• Korean Journal of Veterinary Research
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• v.37 no.4
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• pp.793-807
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• 1997

We tried to develop detection system of porcine reproductive and respiratory syndrome virus(PRRSV) by in situ hybridization(ISH) in the piglets experimentally infected with KPRRS-2, the Korean isolate(12 piglets) or Mn-1b, the American isolate(4 piglets), and in the natural infection suspected 6 piglets. Twelve 30-days-old piglets(two pigs per each inoculated group) were inoculated by nasal instillation of KPRRS-2 virus(total dose $10^{4.5}TCID_{50}$), Six piglets(one pig per each group) were induced contact infection with inoculated piglets, during the experiment, and two piglets were used as control. Inoculated or contacted piglets were euthanized at 1, 3, 5, 7, 14 and 21 days postinoculation(DPI). The respiratory signs such as coughing and nasal discharge were observed on day 3 DPI, and ear cyanosis were on day 5 DPI, including contacted piglets. Through the necropsy, purple discolorization of dorsal part of lung, and hypertrophy of local lymph nodes were observed. The histopathological lesions of lung were interstitial pneumonia characterized by type 2 pneumocyte hyperplasia. We prepared the probe for ISH by RNA isolation from KPRRS-2, RT-PCR, and biotin labeling. We performed the ISH within only 1~2 hours using $Microprobe^{TM}$ capillary action system. As the results, the strong red specific positive signals, means PRRSV distribution, was mainly observed in the cytoplasm of alveolar macrophages. And also signals were detected in some type 2 pneumocytes and bronchiolar epithelium of lung, myocardium, liver, kidney, tonsil, spleen, gastrointestinal mucosa, testis and lymph nodes.

• Journal of Veterinary Science
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• v.21 no.4
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• pp.68.1-68.8
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• 2020

A fluorescent microsphere-based immunochromatographic strip test (FICT) was developed for the rapid, sensitive, and quantitative detection of porcine reproductive and respiratory syndrome virus (PRRSV) antibodies at the pen-side. The assay was based on the formation of a sandwich immune-complex (anti-pig IgG-PRRSV antibodies-NSP7/N), which was validated by a comparison with IDEXX-ELISA using 3325 clinical specimens. The diagnostic specificity, sensitivity, and accuracy of FICT were 97.28, 93.41, and 94.95%, respectively. FICT showed a good correlation with the virus neutralization assay. Overall, a promising pen-side diagnostic tool was developed for the rapid and quantitative detection of PRRSV antibodies within 15 min.

• Korean Journal of Veterinary Service
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• v.37 no.1
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• pp.1-9
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• 2014

Although it has been generally accepted that porcine reproductive and respiratory syndrome virus (PRRSV) induces weak and delayed protective immunity after infection, it is unclear that the same immunological features can be applicable to all PRRS viruses because huge genetic variation exists even among the same genotypes of PRRSV (Type 1 and 2). In the current study, two genetically distinct type 2 PRRSV strains (VR-2332 and JA142) which showed approximately 90% nucleotide homology based on ORF5 sequences were characterized by both in vitro and in vivo assessments to determine the immunological features of the viruses. For in vitro assessment, porcine alveolar macrophages (PAM) were infected with the viruses at $10^{-3}$ multiplicity of infection (MOI) and then supernatants and cells were collected separately at 36 hrs post infection to determine the relative expression levels of IL-$1{\alpha}$, IL-12, TNF-${\alpha}$ and INF-${\alpha}/{\beta}$ by quantitative RT-PCR. In addition, five PRRSV-free pigs were inoculated with either of JA142 or VR2332 for in vivo assessment. Serum samples were collected every week until 6 weeks post challenge. The serum samples were analyzed for the levels of viremia, PRRSV nucleocapsid-specific antibody and virus neutralizing antibody. Based on those assessments, the two viruses showed different patterns of cytokine expression in PAM and immune responses in pigs after infection. These results indicate that genetically distinct PRRSV strains have different immunological features, which might be criteria for virus classification and selection of candidate virus strains for vaccine development in the future.

• Korean Journal of Veterinary Research
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• v.46 no.4
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• pp.363-370
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• 2006

Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease of swine that occurs all over the swine industry worldwide. It was first observed in the Unite States in 1987 then in Europe in 1990. It has been described in Japan and in Korea in 1993. PRRS virus is divided into two distinct types, North American and European, genetically. Based on our limited knowledge there has been no report on the existence of European PRRSV. But according to the government's Korea Customs Service there has been many importations of breeding pigs from Europe. These seem to make an estimate that European PRRSV could be introduced in Korea by inflow of European breeding pigs. We first detected the European PRRSV could be introduced in Korean pig farms by using polymerase chain reaction (PCR). Further, it is also identified that there are not only North American PRRSV antibody but also a European PRRSV antibody. According to the genetical and serological experiment results, the presence of established North American PRRSV in Korea is due to the use of live vaccines made of North American PRRSV strain as well field virus infection, and the European PRRSV is possibly introduced from imported breeding stock.

• Korean Journal of Veterinary Research
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• v.43 no.1
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• pp.129-137
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• 2003

Porcine reproductive and respiratory syndrome (PRRS) is characterized by reproductive failures in sows and respiratory problems in piglets. The nucleocapsid(N) protein, encoded by the open reading frame 7 (ORF7) gene, is known to be the most abundant and antigenic protein in PRRS virus. Therefore, it was suggested that the N protein could be a suitable candidate for the detection of PRRS virus-specific antibodies and diagnosis of PRRS. In the present study, the ORF7 gene encoding the N protein was cloned and expressed as a fusion protein with the glutathione S-transferase (GST) in Escherichia coli. The resulting GST-N recombinant protein was used as an antigen for an indirect sandwich enzyme-linked immunosorbent assay (i-ELISA). Expressed GST-N recombinant protein was migrated at 41 kDa and reacted with ORF7-specific monoclonal antibody by Western blotting. In order to increase the specificity of the ELISA for the detection of PRRS virus-specific antibodes, an i-ELISA was developed using an anti-GST antibody as a capture antibody. The sensitivity and specificity of developed i-ELISA were 92% and 96%, respectively. Based on these results, it was suggested that the i-ELISA is a simple and rapid test for screening a large number of swine sera for the anti-PRRS virus antibodies.

• Microbiology and Biotechnology Letters
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• v.43 no.1
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• pp.1-8
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• 2015

In this study, a DNA-launched reverse genetics system was developed from a type 2 porcine reproductive and respiratory syndrome virus (PRRSV) strain, KNU-12. The complete genome of 15,412 nucleotides was assembled as a single cDNA clone and placed under the eukaryotic CMV promoter. Upon transfection of BHK-tailless pCD163 cells with a full-length cDNA clone, viable and infectious type 2 progeny PRRSV were rescued. The reconstituted virus was found to maintain growth properties similar to those of the parental virus in porcine alveolar macrophage (PAM) cells. With the availability of this type 2 PRRSV infectious clone, we first explored the biological relevance of ORF5a in the PRRSV replication cycle. Therefore, we used a PRRSV reverse genetics system to generate an ORF5a knockout mutant clone by changing the ORF5a translation start codon and introducing a stop codon at the 7^th codon of ORF5a. The ORF5a knockout mutant was found to exhibit a lack of infectivity in both BHK-tailless pCD163 and PAM-pCD163 cells, suggesting that inactivation of ORF5a expression is lethal for infectious virus production. In order to restore the ORF5a gene-deleted PRRSV, complementing cell lines were established to stably express the ORF5a protein of PRRSV. ORF5a-expressing cells were capable of supporting the production of the replicationdefective virus, indicating complementation of the impaired ORF5a gene function of PRRSV in trans.

• Korean Journal of Veterinary Service
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• v.45 no.1
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• pp.63-69
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• 2022

Proliferative and necrotizing pneumonia (PNP) is a form of interstitial pneumonia that occurs in post-weaning pigs. In this study, we investigated the presence of swine influenza virus (SIV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2) and Aujeszky's disease virus (ADV) in PNP lesions in Jeju pigs. Based on the histopathologic criteria for PNP, a total of 50 cases were selected in Jeju pigs between 2008 and 2010. Coupled with histopathological examinations, the presence of ADV and SIV by polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) and PRRSV and PCV2 by immunohistochemical (IHC) methods were investigated. Based on the PCR and RT-PCR methods, ADV and SIV nucleic acids were not detected in all cases. According to IHC, PRRSV was detected in 38 of the 50 cases examined (76%) and PCV2 in 25 cases (50%). PRRSV or PCV2 were detected in 19 (38%) or 6 (12%) cases, respectively. Both PRRSV and PCV2 were identified in other 19 cases (38%). Antigens of PRRSV and PCV2 were commonly observed in the cytoplasm of macrophages and clusters of necrotic cells in alveolar cavities. The results of the present study demonstrate that PRRSV is predominantly associated with PNP in Jeju pigs. Co-infection with PRRSV and PCV2 may enhance the severity of PNP lesions in affected pigs.

• Korean Journal of Veterinary Research
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• v.49 no.3
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• pp.215-220
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• 2009

The prevalence of potentially xenozoonotic viruses in the reproductive tract of female pigs in Korea was investigated by polymerase chain reaction (PCR). These viruses include porcine endogenous retrovirus (PERV), porcine reproductive and respiratory syndrome virus (PRRSV), swine hepatitis E virus (SHEV), porcine lymphotropic herpesvirus (PLHV), and porcine circovirus type 2 (PCV-2). Histopathological examination and PCR analysis were conducted using the ovaries of 70 slaughtered pigs that were collected from 14 farms in Jeju. Histopathologically, infiltrations of mononuclear inflammatory cells around the thick-walled coiled vessels in the ovarian medulla were observed in 15 cases. Based on the PCR method, PERV, PLHV, PRRSV, SHEV, and PCV-2 were detected in 69 (98.6%), 35 (50%), 5 (7.1%), 4 (5.7%), and 1 sample (1.4%), respectively. These results suggest that PERV and PLHV are the major xenozoonotic viruses in the porcine ovary. This study should aid in the development of a monitoring protocol for potential xenozoonotic agents and in the production of germ-free pigs for xenotransplantation.

• Korean Journal of Veterinary Research
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• v.39 no.4
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• pp.751-759
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• 1999

Plaque characteristics of porcine reproductive and respiratory syndrome (PRRS) virus isolates were examined using MARC-145 line cells. The plaque morphology of PRRS virus isolates was variable in size and heterogenic in population. Upon serial passages of the PRRS virus isolates on MARC-145 tells, heterogeneity was maintained but numbers of the large plaque size virus were increased with certain isolates. A PRRS virus isolate with variable plaque sizes was subcloned into 2 populations : small plaque ($H_S$) and large plaque ($H_L$) viruses. Growth kinetics of the subclones were then determined in MARC-145 cells, and production of the structural polypeptides was analyzed by SDS-PAGE. In a comparison of the growth kinetics, the $H_S$ virus showed higher infectivity titers during the first 48 hours but slower to reach the peak titier than $H_L$ virus did. In a nucleotide sequence comparison, differences of 4 nucleotides in open reading frames 5-6 gene were found between $H_S$ and $H_L$ viruses. Both the $H_S$ and $H_L$ clones produced 5 polypeptide bands with molecular weights of 15, 19, 26, 36 and 42 kD. The 5 bands were detected at 48 hours postinoculation (PI) with antisera to $H_L$ and another large plaque virus ($W_L$) and at 72 hours PI with $H_S$ virus antiserum. The present results demonstrate differences of biologic and molecular characteristics between the two PRRS virus plaque clones.

• Korean Journal of Veterinary Service
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• v.43 no.4
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• pp.251-256
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• 2020

Two suckling piglets, 4 days and 10 days of age, showed lethargy and dyspnea after birth and mortality had been increased after incoming gilts from breeding farm. At necropsy, the lungs showed diffuse fail to collapse with rubbery consistency, edematous dilatation of interlobular septa, and lobular consolidation with purple red color. Heart was diffuse pale in color and had several irregular linear-shaped macules or patches. Histopathologically, diffuse interstitial pneumonia with the proliferation of type II pneumocytes was present in the lungs of 2 piglets. Alveolar lumens contained necrotic cellular debris derived from neutrophils and macrophages. Multifocal hemorrhage and necrotizing pneumonia with protozoan tachyzoites were observed in the lungs. Severe multifocal to confluent necrotic myocarditis, necrotic encephalitis, and necrotic adrenalitis with intralesional protozoan tachyzoites were observed in piglets. According to immunohistochemical analysis (IHC), Toxoplasma (T.) gondii tachyzoites antigens were confirmed in lung, heart, brain, and adrenal gland. And porcine reproductive and respiratory syndrome virus (PRRSV) antigens were also detected in the cytoplasm of macrophages in lungs using IHC. Based on the gross, histopathologic and immunohistochemical features, two suckling piglets were diagnosed as co-infection of T. gondii and PRRSV.