Bahan dan metode
Sel, virus, dan reagen
Garis sel Vero E6 diperoleh dari Koleksi
Kultur Tipe Amerika dan dipelihara dalam
medium minimal Eagle (Gibco Invitrogen)
ditambah 10% serum fetal bovine (FBS; Gibco
Invitrogen) dan 1% antibiotik/antimikotik
(Gibco Invitrogen), pada 37 ° C dalam
inkubator CO2 5% yang dilembabkan. Garis sel
Huh7 dikultur dalam medium Eagle yang
dimodifikasi Dulbecco (DMEM; Gibco
Invitrogen) yang dilengkapi dengan 10% FBS
dan 1% antibiotik/antimikotik (Gibco
Invitrogen), pada suhu 37 °C dalam inkubator
CO2 5% yang dilembabkan.
SARS-CoV-2 (nCoV
2019BetaCoV/Wuhan/WIV04/2019)
disebarkan dalam sel Vero E62, dan titer virus
ditentukan oleh 50% dosis infektif kultur
jaringan (TCID50) seperti yang dijelaskan
dalam penelitian kami sebelumnya5. Semua
percobaan infeksi dilakukan di laboratorium
biosafety level-3 (BSL-3).
Benidipine HCI (Selleck Chemicals, no.
S2017), amlodipine besylate (Selleck
Chemicals, S1813), cilnidipine (Selleck
Chemicals, S1293), nicardipine HCl (Selleck
Chemicals, S4181), nifedipine (Selleck
Chemicals, S1808), isradipine (Selleck
Chemicals, S1662), nimodipine (Selleck
Chemicals, S1747), nisoldipine (Selleck
Chemicals, S1748), felodipine (Selleck
Chemicals, S1885), 2APB (Selleck Chemicals,
S6657), BAPTA-AM (Selleck Chemicals, S7534),
dan CQ (Sigma- Aldrich, no.C6628) dibeli dari
perusahaan yang ditunjukkan.
Materials and methods
Cells, virus, and reagents
Vero E6 cell line was obtained from
American Type Culture Collection and
maintained in minimum Eagle’s medium
(Gibco Invitrogen) supplemented with 10%
fetal bovine serum (FBS; Gibco Invitrogen) and
1% antibiotic/ antimycotic (Gibco Invitrogen),
at 37 °C in a humidified 5% CO2 incubator.
Huh7 cell line was cultured in Dulbecco’s
modified Eagle’s medium (DMEM; Gibco
Invitrogen) supplemented with 10% FBS and
1% antibiotic/ antimycotic (Gibco Invitrogen),
at 37 °C in a humidified 5% CO2 incubator.
SARS-CoV-2 (nCoV
2019BetaCoV/Wuhan/WIV04/ 2019) was
propagated in Vero E6 cells2, and viral titer
was determined by 50% tissue culture infective
dose (TCID50) as described in our previous
study5. All the infection experiments were
performed in a biosafety level-3 (BSL-3)
laboratory.
Benidipine HCI (Selleck Chemicals, no.
S2017), amlodipine besylate (Selleck
Chemicals, S1813), cilnidipine (Selleck
Chemicals, S1293), nicardipine HCl (Selleck
Chemicals, S4181), nifedipine (Selleck
Chemicals, S1808), isradipine (Selleck
Chemicals, S1662), nimodipine (Selleck
Chemicals, S1747), nisoldipine (Selleck
Chemicals, S1748), felodipine (Selleck
Chemicals, S1885), 2APB (Selleck Chemicals,
S6657), BAPTA-AM (Selleck Chemicals, S7534),
and CQ (Sigma-Aldrich, no.C6628) were
purchased from indicated companies.
Evaluation of the antiviral activities of the test
compounds
Vero E6 pre-seeded in 48-well dish (1 ×
105 cells/well) were treated with the different
concentration of the indicated compounds for
1 h and infected with SARSCoV-2 at an MOI of
0.05. Two hours later, the virus–drug mixture
was removed and cells were cultured with drug
containing medium. At 24 h p.i., the cell
supernatant was collected and lysed. The viral
RNA extraction and qRTPCR analysis was
described in our previous study5.
Evaluation of the cytotoxicity of the test
compounds
Vero E6 pre-seeded in 96-well dish (5 ×
104 cells/well) were treated with the different
concentration of the indicated compounds,
and 24 h later, the relative numbers of
surviving cells were measured with cell
counting kit-8 (GK10001, GLPBIO) according to
the manufacturer’s instructions.
Immunofluorescence microscopy
To detect the intracellular expression
level of viral NP, cells were fixed with 4%
paraformaldehyde in advance. Fixed cells were
permeabilized with 0.5% Triton X-100 and
blocked with 5% bovine serum albumin (BSA).
Then they were incubated for 2 h with the anti-
sera (1:1000 dilution) against the NP of a bat
SARS-related CoV as the primary antibody,
followed by incubation with Alexa 488- labeled
goat anti-rabbit IgG (Abcam, ab150077; 1:500
dilution). The nuclei were stained with DAPI
(Sigma- Aldrich, no. D9542). The images were
taken by a fluorescence microscopy.
Evaluasi aktivitas antivirus dari senyawa uji
Vero E6 yang diunggulkan sebelumnya
dalam cawan 48-sumur (1 × 105 sel/sumur)
diperlakukan dengan konsentrasi yang
berbeda dari senyawa yang ditunjukkan
selama 1 jam dan terinfeksi dengan SARSCoV-
2 pada MOI 0,05. Dua jam kemudian,
campuran virus-obat dihilangkan dan sel-sel
dikultur dengan media yang mengandung
obat. Pada 24 jam pi, supernatan sel
dikumpulkan dan dilisiskan. Ekstraksi RNA virus
dan analisis qRTPCR dijelaskan dalam
penelitian kami sebelumnya5.
Evaluasi sitotoksisitas senyawa uji
Vero E6 yang diunggulkan sebelumnya
dalam cawan 96-sumur (5 × 104 sel / sumur)
diperlakukan dengan konsentrasi berbeda dari
senyawa yang ditunjukkan, dan 24 jam
kemudian, jumlah relatif sel yang bertahan
diukur dengan penghitungan sel kit-8
(GK10001, GLPBIO) sesuai dengan petunjuk
pabriknya.
Mikroskop imunofluoresensi
Untuk mendeteksi tingkat ekspresi
intraseluler NP virus, sel difiksasi dengan
paraformaldehida 4% terlebih dahulu. Sel-sel
tetap ditembus dengan 0,5% Triton X-100 dan
diblokir dengan 5% albumin serum sapi (BSA).
Kemudian mereka diinkubasi selama 2 jam
dengan anti-sera (pengenceran 1:1000)
terhadap NP dari CoV terkait SARS kelelawar
sebagai antibodi utama, diikuti oleh inkubasi
dengan IgG anti-kelinci kambing berlabel Alexa
488 (Abcam, ab150077; pengenceran 1:500).
Inti diwarnai dengan DAPI (Sigma-Aldrich, no.
D9542). Gambar diambil dengan mikroskop
fluoresensi.
Western blot analysis
For western blot analysis, proteins
were separated by 12% SDS–PAGE and then
transferred onto PVDF membranes (Millipore).
The membranes were blocked with 5% BSA in
TBST (TBS buffer with 0.1% Tween 20) for 1 h
at room temperature. After washed with TBST
for three times, the membranes were
incubated with the anti-NP sera (1:2000
dilution) overnight at 4 °C. After washed with
TBST for three times, the membranes were
incubated with horseradish peroxidase-
conjugated goat anti-rabbit IgG (Proteintech,
China; 1:10000 dilution). Protein bands were
detected by SuperSignal West Pico
Chemiluminescent substrate (Pierce).
Plaque assay
Vero E6 cells (1 × 105 cells/well) were
incubated with supernatant containing SARS-
Cov-2. At 1 h p.i., the supernatant was
removed, and the cells were incubated under
an overlay consisting of DMEM supplemented
with 2% FBS and 0.9% CMC (Calbiochem). At 4
days p.i., the overlay was discarded and cells
were fixed for 30 min in 4% polyoxymethylene,
and stained with crystal violet working
solution.
Analisis Western blot
Untuk analisis western blot, protein
dipisahkan oleh 12% SDS-PAGE dan kemudian
dipindahkan ke membran PVDF (Millipore).
Membran diblokir dengan 5% BSA dalam TBST
(buffer TBS dengan 0,1% Tween 20) selama 1
jam pada suhu kamar. Setelah dicuci dengan
TBST sebanyak tiga kali, membran diinkubasi
dengan serum anti-NP (pengenceran 1:2000)
semalaman pada suhu 4 °C. Setelah dicuci
dengan TBST tiga kali, membran diinkubasi
dengan IgG anti-kelinci terkonjugasi lobak
peroksidase (Proteintech, China; pengenceran
1:10000). Pita protein dideteksi oleh substrat
SuperSignal West Pico Chemiluminescent
(Pierce).
Uji plak
Sel Vero E6 (1 × 105 sel/sumur)
diinkubasi dengan supernatan yang
mengandung SARS-Cov-2. Pada 1 jam pi,
supernatan dihilangkan, dan sel-sel diinkubasi
di bawah lapisan yang terdiri dari DMEM yang
dilengkapi dengan 2% FBS dan 0,9% CMC
(Calbiochem). Pada 4 hari pi, overlay dibuang
dan sel difiksasi selama 30 menit dalam
polioksimetilen 4%, dan diwarnai dengan
larutan kerja kristal violet.