• 한국수의병리학회지
• /
• 제5권2호
• /
• pp.57-62
• /
• 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
• /
• 제38권1호
• /
• pp.55-62
• /
• 1995

한국 마늘 바이러스의 유전자 구조와 병 발생 메카니즘을 연구하기 위하여, 바이러스가 감염된 마늘잎으로부터 바이러스 입자를 분리하고 RNA를 추출하였다. 그 virus RNA를 이용하여 마늘 바이러스 cDNA 유전자 은행을 만들어 일부 clone의 염기 서열을 결정하였다. 여기에서 얻은 cDNA clones 중에서 poly(A) tail을 갖는 clone S81를 분리하고 873 bp의 전체 염기서열을 결정하였다. Clone S81의 염기서열을 다른 식물 바이러스와 비교한 결과 potexvirus의 껍질단백질 부분의 염기서열과 $30{\sim}40%$의 유사성을 보여주었다. Clone S81은 바이러스 RNA의 3' 말단 부위에 해당하고, 껍질단백질의 N-terminal 3개 아미노산이 빠진 open reading frame (ORF) 및 3'-noncoding region을 포함하고 있다. 3' 말단 부분에는 바이러스 복제과정에서 cis-acting element로 작용한다고 여겨지는 hexamer motif와 polyadenylation signal이 존재한다. 이 clone을 probe로 하여 Northern blot을 실시한 결과 genome의 크기는 7.5 knt라는 것을 알 수 있었고 clone S81은 potexvirus의 cDNA clone이라는 결론을 얻었다. 한국 마늘에서 이 바이러스의 분포 양상을 알아보기 위해 껍질단백질에 대한 항체를 만들었다. 먼저 발현벡터를 이용하여 대장균에서 대량으로 발현시키고 affinity chromatography로 껍질단백질을 정제하였다. 그 단백질을 토끼에 주사하여 껍질단백질에 대한 항체를 얻었다. 이 항체를 사용하여 다양한 지역에서 재배되는 마늘잎의 추출액에 대해 immunoblot을 실시하였다. 그 결과 분자량 29,000과 27,000 위치에서 signal을 보였다. 분자량 27,000 단백질이 29,000이 분해되어 생긴 산물인지 알아보기 위하여 그 추출액을 $37^{\circ}C$에서 시간을 달리하여 incubation한 후 immunoblotting하였다. 그 결과도 마찬가지로 같은 위치에서 signal을 보여줬다. 따라서 한국 마늘에는 재배되는 지역에 따라 다소 다르기는 하지만 대체로 두 종류의 potexvirus로 감염되어 있다고 추정된다.

• Journal of Plant Biotechnology
• /
• 제32권4호
• /
• pp.243-250
• /
• 2005

한국 분리주 감자잎말림바이러스 (PLRV)로부터 627bp 크기의 외피단백질 유전자의 ORF (AF296280)를 분리하여 장려품종 감자인 '수미'를 형질전환 하였다. 17계통의 형질전환체를 선발하여 온실과 포장에서 5세대를 증식하면서 PLRV에 대한 저항성이 큰 5계통을 선발 하였다. 도입된 유전자들의 유전적인 안정성을 확인하기위해 PCR, Southern, 그리고 northern blot 분석을 수행하였다. 또한 클론으로 증식된 형질전환 감자들의 특성과 저항성도 검정하였다. 형질전환된 감자들에서 PLRV의 외피단백질 유전자는 안전적으로 발현되며 저항성을 유지하였으며, 유사도가 비교적 낮은 감자 바이러스 Y (PVY)에는 저항성을 나타내지 않았다. 따라서 이러한 저항성은 도입된 유전자와 유사성도가 높은 바이러스에 저항성을 나타내는 homology dependent gene silencing으로 판단되었다. 유망계통 형질전환 감자 계통들의 포장평가를 통해 PLRV에 대한 저항성을 제외한 주요한 농업적 특성과 식물학적 특성은 형질전환 되지 않은 감자와 큰 차이를 보이지 않았다.

• BMB Reports
• /
• 제36권5호
• /
• pp.475-487
• /
• 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
• /
• 제35권6호
• /
• pp.595-603
• /
• 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).

• 대한바이러스학회지
• /
• 제27권2호
• /
• pp.185-195
• /
• 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.

• 한국동물위생학회지
• /
• 제37권1호
• /
• pp.11-18
• /
• 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.