Isolation of SARS-CoV-2:
Isolation of SARS-CoV-2 was performed from nasopharyngeal swabs after
obtaining positive RT-qPCR results by inoculation on VeroB4 (no. ACC-33,
DSMZ) in T25 tissue culture flasks (Sarstedt, Germany) for 1 h at 35° C.
After incubation, the sample was removed and Medium199 (Gibco, USA) with
2.5% fetal calf serum (FCS; Gibco, USA) and a mixture of antibiotics
(streptomycin, vancomycin, penicillin, each 1µg/ml) was added. We
monitored virus cultures daily for cytopathic effects and tested for
specific viral RNA every three days. Isolation was considered successful
when cytopathic effect was 80 to 100% in passage 0 as well as passage 1
and/or Cq value in qPCR was lower than 20. Highly positive supernatants
were harvested, centrifuged at 3,400g for 5 minutes and stored at -80° C
in 10% FCS. A further passage of diverse isolates was performed to
obtain the highest possible concentration, which was Cq 14 on average.
All work involving infectious SARS-CoV-2 was carried out in a BSL3
facility, following the institutional guidelines and regulations. Whole
genome sequencing was carried out by Eurofins Genomics, Germany, and a
frequent local genotype (no. 6893) was used for neutralization assay.
In-house immunofluorescence assays (IFA) were assembled as
described elsewhere . In brief, VeroB4 cells were infected with local
SARS-CoV-2 strain 6893 and fixed on IFA slides after three days using
ice-cold acetone-methanol (1:1).
The LIAISON® SARS-CoV-2 S1/S2 IgG (DiaSorin S.p.A., Saluggia,
Italy) (LIAISON) is a CLIA (Chemiluminescent Immunoassay) which detects
IgG antibodies reactive with the spike protein (S1/S2 domain). The assay
was performed on the LIAISON® XL Analyzer according to the
manufacturer’s instructions. The diagnostic sensitivity was 97.9%
(89.1% - 99.6%; Wilson 95% Cl) according to the manufacturer, the
specificity in laboratory routine was 99.0% (96.4% - 99.7%; Wilson
95% Cl).