Association of SARS-CoV-2 Nucleocapsid Protein Mutations with Patient Demographic and Clinical Characteristics during the Delta and Omicron Waves.

SARS-CoV-2 genomic mutations outside the spike protein that may increase transmissibility and disease severity have not been well characterized. This study identified mutations in the nucleocapsid protein and their possible association with patient characteristics. We analyzed 695 samples from patients with confirmed COVID-19 in Saudi Arabia between 1 April 2021, and 30 April 2022. Nucleocapsid protein mutations were identified through whole genome sequencing. 𝜒2 tests and t tests assessed associations between mutations and patient characteristics. Logistic regression estimated the risk of intensive care unit (ICU) admission or death. Of the 60 mutations identified, R203K was the most common, followed by G204R, P13L, E31del, R32del, and S33del. These mutations were associated with reduced risk of ICU admission. P13L, E31del, R32del, and S33del were also associated with reduced risk of death. By contrast, D63G, R203M, and D377Y were associated with increased risk of ICU admission. Most mutations were detected in the SR-rich region, which was associated with low risk of death. The C-tail and central linker regions were associated with increased risk of ICU admission, whereas the N-arm region was associated with reduced ICU admission risk. Consequently, mutations in the N protein must be observed, as they may exacerbate viral infection and disease severity. Additional research is needed to validate the mutations' associations with clinical outcomes.

SARS-CoV-2 spike gene Sanger sequencing methodology to identify variants of concern.

The global demand for rapid identification of circulating SARS-CoV-2 variants of concern has led to a shortage of commercial kits. Therefore, this study aimed to develop and validate a rapid, cost-efficient genome sequencing protocol to identify circulating SARS-CoV-2 (variants of concern). Sets of primers flanking the SARS-CoV-2 spike gene were designed, verified and then validated using 282 nasopharyngeal positive samples for SARS-CoV-2. Protocol specificity was confirmed by comparing these results with SARS-CoV-2 whole-genome sequencing of the same samples. Out of 282 samples, 123 contained the alpha variant, 78 beta and 13 delta, which were indicted using in-house primers and next-generation sequencing; the numbers of variants found were 100% identical to the reference genome. This protocol is easily adaptable for detection of emerging variants during the pandemic.