Journal: medRxiv

Article Title: Within-host evolution of SARS-CoV-2 in an immunosuppressed COVID-19 patient: a source of immune escape variants

doi: 10.1101/2021.04.30.21256244

Figure Lengend Snippet: ( a ) Schematic overview of the sequence variations in SARS-CoV-2 genomes detected in early (d14) or late (d105) swab samples and isolated viruses. The heatmap illustrates the positions and the frequency of major variations in the viral genome (cut off 15%). The days of isolation are indicated at the right. The heatmap colors represent the variant frequencies. In ORF7b, L14* indicates a frame shift mutation due to a deletion of two nucleotides. ( b ) Immunofluorescence analysis of SARS-CoV-2 infected cell cultures. VeroE6 cells were infected with one of the two virus isolates (d14 or d105) using a multiplicity of infection (moi) of 0.1 plaque forming units (pfu)/cell. At 8 h post infection, the cells were fixed and stained with SARS-CoV-2 N- and S-specific antibodies (red). In addition, F-actin (white) and nuclear DNA (DAPI, blue) were detected. ( c ) Western blot analysis of viral protein expression. Calu-3 cells were infected with a moi of 0.001 pfu/cell with either of the two virus isolates (d14 or d105). Cells were lysed 8h, 24h, 48h and 72h post-infection and analyzed using N- and S-specific antibodies. Detection of β-actin was used as loading control. (Fig.4 continued on the following page) ( d ) Growth of the two patient isolates in VeroE6 and Calu-3 cells. The cells were infected with either of the two patient isolates (d14 or d105) using a moi of 0.001 pfu/cell. At different time points post infection, cell culture supernatants were collected and viral titers were determined by plaque assay on VeroE6 cells. The log-transformed titers are shown as means ± SD of results from three independent experiments. Significance was determined via two-way ANOVA with a Sidak’s multiple comparison test, **p<0.0021, ***p<0.0002, ns=non significant. (E and F) Late SARS-CoV-2 isolate is attenuated in mice. Weight loss ( e ) and survival ( f ) of 8 to 12 weeks-old K18-hACE2 mice intranasally infected with 200 or 2000 pfu of d14 or d105 viruses. Signs of disease and body weight loss were monitored daily for 14 days. The weight loss is visualized as mean ± SEM. Significance was determined via two-way ANOVA with a Sidak’s multiple comparison test, *p<0.0332, **p<0.0021, ***p<0.0002.

Article Snippet: Filtered throat swab samples were inoculated on African green monkey kidney VeroE6 cells (ATCC CRL-1586) (2 x 10 cells) in 4 ml DMEM with 2% FCS and incubated at 37°C and 5% CO2 for 4 to 6 days until cytopathic effect was visible.

Techniques: Sequencing, Isolation, Variant Assay, Mutagenesis, Immunofluorescence, Infection, Virus, Staining, Western Blot, Expressing, Control, Cell Culture, Plaque Assay, Transformation Assay, Comparison

Journal: medRxiv

Article Title: Within-host evolution of SARS-CoV-2 in an immunosuppressed COVID-19 patient: a source of immune escape variants

doi: 10.1101/2021.04.30.21256244

Figure Lengend Snippet: ( a and b ) Detection of neutralizing activity of immune sera against SARS-CoV-2 variants. 100 pfu of the d14 and d105 isolates were incubated for 60 min at room temperature with serial dilutions of the patient sera. Sera obtained from naïve (– ctrl) or convalescent individuals (+ ctrl) served as negative and positive controls. Virus neutralization was determined by plaque assay on VeroE6 cells. Virus titers are indicated as percentages (mean ±SD) of the titer of the untreated virus inoculum. The dotted lines indicate the cutoff value between positive (<50%) and negative (>50%) neutralization. ( a ) Sera from the immunocompromised patient. The times of blood withdrawal are indicated. ( b ) Convalescent sera from COVID-19 patients suffering from mild, moderate or severe disease or human post vaccination (BNT162b2 mRNA) sera. ( c-e ) Neutralization capacity of SARS-CoV-2 antisera using VSV*ΔG(FLuc) vector pseudotyped with the SARS-CoV-2 spike protein and coding for firefly luciferase. The pseudotyped viruses were incubated with serial dilutions of a COVID-19 convalescent serum prior to inoculation of VeroE6 cells. Pseudotyped virus infection was monitored 16 h post infection by measuring the firefly luciferase activity in the cell lysates. ( c ) Neutralization of VSV*ΔG(FLuc) pseudotyped with the early and late SARS-CoV-2 spike variants (d14 and d105) using serial dilutions of a COVID-19 convalescent serum. ( d and e ) Neutralization of VSV*ΔG(FLuc) pseudotyped with the d14 spike protein containing the individual or combined mutations characteristically found in the late d105 and d140 variants. Immune serum from a convalescent COVID-19 patient ( d ) or a vaccinated person ( e ) were analyzed. The neutralization was determined by calculating the NT 50 via a non-linear regression (variable slope, four parameters). Shown are means ± SD (n=3). Statistics were calculated with a one-way ANOVA (Tukey’s multiple comparison test), ns = non-significant, *p<0.0332, **p<0.0021, ***p<0.0002.

Article Snippet: Filtered throat swab samples were inoculated on African green monkey kidney VeroE6 cells (ATCC CRL-1586) (2 x 10 cells) in 4 ml DMEM with 2% FCS and incubated at 37°C and 5% CO2 for 4 to 6 days until cytopathic effect was visible.

Techniques: Activity Assay, Incubation, Virus, Neutralization, Plaque Assay, Plasmid Preparation, Luciferase, Infection, Comparison

Journal: medRxiv

Article Title: Within-host evolution of SARS-CoV-2 in an immunosuppressed COVID-19 patient: a source of immune escape variants

doi: 10.1101/2021.04.30.21256244

Figure Lengend Snippet: Sera were collected from convalescent K18-hACE2 mice at least 21 days post infection with Muc-IMB-1 (n=5), d14 (n=2) or d105 (n=13) virus isolates. ( a ) Anti-SARS-CoV-2 IgG titers of serially diluted sera (mean ±SD) were determined using virus-infected cells and indirect immunofluorescence analysis (IFA). ( b and c ) Neutralization of d14 and d105 virus isolates by convalescent mouse sera obtained after infection with wild-type SARS-CoV-2, d14 and Muc-IMB-1 (anti-wt sera, n=7) ( b ), or with variant d105 virus isolate (anti-d105 sera, n=13) ( c ). Neutralization capacity was determined by incubating 100 pfu of either virus isolate with serial dilutions of the mouse sera. The mixture was then applied to VeroE6 cells and infected cells were stained with N-specific antibodies. The highest dilution for each individual serum causing 50% neutralization was plotted as neutralizing titer. ( d and e ) Neutralization of B.1.1.7 and B.1.351 variants of concern by mouse convalescent sera was determined as described in panels b and c. 100 pfu of the variant viruses were incubated with dilutions of sera from mice infected with wild-type SARS-CoV-2 (wt) ( d ) or d105 isolate ( e ). Neutralization titers are meant as the highest dilution for each individual serum causing 50% reduction of infectivity. Each serum titer (b - e) is shown as mean out of two independent experiments. Significance was determined via a paired t test with *p<0.0332, **p<0.0021, ***p<0.0002. ( f and g ) Convalescent animals are protected against re-challenge infection. Weight loss ( f ) and survival ( g ) of convalescent K18-hACE2 mice (mean ± SEM), challenged one to four months after the prime infection. Animals primarily infected with d14 and Muc-IMB-1 viruses (pooled wt survivors, n=7), or with d105 virus (n=14) were intranasally challenged with 100,000 pfu of d14 or d105 viruses (2 to 7 mice per group, as indicated). As a control, naïve 8 weeks old K18-hACE2 mice were intranasally infected with 100,000 pfu of d14 or d105 isolate viruses (n=2 per group).

Article Snippet: Filtered throat swab samples were inoculated on African green monkey kidney VeroE6 cells (ATCC CRL-1586) (2 x 10 cells) in 4 ml DMEM with 2% FCS and incubated at 37°C and 5% CO2 for 4 to 6 days until cytopathic effect was visible.

Techniques: Infection, Virus, Immunofluorescence, Neutralization, Variant Assay, Staining, Incubation, Control

Journal: bioRxiv

Article Title: Title: Mechanisms of Host Cell Binding and Neurotropism of Zika Virus

doi: 10.1101/350603

Figure Lengend Snippet: (A) NAGylated or unglycosylated ZVBM peptides from strain PRVABC59 and the type strain MR_766 bound Vero cells significantly (* P <0.05) above scrambled ZVBM sequences from PRVABC59, with or without NAG modification. (B) A shorter peptide representing the NTD of PRVABC59 was unable to bind Vero cells above the full-length ZVBM scrambled control; however, a peptide representing the CTD bound Vero cells at a significantly (** P <0.01) higher level than the NTD or scrambled control. (C) Pretreatment of Vero cells with unglycosylated ZVBM peptide (PRVABC59) significantly (* P <0.05) inhibited ZIKV MR_766 replication relative to untreated controls, whereas the unglycosylated scrambled control peptide was not. Similarly, pretreatment with NAGylated ZVBM peptide (PRVABC59) significantly (** P <0.01) inhibited ZIKV PRVABC59 replication relative to untreated controls, whereas the NAGylated scrambled control peptide was not. (D) Unglycosylated PRVABC59 and MR_766 ZVBM peptides bound MDCK cells significantly (* P <0.05) above unglycosylated or NAGylated scrambled controls; however, the NAGylated PRVABC59 ZVBM peptide was not. (E) Pretreatment of ZIKV strain PRVABC59 with annexin V significantly (** P<0.01) inhibited viral replication on Vero cells relative to untreated controls, whereas replication of ZIKV MR_766 following pretreatment with annexin V was unaffected.

Article Snippet: African Green Monkey Kidney (Vero) cells and Madin-Darby Canine Kidney (MDCK) cells were obtained from the American Type Culture Collection.

Techniques: Modification, Control