Laboratory investigation of causes of bovine abortion and stillbirth in the Republic of Korea, 2014-2020.

Bovine abortion is a critical problem in the cattle industry. Identifying causes of abortion is key to establishing appropriate herd management and prevention strategies. We used pathology examinations, detection of etiologic agents, and serology to determine the cause of bovine abortions in Korea. We analyzed 360 abortion and stillbirth cases submitted to the Animal and Plant Quarantine Agency from December 2014 to January 2020. The putative cause of abortion was identified in 140 of 360 (38.9%) cases; 124 of the 140 (88.6%) cases were attributed to infections. The most common etiologic agents detected were bovine viral diarrhea virus (65 of 360; 18.1%), Coxiella burnetii (19 of 360; 5.3%), Leptospira spp. (13 of 360; 3.6%), Listeria monocytogenes (9 of 360; 2.5%), and Neospora caninum (8 of 360; 2.2%). Minor abortifacient pathogens included Brucella abortus (2 of 360; 0.6%), bovine alphaherpesvirus 1 (2 of 360; 0.6%), Akabane virus (2 of 360, 0.6%), and bovine ephemeral fever virus (1 of 360; 0.3%). Non-infectious conditions included congenital anomalies (7 of 360; 1.9%), goiter (7 of 360; 1.9%), and vitamin A deficiency (2 of 360; 0.6%). Our diagnostic rate in cases with placenta submitted (42 of 86; 48.8%) was significantly higher than in cases without placenta (98 of 274; 35.8%), which highlights the value of submitting placentas. Our results confirm the status of the large variety of causative agents associated with abortions in cattle in Korea.

Prevalence of viral agents causing swine reproductive failure in Korea and the development of multiplex real-time PCR and RT-PCR assays.

This study aimed to investigate the prevalence of viral agents causing reproductive failure in pigs in Korea. In addition, two types of multiplex real-time PCR (mqPCR) were developed for the simultaneous detection of Aujeszky's disease virus (ADV) and porcine parvovirus (PPV) in mqPCR and encephalomyocarditis virus (EMCV) and Japanese encephalitis virus (JEV) in reverse transcription mqPCR (mRT-qPCR). A total of 150 aborted fetus samples collected from 2020 to 2022 were analyzed. Porcine reproductive and respiratory syndrome virus was the most prevalent (49/150 32.7%), followed by porcine circovirus type 2 (31/150, 20.7%), and PPV1 (7/150, 4.7%), whereas ADV, EMCV, and JEV were not detected. The newly developed mqPCR and mRT-qPCR could simultaneously detect and differentiate with high sensitivities and specificities. When applied to aborted fetuses, the newly developed mqPCR for PPV was 33.3% more sensitivities than the previously established diagnostic method. Amino acid analysis of the VP2 sequences of PPV isolates revealed considerable similarity to the highly pathogenic Kresse strain. This study successfully evaluated the prevalence of viral agents causing reproductive failure among swine in Korea, the developed mqPCR and mRT-qPCR methods could be utilized as effective and accurate diagnostic methods for the epidemiological surveillance of ADV, PPV, EMCV, and JEV.

First Detection and Genetic Characterization of Swine Orthopneumovirus from Domestic Pig Farms in the Republic of Korea.

Novel swine orthopneumovirus (SOV) infections have been identified in pigs in the USA and some European countries but not in Asian countries, including South Korea, to date. The current study reports the first SOV infections in four domestic pig farms located in four provinces across South Korea. The detection rate of SOV in oral fluid samples using qRT-PCR was 4.4% (14/389), indicating the presence of the virus in pigs at commercial farms in Korea. Two complete genome sequences and one glycoprotein (G) gene sequence were obtained from SOV-positive samples. The complete genome analysis of KSOV-2201 and KSOV-2202 strains showed 98.2 and 95.4% homologies with a previously reported SOV, and the phylogenetic tree exhibited a high correlation with a previously reported SOV strain from the US and a canine pneumovirus (CPnV) strain from China. Based on the genetic analysis of the viral G gene, the murine pneumonia virus (MPV)-like orthopneumoviruses (MLOVs) were divided into two genogroups (G1 and G2). Seventeen CPnVs and two feline pneumoviruses were grouped into G1, while the Korean SOV strains identified in this study were grouped into G2 along with one SOV and two CPnVs. These results will contribute to expanding our understanding of the geographical distribution and genetic characteristics of the novel SOV in the global pig population.

An Advanced Multiplex Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Rapid and Reliable Detection of Porcine Epidemic Diarrhea Virus and Porcine Internal Positive Control.

For rapid and reliable detection of porcine epidemic diarrhea virus (PEDV) from pig clinical samples, a multiplex, real-time, reverse transcription loop-mediated isothermal amplification (mqRT-LAMP) was developed using two sets of primers and assimilating probes specific to the PEDV N gene and the Sus scrofa ß-actin gene, which was used as an endogenous internal positive control (EIPC) to avoid false-negative results. The assay specifically amplified both target genes of PEDV and EIPC in a single reaction without any interference but did not amplify other porcine viral nucleic acids. The limit of detection was 10 copies/µL, 100-fold lower than that of a reverse transcription-polymerase chain reaction (RT-PCR) and equivalent to that of quantitative/real-time RT-PCR (qRT-PCR). This assay has high repeatability and reproducibility with coefficients of variation < 4.0%. The positive signal of the mqRT-LAMP assay was generated within 25 min, demonstrating advantages in rapid detection of PEDV over RT-PCR or qRT-PCR assay, which require at least 2 h turnaround times. In clinical evaluation, the detection rate of PEDV by mqRT-LAMP assay (77.3%) was higher than that of RT-PCR assay (69.7%), and comparable to qRT-PCR (76.8%) with almost 100% concordance (kappa value 0.98). The developed mqRT-LAMP assay can serve as an advanced alternative method for PEDV diagnosis because it has high sensitivity and specificity, rapidity, and reliability even in resource-limited laboratories.

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.

Genetic Characterization and Evolution of Porcine Deltacoronavirus Isolated in the Republic of Korea in 2022.

Porcine deltacoronavirus (PDCoV) is an emerging coronavirus that causes diarrhea in nursing piglets. Since its first outbreak in the United States in 2014, this novel porcine coronavirus has been detected worldwide, including in Korea. However, no PDCoV case has been reported since the last report in 2016 in Korea. In June 2022, the Korean PDCoV strain KPDCoV-2201 was detected on a farm where sows and piglets had black tarry and watery diarrhea, respectively. We isolated the KPDCoV-2201 strain from the intestinal samples of piglets and sequenced the viral genome. Genetically, the full-length genome and spike gene of KPDCoV-2201 shared 96.9-99.2% and 95.8-98.8% nucleotide identity with other global PDCoV strains, respectively. Phylogenetic analysis suggested that KPDCoV-2201 belongs to G1b. Notably, the molecular evolutionary analysis indicated that KPDCoV-2201 evolved from a clade different from that of previously reported Korean PDCoV strains and is closely related to the emergent Peruvian and Taiwanese PDCoV strains. Furthermore, KPDCoV-2201 had one unique and two Taiwanese strain-like amino acid substitutions in the receptor-binding domain of the S1 region. Our findings suggest the possibility of transboundary transmission of the virus and expand our knowledge about the genetic diversity and evolution of PDCoV in Korea.

First report of Porcine respirovirus 1 in South Korea.

Porcine respirovirus 1 (PRV1) is a recently emerging porcine respiratory virus that belongs to the genus Respirovirus of the Paramyxoviridae family. Since its first detection in Hong Kong, China in 2009, PRV1 has been subsequently identified in several American and European countries, suggesting that the emerging virus may have been globally distributed. However, in Asia, the virus has been reported only in China. Here, we report that PRV1 was first detected in pigs from 16 farms located in seven provinces across Korea, with a prevalence of 71.4% based on the tested oral fluid samples, suggesting that the virus is already widespread in Korean pig herds. For further genetic characterization of the Korean PRV1 strains, a complete genome and two F gene sequences were obtained from PRV1-positive samples collected from three different pig farms. Phylogenetic analysis based on the complete genome and F gene sequences showed that all three Korean PRV1 strains were grouped into European lineage 1 and were closely related to strains from Hong Kong (China), Germany and Poland. We could not obtain evidence for the origin of Korean PRV1 because of the limited availability of PRV1 sequences. In conclusion, PRV1 was first identified in Korean pig herds and genetically characterized in the present study. These results contribute to a better understanding of the global geographical distribution and genetic characteristics of PRV1.

Genetic diversity of porcine reproductive and respiratory syndrome virus and evaluation of three one-step real-time RT-PCR assays in Korea.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) has caused huge economic losses in the global swine industry. Frequent genetic variations in this virus cause difficulties in controlling and accurately diagnosing PRRSV. METHODS: In this study, we investigated the genetic characteristics of PRRSV-1 and PRRSV-2 circulating in Korea from January 2018 to September 2021 and evaluated three one-step real-time reverse transcription polymerase chain reaction (RT-PCR) assays. RESULTS: A total of 129 lung samples were collected, consisting of 47 samples for PRRSV-1, 62 samples for PRRSV-2, and 20 PRRSV-negative samples. Nucleotide sequence analysis of open reading frames (ORFs) 5, ORF6, and ORF7 genes from PRRSV samples showed that PRRSV-1 belonged to subgroup A (43/47, 91.49%) and subgroup C (4/47, 8.51%), whereas PRRSV-2 was classified as lineage 1 (25/62, 40.32%), Korean lineage (Kor) C (13/62, 20.97%), Kor B (10/62, 16.13%), lineage 5 (9/62, 14.52%), and Kor A (5/62, 8.06%). Amino acid sequence analysis showed that the neutralizing epitope and T cell epitope of PRRSV-1, and the decoy epitope region and hypervariable regions of PRRSV-2 had evolved under positive selection pressure. In particular, the key amino acid substitutions were found at positions 102 and 104 of glycoprotein 5 (GP5) in some PRRSV-2, and at positions 10 and 70 of membrane protein (M) in most PRRSV-2. In addition, one-step real-time RT-PCR assays, comprising two commercial tests and one test recommended by the World Organization for Animal Health (OIE), were evaluated. CONCLUSION: The results revealed that two of the real-time RT-PCR assays had high sensitivities and specificities, whereas the real-time RT-PCR assay of the OIE had low sensitivity due to mismatches between nucleotides of Korean PRRSVs and forward primers. In this study, we genetically characterized recent PRRSV occurrences and evaluated three one-step real-time RT-PCR assays used in Korea.

Recombination between the Fostera MLV-like Strain and the Strain Belonging to Lineage 1 of Porcine Reproductive and Respiratory Syndrome Virus in Korea.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in the swine industry worldwide. In Korea, Fostera PRRS commercial modified live virus (MLV) vaccines have been used since 2014 to control the PRRSV infection. In this study, two PRRSV-2 strains (20D160-1 and 21R2-63-1) were successfully isolated, and their complete genomic sequences were determined. Genetic analysis showed that the two isolates have recombination events between the P129-like strain derived from the Fostera PRRS MLV vaccine and the strain of lineage 1. The 20D160-1 indicated that partial ORF2 to partial ORF4 of the minor parental KNU-1902-like strain, which belongs to Korean lineage C (Kor C) of lineage 1, was inserted into the major parental P129-like strain. The 21R2-63-1 revealed that partial ORF1b of the P129-like strain was inserted into the backbone of the NADC30-like strain. This study is the first to report natural recombinant strains between Fostera PRRS MLV-like strain and the field strain in Korea. These results may have significant implications for MLV evolution and the understanding of PRRSV genetic diversity, while highlighting the need for continuous surveillance of PRRSV.

Whole-genome sequencing and genetic characteristics of representative porcine reproductive and respiratory syndrome virus (PRRSV) isolates in Korea.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is a macrophage-tropic arterivirus with extremely high genetic and pathogenic heterogeneity that causes significant economic losses in the swine industry worldwide. PRRSV can be divided into two species [PRRSV1 (European) and PRRSV2 (North American)] and is usually diagnosed and genetically differentiated into several lineages based on the ORF5 gene, which constitutes only 5% of the whole genome. This study was conducted to achieve nonselective amplification and whole-genome sequencing (WGS) based on a simplified sequence-independent, single-primer amplification (SISPA) technique with next-generation sequencing (NGS), and to genetically characterize Korean PRRSV field isolates at the whole genome level. METHODS: The SISPA-NGS method coupled with a bioinformatics pipeline was utilized to retrieve full length PRRSV genomes of 19 representative Korean PRRSV strains by de novo assembly. Phylogenetic analysis, analysis of the insertion and deletion (INDEL) pattern of nonstructural protein 2 (NSP2), and recombination analysis were conducted. RESULTS: Nineteen complete PRRSV genomes were obtained with a high depth of coverage by the SISPA-NGS method. Korean PRRSV1 belonged to the Korean-specific subtype 1A and vaccine-related subtype 1C lineages, showing no evidence of recombination and divergent genetic heterogeneity with conserved NSP2 deletion patterns. Among Korean PRRSV2 isolates, modified live vaccine (MLV)-related lineage 5 viruses, lineage 1 viruses, and nation-specific Korean lineages (KOR A, B and C) could be identified. The NSP2 deletion pattern of the Korean lineages was consistent with that of the MN-184 strain (lineage 1), which indicates the common ancestor and independent evolution of Korean lineages. Multiple recombination signals were detected from Korean-lineage strains isolated in the 2010s, suggesting natural interlineage recombination between circulating KOR C and MLV strains. Interestingly, the Korean strain GGYC45 was identified as a recombinant KOR C and MLV strain harboring the KOR B ORF5 gene and might be the ancestor of currently circulating KOR B strains. Additionally, two novel lineage 1 recombinants of NADC30-like and NADC34-like viruses were detected. CONCLUSION: Genome-wide analysis of Korean PRRSV isolates retrieved by the SISPA-NGS method and de novo assembly, revealed complex evolution and recombination in the field. Therefore, continuous surveillance of PRRSV at the whole genome level should be conducted, and new vaccine strategies for more efficient control of the virus are needed.

Detection of a novel porcine circovirus 4 in Korean pig herds using a loop-mediated isothermal amplification assay.

A novel porcine circovirus 4 has been recently identified in China and Korea. A sensitive and specific diagnostic method is urgently required to detect the virus in field samples. We developed a loop-mediated isothermal amplification (LAMP) the assay for the visual detection of PCV4 and evaluated its sensitivity, specificity, and applicability in clinical samples. This assay's results can be directly visualized by the naked eye using hydroxynaphthol blue after incubation for 40 min at 64 °C. The assay specifically amplified PCV4 DNA and no other viral nucleic acids. The sensitivity of the assay was <50 DNA copies/reaction, which was 10 times more sensitive than conventional polymerase chain reaction (cPCR) and comparable to real-time PCR (qPCR). Clinical evaluation revealed that the PCV4 detection rate in individual pig samples and at the farm level was 39.3 % (57/145) and 45.7 % (32/70), respectively, which were higher than cPCR (46 samples, 24 farms) and qPCR (52 samples, 29 farms) results. Cumulatively, owing to the advantages of high sensitivity and specificity, direct visual monitoring of the results, no possibility for cross-contamination, and being a low-cost equipment, the developed LAMP assay will be a valuable tool for the detection of the novel PCV4 in clinical samples, even in resource-limited laboratories.

A simple colorimetric detection of porcine epidemic diarrhea virus by reverse transcription loop-mediated isothermal amplification assay using hydroxynaphthol blue metal indicator.

A simple reverse transcription loop-mediated isothermal amplification combined with visual detection method (vRT-LAMP) assay was developed for rapid and specific detection of porcine epidemic diarrhea virus (PEDV) in this study, which overcomes the shortcomings of previously described RT-LAMP assays that require additional detection steps or pose a risk of cross-contamination. The assay results can be directly detected by the naked eye using hydroxynaphthol blue after incubating for 40 min at 62 °C. The assay specifically amplified PEDV RNA and no other viral nucleic acids. The limit of detection of the assay was less than 50 RNA copies per reaction, which was 100 times more sensitive than conventional reverse transcription polymerase chain reaction (RT-PCR) and comparable to real-time RT-PCR (RRT-PCR). In the clinical evaluation, the PEDV detection rate of vRT-LAMP was higher than that of RRT-PCR, showing 99 % concordance, with a kappa value (95 % confidence interval) of 0.97 (0.93-1.01). Considering the advantages of high sensitivity and specificity, simple and direct visual monitoring of the results, no possibility for cross-contamination, and being able to be used as low-cost equipment, the developed vRT-LAMP assay will be a valuable tool for detecting PEDV from clinical samples, even in resource-limited laboratories.

Temporal lineage dynamics of the ORF5 gene of porcine reproductive and respiratory syndrome virus in Korea in 2014-2019.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the most important pathogen in the Korean swine industry. Despite efforts including improved biosecurity and vaccination protocols, the virus continues to circulate and evolve. Based on phylogenetic analysis of open reading frame 5 (ORF5), Korean PRRSVs are known to form not only globally circulating lineages but also country-specific lineages (Lin Kor A, B, and C). To understand the recent epidemiological status of PRRSV in Korea, a total of 1349 ORF5 sequences of Korean PRRSV isolates from 2014 to 2019 were analyzed. Phylogenetic analysis was conducted using the maximum-likelihood method, and temporal changes in the relative prevalence of lineages were investigated. The analysis showed that PRRSV1 and PRRSV2 were both highly prevalent throughout the years examined. Among the PRRSV1 isolates, subgroup A (90.1%) and vaccine-like subgroup C (9.0%) composed most of the population. For PRRSV2 isolates, vaccine-like lineage 5 (36.3%) was dominant, followed by Lin Kor B (25.9%), Kor C (16.6%), lineage 1 (11.6%), and Kor A (9.1%). The PRRSV2 lineage 1 population increased from 2014 (1.8%) to 2019 (29.6%) in Korea due to the continual spread of sublineage 1.8 (NADC30-like) and introduction of sublineage 1.6 into the country. Additional genetic analysis, including analysis of non synonymous and synonymous mutations, revealed evidence of diversification and positive selection in immunologically important regions of the genome, suggesting that current vaccination is failing and promoting immune-mediated selection. Overall, these findings provide insights into the epidemiological and evolutionary dynamics of cocirculating viral lineages, and constant surveillance of PRRSV occurrence is needed.

Evaluation of the Variability of the ORF34, ORF68, and MLST Genes in EHV-1 from South Korea.

Equine herpesvirus-1 (EHV-1) is an important pathogen in horses. It affects horses worldwide and causes substantial economic losses. In this study, for the first time, we characterized EHV-1 isolates from South Korea at the molecular level. We then aimed to determine the genetic divergences of these isolates by comparing them to sequences in databases. In total, 338 horse samples were collected, and 12 EHV-1 were isolated. We performed ORF30, ORF33, ORF68, and ORF34 genetic analysis and carried out multi-locus sequence typing (MLST) of 12 isolated EHV-1. All isolated viruses were confirmed as non-neuropathogenic type, showing N752 of ORF30 and highly conserved ORF33 (99.7-100%). Isolates were unclassified using ORF68 analysis because of a 118 bp deletion in nucleotide sequence 701-818. Seven EHV-1 isolates (16Q4, 19R166-1, 19R166-6, 19/10/15-2, 19/10/15-4, 19/10/18-2, 19/10/22-1) belonged to group 1, clade 10, based on ORF34 and MLST analysis. The remaining 5 EHV-1 isolates (15Q25-1, 15D59, 16Q5, 16Q40, 18D99) belonged to group 7, clade 6, based on ORF34 and MLST analysis.

Enhanced Virulence of Aeromonas hydrophila Is Induced by Stress and Serial Passaging in Mice.

Aeromonas hydrophila was isolated from an African black-footed penguin (Spheniscus demersus) that died while in zoo captivity. At necropsy, the virulence of A. hydrophila appeared to be enhanced by stress, so was assessed in the presence of in vitro and in vivo stressors and serial passaging in mice. Virulence genes from the isolate were amplified by PCR. In vitro assays were conducted to test the hemolytic activity, cytotoxicity, and effect of stress hormones on A. hydrophila virulence. In vivo assays were conducted to test the stress effect on mortality of A. hydrophila-infected mice and virulence in mice. Two virulence genes coding for hemolysin (ahh1) and aerolysin (aerA) were detected, and the cytotoxic potential of the isolate was demonstrated in baby hamster kidney and Vero cells. Some or all mice inoculated with A. hydrophila and exposed to stress hormones (epinephrine and norepinephrine) or low temperature died, while mice inoculated with A. hydrophila and exposed to fasting or agitation stressors or no stressors survived. We concluded that stress can be fatal in mice experimentally infected with A. hydrophila and that serial passaging in mice dramatically enhances the virulence of A. hydrophila.

Advanced target-specific probe-based real-time loop-mediated isothermal amplification assay for the rapid and specific detection of porcine circovirus 3.

Porcine circovirus type 3 (PCV3) is an emerging viral pathogen that has been identified in pigs with various clinical signs. For rapid and specific detection of PCV3, an advanced real-time loop-mediated isothermal amplification (rLAMP) assay that uses both assimilating probes and swarm primers were developed and evaluated in this study. The assay specifically amplified PCV3 DNA, but it did not amplify other porcine viral nucleic acids. The limit of detection of rLAMP with swarm primers was 50 PCV3 DNA copies/reaction, which was comparable to that of the real-time quantitative polymerase chain reaction (qPCR) and 10 times more sensitive than rLAMP without swarm primers. In an evaluation of clinical samples, the rLAMP assay was able to detect PCV3 DNA within 17.34 ± 4.45 min, which is more rapid than what has been previously reported for the standard qPCR assay (31.78 ± 4.60 min). Detection with rLAMP was largely in agreement with that of the qPCR with a kappa value (95% confidence interval) of 0.98 (0.95-1.00). Taken together, these results suggest that the rLAMP assay presented will be a valuable tool for rapid, specific and reliable diagnosis of PCV3 in clinical samples.

Genetic characterization of canine parvovirus type 2c from domestic dogs in Korea.

Canine parvovirus type 2 (CPV-2) is an aetiological agent that causes acute haemorrhagic enteritis and fatal myocarditis in dogs. Since CPV-2 first emerged in the late 1970s, its rapid evolution has resulted in three antigenic variants: CPV-2a, CPV-2b and CPV-2c. Here, we report, for the first time in Korea, two cases of CPV-2c infection in two dogs with severe diarrhoea. The complete open reading frame (4,269nt) of CPV-2, encoding both non-structural (NS) and structural (VP) proteins, was sequenced. Based on the amino acid Gln present at residue 426 of the VP2 gene, these strains were typed as CPV-2c, and were named Korea CPV-2c_1 and Korea CPV-2c_2. These strains shared 99.48% reciprocal nucleotide sequence identity and had the highest nucleotide identity (99.77%-99.34%) with Asian CPV strains isolated in China, Italy (found in a dog imported from Thailand), and Vietnam from 2013 to 2017. Phylogenetic analysis based on the non-structural (NS1) and capsid (VP2) genes revealed that Korean CPV-2c strains clustered closely to Asian CPV strains, and separately from strains isolated in Europe, South America and North America. Amino acid changes never reported before were observed in NS1 (Thr70Pro, Cys287Tyr), VP1 (Lys17Arg, Phe33Leu) and VP2 (Gln365His, Ala516Val). Additional observed mutations, including Phe267Tyr, Tyr324Ile and Gln370Arg, have been previously reported in the recent CPV-2c strains with Asian origins. These results suggest that the Korean CPV-2c strains were potentially introduced via neighbouring Asian countries.

Co-infection of Dirofilaria immitis and Japanese encephalitis virus in a spotted seal (Phoca largha) in the Republic of Korea.

A 10-year-old male spotted seal presented with loss of appetite and decreased activity. Grossly, the internal organs revealed several filarial nematodes in the right ventricle of the heart and the pulmonary vessels. Histopathological examination of the brain revealed moderate nonsuppurative meningoencephalitis with glial nodules and neuronophagia. Japanese encephalitis virus (JEV) of genotype I was isolated from the brain. All nematodes were identified as Dirofilaria immitis. This is the first clinical case of co-infection with D. immitis and JEV in a seal, suggesting that the seal, may be a dead-end host, like the human and horse, for JEV.

Differential detection of porcine reproductive and respiratory syndrome virus genotypes by a fluorescence melting curve analysis using peptide nucleic acid probe-mediated one-step real-time RT-PCR.

Peptide nucleic acids (PNAs), artificially synthesized DNA analogues, hybridize strongly with DNA and are useful for fluorescence melting curve analyses (FMCA) based on the thermal denaturation of the probe-target duplex. In this study, we developed a PNA-based one-step real-time RT-PCR assay for the differential and qualitative detection of the porcine reproductive and respiratory syndrome virus genotypes PRRSV1 and PRRSV2. The specificity of the assay was analyzed in silico using previously reported primers and probes and was subsequently verified using Korean PRRSV panels and clinical samples. Seven clinical samples showing low curves with high Ct values were confirmed as negative by FMCA. The sensitivities of one-step real-time PCR for PRRSV1 and PRRSV2 were 15 and 11 copies, respectively, and the results were in 100% agreement with those of conventional RT-PCR combined with nested PCR using clinical samples. Therefore, the assay is highly specific for the detection of current PRRSV1 and PRRSV2 without non-specific amplification by FMCA.

Analysis of Five Arboviruses and Culicoides Distribution on Cattle Farms in Jeollabuk-do, Korea.

Arthropod-borne viruses (Arboviruses) are transmitted by arthropods such as Culicoides biting midges and cause abortion, stillbirth, and congenital malformation in ruminants, apparently leading to economic losses to farmers. To monitor the distribution of Culicoides and to determine their relationship with different environmental conditions (temperature, humidity, wind speed, and altitude of the farms) on 5 cattle farms, Culicoides were collected during summer season (May-September) in 2016 and 2017, and analyzed for identification of species and detection of arboviruses. About 35% of the Culicoides were collected in July and the collection rate increased with increase in temperature and humidity. The higher altitude where the farms were located, the more Culicoides were collected on inside than outside. In antigen test of Culicoides against 5 arboviruses, only Chuzan virus (CHUV) (2.63%) was detected in 2016. The Akabane virus (AKAV), CHUV, Ibaraki virus and Bovine ephemeral fever virus (BEFV) had a positive rate of less than 1.8% in 2017. In antigen test of bovine whole blood, AKAV (12.96%) and BEFV (0.96%) were positive in only one of the farms. As a result of serum neutralization test, antibodies against AKAV were generally measured in all the farms. These results suggest that vaccination before the season in which the Culicoides are active is probably best to prevent arbovirus infections.