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This raises concerns of disease control, transmissibility, and severity. Numerous substitutions have been identified to increase viral fitness within the nucleocapsid and nonstructural proteins, in addition to spike mutations. Therefore, we sought to generate infectious viruses carrying only the variant-specific spike mutations in an identical backbone to evaluate the impact of spike and non-spike mutations in the virus life cycle. We used en passant mutagenesis to generate recombinant viruses carrying spike mutations of B.1 and B.1.617.2 variants using SARS-CoV-2- bacterial artificial chromosome (BAC). Neutralization assays using clinical sera yielded comparable results between recombinant viruses and corresponding clinical isolates. Non-spike mutations for both variants neither seemed to effect neutralization efficiencies with monoclonal antibodies nor the response to treatment with inhibitors. However, live-cell imaging and microscopy revealed differences, such as persisting syncytia and pronounced cytopathic effect formation, as well as their progression between BAC-derived viruses and clinical isolates in human lung epithelial cell lines and primary bronchial epithelial cells. Complementary RNA analyses further suggested a potential role of non-spike mutations in infection kinetics.<\/jats:p>","DOI":"10.3390\/v14092017","type":"journal-article","created":{"date-parts":[[2022,9,14]],"date-time":"2022-09-14T00:15:02Z","timestamp":1663114502000},"page":"2017","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Molecular Analyses of Clinical Isolates and Recombinant SARS-CoV-2 Carrying B.1 and B.1.617.2 Spike Mutations Suggest a Potential Role of Non-Spike Mutations in Infection Kinetics"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0010-7417","authenticated-orcid":false,"given":"Andrei","family":"Veleanu","sequence":"first","affiliation":[{"name":"Institute for Medical Virology, University Hospital, Goethe University Frankfurt, D-60596 Frankfurt am Main, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maximilian A.","family":"Kelch","sequence":"additional","affiliation":[{"name":"Institute for Medical Virology, University Hospital, Goethe University Frankfurt, D-60596 Frankfurt am Main, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chengjin","family":"Ye","sequence":"additional","affiliation":[{"name":"Texas Biomedical Research Institute, San Antonio, TX 78227-5302, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Melanie","family":"Flohr","sequence":"additional","affiliation":[{"name":"Institute for Medical Virology, University Hospital, Goethe University Frankfurt, D-60596 Frankfurt am Main, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0904-3376","authenticated-orcid":false,"given":"Alexander","family":"Wilhelm","sequence":"additional","affiliation":[{"name":"Institute for Medical Virology, University Hospital, Goethe University Frankfurt, D-60596 Frankfurt am Main, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5417-9307","authenticated-orcid":false,"given":"Marek","family":"Widera","sequence":"additional","affiliation":[{"name":"Institute for Medical Virology, University Hospital, Goethe University Frankfurt, D-60596 Frankfurt am Main, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7084-0804","authenticated-orcid":false,"given":"Luis","family":"Martinez-Sobrido","sequence":"additional","affiliation":[{"name":"Texas Biomedical Research Institute, San Antonio, TX 78227-5302, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1802-7565","authenticated-orcid":false,"given":"Sandra","family":"Ciesek","sequence":"additional","affiliation":[{"name":"Institute for Medical Virology, University Hospital, Goethe University Frankfurt, D-60596 Frankfurt am Main, Germany"},{"name":"German Centre for Infection Research (DZIF), Partner Site Frankfurt am Main, D-60596 Frankfurt am Main, Germany"},{"name":"Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, Theodor Stern Kai 7, D-60595 Frankfurt am Main, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5399-9263","authenticated-orcid":false,"given":"Tuna","family":"Toptan","sequence":"additional","affiliation":[{"name":"Institute for Medical Virology, University Hospital, Goethe University Frankfurt, D-60596 Frankfurt am Main, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,9,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"262","DOI":"10.1016\/j.virusres.2014.05.026","article-title":"Coronavirus reverse genetic systems: Infectious clones and replicons","volume":"189","author":"Almazan","year":"2014","journal-title":"Virus Res."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"10900","DOI":"10.1128\/JVI.00385-06","article-title":"Construction of a severe acute respiratory syndrome coronavirus infectious cDNA clone and a replicon to study coronavirus RNA synthesis","volume":"80","author":"Almazan","year":"2006","journal-title":"J. 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