key: cord-010053-kniq2mbw
authors: Lee, Sunhee; Lee, Dong‐Uk; Noh, Yun‐Hee; Lee, Seung‐Chul; Choi, Hwan‐Won; Yang, Hyoung‐Seok; Seol, Jun‐Ho; Mun, Seong Hwan; Kang, Won‐Myoung; Yoo, Hyekyung; Lee, Changhee
title: Molecular characteristics and pathogenic assessment of porcine epidemic diarrhoea virus isolates from the 2018 endemic outbreaks on Jeju Island, South Korea
date: 2019-05-20
journal: Transbound Emerg Dis
DOI: 10.1111/tbed.13219
sha: 
doc_id: 10053
cord_uid: kniq2mbw

Since the 2013–2014 incursion of the virulent G2b porcine epidemic diarrhoea virus (PEDV) pandemic strains in South Korea, frequent moderate‐scale regional outbreaks have recurred. In particular, areas of Jeju Island with extensive swine production have faced repeated epidemics since the re‐emergence in 2014. The current study reports the complete genome sequences and molecular characterization of the representative PEDV strains responsible for the 2018 endemic outbreaks on Jeju Island. All isolates were determined to belong genetically to the highly pathogenic pandemic G2b group. Full‐length genome sizes of four isolates differed from that of the G2b epidemic field strain due to insertion or deletion (DEL) mutations in the non‐structural protein (nsp)‐ or spike (S) protein‐coding regions. The 2018 Jeju isolates shared 96.7%–98.7% and 98.5%–99.4% identity at the S gene and whole‐genome levels, respectively, compared to global G2b PEDV strains. Genetic and phylogenetic analyses indicated that the 2018 isolates were closest to the 2014 G2b re‐emergent Jeju strains, but appeared to have undergone substantial rapid independent evolution. Among the isolates, a notable nsp3 DEL variant strain, KOR/KNU‐1807/2018, was isolated and propagated by continuous passages in Vero cells, and displayed typical PEDV‐induced syncytia formation. Genomic sequencing identified a unique 8‐nt DEL in the extreme C‐terminal region of the S gene at the 4th passage (KNU‐1807‐P4) compared to its original sample. This DEL resulted in the premature termination of S by nine amino acid residues (EVFEKVHVQ), which contained a KxHxx motif that is a potential endoplasmic reticulum retrieval signal. In vivo animal studies showed that variant strain KNU‐1807 had decreased virulence in suckling piglets. These results advance our knowledge regarding the genetic variation and pathogenicity of the G2b PEDV endemic strains prevalent in Jeju swine herds in South Korea.

Since its emergence in South Korea in 1992, small-to large-scale PEDV epizootics have occurred annually throughout the nation, leading to substantial economic losses in domestic pig production (Lee, 2015 (Lee, , 2019 . The 2013 PED pandemic that ravaged the United States (Stevenson et al., 2013) also struck the Korean Peninsula and decimated more than 40% of the pig farms across the country during 2013-2014 (Lee, 2015 (Lee, , 2019 . Subsequently, in late March 2014, the virus invaded Jeju Island located 80 km from the South Korea mainland at the closest point, which had maintained PEDV-naïve status for a decade, causing massive neonatal mortality in provincial herds. Genetic and phylogenetic analyses revealed that the re-emergent Jeju Island PEDV isolates were most closely related to the pandemic genogroup 2b (G2b) strains that were responsible for the 2013-2014 global outbreaks, suggesting a direct introduction of the virus from the mainland of South Korea via unknown contaminating sources . Even with province-wide vaccination or intentional virus-exposure practices being implemented in order to provide herd immunity around the areas that contain dense swine populations, PEDV has continued to plague the provincial pork industry. Since several pig farms have experienced recurrent PEDV outbreaks within a single year, PED has become endemic on Jeju Island . In this study, we determined the complete genome sequences of field isolates on Jeju Island to investigate the diversity of the PEDVs responsible for the ongoing endemic outbreaks. Additionally, we isolated and serially cultured a novel PEDV strain, KOR/KNU-1807/2018, in cell culture and investigated its genotypic and phenotypic characteristics in vitro and in vivo.

In early 2018, mild sporadic suspect-PEDV outbreaks with low mortality rates in newborn piglets occurred on several farms in the Hallim and Daejeong areas of Jeju Province. Small intestine (SI) specimens were collected at 18 different pig farms located in those districts from January through June 2018 from dead piglets that had acute diarrhoea. Intestinal homogenates were prepared as 10% (wt/vol) suspensions in phosphate-buffered saline (PBS) using a MagNA Lyser Instrument (Roche Diagnostics, Mannheim, Germany) with three rounds of 15 s at a force of 8,000 g. The suspensions were then vortexed and centrifuged for 10 min at 4,500 g (Hanil Centrifuge FLETA5, Incheon, South Korea). The clarified supernatants were initially subjected to RT-PCR analysis using an i-TGE/PED Detection Kit (iNtRON Biotechnology) according to the manufacturer's instructions. PEDV-positive samples were filtered through a 0.22-μm-pore syringe filter (Millipore) and stored at −80°C until subsequent sequencing analysis and virus isolation were performed.

The S glycoprotein gene sequences of the virus isolates were determined by traditional Sanger methods. Two overlapping cDNA fragments spanning the entire S gene of each isolate were amplified by RT-PCR as previously described (Lee, Park, Kim, & Lee, 2010) . The individual cDNA amplicons were gel-purified, cloned using the pGEM-T Easy Vector System (Promega) and sequenced in both directions using two commercial vector-specific T7 and SP6 primers and gene-specific primers. In addition, the complete genomes of representative PEDV field strains were also sequenced. Ten overlapping cDNA fragments spanning the entire genome of each virus strain were RT-PCR-amplified as described previously (Lee, Kim, & Lee, 2015; and each PCR product was sequenced as described above.

The 5′ and 3′ ends of the genomes of the individual isolates were determined by rapid amplification of cDNA ends (RACE) as described previously . The full-length S gene or whole-genome sequences of the 2018 viruses have been deposited in the GenBank database under the accession numbers shown in Figure 2a.

The sequences of 66 fully sequenced S genes and 39 complete genomes of global PEDV isolates were independently used in sequence alignments and phylogenetic analyses. Multiple sequence alignments were generated using the ClustalX 2.0 program (Thompson, Gibson, Plewniak, Jeanmougin, & Higgins, 1997) and the percentages of nucleotide sequence divergences were assessed using the same software. Phylogenetic trees were constructed from the aligned nucleotide or amino acid sequences using the neighbourjoining method and subsequently subjected to bootstrap analysis with 1,000 replicates to determine the percentage reliability values of each internal node of the tree (Saitou & Nei, 1987) . All phylogenetic trees were generated using Mega 4.0 software (Tamura, Dudley, Nei, & Kumar, 2007) .

Porcine epidemic diarrhoea virus isolation was performed using Vero cells in the presence of trypsin (USB) as described previously . Briefly, inocula were prepared by adding trypsin (USB) to intestinal suspensions to a final concentration of 10 μg/ml. Confluent Vero cells grown in 6-well plates were washed with PBS and inoculated with 400 μl of each trypsincontaining inoculum. After incubating at 37°C for 1 hr to allow for viral adsorption, 2 ml of virus growth medium consisting of alpha minimum essential medium (α-MEM; Invitrogen) supplemented with antibiotic-antimycotic solutions (100×; Invitrogen), 0.3% tryptose phosphate broth (TPB; Sigma), 0.02% yeast extract (Difco), 10 mM HEPES (Invitrogen) and 5 μg/ml of trypsin was added to each well. The inoculated cells were maintained at 37°C under 5% CO 2 and monitored daily for cytopathic effects (CPE). When ~70% of cells showed CPE, the infected cells were subjected to three rounds of freezing and thawing. The culture supernatants were then collected and centrifuged for 10 min at 400 g and filtered through a 0.22-μm pore filter. The clarified supernatants were aliquoted and stored at −80°C as passage 1 (P1) viral stocks for use in plaque purification and subsequent serial passaging. If CPE and RT-PCR results were negative after five blind passages, virus isolation was considered negative for those samples.

Vero cells grown on microscope coverslips placed in 6-well tissue culture plates were mock infected or infected with PEDV at a multiplicity of infection (MOI) of 0.1. The virus-infected cells were cultured until 24 hr, fixed with 4% paraformaldehyde for 10 min at room temperature (RT) and permeabilized with 0.2% Triton X-100 in PBS at RT for 10 min. The cells were blocked with 1% bovine serum albumin (BSA) in PBS for 30 min at RT and then incubated for 2 hr with a monoclonal antibody (MAb) specific for PEDV N protein (ChoogAng Vaccine Laboratories). After being washed five times with PBS, the cells were incubated for 1 hr at RT with a goat anti-mouse secondary antibody conjugated to Alexa Fluor 488 (Invitrogen) followed by counterstaining with 4′,6-diamidino-2phenylindole (DAPI; Sigma). The coverslips were mounted onto glass microscope slides using mounting buffer and the stained cells were visualized using a fluorescence Leica DM IL LED microscope (Leica).

Vero cells were infected with each passage of KNU-1807 virus stock in the presence of trypsin as described above. The culture supernatants were collected at 24 or 48 hr post-infection (hpi) when 70% CPE had commonly developed. For growth kinetics experiments, supernatants were harvested from cells infected with each selected passage virus at various time points (6, 12, 24, 36 and 48 hpi) and stored at −80°C. Virus titres were measured by end-point titration in 96-well plates using 10-fold serial dilutions of the samples in triplicate for each dilution to determine the amount of virus required to produce CPE in 50% of the inoculated Vero cells. The 50% tissue culture infectious dose (TCID 50 ) per ml of virus stock was calculated using the Reed-Muench method (Reed & Muench, 1938) .

Viral RNA was extracted from virus supernatants from infected Vero cells and faecal specimens using an i-TGE/PED Detection Kit according to the manufacturer's protocol. Quantitative real-time RT-PCR was performed using a One Step PrimeScript RT-PCR Kit (TaKaRa) and primers (forward primer 5′-ACGTCCCTTTACTTTCAATTCACA-3′, reverse primer 5′-TATACTTGGTACACACATCCAGAGTCA-3′) and a probe (5′-FAM-TGAGTTGATTACTGGCACGCCTAAACCAC-BHQ1-3′) described elsewhere (Kim et al., 2007; Sagong & Lee, 2011) . Amplification of the reaction mixtures was performed using a Thermal Cycler Dice Real Time System (TaKaRa) and

the results were analysed using software as described previously Sagong & Lee, 2011 ).

The in vivo swine studies were performed at the ChoongAng Vaccine Laboratory Animal Facility under the guidelines established by its Institutional Animal Care and Use Committee. A total of nine 3-dayold suckling piglets were obtained from commercial cross-bred sows (Great Yorkshire × Dutch Landrace) at a conventional breeding farm with a good health record and either vaccinated against PEDV or no known prior PED outbreak. All animals were confirmed negative for PEDV, transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus and porcine rotaviruses by virus-specific RT-PCR analysis of rectal swabs and determined to be free of antibodies to PEDV, TGEV and porcine reproductive and respiratory syndrome virus (PRRSV) by serum neutralization tests as described previously and a commercial PRRSV antibody ELISA kit (HerdChek PRRS X3;

IDEXX Laboratories). Pigs were randomly assigned to three experimental groups: the highly virulent KNU-141112-P5-inoculated group (n = 3) , the KNU-1807-P10-inoculated group (n = 4) and the sham-inoculated control group (n = 2). Animals were fed commercial milk replacer (3-4 times daily) and had ad libitum access to water for the 5-day duration of the study. Following a 2-day acclimation period, piglets (5-day old) in the virus-inoculated groups received an oral 1-ml dose of 10 3.0 TCID 50 /ml of the appropriate virus, which was equivalent to 100 median pig diarrhoea dose PDD 50 of KNU-141112 (Baek et al., 2016; Lee et al., 2015 . The sham-inoculated pigs were administered with cell culture media as a placebo.

Animals were monitored three times daily throughout the experiment for clinical signs of vomiting and diarrhoea and for mortality.

Stool samples from the pigs in all groups were collected prior to inoculation and thereon daily using 16-inch cotton-tipped swabs. The PEDV faecal shedding titres were determined by real-time RT-qPCR as described above. A PEDV isolate with a known infectivity titre was 10-fold serially diluted to generate a standard curve in each PCR 

At necropsy, small intestine tissue specimens (<3 mm thick) were collected from each piglet, fixed in 10% formalin for 24 hr at RT, and embedded in paraffin according to standard laboratory procedures. The formalin-fixed paraffin-embedded tissues were cut at 5-8-μm thick sections using a microtome (Leica), floated in a 40°C

water bath containing distilled water, and transferred to glass slides. The tissues were then deparaffinized in xylene for 5 min and rehydrated in decreasing concentrations of ethanol (100%, 95%, 90%, 80% and 70%, respectively) for 3 min each. The deparaffinized intestinal tissue sections were stained with haematoxylin and eosin (Sigma) for histopathology or subjected to immunohistochemistry (IHC) using PEDV N-specific MAb as described previously . Villous height and crypt depth were also measured throughout the H&E-stained jejunal sections and the mean ratio of jejunal villous height to crypt depth (VH:CD) was calculated as described previously (Jung, Kim, Ha, Choi, & Chae, 2006 ).

All values are expressed as the means ± standard deviation of the means (SDM). All statistical significances were evaluated by a 

We in Vero cells, reaching a titre >10 6 TCID 50 /ml by 12 hpi (Figure 3b ).

In addition, we sequenced the coding region of nsp3 and the entire S gene of strain KNU-1807 at the first productive passages of P4 and Table 4 ). These genetic alterations that occurred in P4 remained unchanged through P10 and no additional variations emerged in the S-coding region during the subsequent serial passages. The partial or complete genome sequences of all the cell culture-passaged viruses were compared to the original KNU-1807-SI and the results are summarized in Table 4 .

Since the KNU-1807 strain with the truncated S cytoplasmic tail (0) 100 (0) nsp10 (405) 100 (0) 100 (0) 100 (0) 100 (0) 100 (0) 100 (0) 99.2 (1) nsp11 (54) 100 (0) 100 (0) 100 (0) 100 (0) 100 (0) 100 (0) 100 (0) nsp12 ( The number of individual differences in the 5′-UTR, protein-coding region, and 3′-UTR, respectively.

F I G U R E 2 Phylogenetic analyses based on the nucleotide sequences of the spike (S) genes (a) and full-length genomes (b) of the PEDV strains. A region of the S gene and the complete genome sequence of TGEV were included as the outgroups in each tree. Multiple sequence alignments were performed using ClustalX software and phylogenetic trees were constructed from the aligned nucleotide sequences using the neighbour-joining method. Numbers at each branch are bootstrap values greater than 50% based on 1,000 replicates. The names of the strains, countries and dates ( 

In the current study, determined the complete genome sequences Similar to other coronavirus replicase-encoded nsps, many PEDV nsps (nsp1, nsp3, nsp5, nsp7, nsp14, nsp15 and nsp16) function as interferon (IFN) antagonists that modulate the innate immune response (Wang et al., 2016; Zhang, Shi, & Yoo, 2016) . Along with variations extensively dispersed throughout the S gene, it would be interesting to identify mutations in these nsp genes, including

INDELs that possibly contribute to the pathogenesis of PEDV. A majority of the non-silent point mutations, which appeared to have resulted from their continuous accumulation in the field over the past 3-4 years, occurred in the ORF1ab region encoding 16 nsps, particularly in nsp3. These 29-41 variations seem to be significant since the cell culture-attenuated G2b strain contained only 4-aa changes over 100 serial passages when compared to the virulent parental strain . Although no INDELs arose in the attenuated G2b virus S DEL5/ORF3 , the attenuated G1a-derived vaccine strains possess an 8-aa DEL in nsp3, which overlaps or is located 12 or 18 aa downstream of those found in the recent G2b field TA B L E 4 Nucleotide and amino acid changes of KNU-1807 during serial passages in cell culture strains, implicating the potential involvement of the nsp3 DEL in attenuation ( Figure 1b) . Although nsp3 critically acts as a PL pro that post-transcriptionally cleaves replicase polyproteins into functional nsps, the DELs present in the Glu-rich acidic region of nsp3 have no effect on its own roles and therefore are dispensable for the replication of coronaviruses, including PEDV (Lee & Lee, 2018; Lei, Kusov, & Hilgenfeld, 2018) . Consistent with our previous study (Lee & Lee, 2018) , this tolerance for the nsp3 DEL during PEDV replication was reproduced in the current study as shown in 

to contain a homologous 3-aa DEL in nsp3, consistent with a typical farm-to-farm transmission of PEDV via lax biosecurity.

In addition to genetic drift under field conditions, an outstanding DEL in the cytosolic endodomain of S was identified in the novel nsp3-DEL PEDV KNU-1807 isolate since the first productive passage in cell culture (P4) and subsequently retained, thereby resulting in a 9-aa DEL at the end of the KNU-1807-P4 S protein. Like other coronaviruses, the PEDV S glycoprotein can be functionally divided into two subdomains, S1 responsible for binding to cell receptor(s) and S2 involved in direct fusion between the viral and cellular membranes (Lee, 2015) . The last 5-aa (KVHVQ) of the cytoplasmic tail of S is known to be a potential ER retrieval signal with its KxHxx motif and loss of this motif increases cell fusion activity by defecting the ER retention of S protein and promoting their transport to the cell surface Lin et al., 2017; Shirato et al., 2011) . Since KNU-1807-P4 exhibited a premature termination of the S protein by 9-aa residues (EVFEKVHVQ) that included the ER retrieval motif, it would be anticipated that this strain would demonstrate increased fusion activity in vitro. Our data revealed that the KNU-1807 strain in the S gene, resulting in a stop codon and a 9-aa (EVFEKVHVQ)

DEL. The S C-DEL9 identified in this study differed from aforementioned strains in that the early termination was caused by a combination of a novel nt DEL and a −1 frameshift event that occurred in the most primitive cell culture passage P4. It is possible that the S DEL naturally emerged during the initial culturing in the Vero cell via an unknown mechanism. Indeed, the occurrence of a large 197-aa S DEL in the N-terminus of the S protein that was absent in the original sample has been reported during the first cell culture passage of the US G2b PC177 strain (Oka et al., 2014) .

We were consistently unable to detect the same S C-DEL9 in the original KNU-1807-SI sample by deep genome sequencing (data not shown). However, since coronaviruses are known to innately exist as quasispecies or as mixed populations of several strains (Zhang et al., 2007) , we cannot exclude the possibility that this variant was initially present as a minor proportion in the clinical sample and was able to replicate more competently in cell culture instead of the emergence of the extraordinarily DEL during the earliest stages of cell adaption. Similarly, a Chinese field G2b strain, FL2013, that naturally contains a unique 21-nt DEL in the extreme C-terminus of its S protein, leading to a 7-aa (FEKVHVQ ) S C-DEL pattern, has been found to have reduced virulence to newborn piglets (Zhang et al., 2015) . Thus, it is important to further surveil whether S C-DEL variant strains naturally circulate in Korean pig populations.

Since a growing body of evidence proposes the early termination of the S protein and the extinction of its ER retention signal as being a marker for PEDV attenuation, we investigated whether the virulence of the KNU-1807 virus would be markedly less than that previously reported for PEDV pathogenesis. Our data revealed that despite similarities of macroscopic and microscopic small intestine lesions, the KNU-1807 virus possessed weakened pathogenicity in experimentally inoculated piglets compared to the virulent KNU-141112 strain in terms of disease severity of clinical presentation, including the mortality rate and onset of virus shedding, indicating the involvement of the extreme C-terminal region of the S protein in virulence. However, we were unable to observe a fully attenuated phenotypes for KNU-1807 in inoculated animals compared with those seen in our previous study .

This suggested that DEL mutations in several parts of the S protein In conclusion, PEDV continues to endemically affect pig farms on Jeju Island, giving rise to moderate-to-severe clinical disease associated with PED in infected piglets. However, the loss of neonates to death varies among litters and between farms and is less than that reported for severe G2b epidemics, which approach 100% mortality in newborn piglets. This mild-to-moderate outbreak scenario seen on Jeju Island may be due to herd immunity developed from vaccination and intentional infection since the re-emergence of PEDV in the area. Another possibility is that genetic variation and/or DEL in nsps and S protein have arisen under field circumstances leading to the evasion of host immune defenses, such as IFN and neutralizing antibodies, and consequently alter viral pathogenicity, leading to endemic and low-pathogenic outbreaks in the field. Thus, cutting-edge research using reverse genetics will be necessary to provide fundamental insights into the specific role of nsp and S gene mutations in PEDV pathogenesis. More importantly, the current study confirmed that the contemporary field G2b isolates have nearly 4% amino acid sequence divergence in the S protein compared to the 2013-2014 domestic G2b strains. This mutation rate is approximately twice as high as that in recent mainland strains of PEDV (Lee & Lee, 2018) .

Furthermore, the evolutionary rate estimated for the S gene of PEDV G2b Jeju Island strains was 14.80 × 10 -4 substitutions/site/ year, whereas that for the G2b mainland strains was 7.18 × 10 -4 substitutions/site/year (Lee & Lee, 2018) . These results indicated that ongoing genetic drift appears to be faster on Jeju Island than 

The authors declare that they have no conflict of interest.

Changhee Lee https://orcid.org/0000-0002-5930-5461

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