Evaluation of Rapid Lateral-Flow Tests Directed against the SARS-CoV-2 Nucleoprotein Using Viral Suspensions Belonging to Different Lineages of SARS-CoV-2.

Within the successive waves that occurred during the SARS-CoV-2 pandemic, recommendations arose to test symptomatic and contact subjects by using rapid antigen devices directed against the viral nucleocapsid protein with the aim to isolate contagious patients without delay. The objective of this study was to evaluate the ability of four rapid lateral-flow tests (RLFT) that were commercially available on the French market in 2022 to recognize various strains of SARS-CoV-2. Series of five-fold dilutions of seven viral suspensions belonging to different lineages of SARS-CoV-2 (19A, 20A, Alpha, Beta, Gamma, Delta and Omicron) were used to evaluate the analytical sensitivity of four commercially available RLFTs (manufacturers: Abbott, AAZ, Becton-Dickinson and Biospeedia). Cell culture and quantitative RT-PCR were used as references. Excellent correlations were observed for each lineage strain between the viral titer obtained via cell culture and the number of RNA copies measured by quantitative RT-PCR. Although the four tests were able to recognize all the tested variants, significant differences in terms of sensitivity were observed between the four RLFTs. Despite the limitation represented by the small number of devices and clinical isolates that were tested, this study contributed by rapidly comparing the sensitivity of SARS-CoV-2 RLFTs in the Omicron era.

Accumulation of mutations in antibody and CD8 T cell epitopes in a B cell depleted lymphoma patient with chronic SARS-CoV-2 infection.

Antibodies against the spike protein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can drive adaptive evolution in immunocompromised patients with chronic infection. Here we longitudinally analyze SARS-CoV-2 sequences in a B cell-depleted, lymphoma patient with chronic, ultimately fatal infection, and identify three mutations in the spike protein that dampen convalescent plasma-mediated neutralization of SARS-CoV-2. Additionally, four mutations emerge in non-spike regions encoding three CD8 T cell epitopes, including one nucleoprotein epitope affected by two mutations. Recognition of each mutant peptide by CD8 T cells from convalescent donors is reduced compared to its ancestral peptide, with additive effects resulting from double mutations. Querying public SARS-CoV-2 sequences shows that these mutations have independently emerged as homoplasies in circulating lineages. Our data thus suggest that potential impacts of CD8 T cells on SARS-CoV-2 mutations, at least in those with humoral immunodeficiency, warrant further investigation to inform on vaccine design.

Comparison of the SARS-CoV-2 BD Veritor Nasal Antigen Test with Nasopharyngeal Reverse Transcription-PCR in Symptomatic Patients.

This study evaluated the BD Veritor system for rapid detection of SARS-CoV-2, an immunochromatographic point-of-care test, by comparing it with a standard reverse transcription-PCR (RT-PCR) methodology using samples from symptomatic patients. Samples from 146 symptomatic and 2 asymptomatic patients between the 1st and the 40th day of infection were evaluated. The nasopharyngeal and/or oropharyngeal swabs were inserted in a tube containing 0.9% saline solution and stored at refrigerator temperature until the moment of use. The samples were first tested with the Xpert Xpress SARS-CoV-2 (GeneXpert) kit (RT-PCR method), and the cycle thresholds (CTs) for the E and N2 genes encoding the SARS-CoV-2 envelope and nucleoprotein, respectively, were established. Subsequently, the same samples were tested using the Veritor rapid test. We analyzed the CTs of the N2 gene, which is detected in both methodologies, and observed sensitivities of 100%, 98.8%, 89.6%, and 82.7% for the CTs of <25, <27, and <30 and all the CTs, respectively. The greatest sensitivity was observed when we performed the test on patients within 5 days of symptom onset. The BD Veritor system's workflow is simple and fast, taking approximately 16 min from sample preparation to obtaining the test result. In addition to its satisfactory sensitivity, with results that correlate with those of the RT-PCR, the BD Veritor analyzer instrument reduces the subjectivity of unaided visual readings and consequent potential variation in result interpretation. Therefore, our results showed that the BD Veritor diagnostic test can provide a rapid and accurate diagnosis for SARS-CoV-2. IMPORTANCE This study provides important and useful information, especially for diagnostic laboratories, since the results show that the BD Veritor system can provide a fast and safe point-of-care antigen diagnostic test for rapid detection of COVID-19 that has high sensitivity, reproducibility, and accuracy.

SARS-CoV-2-Seronegative Subjects Target CTL Epitopes in the SARS-CoV-2 Nucleoprotein Cross-Reactive to Common Cold Coronaviruses.

The beta-coronavirus SARS-CoV-2 induces severe disease (COVID-19) mainly in elderly persons with risk factors, whereas the majority of patients experience a mild course of infection. As the circulating common cold coronaviruses OC43 and HKU1 share some homologous sequences with SARS-CoV-2, beta-coronavirus cross-reactive T-cell responses could influence the susceptibility to SARS-CoV-2 infection and the course of COVID-19. To investigate the role of beta-coronavirus cross-reactive T-cells, we analyzed the T-cell response against a 15 amino acid long peptide (SCoV-DP15: DLSPRWYFYYLGTGP) from the SARS-CoV-2 nucleoprotein sequence with a high homology to the corresponding sequence (QLLPRWYFYYLGTGP) in OC43 and HKU1. SCoV-DP15-specific T-cells were detected in 4 out of 23 (17.4%) SARS-CoV-2-seronegative healthy donors. As HIV-1 infection is a potential risk factor for COVID-19, we also studied a cohort of HIV-1-infected patients on antiretroviral therapy. 44 out of these 116 HIV-1-infected patients (37.9%) showed a specific recognition of the SCoV-DP15 peptide or of shorter peptides within SCoV-DP15 by CD4+ T-cells and/or by CD8+ T-cells. We could define several new cross-reactive HLA-I-restricted epitopes in the SARS-CoV-2 nucleoprotein such as SPRWYFYYL (HLA-B*07, HLA-B*35), DLSPRWYFYY (HLA-A*02), LSPRWYFYY (HLA-A*29), WYFYYLGTGP and WYFYYLGT. Epitope specific CD8+ T-cell lines recognized corresponding epitopes within OC43 and HKU1 to a similar degree or even at lower peptide concentrations suggesting that they were induced by infection with OC43 or HKU1. Our results confirm that SARS-CoV-2-seronegative subjects can target SARS-CoV-2 not only by beta-coronavirus cross-reactive CD4+ T-cells but also by cross-reactive CD8+ cytotoxic T-cells (CTL). The delineation of cross-reactive T-cell epitopes contributes to an efficient epitope-specific immunomonitoring of SARS-CoV-2-specific T-cells. Further prospective studies are needed to prove a protective role of cross-reactive T-cells and their restricting HLA alleles for control of SARS-CoV-2 infection. The frequent observation of SARS-CoV-2-reactive T-cells in HIV-1-infected subjects could be a reason that treated HIV-1 infection does not seem to be a strong risk factor for the development of severe COVID-19.

SARS CoV-2 Nucleoprotein Enhances the Infectivity of Lentiviral Spike Particles.

The establishment of SARS CoV-2 spike-pseudotyped lentiviral (LV) systems has enabled the rapid identification of entry inhibitors and neutralizing agents, alongside allowing for the study of this emerging pathogen in BSL-2 level facilities. While such frameworks recapitulate the cellular entry process in ACE2+ cells, they are largely unable to factor in supplemental contributions by other SARS CoV-2 genes. To address this, we performed an unbiased ORF screen and identified the nucleoprotein (N) as a potent enhancer of spike-pseudotyped LV particle infectivity. We further demonstrate that the spike protein is better enriched in virions when the particles are produced in the presence of N protein. This enrichment of spike renders LV particles more infectious as well as less vulnerable to the neutralizing effects of a human IgG-Fc fused ACE2 microbody. Importantly, this improvement in infectivity is observed with both wild-type spike protein as well as the D614G mutant. Our results hold important implications for the design and interpretation of similar LV pseudotyping-based studies.

SARS-CoV-2 samples may escape detection because of a single point mutation in the N gene.

We found that a single nucleotide polymorphism (SNP) in the nucleoprotein gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from a patient interfered with detection in a widely used commercial assay. Some 0.2% of the isolates in the EpiCoV database contain this SNP. Although SARS-CoV-2 was still detected by the other probe in the assay, this underlines the necessity of targeting two independent essential regions of a pathogen for reliable detection.

Ancestral origin, antigenic resemblance and epidemiological insights of novel coronavirus (SARS-CoV-2): Global burden and Bangladesh perspective.

SARS-CoV-2, a new coronavirus strain responsible for COVID-19, has emerged in Wuhan City, China, and continuing its global pandemic nature. The availability of the complete gene sequences of the virus helps to know about the origin and molecular characteristics of this virus. In the present study, we performed bioinformatic analysis of the available gene sequence data of SARS-CoV-2 for the understanding of evolution and molecular characteristics and immunogenic resemblance of the circulating viruses. Phylogenetic analysis was performed for four types of representative viral proteins (spike, membrane, envelope and nucleoprotein) of SARS-CoV-2, HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HKU1, MERS-CoV, HKU4, HKU5 and BufCoV-HKU26. The findings demonstrated that SARS-CoV-2 exhibited convergent evolutionary relation with previously reported SARS-CoV. It was also depicted that SARS-CoV-2 proteins were highly similar and identical to SARS-CoV proteins, though proteins from other coronaviruses showed a lower level of resemblance. The cross-checked conservancy analysis of SARS-CoV-2 antigenic epitopes showed significant conservancy with antigenic epitopes derived from SARS-CoV. Descriptive epidemiological analysis on several epidemiological indices was performed on available epidemiological outbreak information from several open databases on COVID-19 (SARS-CoV-2). Satellite-derived imaging data have been employed to understand the role of temperature in the environmental persistence of the virus. Findings of the descriptive analysis were used to describe the global impact of newly emerged SARS-CoV-2, and the risk of an epidemic in Bangladesh.