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.

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.

Experiencing social exclusion changes gut microbiota composition.

Gut microbiota is suggested to regulate the host's mental health via the gut-brain axis. In this study, we investigated the relationship between the microbiome and psychological pain due to social exclusion. Adult individuals with (n = 14) and without (n = 25) social exclusion experience were assessed for the psychological status using self-reported questionnaires: Beck Anxiety Inventory (BAI), Beck Depression Inventory, and the UCLA Loneliness Scale. The gut microbiota was analyzed by 16 S rRNA gene sequencing and bioinformatics. The exclusion group had a 1.70-fold higher total BAI score and 2.16-fold higher levels of anxiety-related physical symptoms (p < 0.05). The gut microbial profiles also differed between the two groups. The exclusion group showed higher probability of having Prevotella-enriched microbiome (odds ratio, 2.29; 95% confidence interval, 1.65-2.75; p < 0.05), a significantly reduced Firmicutes/Bacteroidetes ratio, and decreased abundance of Faecalibacterium spp. (p < 0.05) which was associated with the duration and intensity of social exclusion (p < 0.05). Our results indicate that the psychological pain due to social exclusion is correlated with the gut microbiota composition, suggesting that targeting social exclusion-related microorganisms can be a new approach to solving psychological problems and related social issues.

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.

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.