Genotypic characterization of novel S-DEL variants of porcine epidemic diarrhea virus identified in South Korea.

The highly pathogenic genotype 2b (HP-G2b) of porcine epidemic diarrhea virus (PEDV), which caused a pandemic in 2013-2014, evolved in South Korea and became endemic, affecting the domestic pig industry. This study describes the genotypic traits of novel HP-G2b PEDV strains identified on affected farms experiencing low disease severity with < 10% neonatal mortality. Nucleotide sequencing revealed common deletion patterns, termed S-DEL2, resulting in a two-amino-acid deletion at positions 60 and 61, 61 and 62, or 63 and 64 in the N-terminal domain of the spike (S) protein of all isolates. The S barcode profiles of S-DEL2 variants differed from each other and shared 96.0-99.4% and 98.5-99.6% nt sequence identity with other South Korean HP-G2b PEDV strains in the S gene and in the complete genome sequence, respectively. Genetic and phylogenetic analysis showed that the S-DEL2 strains belonged to diverse domestic clades: CK, CK.1, CK.2, or NC. The emergence of novel S-DEL2 strains suggests that continuous evolution of PEDV occurs under endemic circumstances, resulting in genetic diversity and distinct clinical presentations. This study advances our knowledge regarding the genetic and pathogenic heterogeneity of PEDV and emphasizes the importance of active monitoring and surveillance to identify novel variants and determine their genotypic and phenotypic characteristics.

Coinfection with porcine epidemic diarrhea virus and Clostridium perfringens type A enhances disease severity in weaned pigs.

Clostridium perfringens is a constituent of the normal gut microbiome in pigs; however, it can potentially cause pre- and post-weaning diarrhea. Nevertheless, the importance of this bacterium as a primary pathogen of diarrhea in piglets needs to be better understood, and the epidemiology of C. perfringens in Korean pig populations is unknown. To study the prevalence and typing of C. perfringens, 203 fecal samples were collected from diarrheal piglets on 61 swine farms during 2021-2022 and examined for the presence of C. perfringens and enteric viruses, including porcine epidemic diarrhea virus (PEDV). We determined that the most frequently identified type of C. perfringens was C. perfringens type A (CPA; 64/203, 31.5%). Among the CPA infections, single infections with CPA (30/64, 46.9%) and coinfections with CPA and PEDV (29/64, 45.3%) were the most common in diarrheal samples. Furthermore, we conducted animal experiments to investigate the clinical outcome of single infections and coinfections with highly pathogenic (HP)-PEDV and CPA in weaned piglets. The pigs infected with HP-PEDV or CPA alone showed mild or no diarrhea, and none of them died. However, animals that were co-inoculated with HP-PEDV and CPA showed more-severe diarrheal signs than those of the singly infected pigs. Additionally, CPA promoted PEDV replication in coinfected piglets, with high viral titers in the feces. A histopathological examination revealed more-severe villous atrophy in the small intestine of coinfected pigs than in singly infected pigs. This indicates a synergistic effect of PEDV and CPA coinfection on clinical disease in weaned piglets.

Trypsin enhances SARS-CoV-2 infection by facilitating viral entry.

Coronaviruses infect cells by cytoplasmic or endosomal membrane fusion, driven by the spike (S) protein, which must be primed by proteolytic cleavage at the S1/S2 furin cleavage site (FCS) and the S2' site by cellular proteases. Exogenous trypsin as a medium additive facilitates isolation and propagation of several coronaviruses in vitro. Here, we show that trypsin enhances severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in cultured cells and that SARS-CoV-2 enters cells via either a non-endosomal or an endosomal fusion pathway, depending on the presence of trypsin. Interestingly, trypsin enabled viral entry at the cell surface and led to more efficient infection than trypsin-independent endosomal entry, suggesting that trypsin production in the target organs may trigger a high level of replication of SARS-CoV-2 and cause severe tissue injury. Extensive syncytium formation and enhanced growth kinetics were observed only in the presence of exogenous trypsin when cell-adapted SARS-CoV-2 strains were tested. During 50 serial passages without the addition of trypsin, a specific R685S mutation occurred in the S1/S2 FCS (681PRRAR685) that was completely conserved but accompanied by several mutations in the S2 fusion subunit in the presence of trypsin. These findings demonstrate that the S1/S2 FCS is essential for proteolytic priming of the S protein and fusion activity for SARS-CoV-2 entry but not for viral replication. Our data can potentially contribute to the improvement of SARS-CoV-2 production for the development of vaccines or antivirals and motivate further investigations into the explicit functions of cell-adaptation-related genetic drift in SARS-CoV-2 pathogenesis.

Development of the new microsatellite markers of Lucilia sericata (Diptera: Calliphoridae) from Korea.

BACKGROUND: Lucilia sericata is a medical and veterinary important insect species because its larvae feed on tissues of vertebrates including humans. Very few microsatellite makers have been reported from the species to illuminate its genetic variability and population genetic structure. METHODS AND RESULTS: In this study, L. sericata samples were collected from four different localities in Korea to develop the microsatellite markers to provide basic information on the genetic variability and population genetic structure in Korea of this species. In total, ten new microsatellite markers were sequenced and analyzed. Genetic diversity was performed using these microsatellite markers. The observed heterozygosity varied from 0.205 to 0.824, with an average of 0.546. The expected heterozygosity ranged from 0.579 to 0.886, with an average of 0.804. PIC value varied from 0.553 to 0.876. CONCLUSIONS: The markers developed in the present study are expected as informative for estimating genetic diversity of L. sericata.

Thiol-disulfide status regulates quality control of prion protein at the plasma membrane.

Rapid endoplasmic reticulum (ER) stress-induced export (RESET) is undoubtedly beneficial in that it eliminates misfolded prion protein (PrP) from a stressed ER. Considering that RESET induces rapid endocytosis of misfolded PrP for degradation, it is questionable whether RESET is beneficial when its product amount overwhelms the capacity of subsequent clearance pathways. We require a strategy to monitor the endocytic flux rate of misfolded PrPs. Here, we stabilized misfolded PrPs by inserting red fluorescent protein (RFP) and indirectly determined this rate by monitoring the lysosomal free RFP. We discovered a surveillance mechanism that limits endocytosis of misfolded PrPs through plasma membrane quality control (pmQC). pmQC was regulated by the thiol-disulfide status of misfolded PrPs and consequently accumulates nonpathogenic PrP variants at the plasma membrane. This variant alleviated prion proteotoxicity induced by persistent RESET. Thus, PrP endocytosis is regulated by pmQC to ensure the safety of endolysosomal pathway from persistent internalization of misfolded PrP.-Lee, D., Lee, S., Shin, Y., Song, Y., Kang, S.-W. Thiol-disulfide status regulates quality control of prion protein at the plasma membrane.

Single octapeptide deletion selectively processes a pathogenic prion protein mutant on the cell surface.

The number of octapeptide repeats has been considered to correlate with clinical and pathogenic phenotypes of prion diseases resulting from aberrant metabolism of prion protein (PrP). However, it is still poorly understood how this motif affects PrP metabolism. Here, we discover homozygous single octapeptide repeat deletion mutation in the PRNP gene encoding PrP in HeLa cells. The level of PrP proves to be unaffected by this mutation alone, but selectively reduced by additional pathogenic mutations within internal hydrophobic region of PrP. The pattern and relative amount of newly synthesized A117V mutant is unaffected, whereas the mutant appears to be differentially distributed and processed on the cell surface by single octapeptide deletion. This study provides an insight into a novel mutant-specific metabolism of PrP on the cell surface.

Porcine epidemic diarrhea virus: an update overview of virus epidemiology, vaccines, and control strategies in South Korea.

Porcine epidemic diarrhea virus (PEDV) has posed significant financial threats to the domestic pig industry over the last three decades in South Korea. PEDV infection will mostly result in endemic persistence in the affected farrow-to-finish (FTF) herds, leading to endemic porcine epidemic diarrhea (PED) followed by year-round recurrent outbreaks. This review aims to encourage collaboration among swine producers, veterinarians, and researchers to offer answers that strengthen our understanding of PEDV in efforts to prevent and control endemic PED and to prepare for the next epidemics or pandemics. We found that collaboratively implementing a PED risk assessment and customized four-pillar-based control measures is vital to interrupt the chain of endemic PED in affected herds: the former can identify on-farm risk factors while the latter aims to compensate for or improve weaknesses via herd immunity stabilization and virus elimination. Under endemic PED, long-term virus survival in slurry and asymptomatically infected gilts ("Trojan Pigs") that can transmit the virus to farrowing houses are key challenges for PEDV eradication in FTF farms and highlight the necessity for active monitoring and surveillance of the virus in herds and their environments. This paper underlines the current knowledge of molecular epidemiology and commercially available vaccines, as well as the risk assessment and customized strategies to control PEDV. The intervention measures for stabilizing herd immunity and eliminating virus circulation may be the cornerstone of establishing regional or national PED eradication programs.

A case study on DNA barcoding for pet food mislabeling in South Korea.

Due to the close relationship between pets and humans, pet owners are highly invested in proper diets for their pets. Even though pet food mislabeling is concerning, there are few studies on this topic. This study investigated pet food mislabeling in South Korea's market based on DNA barcoding. In total, 10 pet food products were purchased, and 200 sequences of the partial Cytochrome c oxidase subunit 1 (COI) gene were generated from clones of the samples. The obtained sequences were compared to available public databases to identify species present in the ingredients. The data analyses showed that the labeled species were consistent with species detected by COI sequences in 6 of the products. However, the expected species were not detected in 4 products, revealing possible mislabeling in these samples. Our findings indicated that DNA barcoding might represent a promising tool to detect pet food mislabeling.

Development of a Next-Generation Vaccine Platform for Porcine Epidemic Diarrhea Virus Using a Reverse Genetics System.

For the past three decades, the porcine epidemic diarrhea virus (PEDV) has remained an enormous threat to the South Korean swine industry. The scarcity of an effective method for manipulating viral genomes has impeded research progress in PEDV biology and vaccinology. Here, we report the development of reverse genetics systems using two novel infectious full-length cDNA clones of a Korean highly pathogenic-G2b strain, KNU-141112, and its live attenuated vaccine strain, S DEL5/ORF3, in a bacterial artificial chromosome (BAC) under the control of a eukaryotic promoter. Direct transfection of cells with each recombinant BAC clone induced cytopathic effects and produced infectious progeny. The reconstituted viruses, icKNU-141112 and icS DEL5/ORF3, harboring genetic markers, displayed phenotypic and genotypic properties identical to their respective parental viruses. Using the DNA-launched KNU-141112 infectious cDNA clone as a backbone, two types of recombinant viruses were generated. First, we edited the open reading frame 3 (ORF3) gene, as cell-adapted strains lose full-length ORF3, and replaced this region with an enhanced green fluorescent protein (EGFP) gene to generate icPEDV-EGFP. This mutant virus presented parental virus-like growth kinetics and stably retained robust EGFP expression, indicating that ORF3 is dispensable for PEDV replication in cell culture and is a tolerant location for exogeneous gene acceptance. However, the plaque size and syncytia phenotypes of ORF3-null icPEDV-EGFP were larger than those of icKNU-141112 but similar to ORF3-null icS DEL5/ORF3, suggesting a potential role of ORF3 in PEDV cytopathology. Second, we substituted the spike (S) gene with a heterologous S protein, designated S51, from a variant of interest (VOI), which was the most genetically and phylogenetically distant from KNU-141112. The infectious recombinant VOI, named icPEDV-S51, could be recovered, and the rescued virus showed indistinguishable growth characteristics compared to icKNU-141112. Virus cross-neutralization and structural analyses revealed antigenic differences in S between icKNU-141112 and icPEDV-S51, suggesting that genetic and conformational changes mapped within the neutralizing epitopes of S51 could impair the neutralization capacity and cause considerable immune evasion. Collectively, while the established molecular clones afford convenient, versatile platforms for PEDV genome manipulation, allowing for corroborating the molecular basis of viral replication and pathogenesis, they also provide key infrastructural frameworks for developing new vaccines and coronaviral vectors.

Role of redox-sensitive catalytic interaction with ADAM10 in mutant-selective extracellular shedding of prion protein.

Misfolded glycosylphosphatidylinositol-anchored prion protein (PrP) is primarily degraded in lysosomes but is often rapidly removed from the cell surface before endocytosis in a preemptive manner. However, this mechanism is poorly understood. In this study, we discovered a disease-causing prion mutation (Q212P) that exceptionally promoted the extracellular release of PrP. Spatiotemporal analyses combined with genome editing identified the role of sheddase ADAM10 in Q212P shedding from the cell surface. ADAM10 was observed to catalytically interacts with Q212P but non-catalytically with wild-type PrP (wtPrP). This intrinsic difference in the interaction of ADAM10 between Q212P and wtPrP allowed Q212P to selectively access the sheddase activity of ADAM10 in a redox-sensitive manner. In addition, redox perturbation instigated the latent misfolding propensity of Q212P and disrupted the catalytic interaction between PrP and ADAM10, resulting in the accumulation of misfolded PrP on the cell surface. Upon recovery, active ADAM10 was able to reversibly release the surface Q212P. However, it might prove detrimental if unregulated resulting in unexpected proteotoxicity. This study provides a molecular basis of the mutant-selective shedding of PrP by demonstrating the catalytic interaction of ADAM10 with Q212P.

Mutant-selective topologic conversion facilitates selective degradation of a pathogenic prion isoform.

Regulating protein import across the endoplasmic reticulum (ER) membrane occasionally results in the synthesis of topologically unnatural variants, and their accumulation often leads to proteotoxicity. However, since this is a regulated process, it is questionable whether the topological rearrangement really has adverse consequences. In the present study, we provide an insight into the functional benefit of translocational regulation by illustrating mutant-selective topologic conversion (MSTC) and demonstrate that MSTC contributes to selective degradation of a membrane-anchored prion protein isoform (ctmPrP). We find that ctmPrP is inherently short-lived and topologically competent for degradation rather than accumulation. MSTC achieves, cotranslationally, the unique topology of ctmPrP during translocation, facilitating selective ctmPrP degradation from the ER via the proteasome-dependent pathway before entering the secretory pathway. At this time, the N-terminal polycationic cluster is essential for MSTC, and its cytosolic exposure acquires "ERAD-degron"-like activity for ctmPrP. Bypassing MSTC delays ctmPrP degradation, thus increasing prion proteotoxicity. Thus, topological rearrangement is used for the MSTC as a part of the protein quality control pathway to ensure the safety of the secretory pathway from misfolded PrP.