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

virus (PEDV), porcine deltacoronavirus, and swine acute reaching up to 100%. Intestinal luminal contents were
diarrhoea syndrome coronavirus. 8-11 Despite the availability collected from PEDV-positive piglets exhibiting clinical
of vaccines for immunization control, frequent outbreaks of signs of severe diarrhea and vomiting, cryopreserved at
viral enteric diseases continue to occur, with more than 50% −80°C, and processed for viral isolation.
of cases attributed to PEDV infections. Notably, PEDV
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exhibits a high mutation rate, enabling variant strains to 2.2. Virus isolation, plaque purification, and
evade immune protection conferred by existing vaccines propagation in Vero cells
derived from original strains. This highlights the necessity Before virus isolation, intestinal luminal contents were
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for continuous monitoring and analysis of the biological homogenized in an equal volume of sterile 1× phosphate-
characteristics of circulating PEDV strains to optimize buffered saline (PBS, pH 7.4; Solarbio, China). Following
prevention strategies. homogenization, particulate debris was removed through

PEDV is an enveloped, single-stranded positive-sense centrifugation (11,000 g, 4°C, 10 min; 5804 R, Eppendorf,
RNA virus belonging to the Alphacoronavirus genus, with Germany), and supernatants were clarified through
a genome length of approximately 28 kb. The 5’ end of sterile 0.22-μm syringe filters (Millipore, United States
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its genome contains a cap structure in the untranslated of America). Vero cells (ATCC number: CCL-81; ATCC,
region, while the 3’ end contains a polyadenylated tail. USA) were utilized for PEDV isolation.
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The PEDV genome contains seven open reading frames Virus isolation, plaque purification, and propagation
(ORFs): ORF1a, ORF1b, and ORF2–6, encoding a total of were conducted as previously described with some
four structural proteins (spike [S], envelope [E], membrane modifications. Briefly, Vero cells were cultured in a
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[M], and nucleocapsid [N]) and 16 non-structural proteins six-well plate until reaching 90% confluency. The cells
(nsp1–nsp16). Porcine epidemic diarrhea (PED) was first were washed three times with sterilized 1× PBS buffer.
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reported in the United Kingdom in 1971, and the pathogen Subsequently, 350 μL of Dulbecco’s Modified Eagle Medium
PEDV was first identified and isolated in Belgium in 1978, (DMEM; Solarbio, China) maintenance medium (serum-
with the isolate named CV777. Between 1970 and 1980, free DMEM medium supplemented with 7 μg/mL trypsin
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PEDV spread across Europe, causing significant mortality (Sigma, USA) and 200 μL of filtered sample were added to
among piglets. From 1980 to 2010, the disease occurred each well. Control wells received an equivalent volume of
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sporadically, with occasional reports from several countries, DMEM maintenance medium. The plates were incubated at
including the United Kingdom, Germany, and Italy. In 37℃ in a 5% CO atmosphere (Thermo, USA). Cells were
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China, PED was first reported in 1973. In October 2010, observed daily for cytopathic effects (CPE). When CPE
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a large-scale PED outbreak emerged across pig farms reached 80%, the plates were subjected to two freeze-thaw
nationwide, with mortality rates among neonatal piglets cycles. The supernatant was collected after centrifugation at
reaching 50–100% in affected farms, causing substantial 11,000 × g for 10 min at 4℃, and stored at −80℃, followed
economic losses to the Chinese swine industry. Subsequent by further examination using quantitative real-time (RT)-
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investigations revealed that variant PEDV strains had evolved polymerase chain reaction (PCR) as described below.
to evade the immune protection conferred by CV777-
derived vaccines, indicating the importance of monitoring For virus plaque purification, supernatants from virus-
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circulating PEDV strains and characterizing their biological infected Vero cells were serially diluted and inoculated
properties to inform effective control strategies. onto fresh Vero cell monolayers. Cells were incubated with
the diluted virus in maintenance medium for 1.5 h at 37°C
To investigate the biological characteristics of current under 5% CO . After incubation, the medium was aspirated,
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circulating PEDV strains in Chinese pig farms, this and cells were overlaid with 2 mL of maintenance medium
study isolated viruses from diarrheic piglets obtained containing 1.25% low-melting-point (LM), genetically
from a swine facility in Chongqing in 2021. The isolates quality-tested (GQT) agarose (Gibco, USA) to immobilize
underwent comprehensive genomic characterization and viral particles. Following a 24-h incubation, cells were
in vivo pathogenicity assessment. These findings enhance stained with 2 mL of maintenance medium supplemented
our understanding of PEDV pathogenesis and establish the with 1.25% LM GQT Agarose and 0.01% Neutral Red
foundation for vaccine development. solution (Sigma, USA). Plaques were isolated using sterile
pipette tips and transferred into microcentrifuge tubes
2. Materials and methods containing 0.5 mL of maintenance medium.

2.1. Clinical samples collection The purified plaques, named as CHN-CQ-2021, were
In early March 2021, an outbreak of PEDV was reported propagated in Vero cells. Vero cells were cultured in T175
in swine herds in Chongqing, China, with mortality rates flasks until reaching 90% confluency, washed 3 times with

Volume X Issue X (2025) 111 doi: 10.36922/MI025260059