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1.
J Mol Diagn ; 24(9): 963-976, 2022 Sep.
Article in English | MEDLINE | ID: covidwho-2083089

ABSTRACT

Amplicon-based sequencing methods are central in characterizing the diversity, transmission, and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but need to be rigorously assessed for clinical utility. Herein, we validated the Swift Biosciences' SARS-CoV-2 Swift Normalase Amplicon Panels using remnant clinical specimens. High-quality genomes meeting our established library and sequence quality criteria were recovered from positive specimens, with 95% limit of detection of 40.08 SARS-CoV-2 copies/PCR. Breadth of genome recovery was evaluated across a range of CT values (11.3 to 36.7; median, 21.6). Of 428 positive samples, 413 (96.5%) generated genomes with <10% unknown bases, with a mean genome coverage of 13,545× ± SD 8382×. No genomes were recovered from PCR-negative specimens (n = 30) or from specimens positive for non-SARS-CoV-2 respiratory viruses (n = 20). Compared with whole-genome shotgun metagenomic sequencing (n = 14) or Sanger sequencing for the spike gene (n = 11), pairwise identity between consensus sequences was 100% in all cases, with highly concordant allele frequencies (R2 = 0.99) between Swift and shotgun libraries. When samples from different clades were mixed at varying ratios, expected variants were detected even in 1:99 mixtures. When deployed as a clinical test, 268 tests were performed in the first 23 weeks, with a median turnaround time of 11 days, ordered primarily for outbreak investigations and infection control.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/genetics , Genome, Viral , Humans , RNA, Viral/genetics , SARS-CoV-2/genetics , Whole Genome Sequencing/methods
2.
Proc Natl Acad Sci U S A ; 119(5)2022 02 01.
Article in English | MEDLINE | ID: covidwho-1642084

ABSTRACT

Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants pose a challenge to controlling the COVID-19 pandemic. Previous studies indicate that clinical samples collected from individuals infected with the Delta variant may contain higher levels of RNA than previous variants, but the relationship between levels of viral RNA and infectious virus for individual variants is unknown. We measured infectious viral titer (using a microfocus-forming assay) and total and subgenomic viral RNA levels (using RT-PCR) in a set of 162 clinical samples containing SARS-CoV-2 Alpha, Delta, and Epsilon variants that were collected in identical swab kits from outpatient test sites and processed soon after collection. We observed a high degree of variation in the relationship between viral titers and RNA levels. Despite this, the overall infectivity differed among the three variants. Both Delta and Epsilon had significantly higher infectivity than Alpha, as measured by the number of infectious units per quantity of viral E gene RNA (5.9- and 3.0-fold increase; P < 0.0001, P = 0.014, respectively) or subgenomic E RNA (14.3- and 6.9-fold increase; P < 0.0001, P = 0.004, respectively). In addition to higher viral RNA levels reported for the Delta variant, the infectivity (amount of replication competent virus per viral genome copy) may be increased compared to Alpha. Measuring the relationship between live virus and viral RNA is an important step in assessing the infectivity of novel SARS-CoV-2 variants. An increase in the infectivity for Delta may further explain increased spread, suggesting a need for increased measures to prevent viral transmission.


Subject(s)
COVID-19/epidemiology , Gene Expression Regulation, Viral , Genome, Viral , RNA, Viral/genetics , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Animals , COVID-19/pathology , COVID-19/transmission , COVID-19/virology , Cell Line, Tumor , Chlorocebus aethiops , Coronavirus Envelope Proteins/genetics , Coronavirus Envelope Proteins/metabolism , Hepatocytes/metabolism , Hepatocytes/virology , Humans , RNA, Viral/metabolism , SARS-CoV-2/classification , SARS-CoV-2/metabolism , Vero Cells , Viral Load , Virulence
3.
EMBO Mol Med ; 14(2): e15290, 2022 02 07.
Article in English | MEDLINE | ID: covidwho-1551925

ABSTRACT

With the COVID-19 pandemic caused by SARS-CoV-2 now in its second year, there remains an urgent need for diagnostic testing that can identify infected individuals, particularly those who harbor infectious virus. Various RT-PCR strategies have been proposed to identify specific viral RNA species that may predict the presence of infectious virus, including detection of transcriptional intermediates (e.g., subgenomic RNA [sgRNA]) and replicative intermediates (e.g., negative-strand RNA species). Using a novel primer/probe set for detection of subgenomic (sg)E transcripts, we successfully identified 100% of specimens containing culturable SARS-CoV-2 from a set of 126 clinical samples (total sgE CT values ranging from 12.3 to 37.5). This assay showed superior performance compared to a previously published sgRNA assay and to a negative-strand RNA assay, both of which failed to detect target RNA in a subset of samples from which we isolated live virus. In addition, total levels of viral RNA (genome, negative-strand, and sgE) detected with the WHO/Charité primer-probe set correlated closely with levels of infectious virus. Specifically, infectious virus was not detected in samples with a CT above 31.0. Clinical samples with higher levels of viral RNA also displayed cytopathic effect (CPE) more quickly than those with lower levels of viral RNA. Finally, we found that the infectivity of SARS-CoV-2 samples is significantly dependent on the cell type used for viral isolation, as Vero E6 cells expressing TMRPSS2 extended the analytical sensitivity of isolation by more than 3 CT compared to parental Vero E6 cells and resulted in faster isolation. Our work shows that using a total viral RNA Ct cutoff of > 31 or specifically testing for sgRNA can serve as an effective rule-out test for the presence of culturable virus.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Pandemics , Polymerase Chain Reaction , RNA, Viral/genetics
4.
Immunity ; 54(9): 2143-2158.e15, 2021 09 14.
Article in English | MEDLINE | ID: covidwho-1364125

ABSTRACT

Neutralizing antibodies (NAbs) are effective in treating COVID-19, but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment during prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. Real-time imaging revealed that the virus spread sequentially from the nasal cavity to the lungs in mice and thereafter systemically to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct neutralization, depletion studies indicated that Fc effector interactions of NAbs with monocytes, neutrophils, and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.


Subject(s)
Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , Brain/pathology , COVID-19/immunology , Lung/pathology , SARS-CoV-2/physiology , Testis/pathology , Angiotensin-Converting Enzyme 2/genetics , Animals , Antibodies, Neutralizing/genetics , Antibodies, Viral/genetics , Brain/virology , COVID-19/therapy , Cells, Cultured , Disease Models, Animal , Humans , Immunoglobulin Fc Fragments/genetics , Luciferases/genetics , Luminescent Measurements , Lung/virology , Male , Mice , Mice, Transgenic , Testis/virology
5.
Science ; 373(6557): 931-936, 2021 08 20.
Article in English | MEDLINE | ID: covidwho-1319369

ABSTRACT

There is an urgent need for antiviral agents that treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We screened a library of 1900 clinically safe drugs against OC43, a human beta coronavirus that causes the common cold, and evaluated the top hits against SARS-CoV-2. Twenty drugs significantly inhibited replication of both viruses in cultured human cells. Eight of these drugs inhibited the activity of the SARS-CoV-2 main protease, 3CLpro, with the most potent being masitinib, an orally bioavailable tyrosine kinase inhibitor. X-ray crystallography and biochemistry show that masitinib acts as a competitive inhibitor of 3CLpro. Mice infected with SARS-CoV-2 and then treated with masitinib showed >200-fold reduction in viral titers in the lungs and nose, as well as reduced lung inflammation. Masitinib was also effective in vitro against all tested variants of concern (B.1.1.7, B.1.351, and P.1).


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus OC43, Human/drug effects , Cysteine Proteinase Inhibitors/pharmacology , SARS-CoV-2/drug effects , Thiazoles/pharmacology , A549 Cells , Animals , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/therapeutic use , Benzamides , COVID-19/virology , Catalytic Domain , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Coronavirus OC43, Human/physiology , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/metabolism , HEK293 Cells , Humans , Inhibitory Concentration 50 , Mice , Mice, Transgenic , Microbial Sensitivity Tests , Piperidines , Pyridines , SARS-CoV-2/enzymology , SARS-CoV-2/physiology , Thiazoles/chemistry , Thiazoles/metabolism , Thiazoles/therapeutic use , Viral Load/drug effects , Virus Replication/drug effects
7.
Gastroenterology ; 160(7): 2435-2450.e34, 2021 06.
Article in English | MEDLINE | ID: covidwho-1116737

ABSTRACT

BACKGROUND & AIMS: Given that gastrointestinal (GI) symptoms are a prominent extrapulmonary manifestation of COVID-19, we investigated intestinal infection with SARS-CoV-2, its effect on pathogenesis, and clinical significance. METHODS: Human intestinal biopsy tissues were obtained from patients with COVID-19 (n = 19) and uninfected control individuals (n = 10) for microscopic examination, cytometry by time of flight analyses, and RNA sequencing. Additionally, disease severity and mortality were examined in patients with and without GI symptoms in 2 large, independent cohorts of hospitalized patients in the United States (N = 634) and Europe (N = 287) using multivariate logistic regressions. RESULTS: COVID-19 case patients and control individuals in the biopsy cohort were comparable for age, sex, rates of hospitalization, and relevant comorbid conditions. SARS-CoV-2 was detected in small intestinal epithelial cells by immunofluorescence staining or electron microscopy in 15 of 17 patients studied. High-dimensional analyses of GI tissues showed low levels of inflammation, including down-regulation of key inflammatory genes including IFNG, CXCL8, CXCL2, and IL1B and reduced frequencies of proinflammatory dendritic cells compared with control individuals. Consistent with these findings, we found a significant reduction in disease severity and mortality in patients presenting with GI symptoms that was independent of sex, age, and comorbid illnesses and despite similar nasopharyngeal SARS-CoV-2 viral loads. Furthermore, there was reduced levels of key inflammatory proteins in circulation in patients with GI symptoms. CONCLUSIONS: These data highlight the absence of a proinflammatory response in the GI tract despite detection of SARS-CoV-2. In parallel, reduced mortality in patients with COVID-19 presenting with GI symptoms was observed. A potential role of the GI tract in attenuating SARS-CoV-2-associated inflammation needs to be further examined.


Subject(s)
COVID-19/virology , Gastrointestinal Diseases/virology , Immunity, Mucosal , Intestinal Mucosa/virology , SARS-CoV-2/pathogenicity , Aged , Aged, 80 and over , COVID-19/diagnosis , COVID-19/immunology , COVID-19/mortality , Case-Control Studies , Cells, Cultured , Cytokines/blood , Female , Gastrointestinal Diseases/diagnosis , Gastrointestinal Diseases/immunology , Gastrointestinal Diseases/mortality , Host-Pathogen Interactions , Humans , Inflammation Mediators/blood , Intestinal Mucosa/immunology , Italy , Male , Middle Aged , New York City , Prognosis , Risk Assessment , Risk Factors , SARS-CoV-2/immunology , Viral Load
8.
Cell ; 183(5): 1325-1339.e21, 2020 11 25.
Article in English | MEDLINE | ID: covidwho-838593

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently identified coronavirus that causes the respiratory disease known as coronavirus disease 2019 (COVID-19). Despite the urgent need, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis. Here, we comprehensively define the interactions between SARS-CoV-2 proteins and human RNAs. NSP16 binds to the mRNA recognition domains of the U1 and U2 splicing RNAs and acts to suppress global mRNA splicing upon SARS-CoV-2 infection. NSP1 binds to 18S ribosomal RNA in the mRNA entry channel of the ribosome and leads to global inhibition of mRNA translation upon infection. Finally, NSP8 and NSP9 bind to the 7SL RNA in the signal recognition particle and interfere with protein trafficking to the cell membrane upon infection. Disruption of each of these essential cellular functions acts to suppress the interferon response to viral infection. Our results uncover a multipronged strategy utilized by SARS-CoV-2 to antagonize essential cellular processes to suppress host defenses.


Subject(s)
COVID-19/metabolism , Host-Pathogen Interactions , Protein Biosynthesis , RNA Splicing , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , A549 Cells , Animals , COVID-19/virology , Chlorocebus aethiops , HEK293 Cells , Humans , Interferons/metabolism , Protein Transport , RNA, Messenger/metabolism , RNA, Ribosomal, 18S/metabolism , RNA, Small Cytoplasmic/chemistry , RNA, Small Cytoplasmic/metabolism , Signal Recognition Particle/chemistry , Signal Recognition Particle/metabolism , Vero Cells , Viral Nonstructural Proteins/chemistry
9.
Clin Transl Immunology ; 9(10): e1189, 2020.
Article in English | MEDLINE | ID: covidwho-837988

ABSTRACT

OBJECTIVES: There is an incomplete understanding of the host humoral immune response to severe acute respiratory syndrome (SARS)-coronavirus (CoV)-2, which underlies COVID-19, during acute infection. Host factors such as age and sex as well as the kinetics and functionality of antibody responses are important factors to consider as vaccine development proceeds. The receptor-binding domain of the CoV spike (RBD-S) protein mediates host cell binding and infection and is a major target for vaccine design to elicit neutralising antibodies. METHODS: We assessed serum anti-SARS-CoV-2 RBD-S IgG, IgM and IgA antibodies by a two-step ELISA and neutralising antibodies in a cross-sectional study of hospitalised COVID-19 patients of varying disease severities. Anti-RBD-S IgG levels were also determined in asymptomatic seropositives. RESULTS: We found equivalent levels of anti-RBD-S antibodies in male and female patients and no age-related deficiencies even out to 93 years of age. The anti-RBD-S response was evident as little as 6 days after onset of symptoms and for at least 5 weeks after symptom onset. Anti-RBD-S IgG, IgM and IgA responses were simultaneously induced within 10 days after onset, with anti-RBD-S IgG sustained over a 5-week period. Anti-RBD-S antibodies strongly correlated with neutralising activity. Lastly, anti-RBD-S IgG responses were higher in symptomatic COVID-19 patients during acute infection compared with asymptomatic seropositive donors. CONCLUSION: Our results suggest that anti-RBD-S IgG reflect functional immune responses to SARS-CoV-2, but do not completely explain age- and sex-related disparities in COVID-19 fatalities.

10.
PLoS Biol ; 18(10): e3000896, 2020 10.
Article in English | MEDLINE | ID: covidwho-810274

ABSTRACT

The ongoing COVID-19 pandemic has created an unprecedented need for rapid diagnostic testing. The World Health Organization (WHO) recommends a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing. The goal of this study was to determine whether SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that omits the RNA extraction step altogether. The direct RT-qPCR approach correctly identified 92% of a reference set of blinded NP samples (n = 155) demonstrated to be positive for SARS-CoV-2 RNA by traditional clinical diagnostic RT-qPCR that included an RNA extraction. Importantly, the direct method had sufficient sensitivity to reliably detect those patients with viral loads that correlate with the presence of infectious virus. Thus, this strategy has the potential to ease supply choke points to substantially expand COVID-19 testing and screening capacity and should be applicable throughout the world.


Subject(s)
Betacoronavirus/genetics , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , RNA, Viral/genetics , Reagent Kits, Diagnostic/standards , Reverse Transcriptase Polymerase Chain Reaction/standards , Betacoronavirus/pathogenicity , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques/standards , Coronavirus Infections/virology , DNA Primers/standards , Humans , Nasopharynx/virology , Pandemics , Pneumonia, Viral/virology , SARS-CoV-2 , Sensitivity and Specificity , United States , Viral Load
11.
EMBO Mol Med ; 12(6): e12634, 2020 06 08.
Article in English | MEDLINE | ID: covidwho-197868

ABSTRACT

Current demand for SARS-CoV-2 testing is straining material resource and labor capacity around the globe. As a result, the public health and clinical community are hindered in their ability to monitor and contain the spread of COVID-19. Despite broad consensus that more testing is needed, pragmatic guidance toward realizing this objective has been limited. This paper addresses this limitation by proposing a novel and geographically agnostic framework (the 4Ps framework) to guide multidisciplinary, scalable, resource-efficient, and achievable efforts toward enhanced testing capacity. The 4Ps (Prioritize, Propagate, Partition, and Provide) are described in terms of specific opportunities to enhance the volume, diversity, characterization, and implementation of SARS-CoV-2 testing to benefit public health. Coordinated deployment of the strategic and tactical recommendations described in this framework has the potential to rapidly expand available testing capacity, improve public health decision-making in response to the COVID-19 pandemic, and/or to be applied in future emergent disease outbreaks.


Subject(s)
Coronavirus Infections/diagnosis , Global Health , Pneumonia, Viral/diagnosis , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , Humans , Pandemics , Pneumonia, Viral/epidemiology , Pneumonia, Viral/virology , RNA, Viral/analysis , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2 , Strategic Planning
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