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Microbes & Immunity Isolation and identification of PEDV strain CHN-CQ-2021

sterile 1× PBS (pH 7.4), and inoculated with 1 mL of PEDV A 10-fold serially diluted PEDV (100 μL/well) was
CHN-CQ-2021 diluted in 50 mL of maintenance medium. inoculated onto the cell monolayer, with eight replicates per
Cells and supernatant were maintained at 37°C under 5% dilution. Cells were maintained in a humidified incubator at
CO and monitored for CPE. When CPE became evident 37°C with 5% CO for 5–7 days. Viral titers were quantified
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2
(approximately 2 days post-infection), the cultures were by observing CPE and calculated using the Reed–Muench
subjected to two freeze-thaw cycles. The resulting cell method, expressed as 50% tissue culture infectious dose
lysates and supernatants were harvested for viral titer (TCID )/mL. Plaque-forming units (PFUs) were derived
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50
determination as described below. using Equation I as previously described, and PFU values
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2.3. Immunofluorescence assay (IFA) were used to determine the MOI.
The IFA experiment was performed as previously described PFU = 0.7 ×TCID 50 (I)
with some modifications. Briefly, Vero cells were seeded 2.6. Measurement of PEDV (CHN-CQ-2021) growth
22
in 12-well plates and cultured overnight, followed by
infection with PEDV (CHN-CQ-2021) at a multiplicity of Vero cells were seeded into 35-mm cell culture dishes and
infection (MOI) of 0.1. At 16–24 h post-PEDV infection, infected with PEDV at an MOI of 0.1 when the cell density
cells were fixed with 4% paraformaldehyde for 15 min, reached 90%. Cells cultured with DMEM maintenance
permeabilized with 0.5% Triton X-100 for 10 min at room medium without viral inoculation served as controls.
temperature, and blocked with 3% bovine serum albumin Every 4 h post-inoculation (hpi), PEDV-infected cells
(BSA). Subsequently, cells were sequentially incubated and control cells were observed, and then, the cells were
with PEDV-specific antiserum (M100048, Zoonogen, harvested and stored at −80℃. After two freeze-thaw
China) and Cy3-conjugated secondary antibody (A10521, cycles, supernatants were harvested to determine viral
Thermo, USA) in the dark at room temperature for 1 h. titers as described above, and a growth curve of PEDV
After three washes with 1× PBS, nuclei were counterstained CHN-CQ-2021 was generated.
with 4’,6-diamidino-2-phenylindole (Solarbio, China) 2.7. Genomic cloning and phylogenetic analysis of
for 5 min. Fluorescence signals were visualized using an the whole genome and S genes
inverted fluorescence microscope (Leica, Germany).
Viral RNA was extracted from PEDV cell lysates using
2.4. Electron microscopic observation an RNeasy kit (R4111-03, Magen, China) according to
Virus morphology was observed using transmission the manufacturer’s instructions, followed by DNase I
electron microscopy (TEM; JEM-1400, JEOL, Japan) as treatment to remove genomic DNA contamination. The
previously described, with some modifications. Briefly, specific primers (Sangon Company, China; Table 1) for
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PEDV was propagated in Vero cells. Cell debris was removed amplification of the PEDV whole genome were designed
through centrifugation at 11,000 × g (4℃, 30 min). The with reference to the published sequence (GenBank:
clarified supernatant was mixed with 7% PEG6000 (Solarbio, JX647847) and synthesized. The reaction mixture (50 μL
China) and incubated overnight at 4℃ with continuous total volume) contained 1 μL PrimeScript 1-Step Enzyme
agitation. The mixture was then centrifuged at 11,000 × g Mix, 25 μL 2× One-Step Buffer, 2.5 μM each of forward and
(4℃, 1 h), and the pelleted virions were resuspended in reverse primers, 1 μg viral RNA, and RNase-free H O. The
2
2 mL of 1× PBS and layered onto a discontinuous sucrose PCR reaction conditions were as follows: 50°C for 30 min;
gradient (20–60%). After ultracentrifugation at 177,600 × 94°C for 2 min; followed by 34 cycles of 94°C for 30 s, 55°C
g (4℃, 2 h) using an ultracentrifuge (Himac CP 100WX, for 30 s, and 72°C for 2.5 min; with a final extension at 72°C
Hitachi Koki, Japan), distinct viral bands were carefully for 10 min. Subsequently, the PCR products were subjected
collected. Viral band was diluted in 1× PBS and subjected to 1% agarose gel electrophoresis, and target bands were
to additional ultracentrifugation (177,600 × g, 4℃, 2 h) excised and purified using a DNA gel extraction kit (UE-
for sucrose removal. Purified virions were resuspended GX-50, US Everbright, China). Purified gene fragments
in minimal sterile 1× PBS and negatively stained with were then sent for sequencing (Sangon Biotech, China).
3% (w/v) phosphotungstic acid (pH 6.8; Sigma, USA) for Viral genome sequences were assembled using DNAStar
2 min. Excess liquid was blotted using filter paper, and grids Lasergene 7.0 (Version 7.0, DNASTAR Inc., USA). The
were air-dried before observation under TEM at 80 kV. S gene and the whole genome of CHN-CQ-2021 were
aligned with representative PEDV strains retrieved from
2.5. Infectious-virus titrations by a 50% tissue the National Center for Biotechnology Information.
culture infectious dose (TCID ) assay Phylogenetic trees were constructed by the neighbor-
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Vero cells were seeded into 96-well plates and cultured joining method using MEGA 5 software available online
overnight, followed by two washes with sterile 1× PBS. (http://www.megasoftware.net/).

Volume X Issue X (2025) 112 doi: 10.36922/MI025260059