Implementation of an in-house real-time reverse transcription-PCR assay for the rapid detection of the SARS-CoV-2 Marseille-4 variant (preprint)

IntroductionThe SARS-CoV-2 pandemic has been associated with the occurrence since summer 2020 of several viral variants that overlapped or succeeded each other in time. Those of current concern harbor mutations within the spike receptor binding domain (RBD) that may be associated with viral escape to immune responses. In our geographical area a viral variant we named Marseille-4 harbors a S477N substitution in this RBD. Materials and methodsWe aimed to implement an in-house one-step real-time reverse transcription-PCR (qPCR) assay with a hydrolysis probe that specifically detects the SARS-CoV-2 Marseille-4 variant. ResultsAll 6 cDNA samples from Marseille-4 variant strains identified in our institute by genome next-generation sequencing (NGS) tested positive using our Marseille-4 specific qPCR, whereas all 32 cDNA samples from other variants tested negative. In addition, 39/42 (93%) respiratory samples identified by NGS as containing a Marseille-4 variant strain and 0/26 samples identified as containing non-Marseille-4 variant strains were positive. Finally, 1,585/2,889 patients SARS-CoV-2-diagnosed in our institute, 10/277 (3.6%) respiratory samples collected in Algeria, and none of 207 respiratory samples collected in Senegal, Morocco, or Lebanon tested positive using our Marseille-4 specific qPCR. DiscussionOur in-house qPCR system was found reliable to detect specifically the Marseille-4 variant and allowed estimating it is involved in more than half of our SARS-CoV-2 diagnoses since December 2020. Such approach allows the real-time surveillance of SARS-CoV-2 variants, which is warranted to monitor and assess their epidemiological and clinical characterics based on comprehensive sets of data.

Introduction into the Marseille geographical area of a mild SARS-CoV-2 variant originating from sub-Saharan Africa (preprint)

BACKGROUNDIn Marseille, France, the COVID-19 incidence evolved unusually with several successive epidemic episodes. The second outbreak started in July, was associated with North Africa, and involved travelers and an outbreak on passenger ships. This suggested the involvement of a new viral variant. METHODSWe sequenced the genomes from 916 SARS-CoV-2 strains from COVID-19 patients in our institute. The patients demographic and clinical features were compared according to the infecting viral variant. RESULTSFrom June 26th to August 14th, we identified a new viral variant (Marseille-1). Based on genome sequences (n=89) or specific qPCR (n=53), 142 patients infected with this variant were detected. It is characterized by a combination of 10 mutations located in the nsp2, nsp3, nsp12, S, ORF3a, ORF8 and N/ORF14 genes. We identified Senegal and Gambia, where the virus had been transferred from China and Europe in February-April as the sources of the Marseille-1 variant, which then most likely reached Marseille through Maghreb when French borders reopened. In France, this variant apparently remained almost limited to Marseille. In addition, it was significantly associated with a milder disease compared to clade 20A ancestor strains. CONCLUSIONOur results demonstrate that SARS-CoV-2 can genetically diversify rapidly, its variants can diffuse internationally and cause successive outbreaks.

Computational Approach Revealed Potential Affinity of Antiasthmatics Against Receptor Binding Domain of 2019n-Cov Spike Glycoprotein (preprint)

The novel COVID-19 pandemic is now a health threat, with a deep-felt impact worldwide. The new coronavirus 2019 (2019 n-Cov) binds to host human receptors through Receptor Binding Domain RBD of Spike glycoprotein (S), making it a prominent drug target. The present study aims to identify new potential hits that can inhibit the S protein using in silico approaches. Several natural and synthetics compounds (antiasthmatics, Antiviral, Antimalarial, Antibacterial, Anti-Inflammatory, cyclic peptide, and cyclic bis) were screened by molecular docking using AutoDock Vina. Additionally, we tested calcitriol and three known drugs (Azithromycin, HydroxyChloroquine, and Chloroquine ) against the spike protein to found if they have any direct interaction. Our finding consists of 4 potential synthetic compounds from PubChem database, known for their antiasthmatic effects, that show highly binding energies each (-8.6 kcal/mol, 7.7kcal/mol, -7.2 kcal/mol and -7.0 kcal/mol). Another 5 natural compounds from the South African natural sources database (SANCDB) that bind to RBD of Spike with significant energy each: (Marchantin C with -7.3 kcal/mol, Riccardin C with -7.0 kcal/mol, Digitoxigenin-glucoside with -6.9 kcal/mol, D-Friedoolean-14-en-oic acid with -6.8 kcal/mol and, Spongotine A with -6.7 kcal/mol). The FaF-Drugs server was used to evaluate the drug-like properties of the identified compounds. Additionally, Calcitriol, Azithromycin, and HydroxyChloroquine have an appreciable binding affinity to 2019-nCoV S, suggesting a possible mechanism of action. Using in silico approaches like molecular docking and pharmacokinetic properties, we showed new potential inhibitors. Our findings need further analysis, and chemical design for more effective derivatives of these compounds speculated to disrupt the viral recognition of host receptors.