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1.
Cell Rep Med ; 3(3): 100564, 2022 Mar 15.
Article in English | MEDLINE | ID: covidwho-1740281

ABSTRACT

We report on the sequencing of 74,348 SARS-CoV-2 positive samples collected across the United States and show that the Delta variant, first detected in the United States in March 2021, made up the majority of SARS-CoV-2 infections by July 1, 2021 and accounted for >99.9% of the infections by September 2021. Not only did Delta displace variant Alpha, which was the dominant variant at the time, it also displaced the Gamma, Iota, and Mu variants. Through an analysis of quantification cycle (Cq) values, we demonstrate that Delta infections tend to have a 1.7× higher viral load compared to Alpha infections (a decrease of 0.8 Cq) on average. Our results are consistent with the hypothesis that the increased transmissibility of the Delta variant could be due to the ability of the Delta variant to establish a higher viral load earlier in the infection as compared to the Alpha variant.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/epidemiology , Humans , SARS-CoV-2/genetics , United States/epidemiology , Viral Load/genetics
2.
Cytokine ; 143: 155525, 2021 07.
Article in English | MEDLINE | ID: covidwho-1628419

ABSTRACT

Interferon gamma (IFN-γ) is a crucial cytokine in host immune response to hepatitis B virus (HBV) infection. This study aimed to determine whether a functional polymorphism +874T/A in IFN-γ gene linked to high and low producer phenotypes [IFN-γ (+874Thigh â†’ Alow)] may alter the outcomes of chronic HBV infection in Tunisian population. The +874T/A was analysed by ARMS-PCR method in the group of 200 patients chronically infected with HBV and 200 healthy controls. We observed that minor +874A allele, minor +874AA and +874TA genotypes were significantly more frequent in the chronic hepatitis B group in comparison to the control group [49 vs. 31%, P < 10-4; 24 vs. 13%, P < 10-4; 52 vs. 38%, P < 10-4; respectively]. Besides, they were associated with susceptibility to hepatitis B infection [OR = 2.15, 3.87 and 2.84, respectively]. The minor +874A allele and +874AA genotype were statistically more representative in the sub-group of patients with high viral DNA load when compared with the sub-group of patients with low HBV DNA load [(57% vs. 43%, P = 0.003, OR = 1.79); (33% vs. 14%, P = 0.003, OR = 3.59), respectively]. Collectively, our study suggests an association between the IFN-γ +874T/A SNP and persistence of HBV by the enhancement of HBV DNA replication.


Subject(s)
DNA Replication , Genetic Association Studies , Genetic Predisposition to Disease , Hepatitis B virus/physiology , Hepatitis B, Chronic/genetics , Interferon-gamma/genetics , Polymorphism, Single Nucleotide/genetics , Virus Replication/physiology , Adult , Alleles , Case-Control Studies , DNA, Viral/genetics , Female , Gene Frequency/genetics , Hepatitis B, Chronic/virology , Humans , Male , Viral Load/genetics
3.
Nat Commun ; 13(1): 460, 2022 01 24.
Article in English | MEDLINE | ID: covidwho-1651070

ABSTRACT

The SARS-CoV-2 Delta variant has spread rapidly worldwide. To provide data on its virological profile, we here report the first local transmission of Delta in mainland China. All 167 infections could be traced back to the first index case. Daily sequential PCR testing of quarantined individuals indicated that the viral loads of Delta infections, when they first become PCR-positive, were on average ~1000 times greater compared to lineage A/B infections during the first epidemic wave in China in early 2020, suggesting potentially faster viral replication and greater infectiousness of Delta during early infection. The estimated transmission bottleneck size of the Delta variant was generally narrow, with 1-3 virions in 29 donor-recipient transmission pairs. However, the transmission of minor iSNVs resulted in at least 3 of the 34 substitutions that were identified in the outbreak, highlighting the contribution of intra-host variants to population-level viral diversity during rapid spread.


Subject(s)
COVID-19/transmission , Contact Tracing/methods , Disease Outbreaks/prevention & control , SARS-CoV-2/isolation & purification , Animals , COVID-19/epidemiology , COVID-19/virology , Chlorocebus aethiops , Humans , RNA-Seq/methods , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Time Factors , Vero Cells , Viral Load/genetics , Viral Load/physiology , Virus Replication/genetics , Virus Replication/physiology , Virus Shedding/genetics , Virus Shedding/physiology
4.
J Virol ; 95(22): e0096621, 2021 10 27.
Article in English | MEDLINE | ID: covidwho-1561933

ABSTRACT

The high pathogenicity of SARS-CoV-2 requires it to be handled under biosafety level 3 conditions. Consequently, Spike protein-pseudotyped vectors are a useful tool to study viral entry and its inhibition, with retroviral, lentiviral (LV), and vesicular stomatitis virus (VSV) vectors the most commonly used systems. Methods to increase the titer of such vectors commonly include concentration by ultracentrifugation and truncation of the Spike protein cytoplasmic tail. However, limited studies have examined whether such a modification also impacts the protein's function. Here, we optimized concentration methods for SARS-CoV-2 Spike-pseudotyped VSV vectors, finding that tangential flow filtration produced vectors with more consistent titers than ultracentrifugation. We also examined the impact of Spike tail truncation on transduction of various cell types and sensitivity to convalescent serum neutralization. We found that tail truncation increased Spike incorporation into both LV and VSV vectors and resulted in enhanced titers but had no impact on sensitivity to convalescent serum. In addition, we analyzed the effect of the D614G mutation, which became a dominant SARS-CoV-2 variant early in the pandemic. Our studies revealed that, similar to the tail truncation, D614G independently increases Spike incorporation and vector titers, but this effect is masked by also including the cytoplasmic tail truncation. Therefore, the use of full-length Spike protein, combined with tangential flow filtration, is recommended as a method to generate high titer pseudotyped vectors that retain native Spike protein functions. IMPORTANCE Pseudotyped viral vectors are useful tools to study the properties of viral fusion proteins, especially those from highly pathogenic viruses. The Spike protein of SARS-CoV-2 has been investigated using pseudotyped lentiviral and VSV vector systems, where truncation of its cytoplasmic tail is commonly used to enhance Spike incorporation into vectors and to increase the titers of the resulting vectors. However, our studies have shown that such effects can also mask the phenotype of the D614G mutation in the ectodomain of the protein, which was a dominant variant arising early in the COVID-19 pandemic. To better ensure the authenticity of Spike protein phenotypes when using pseudotyped vectors, we recommend using full-length Spike proteins, combined with tangential flow filtration methods of concentration if higher-titer vectors are required.


Subject(s)
Genetic Vectors/physiology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Animals , Antibodies, Neutralizing/immunology , Cell Line , Genetic Vectors/genetics , Genetic Vectors/immunology , Humans , Lentivirus/genetics , Mutation , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Vesicular stomatitis Indiana virus/genetics , Viral Load/genetics
5.
Lab Med ; 52(6): e154-e158, 2021 Nov 02.
Article in English | MEDLINE | ID: covidwho-1559980

ABSTRACT

OBJECTIVE: This study aims to evaluate the performance of an antigen-based rapid diagnostic test (RDT) for the detection of the SARS-CoV-2 virus. METHODS: A cross-sectional study was conducted on 677 patients. Two nasopharyngeal swabs and 1 oropharyngeal swab were collected from patients. The RDT was performed onsite by a commercially available immune-chromatographic assay on the nasopharyngeal swab. The nasopharyngeal and oropharyngeal swabs were examined for SARS-CoV-2 RNA by real-time reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay. RESULTS: The overall sensitivity of the SARS-CoV-2 RDT was 34.5% and the specificity was 99.8%. The positive predictive value and negative predictive value of the test were 96.6% and 91.5%, respectively. The detection rate of RDT in RT-qPCR positive results was high (45%) for cycle threshold values <25. CONCLUSION: The utility of RDT is in diagnosing symptomatic patients and may not be particularly suited as a screening tool for patients with low viral load. The low sensitivity of RDT does not qualify its use as a single test in patients who test negative; RT-qPCR continues to be the gold standard test.


Subject(s)
Antigens, Viral/genetics , COVID-19 Serological Testing/standards , COVID-19/diagnosis , Chromatography, Affinity/methods , RNA, Viral/genetics , SARS-CoV-2/genetics , Adolescent , Aged , Aged, 80 and over , Automation, Laboratory , COVID-19/immunology , COVID-19/virology , COVID-19 Serological Testing/methods , Cross-Sectional Studies , Female , Humans , Male , Middle Aged , Nasopharynx/virology , Oropharynx/virology , Reagent Kits, Diagnostic , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/immunology , Sensitivity and Specificity , Viral Load/genetics
6.
J Med Virol ; 93(12): 6837-6840, 2021 12.
Article in English | MEDLINE | ID: covidwho-1544319

ABSTRACT

BACKGROUND: Gargle samples have been proposed as a noninvasive method for detection of SARS-CoV-2 RNA. The clinical performance of gargle specimens diluted in Cobas® PCR Media and in Cobas® Omni Lysis Reagent was compared to oropharyngeal/nasopharyngeal swab (ONPS) for the detection of SARS-CoV-2 RNA. STUDY DESIGN: Participants were recruited prospectively in two COVID-19 screening clinics. In addition to the ONPS, participants gargled with 5 ml of natural spring water split in the laboratory as follows: 1 ml was added to 4.3 ml of polymerase chain reaction (PCR) media and 400 µl was added to 200 µl of lysis buffer. Testing was performed with the Cobas® SARS-CoV-2 test on the Cobas® 6800 or 8800 platforms. RESULTS: Overall, 134/647 (20.7%) participants were considered infected because the ONPS or at least one gargle test was positive. ONPS had, respectively, a sensitivity of 96.3% (95% confidence interval [CI]: 91.3-98.5); both gargle processing methods were slightly less but equally sensitive (90.3% [95% CI: 83.9-94.3]). When ONPS and gargle specimens were both positive, the mean cycle threshold (Ct ) was significantly higher for gargles, suggesting lower viral loads. CONCLUSION: Gargle specimens directly added in PCR Media provide a similar clinical sensitivity to chemical lysis, both having a slightly, not significantly, lower sensitivity to ONPS.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , COVID-19/virology , Nasopharynx/virology , Oropharynx/virology , SARS-CoV-2/genetics , Diagnostic Tests, Routine/methods , Humans , Mass Screening/methods , Prospective Studies , RNA, Viral/genetics , Saliva/virology , Specimen Handling/methods , Viral Load/genetics
7.
J Med Virol ; 93(12): 6794-6797, 2021 12.
Article in English | MEDLINE | ID: covidwho-1544304

ABSTRACT

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) has affected all inhabited continents, and India is currently experiencing a devastating second wave of coronavirus disease-2019 (COVID-19). Here, we examined the duration of clearance of SARS-CoV-2 in respiratory samples from 207 infected cases by real-time reverse-transcription polymerase chain reaction (RT-PCR). A substantial proportion of COVID-19 positive cases with cycle threshold (Ct) values more than or equal to 31 (45.7%) were subsequently tested negative for SARS-CoV-2 RNA within 7 days of initial detection of the viral load. A total of 60% of all the patients with COVID-19, irrespective of their Ct values, cleared SARS-CoV-2 RNA within 14 days of the initial detection. Longitudinal assessment of RT-PCR test results in individuals requiring 15-30 days to clear SARS-CoV-2 RNA showed a significant reduction of the viral load in samples with high or intermediate viral loads (Ct values ≤ 25 and between 26 and 30, respectively) but the follow-up group with low viral RNA (Ct values ≥ 31) exhibited a stable viral load. Together, these results suggest that COVID-19 positive cases with Ct values more than or equal to 31 require reduced duration to clear SARS-CoV-2, and thus, a shorter isolation period for this group might be considered to facilitate adequate space in the COVID Care Centres and reduce the burden on healthcare infrastructure.


Subject(s)
COVID-19/virology , SARS-CoV-2/genetics , Viral Load/genetics , Adult , Aged , COVID-19 Testing/methods , Diagnostic Tests, Routine/methods , Female , Humans , India , Longitudinal Studies , Male , Middle Aged , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , Sensitivity and Specificity , Serologic Tests/methods , Young Adult
8.
J Med Virol ; 93(12): 6778-6781, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1544295

ABSTRACT

A high-throughput, fully automated antigen detection test for SARS-CoV-2 is a viable alternative to reverse-transcription polymerase chain reaction (RT-qPCR) for mass screening during outbreaks. In this study, we compared RT-qPCR for viral load and the VITROS® SARS-CoV-2 Antigen Test with reference to the results of the LUMIPULSE® SARS-CoV-2 Ag Test. Of 128 nasopharyngeal swab specimens taken from patients suspected of being infected with SARS-CoV-2, 49 were positive and 79 were negative according to RT-qPCR. Consistent dose-dependent detection with VITROS® assay was successfully achieved when using nasopharyngeal swab specimens with Ct values of 32.0 or lesser, whereas the CLEIA-based LUMIPULSE® assay was able to detect lower viral loads compared with the VITROS® assay. Our results show that the performance of the VITROS® assay was satisfactory for the diagnosis of contagious COVID-19 patients in the clinical setting. Highlights The performance of the VITROS® SARS-CoV-2 Antigen Test was sufficient for the diagnosis of contagious COVID-19. This test showed high sensitivity and specificity in the detection of SARS-CoV-2 in samples with a Ct value of 32 or less.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19 Testing/methods , COVID-19/diagnosis , COVID-19/immunology , Immunoenzyme Techniques/methods , Immunologic Tests/methods , SARS-CoV-2/immunology , Antigens, Viral/genetics , Antigens, Viral/immunology , COVID-19/virology , Humans , Mass Screening/methods , Nasopharynx/immunology , Nasopharynx/virology , RNA, Viral/genetics , RNA, Viral/immunology , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Sensitivity and Specificity , Viral Load/genetics , Viral Load/immunology
9.
Bull Exp Biol Med ; 172(1): 53-56, 2021 Nov.
Article in English | MEDLINE | ID: covidwho-1520385

ABSTRACT

The antiviral activity of recombinant human IFN-lambda type 1 (IFNλ-1) against culture strain of SARS-CoV-2 virus was determined by infecting a highly sensitive VeroE6 coronavirus cell culture after preincubation test (the cell monolayer was incubated with 4-fold dilutions of IFNλ-1 in a concentration range of 0.16-42,500 ng/ml in a culture medium for 12 h at 37°C) and without preincubation (simultaneous addition of different concentrations of IFNλ-1 and SARS-CoV-2 infection in a dose of 102 TCID50). The created recombinant human IFNλ-1 demonstrated obvious antiviral activity against SARS-CoV-2 virus in vitro. In the tests with and without preincubation, IFNλ-1 exhibited significant activity, although somewhat lower in variant with simultaneous addition of IFNλ-1 and virus to the cell culture. It should be noted that the antiviral effect of IFNλ-1 was observed in a wide range of concentrations.


Subject(s)
Antiviral Agents/pharmacology , Interferons/pharmacology , Recombinant Proteins/pharmacology , SARS-CoV-2/drug effects , Viral Load/drug effects , Virus Replication/drug effects , Animals , Antiviral Agents/isolation & purification , COVID-19/drug therapy , COVID-19/virology , Chlorocebus aethiops , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Interferons/biosynthesis , Interferons/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , SARS-CoV-2/genetics , SARS-CoV-2/growth & development , Vero Cells , Viral Load/genetics
11.
J Mol Diagn ; 23(12): 1691-1698, 2021 12.
Article in English | MEDLINE | ID: covidwho-1433559

ABSTRACT

Population testing for severe acute respiratory syndrome coronavirus 2 is necessary because of the potential for viral transmission from asymptomatic cases, yet the scarcity of reagents and equipment has increased the cost-prohibitive implementation of screening campaigns at institutions of higher education. Significant analytical sensitivities of nucleic acid amplification methods permit sample pooling to increase testing capacity. Statistical models compared optimal testing configurations for pools of 3, 5, and 10 samples. Assessment of pooling using the TaqPath COVID-19 Combo Kit multiplex assay (ORF1ab, N, and S gene targets) involved a limit-of-detection study, matrix-effect study, and clinical comparison of neat with pooled samples. A limit of detection of 135.02 (ORF1ab; 95% CI, 117.21-155.52), 373.92 (N; 95% CI, 257.05-437.64), and 1001.32 (S; 95% CI, 896.62-1118.33) gene copy equivalents per milliliter was resolved. Seventy-two randomly selected samples showed slight suppression owing to a negative sample matrix. The resulting mean cycle threshold shifts were 2.09 (ORF1ab), 1.76 (N), and 2.31 (S) for the 3-sample pool, 2.83 (ORF1ab), 2.45 (N), and 3.24 (S) for the 5-sample pool, and 3.99 (ORF1ab), 3.46 (N), and 4.07 (S) for the 10-sample pool. Despite a quantitative sensitivity loss trend, the qualitative result was unaffected in each pool. According to the range of disease prevalence observed at the testing site (0.03% to 7.32%), a pool of five samples was deemed an optimal and cost-effective option for monitoring the Northeastern University community.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , SARS-CoV-2/genetics , Diagnostic Tests, Routine/methods , Humans , Nucleic Acid Amplification Techniques/methods , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Retrospective Studies , Sensitivity and Specificity , Specimen Handling , Viral Load/genetics
12.
PLoS One ; 16(9): e0255981, 2021.
Article in English | MEDLINE | ID: covidwho-1416868

ABSTRACT

BACKGROUND: Hospitalization of patients infected with the severe acute respiratory syndrome virus 2 (SARS-CoV-2) have remained considerable worldwide. Patients often develop severe complications and have high mortality rates. The cycle threshold (Ct) value derived from nasopharyngeal swab samples using real time polymerase chain reaction (RT-PCR) may be a useful prognostic marker in hospitalized patients with SARS-CoV-2 infection, however, its role in predicting the course of the pandemic has not been evaluated thus far. METHODS: We conducted a retrospective cohort study which included all patients who had a nasopharyngeal sample positive for SARS-CoV-2 between April 4 -June 5, 2020. The Ct value was used to estimate the number of viral particles in a patient sample. The trend in initial viral load on admission on a population level was evaluated. Moreover, patient characteristics and outcomes stratified by viral load categories were compared and initial viral load was assessed as an independent predictor of intubation and in-hospital mortality. RESULTS: A total of 461 hospitalized patients met the inclusion criteria. This study consisted predominantly of acutely infected patients with a median of 4 days since symptom onset to PCR. As the severity of the pandemic eased, there was an increase in the percentage of samples in the low initial viral load category, coinciding with a decrease in deaths. Compared to an initial low viral load, a high initial viral load was an independent predictor of in-hospital mortality (OR 5.5, CI 3.1-9.7, p < 0.001) and intubation (OR 1.82 CI 1.07-3.11, p = 0.03), while an initial intermediate viral load was associated with increased risk of inpatient mortality (OR 1.9, CI 1.14-3.21, p = 0.015) but not with increased risk for intubation. CONCLUSION: The Ct value obtained from nasopharyngeal samples of hospitalized patients on admission may serve as a prognostic marker at an individual level and may help predict the course of the pandemic when evaluated at a population level.


Subject(s)
COVID-19/diagnosis , Hospitalization/statistics & numerical data , Nasopharynx/virology , SARS-CoV-2/genetics , Viral Load/genetics , Adolescent , Adult , Aged , COVID-19/epidemiology , COVID-19/virology , Female , Humans , Male , Middle Aged , Pandemics/prevention & control , Real-Time Polymerase Chain Reaction/methods , Retrospective Studies , SARS-CoV-2/physiology , Severity of Illness Index , Viral Load/statistics & numerical data , Young Adult
13.
Virus Res ; 305: 198563, 2021 11.
Article in English | MEDLINE | ID: covidwho-1415831

ABSTRACT

This study compared the lethality of severe acute respiratory syndrome coronavirus 2 variants belonging to the S, V, L, G, GH, and GR clades using K18-human angiotensin-converting enzyme 2 heterozygous mice. To estimate the 50% lethal dose (LD50) of each variant, increasing viral loads (100-104 plaque-forming units [PFU]) were administered intranasally. Mouse weight and survival were monitored for 14 days. The LD50 of the GH and GR clades was significantly lower than that of other clades at 50 PFU. These findings suggest that the GH and GR clades, which are prevalent worldwide, are more virulent than the other clades.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/mortality , Receptors, Virus/genetics , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Viral Load/genetics , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/metabolism , Animals , Base Sequence , Body Weight , COVID-19/pathology , COVID-19/virology , Chlorocebus aethiops , Gene Expression , Humans , Lethal Dose 50 , Male , Mice , Mice, Transgenic , Phylogeny , Receptors, Virus/metabolism , SARS-CoV-2/classification , SARS-CoV-2/metabolism , Severity of Illness Index , Survival Analysis , Transgenes , Vero Cells , Viral Plaque Assay , Virulence
14.
Life Sci ; 284: 119881, 2021 Nov 01.
Article in English | MEDLINE | ID: covidwho-1347741

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an infectious disease that has spread worldwide. Current treatments are limited in both availability and efficacy, such that improving our understanding of the factors that facilitate infection is urgently needed to more effectively treat infected individuals and to curb the pandemic. We and others have previously demonstrated the significance of interactions between the SARS-CoV-2 spike protein, integrin α5ß1, and human ACE2 to facilitate viral entry into host cells in vitro. We previously found that inhibition of integrin α5ß1 by the clinically validated small peptide ATN-161 inhibits these spike protein interactions and cell infection in vitro. In continuation with our previous findings, here we have further evaluated the therapeutic potential of ATN-161 on SARS-CoV-2 infection in k18-hACE2 transgenic (SARS-CoV-2 susceptible) mice in vivo. We discovered that treatment with single or repeated intravenous doses of ATN-161 (1 mg/kg) within 48 h after intranasal inoculation with SARS-CoV-2 lead to a reduction of lung viral load, viral immunofluorescence, and improved lung histology in a majority of mice 72 h post-infection. Furthermore, ATN-161 reduced SARS-CoV-2-induced increased expression of lung integrin α5 and αv (an α5-related integrin that has also been implicated in SARS-CoV-2 interactions) as well as the C-X-C motif chemokine ligand 10 (Cxcl10), further supporting the potential involvement of these integrins, and the anti-inflammatory potential of ATN-161, respectively, in SARS-CoV-2 infection. To the best of our knowledge, this is the first study demonstrating the potential therapeutic efficacy of targeting integrin α5ß1 in SARS-CoV-2 infection in vivo and supports the development of ATN-161 as a novel SARS-CoV-2 therapy.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/drug therapy , COVID-19/prevention & control , Oligopeptides/therapeutic use , SARS-CoV-2/physiology , Alanine Transaminase/metabolism , Animals , Aspartate Aminotransferases/metabolism , COVID-19/virology , Genome, Viral , Humans , Integrins/metabolism , Liver/enzymology , Liver/pathology , Lung/pathology , Lung/virology , Male , Mice, Inbred C57BL , Mice, Transgenic , Oligopeptides/pharmacology , SARS-CoV-2/genetics , Staining and Labeling , Viral Load/genetics
15.
mBio ; 12(4): e0058721, 2021 08 31.
Article in English | MEDLINE | ID: covidwho-1327613

ABSTRACT

Since the D614G substitution in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, the variant strain has undergone a rapid expansion to become the most abundant strain worldwide. Therefore, this substitution may provide an advantage for viral spreading. To explore the mechanism, we analyzed 18 viral isolates containing S proteins with either G614 or D614 (S-G614 and S-D614, respectively). The plaque assay showed a significantly higher virus titer in S-G614 than in S-D614 isolates. We further found increased cleavage of the S protein at the furin substrate site, a key event that promotes syncytium formation, in S-G614 isolates. The enhancement of the D614G substitution in the cleavage of the S protein and in syncytium formation has been validated in cells expressing S protein. The effect on the syncytium was abolished by furin inhibitor treatment and mutation of the furin cleavage site, suggesting its dependence on cleavage by furin. Our study pointed to the impact of the D614G substitution on syncytium formation through enhanced furin-mediated S cleavage, which might increase the transmissibility and infectivity of SARS-CoV-2 strains containing S-G614. IMPORTANCE Analysis of viral genomes and monitoring of the evolutionary trajectory of SARS-CoV-2 over time has identified the D614G substitution in spike (S) as the most prevalent expanding variant worldwide, which might confer a selective advantage in transmission. Several studies showed that the D614G variant replicates and transmits more efficiently than the wild-type virus, but the mechanism is unclear. By comparing 18 virus isolates containing S with either D614 or G614, we found significantly higher virus titers in association with higher furin protease-mediated cleavage of S, an event that promotes syncytium formation and virus infectivity, in the S-G614 viruses. The effect of the D614G substitution on furin-mediated S cleavage and the resulting enhancement of the syncytium phenotype has been validated in S-expressing cells. This study suggests a possible effect of the D614G substitution on S of SARS-CoV-2; the antiviral effect through targeting furin protease is worthy of being investigated in proper animal models.


Subject(s)
COVID-19/transmission , Furin/metabolism , Giant Cells/virology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Substitution/genetics , Animals , COVID-19/pathology , Cell Line , Chlorocebus aethiops , Furin/antagonists & inhibitors , Genetic Fitness/genetics , Genome, Viral/genetics , HEK293 Cells , Humans , SARS-CoV-2/isolation & purification , Vero Cells , Viral Load/genetics , Virus Replication/genetics
16.
J Med Virol ; 93(12): 6794-6797, 2021 12.
Article in English | MEDLINE | ID: covidwho-1310533

ABSTRACT

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) has affected all inhabited continents, and India is currently experiencing a devastating second wave of coronavirus disease-2019 (COVID-19). Here, we examined the duration of clearance of SARS-CoV-2 in respiratory samples from 207 infected cases by real-time reverse-transcription polymerase chain reaction (RT-PCR). A substantial proportion of COVID-19 positive cases with cycle threshold (Ct) values more than or equal to 31 (45.7%) were subsequently tested negative for SARS-CoV-2 RNA within 7 days of initial detection of the viral load. A total of 60% of all the patients with COVID-19, irrespective of their Ct values, cleared SARS-CoV-2 RNA within 14 days of the initial detection. Longitudinal assessment of RT-PCR test results in individuals requiring 15-30 days to clear SARS-CoV-2 RNA showed a significant reduction of the viral load in samples with high or intermediate viral loads (Ct values ≤ 25 and between 26 and 30, respectively) but the follow-up group with low viral RNA (Ct values ≥ 31) exhibited a stable viral load. Together, these results suggest that COVID-19 positive cases with Ct values more than or equal to 31 require reduced duration to clear SARS-CoV-2, and thus, a shorter isolation period for this group might be considered to facilitate adequate space in the COVID Care Centres and reduce the burden on healthcare infrastructure.


Subject(s)
COVID-19/virology , SARS-CoV-2/genetics , Viral Load/genetics , Adult , Aged , COVID-19 Testing/methods , Diagnostic Tests, Routine/methods , Female , Humans , India , Longitudinal Studies , Male , Middle Aged , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , Sensitivity and Specificity , Serologic Tests/methods , Young Adult
17.
J Med Virol ; 93(12): 6778-6781, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1281221

ABSTRACT

A high-throughput, fully automated antigen detection test for SARS-CoV-2 is a viable alternative to reverse-transcription polymerase chain reaction (RT-qPCR) for mass screening during outbreaks. In this study, we compared RT-qPCR for viral load and the VITROS® SARS-CoV-2 Antigen Test with reference to the results of the LUMIPULSE® SARS-CoV-2 Ag Test. Of 128 nasopharyngeal swab specimens taken from patients suspected of being infected with SARS-CoV-2, 49 were positive and 79 were negative according to RT-qPCR. Consistent dose-dependent detection with VITROS® assay was successfully achieved when using nasopharyngeal swab specimens with Ct values of 32.0 or lesser, whereas the CLEIA-based LUMIPULSE® assay was able to detect lower viral loads compared with the VITROS® assay. Our results show that the performance of the VITROS® assay was satisfactory for the diagnosis of contagious COVID-19 patients in the clinical setting. Highlights The performance of the VITROS® SARS-CoV-2 Antigen Test was sufficient for the diagnosis of contagious COVID-19. This test showed high sensitivity and specificity in the detection of SARS-CoV-2 in samples with a Ct value of 32 or less.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19 Testing/methods , COVID-19/diagnosis , COVID-19/immunology , Immunoenzyme Techniques/methods , Immunologic Tests/methods , SARS-CoV-2/immunology , Antigens, Viral/genetics , Antigens, Viral/immunology , COVID-19/virology , Humans , Mass Screening/methods , Nasopharynx/immunology , Nasopharynx/virology , RNA, Viral/genetics , RNA, Viral/immunology , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Sensitivity and Specificity , Viral Load/genetics , Viral Load/immunology
18.
Ann Clin Microbiol Antimicrob ; 20(1): 39, 2021 May 24.
Article in English | MEDLINE | ID: covidwho-1242238

ABSTRACT

BACKGROUND: There is limited and controverting evidence looking at possible associations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA copies and patient variables in large cohorts of symptomatic and asymptomatic patients. METHODS: We studied 2275 symptomatic and asymptomatic patients from Colombia with coronavirus disease 2019 (COVID-19) and analyzed the associations between RT-PCR cycle threshold (Ct) value with gender, age, comorbidities, symptomatology, and disease severity. RESULTS: 15.4 % of the samples (n = 428) reported at least one comorbidity. There were 2011 symptomatic cases (72.4 %), being the most common reported symptom cough (57.2 %, n = 1586). Respiratory distress was present in 21.4 % of patients (n = 595), and 435 patients (15.6 %) required hospital admission. We observed that patients with no prior medical history harbored higher RNA copies than patients with comorbidities (p = 0.02). No significant differences in RNA copies were observed between symptomatic and asymptomatic patients (p = 0.82). Strong correlations were detected between Ct values and the presence of odynophagia (p = 0.03), diarrhea (p = 0.04), and headache (p = 0.0008). An inverse association was found between RNA copy number and markers of disease severity, namely, respiratory distress (P < 0.0001) and hospitalization requirement (P < 0.0001). CONCLUSIONS: SARS-CoV-2 RT-PCR cycle thresholds reveal strong associations with a prior medical history, specific symptomatology, and disease severity markers. Further research controlling potential confounding variables needs to be conducted to evaluate the nature and usefulness of these associations in managing COVID-19 patients.


Subject(s)
COVID-19/pathology , RNA, Viral/blood , SARS-CoV-2/genetics , Viral Load/genetics , Adolescent , Adult , Aged , Aged, 80 and over , COVID-19/diagnosis , COVID-19 Nucleic Acid Testing , Child , Child, Preschool , Colombia , Comorbidity , Female , Humans , Infant , Male , Middle Aged , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction , Young Adult
19.
Hum Genomics ; 15(1): 29, 2021 05 17.
Article in English | MEDLINE | ID: covidwho-1232439

ABSTRACT

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a global health problem that causes millions of deaths worldwide. The clinical manifestation of COVID-19 widely varies from asymptomatic infection to severe pneumonia and systemic inflammatory disease. It is thought that host genetic variability may affect the host's response to the virus infection and thus cause severity of the disease. The SARS-CoV-2 virus requires interaction with its receptor complex in the host cells before infection. The transmembrane protease serine 2 (TMPRSS2) has been identified as one of the key molecules involved in SARS-CoV-2 virus receptor binding and cell invasion. Therefore, in this study, we investigated the correlation between a genetic variant within the human TMPRSS2 gene and COVID-19 severity and viral load. RESULTS: We genotyped 95 patients with COVID-19 hospitalised in Dr Soetomo General Hospital and Indrapura Field Hospital (Surabaya, Indonesia) for the TMPRSS2 p.Val160Met polymorphism. Polymorphism was detected using a TaqMan assay. We then analysed the association between the presence of the genetic variant and disease severity and viral load. We did not observe any correlation between the presence of TMPRSS2 genetic variant and the severity of the disease. However, we identified a significant association between the p.Val160Met polymorphism and the SARS-CoV-2 viral load, as estimated by the Ct value of the diagnostic nucleic acid amplification test. Furthermore, we observed a trend of association between the presence of the C allele and the mortality rate in patients with severe COVID-19. CONCLUSION: Our data indicate a possible association between TMPRSS2 p.Val160Met polymorphism and SARS-CoV-2 infectivity and the outcome of COVID-19.


Subject(s)
COVID-19/genetics , Genetic Predisposition to Disease/genetics , Polymorphism, Single Nucleotide , SARS-CoV-2/isolation & purification , Serine Endopeptidases/genetics , Adult , Alleles , COVID-19/diagnosis , COVID-19/virology , Cross-Sectional Studies , Female , Gene Frequency , Genotype , Humans , Indonesia , Male , Middle Aged , SARS-CoV-2/physiology , Viral Load/genetics
20.
J Gen Virol ; 102(3)2021 03.
Article in English | MEDLINE | ID: covidwho-1093630

ABSTRACT

Understanding the pathogenesis of the SARS-CoV-2 infection is key to developing preventive and therapeutic strategies against COVID-19, in the case of severe illness but also when the disease is mild. The use of appropriate experimental animal models remains central in the in vivo exploration of the physiopathology of infection and antiviral strategies. This study describes SARS-CoV-2 intranasal infection in ferrets and hamsters with low doses of low-passage SARS-CoV-2 clinical French isolate UCN19, describing infection levels, excretion, immune responses and pathological patterns in both animal species. Individual infection with 103 p.f.u. SARS-CoV-2 induced a more severe disease in hamsters than in ferrets. Viral RNA was detected in the lungs of hamsters but not of ferrets and in the brain (olfactory bulb and/or medulla oblongata) of both species. Overall, the clinical disease remained mild, with serological responses detected from 7 days and 10 days post-inoculation in hamsters and ferrets respectively. The virus became undetectable and pathology resolved within 14 days. The kinetics and levels of infection can be used in ferrets and hamsters as experimental models for understanding the pathogenicity of SARS-CoV-2, and testing the protective effect of drugs.


Subject(s)
Antibodies, Viral/immunology , COVID-19/virology , Cricetinae , Disease Models, Animal , Ferrets , Animals , Brain/virology , COVID-19/immunology , COVID-19/pathology , COVID-19/physiopathology , Disease Progression , Immunoglobulin G/immunology , Lung/pathology , Lung/virology , Nose , Real-Time Polymerase Chain Reaction , SARS-CoV-2/genetics , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification , SARS-CoV-2/pathogenicity , Viral Load/genetics
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