SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected, hospitalized COVID-19 patients

Abstract

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.

Functional importance of the D614G mutation in the SARS-CoV-2 spike protein

“…SARS-CoV-2 acquired a furin cleavage site not found in SARS-CoV-1 or any other known sarbecovirus. Studies have shown that acquisition of this furin cleavage site could be beneficial to the virus, because mutations at this site decrease viral infectivity [ 4 , 14 , 21 , 58 ], but in other contexts or other mutations of the furin cleavage site, infectivity is increased [ 4 , 35 , 58 , 59 ]. We currently do not know whether viruses without the furin cleavage site would replicate more efficiently in vivo .…”

“…One caveat is that visualization of isogenic D614 and G614 variant SARS-CoV-2 by scanning EM and transmission EM did not reveal significant differences in spike density [ 48 ], which cannot be explained by the masking effect of contaminating S protein. Because many studies have observed deletion of the furin cleavage site in live SARS-CoV-2 in few as two to three passages in Vero cells [ [58] , [59] , [60] , [61] ], care must be taken to ensure that studies of live viruses are not impacted by artifacts of this adaptation. In line with our own observations on VLPs and MLV PVs, Turonova et al visualized live SARS-CoV-2 G614 virions by cryo-EM and observed much greater spike density than was reported in prior studies with D614 viruses [ 61 , 62 ].…”

SARS-CoV-2 entry into human airway organoids is serine protease-mediated and facilitated by the multibasic cleavage site

“…While mutations in the SARS-CoV-2 MBCS decreased airway cell infectivity, they increased infectivity on VeroE6 cells. Several groups have reported mutations or deletions in or around the SARS-CoV-2 MBCS that arise in cell culture on VeroE6 cells (Klimstra et al, 2020;Lau et al, 2020;Ogando et al, 2020), indicating that the lack of a MBCS creates a selective advantage in cell culture on VeroE6 cells. The mechanism behind this remains unknown.…”

Variable Performance in 6 Commercial SARS-CoV-2 Antibody Assays May Affect Convalescent Plasma and Seroprevalence Screening

“… 3-10 In addition, neutralizing antibodies to both viral spike and nucleocapsid proteins have been found in patients. 5 , 10 , 11 The longevity of this antibody response is also still under investigation. Preliminary work found that the half-lives of antibodies recognizing the nucleocapsid, spike protein, and receptor binding domain of the spike protein are 52, 81, and 83 days, respectively, with an estimated time to negativity of 50% of the seropositive population of 195, 532, and 260 days, respectively.…”