 Nimotrelvir, an oral antiviral agent that targets a SARS-CoV-2 main protease, 3CL-PRO, is clinically useful against infection with SARS-CoV-2 including its Armachron variants. Since most Armachron subvariants have reduced sensitivity to many monoclonal antibody therapies, potential SARS-CoV-2 resistance to Nimotrelvir is a major public health concern. Several amino acid substitutions have been identified as being responsible for reduced susceptibility to Nimotrelvir. Among them, we selected L50FE-166V and L50FE-166AL-167F in the 3CL-PRO because these combinations of substitutions are unlikely to affect virus fitness. We prepared and characterized Delta variants possessing NSP5L50FE-166V and NSP5L50FE-166AL-167F. Both mutant viruses showed decreased susceptibility to Nimotrelvir and their growth in Vero E60M-P-RSS2 cells. This article was authored by Moki Kizo, Yui Furusawa, Ryota Yuraki, and others. We are article.tv, links in the description below.