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).