• Journal of Veterinary Science
• /
• v.21 no.2
• /
• pp.22.1-22.9
• /
• 2020

Rabid raccoon dogs (Nyctereutes procyonoides koreensis) have been responsible for animal rabies in South Korea since the 1990s. A recombinant rabies vaccine strain, designated as ERAGS, was constructed for use as a bait vaccine. Therefore, new means of differentiating ERAGS from other rabies virus (RABV) strains will be required in biological manufacturing and diagnostic service centers. In this study, we designed two specific primer sets for differentiation between ERAGS and other RABVs based on mutation in the RABV glycoprotein gene. Polymerase chain reaction analysis of the glycoprotein gene revealed two DNA bands of 383 bp and 583 bp in the ERAGS strain but a single DNA band of 383 bp in the field strains. The detection limits of multiplex reverse transcription polymerase chain reaction (RT-PCR) were 80 and 8 FAID₅₀/reaction for the ERAGS and Evelyn-Rokitnicki-Abelseth strains, respectively. No cross-reactions were detected in the non-RABV reference viruses, including canine distemper virus, parvovirus, canine adenovirus type 1 and 2, and parainfluenza virus. The results of multiplex RT-PCR were 100% consistent with those of the fluorescent antibody test. Therefore, one-step multiplex RT-PCR is likely useful for differentiation between RABVs with and those without mutation at position 333 of the RABV glycoprotein gene.

• The Plant Pathology Journal
• /
• v.22 no.2
• /
• pp.131-138
• /
• 2006

Two strains of Cucumber mosaic virus (CMV) isolated in Kuwait were confirmed their infectivity based on symptomatology and host range on different cultivars of tomato (Lycopersicon esculentum), tobacco(Nicotiana tabacum L.) and squash (Cucurbita pepo). The pattern of symptoms differed for the two CMV strains in tomato and tobacco, showing severe stunting and mosaic symptoms with one strain designated KU2, and almost symptomless with the other strain designated KU1. A satellite RNA 5 (sat-RNA) was found to be associated with the KU1 strain and was characterized as a benign viral satellite RNA. Using reverse transcription and polymerase chain reaction (RT-PCR) with sat-RNA specific primers, an amplified PCR product of about 160bp was determined and analyzed by gel electrophoresis. This naturally occurring benign viral satellite RNA was successfully used as a biological control agent to protect tomato plants against the severe KU2 strain. Tomato plants grown in plant-growth chambers, were preinoculated with KU1 containing the benign viral satellite and then challenge inoculated with the severe KU2 strain at different time intervals. All plants challenged three weeks after preinoculation showed nearly complete protection from subsequent infection by the severe strain. This biological control technology using plant viruses was found protective and could be successfully established sooner after the preinoculation.

• Journal of Plant Biology
• /
• v.37 no.3
• /
• pp.349-355
• /
• 1994

Viral RNA was extracted from a purified orchid strain of tobacco mosaic virus (TMV-O) from Cymbidium "Grace Kelly". Polyadenylated viral RNAs were primed with Not I-oligo (dT) primer-adapter. First-strand cDNAs were reversely transcribed by Moloney murine leukaemia virus reverse transcriptase (RNAse H-), and then second-strand cDNAs were synthesized by RNase H and DNA polymerase I. The resulting double-stranded cDNAs were ligated into pSPORT1 vector and transformed into competent E. coli strain JM109 cells. The size of cDNAs within the recombinant plasmids was ranging from 0.9 to 3.9 kb. Among the selected clones, pTMO-0205 and -0210 covered the 3' half and the 5' half of the viral genomic RNA, respectively, which were covering more than 99% of the viral genemo size based on sequencing analysis. Two cDNA fragments which were 3.1 kb BamHI and NotI fragement released from pTMO-0.205 and 3.3 kb SalI and BamHI fragment released from pTMO-0210 were ligated with T4 DNA ligase. The clone was almost entire length, lacking only 31 nucleotides from the 5' terminus based on the sequencing result. This method was shown to be efficiently applicable to other plant viral gnomic RNA for the construction of cDNA.n of cDNA.

• Journal of Microbiology and Biotechnology
• /
• v.4 no.3
• /
• pp.183-190
• /
• 1994

Cell-free extracts prepared from cultured insect cells, Spodoptera. frugiperda, were analyzed for activation of early gene transcription of an insect baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV). The template DNA used for in vitro transcription assays contained promoter sites for the baculovirus genes that have been classified as immediate early ($\alpha$) or early genes. These genes are located in the HindIII-K/Q region of the AcNPV genome. Nuclei isolated from the AcNPV-infected Spodoptera frugiperda cells were also used for in vitro transcription analysis by RNase-mapping the labeled RNA synthesized from in vitro run-on reaction in the isolated nuclei. The genes studied by this technique were p26 and pl0 genes which were classified as delayed early and late gene, respectively. We found that transcription of the genes from the HindIII-K region was accurately initiated and unique in the whole cell extract obtained from uninfected cells, although abundance of the in vitro transcripts was reverse to that of in vivo RNA. With isolated nuclei transcription of the p26 gene was inhibited by $\alpha$-amanitin suggesting that the p26 gene was transcribed by host RNA polymerase II. However, transcription of the pl0 gene in isolated nuclei was not inhibited by $\alpha$-amanitin, but rather stimulated by the inhibitor. We also found that the synthesis of $\alpha$-amanitin-resistant RNA polymerase was begun before 6 hr p.i., the time point at which the onset of viral DNA replication as well as the appearance of a-amanitin-resistant viral transcripts were detected. These studies give us strong evidence to support the previous data that early genes of AcNPV were transcribed by host RNA polymerease III, while transcription of late genes was mediated at least by a novel $\alpha$-amanitin-resistant RNA polymerase.

• Journal of Microbiology and Biotechnology
• /
• v.31 no.3
• /
• pp.358-367
• /
• 2021

The World Health Organization (WHO) has declared the coronavirus disease 2019 (COVID-19) as an international health emergency. Current diagnostic tests are based on the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method, which is the gold standard test that involves the amplification of viral RNA. However, the RT-qPCR assay has limitations in terms of sensitivity and quantification. In this study, we tested both qPCR and droplet digital PCR (ddPCR) to detect low amounts of viral RNA. The cycle threshold (CT) of the viral RNA by RT-PCR significantly varied according to the sequences of the primer and probe sets with in vitro transcript (IVT) RNA or viral RNA as templates, whereas the copy number of the viral RNA by ddPCR was effectively quantified with IVT RNA, cultured viral RNA, and RNA from clinical samples. Furthermore, the clinical samples were assayed via both methods, and the sensitivity of the ddPCR was determined to be equal to or more than that of the RT-qPCR. However, the ddPCR assay is more suitable for determining the copy number of reference materials. These findings suggest that the qPCR assay with the ddPCR defined reference materials could be used as a highly sensitive and compatible diagnostic method for viral RNA detection.

• Journal of Microbiology and Biotechnology
• /
• v.19 no.10
• /
• pp.1103-1108
• /
• 2009

The human immunodeficiency virus (HIV)-l protein Tat acts as a transcription transactivator that stimulates expression of the infected viral genome. It is released from infected cells and can similarly affect neighboring cells. The nucleocapsid is an important protein that has a related significant role in early mRNA expression, and which contributes to the rapid viral replication that occurs during HIV-1 infection. To investigate the interaction between the Tat and nucleocapsid proteins, we utilized cDNA micro arrays using pTat and flag NC cotransfection in HEK 293T cells and reverse transcription-polymerase chain reaction to validate the micro array data. Four upregulated genes and nine downregulated genes were selected as candidate genes. Gene ontology analysis was conducted to define the biological process of the input genes. A proteomic approach using PathwayStudio determined the relationship between Tat and nucleocapsid; two automatically built pathways represented the interactions between the upregulated and downregulated genes. The results indicate that the up- and downregulated genes regulate HIV-1 replication and proliferation, and viral entry.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.50 no.5
• /
• pp.621-623
• /
• 2017

Mariculture of rainbow trout Onchorhynchus mykiss has been initiated in or around olive flounder Paralichthys olivaceus farms, where viral hemorrhagic septicemia virus (VHSV) is often detected in some fish. In the present study, we investigated VHSV infection in seawater-reared rainbow trout because VHSV has never been detected in salmonids in Korea. A total of 104 adult fish were tested for the presence of VHSV by reverse transcription-polymerase chain reaction, followed by virus isolation with the fathead minnow caudal trunk cell line. Cytopathic effects were observed in two samples but the virus was identified as infectious hematopoietic necrosis virus. Thus, VHSV was not isolated from seawater-reared rainbow trout.

• Korean Journal of Veterinary Service
• /
• v.39 no.1
• /
• pp.35-39
• /
• 2016

The purpose of this study was to survey on infection status of pathogens of diarrhea from Korean indigenous goat. A total of 800 fecal samples was collected from 50 farms from January to October 2015 and was tested by automatic biochemical machine and polymerase chain reaction (PCR). The overall detection ratio of bacterial pathogens was 22.4% and viral pathogens was 16.3%, respectively. The detection ratio of Escherichia coli (E. coli), Salmonella spp., bovine viral diarrhea virus (BVDV), rotavirus and coronavirus were 21.5%, 0.9%, 7.6%, 5.6% and 3.0%, respectively. In the rates of mixed detection, single was 78.2%, double 8.4%, triple 11.6% and quadruple 1.8% in each sample and 38%, 12%, 16%, 20% in each farm, respectively.

• Journal of Practical Agriculture & Fisheries Research
• /
• v.5 no.1
• /
• pp.57-63
• /
• 2003

The bovine rotavirus(BRV), bovine coronavirus(BCV) and bovine viral diarrhea virus(BVDV) are main viruses of bovine viral diarrhea disease. These viruses could be rapidly amplified by the reverse transcriptase polymerase chain reaction(RT-PCR). This study was conducted to develop rapid and accurate diagnostic methods of these viral diseases by multiplex RT-PCR. Specific primers were designed based on the sequences reported by Chang KO et. al. (1997) and Schroeder BA, et. al. (1990), RNA were prepared from the cultured viruses, first-stranded DNAs were synthesised by reverse transcriptase. PCR were conducted to amplify specific regions of the viruses by multiplex. Three bands such as 1,062bp for BRV, 458bp for BCV, and 300bp for BVDV were successfully produced by multiplex RT-PCR. In conclusion, this result suggested that these viruses could be diagnosed rapidly and accurately by multiplex RT-PCR.

• Journal of Environmental Health Sciences
• /
• v.39 no.3
• /
• pp.230-238
• /
• 2013

Objectives: For our survey of the incidence of influenza viruses among respiratory viral infections in Seoul, we evaluated their prevalence among infectious acute respiratory viral patients in Seoul from 2010 to 2012 through regular surveillance. Methods: For influenza virus detection, we conducted real-time PCR analyses on 2,544 throat specimens collected from patients with respiratory viral infections in Seoul between 2010 and 2012. They were collected and then tested for the presence of influenza viruses through reverse transcription (RT) - polymerase chain reaction (PCR). Results: 19.1% (486/2,544) of the throat specimens were determined to be positive for influenza viruses. The incidences of influenza viral infection in the case of respiratory viral infections through regular surveillance in Seoul were 23.0% (212/923) in 2010, 6.4% (47/738) in 2011, and 25.7% (227/883) in 2012, and 10.8% (275/2,544) of type A, and 8.3% (211/2,544) type B influenza viruses. In addition, the greatest prevalence was in the 20-49 age group (51.6% ), which shows that influenza viruses constituted a major causative agent of acute respiratory viral infections. Conclusions: The distributions of influenza viruses and the epidemiologic patterns of the viral pathogen in acute respiratory viral infectious patients may provide potentially effective data for epidemiological studies in Seoul, Korea.