Characterization of SARS-CoV-2 East Java isolate, Indonesia

Subject characteristics

Subject data was retrieved from Dr Soetomo General Hospital medical records. The first subject, (#35 subject), was a local transmission case of a 49-year-old man with a normal body mass index (22.9 kg/m²), who was discharged on the 15th day of hospital care. The second subject, (#6 subject), was a local transmission case of a 38-year-old woman with an overweight body mass index (25.2 kg/m²) and who rapidly tested positive for IgG with IgM antibodies. Subject #6 was admitted to inpatient care and deteriorated until being declared brain dead after six days of hospitalization due to respiratory failure. The third subject, (#11 subject), was a local transmission case of a 64-year-old woman with a normal body mass index (19.5 kg/m²) and a history of breast cancer, and was in remission. Subject #11 was discharged after 26 days of hospitalization.

Specimen collection

There were 54 patients suspected with SARS-COV-2 infection and of these 27 patients were diagnosed COVID-19 positive during March to June 2020. A SARS-CoV-2 sample source was retrieved from COVID-19 confirmed inpatients in the Dr Soetomo General Hospital and three isolates originating from three individual subjects from Surabaya, Indonesia were obtained. Each subject was asked for informed consent. A nasopharyngeal swab was performed to obtain a SARS-CoV-2 sample using a sterile cotton swab. The sample cotton swab was then submerged in virus transport medium (VTM) containing a sterile filtered solution of Minimum Essential Media (MEM) (Gibco, USA), 1x penicillin-streptomycin (Gibco, USA), and 1x amphotericin B (Gibco, USA) as reported before.²⁴ The sample was then immediately transferred, in a 4°C cool box containing an icepack, to the Research Center for Vaccine Technology and Development, Institute of Tropical Diseases Laboratory, Biosafety Level-3 (BSL-3) facility in Universitas Airlangga, Surabaya, East Java, Indonesia.²⁵

SARS-CoV-2 isolation

There were only three samples from subjects #6, #11, #35 with good growth characteristics that provided isolates. Serial blind passage and cytopathic effect observation in the Vero E6 cell line were performed for virus isolation. The virus isolation that was performed in the Vero E6 cell line (ATCC, USA) was seeded on a T25 flask (Corning, USA) with a 2 × 10⁶ cell count and cultured with a MEM (Gibco, USA), supplemented with 10% Fetal Bovine Serum (FBS) (Gibco, USA) until it reached 80% confluency (37°C and 5% CO₂), (Figure 1), by introducing a 1 ml sample of VTM to the Vero E6 cell line culture for a one-hour incubation. After incubation, 4 ml of fresh MEM (Gibco, USA), supplemented with 10% FBS (Gibco, USA) were added. The cell was labeled as inoculation culture and observed daily through a phase contrast inverted microscope TMS (Nikon, Japan). After three days of inoculation, blind passage was performed to a fresh monolayer Vero E6 cell culture by introducing a 1 ml supernatant medium from infected cells with a one-hour incubation before adding 4 ml of fresh MEM supplemented with 10% FBS medium. Viral growth was analyzed using cytopathogenic effect (CPE) and plaque forming unit (PFU) assay. TCID50 evaluation of #35 isolate through plaque forming assay and Reed and Muench method was as follows: log10 50% end point dilution = log10 of dilution showing a mortality next above 50% - (difference of logarithms × logarithm of dilution factor). Generally, the following formula is used to calculate “difference of logarithms” (difference of logarithms is also known as “proportionate distance” or “interpolated value”): Difference of logarithms = [(mortality at dilution next above 50%)-50%] / [(mortality next above 50%) - (mortality next below 50%)].²⁶

Figure 1.

(A) Cytopathic effect of infected Vero E6 cell in third blind passage on the eighth day post passage (40× magnification); (B) Cytopathic effect of infected Vero E6 cell in 15th blind passage on second day post passage (40× magnification); (C) Uninfected Vero E6 cell in 40× magnification; (D) Plaque-Forming Unit (PFU) evaluation (Nikon TMS inverted microscope, Japan).

SARS-CoV-2 morphology

The entire electron microscopy sample preparation was performed in a biosafety class II, type A2 biosafety cabinet (Nuaire, USA). SARS-CoV-2 morphology was assessed using a scanning electron microscope (SEM) and a scanning transmission electron microscope (STEM). Sample preparation for SEM was performed by collecting and filtering 1 ml of UV-inactivated infected medium from the virus culture during the occurrence of cytopathic effect through a 1 μm pore syringe nitrocellulose filter (Merck, USA) to trap the virus. Fixation, Periodic Acid-Schiff (PAS), was performed by filtering 2 ml of 2% glutaraldehyde (Serva, USA) through the virus-entrapped syringe filter and incubating at 4°C for four hours. The fixated syringe filter then went through a dehydration process with cold ethanol (Merck, USA), at a gradient of 50%, 70%, 85%, 95% ethanol, and absolute ethanol with 15 minutes of incubation for each sequential dehydration process. Glutaraldehyde fixation and 50–75% ethanol dehydration sequences were performed using a 4°C refrigerator in an airtight container. The 85% and absolute ethanol dehydration processes were carried out at room temperature. The nitrocellulose membrane filter was then extracted from the syringe filter using pliers and air-dried in a biosafety cabinet overnight. A FEI Quanta 650 FEG (FEI, USA) electron microscope in low vacuum SEM mode (80 Pa; 10 kV) at 80,000× magnification and Modular Automated Processing System (MAPS) software version 3.14.11 (ThermoFisher Scientific, Waltham, Massachusetts, United States) was used for sample observation.

STEM sample preparation was performed by collecting and pipetting 50μl UV-inactivated infected mediums from virus culture during the occurrence of cytopathic effect to parafilm. Immobilization of a SARS-CoV-2 viral particle was conducted on a formvar (SPI-chem, USA) coated copper electron microscope grid (EMS, USA). Formvar grid coating was performed according to UK standards for microbiology investigation protocols.²⁷ The formvar-coated grid was pushed to an infected medium droplet on parafilm and incubated for 20 minutes. Excess infected medium was absorbed by filter paper, air-dried, and fixated with 2% glutaraldehyde in an airtight container (4°C, four hours). Excess glutaraldehyde was absorbed with filter paper and the virus-seeded grid was air-dried in a biosafety cabinet overnight. Negative staining was performed by utilizing 2% phosphotungstic acid (EMS, USA) with a one-minute incubation before observation. STEM sample observation was conducted using a FEI Quanta 650 FEG (FEI, USA) electron microscope through the STEM mode (1.5°C, 676 psi, 30kV) with MAPS software.²⁸

Reverse transcription-polymerase chain reaction (RT-PCR) and gel electrophoresis

Identification of SARS-CoV-2 virus culture was performed by total RNA extraction utilizing Trizol reagent (Thermo Fisher, USA) according to the manufacturer’s protocol. A Qubit™ RNA BR Assay Kit (Thermo Fisher Scientific, USA) with a Qubit™ fluorometer was used according to the kit manual.²⁹ RT-PCR was performed from extracted RNA samples utilizing the Goscript^® RT-PCR system (Promega, USA) with SARS-CoV-2 receptor-binding domain spike gene primers adopted from previous studies (forward: 5′-CCACAGACACTTGAGATTC-3′ and reverse: 5′-GCAACTGAATTTTCTGCACCA-3′) and according to supplied protocol by Lau et al.³⁰

Conventional PCR was performed from RT-PCR cDNA using the previously described primer with GoTaq^® green master mix (Promega, USA) in a Thermal Cycler XP machine (Bioer Technology, China). Conventional PCR conditions were conducted through initial denaturation (95°C, five minutes), amplification (45 cycles of ten seconds, 95°C), denaturation (ten seconds, 62°C) annealing, (ten seconds, 72°C) elongation, final elongation (72°C, five minutes), and PCR reaction termination (4°C, 30 minutes). PCR products were detected by performing agarose gel electrophoresis in 2% agarose gel (INTRON Biotechnology, South Korea) in 0.5x Tris-Boric-EDTA (TBE) Buffer (BIOWORLD, USA) with 110 volts for 60 minutes, the DNA band was stained with ethidium bromide (TCI, Japan), and visualized through the Gel Doc XR+ gel documentation system (Bio-Rad, USA). The PCR product length was confirmed with Image Lab software for PC version 6.1 (SOFT-LIT-170-9690-ILSPC-V-6-1, Bio-Rad Laboratories Inc, Hercules, California, US, https://www.bio-rad.com/en-id/product/image-lab-software?ID=KRE6P5E8Z) in alignment with a Thermal Cycler XP machine (Roche, USA).^31,32

Whole genome sequencing

Whole genome sequencing protocol was performed by targeted sequencing after gene amplification using ARTIC V3 primer sets RT-PCR before proceeding to PCR clean up, and processed with Nanopore sequencing kits in the GRIDION platform, according to provided protocols supplied with the kits.³³ Whole-genome sequencing was performed by Genetika Science Indonesia Ltd through EPI2ME Labs software version 21.05 (https://labs.epi2me.io/wfindex) and trimming through Medaka software version 1.3.4 (https://nanoporetech.github.io/medaka/). Gene analysis was performed by RAMPART software version 1.7.1 (https://artic.network/ncov-2019/ncov2019-using-rampart.html) for mutation analysis and identification of the spike gene and the Wuhan Hu-1 as the reference sequence.³⁴

Immunocytochemistry

Staining of Vero E6 SARS-CoV-2 infected cells were performed with immunized rabbit antibody serum, and visualization with diaminobenzidine (DAB) was performed with horseradish peroxidase (HRP) conjugated anti-rabbit IgG. Unspecific binding blocking was performed with 0.5% bovine serum albumin. The positive infected cells that were stained brown were observed by an inverted light microscope (Nikon TMS, Japan).³⁵

Western blot analysis

Viral protein was extracted from infected Vero E6 SARS-CoV-2 culture by RIPA buffer (ThermoFisher Scientific, US) and PMSF extraction (Sigma-Aldrich, US). The sample was centrifuged at 3000×g at 4°C for one hour, and the samples were subjected to polyacrylamide gel electrophoresis (PAGE).

Samples were diluted and mixed with a Laemmli loading solution (Bio-Rad Laboratories Inc, US) and were denatured at 100°C for five minutes before loading on the polyacrylamide gel. Viral proteins were separated on 12% gradient polyacrylamide gels. The transfer of proteins to the PVDF membrane (Sigma-Aldrich, US) was performed with a Trans-Blot Turbo system (Bio-Rad Laboratories Inc, US) for seven minutes.³⁶

Western blot was performed by using antibody serum from an immunized rabbit with visualization and with HRP-conjugated anti-rabbit IgG polyclonal antibody with 1:10.000 concentration (Cat. no. 611-103-122, Rockland, USA) and DAB (Rockland, USA). A comparison of the western blot was made with convalescent serum that was collected from a volunteer COVID-19 convalescent subject with visualization by HRP-conjugated anti-human IgG polyclonal antibody with 1:10.000 concentration (Cat. no. 611-103-123, Rockland, USA) and DAB with 1X concentration (DAB-10, Rockland, USA). This human serum was used to demonstrate human immune response to SARS-CoV-2.

Ethics statement

Ethical clearance for this study was approved by the Institutional Review Board of the Dr Soetomo General Hospital, Surabaya (IRB Number IRB00008635), Ethical Clearance No. 0099/LOE/301.4.2/VIII/2020. During hospitalization, the subject or subject’s guardian was informed about the study and gave their written informed consent for participating in this study and use for publication.

Virus isolation and culture

SARS-CoV-2 CPE appeared in the form of rounding, elevation, and detachment of Vero E6 monolayer cell culture as reported previously.³⁸ Virus passage was performed periodically until stable cytopathic effect patterns were established (5–6 days’ interval) (see Figure 1).

Growth characteristics and viral morphology

Growth characteristics suggested a more adapted virus culture along with the blind passage progression and intervals with shorter CPE formation times. Moreover, TICD50 evaluation of #35 isolate through plaque forming assay and Reed and Muench method showed 10^13.76 TICD50.

Scanning transmission electron microscopy

FEI Quanta 650 FEG electron microscopy was performed in low vacuum SEM mode (80 Pa; 10 kV) at 80,000× magnification, and MAPS (Modular Automated Processing System) software observation was able to find several ovoid-shaped multilobulated viral particles that were observed in SEM, with an estimated diameter ranging from 125.8-199.1 nm between filter membrane fibers. Further analysis in STEM showed SARS-CoV-2 viral particles with its surface spike proteins and internal diameter ranging from 120–200 nm (see Figure 2).

Figure 2.

The result of scanning transmission electron microscopy (STEM) (A, B) uninfected cells medium acted as a blank control; (C, D) the morphology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected patients under scanning electron microscopy (SEM) 80,000× and scanning transmission electron microscopy (STEM) (FEI Quanta 650 FEG (FEI, USA).

Reverse transcription-polymerase chain reaction (RT-PCR)

The visualization of a 398-base pair size band fragment of Receptor Binding Domain (RBD) sequences confirmed SARS-CoV-2 presence. (Roche, USA) (see Figure 3 and Raw RT-PCR Results of SARS-COV-2³⁹).

Figure 3.

(A) Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S1/S2 cleavage site Receptor Binding Domain (RBD) detected by two-step reverse transcription-polymerase chain reaction (RT-PCR), DNA electrophoresis ethidium bromide staining, and visualized through the Gel Doc XR+ gel documentation system (Bio-Rad, USA).

Spike gene analysis

Spike gene analysis of three isolates from three patients found that only the D614G mutation was detected among the isolates, although in #35 isolates D215Y and E484D mutations were also present. Based on whole genome analysis, those three isolates were included in clade 20A, and two isolates were included in lineage B.1.6, with one isolate belonging to lineage B.1.4.7 (Table 1).

Isolation and characterization of protein immunogenic of SARS-CoV-2

Immunocytochemistry staining suggested that antibody serum from immunized rabbit and human natural infection serum could detect SARS-CoV-2 infected cells in virus culture, indicated by brown stained cells that were observed in light-inverted microscopy with 100x magnification (Nikon TMS, Tokyo, Japan) (see Figure 4).

Figure 4.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Vero E6 infected cells, immunocytochemistry using (A) uninfected Vero E6 cells as control; (B) rabbit antibody serum, (C) human antibody serum (100× magnification).

Comparison of western blot staining with antibody serum from immunized rabbit and human natural infection serum that was collected from a volunteer COVID-19 convalescent subject showed several main immunogenic proteins that were detected, including spike glycoprotein (S), nucleocapsid protein (N), membrane glycoprotein (M), accessory 3a protein, and envelope protein (E). Both western blot comparisons between rabbit antibody serum and human convalescent serum from the second wave of COVID-19 in Surabaya showed a relatively similar protein immunogenic capacity (see Figure 5).^37,40

Figure 5.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) western blot staining with anti-SARS-CoV-2 Receptor Binding Domain (RBD) monoclonal antibody for detecting S1 protein (MAB10540, RnD System) (8% gel); (A): western blot staining with anti-SARS-CoV-2 S2 monoclonal antibody (MAB10557, RnD System) (8% gel); (B): western blot staining with rabbit antibody serum (12% gel); (C): western blot staining with human convalescent serum from the COVID-19 infection (8% gel); (D): (S) labeled for spike protein, (N) labeled for nucleoprotein protein, (M) labeled for membrane protein, (E) labeled for envelope protein.

Underlying data

Figshare: Underlying data for ‘Characterization of SARS-CoV-2 East Java isolate, Indonesia’, https://doi.org/10.6084/m9.figshare.14703567.v1.³⁷

This project contains the following underlying data:

‘Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) western blot staining with anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Receptor Binding Domain (RBD) monoclonal antibody for detecting S1 protein’

Figshare: https://doi.org/10.6084/m9.figshare.14703579.v1.³⁹

This project contains the following underlying data:

‘Raw RT-PCR Results of SARS-COV-2’

Figshare: https://doi.org/10.6084/m9.figshare.14703582.v1.⁴⁰

This project contains the following underlying data:

‘Gels for the Western blot (Raw) Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) western blot staining with anti-Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Receptor Binding Domain (RBD) monoclonal antibody for detecting S1 protein’

Data are available under the terms under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).