PMID- 32607507 OWN - NLM STAT- PubMed-not-MEDLINE LR - 20210110 DP - 2020 Jun 22 TI - SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected, hospitalized COVID-19 patients. LID - 2020.06.19.154930 [pii] LID - 10.1101/2020.06.19.154930 [doi] AB - SARS-CoV-2, the causative agent of COVID-19, emerged at the end of 2019 and by mid-June 2020, the virus has spread to at least 215 countries, caused more than 8,000,000 confirmed infections and over 450,000 deaths, and overwhelmed healthcare systems worldwide. Like SARS-CoV, which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low passage (P) strains of SARS-CoV-2 (Wash1: P4 and Munich: P1) were cultured twice in Vero-E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1: P6 and minor variants in the Munich: P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero-E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies we investigated the development of neutralizing antibody titers in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies. FAU - Klimstra, William B AU - Klimstra WB AD - Center for Vaccine Research, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. AD - Department of Microbiology and Molecular Genetics, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. AD - Department of Immunology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. FAU - Tilston-Lunel, Natasha L AU - Tilston-Lunel NL AD - Center for Vaccine Research, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. AD - Department of Microbiology and Molecular Genetics, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. FAU - Nambulli, Sham AU - Nambulli S AD - Center for Vaccine Research, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. AD - Department of Microbiology and Molecular Genetics, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. FAU - Boslett, James AU - Boslett J AD - Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA. FAU - McMillen, Cynthia M AU - McMillen CM AD - Center for Vaccine Research, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. FAU - Gilliland, Theron AU - Gilliland T AD - Center for Vaccine Research, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. FAU - Dunn, Matthew D AU - Dunn MD AD - Center for Vaccine Research, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. FAU - Sun, Chengqun AU - Sun C AD - Center for Vaccine Research, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. FAU - Wheeler, Sarah E AU - Wheeler SE AD - Department of Pathology, University of Pittsburgh School of Public Health, Pittsburgh, Pennsylvania, USA. FAU - Wells, Alan AU - Wells A AD - Department of Pathology, University of Pittsburgh School of Public Health, Pittsburgh, Pennsylvania, USA. FAU - Hartman, Amy L AU - Hartman AL AD - Center for Vaccine Research, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. AD - Department of Infectious Diseases and Microbiology, University of Pittsburgh School of Public Health, Pittsburgh, Pennsylvania, USA. FAU - McElroy, Anita K AU - McElroy AK AD - Center for Vaccine Research, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. AD - University of Pittsburgh, Division of Pediatric Infectious Disease, Pittsburgh, Pennsylvania, USA. FAU - Reed, Douglas S AU - Reed DS AD - Center for Vaccine Research, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. AD - Department of Immunology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. FAU - Rennick, Linda J AU - Rennick LJ AD - Center for Vaccine Research, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. AD - Department of Microbiology and Molecular Genetics, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. FAU - Duprex, W Paul AU - Duprex WP AD - Center for Vaccine Research, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. AD - Department of Microbiology and Molecular Genetics, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. LA - eng GR - T32 AI060525/AI/NIAID NIH HHS/United States GR - UL1 TR001857/TR/NCATS NIH HHS/United States PT - Preprint DEP - 20200622 PL - United States TA - bioRxiv JT - bioRxiv : the preprint server for biology JID - 101680187 UIN - J Gen Virol. 2020 Aug 21;:. PMID: 32821033 PMC - PMC7325173 COIS- Conflicts of interest The authors declare that there are no conflicts of interest. EDAT- 2020/07/02 06:00 MHDA- 2020/07/02 06:01 PMCR- 2020/06/30 CRDT- 2020/07/02 06:00 PHST- 2020/07/02 06:00 [entrez] PHST- 2020/07/02 06:00 [pubmed] PHST- 2020/07/02 06:01 [medline] PHST- 2020/06/30 00:00 [pmc-release] AID - 2020.06.19.154930 [pii] AID - 10.1101/2020.06.19.154930 [doi] PST - epublish SO - bioRxiv [Preprint]. 2020 Jun 22:2020.06.19.154930. doi: 10.1101/2020.06.19.154930.