Abstract
The development of prophylactic agents against the SARS-CoV-2 virus is a public health priority in the search for new surrogate markers of active virus replication. Early detection markers are needed to follow disease progression and foresee patient negativization. Subgenomic RNA transcripts (with a focus on sgN) were evaluated in oro/nasopharyngeal swabs from COVID-19-affected patients with an analysis of 315 positive samples using qPCR technology. Cut-off Cq values for sgN (Cq < 33.15) and sgE (Cq < 34.06) showed correlations to high viral loads. The specific loss of sgN in home-isolated and hospitalized COVID-19-positive patients indicated negativization of patient condition, 3–7 days from the first swab, respectively. A new detection kit for sgN, gene E, gene ORF1ab, and gene RNAse P was developed recently. In addition, in vitro studies have shown that 2’-O-methyl antisense RNA (related to the sgN sequence) can impair SARS-CoV-2 N protein synthesis, viral replication, and syncytia formation in human cells (i.e., HEK-293T cells overexpressing ACE2) upon infection with VOC Alpha (B.1.1.7)-SARS-CoV-2 variant, defining the use that this procedure might have for future therapeutic actions against SARS-CoV-2.
Highlights
SARS-CoV-2 is the human coronavirus (CoV) responsible for the CoV disease 19 (COVID-19) pandemic
We demonstrate that the new SARS-CoV-2 Viral3 kit can identify ‘true negative’ COVID-19-free people through analysis of an independent cohort of 12 samples (Supplementary Table S2)
We show here that in vitro it is possible to impair this process using 2 -O-methyl antisense RNA oligos, with therapeutic benefits seen through reduction of viral load in SARS-CoV-2-infected cells, further inhibiting syncytia formation and subsequent virus propagation (Figure 5A)
Summary
SARS-CoV-2 is the human coronavirus (CoV) responsible for the CoV disease 19 (COVID-19) pandemic. Human CoVs are members of the Nidovirales order and belong to the Coronaviridae family. Seven species of human CoVs have been described: HCoV-NL63, HCoV-229E, HCoV-OC43, HCoVHKU1, SARS-CoV, MERS-CoV, and SARSCoV-2. Like other CoVs, SARS-CoV-2 is an enveloped virus with a positive-sense, singlestranded RNA genome. SARS-CoV-2 belongs to the genus betacoronavirus, together with SARS-CoV and MERS-CoV (with 80% and 50% identity, respectively) [1]. Coronaviruses, including SARS-CoV-2, have the largest genomes (26–32 kb) among all of the RNA virus families, which are flanked by 5 and 3 untranslated regions. The viral genomic RNA (gRNA) is translated to produce nonstructural proteins from two large open reading frames (ORFs), ORF1a, and ORF1b, via proteolytic cleavage. 15 nonstructural proteins make up the viral replication and transcription complex [3]. Nsp (which harbors RNA-dependent RNA polymerase; RdRp) leads the viral replication and transcription mechanisms by using viral RNA as the template
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