scholarly journals ddPCR increases detection of SARS-CoV-2 RNA in patients with low viral loads

2021 ◽  
Author(s):  
Agnès Marchio ◽  
Christophe Batejat ◽  
Jessica Vanhomwegen ◽  
Maxence Feher ◽  
Quentin Grassin ◽  
...  
Keyword(s):  
Upper Airway ◽  
Digital Pcr ◽  
N Gene ◽  
Rna Sequences ◽  
Subgenomic Rna ◽  
N Protein ◽  
Viral Loads ◽  
Gene Coding ◽  
Diagnosis Method

AbstractRT-qPCR detection of SARS-CoV-2 RNA still represents the method of reference to diagnose and monitor COVID-19. From the onset of the pandemic, however, doubts have been expressed concerning the sensitivity of this molecular diagnosis method. Droplet digital PCR (ddPCR) is a third-generation PCR technique that is particularly adapted to detecting low-abundance targets. We developed two-color ddPCR assays for the detection of four different regions of SARS-CoV-2 RNA, including non-structural (IP4-RdRP, helicase) and structural (E, N) protein-encoding sequences. We observed that N or E subgenomic RNAs are generally more abundant than IP4 and helicase RNA sequences in cells infected in vitro, suggesting that detection of the N gene, coding for the most abundant subgenomic RNA of SARS-CoV-2, increases the sensitivity of detection during the highly replicative phase of infection. We investigated 208 nasopharyngeal swabs sampled in March-April 2020 in different hospitals of Greater Paris. We found that 8.6% of informative samples (n = 16/185, P < 0.0001) initially scored as “non-positive” (undetermined or negative) by RT-qPCR were positive for SARS-CoV-2 RNA by ddPCR. Our work confirms that the use of ddPCR modestly, but significantly, increases the proportion of upper airway samples testing positive in the framework of first-line diagnosis of a French population.

scholarly journals Specific viral RNA drives the SARS CoV-2 nucleocapsid to phase separate

2020 ◽  
Author(s):  
Christiane Iserman ◽  
Christine Roden ◽  
Mark Boerneke ◽  
Rachel Sealfon ◽  
Grace McLaughlin ◽  
...  
Keyword(s):  
Small Molecules ◽  
Mammalian Cells ◽  
Upper Airway ◽  
Dependent Manner ◽  
Rna Sequences ◽  
Concentration Dependent Manner ◽  
Health Crisis ◽  
Viral Packaging ◽  
N Protein ◽  
Human Body Temperature

AbstractA mechanistic understanding of the SARS-CoV-2 viral replication cycle is essential to develop new therapies for the COVID-19 global health crisis. In this study, we show that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with the viral genome, and propose a model of viral packaging through LLPS. N-protein condenses with specific RNA sequences in the first 1000 nts (5’-End) under physiological conditions and is enhanced at human upper airway temperatures. N-protein condensates exclude non-packaged RNA sequences. We comprehensively map sites bound by N-protein in the 5’-End and find preferences for single-stranded RNA flanked by stable structured elements. Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules thus presenting screenable processes for identifying antiviral compounds effective against SARS-CoV-2.

scholarly journals Epidemiological Significance of SARS-CoV-2 RNA Dynamic in Naso-Pharyngeal Swabs

2021 ◽  
Vol 9 (6) ◽  
pp. 1264
Author(s):  
Paolo Calistri ◽  
Maria Luisa Danzetta ◽  
Laura Amato ◽  
Francesca Cito ◽  
Alessandra Di Di Giuseppe ◽  
...  
Keyword(s):  
Pearson Correlation ◽  
Pairwise Comparison ◽  
Inverse Correlation ◽  
Indirect Detection ◽  
N Gene ◽  
N Protein ◽  
Viral Loads ◽  
Protein Encoding ◽  
And Gender ◽  
Epidemiological Significance

From 16 March to 15 December 2020, 132,357 naso-pharyngeal/oropharyngeal swabs were collected in the province of Teramo, Abruzzo Region, Italy, and tested for the presence of SARS-CoV-2 genomic RNA by a commercially available molecular assay. A total of 12,880 swabs resulted positive. For 8212 positive patients (4.150 women and 4.062 men) the median age was statistically different between women (median: 49.55 ± 23.9 of SD) and men (median: 48.35 ± 23.5 of SD) while no differences were found in the comparison between the cycle threshold for the N protein-encoding gene (CT N) median values and gender. Differences were observed in the CT N gene median values of swabs collected from March to September as well as in the pairwise comparison between September and October and between November and December. The CT N gene median values observed in specific periods characterizing the SARS-CoV-2 epidemic in 2020 were also compared with the incidence of COVID-19 cases; a strong inverse correlation was highlighted (Pearson correlation coefficient = −0.978). Our findings confirm the usefulness of the CT N values as an indirect detection parameter to monitor viral loads in the population.

scholarly journals SARS CoV-2 Nucleocapsid Protein-mediated Enhancement of Spike pseudotyped Lentivirus Infectivity and Loss of Sensitivity to Neutralisation

2021 ◽  
Author(s):  
Tarun Mishra ◽  
M Sreepadmanabh ◽  
Ajit Chande
Keyword(s):  
Neutralizing Antibodies ◽  
Lentiviral Vector ◽  
Virus Production ◽  
Rapid Screening ◽  
Vector System ◽  
N Gene ◽  
N Protein ◽  
Entry Inhibitors ◽  
Screening And Identification

BACKGROUND: The causative agent of the ongoing COVID-19 pandemic, SARS CoV-2, is a highly pathogenic virus requiring specialized biocontainment facilities. However, pseudotyping-based approaches using the spike glycoprotein have allowed for their study in BSL-2 level laboratories, enabling the rapid screening and identification of neutralizing antibodies, entry inhibitors, host factors, and therapeutic agents. However, this minimalist approach fails to capture the possible contributions and roles of other SARS CoV-2 genes in the entry process. OBJECTIVES: To determine the relative effects of structural and non-structural genes of the SARS CoV-2 on the infectivity of spike-pseudotyped particles, using a lentiviral vector system. METHODS AND RESULTS: An unbiased co-transfection screen of twenty-four SARS CoV-2 genes revealed the nucleocapsid (N) protein as a prime promoter of spike-pseudotyped lentivirus infectivity, as assayed by transduction of an ACE2+ cell line. The spike protein was also observed to be enriched in virions when augmented by the presence of the N gene during virus production. Further, N-enhanced spike-pseudoviruses exhibited a lowered sensitivity to neutralisation by an IgG-Fc fused ACE2 microbody. These results highlight the broad importance of incorporating specific accessory genes during spike-pseudovirus preparation, which may help better recapitulate a physiologically relevant in vitromodel for SARS CoV-2 infectivity.

scholarly journals Sonchus Yellow Net Rhabdovirus Nuclear Viroplasms Contain Polymerase-Associated Proteins

1998 ◽  
Vol 72 (7) ◽  
pp. 5669-5679 ◽  
Author(s):  
Claudia R. F. Martins ◽  
Jennifer A. Johnson ◽  
Diane M. Lawrence ◽  
Tae-Jin Choi ◽  
Anna-Maria Pisi ◽  
...  
Keyword(s):  
Potato Virus X ◽  
Antibody Detection ◽  
N Gene ◽  
Rna Sequences ◽  
N Protein ◽  
Associated Proteins ◽  
Multiple Regions ◽  
Infected Cells ◽  
In Situ Hybridizations ◽  
Pvx Vector

ABSTRACT We have initiated a study of the cytopathology of nucleorhabdoviruses by analyzing the subcellular localization of sonchus yellow net virus (SYNV) genomic and antigenomic RNAs and the encoded polymerase proteins. In situ hybridizations demonstrated that the minus-strand genomic RNA sequences are restricted to the nuclei of infected cells, while the complementary plus-strand antigenomic RNA sequences are present in both the nuclei and the cytoplasm. Immunofluorescence and immunogold labeling experiments also revealed that the nucleocapsid (N) protein and phosphoprotein (M2) are primarily localized to discrete regions within the nuclei and in virus particles that accumulate in perinuclear spaces. The N protein antiserum specifically labeled the nuclear viroplasms, whereas the M2 antiserum was more generally distributed throughout the nuclei. Antibody detection also indicated that the polymerase (L) protein is present in small amounts in the viroplasm. When the N and M2 proteins were expressed individually from the heterologous potato virus X (PVX) vector, both proteins preferentially accumulated in the nuclei. In addition, viroplasm-like inclusions formed in the nuclei of cells infected with the PVX vector containing the N gene. Fusions of the carboxy terminus of β-glucuronidase to N and M2 resulted in staining of the nuclei of infected cells following expression from the PVX vector. Deletion analyses suggested that multiple regions of the N protein contain signals that are important for nuclear localization.

scholarly journals SARS-CoV-2 subgenomic RNA kinetics in longitudinal clinical samples

2021 ◽  
Author(s):  
Renu Verma ◽  
Eugene Kim ◽  
Giovanny Joel Martinez-Colón ◽  
Prasanna Jagannathan ◽  
Arjun Rustagi ◽  
...  
Keyword(s):  
Decay Rates ◽  
N Gene ◽  
Clinical Samples ◽  
Genomic Rna ◽  
Decay Kinetics ◽  
Subgenomic Rna ◽  
Rate Of Increase ◽  
Nasal Swabs ◽  
Few Data

AbstractBackgroundGiven the persistence of viral RNA in clinically recovered COVID-19 patients, subgenomic RNAs (sgRNA) have been reported as potential molecular viability markers for SARS-CoV-2. However, few data are available on their longitudinal kinetics, compared with genomic RNA (gRNA), in clinical samples.MethodsWe analyzed 536 samples from 205 patients with COVID-19 from placebo-controlled, outpatient trials of Peginterferon Lambda-1a (Lambda; n=177) and favipiravir (n=359). Nasal swabs were collected at three time points in the Lambda (Day 1, 4 and 6) and favipiravir (Day 1, 5, and 10) trials. N-gene gRNA and sgRNA were quantified by RT-qPCR. To investigate the decay kinetics in vitro, we measured gRNA and sgRNA in A549ACE2+ cells infected with SARS-CoV-2, following treatment with remdesivir or DMSO control.ResultsAt six days in the Lambda trial and ten days in the favipiravir trial, sgRNA remained detectable in 51.6% (32/62) and 49.5% (51/106) of the samples, respectively. Cycle threshold (Ct) values for gRNA and sgRNA were highly linearly correlated (Pearson’s r=0.87) and the rate of increase did not differ significantly in Lambda (1.36 cycles/day vs 1.36 cycles/day; p = 0.97) or favipiravir (1.03 cycles/day vs 0.94 cycles/day; p=0.26) trials. From samples collected 15-21 days after symptom onset, sgRNA was detectable in 48.1% (40/83) of participants. In SARS-CoV-2 infected A549ACE2+ cells treated with remdesivir, the rate of Ct increase did not differ between gRNA and sgRNA.ConclusionsIn clinical samples and in vitro, sgRNA was highly correlated with gRNA and did not demonstrate different decay patterns to support its application as a viability marker.SummaryWe observed prolonged detection of subgenomic RNA in nasal swabs and equivalent decay rates to genomic RNA in both longitudinal nasal swabs and in remdesivir-treated A549ACE2+ cells infected with SARS-CoV-2. Taken together, these findings suggest that subgenomic RNA from SARS-CoV-2 is comparably stable to genomic RNA and that its detection is therefore not a more reliable indicator of replicating virus.

scholarly journals SARS-CoV-2 subgenomic RNA kinetics in longitudinal clinical samples

2021 ◽  
Author(s):  
Renu Verma ◽  
Eugene Kim ◽  
Giovanny Joel Martinez-Colón ◽  
Prasanna Jagannathan ◽  
Arjun Rustagi ◽  
...  
Keyword(s):  
N Gene ◽  
Clinical Samples ◽  
Decay Kinetics ◽  
Subgenomic Rna ◽  
Rate Of Increase ◽  
Nasal Swabs ◽  
Highly Correlated ◽  
Few Data ◽  
Viability Markers

Abstract Background Given the persistence of viral RNA in clinically recovered COVID-19 patients, subgenomic RNAs (sgRNA) have been reported as potential molecular viability markers for SARS-CoV-2. However, few data are available on their longitudinal kinetics, compared with genomic RNA (gRNA), in clinical samples. Methods We analyzed 536 samples from 205 patients with COVID-19 from placebo-controlled, outpatient trials of Peginterferon Lambda-1a (Lambda; n=177) and favipiravir (n=359). Nasal swabs were collected at three time points in the Lambda (Day 1, 4 and 6) and favipiravir (Day 1, 5, and 10) trials. N-gene gRNA and sgRNA were quantified by RT-qPCR. To investigate the decay kinetics in vitro, we measured gRNA and sgRNA in A549 ACE2+ cells infected with SARS-CoV-2, following treatment with remdesivir or DMSO control. Results At six days in the Lambda trial and ten days in the favipiravir trial, sgRNA remained detectable in 51.6% (32/62) and 49.5% (51/106) of the samples, respectively. Cycle threshold (Ct) values for gRNA and sgRNA were highly linearly correlated (marginal R 2, 0.83) and the rate of increase did not differ significantly in Lambda (1.36 cycles/day vs 1.36 cycles/day; p = 0.97) or favipiravir (1.03 cycles/day vs 0.94 cycles/day; p=0.26) trials. From samples collected 15-21 days after symptom onset, sgRNA was detectable in 48.1% (40/83) of participants. In SARS-CoV-2 infected A549 ACE2+ cells treated with remdesivir, the rate of Ct increase did not differ between gRNA and sgRNA. Conclusions In clinical samples and in vitro, sgRNA was highly correlated with gRNA and did not demonstrate different decay patterns to support its application as a viability marker.

scholarly journals Insertion of a New Transcriptional Unit into the Genome of Mouse Hepatitis Virus

1999 ◽  
Vol 73 (7) ◽  
pp. 6128-6135 ◽  
Author(s):  
Bilan Hsue ◽  
Paul S. Masters
Keyword(s):  
Relative Efficiency ◽  
Untranslated Region ◽  
Hepatitis Virus ◽  
Consensus Sequence ◽  
Mouse Hepatitis Virus ◽  
Transcriptional Unit ◽  
N Gene ◽  
Subgenomic Rna ◽  
N Protein ◽  
Intergenic Sequences

ABSTRACT The subgenomic mRNAs of the coronavirus mouse hepatitis virus (MHV) are composed of a leader sequence, identical to the 5′ 70 nucleotides of the genome, joined at distant downstream sites to a stretch of sequence that is identical to the 3′ end of the genome. The points of fusion occur at intergenic sequences (IGSs), loci on the genome that contain a tract of sequence homologous to the 3′ end of the leader RNA. We have constructed a mutant of MHV-A59 containing an extra IGS inserted into the genome immediately downstream of the 3′-most gene, that encoding the nucleocapsid (N) protein. We show that in cells infected with the mutant, there is synthesis of an additional leader-containing subgenomic RNA of the predicted size. Our study demonstrates that (i) an IGS can be a sufficient cis-acting element to dictate MHV transcription, (ii) the relative efficiency of an IGS must be influenced by factors other than the nucleotides immediately adjacent to the 5′AAUCUAAAC3′ core consensus sequence or its position relative to the 3′ end of the genome, (iii) a downstream IGS can exert a polar attenuating effect on upstream IGSs, and (iv) unknown factors prevent the insertion of large exogenous elements between the N gene and the 3′ untranslated region of MHV. These results confirm and extend conclusions previously derived from the analysis of defective interfering RNAs.

scholarly journals Assessment of SARS-CoV-2 infectivity of upper respiratory specimens from COVID-19 patients by virus isolation using VeroE6/TMPRSS2 cells

2021 ◽  
Vol 8 (1) ◽  
pp. e000830
Author(s):  
Souichi Yamada ◽  
Shuetsu Fukushi ◽  
Hitomi Kinoshita ◽  
Makoto Ohnishi ◽  
Tadaki Suzuki ◽  
...  
Keyword(s):  
Virus Isolation ◽  
Laboratory Diagnostics ◽  
Viral Isolation ◽  
Viral Loads ◽  
A Cell ◽  
Blind Passage ◽  
Upper Respiratory ◽  
Novel Coronavirus ◽  
Respiratory Specimens

BackgroundAn outbreak of novel coronavirus (SARS-CoV-2)-associated respiratory infectious diseases (COVID-19) emerged in 2019 and has spread rapidly in humans around the world. The demonstration of in vitro infectiousness of respiratory specimens is an informative surrogate for SARS-CoV-2 transmission from patients with COVID-19; accordingly, viral isolation assays in cell culture are an important aspect of laboratory diagnostics for COVID-19.MethodsWe developed a simple and rapid protocol for isolating SARS-CoV-2 from respiratory specimens using VeroE6/TMPRSS2 cells, a cell line that is highly susceptible to the virus. We also investigated a correlation between isolation of SARS-CoV-2 and viral load detected by real-time RT-PCR (rRT-PCR) using N2 primer/probe set that has been developed for testing of COVID-19 in Japan.ResultsThe SARS-CoV-2 isolation protocol did not require blind passage of inoculated cells and yielded the results of viral isolation within 7 days after inoculation. Specimens with cycle threshold (Ct) values of <20.2, determined by rRT-PCR, were predicted to be isolation-positive. On the other hand, 6.9% of specimens with Ct values >35 were virus isolation-positive, indicating that low viral loads (high Ct values) in upper respiratory specimens do not always indicate no risk of containing transmissible virus.ConclusionIn combination with rRT-PCR, the SARS-CoV-2 isolation protocol provides a means for assessing the potential risk of transmissible virus in upper respiratory specimens.

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