CoV-2. Results The framework utilises an adaptable whole-of-system approach towards understanding how interconnected elements in the public health application of pathogen genomics contribute to public health processes and outcomes. The three phases of the PG-PHASE Framework are designed to support understanding of WGS laboratory processes, analysis, reporting and data sharing, and how genomic data are utilised in public health practice across all stages, from the decision to send an isolate or sample for sequencing to the use of sequence data in public health surveillance, investigation and decision-making. Importantly, the phases can be used separately or in conjunction, depending on the need of the evaluator. Subsequent to conducting evaluation underpinned by the framework, avenues may be developed for strategic investment or interventions to improve utilisation of whole genome sequencing. Conclusions Comprehensive evaluation is critical to support health departments, public health laboratories and other stakeholders to successfully incorporate microbial genomics into public health practice. The PG-PHASE Framework aims to assist public health laboratories, health departments and authorities who are either considering transitioning to whole genome sequencing or intending to assess the integration of WGS in public health practice, including the capacity to detect and respond to outbreaks and associated costs, challenges and facilitators in the utilisation of microbial genomics and public health impacts. 

    COVID-19 MESHD risk haplogroups differ between populations, deviate from Neanderthal haplotypes and compromise risk assessment in non-Europeans

    Authors: Inken Wohlers; Verónica Calonga-Solís; Jan-Niklas Jobst; Hauke Busch; Bruno Frank; Maren Eggers; Janis A Mueller; Jan Muench; Xiao Zhang; Xiao-Hui Yu; Xiang-Wei Kong; Qian-Ying Zhu; Miao Xu; Nan-Shan Zhong; Yi-Xin Zeng; Guo-Kai Feng; Chang-Wen Ke; Jin-Cun Zhao; Mu-Sheng Zeng; Aishun Jin; Drew W. Barron-Kraus; Harrison C. Shrock; - UFCOVID Interventions Team; Justin Lessler; Carl D. Laird; Derek A.T. Cummings

    doi:10.1101/2020.11.02.365551 Date: 2020-11-03 Source: bioRxiv

    Recent genome wide association studies ( GWAS MESHD) have identified genetic risk factors for developing severe COVID-19 MESHD symptoms. The studies reported a 1bp insertion rs11385942 on chromosome 3 and furthermore two single nucleotide variants (SNVs) rs35044562 and rs67959919, all three correlated with each other. Zeberg and Paabo subsequently traced them back to Neanderthal origin. They found that a 49.4 kb genomic region including the risk allele of rs35044562 is inherited from Neanderthals of Vindija in Croatia. Here we add a differently focused evaluation of this major genetic risk factor to these recent analyses. We show that (i) COVID-19 MESHD-related genetic factors of Neanderthals deviate from those of modern humans and that (ii) they differ among world-wide human populations, which compromises risk prediction in non-Europeans. Currently, caution is thus advised in the genetic risk assessment of non-Europeans during this world-wide COVID-19 pandemic MESHD.

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MeSH Disease
HGNC Genes
SARS-CoV-2 Proteins

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