Sunhee Lee

Outbreak-Related Porcine Epidemic Diarrhea Virus Strains Similar to US Strains, South Korea, 2013

In late 2013, outbreaks of porcine epidemic diarrhea virus (PEDV) infection recurred in South Korea. Genetic and phylogenetic analyses showed that isolates from the outbreaks were most closely related to emergent US strains of PEDV. These US strain–like PEDV variants are prevalent in South Korea and responsible for recent outbreaks in the country.

Complete Genome Characterization of Korean Porcine Deltacoronavirus Strain KOR/KNU14-04/2014

ABSTRACT In April 2014, porcine deltacoronavirus (PDCoV) was first identified in feces from diarrheic piglets in South Korea and found to be closely related to other PDCoV strains. The complete genome of the Korean PDCoV strain, KOR/KNU14-04/2014, was sequenced and analyzed to characterize PDCoV circulating in South Korea.

Isolation and characterization of a Korean porcine epidemic diarrhea virus strain KNU-141112.

Abstract Severe outbreaks of porcine epidemic diarrhea virus (PEDV) have re-emerged in Korea and rapidly swept across the country, causing tremendous economic losses to producers and customers. Despite the availability of PEDV vaccines in the domestic market, the disease continues to plague the Korean pork industry, raising issues regarding their protective efficacy and new vaccine development. Therefore, PEDV isolation in cell culture is urgently needed to develop efficacious vaccines and diagnostic assays and to conduct further studies on the virus biology. In the present study, one Korean PEDV strain, KOR/KNU-141112/2014, was successfully isolated and serially propagated in Vero cells for over 30 passages. The in vitro and in vivo characteristics of the Korean PEDV isolate were investigated. Virus production in cell culture was confirmed by cytopathology, immunofluorescence, and real-time RT-PCR. The infectious virus titers of the viruses during the first 30 passages ranged from 105.1 to 108.2 TCID50 per ml. The inactivated KNU-141112 virus was found to mediate potent neutralizing antibody responses in immunized guinea pigs. Animal studies showed that KNU-141112 virus causes severe diarrhea and vomiting, fecal shedding, and acute atrophic enteritis, indicating that strain KNU-141112 is highly enteropathogenic in the natural host. In addition, the entire genomes or complete S genes of KNU-141112 viruses at selected cell culture passages were sequenced to assess the genetic stability and relatedness. Our genomic analyses indicated that the Korean isolate KNU-141112 is genetically stable during the first 30 passages in cell culture and is grouped within subgroup G2b together with the recent re-emergent Korean strains.

Full-genome sequence analysis of a variant strain of porcine epidemic diarrhea virus in South Korea.

ABSTRACT In March of 2014, a variant of novel porcine epidemic diarrhea virus (PEDV) was first identified in South Korea and found to be most closely related to the U.S. variant strain OH851. The complete genome of the KOR/KNU-1406/2014 strain was sequenced and analyzed to investigate the U.S.-strain-like variant circulating in South Korea.

Functional characterization and proteomic analysis of the nucleocapsid protein of porcine deltacoronavirus.

Abstract Porcine deltacoronavirus (PDCoV) is a newly discovered enterotropic swine coronavirus that causes enteritis and diarrhea in piglets. Like other coronaviruses, PDCoV commonly contains 4 major structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. Among these, the N protein is known to be the most abundant and multifunctional viral component. Therefore, as the first step toward understanding the biology of PDCoV, the present study investigated functional characteristics and expression dynamics of host proteins in a stable porcine cell line constitutively expressing the PDCoV N protein. Similar to N proteins of other coronaviruses, the PDCoV N protein was found to interact with itself to form non-covalently linked oligomers and was mainly localized to the nucleolus. We then assessed alterations in production levels of proteins in the N-expressing PK (PK-PDCoV-N) cells at different time points by means of proteomic analysis. According to the results of high-resolution two-dimensional gel electrophoresis, a total of 43 protein spots were initially found to be differentially expressed in PK-PDCoV-N cells in comparison with control PK cells. Of these spots, 10 protein spots showed a statistically significant alteration, including 8 up-regulated and 2 down-regulated protein spots and were picked for subsequent protein identification by peptide mass fingerprinting following matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The affected cellular proteins that we identified in this study were classified into the functional groups involved in various cellular processes such as cell division, metabolism, the stress response, protein biosynthesis and transport, cytoskeleton networks and cell communication. Notably, two members of the heat shock protein 70 family were found to be up-regulated in PK-PDCoV-N cells. These proteomic data will provide insights into the specific cellular response to the N protein during PDCoV infection.

Efficacy of an inactivated genotype 2b porcine epidemic diarrhea virus vaccine in neonatal piglets

Abstract Massive outbreaks of porcine epidemic diarrhea virus (PEDV) recurred in South Korea in 2013–2014 and affected approximately 40% of the swine breeding herds across the country, incurring a tremendous financial impact on producers and consumers. Despite the nationwide use of commercially available attenuated and inactivated vaccines in South Korea, PEDV has continued to plague the domestic pork industry, raising concerns regarding their protective efficacies and the need for new vaccine development. In a previous study, we isolated and serially cultivated a Korean PEDV epidemic strain, KOR/KNU-141112/2014, in Vero cells. With the availability of a cell culture-propagated PEDV strain, we are able to explore vaccination and challenge studies on pigs. Therefore, the aim of the present study was to produce an inactivated PEDV vaccine using the KNU-141112 strain and evaluate its effectiveness in neonatal piglets. Pregnant sows were immunized intramuscularly with the inactivated adjuvanted monovalent vaccine at six and three weeks prior to farrowing. Six-day-old piglets born to vaccinated or unvaccinated sows were challenged with the homogeneous KNU-141112 virus. The administration of the inactivated vaccine to sows greatly increased the survival rate of piglets challenged with the virulent strain, from 0% to approximately 92% (22/24), and significantly reduced diarrhea severity including viral shedding in feces. In addition, litters from unvaccinated sows continued to lose body weight throughout the experiment, whereas litters from vaccinated sows started recovering their daily weight gain at 7 days after the challenge. Furthermore, strong neutralizing antibody responses to PEDV were verified in immunized sows and their offspring, but were absent in the unvaccinated controls. Altogether, our data demonstrated that durable lactogenic immunity was present in dams administrated with the inactivated vaccine and subsequently conferred critical passive immune protection to their own litters against virulent PEDV infection.

Genetic characteristics, pathogenicity, and immunogenicity associated with cell adaptation of a virulent genotype 2b porcine epidemic diarrhea virus

Abstract Porcine epidemic diarrhea virus (PEDV) has emerged or re-emerged worldwide, posing a significant financial threat to major pig-producing countries. In the present study, a virulent Korean pandemic PEDV strain, KNU-141112, was serially propagated in Vero cells for up to 100 passages. Through cell culture adaptation, we obtained four distinct deletion (DEL) mutants by plaque purification followed by nucleotide sequencing of the spike (S)/ORF3 gene-coding region, which were designated KNU-141112-S DEL2, −S DEL5, −S DEL2/ORF3, and −S DEL5/ORF3. Further whole genome sequencing identified 12 or 14 amino acid changes in the cell-adapted DEL strains. Animal inoculation studies revealed that the virulence of both S DEL2/ORF3 and S DEL5/ORF3 viruses with a large 46-nt deletion in the intergenic portion of S and ORF3 was remarkably diminished, indicating viral attenuation in the natural host. Furthermore, these cell-adapted strains elicited potent neutralizing antibody responses in immunized pigs. Taken together, our data indicate that the cell-attenuated S DEL2/ORF3 and S DEL5/ORF3 strains are promising candidates for the development of a safe and effective live PEDV vaccine.

Pathogenicity and genetic characteristics associated with cell adaptation of a virulent porcine reproductive and respiratory syndrome virus nsp2 DEL strain CA-2

Porcine reproductive and respiratory syndrome virus (PRRSV) is the most common and world-widespread viral pathogen of swine. We previously reported genomic sequences and pathogenicity of type 2 Korean PRRSV strains belonging to the virulent lineage 1 family, which contain remarkable amino acid deletions in nonstructural protein 2 (nsp2 DEL) compared to VR-2332. Here, a virulent type 2 Korean PRRSV nsp2 DEL strain, CA-2, was serially propagated in MARC-145 cells for up to 100 passages (CA-2-P100). As the passage number increased, the phenotypic characteristics of cell-adapted CA-2 strains were altered, in terms of higher viral titers and larger plaque sizes compared to the parental virus. Pro-inflammatory cytokine genes, including TNF-α, IL-8, MCP-1, and MCP-2, were found to be significantly down-regulated in PAM cells with the CA-2-P100 strain compared to its parental nsp2 DEL virus. Animal inoculation studies demonstrated that the virulence of CA-2-P100 was reduced significantly, with showing normal weight gain, body temperatures, and lung lesions comparable to the control group. Furthermore, high-passage CA-2-P100 showed declined and transient viremia kinetics, as well as delayed and low PRRSV-specific antibody responses in infected pigs. In addition, we determined whole genome sequences of low to high-passage derivatives of CA-2. The nsp2 DEL pattern was conserved for 100 passages, whereas no other deletions or insertions arose during the cell adaptation process. However, CA-2-P100 possessed 54 random nucleotide substitutions that resulted in 27 amino acid changes distributed throughout the genome, suggesting that these genetic drifts provide a possible molecular basis correlated with the cell-adapted features in vitro and the attenuated phenotype in vivo. Taken together, our data indicate that the cell-attenuated CA-2-P100 strain is a promising candidate for developing a safe and effective live PRRSV vaccine.

Genomic and antigenic characterization of porcine epidemic diarrhoea virus strains isolated from South Korea, 2017

Summary Porcine epidemic diarrhoea virus (PEDV) is a globally emerging and re‐emerging enteric coronavirus in pigs causing serious economic threats to the world swine industry. Since the re‐emergence of massive PEDV outbreaks in South Korea in 2013−2014, domestic pig farms have continued to experience PED epidemics or endemics. This study represents the molecular characterization of PEDV isolates identified in diarrhoeic animals collected across the country in 2017. Initial sequencing analysis of the full‐length S genes revealed that 70% of the 2017 isolates (7/10) belong to the G2b subgroup, while the remaining isolates were classified as G1b. The data indicated that both variant G1b and global epidemic G2b strains were responsible for current PED outbreaks in South Korea. The 2017 G1b and G2b isolates shared 98.7%–99.4% and 98.1%–99.2% amino acid sequence identity at the S gene level and 99.3% and 99.0%–99.6% nucleotide sequence homology at the genome level compared to the corresponding Korean prototype G1b and G2b strains, respectively. In an interesting manner, one G2b‐like KNU‐1705 strain was found to possess a large 39‐nucleotide deletion in the ORF1a region theoretically encoding nonstructural protein 3. Phylogenetic analysis based on the entire genome and spike protein sequences indicated that the 2017 isolates were most closely related to other global G1b or G2b strains but formed different branches within the same genogroup. These results indicate that PEDVs undergo continuous evolution in the field. In addition, one 2017 PEDV strain, KOR/KNU‐1705/2017, was successfully isolated and propagated in Vero cells. The antisera raised against the Korean prototype 2014 G2b strain efficiently neutralized KNU‐1705 virus infection, suggesting antigenic homology between the 2014 and 2017 PEDV strains. Our data advance the understanding of the molecular epidemiology and antigenicity of PEDV circulating in South Korea.

Phenotypic and genotypic analyses of an attenuated porcine reproductive and respiratory syndrome virus strain after serial passages in cultured porcine alveolar macrophages

The porcine reproductive and respiratory syndrome virus (PRRSV) is a globally ubiquitous swine viral pathogen that causes major economic losses worldwide. We previously reported an over-attenuated phenotype of cell-adapted PRRSV strain CA-2-P100 in vivo. In the present study, CA-2-P100 was serially propagated in cultured porcine alveolar macrophage (PAM) cells for up to 20 passages to obtain the derivative strain CA-2-MP120. Animal inoculation studies revealed that both CA-2-P100 and CA-2-MP120 had decreased virulence, eliciting weight gains, body temperatures, and histopathologic lesions similar to those in the negative control group. However, compared to CA-2-P100 infection, CA-2-MP120 yielded consistently higher viremia kinetics and enhanced antibody responses in pigs. All pigs inoculated with CA-2-MP120 developed viremia and seroconverted to PRRSV. During 20 passages in PAM cells, CA-2-MP120 acquired 15 amino acid changes that were mostly distributed in nsp2 and minor structural protein-coding regions. Among these changes, 6 mutations represented reversions to the sequences of the reference CA-2 and parental CA-2-P20 strains. These genetic drifts may be hypothetical molecular markers associated with PRRSV macrophage tropism and virulence. Our results indicate that the PAM-passaged CA-2-MP120 strain is a potential candidate for developing a live, attenuated PRRSV vaccine.