PMID- 32821033 OWN - NLM STAT- MEDLINE DCOM- 20201221 LR - 20240211 IS - 1465-2099 (Electronic) IS - 0022-1317 (Print) IS - 0022-1317 (Linking) VI - 101 IP - 11 DP - 2020 Nov TI - SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected hospitalized COVID-19 patients. PG - 1156-1169 LID - 10.1099/jgv.0.001481 [doi] AB - Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), emerged at the end of 2019 and by mid-June 2020 the virus had 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 severe acute respiratory syndrome coronavirus (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 that 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 titres 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, Chengun 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 - Division of Pediatric Infectious Disease, University of Pittsburgh, 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 - Journal Article PT - Research Support, N.I.H., Extramural DEP - 20200821 PL - England TA - J Gen Virol JT - The Journal of general virology JID - 0077340 RN - 0 (Antibodies, Neutralizing) RN - 0 (Immunoglobulin A) RN - 0 (Immunoglobulin G) RN - 0 (RNA, Viral) RN - EC 3.4.21.75 (Furin) SB - IM UOF - bioRxiv. 2020 Jun 22;:. PMID: 32607507 MH - Adaptation, Physiological MH - Animals MH - Antibodies, Neutralizing/immunology MH - COVID-19/epidemiology/immunology/*metabolism/*virology MH - Chlorocebus aethiops MH - Furin/immunology/*metabolism MH - Genetic Variation MH - Hospitalization MH - *Host-Pathogen Interactions/immunology MH - Humans MH - Immunoglobulin A/immunology MH - Immunoglobulin G/immunology MH - Neutralization Tests MH - Proteolysis MH - RNA, Viral MH - SARS-CoV-2/*physiology MH - Sequence Analysis, RNA MH - Vero Cells MH - Viral Load MH - *Virus Replication PMC - PMC7879561 OTO - NOTNLM OT - COVID-19 OT - SARS-CoV-2 OT - adaptation OT - clinical isolates OT - furin cleavage OT - neutralization COIS- The authors declare that there are no conflicts of interest. EDAT- 2020/08/22 06:00 MHDA- 2020/12/22 06:00 PMCR- 2020/08/21 CRDT- 2020/08/22 06:00 PHST- 2020/08/22 06:00 [pubmed] PHST- 2020/12/22 06:00 [medline] PHST- 2020/08/22 06:00 [entrez] PHST- 2020/08/21 00:00 [pmc-release] AID - 001481 [pii] AID - 10.1099/jgv.0.001481 [doi] PST - ppublish SO - J Gen Virol. 2020 Nov;101(11):1156-1169. doi: 10.1099/jgv.0.001481. Epub 2020 Aug 21.