Methods
Vietnam established a National Steering Committee on Prevention and
Control of COVID-19 on 28 January 2020, six days after the first cases
of COVID-19 were identified in the country (2). Subsequent guidelines
issued by the Steering Committee on 19 February 2020 called for
collection nasopharyngeal and oropharyngeal (NP/OP) swabs from suspected
cases and close contacts (F1) of confirmed cases (F0) and these
guidelines were harmonized with those from WHO in March 2020 (7).
Confirmed cases of COVID-19 underwent repeated sampling every three days
during hospitalization until they recovered clinically and had at least
three negative results by real-time RT-PCR for SARS-CoV-2.
Realtime RT-PCR testing : NP/OP swabs were placed into viral
transport medium (VTM) and maintained at 4 °C during transport to the
NIC at NIHE during 24-48 hours (8). RNA was isolated from NP/OP using
the viral RNA extraction kit (Qiagen, Hilden, Germany) according to the
manufacturer’s instructions in biosafety level 3 (BSL3) containment
laboratories. Realtime RT-PCR was conducted using SuperScript III One
-Step RT-PCR system with Platinum Taq High Fidelity DNA Polymerase
(Invitrogen, Carlsbad, CA-USA) with target of E, RdRp and N genes
following WHO recommendations. We defined confirmed cases as those with
cycle threshold (Ct) values less than 37 for at least two target genes
(E and RdRp or E and N) of SARS-CoV-2 (9).
Viral isolation: Vero E6 cells were maintained in Eagle’s minimal
essential medium (EMEM) containing 5% (v/v) newborn calf serum (NCS);
100ul of real-time RT-PCR positive samples were inoculated onto Vero E6
cells and incubated at 37 °C. Viral growth was monitored by daily
observation of cytopathic effect. All experiments with SARS-CoV-2
viruses were performed in biosafety level 3 (BSL3) containment
laboratories (10).