Bahman Aghcheli - Department of Microbiology, School of Medicine, Golestan University of Medical Sciences, Gorgan, Iran.
Alireza Tahamtan - Department of Microbiology, School of Medicine, Golestan University of Medical Sciences, Gorgan, Iran.
Hadi Razavi Nikoo - Department of Microbiology, School of Medicine, Golestan University of Medical Sciences, Gorgan, Iran.
Zahra Bazi - Department of Biotechnology, Golestan University of Medical Sciences, Gorgan, Iran.
Mohammad Reza Kalani - Senior Scientist, Enterprise-TTM, University of Pittsburgh Medical Center, Pittsburgh, PA.
Abdolvahab Moradi - Department of Microbiology, School of Medicine, Golestan University of Medical Sciences, Gorgan, Iran.

Background: Natural selection such as mutations is known as a constant process for viral fitness and selective adaptation. Understanding the effects of each mutation, especially on structural proteins in the viral life cycle, is important in tracking the viruses behavior. Here, we evaluated the effects of mutations in SARS-CoV-۲ nucleoprotein (N) and spike (S) genes on the protein stability, immunogenicity, and pathogenicity in Iranian COVID-۱۹ patients from Golestan province. Methods: In this study, ۸ SARS-CoV-۲ RNA samples were enrolled from referral hospitals in Golestan province. These samples were confirmed using a real-time RT-PCR assay targeting the SARS-CoV-۲ nucleoprotein (N) and ORF۱ab genes (Pishtazteb, Iran). Next-generation sequencing (NGS) was done on samples and subsequent sequences were retrieved from Global Initiative on Sharing All Influenza Data (GISAID) EpiCoV database. Structural analysis was performed between wild type (Wuhan accession number: NC_۰۴۵۵۱۲.۲) and mutant N and S proteins to evaluate their stability, immunogenicity, and pathogenicity via bioinformatics servers such as Dynamut, Prodigy, IEDB, and software’s (Mega XI and Pymol II.V.II visualizer). Results: Amino acid codon changes in N and S proteins show that mutations could alter the translation efficiency. Normal Mode Analysis (NMA) by Dynamut server shows that stability and flexibility are changed by the mutations of these proteins. Immunogenicity analysis indicates the potential effects of some mutations such as P۶۸۱H, Q۶۷۵R, L۶۹۹I, and D۳L on immune escape. Interaction complex binding energy and affinity are higher in the mutant type compared to the Wuhan wild type, indicating higher pathogenicity. Conclusion: The results indicate that there are some important mutations in N and S genes that affect the virus behavior in the infectivity. Regarding the sample size limitation and various mutations in SARS-CoV-۲ variants, other studies using whole-genome sequencing with larger sample sizes will be required. Therefore, continuous monitoring of the SARS-CoV-۲ genome seems important.

SARS-CoV-۲, COVID-۱۹, mutations, spike, nucleoprotein, Bioinformatics analysis