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.

Comparison of the pathogenicity of porcine reproductive and respiratory syndrome virus (PRRSV)-1 and PRRSV-2 in pregnant sows.

To date, few studies related to the evaluation of the pathogenicity of different PRRSV isolates using a reproductive model have been undertaken, and the main focus has remained on respiratory models using young pigs. This study aimed to evaluate the pathogenicity of two PRRSV-1 isolates (D40 and CBNU0495) and two PRRSV-2 isolates (K07-2273 and K08-1054) in a reproductive model. Pregnant sows were experimentally infected with PRRSV at gestational day 93 or used as an uninfected negative control. Sera were collected at 0, 3, 7, 14, and 19 days post-challenge (dpc) for virological and serological assays. At 19 dpc, all sows were euthanized, and their fetuses were recovered by performing cesarean section and immediately euthanized for sample collection. Here, compared to the other isolates, the CBNU0495 isolate replicated most efficiently in the pregnant sows, and K07-2273 produced the highest rate of reproductive failure even though it did not replicate as efficiently as the other isolates in sows and fetuses, indicating that vertical transmission and reproductive failure due to PRRSV infection do not have any significant correlation with the viral loads in samples from sows and fetuses. Similarly, the viral loads and the histopathological lesions did not show any correlation with each other, as the PRRSV-2-infected groups displayed more prominent and frequent histopathological lesions with lower viral loads than the PRRSV-1-infected groups. However, viral loads in the myometrium/endometrium might be related to the spreading of PRRSV in the fetuses, which affected the birth weight of live fetuses. This study contributes to a better understanding of the pathogenicity of the most prevalent Korean PRRSVs in a reproductive model.

Prevalence of porcine parvovirus 1 through 7 (PPV1-PPV7) and co-factor association with PCV2 and PRRSV in Korea.

BACKGROUND: Classical porcine parvovirus (PPV1) and novel porcine parvoviruses designated porcine parvovirus 2 through 7 (PPV2-PPV7) are widespread in pig populations. The objective of this study was to investigate the prevalence rates of PPV1-PPV7 in Korea by detecting PPVs in serum, lung and fecal samples and to elucidate the association of PPVs with porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory virus (PRRSV), major pathogens involved in porcine respiratory disease complex (PRDC). A total of 286 serum, 481 lung, and 281 fecal samples collected from 2018 to 2020 were analyzed. RESULTS: The results showed that PPVs are widespread in Korea; the highest detection rates were found in lung samples and ranged from 7.9% (PPV1) to 32.6% (PPV2). Regarding age groups, fattening pigs had the highest detection rates of PPVs, ranging from 6.4% (PPV1) to 36.5% (PPV6); this finding suggests the chronic nature of PPV infections and the continual circulation of these viruses. When compared with PCV2- and PRRSV-negative lung samples, PCV2-positive samples with or without PRRSV positivity had significantly higher detection levels of PPV1 and PPV6. In contrast, the prevalence of PPV2 and PPV7 was significantly higher in PRRSV-infected lung samples regardless of PCV2 detection. PPV5 was detected significantly more frequently in samples  with both PCV2 and PRRSV positivity. CONCLUSIONS: This study could offer a better understanding of the role of PPVs in PCV2 and/or PRRSV infection though further studies are needed to experimentally assess the impact of PPVs in coinfections.

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.

Integrated time-serial transcriptome networks reveal common innate and tissue-specific adaptive immune responses to PRRSV infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection is the most important viral disease causing severe economic losses in the swine industry. However, mechanisms underlying gene expression control in immunity-responsible tissues at different time points during PRRSV infection are poorly understood. We constructed an integrated gene co-expression network and identified tissue- and time-dependent biological mechanisms of PRRSV infection through bioinformatics analysis using three tissues (lungs, bronchial lymph nodes [BLNs], and tonsils) via RNA-Seq. Three groups with specific expression patterns (i.e., the 3-dpi, lung, and BLN groups) were discovered. The 3 dpi-specific group showed antiviral and innate-immune signalling similar to the case for influenza A infection. Moreover, we observed adaptive immune responses in the lung-specific group based on various cytokines, while the BLN-specific group showed down-regulated AMPK signalling related to viral replication. Our study may provide comprehensive insights into PRRSV infection, as well as useful information for vaccine development.

Evaluation of local and systemic immune responses in pigs experimentally challenged with porcine reproductive and respiratory syndrome virus.

The host-associated defence system responsible for the clearance of porcine reproductive and respiratory syndrome virus (PRRSV) from infected pigs is currently poorly understood. To better understand the dynamics of host-pathogen interactions, seventy-five of 100 pigs infected with PRRSV-JA142 and 25 control pigs were euthanized at 3, 10, 21, 28 and 35 days post-challenge (dpc). Blood, lung, bronchoalveolar lavage (BAL) and bronchial lymph node (BLN) samples were collected to evaluate the cellular immune responses. The humoral responses were evaluated by measuring the levels of anti-PRRSV IgG and serum virus-neutralizing (SVN) antibodies. Consequently, the highest viral loads in the sera and lungs of the infected pigs were detected between 3 and 10 dpc, and these resulted in moderate to mild interstitial pneumonia, which resolved accompanied by the clearance of most of the virus by 28 dpc. At peak viremia, the frequencies of alveolar macrophages in infected pigs were significantly decreased, whereas the monocyte-derived DC/macrophage and conventional DC frequencies were increased, and these effects coincided with the early induction of local T-cell responses and the presence of proinflammatory cytokines/chemokines in the lungs, BAL, and BLN as early as 10 dpc. Conversely, the systemic T-cell responses measured in the peripheral blood mononuclear cells were delayed and significantly induced only after the peak viremic stage between 3 and 10 dpc. Taken together, our results suggest that activation of immune responses in the lung could be the key elements for restraining PRRSV through the early induction of T-cell responses at the sites of virus replication.

Effect of polymorphisms in porcine guanylate-binding proteins on host resistance to PRRSV infection in experimentally challenged pigs.

Guanylate-binding proteins (GBP1 and GBP5) are known to be important for host resistance against porcine reproductive and respiratory syndrome virus (PRRSV) infection. In this study, the effects of polymorphisms in GBP1 (GBP1E2 and WUR) and GBP5 on host immune responses against PRRSV were investigated to elucidate the mechanisms governing increased resistance to this disease. Seventy-one pigs [pre-genotyped based on three SNP markers (GBP1E2, WUR, and GBP5)] were assigned to homozygous (n = 36) and heterozygous (n = 35) groups and challenged with the JA142 PRRSV strain. Another group of nineteen pigs was kept separately as a negative control group. Serum and peripheral blood mononuclear cells (PBMCs) were collected at 0, 3, 7, 14, 21 and 28 days post-challenge (dpc). Viremia and weight gain were measured in all pigs at each time point, and a flow cytometry analysis of PBMCs was performed to evaluate T cell activation. In addition, 15 pigs (5 pigs per homozygous, heterozygous and negative groups) were sacrificed at 3, 14 and 28 dpc, and the local T cell responses were evaluated in the lungs, bronchoalveolar lavage cells (BALc), lymph nodes and tonsils. The heterozygous pigs showed lower viral loads in the serum and lungs and higher weight gains than the homozygous pigs based on the area under the curve calculation. Consistently, compared with the homozygous pigs, the heterozygous pigs exhibited significantly higher levels of IFN-α in the serum, proliferation of various T cells (γδT, Th1, and Th17) in PBMCs and tissues, and cytotoxic T cells in the lungs and BALc. These results indicate that the higher resistance in the pigs heterozygous for the GBP1E2, WUR and GBP5 markers could be mediated by increased antiviral cytokine (IFN-α) production and T cell activation.

Characterization of Clostridium novyi isolated from a sow in a sudden death case in Korea.

BACKGROUND: Multifocal spherical nonstaining cavities and gram-positive, rod-shaped, and endospore-forming bacteria were found in the liver of a sow that died suddenly. Clostridium novyi type B was identified and isolated from the sudden death case, and the isolate was characterized by molecular analyses and bioassays in the current study. RESULTS: C. novyi was isolated from the liver of a sow that died suddenly and was confirmed as C. novyi type B by differential PCR. The C. novyi isolate fermented glucose and maltose and demonstrated lecithinase activity, and the cell-free culture supernatant of the C. novyi isolate exhibited cytotoxicity toward Vero cells, demonstrating that the isolate produces toxins. In addition, whole-genome sequencing of the C. novyi isolate was performed, and the complete sequences of the chromosome (2.29 Mbp) and two plasmids (134 and 68 kbp) were identified for the first time. Based on genome annotation, 7 genes were identified as glycosyltransferases, which are known as alpha toxins; 23 genes were found to be related to sporulation; 12 genes were found to be related to germination; and 20 genes were found to be related to chemotaxis. CONCLUSION: C. novyi type B was isolated from a sow in a sudden death case and confirmed by biochemical and molecular characterization. Various virulence-associated genes were identified for the first time based on whole-genome sequencing.

Evaluation of the Inhibitory Effects of (E)-1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(naphthalen-1-yl)prop-2-en-1-one (DiNap), a Natural Product Analog, on the Replication of Type 2 PRRSV In Vitro and In Vivo.

DiNap [(E)-1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(naphthalen-1-yl)prop-2-en-1-one], an analog of a natural product (the chalcone flavokawain), was synthesized and characterized in this study. Porcine reproductive and respiratory syndrome virus (PRRSV) is the most challenging threat to the swine industry worldwide. Currently, commercially available vaccines are ineffective for controlling porcine reproductive and respiratory syndrome (PRRS) in pigs. Therefore, a pharmacological intervention may represent an alternative control measure for PRRSV infection. Hence, the present study evaluated the effects of DiNap on the replication of VR2332 (a prototype strain of type 2 PRRSV). Initially, in vitro antiviral assays against VR2332 were performed in MARC-145 cells and porcine alveolar macrophages (PAMs). Following this, a pilot study was conducted in a pig model to demonstrate the effects of DiNap following VR2332 infection. DiNap inhibited VR2332 replication in both cell lines in a dose-dependent manner, and viral growth was completely suppressed at concentrations ≥0.06 mM, without significant cytotoxicity. Consistent with these findings, in the pig study, DiNap also reduced viral loads in the serum and lungs and enhanced the weight gain of pigs following VR2332 infection, as indicated by comparison of the DiNap-treated groups to the untreated control (NC) group. In addition, DiNap-treated pigs had fewer gross and microscopic lesions in their lungs than NC pigs. Notably, virus transmission was also delayed by approximately 1 week in uninfected contact pigs within the same group after treatment with DiNap. Taken together, these results suggest that DiNap has potential anti-PRRSV activity and could be useful as a prophylactic or post-exposure treatment drug to control PRRSV infection in pigs.

The prevalence and genetic characteristics of porcine circovirus type 2 and 3 in Korea.

BACKGROUND: Porcine circovirus-associated diseases (PCVAD), caused by porcine circovirus type 2 (PCV2), threaten the pig industry worldwide. Five genotypes of PCV2 were recently identified: PCV2a, PCV2b, PCV2c, PCV2d and PCV2e. In addition, a novel porcine circovirus from a case of a sow with dermatitis, nephropathy syndrome and reproductive failure has been identified based on metagenomic analysis and classified as porcine circovirus type 3 (PCV3). Therefore, the current study was conducted to determine the prevalence and genetic characteristics of PCV2 and PCV3 in clinical samples. RESULTS: A total of 471 samples (161 tissue samples of lungs and lymph nodes from 34 farms and 310 serum samples from 47 farms) were tested for PCV2. Among them, 171 samples from 59 farms that had been positive for PCV2 were genotyped. Another 690 samples (296 tissue samples of lungs and lymph nodes from 91 farms, 108 samples of aborted foetuses from 26 farms, and 286 serum samples from 47 farms) were tested for PCV3. Based on PCV2 genotyping results, PCV2d was the most prevalent genotype (107 of 171 samples), and co-infections with combinations of PCV2a, 2b and 2d were identified in 48 samples from 17 farms. A total of 14 samples from 11 farms were also positive for both PCV2 and PCV3. For PCV3, 57 samples (9.8%) from 32 farms (23.2%) were positive. Among the 108 aborted foetuses from 26 farms, only 2 samples were positive for PCV3. Based on sequence comparisons, PCV2d shares 89.6-91.0% and 93.2-94.3% homology with PCV2a and PCV2b, respectively; 98.6-100% homology is shared among PCV2d strains. The PCV3 strains identified in this study share 98.0-99.5% homology. CONCLUSIONS: Our study concludes that PCV2d has become the most predominant genotype in Korea. PCV3 was also identified in clinical samples, though no significant association with clinical symptoms was observed in PCV3-positive cases.

Porcine Astrovirus Infection in Brains of Pigs in Korea.

Recently, neurological diseases associated with astroviruses (AstVs) have been reported in pigs, ruminants, minks, and humans. In 2017, neuro-invasive porcine astrovirus (Ni-PAstV) 3 was detected in the central nervous system (CNS) of pigs with encephalomyelitis in Hungary and the USA. In the process of diagnosing domestic pigs exhibiting neurological signs, histopathologic lesions of non-suppurative encephalomyelitis with meningitis, neuronal vacuolation, and gliosis were detected, and PAstV was identified using reverse transcriptase PCR in CNS samples of four pigs in three farms from August to September in 2020, South Korea. Subsequently, the ORF2 region was successfully acquired from three brain samples, facilitating subsequent analysis. Four genotypes of PAstV (PAstV1, 3, 4, and 5) were detected, and coinfection of PAstV with multiple genotypes was observed in brain samples. This is the first study to report Ni-PAstV infection in pigs in South Korea.

Intradermal Inoculation of Inactivated Foot-and-Mouth Disease Vaccine Induced Effective Immune Responses Comparable to Conventional Intramuscular Injection in Pigs.

All pigs in the Republic of Korea are given the foot-and-mouth disease virus (FMDV) vaccine intramuscularly (IM) as part of the country's vaccination policy. However, the IM administration of the FMDV vaccine to pig results in residual vaccine components in the muscle and undesirable changes in muscle and soft tissues, causing economic losses in swine production. In this study, we evaluated whether intradermal (ID) vaccination could be proposed as an alternative to IM administration. ID vaccination (0.2 mL on each side of the neck muscle) and IM vaccination (2 mL on each side of the neck muscle) were performed twice, separated by 14 days, using a commercial FMD vaccine in specific-pathogen-free pigs. We observed growth performance, gross and microscopic lesions at the inoculation site, FMDV-specific antibodies, and neutralizing antibodies for 35 days after vaccination. Side effects on the skin grossly appeared following ID administration, but most were reduced within two weeks. All ID-vaccinated pigs showed inflammatory lesions limited to the dermis, but IM-vaccinated pigs had abnormal undesirable changes and pus in the muscle. ID-vaccinated pigs performed comparably to IM-vaccinated pigs in terms of growth, FMD virus-specific antibodies, protection capability against FMDV, and T-cell induction. This study demonstrated that the ID inoculation of the inactivated FMD vaccine induced immune responses comparable to an IM injection at 1/10 of the inoculation dose and that the inoculation lesion was limited to the dermis, effectively protecting against the formation of abnormal undesirable changes in muscle and soft tissues.

Detection of various DNA and RNA viruses in bats in Yamaguchi Prefecture, Japan.

Bats are important natural hosts of various zoonotic viruses, including Ebola virus, Lyssa virus, and severe acute respiratory syndrome coronavirus (SARS-CoV). Although investigation of bats is valuable for predicting emerging infectious diseases from these animals, few surveys of bat-derived viruses have been conducted in Japan. In the present study, samples were collected from a total of 132 bats of 4 different species from 4 different locations within Yamaguchi Prefecture; these sample were employed for comprehensive detection of bat-derived viruses by polymerase chain reaction (PCR) and reverse transcription (RT)-PCR using primers universal for each of 4 different viral classes. As a result of PCR and RT-PCR, various herpesviruses, astroviruses, coronaviruses, and adenoviruses were identified from a total of 80 bats. The detected herpesviruses belong to the Betaherpesvirinae or Gammaherpesvirinae subfamily, the detected adenoviruses to the genus Mastadenovirus, the detected astroviruses to the genus Mamastrovirus; and the detected coronaviruses belong to the genus Alphacoronavirus. The detected sequences of 12 strains of 4 families showed 100 % amino acid identity with viruses previously detected either in China or South Korea. These findings expand our understanding of viruses carried by bats, and provide insights into the nature of bat-derived viruses in Japan.

First Trial of a Novel Caseous Lymphadenitis Inactivated Vaccine in South Korea: Experimental Evaluation across Various Animal Models.

Caseous lymphadenitis (CLA) is a chronic and subclinical bacterial disease of ruminants caused by Corynebacterium pseudotuberculosis (C. pseudotuberculosis) infection. Until 2014, there were no reports of CLA outbreaks in South Korea; however, the prevalence of CLA cases has steadily increased. In this study, we used recently obtained field isolates to develop the first inactivated CLA vaccine in South Korea and evaluated it in various animal models. The inactivated vaccine was evaluated for virulence and effectiveness. Mice were tested for virulence and immunization challenges, and guinea pigs and Korean Native Black Goats (KNBGs) evaluated various vaccine concentrations to determine the optimal dose and effectiveness. In the case of KNBGs, clinical symptoms were not observed after vaccination. In addition, CLA-specific IgG was detected at a significantly (p < 0.05) high level and was maintained. In histopathological evaluations, inflammation was predominantly observed in the prefemoral lymph nodes in the non-vaccinated+CHAL group. The genetic diversity of C. pseudotuberculosis, which has become widespread in South Korea, is less than 0.5% our vaccine is expected to prevent infection by a wide range of strains effectively. In summary, our CLA vaccine can potentially prevent CLA and foster the growth of South Korea's domestic KNBG industry.

Detection and Genetic Characterization of Astroviruses in Brain Tissues of Wild Raccoon Dogs.

Astroviruses (AstVs) have been detected in a wide range of animal species, including mammals and birds. Recently, a novel AstV associated with neurological symptoms has been detected in the brains of some mammals. Raccoon dog AstV has been reported recently in China. However, there have been no reports in South Korea. Therefore, the present study aimed to detect and genetically characterize AstVs in the intestine and brain tissues of 133 wild raccoon dogs collected in Korea between 2017 and 2019. Of the seven raccoon dogs, AstVs were detected in six intestinal tissues and four brain tissues. Analysis of the capsid protein amino acid sequences of raccoon dog AstVs detected in Korea revealed a high similarity to canine AstVs, suggesting possible interspecies transmission between raccoon dogs and dogs. Phylogenetic and capsid protein amino acid sequence analysis of raccoon dog AstVs detected in the brain the 17-148B strain belonging to the HMO clade and exhibiting conserved sequences found in neurotropic AstVs (NT-AstVs), indicating their potential as NT-AstVs. However, the pathogenicity and transmission routes of the raccoon dog AstV detected in Korea have not yet been elucidated, so further research and continued surveillance for AstV in wild raccoon dogs are needed.

A standardized method to study immune responses using porcine whole blood.

BACKGROUND: Peripheral blood mononuclear cells (PBMCs) are commonly used to assess in vitro immune responses. However, PBMC isolation is a time-consuming procedure, introduces technical variability, and requires a relatively large volume of blood. By contrast, whole blood assay (WBA) is faster, cheaper, maintains more physiological conditions, and requires less sample volume, laboratory training, and equipment. OBJECTIVES: Herein, this study aimed to develop a porcine WBA for in vitro evaluation of immune responses. METHODS: Heparinized whole blood (WB) was diluted (non-diluted, 1/2, 1/8, and 1/16) in RPMI-1640 media, followed by phorbol myristate acetate and ionomycin. After 24 h, cells were stained for interferon (IFN)-γ secreting T-cells followed by flow cytometry, and the supernatant was analyzed for tumor necrosis factor (TNF)-α. In addition, diluted WB was stimulated by lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I:C), reference strain KCTC3557 (RS), field isolate (FI), of heat-killed (HK) Streptococcus suis, and porcine reproductive and respiratory syndrome virus (PRRSV). RESULTS: The frequency of IFN-γ+CD3+ T-cells and concentration of TNF-α in the supernatant of WB increased with increasing dilution factor and were optimal at 1/8. WB TNF-α and interleukin (IL)-10 cytokine levels increased significantly following stimulation with LPS or poly I:C. Further, FI and RS induced IL-10 production in WB. Additionally, PRRSV strains increased the frequency of IFN-γ+CD4-CD8+ cells, and IFN-γ was non-significantly induced in the supernatant of re-stimulated samples. CONCLUSIONS: We propose that the WBA is a rapid, reliable, and simple method to evaluate immune responses and WB should be diluted to trigger immune cells.

First Isolation of Pseudogymnoascus destructans, the Fungal Causative Agent of White-Nose Syndrome, in Korean Bats (Myotis petax)

White-nose syndrome (WNS), caused by Pseudogymnoascus destructans (Pd), is a lethal fungal disease that affects hibernating bats in North America. Recently, the presence of Pd was reported in countries neighboring Korea. However, Pd has not been investigated in Korea. Therefore, this study aimed to identify the presence of Pd in Korean bats. Altogether, wings from 241 bats were collected from 13 cities and cultured. A total of 79 fungal colonies were isolated, and two isolates were identified as Pd using polymerase chain reaction. Of the nine bat species captured in 13 cities, Pd was isolated only from Myotis petax in Goryeong. Atypical, curved conidia were observed in two isolated fungal colonies. Although histological lesions were not observed by hematoxylin and eosin or periodic acid−Schiff staining, fungal invasion was observed in the tissue sections. Taken together, these results confirmed the presence of Pd in Korean bats and suggest the possibility of WNS outbreaks in Korean bats. This is the first report of the isolation and molecular analysis of Pd from Korean bats.

Insight into the Economic Effects of a Severe Korean PRRSV1 Outbreak in a Farrow-to-Nursery Farm.

Porcine reproductive and respiratory syndrome (PRRS) is a disease that has inflicted economic losses in the swine industry. The causative agent, porcine reproductive and respiratory syndrome virus (PRRSV), is known to have a high genetic diversity which leads to heterogeneous pathogenicity. To date, the impact of PRRS outbreaks on swine production and the economy of the swine industry in South Korea has been rarely reported. In this study, we compare the reproductive performance in the breeding-farrowing phase and growth performance in the nursery phase, in two 27-week periods, one before and one after a PRRSV1 outbreak on a 650-sow farrow-to-nursery farm caused by a Korean PRRSV1 isolate which was genetically distinct from vaccine strains or other global strains. The reproductive performance of sows and the growth performance of nursery pigs were compared using row data consisting of 1907 mating records, 1648 farrowing records, and 17,129 weaning records from 32 breeding batches. The following variables were significantly different between the pre-PRRS outbreak period and the post-PRRS outbreak period: the farrowing rate (−7.1%, p < 0.0001), the abortion rate (+3.9%, p < 0.0001), the return rate (+2.9%, p = 0.0250), weaning to estrus interval days (+1.9 days, p < 0.0001), total piglets born (−1.2 pigs/litter, p < 0.0001), piglets born alive (−2.2 pigs/litter, p < 0.0001), weaned piglets (−2.7 pigs/litter, p < 0.0001), pre-weaning mortality (+7.4%, p < 0.0001), weaning weight (−0.9 kg/pig, p = 0.0015), the mortality rate (+2.8%, p < 0.0001), average daily gain (−69.8 g/d, p < 0.0001), and the feed conversion ratio (+0.26, p = 0.0036). Economic losses for a period of 27 weeks after a PRRS outbreak were calculated at KRW 99,378 (USD 82.8) per mated female for the breeding-farrowing phase, KRW 8,968 (USD 7.5) per pig for the nursery growth phase, and KRW 245,174 (USD 204.3) per sow in the post-outbreak period. In conclusion, the farrow-to-nursery farm in our study suffered extensive production and economic losses as a result of a PRRSV1 outbreak.

Pathological Evaluation of Porcine Circovirus 2d (PCV2d) Strain and Comparative Evaluation of PCV2d and PCV2b Inactivated Vaccines against PCV2d Infection in a Specific Pathogen-Free (SPF) Yucatan Miniature Pig Model.

Porcine circovirus type 2 (PCV2) is an economically important swine pathogen that causes porcine circovirus-associated diseases (PCVADs). The objective of this study was to evaluate the use of specific pathogen-free Yucatan miniature pigs (YMPs) as an experimental model for PCV2d challenge and vaccine assessment because PCV2-negative pigs are extremely rare in conventional swine herds in Korea. In the first experiment, every three pigs were subjected to PCV2d field isolate or mock challenge. During three weeks of experiments, the PCV2d infection group exhibited clinical outcomes of PCVAD with high viral loads, lymphoid depletion, and detection of PCV2d antigens in lymphoid organs by immunohistochemistry. In the second experiment, three groups of pigs were challenged with PCV2d after immunization for three weeks: a nonvaccinated group (three pigs), a PCV2b-Vac group vaccinated with a commercial PCV2b-based inactivated vaccine SuiShot® Circo-ONE (five pigs), and a PCV2d-Vac group vaccinated with an experimental PCV2d-based inactivated vaccine (five pigs). During the three weeks of the challenge period, nonvaccinated pigs showed similar clinical outcomes to those observed in the PCV2d infection group from the first experiment. In contrast, both the PCV2b and PCV2d vaccinations produced good levels of protection against PCV2d challenge, as evidenced by reduced viral loads, improved growth performance, high virus-neutralizing antibody titers, and less development of PCV2-associated pathological lesions. Taken together, these data suggest that YMPs could be an alternative model for PCV2 challenge experiments, and these animals displayed typical clinical and pathological features and characteristics of protective immunity induced by the vaccines that were consistent with those resulting from PCV2 infections in conventional pigs.