• Korean Journal of Veterinary Pathology
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• v.5 no.2
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• pp.57-62
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• 2001

Recently various molecular diagnostic techniques have been used to identify rabbit hemorrhagic disease virus (RHDV), a causative agent responsible for acute hepatitis and disseminated intravascular coagulation in rabbit. But they were hard to perform and time consuming. To detect RHDV in a rapid and easy way, we developed biotinylated oligonucleotide probe within ORF 1 region encoding the polyprotein of RHDV in formalin-fixed and paraffin-embedded tissues from various tissues of 20 rabbits naturally infected with RHDV, Our in situ hybridization (ISH) was quickly carried out within two hours by MicroProbe capillary action system. The ISH produced a positive reaction in liver, kidney and lung. In conclusion, ISH with a biotintlated oligonucleotide probe provided a useful diagnostic method for detecting RHDV.

• Applied Biological Chemistry
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• v.38 no.1
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• pp.55-62
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• 1995

To understand the molecular structure of Korean garlic viruses, cDNA cloning of virus genomic RNA was attempted. Virus particles were isolated from virus-infected garlic leaves and a cDNA library was constructed from garlic virus RNA. One of these clones, S81, selected by random sequencing has been identified as a member of potexvirus group other than potyvirus and carlavirus. The clone is 873 bp long contains most of the coat protein (CP) coding region and 3'-noncoding region including poly(A) tail. A putative polyadenylation signal sequence (AAUAAA) and the hexanucleotide motif (ACUUAA), a replicational cis-acting element conserved in the 3'-noncoding region of potexvirus RNAs are noticed. The clone S81 shows about 30-40% identity in both nucleotide and amino acid sequences with CPs of potexviruses. The genome size of the virus was analysed to be 7.46 knt by Northern blot analysis, which was longer than those of other potexviruses. The open reading frame encoding CP was expressed as a fusion protein (S81CP) in Escherichia coli and the recombinant protein was purified by immobilized metal binding affinity chromatography. Polyclonal antibody was raised against S81CP in rabbit to examine the occurrence of garlic potexvirus in Korean garlic plants by immunoblot analysis. Two virus protein bands of Mr 27,000 and 29,000 from garlic leaf extract of various cultivars reacted with the antibody. It was shown that Mr 27,000 band might not be a degradation product of Mr 29,000 band, suggesting that two types of potexvirus different in size of coat protein could exist in Korean garlic plants.

• Journal of Plant Biotechnology
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• v.32 no.4
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• pp.243-250
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• 2005

The coat protein gene (AF296280) of the Korean isolate Potato leafroll virus (PLRV) was cloned and the open reading frame (627 bp) was transformed into potato (Solanum tuberosum cv. Superior). Out of seventeen individual transgenic lines, five lines were identified to confer resistance to PLRV through the five generation's selection program in the greenhouse as well as isolated trial field. Successful introduction and genetic stability of coat protein gene in the genome of potato were confirmed by polymerase chain reaction (PCR), Southern blot hybridization and northern blot hybridization. Some of the transgenic lines were highly resistant to PLRV but did not show any resistance to less homologous Potato virus Y (PVY). Our results suggest that the resistance to PLRV is due to homology dependent gene silencing by sense strand coat protein gene. In addition, the results of field test through five generations showed that there were no significant differences comparing to nontransgenic potatoes in the morphological aspect of shoot as well as tuber, Ho remarkable differences were also observed in the major agronomic characters and yields except for the resistance to PLRV.

• BMB Reports
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• v.36 no.5
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• pp.475-487
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• 2003

A gene that encodes a homologue to baculoviral p74, an envelope-associated viral structural protein, has been identified and sequenced on the genome of Choristoneura fumiferana granulovirus (ChfuGV). A part of the ChfuGV p74 gene was located on an 8.9 kb BamHI subgenomic fragment using different sets of degenerated primers. These were designed using the results of the protein sequencing of a major 74 kDa structural protein that is associated with the occlusion-derived virus (ODV). The gene has a 1992 nucleotide (nt) open-reading frame (ORF) that encodes a protein with 663 amino acids with a predicted molecular mass of 74,812 Da. Comparative studies revealed the presence of two major conserved regions in the ChfuGV p74 protein. This study also shows that all of the p74 proteins contain two putative transmembrane domains at their C-terminal segments. At the nucleotide sequence level, two late promoter motifs (TAAG and GTAAG) were located upstream of the first ATG of the p74 gene. The gene contained a canonical poly(A) signal, AATAAA, at its 3' non-translated region. A phylogenetic tree for baculoviral p74 was constructed using a maximum parsimony analysis. The phylogenetic estimation demonstrated that ChfuGV p74 is related the closest to those of Cydia pomonella granulovirus (CpGV) and Phthorimaea operculella granulovirus (PhopGV).

• BMB Reports
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• v.35 no.6
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• pp.595-603
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• 2002

A gene that encodes a homologue to baculoviral ODVP-6E/ODV-E56, a baculoviral envelope-associated viral structural protein, has been identified and sequenced on the genome of Choristoneura fumiferana granulovirus (ChfuGV). The ChfuGV odvp-6e/odv-e56 gene was located on an 11-kb BamHI subgenomic fragment using different sets of degenerated primers, which were designed using the results of the protein sequencing of a major 39 kDa structural protein that is associated with the occlusion-derived virus (ODV). The gene has a 1062 nucleotide (nt) open-reading frame (ORF) that encodes a protein with 353 amino acids with a predicated molecular mass of 38.5 kDa. The amino acid sequence data that was derived from the nucleotide sequence in ChfuGV was compared to those of other baculoviruses. ChfuGV ODVP-6E/ODV-E56, along with othe baculoviral ODVP-6E/ODV-E56 proteins, all contained two putative transmembrane domains at their C-terminus. Several putative N-and O-glycosylation, N-myristoylation, and phosphorylation sites were detected in the ChfuGV ODVP-6E/ODV-E56 protein. A similar pattern was detected when a hydrophobicity-plots comparison was performed on ChfuGV ODVP-6E/ODV-E56 with other baculoviral homologue proteins. At the nucleotide level, a late promoter motif (GTAAG) was located at -14 nt upstream to the start codon of the GhfuGV odvp-6e/odv-e56 gene. a slight variant of the polyadenylation signal, AATAAT, was detected at the position +10 nt that is downstream from the termination signal. A phylogenetic tree for baculoviral ODVP-6E/ODV-E56 was constructed using a maximum parsimony analysis. The phylogenetic estimation demonstrated that ChfuGV ODVP-6E/ODV-E56 is most closely related to those of Cydia pomonella granulovirus (CpGV) and Plutella xylostella granulovirus (PxGV).

• The Journal of Korean Society of Virology
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• v.27 no.2
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• pp.185-195
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• 1997

Sequence comparison of the RNA-dependent RNA polymerase of human caliciviruses (HuCVs) from Korean children with gastroenteritis revealed significant genetic variation among them. cDNA clones were produced from the HuCVs collected from pediatric population during a period of 1987-1994. The application of reverse transcription-polymerase chain reaction (RT-PCR) using primers directed to the RNA-dependent RNA polymerase region within ORF1 of Norwalk virus (NV) showed that 13.7% of HuCVs yielded PCR products of similar size to the NV prototype, NV8FIIa/68/US, with exceptions of HuCV 185/87/Korea and HuCV 1115/90/Korea. Computer analyses showed that the PCR products had a continuous protein encoding frame on the positive strand, and contained GLPSG and YGDD amino acid motifs at the predicted distance from primers. Alignment of the amino acid sequences of HuCVs with previously published sequences for Snow Mountain agent (SMA), NV, and Sapporo/82/Japan indicated that these strains can be divided into four major genogroups. There were 10 (45%) SMA-like CVs, one (4.5%) NV-like HuCVs, two (9%) Sapporo-like HuCVs, and nine (41%) unidentified HuCVs. This fourth genogroup should be investigated further. HuCV 185/87/Korea and HuCV 1115/90/Korea, Sapporo-like CVs, were genetically distinct from previously characterized HuCVs and more closely related to known animal CVs. One of the animal CV-like strain, HuCV 185/87/Korea, showed nucleotide and amino acid homology of only 67% and 73% with the prototype Sapporo/82/Japan. Further characterization of animal and human CV genomes and studies of possible cross-transmission of CVs from animals to humans are likely to be beneficial in understanding the epidemiology of HuCVs.

• Korean Journal of Veterinary Service
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• v.37 no.1
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• pp.11-18
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• 2014

Porcine reproductive and respiratory syndrome (PRRS) causes a significant economic loss in the swine industry not only in Korea but also all over the world. Andong and Hapcheon region were selected for Area Regional Control (ARC) programme to reduce the shedding of PRRS virus (PRRSV) and decrease PRRS outbreaks. Before conducting the PRRS ARC, sera of pigs were tested for both antibody using ELISA and antigen using RT-PCR, then phylogenetic classifications was analysed. Pigs of 138/275 (50.2%) in Andong and 352/425 (82.8%) in Hapcheon were seropositive. Also, the RT-PCR results revealed that 27 heads (8.2%) in Andong, 112 heads (22.0%) in Hapcheon were positive for PRRSV antigen. PRRSVs were mainly detected between the ages of 40 to 60 days. PRRSV ORF5 regions were used to determine genetic clusters based on previous report. All PRRSV type I detected in both Andong and Hapcheon were classified as Cluster I. The PRRSV type II isolates in Andong were assorted to Cluster II, whereas the PRRSV type II isolates in Hapcheon were the viruses were unassembled into any cluster except one identified to Cluster III. Phylogenetic analysis indicated that new clusters of PRRSVs type II were prevalent in Hapcheon.