ABSTRACT
The emergence of SARS-CoV-2 in 2019 led to a severe pandemic situation. Treatment options are limited and the efficacy of vaccines decreases due to mutations in SARS-CoV-2 strains. Therefore, new treatment options are urgently needed, and computational compound screenings are used to predict drugs quickly. One of these screenings revealed farnesyltransferase inhibitors (FTIs) as potential candidates. We demonstrated that the FTIs lonafarnib and tipifarnib have an impact on SARS-CoV-2 Wildtype and the Delta variant. Both FTIs dose-dependently reduced morphological changes and the formation of cytopathic effects in SARS-CoV-2 infected Calu-3 cells.
ABSTRACT
PURPOSE: Omicron is rapidly spreading as a new SARS-CoV-2 variant of concern (VOC). The question whether this new variant has an impact on SARS-CoV-2 rapid antigen test (RAT) performance is of utmost importance. To obtain an initial estimate regarding differences of RATs in detecting omicron and delta, seven commonly used SARS-CoV-2 RATs from different manufacturers were analysed using cell culture supernatants and clinical specimens. METHODS: For this purpose, cell culture-expanded omicron and delta preparations were serially diluted in Dulbecco's modified Eagle's Medium (DMEM) and the Limit of Detection (LoD) for both VOCs was determined. Additionally, clinical specimens stored in viral transport media or saline (n = 51) were investigated to complement in vitro results with cell culture supernatants. Ct values and RNA concentrations were determined via quantitative reverse transcription polymerase chain reaction (RT-qPCR). RESULTS: The in vitro determination of the LoD showed no obvious differences in detection of omicron and delta for the RATs examined. The LoD in this study was at a dilution level of 1:1,000 (corresponding to 3.0-5.6 × 106 RNA copies/mL) for tests I-V and at a dilution level of 1:100 (corresponding to 3.7-4.9 × 107 RNA copies/mL) for tests VI and VII. Based on clinical specimens, no obvious differences were observed between RAT positivity rates when comparing omicron to delta in this study setting. Overall positivity rates varied between manufacturers with 30-81% for omicron and 42-71% for delta. Test VII was only conducted in vitro with cell culture supernatants for feasibility reasons. In the range of Ct < 23, positivity rates were 50-100% for omicron and 67-93% for delta. CONCLUSION: In this study, RATs from various manufacturers were investigated, which displayed no obvious differences in terms of analytical LoD in vitro and RAT positivity rates based on clinical samples comparing the VOCs omicron and delta. However, differences between tests produced by various manufacturers were detected. In terms of clinical samples, a focus of this study was on specimens with high virus concentrations. Further systematic, clinical and laboratory studies utilizing large datasets are urgently needed to confirm reliable performance in terms of sensitivity and specificity for all individual RATs and SARS-CoV-2 variants.
ABSTRACT
BACKGROUND: During the ongoing Covid-19 pandemic caused by the emerging virus SARS-CoV-2, research in the field of coronaviruses has expanded tremendously. The genome of SARS-CoV-2 has rapidly acquired numerous mutations, giving rise to several Variants of Concern (VOCs) with altered epidemiological, immunological, and pathogenic properties. METHODS: As cell culture models are important tools to study viruses, we investigated replication kinetics and infectivity of SARS-CoV-2 in the African Green Monkey-derived Vero E6 kidney cell line and the two human cell lines Caco-2, a colon epithelial carcinoma cell line, and the airway epithelial carcinoma cell line Calu-3. We assessed viral RNA copy numbers and infectivity of viral particles in cell culture supernatants at different time points ranging from 2 to 96 h post-infection. RESULTS: We here describe a systematic comparison of growth kinetics of the five SARS-CoV-2 VOCs Alpha/B.1.1.7, Beta/B.1.351, Gamma/P.1, Delta/B.1.617.2, and Omicron/B.1.1.529 and a non-VOC/B.1.1 strain on three different cell lines to provide profound information on the differential behaviour of VOCs in different cell lines for researchers worldwide. We show distinct differences in viral replication kinetics of the SARS-CoV-2 non-VOC and five VOCs on the three cell culture models Vero E6, Caco-2, and Calu-3. CONCLUSION: This is the first systematic comparison of all SARS-CoV-2 VOCs on three different cell culture models. This data provides support for researchers worldwide in their experimental design for work on SARS-CoV-2. It is recommended to perform virus isolation and propagation on Vero E6 while infection studies or drug screening and antibody-based assays should rather be conducted on the human cell lines Caco-2 and Calu-3.
Subject(s)
COVID-19 , Carcinoma , Caco-2 Cells , Cell Culture Techniques , Chlorocebus aethiops , Humans , Kinetics , Pandemics , SARS-CoV-2/geneticsABSTRACT
Rapid antigen tests (RATs) are an integral part of SARS-CoV-2 containment strategies. As emerging variants of concern (VOCs) displace the initially circulating strains, it is crucial that RATs do not fail to detect these new variants. In this study, four RATs for nasal swab testing were investigated using cultured strains of B.1.1 (non-VOC), B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta). Based on dilution series in cell culture medium and pooled saliva, the limit of detection of these RATs was determined in a laboratory setting. Further investigations on cross-reactivity were conducted using recombinant N-protein from seasonal human coronaviruses (hCoVs). RATs evaluated showed an overall comparable performance with cultured strains of the non-VOC B.1.1 and the VOCs Alpha, Beta, Gamma, and Delta. No cross-reactivity was detected with recombinant N-protein of the hCoV strains HKU1, OC43, NL63, and 229E. A continuous evaluation of SARS-CoV-2 RAT performance is required, especially with regard to evolving mutations. Moreover, cross-reactivity and interference with pathogens and other substances on the test performance of RATs should be consistently investigated to ensure suitability in the context of SARS-CoV-2 containment.
ABSTRACT
SARS-CoV-2 variants of concern (VOC) should not escape molecular surveillance. We investigated if SARS-CoV-2 rapid antigen tests (RATs) could detect B.1.1.7 and B.1.351 VOCs in certain laboratory conditions. Infectious cell culture supernatants containing B.1.1.7, B.1.351 or non-VOC SARS-CoV-2 were respectively diluted both in DMEM and saliva. Dilutions were analysed with Roche, Siemens, Abbott, nal von minden and RapiGEN RATs. While further studies with appropriate real-life clinical samples are warranted, all RATs detected B.1.1.7 and B.1.351, generally comparable to non-VOC strain.
Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19 Serological Testing , Germany , HumansABSTRACT
BACKGROUND: Fast, reliable and easy to handle methods are required to facilitate urgently needed point-of-care testing (POCT) in the current coronavirus pandemic. Life-threatening severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread all over the world, infecting more than 33,500,000 people and killing over 1 million of them as of October 2020. Infected individuals without any symptoms might still transfer the virus to others underlining the extraordinary transmissibility of this new coronavirus. In order to identify early infections effectively, treat patients on time and control disease spreading, rapid, accurate and onsite testing methods are urgently required. RESULTS: Here we report the development of a loop-mediated isothermal amplification (LAMP) based method to detect SARS-CoV-2 genes ORF8 and N directly from pharyngeal swab samples. The established reverse transcription LAMP (RT-LAMP) assay detects SARS-CoV-2 directly from pharyngeal swab samples without previous time-consuming and laborious RNA extraction. The assay is sensitive and highly specific for SARS-CoV-2 detection, showing no cross reactivity when tested on 20 other respiratory pathogens. The assay is 12 times faster and 10 times cheaper than routine reverse transcription real-time polymerase chain reaction, depending on the assay used. CONCLUSION: The fast and easy to handle RT-LAMP assay amplifying specifically the genomic regions ORF8 and N of SARS-CoV-2 is ideally suited for POCT at e.g. railway stations, airports or hospitals. Given the current pandemic situation, rapid, cost efficient and onsite methods like the here presented RT-LAMP assay are urgently needed to contain the viral spread.