SARS-CoV-2 genomic diversity and the implications for qRT-PCR diagnostics and transmission.
Ontology highlight
ABSTRACT:
SUBMITTER: Sapoval N
PROVIDER: S-EPMC8015855 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The COVID-19 pandemic has sparked an urgent need to uncover the underlying biology of this devastating disease. Though RNA viruses mutate more rapidly than DNA viruses, there are a relatively small number of single nucleotide polymorphisms (SNPs) that differentiate the main SARS-CoV-2 clades that have spread throughout the world. In this study, we investigated over 7,000 SARS-CoV-2 datasets to unveil both intrahost and interhost diversity. Our intrahost and interhost diversity analyses yielded three major observations. First, the mutational profile of SARS-CoV-2 highlights iSNV and SNP similarity, albeit with high variability in C>T changes. Second, iSNV and SNP patterns in SARS-CoV-2 are more similar to MERS-CoV than SARS-CoV-1. Third, a significant fraction of small indels fuel the genetic diversity of SARS-CoV-2. Altogether, our findings provide insight into SARS-CoV-2 genomic diversity, inform the design of detection tests, and highlight the potential of iSNVs for tracking the transmission of SARS-CoV-2.
| S-EPMC7337385 | biostudies-literature
Project description:Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) is the gold standard method for SARS-CoV-2 detection, and several qRT-PCR kits have been established targeting different genes of the virus. Due to the high mutation rate of these genes, false negative results arise thus complicating the interpretation of the diagnosis and increasing the need of alternative targets. In this study, an alternative approach for the detection of SARS-CoV-2 viral RNA targeting the membrane (M) gene of the virus using qRT-PCR was described. Performance evaluation of this newly developed in-house assay against commercial qRT-PCR kits was done using clinical oropharyngeal specimens of COVID-19 positive patients. The limit of detection was determined using successive dilutions of known copies of SARS-CoV-2 pseudovirus. The M gene based assay was able to detect a minimum of 100 copies of virus/mL indicating its capacity to detect low viral load. The assay showed comparable accuracy, sensitivity and specificity with commercially available kits while detecting all the variants efficiently. The study concluded that the in-house M gene based assay might be an effective alternative for the currently available commercial qRT-PCR kits.
| S-EPMC10547753 | biostudies-literature
Project description:BackgroundThe novel respiratory virus SARS-CoV-2, responsible for over 380,000 COVID-19 related deaths, has caused significant strain on healthcare infrastructure and clinical laboratories globally. The pandemic's initial challenges include broad diagnostic testing, consistent reagent supply lines, and access to laboratory instruments and equipment. In early 2020, primer/probe sets distributed by the CDC utilized the same fluorophore for molecular detection - requiring multiple assays to be run in parallel - consuming valuable and limited resources.MethodsNasopharyngeal swabs submitted to UW Virology for SARS-CoV-2 clinical testing were extracted, amplified by our laboratory developed test (LDT) - a CDC-based quantitative reverse transcriptase PCR reaction - and analyzed for agreement between the multiplexed assay. Laboratory- confirmed respiratory infection samples were included to evaluate assay cross-reaction specificity.ResultsTriplexing correctly identified SARS-CoV-2 in 98.4% of confirmed positive or inconclusive patient samples by single-plex LDT (n?=?183/186). All 170 SARS-CoV-2 negative samples tested by single-plex LDT were negative by triplexing. Other laboratory-confirmed respiratory infections did not amplify for SARS-CoV-2 in the triplex reaction.ConclusionsMultiplexing two virus-specific gene targets and an extraction control was found to be comparable to running parallel assays independently, while significantly improving assay throughput.
| S-EPMC7278635 | biostudies-literature
Project description:The protocols herein outline the use of qRT-PCR to detect the presence of SARS-CoV-2 genomic RNA in patient samples. In order to cope with potential fluctuations in supply chain and testing demands and to enable expedient adaptation of reagents and assays on hand, we include details for three parallel methodologies (one- and two-step singleplex and one-step multiplex assays). The diagnostic platforms described can be easily adapted by basic science research laboratories for SARS-CoV-2 diagnostic testing with relatively short turnaround time. For complete details on the use and execution of this protocol, please refer to Vanuytsel et al. (2020).
| S-EPMC7490627 | biostudies-literature
Project description: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.
| S-EPMC8128293 | biostudies-literature
Project description:Here we describe a SARS-CoV-2 variant with diminished amplification of the ORF ORF1ab target in the Cobas® dual-target SARS-CoV-2 assay resulting in a discrepancy of Ct-values (Ct-value 20.7 for the E-gene and Ct-value 30.2 for ORF1ab). Five unique nucleotide mutations were identified in ORF1ab: C11450A (nsp10) C14178T (RdRp), G15006T (RdRp), G18394T (Hel), and G20995T (Hel). This case highlights the importance of surveillance of genomic regions used in molecular diagnostics and the importance of the public release of target regions used to update commercial and in-house developed SARS-CoV-2 PCR tests. This work underpins the importance of using dual-targets in molecular diagnostic assays to limit the change of false-negative results due to primer and/or probe mismatches.
| S-EPMC8634734 | biostudies-literature
Project description:The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Meanwhile, increased demand for testing has led to a shortage of reagents and supplies and compromised the performance of diagnostic laboratories in many countries. Both the World Health Organization (WHO) and the Center for Disease Control and Prevention (CDC) recommend multi-step RT-PCR assays using multiple primer and probe pairs, which might complicate the interpretation of the test results, especially for borderline cases. In this study, we describe an alternative RT-PCR approach for the detection of SARS-CoV-2 RNA that can be used for the probe-based detection of clinical isolates in diagnostics as well as in research labs using a low-cost SYBR green method. For the evaluation, we used samples from patients with confirmed SARS-CoV-2 infections and performed RT-PCR assays along with successive dilutions of RNA standards to determine the limit of detection. We identified an M-gene binding primer and probe pair highly suitable for the quantitative detection of SARS-CoV-2 RNA for diagnostic and research purposes.
| S-EPMC7352576 | biostudies-literature
Project description:ObjectivesSARS-CoV-2 can be transmitted by aerosols, and the ocular surface may be an important route of transmission. Little is known about protective antibody responses to SARS-CoV-2 in tears after infection or vaccination. We analysed the SARS-CoV-2-specific IgG and IgA responses in human tears after either COVID-19 infection or vaccination.MethodsWe measured the antibody responses in 16 subjects with COVID-19 infection for an average of 7 months before, and 15 subjects before and 2 weeks post-Comirnaty (Pfizer-BioNtech) vaccination. Plasma, saliva and basal tears were collected. Eleven pre-pandemic individuals were included as healthy controls.ResultsIgG antibodies to spike and nucleoprotein were detected in tears, saliva and plasma from subjects with prior SARS-CoV-2 infection in comparison with uninfected controls. While receptor-binding domain (RBD)-specific antibodies were detected in plasma, minimal RBD-specific antibodies were detected in tears and saliva. By contrast, high levels of IgG antibodies to spike and RBD, but not nucleoprotein, were induced in tears, saliva and plasma of subjects receiving 2 doses of the Comirnaty vaccine. Increased levels of IgA1 and IgA2 antibodies to SARS-CoV-2 antigens were detected in plasma following infection or vaccination but were unchanged in tears and saliva. Comirnaty vaccination induced high neutralising Abs in the plasma, but limited neutralising antibodies were detected in saliva or tears.ConclusionBoth infection and vaccination induce SARS-CoV-2-specific IgG antibodies in tears. RBD-specific IgG antibodies in tears were induced by vaccination but were not present 7 months post-infection. This suggests the neutralising antibodies may be low in the tears late following infection.
| S-EPMC8559894 | biostudies-literature
Project description:Family clusters have contributed significantly to the onward spread of SARS-CoV-2. However, the dynamics of viral transmission in this setting remain incompletely understood. We describe the clinical and viral-phylogenetic characteristics of a family cluster of SARS-CoV-2 infections with a high attack rate, and explore how whole-genome sequencing (WGS) can inform outbreak investigations in this context. In this cluster, the first symptomatic case was a 22-month-old infant who developed rhinorrhoea and sneezing 2 days prior to attending a family gathering. Subsequently, seven family members in attendance at this event were diagnosed with SARS-CoV-2 infections, including the infant described. WGS revealed indistinguishable SARS-CoV-2 genomes recovered from the adults at the gathering, which were closely related genetically to B.1 lineage viruses circulating in the local community. However, a divergent viral sub-lineage was recovered from the infant and another child, each harbouring a distinguishing spike substitution (N30S). This suggested that the infant was unlikely to be the primary case, despite displaying symptoms first, and additional analysis of her nasopharyngeal swab revealed a picornavirus co-infection to account for her early symptoms. Our findings demonstrate how WGS can elucidate the transmission dynamics of SARS-CoV-2 infections within household clusters and provide useful information to support outbreak investigations. Additionally, our description of SARS-CoV-2 viral lineages and notable variants circulating in Ireland to date provides an important genomic-epidemiological baseline in the context of vaccine introduction.
| S-EPMC8479960 | biostudies-literature
Project description:BackgroundMore than a year after its first appearance in December 2019, the COVID-19 pandemic is still on a rampage in many parts of the world. Although several vaccines have been approved for emergency use, the emergence and rapid spread of new SARS-CoV-2 variants have sparked fears of vaccine failure due to immune evasion. Massive viral genome sequencing has been recommended to track the genetic changes that could lead to adverse consequences.MethodsWe sequenced SARS-CoV-2 respiratory isolates from the National Public Health Laboratory, Malaysia and examined them together with viral genomes deposited in GISAID by other Malaysian researchers, to understand the evolutionary trend of the virus circulating in the country. We studied the distribution of virus lineages and site-wise mutations, analysed genetic clustering with the goeBURST full Minimum Spanning Tree algorithm, examined the trend of viral nucleotide diversity over time and performed nucleotide substitution association analyses.ResultsWe identified 22 sub-lineages, 13 clonal complexes, 178 sequence types and seven sites of linkage disequilibrium in 277 SARS-CoV-2 genomes sequenced between January and December 2020. B.1.524 was the largest lineage group. The number of mutations per genome ranged from 0 to 19. The mean genomic diversity value over 12 months was 3.26 × 10-4. Of 359 mutations detected, 60.5% of which were non-synonymous, the most frequent were in the ORF1ab (P4715L), S (D614G and A701V) and N (S194L) genes.ConclusionThe SARS-CoV-2 virus accumulated an abundance of mutations in the first year of the COVID-19 pandemic in Malaysia. Its overall genetic diversity, however, is relatively low compared to other Asian countries with larger populations. Continuous genomic and epidemiological surveillance will help to clarify the evolutionary processes determining viral diversity and impacting on human health.
| S-EPMC8571957 | biostudies-literature