Identification of a novel risk factor for chronic wasting disease (CWD) in elk: S100G single nucleotide polymorphism (SNP) of the prion protein gene (PRNP).

Prion diseases are fatal and malignant infectious encephalopathies induced by the pathogenic form of prion protein (PrPSc) originating from benign prion protein (PrPC). A previous study reported that the M132L single nucleotide polymorphism (SNP) of the prion protein gene (PRNP) is associated with susceptibility to chronic wasting disease (CWD) in elk. However, a recent meta-analysis integrated previous studies that did not find an association between the M132L SNP and susceptibility to CWD. Thus, there is controversy about the effect of M132L SNP on susceptibility to CWD. In the present study, we investigated novel risk factors for CWD in elk. We investigated genetic polymorphisms of the PRNP gene by amplicon sequencing and compared genotype, allele, and haplotype frequencies between CWD-positive and CWD-negative elk. In addition, we performed a linkage disequilibrium (LD) analysis by the Haploview version 4.2 program. Furthermore, we evaluated the 3D structure and electrostatic potential of elk prion protein (PrP) according to the S100G SNP using AlphaFold and the Swiss-PdbViewer 4.1 program. Finally, we analyzed the free energy change of elk PrP according to the S100G SNP using I-mutant 3.0 and CUPSAT. We identified 23 novel SNP of the elk PRNP gene in 248 elk. We found a strong association between PRNP SNP and susceptibility to CWD in elk. Among those SNP, S100G is the only non-synonymous SNP. We identified that S100G is predicted to change the electrostatic potential and free energy of elk PrP. To the best of our knowledge, this was the first report of a novel risk factor, the S100G SNP, for CWD.

Incomplete glycosylation during prion infection unmasks a prion protein epitope that facilitates prion detection and strain discrimination.

The causative factors underlying conformational conversion of cellular prion protein (PrPC) into its infectious counterpart (PrPSc) during prion infection remain undetermined, in part because of a lack of monoclonal antibodies (mAbs) that can distinguish these conformational isoforms. Here we show that the anti-PrP mAb PRC7 recognizes an epitope that is shielded from detection when glycans are attached to Asn-196. We observed that whereas PrPC is predisposed to full glycosylation and is therefore refractory to PRC7 detection, prion infection leads to diminished PrPSc glycosylation at Asn-196, resulting in an unshielded PRC7 epitope that is amenable to mAb recognition upon renaturation. Detection of PRC7-reactive PrPSc in experimental and natural infections with various mouse-adapted scrapie strains and with prions causing deer and elk chronic wasting disease and transmissible mink encephalopathy uncovered that incomplete PrPSc glycosylation is a consistent feature of prion pathogenesis. We also show that interrogating the conformational properties of the PRC7 epitope affords a direct means of distinguishing different prion strains. Because the specificity of our approach for prion detection and strain discrimination relies on the extent to which N-linked glycosylation shields or unshields PrP epitopes from antibody recognition, it dispenses with the requirement for additional standard manipulations to distinguish PrPSc from PrPC, including evaluation of protease resistance. Our findings not only highlight an innovative and facile strategy for prion detection and strain differentiation, but are also consistent with a mechanism of prion replication in which structural instability of incompletely glycosylated PrP contributes to the conformational conversion of PrPC to PrPSc.

Identification of the prion-related protein gene (PRNT) sequences in various species of the Cervidae family.

Chronic wasting disease (CWD) is caused by abnormal deleterious prion protein (PrPSc), and transmissible spongiform encephalopathy occurs in the Cervidae family. In recent studies, the susceptibility of prion disease has been affected by polymorphisms of the prion gene family. However, the study of the prion-related protein gene (PRNT) is rare, and the DNA sequence of this gene was not fully reported in all Cervidae families. In the present study, we amplified and first identified PRNT DNA sequences in the Cervidae family, including red deer, elk, sika deer and Korean water deer, using polymerase chain reaction (PCR). We aligned nucleotide sequences of the PRNT gene and the amino acid sequences of prion-related protein (Prt) protein among several species. In addition, we performed phylogenetic analysis to measure the evolutionary relationships of the PRNT gene in the Cervidae family. Furthermore, we performed homology modeling of the Prt protein using SWISS-MODEL and compared the structure of Prt protein between sheep and the Cervidae family using the Swiss-PdbViewer program. We obtained much longer PRNT sequences of red deer compared to the PRNT gene sequence registered in GenBank. Korean water deer denoted more close evolutionary distances with goats and cattle than the Cervidae family. We found 6 Cervidae family-specific amino acids by the alignment of Prt amino acid sequences. There are significantly different distributions of hydrogen bonds and the atomic distance of the N-terminal tail and C-terminal tail between sheep and the Cervidae family. We also detected the mRNA expression of PRNT gene in 3 tissues investigated. To our knowledge, this report is the first genetic study of the PRNT gene in the Cervidae family.

African Swine Fever Virus in Pork Brought into South Korea by Travelers from China, August 2018.

We tested samples of pork products confiscated from travelers to South Korea for African swine fever virus (ASFV). We detected ASFV in 4 food items confiscated from travelers from Shenyang, China, in August 2018. Surveillance of pork products at country entry points is needed to mitigate the risk for ASFV introduction.

Quinacrine promotes replication and conformational mutation of chronic wasting disease prions.

Quinacrine's ability to reduce levels of pathogenic prion protein (PrP(Sc)) in mouse cells infected with experimentally adapted prions led to several unsuccessful clinical studies in patients with prion diseases, a 10-y investment to understand its mechanism of action, and the production of related compounds with expectations of greater efficacy. We show here, in stark contrast to this reported inhibitory effect, that quinacrine enhances deer and elk PrP(Sc) accumulation and promotes propagation of prions causing chronic wasting disease (CWD), a fatal, transmissible, neurodegenerative disorder of cervids of uncertain zoonotic potential. Surprisingly, despite increased prion titers in quinacrine-treated cells, transmission of the resulting prions produced prolonged incubation times and altered PrP(Sc) deposition patterns in the brains of diseased transgenic mice. This unexpected outcome is consistent with quinacrine affecting the intrinsic properties of the CWD prion. Accordingly, quinacrine-treated CWD prions were comprised of an altered PrP(Sc) conformation. Our findings provide convincing evidence for drug-induced conformational mutation of prions without the prerequisite of generating drug-resistant variants of the original strain. More specifically, they show that a drug capable of restraining prions in one species/strain setting, and consequently used to treat human prion diseases, improves replicative ability in another and therefore force reconsideration of current strategies to screen antiprion compounds.

Structural effects of PrP polymorphisms on intra- and interspecies prion transmission.

Understanding the molecular parameters governing prion propagation is crucial for controlling these lethal, proteinaceous, and infectious neurodegenerative diseases. To explore the effects of prion protein (PrP) sequence and structural variations on intra- and interspecies transmission, we integrated studies in deer, a species naturally susceptible to chronic wasting disease (CWD), a burgeoning, contagious epidemic of uncertain origin and zoonotic potential, with structural and transgenic (Tg) mouse modeling and cell-free prion amplification. CWD properties were faithfully maintained in deer following passage through Tg mice expressing cognate PrP, and the influences of naturally occurring PrP polymorphisms on CWD susceptibility were accurately reproduced in Tg mice or cell-free systems. Although Tg mice also recapitulated susceptibility of deer to sheep prions, polymorphisms that provided protection against CWD had distinct and varied influences. Whereas substitutions at residues 95 and 96 in the unstructured region affected CWD propagation, their protective effects were overridden during replication of sheep prions in Tg mice and, in the case of residue 96, deer. The inhibitory effects on sheep prions of glutamate at residue 226 in elk PrP, compared with glutamine in deer PrP, and the protective effects of the phenylalanine for serine substitution at the adjacent residue 225, coincided with structural rearrangements in the globular domain affecting interaction between α-helix 3 and the loop between ß2 and α-helix 2. These structure-function analyses are consistent with previous structural investigations and confirm a role for plasticity of this tertiary structural epitope in the control of PrP conversion and strain propagation.

Epigenetic dominance of prion conformers.

Although they share certain biological properties with nucleic acid based infectious agents, prions, the causative agents of invariably fatal, transmissible neurodegenerative disorders such as bovine spongiform encephalopathy, sheep scrapie, and human Creutzfeldt Jakob disease, propagate by conformational templating of host encoded proteins. Once thought to be unique to these diseases, this mechanism is now recognized as a ubiquitous means of information transfer in biological systems, including other protein misfolding disorders such as those causing Alzheimer's and Parkinson's diseases. To address the poorly understood mechanism by which host prion protein (PrP) primary structures interact with distinct prion conformations to influence pathogenesis, we produced transgenic (Tg) mice expressing different sheep scrapie susceptibility alleles, varying only at a single amino acid at PrP residue 136. Tg mice expressing ovine PrP with alanine (A) at (OvPrP-A136) infected with SSBP/1 scrapie prions propagated a relatively stable (S) prion conformation, which accumulated as punctate aggregates in the brain, and produced prolonged incubation times. In contrast, Tg mice expressing OvPrP with valine (V) at 136 (OvPrP-V136) infected with the same prions developed disease rapidly, and the converted prion was comprised of an unstable (U), diffusely distributed conformer. Infected Tg mice co-expressing both alleles manifested properties consistent with the U conformer, suggesting a dominant effect resulting from exclusive conversion of OvPrP-V136 but not OvPrP-A136. Surprisingly, however, studies with monoclonal antibody (mAb) PRC5, which discriminates OvPrP-A136 from OvPrP-V136, revealed substantial conversion of OvPrP-A136. Moreover, the resulting OvPrP-A136 prion acquired the characteristics of the U conformer. These results, substantiated by in vitro analyses, indicated that co-expression of OvPrP-V136 altered the conversion potential of OvPrP-A136 from the S to the otherwise unfavorable U conformer. This epigenetic mechanism thus expands the range of selectable conformations that can be adopted by PrP, and therefore the variety of options for strain propagation.

Disease Course of Korean African Swine Fever Virus in Domestic Pigs Exposed Intraorally, Intranasally, Intramuscularly, and by Direct Contact with Infected Pigs.

African swine fever (ASF) is a fatal contagious disease affecting swine. The first Korean ASF virus (ASFV) isolate (Korea/Pig/Paju1/2019) was used to compare the disease course of ASFV in pigs inoculated via the four routes. In the challenge experiment, domestic pigs were infected via the intraoral (IO) and intranasal (IN) routes with a 106 50% hemadsorbing dose (HAD50) and an intramuscular (IM) injection of 103 HAD50. In the direct contact (DC) group, five naïve pigs were brought into direct contact with two IM-ASFV-infected pigs. IO-, IN-, and IM-inoculated pigs showed similar disease courses, whereas DC pigs had comparable ASF syndrome after a 7-day latent period. The disease course in the DC route, one of the most common routes of infection, was not significantly different from that in the IO and IN routes. IM and DC groups differed in terms of the severity of fever and hemorrhagic lesions in the lymph nodes and spleen, indicating that the IM route, suitable for early vaccine development trials, is not appropriate for studying the ASFV infection mechanism, including early stage of infection, and IO and IN challenges with a designated dose can be alternatives in trials for assessing ASFV pathogenicity and vaccine efficacy investigations.

Characterization of conformation-dependent prion protein epitopes.

Whereas prion replication involves structural rearrangement of cellular prion protein (PrP(C)), the existence of conformational epitopes remains speculative and controversial, and PrP transformation is monitored by immunoblot detection of PrP(27-30), a protease-resistant counterpart of the pathogenic scrapie form (PrP(Sc)) of PrP. We now describe the involvement of specific amino acids in conformational determinants of novel monoclonal antibodies (mAbs) raised against randomly chimeric PrP. Epitope recognition of two mAbs depended on polymorphisms controlling disease susceptibility. Detection by one, referred to as PRC5, required alanine and asparagine at discontinuous mouse PrP residues 132 and 158, which acquire proximity when residues 126-218 form a structured globular domain. The discontinuous epitope of glycosylation-dependent mAb PRC7 also mapped within this domain at residues 154 and 185. In accordance with their conformational dependence, tertiary structure perturbations compromised recognition by PRC5, PRC7, as well as previously characterized mAbs whose epitopes also reside in the globular domain, whereas conformation-independent epitopes proximal or distal to this region were refractory to such destabilizing treatments. Our studies also address the paradox of how conformational epitopes remain functional following denaturing treatments and indicate that cellular PrP and PrP(27-30) both renature to a common structure that reconstitutes the globular domain.

Data-driven risk assessment of the incursion of African swine fever virus via pig products brought illegally into South Korea by travelers based on the temporal relationship between outbreaks in China.

Since 2018, Asian countries have been affected by the African swine fever (ASF) virus, with major socioeconomic consequences. Moreover, the number of people traveling in Asian countries has been increasing, leading to an inevitable increase in the risk of ASF spread through livestock products carried by travelers. China and South Korea have close geo-economic ties and numerous international travelers. After the ASF outbreak in China in 2018, many illegally imported pig products (IIPPs) that were confiscated from travelers from China at the port of entry in South Korea tested positive for ASF. The detection of ASF virus (ASFV)-positive IIPPs highlights the need to further assess the risk of incursion by travelers and review the existing prevention strategies. Here, we investigated the temporal relationship between ASF outbreaks in China and the detection of ASFV-positive IIPPs in randomly confiscated samples from all ports of entry, such as flights and ships to South Korea, from 2018 to 2019 using a cross-correlation analysis. Based on the significantly correlated temporal lags between the bivariate time-series data, a risk assessment model, using the Bayesian framework, was built to estimate the distribution of the parameters for the risk assessment model and the monthly probability of ASF being introduced via IIPPs from China to South Korea. ASF outbreaks in China were significantly associated with the detection of ASFV-positive IIPPs in South Korea 5 months later. Hence, the monthly probability of ASFV-infected pig products imported from China via a traveler to South Korea was estimated to be 2.00 × 10-5, corresponding to a 0.98 mean monthly probability of at least one ASF-infected pig product arriving at ports of entry via travelers, from 2018 to 2019. To our knowledge, this study is the first attempt to estimate the risk of ASF introduction via pig products carried by international travelers to all ports from neighboring countries in the Asian region using commonly exchanged observed data. The data presented in this study can be used to refine the intervention strategies to combat the spread of transboundary animal diseases.

An Improved Duplex Real-Time Quantitative RT-PCR Assay with a Canine Endogenous Internal Positive Control for More Sensitive and Reliable Detection of Canine Parainfluenza Virus 5.

A duplex real-time quantitative reverse transcription-polymerase chain reaction (dqRT-PCR) assay was successfully developed to simultaneously detect canine parainfluenza virus 5 (CPIV5) and a canine endogenous internal positive control (EIPC) in canine clinical samples. Two sets of primers and probes for the CPIV5 L and canine 16S rRNA genes were included in the dqRT-PCR assay to detect CPIV and monitor invalid results throughout the qRT-PCR process. The developed dqRT-PCR assay specifically detected CPIV5 but no other canine pathogens. Furthermore, 16S rRNA was stably amplified by dqRT-PCR assay in all samples containing canine cellular materials. The assay's sensitivity was determined as below ten RNA copies per reaction, with CPIV5 L gene standard RNA and 1 TCID50/mL with the CPIV5 D008 vaccine strain, which was 10-fold higher than that of the previous HN gene-specific qRT-PCR (HN-qRT-PCR) assays and was equivalent to that of the previous N gene-specific qRT-PCR (N-qRT-PCR) assays, respectively. Moreover, the Ct values of the CPIV5-positive samples obtained using the dqRT-PCR assay were lower than those obtained using the previous HN- and N-qRT-PCR assays, indicating that the diagnostic performance of the dqRT-PCR assay was superior to those of previous HN- and N-qRT-PCR assays. The calculated Cohen's kappa coefficient values (95% confidence interval) between dqRT-PCR and the HN- or N-specific qRT-PCR assays were 0.97 (0.90-1.03) or 1.00 (1.00-1.00), respectively. In conclusion, the newly developed dqRT-PCR assay with high sensitivity, specificity, and reliability will be a promising diagnostic tool for the detection of CPIV5 in clinical samples and useful for etiological and epidemiological studies of CPIV5 infection in dogs.

Simple and Rapid Colorimetric Detection of Canine Parainfluenza Virus 5 (Orthorubulavirus mammalis) Using a Reverse-Transcription Loop-Mediated Isothermal Amplification Assay.

Despite its many advantages, a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay has yet to be developed for canine parainfluenza virus 5 (CPIV5). In this study, a visual RT-LAMP (vRT-LAMP) assay was developed for the rapid detection of CPIV5 in clinical samples. At a constant reaction temperature of 62 °C, the assay was completed within 40 min, and the results could be directly detected with the naked eye using a hydroxynaphthol blue (HNB) metal indicator without any additional detection apparatuses. The assay specifically amplified CPIV5 RNA with a limit of detection of 10 RNA copies/reaction, which was 10-fold more sensitive than the previously reported conventional reverse-transcription polymerase chain reaction (cRT-PCR) assay and was comparable to the previously reported real-time RT-PCR (qRT-PCR) assay. In a clinical evaluation using 267 nasopharyngeal swab samples collected from hospitalized dogs with respiratory symptoms, the CPIV5 detection rate using the vRT-LAMP assay was 5.24% (14/267), which was higher than that of the cRT-PCR assay (4.49%, 12/267) and consistent with that of the qRT-PCR assay, demonstrating 100% concordance with a kappa coefficient value (95% confidence interval) of 1 (1.00-1.00). The discrepancies in the results of the assays were confirmed to be attributed to the low sensitivity of the cRT-PCR assay. Owing to the advantages of a high specificity, rapidity, and simplicity, the developed vRT-LAMP assay using an HNB metal indicator will be a valuable diagnostic tool for the detection of CPIV5 in canine clinical samples, even in resource-limited laboratories.

Genetic Profile of African Swine Fever Viruses Circulating at Pig Farms in South Korea during the Outbreaks between 2022 and April 2023.

Fifteen pig farms were affected by African swine fever (ASF) in South Korea during the outbreaks between 2022 and April 2023. The ASF virus (ASFV) genome was directly extracted from the blood and tissue samples of 15 ASFV-positive pig farms to analyze the genetic characteristics. Phylogenetic analysis revealed that the 15 strains belonged to p72 genotype II and CD2v serogroup 8, which were the central variable region (CVR) I variants of the B602L gene. Fourteen strains were intergenic region (IGR) II variants, containing an additional tandem repeat sequence (TRS), between I73L and I329R, with the exception of one strain from an ASFV-infected pig farm reported on 22 January 2023, which was an IGR I variant. In addition, a single-nucleotide polymorphism (SNP) was detected at position 107 from the start of the IGR between A179L and A137R in six isolates. The findings of this study suggest that the sources of the virus at the pig farms from which these variants originated differed from those of other pig farms.

Tailored Multiplex Real-Time RT-PCR with Species-Specific Internal Positive Controls for Detecting SARS-CoV-2 in Canine and Feline Clinical Samples.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have been frequently reported in companion dogs and cats worldwide during the ongoing coronavirus disease. However, RT-qPCR methods developed for humans have been used for the diagnosis of SARS-CoV-2 infections in suspected companion dogs and cats owing to the lack of the companion animal-tailored methods. Therefore, we developed a multiplex RT-qPCR (mRT-qPCR) using newly designed primers and probes targeting RdRp and N genes of all currently circulating SARS-CoV-2 variants as well as the canine or feline 16S rRNA gene as an endogenous internal positive control (EIPC) for reliable diagnosis of SARS-CoV-2 infection from suspected dogs and cats. The developed mRT-qPCR assay specifically detected the target genes of SARS-CoV-2 but no other canine or feline pathogens. Furthermore, canine and feline EIPCs were stably amplified by mRT-qPCR in samples containing canine- or feline-origin cellular materials. This assay has high repeatability and reproducibility, with an optimal limit of detection (<10 RNA copies per reaction) and coefficients of variation (<1.0%). The detection rate of SARS-CoV-2 of the developed mRT-qPCR was 6.6% for canine and feline nasopharyngeal samples, which was consistent with that of a commercial mRT-qPCR kit for humans. Collectively, the newly developed mRT-qPCR with canine and feline EIPC can efficiently diagnose and evaluate the viral load in field specimens and will be a valuable tool for etiological diagnosis, epidemiological study, and controlling SARS-CoV-2 infections in canine and feline populations.

Demonstration of SARS-CoV-2 Exposure in Korean Native Cattle and Korean Native Black Goats in Korea.

The COVID-19 pandemic is caused by the zoonotic SARS-CoV-2 virus. A wide range of animals that interact with humans have been investigated to identify potential infections. As the extent of infection became more apparent, extensive animal monitoring became necessary to assess their susceptibility. This study analyzed nasal swabs and blood samples collected from randomly selected Korean native cattle and Korean native black goats. The tests conducted included real-time qPCR to detect SARS-CoV-2 antigens, an ELISA to detect antibodies, and a plaque reduction neutralization test (PRNT) to determine the presence of neutralizing antibodies. Among the 1798 animals tested (consisting of 1174 Korean native cattle and 624 Korean native black goats), SARS-CoV-2 viral RNA was detected in one Korean native cattle and one Korean native black goat. ELISA testing revealed positive results for antibodies in 54 Korean native cattle (4.60%) and 16 Korean native black goats (2.56%), while PRNTs yielded positive results in 51 Korean native cattle (4.34%) and 14 Korean native black goats (2.24%). The presence of SARS-CoV-2 antigens and/or antibodies was identified in animals on farms where farmworkers were already infected. It is challenging to completely rule out the possibility of reverse zoonotic transmission from humans to livestock in Korea, although the transmission is not to the same extent as it is in highly susceptible animal species like minks, cats, and dogs. This is due to the limited geographical area and the dense, intensive farming practices implemented in these regions. In conclusion, continuous viral circulation between humans and animals is inevitable, necessitating ongoing animal monitoring to ensure public health and safety.

Complete Genomic Characterization of Lumpy Skin Disease Virus Isolates from Beef Cattle in Lopburi Province, Central Thailand, during 2021-2022.

Lumpy skin disease (LSD) is a viral infection that impacts the cattle industry. The most efficient approach to prevent disease involves the utilization of live-attenuated LSD vaccines (LAVs), which stands out as the most successful method. However, LAVs might be subjected to changes to their genomes during replication that increase viral infectivity or virulence. The objective of this study was to monitor alterations in the genetic characteristics of the lumpy skin disease virus (LSDV) in beef cattle following the administration of LAVs in Lopburi Province of Central Thailand. A total of four skin samples from LSD cases were collected from non-vaccinated animals that exhibited LSD clinical symptoms from two distinct districts, spanning three subdistricts within the region. The samples of cattle were analyzed using real-time PCR targeting the LSDV074 p32 gene, the virus was isolated, and the entire genome sequences were evaluated through a single nucleotide polymorphisms (SNPs) analysis, and phylogenetic trees were assembled. The investigations revealed that LSDVs from two isolates from Chai Badan district exhibited significant mutations in the open reading frame (ORF) 023 putative protein, while another two isolates from Lam Sonthi district had a change in the untranslated region (UTR). For a result, the most proficient disease diagnosis and control should be evaluated on viral genetics on a regular basis.

A Robust Quadruple Protein-Based Indirect ELISA for Detection of Antibodies to African Swine Fever Virus in Pigs.

African swine fever (ASF) emerged in domestic pigs and wild boars in China in 2018 and rapidly spread to neighboring Asian countries. Currently, no effective vaccine or diagnostic tests are available to prevent its spread. We developed a robust quadruple recombinant-protein-based indirect enzyme-linked immunosorbent assay (QrP-iELISA) using four antigenic proteins (CD2v, CAP80, p54, and p22) to detect ASF virus (ASFV) antibodies and compared it with a commercial kit (IDvet) using ASFV-positive and -negative serum samples. The maximum positive/negative value was 24.033 at a single antigen concentration of 0.25 µg/mL and quadruple ASFV antigen combination of 1 µg/mL at a 1:100 serum dilution. Among 70 ASFV-positive samples, 65, 67, 65, 70, 70, and 14 were positive above the cut-offs of 0.121, 0.121, 0.183, 0.065, 0.201, and 0.122, for CD2v, CAP80, p54, p22-iELISA, QrP-iELISA, and IDvet, respectively, with sensitivities of 92.9%, 95.7%, 92.9%, 100%, 100%, and 20%, respectively, all with 100% specificity. The antibody responses in QrP-iELISA and IDvet were similar in pigs infected with ASFV I. QrP-iELISA was more sensitive than IDvet for early antibody detection in pigs infected with ASFV II. These data provide a foundation for developing advanced ASF antibody detection kits critical for ASF surveillance and control.

Epidemiological and Molecular Approaches for a Fatal Feline Panleukopenia Virus Infection of Captive Siberian Tigers (Panthera tigris altaica) in the Republic of Korea.

Feline panleukopenia virus (FPV), a member of the species Protoparvovirus carnivoran1, is one of the most fatal pathogens of domestic and wild carnivores. The virus endemically infects domestic carnivores worldwide and its cross-species transmission threatens endangered wild carnivores, including Siberian tigers. In this study, a fatal FPV infection in endangered Siberian tigers was investigated to trace the origin of the virus and elucidate the reason behind FPV's infection of the vaccinated tigers. Our genetic characterization and phylogenetic analysis revealed that the virus detected in the infected tigers, designated as the KTPV-2305 strain, was closely related to FPV strains circulating in Korean cats, suggesting that it might have been transmitted from stray cats wandering around the zoo. Compared with the prototype FPV reference strains, the KTPV-2305 strain carried three distinct amino acid (aa) mutations in the VP2 protein sequence (I101T, I232V, and L562V) in this study. These three mutations are commonly found in most global FPV strains, including Korean strains, indicating that these mutations are common evolutionary characteristics of currently circulating global FPVs. The reason why the vaccinated tigers were infected with FPV was most likely the insufficient protective immunity of the affected tigress or vaccine failure triggered by the interference of maternal-derived antibodies in the affected tiger cubs. These findings suggest that improved vaccination guidelines are urgently needed to save the lives of wild carnivores from this fatal virus.

Pathogenicity and Pathological Characteristics of African Swine Fever Virus Strains from Pig Farms in South Korea from 2022 to January 2023.

Since the first African swine fever (ASF) outbreak occurred at a pig farm in South Korea in September 2019, as of 31 January 2023, 31 ASF cases have occurred at pig farms, while 2799 ASF virus (ASFV)-infected wild boars have been identified. The circulation of ASFV in wild boar populations poses a high risk of spillover to pig farms in the country. However, information on the changes in the pathogenicity of Korean ASFV strains from wild boars is not available. Investigating the pathogenicity of ASFV strains from pig farms is the only way to predict their alterations. In a previous study, no changes in the pathogenicity of ASFV strains circulating during 2019-2021 were identified through animal experiments. In this study, we chose two ASFV strains with potentially reduced pathogenicity among ten viruses obtained from pig premises from 2022 to January 2023 and estimated their pathogenicities and pathological characteristics. All the inoculated pigs died 8-10 days post-inoculation after showing pyrexia, depression, anorexia, and recumbency together with the common pathological lesions of enlarged hemorrhagic lymph nodes and splenomegaly with infarction. These results support that the pathogenicity among ASFV isolates in South Korea still remained unchanged during the study period.

Endoproteolysis of cellular prion protein by plasmin hinders propagation of prions.

Many questions surround the underlying mechanism for the differential metabolic processing observed for the prion protein (PrP) in healthy and prion-infected mammals. Foremost, the physiological α-cleavage of PrP interrupts a region critical for both toxicity and conversion of cellular PrP (PrP C ) into its misfolded pathogenic isoform (PrP Sc ) by generating a glycosylphosphatidylinositol (GPI)-anchored C1 fragment. During prion diseases, alternative ß-cleavage of PrP becomes prominent, producing a GPI-anchored C2 fragment with this particular region intact. It remains unexplored whether physical up-regulation of α-cleavage can inhibit disease progression. Furthermore, several pieces of evidence indicate that a disintegrin and metalloproteinase (ADAM) 10 and ADAM17 play a much smaller role in the α-cleavage of PrP C than originally believed, thus presenting the need to identify the primary protease(s) responsible. For this purpose, we characterized the ability of plasmin to perform PrP α-cleavage. Then, we conducted functional assays using protein misfolding cyclic amplification (PMCA) and prion-infected cell lines to clarify the role of plasmin-mediated α-cleavage during prion propagation. Here, we demonstrated an inhibitory role of plasmin for PrP Sc formation through PrP α-cleavage that increased C1 fragments resulting in reduced prion conversion compared with non-treated PMCA and cell cultures. The reduction of prion infectious titer in the bioassay of plasmin-treated PMCA material also supported the inhibitory role of plasmin on PrP Sc replication. Our results suggest that plasmin-mediated endoproteolytic cleavage of PrP may be an important event to prevent prion propagation.