Isolation and characterization of severe acute respiratory syndrome coronavirus 2 in Turkey

Abstract

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and associated with severe respiratory illness emerged in Wuhan, China, in late 2019. The virus has been able to spread promptly across all continents in the world. The current pandemic has posed a great threat to public health concern and safety. Currently, there are no specific treatments or licensed vaccines available for COVID-19. We isolated SARS-CoV-2 from the nasopharyngeal sample of a patient in Turkey with confirmed COVID-19. We determined that the Vero E6 and MA-104 cell lines are suitable for supporting SARS-CoV-2 that supports viral replication, development of cytopathic effect (CPE) and subsequent cell death. Phylogenetic analyses of the whole genome sequences showed that the hCoV-19/Turkey/ERAGEM-001/2020 strain clustered with the strains primarily from Australia, Canada, England, Iran and Kuwait and that the cases in the nearby clusters were reported to have travel history to Iran and to share the common unique nucleotide substitutions.

Conflict of interest statement

Gen Era Diagnostics Inc. provided support in the form of salaries for authors [CH and CC], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1. Isolation of hCoV-19/Turkey/ERAGEM-001/2020 in Vero E6 cells from the nasopharyngeal sample of a patient with COVID-19 in Turkey in 2020.

Phase contrast microscopy (Leica, DMi1) of culture containing (A) mock-infected Vero E6 cells, (B) Vero E6 cells at 24 h post-infection, (C) Vero E6 cells at 48 h post-infection (D), and Vero E6 cells at 72 h post-infection. Scale bars = 100 μm. (E) RT-PCR amplification of viral RNA from Vero E6 cells infected with hCoV-19/Turkey/ERAGEM-001/2020 (1% gel). (1) Molecular weight marker 1 kb (MW). Target amplicons: (2) SARS-CoV-2 NP1 amplified with 2019-nCoV_N1 forward and 2019-nCoV_N3 reverse primers, 469 bp. (3) SARS-CoV-2 NP2 amplified with 2019-nCoV_N1 forward and 2019-nCoV_N2 reverse primers, 945 bp, and (4) SARS-CoV-2 NP3 amplified with2019-nCoV_N3 forward 2019-nCoV and 2019-nCoV N2 reverse primers, 549 bp, 1% gel. Plaque morphology for SARS-CoV-2 on Vero E6 cells at 72 h post-infection. (F) SARS-CoV-2 stock was plaqued on Vero E6 cells and visualized by crystal violet staining. (G) An uninfected control; the dilution factor of SARS-CoV-2 used for infection was a 10−4.

Fig 2. Passaging of hCoV-19/Turkey/ERAGEM-001/2020 in Vero E6 cells led to an increase in the viral titer.

The viral supernatant was collected at 48 h post-infection in each passage and tested by a focus forming assay (FFA) for titration of the virus at the 2nd to the 4th passages. Fluorescent foci in each well were counted, and the virus titers were calculated and expressed as fluorescent focus units per ml (FFU/ml). A Mann-Whitney U test was performed for comparisons between different passage numbers of virus where p

Fig 3. MA-104 cells supported replication of hCoV-19/Turkey/ERAGEM-001/2020.

Phase contrast microscopy (Leica, DMi1) of cultures containing (A) mock-infected MA-104 cells, (B) MA-104 cells at 24 h post-infection, (C) MA-104 cells at 48 h post-infection, and (D) MA-104 cells at 72 h post-infection. Scale bars = 100 μm.

Fig 4. Susceptibility of different cell lines to infection with hCoV-19/Turkey/ERAGEM-001/2020.

The cell lines were infected with at an MOI of 0.1, washed after adsorption, and the cell monolayers were overlaid with virus medium containing 1% CMC and incubated at 37°C with 5% CO2 for 48 h. After fixation with 10% formaldehyde, the cells were permeabilized with 0.1% Triton X-100 and blocked with 5% skim milk in PBS. The wells were then incubated with a human antibody to the SARS-CoV-2 nucleocapsid protein (1:2500) (GenScript; HC2003) for 1 h in TBST (100 mM Tris–HCl pH 8.0, 1.5 M NaCl, 1% Tween 20) at 37°C, followed by washing three times, after which the cells were incubated for 1 h with goat anti-human IgG conjugated to FITCH (Southern Biotech, USA). The antibody-labeled cells were detected and analyzed by immunofluorescence microscopy (Leica, DFC450C). Scale bars = 200 μm.

Fig 5. Western blot assay to examine SARS-CoV-2 proteins production.

The hCoV-19/Turkey/ERAGEM-001/2020 strain was used to infect the cell lines at an MOI of 0.5. The whole cell lysate samples were collected at 24 h post-infection. The Western blot assay was performed to examine the production of viral proteins using a rabbit polyclonal antibody to the SARS-CoV-2 spike glycoprotein (S) (1/1000) (Abcam; ab272504) and a human antibody to the SARS-CoV-2 nucleocapsid protein (NP) (1:2500) (GenScript; HC2003). The membrane was reacted with the ECL substrate solution (Pierce ECL, USA). The membrane was exposed to an autoradiograph film (KODAK X-OMAT, Sigma Germany), and was developed using a Kodak developer (X-OMAT 1000A, Sigma Germany). The arrows indicate that the bands at approximately 180 kDa (Fig 5A) and 48 kDa (Fig 5B) represent S and NP, respectively.

Fig 6. Multi-step growth kinetics of hCoV-19/Turkey/ERAGEM-001/2020.

Vero E6 and MA-104 cell lines were infected at an MOI of 0.1. (A) The culture supernatants were harvested from at different time points (6, 12, 18, 24, 48 and 72 h) and stored at -80°C. The Vero E6 cells were then inoculated with 10-fold serial dilutions of the samples in triplicate per dilution. The virus titers were determined by a focus forming assay (FFA) at the indicated intervals and were assayed in triplicate. The fluorescent foci in each well were counted, and the virus titers were calculated and expressed as fluorescent focus units per ml (FFU/ml). The standard bars represent standard deviations of the mean of the results. (B) The infected cells (Vero E6 supernatant, upper panel, and MA-104 supernatant, lower panel) were stained with the FITC-labeled goat anti-human IgG at the indicated time points and examined under a fluorescence microscope (Leica, DFC450C); for each group, the representative image out of three replicates is shown. Scale bars = 200 μm.

Fig 7. Phylogenetic analysis of the full-length genomes of SARS-CoV-2.

(A) A phylogenetic tree of known sequences of SARS-CoV-2 (n = 3970) and the location of our cluster. (B) The sub-tree includes the strain of hCoV-19/Turkey/ERAGEM-001/2020, and other strains from Turkey are highlighted in red. The cases that were known to have G1397A, T28688C, and G29742T mutations and an Iran travel history or direct contact with the Iran travelers are highlighted in green.