scholarly journals Clinical severe acute respiratory syndrome coronavirus 2 isolation and antiviral testing

2021 ◽  
Vol 29 ◽  
pp. 204020662110610
Author(s):  
Gregory Mathez ◽  
Valeria Cagno
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Rna Virus ◽  
Clinical Samples ◽  
Viral Titer ◽  
Cell Adaptation

Severe acute respiratory syndrome coronavirus 2 is an RNA virus currently causing a pandemic. Due to errors during replication, mutations can occur and result in cell adaptation by the virus or in the rise of new variants. This can change the attachment receptors' usage, result in different morphology of plaques, and can affect as well antiviral development. Indeed, a molecule can be active on laboratory strains but not necessarily on circulating strains or be effective only against some viral variants. Experiments with clinical samples with limited cell adaptation should be performed to confirm the efficiency of drugs of interest. In this protocol, we present a method to culture severe acute respiratory syndrome coronavirus 2 from nasopharyngeal swabs, obtain a high viral titer while limiting cell adaptation, and assess antiviral efficiency.

scholarly journals 423. SARS-CoV-2 NGS Assay Powered by Biotia COVID-DX Software

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S278-S279
Author(s):  
Dorottya Nagy-Szakal ◽  
Mara Couto-Rodriguez ◽  
Joseph Barrows ◽  
Heather L Wells ◽  
Marilyne Debieu ◽  
...  
Keyword(s):  
Genetic Variants ◽  
Viral Genome ◽  
Board Member ◽  
Limit Of Detection ◽  
Clinical Samples ◽  
Analysis Tool ◽  
Viral Titer ◽  
Target Enrichment ◽  
Library Preparation ◽  
Software Analysis

Abstract Background COVID-19 had spread quickly, causing an international public health emergency with an alarming global shortage of COVID-19 diagnostic tests. We developed and clinically validated a next-generation sequencing (NGS)-based target enrichment assay with the COVID-DX Software tailored for the detection, characterization, and surveillance of the SARS-CoV-2 viral genome. Methods The SARS-CoV-2 NGS assay consists of components including library preparation, target enrichment, sequencing, and a COVID-DX Software analysis tool. The NGS library preparation starts with extracted RNA from nasopharyngeal (NP) swabs followed by cDNA synthesis and conversion to Illumina TruSeq-compatible libraries using the Twist Library Preparation Kit via Enzymatic Fragmentation and Unique Dual Indices (UDI). The library is then enriched for SARS-CoV-2 sequences using a panel of dsDNA biotin-labeled probes, specifically designed to target the SARS-CoV-2 genome, then sequenced on an Illumina NextSeq 550 platform. The COVID-DX Software analyzes sequence results and provides a clinically oriented report, including the presence/absence of SARS-CoV-2 for diagnostic use. An additional research use only report describes the assay performance, estimated viral titer, coverage across the viral genome, genetic variants, and phylogenetic analysis. Results The SARS-CoV-2 NGS Assay was validated on 30 positive and 30 negative clinical samples. To measure the sensitivity and specificity of the assay, the positive and negative percent agreement (PPA, NPA) was defined in comparison to an orthogonal EUA RT-PCR assay (PPA [95% CI]: 96.77% [90.56%-100%] and NPA [95% CI]: 100% [100%-100%]). Data reported using our assay defined the limit of detection to be 40 copies/ml using heat-inactivated SARS-CoV-2 viral genome in clinical matrices. In-silico analysis provided >99.9% coverage across the SARS-CoV-2 viral genome and no cross-reactivity with evolutionarily similar respiratory pathogens. Conclusion The SARS-CoV-2 NGS Assay powered by the COVID-DX Software can be used to detect the SARS-CoV-2 virus and provide additional insight into viral titer and genetic variants to track transmission, stratify risk, predict outcome and therapeutic response, and control the spread of infectious disease. Disclosures Dorottya Nagy-Szakal, MD PhD, Biotia (Employee) Mara Couto-Rodriguez, MS, Biotia (Employee) Joseph Barrows, MS, Biotia, Inc. (Employee, Shareholder) Heather L. Wells, MPH, Biotia (Consultant) Marilyne Debieu, PhD, Biotia (Employee) Courteny Hager, BS, Biotia (Employee) Kristin Butcher, MS, Twist Bioscience (Employee) Siyuan Chen, PhD, Twist Bioscience (Employee) Christopher Mason, PhD, Biotia (Board Member, Employee, Shareholder) Niamh B. O’Hara, PhD, Biotia (Board Member, Employee, Shareholder)Twist (Other Financial or Material Support, I am CEO of Biotia and Biotia has business partnership with Twist)

scholarly journals SARS-CoV-2 within-host diversity and transmission

Science ◽  
2021 ◽  
Vol 372 (6539) ◽  
pp. eabg0821 ◽  
Author(s):  
Katrina A. Lythgoe ◽  
Matthew Hall ◽  
Luca Ferretti ◽  
Mariateresa de Cesare ◽  
George MacIntyre-Cockett ◽  
...  
Keyword(s):  
United Kingdom ◽  
Severe Acute Respiratory Syndrome ◽  
Phylogenetic Tree ◽  
Immune Escape ◽  
Clinical Samples ◽  
Diversity Patterns ◽  
Host Diversity ◽  
Viral Loads ◽  
The United Kingdom ◽  
Low Levels

Extensive global sampling and sequencing of the pandemic virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have enabled researchers to monitor its spread and to identify concerning new variants. Two important determinants of variant spread are how frequently they arise within individuals and how likely they are to be transmitted. To characterize within-host diversity and transmission, we deep-sequenced 1313 clinical samples from the United Kingdom. SARS-CoV-2 infections are characterized by low levels of within-host diversity when viral loads are high and by a narrow bottleneck at transmission. Most variants are either lost or occasionally fixed at the point of transmission, with minimal persistence of shared diversity, patterns that are readily observable on the phylogenetic tree. Our results suggest that transmission-enhancing and/or immune-escape SARS-CoV-2 variants are likely to arise infrequently but could spread rapidly if successfully transmitted.

scholarly journals Evaluation of mobile real-time polymerase chain reaction tests for the detection of severe acute respiratory syndrome coronavirus 2

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chukwunonso Onyilagha ◽  
Henna Mistry ◽  
Peter Marszal ◽  
Mathieu Pinette ◽  
Darwyn Kobasa ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Point Of Care ◽  
Middle Eastern ◽  
Turnaround Time ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Diarrhea Virus ◽  
Highly Sensitive ◽  
Transmissible Gastroenteritis

AbstractThe coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), calls for prompt and accurate diagnosis and rapid turnaround time for test results to limit transmission. Here, we evaluated two independent molecular assays, the Biomeme SARS-CoV-2 test, and the Precision Biomonitoring TripleLock SARS-CoV-2 test on a field-deployable point-of-care real-time PCR instrument, Franklin three9, in combination with Biomeme M1 Sample Prep Cartridge Kit for RNA 2.0 (M1) manual extraction system for rapid, specific, and sensitive detection of SARS-COV-2 in cell culture, human, and animal clinical samples. The Biomeme SARS-CoV-2 assay, which simultaneously detects two viral targets, the orf1ab and S genes, and the Precision Biomonitoring TripleLock SARS-CoV-2 assay that targets the 5′ untranslated region (5′ UTR) and the envelope (E) gene of SARS-CoV-2 were highly sensitive and detected as low as 15 SARS-CoV-2 genome copies per reaction. In addition, the two assays were specific and showed no cross-reactivity with Middle Eastern respiratory syndrome coronavirus (MERS-CoV), infectious bronchitis virus (IBV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis (TGE) virus, and other common human respiratory viruses and bacterial pathogens. Also, both assays were highly reproducible across different operators and instruments. When used to test animal samples, both assays equally detected SARS-CoV-2 genetic materials in the swabs from SARS-CoV-2-infected hamsters. The M1 lysis buffer completely inactivated SARS-CoV-2 within 10 min at room temperature enabling safe handling of clinical samples. Collectively, these results show that the Biomeme and Precision Biomonitoring TripleLock SARS-CoV-2 mobile testing platforms could reliably and promptly detect SARS-CoV-2 in both human and animal clinical samples in approximately an hour and can be used in remote areas or health care settings not traditionally serviced by a microbiology laboratory.

scholarly journals Multiplex Real-Time Reverse-Transcription Polymerase Chain Reaction Assays for Diagnostic Testing of Severe Acute Respiratory Syndrome Coronavirus 2 and Seasonal Influenza Viruses: A Challenge of the Phase 3 Pandemic Setting

2020 ◽  
Author(s):  
Fabiola Mancini ◽  
Fabrizio Barbanti ◽  
Maria Scaturro ◽  
Stefano Fontana ◽  
Angela Di Martino ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Reverse Transcription ◽  
Seasonal Influenza ◽  
Influenza Viruses ◽  
Activity Period ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Polymerase Chain

Abstract Background Pandemic coronavirus disease 2019 (COVID-19) disease represents a challenge for healthcare structures. The molecular confirmation of samples from infected individuals is crucial and therefore guides public health decision making. Clusters and possibly increased diffuse transmission could occur in the context of the next influenza season. For this reason, a diagnostic test able to discriminate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from influenza viruses is urgently needed. Methods A multiplex real-time reverse-transcription polymerase chain reaction (PCR) assay was assessed using 1 laboratory protocol with different real-time PCR instruments. Overall, 1000 clinical samples (600 from samples SARS-CoV-2–infected patients, 200 samples from influenza-infected patients, and 200 negative samples) were analyzed. Results The assay developed was able to detect and discriminate each virus target and to intercept coinfections. The limit of quantification of each assay ranged between 5 and 10 genomic copy numbers, with a cutoff value of 37.7 and 37.8 for influenza and SARS-CoV-2 viruses, respectively. Only 2 influenza coinfections were detected in COVID-19 samples. Conclusions This study suggests that multiplex assay is a rapid, valid, and accurate method for the detection of SARS-CoV-2 and influenza viruses in clinical samples. The test may be an important diagnostic tool for both diagnostic and surveillance purposes during the seasonal influenza activity period.

scholarly journals Detectable Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Human Breast Milk of a Mildly Symptomatic Patient With Coronavirus Disease 2019 (COVID-19)

2020 ◽  
Author(s):  
Patrick C K Tam ◽  
Kathleen M Ly ◽  
Max L Kernich ◽  
Nicola Spurrier ◽  
Diana Lawrence ◽  
...  
Keyword(s):  
Case Report ◽  
Breast Milk ◽  
Severe Acute Respiratory Syndrome ◽  
Human Milk ◽  
Symptomatic Patient ◽  
Clinical Samples ◽  
Human Breast Milk ◽  
Causative Pathogen ◽  
Human Breast ◽  
Novel Coronavirus

Abstract SARS-CoV-2 is a novel coronavirus and causative pathogen to the pandemic illness COVID-19. Although RNA has been detected in various clinical samples, no reports to date have documented SARS-CoV-2 in human milk. This case report describes an actively breastfeeding patient with COVID-19 infection with detectable viral RNA in human milk.

scholarly journals Identification of Common Deletions in the Spike Protein of Severe Acute Respiratory Syndrome Coronavirus 2

2020 ◽  
Vol 94 (17) ◽  
Author(s):  
Zhe Liu ◽  
Huanying Zheng ◽  
Huifang Lin ◽  
Mingyue Li ◽  
Runyu Yuan ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Virus Replication ◽  
Receptor Binding ◽  
Cleavage Site ◽  
Spike Protein ◽  
Clinical Samples ◽  
Receptor Binding Domain ◽  
Flanking Sequence

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus first identified in December 2019. Notable features that make SARS-CoV-2 distinct from most other previously identified betacoronaviruses include a receptor binding domain and a unique insertion of 12 nucleotides or 4 amino acids (PRRA) at the S1/S2 boundary. In this study, we identified two deletion variants of SARS-CoV-2 that either directly affect the polybasic cleavage site itself (NSPRRAR) or a flanking sequence (QTQTN). These deletions were verified by multiple sequencing methods. In vitro results showed that the deletion of NSPRRAR likely does not affect virus replication in Vero and Vero-E6 cells; however, the deletion of QTQTN may restrict late-phase viral replication. The deletion of QTQTN was detected in 3 of 68 clinical samples and 12 of 24 in vitro-isolated viruses, while the deletion of NSPRRAR was identified in 3 in vitro-isolated viruses. Our data indicate that (i) there may be distinct selection pressures on SARS-CoV-2 replication or infection in vitro and in vivo; (ii) an efficient mechanism for deleting this region from the viral genome may exist, given that the deletion variant is commonly detected after two rounds of cell passage; and (iii) the PRRA insertion, which is unique to SARS-CoV-2, is not fixed during virus replication in vitro. These findings provide information to aid further investigation of SARS-CoV-2 infection mechanisms and a better understanding of the NSPRRAR deletion variant observed here. IMPORTANCE The spike protein determines the infectivity and host range of coronaviruses. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has two unique features in its spike protein, the receptor binding domain and an insertion of 12 nucleotides at the S1/S2 boundary resulting in a furin-like cleavage site. Here, we identified two deletion variants of SARS-CoV-2 that either directly affect the furin-like cleavage site itself (NSPRRAR) or a flanking sequence (QTQTN), and we investigated these deletions in cell isolates and clinical samples. The absence of the polybasic cleavage site in SARS-CoV-2 did not affect virus replication in Vero or Vero-E6 cells. Our data indicate the PRRAR sequence and the flanking QTQTN sequence are not fixed in vitro; thus, there appears to be distinct selection pressures on SARS-CoV-2 sequences in vitro and in vivo. Further investigation of the mechanism of generating these deletion variants and their infectivity in different animal models would improve our understanding of the origin and evolution of this virus.

CORONAVIRUS: Pathology, Immunology and Therapies.

2020 ◽  
pp. 1-17
Keyword(s):  
Immune System ◽  
Virus Infection ◽  
Severe Acute Respiratory Syndrome ◽  
Effective Treatment ◽  
Common Cold ◽  
Rna Virus ◽  
Clinical Protocols ◽  
Biochemical Mechanism ◽  
The Family

Abstract Coronavirus is a family of positive single-stranded RNA virus belonging to the family of coronaviridae. Coronavirus-19 infection (COVID-19) has appeared in 2019 and so there is no effective treatment that can eradicate it. The objective of this review is to present data on cellular and molecular characteristic of virus infection and also elucidate all molecular associated events with covid-19 infection in patients. The infection in humans can cause diseases ranging from a common cold to more serious diseases such as severe acute respiratory syndrome (SARS). The disease that it transmits (Covid-19) cannot be cured with conventional treatments. However, a large number of protocols have been implemented based on the sequels that it produces. In this review we summarize 1) the role of immune system against this pathogen as well as the biochemical mechanism by which squealed is responsible for disease progression 2) the possibility or not that patients who have suffered the disease have antibodies against the virus and 3) the clinical protocols used in order to mitigate induced-damage by virus.

scholarly journals Reducing false-positive SARS-CoV-2 diagnoses using long-range RT-qPCR

2021 ◽  
Author(s):  
Aartjan J.W. te Velthuis ◽  
Dovile Juozapaite ◽  
Charlotte Rigby ◽  
Ingrida Olendraite ◽  
Pankaj Mathur ◽  
...  
Keyword(s):  
Rna Virus ◽  
Quantitative Polymerase Chain Reaction ◽  
Diagnostic Testing ◽  
Genetic Material ◽  
Viral Rna ◽  
Clinical Samples ◽  
Virus Infections ◽  
Molecular Method ◽  
Polymerase Chain ◽  
Positive Results

Quantitative polymerase chain reaction (qPCR) is a sensitive molecular method for the detection of genetic material and regarded as the gold-standard for diagnostic testing. To detect respiratory RNA virus infections, a reverse transcription (RT) step is implemented to create cDNA molecules that can serve as template in the qPCR step. However, positive RT-qPCR results can be found long after patient recovery, in part because the RT-qPCR can detect residual viral RNA genome fragments. To minimize the detection of such fragments, we here modified the RT-qPCR assay by replacing the routinely used random hexamers with an oligonucleotide that binds to the 3' end of the viral genome. We demonstrate that this method allows us to distinguish between infectious and non-infectious samples. Moreover, in clinical samples obtained over 15 days after the onset of symptoms, we observe that the modified RT-qPCR protocol yields significantly fewer positive results compared to a commercial RT-qPCR test. No significantly different results were found compared to the commercial test when SARS-CoV-2 clinical samples were tested within 5 days of the onset of symptoms, suggesting that the modification has a similar sensitivity for detecting infectious viral RNA. Overall, these findings may help differentiate between incorrectly-positive, persistently positive, and reinfection cases in COVID-19 patients.

scholarly journals Covid-19 Vaccination: A Bird’s Eye View

2021 ◽  
Vol 6 (1) ◽  
pp. 1-2
Author(s):  
Tahir Sultan Shamsi ◽  
◽  
Mehjabeen Imam ◽  
Keyword(s):  
Social Media ◽  
Severe Acute Respiratory Syndrome ◽  
Rna Virus ◽  
Electronic Media ◽  
Daily Basis ◽  
Scientific Journals ◽  
Positive Strand ◽  
Positive Strand Rna Virus ◽  
The Masses ◽  
Positive Strand Rna

Covid-19 pandemic plagued this world since the beginning of 2020 AD. It is caused by a new positive-strand RNA virus of coronaviridae family [1]. It causes Coronavirus disease 2019 (hence the name COVID-19). It is a contagious disease predominantly causes severe acute respiratory syndrome, hence the name SARS-CoV-2. It started from Wuhan, China, in December 2019. Since then, it has spread globally. It is reported to be a new virus therefore it’s properties, pathogenesis, virulence, immunogenicity, variants, and how will host body will react to this virus was unknown. Despite of 22 months since this virus started to spread worldwide, researchers and clinicians continued to learn about it on daily basis. Newer information about it poured in daily in scientific journals as well as in print / electronic media. Mostly, newer information continued to negate earlier information. Social media disinformation continued to confuse the masses.

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