• 대한수의학회지
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• 제61권2호
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• pp.13.1-13.8
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• 2021

Mammalian reovirus (MRV) causes respiratory and intestinal disease in mammals. Although MRV isolates have been reported to circulate in several animals, there are no reports on Korean MRV isolates from wildlife. We investigated the biological and molecular characteristics of Korean MRV isolates based on the nucleotide sequence of the segment 1 gene. In total, 144 swabs from wild animals were prepared for virus isolation. Based on virus isolation with specific cytopathic effects, indirect fluorescence assays, electron microscopy, and reverse transcription-polymerase chain reaction, only one isolate was confirmed to be MRV from a Korean roe deer (Capreolus pygargus). The isolate exhibited a hemagglutination activity level of 16 units with pig erythrocytes and had a maximum viral titer of 10^5.7 50% tissue culture infectious dose (TCID₅₀)/mL in Vero cells at 5 days after inoculation. The nucleotide and amino-acid sequences of the partial segment S1 of the MReo2045 isolate were determined and compared with those of other MRV strains. The MReo2045 isolate had nucleotide sequences similar to MRV-3 and was most similar (96.1%) to the T3/Bat/Germany/342/08 strain, which was isolated in Germany in 2008. The MReo2045 isolate will be useful as an antigen for sero-epidemiological studies and developing diagnostic tools.

• 대한수의학회지
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• 제62권1호
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• pp.10.1-10.9
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• 2022

Feline panleukopenia virus (FPV) causes fatal leukopenia and severe hemorrhagic diarrhea in cats. Although FPV isolates have been reported worldwide from several animals, the biological and genetic features of South Korean FPVs remain unclear. We characterized molecularly South Korean FPV isolates. Crandell-Rees feline kidney (CRFK) cells were used to isolate FPV from 60 organ homogenates. The isolates were confirmed to be FPVs via analyses of cytopathic effects, immunofluorescence studies, electron microscopy, and polymerase chain reaction. Viral genetic analyses used the full VP2 sequences. Eight isolates propagated in CRFK cells were confirmed to be FPVs. All isolates yielded viral titers ranging from 10^4.5 to 10^6.0 TCID₅₀/mL 5 days after inoculation into CRFK cells and exhibited hemagglutination titers ranging from 2⁷ to 2¹² (using pig erythrocytes). The Korean FPV isolates grew well in cat cells such as CRFK and Fcwf-4 cells. The FPV isolates were most similar to the KS42 strain isolated from a Korean cat in 2008. The FPV isolates will serve as useful antigens in future sero-epidemiological studies and will aid in the development of diagnostic tools.

• Food Science and Biotechnology
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• 제17권3호
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• pp.651-654
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• 2008

In order to enhance the efficacy of norovirus detection by reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR, this study developed a norovirus mRNA concentration method using poly oligo dT-conjugated magnetic beads. An efficient norovirus detection protocol was performed on commercial ham using 2 viral elution buffers (glycine buffer and Tris beef extract buffer) and 2 concentration solutions [polyethylene glycol (PEG) and zirconium hydroxide]. The different approaches were verified by RT-PCR and nested PCR. This method was performed on ham in less than 8 hr by artificial inoculation of serial dilutions of the virus ranging from 1,000 to 1 RT-PCR unit/mL. The viral extraction and concentration method had 10-fold higher sensitivity using the combination of Tris beef extract buffer and PEG as compared to glycine buffer and zirconium hydroxide. This method proved that RT-PCR and nested PCR have the sensitive ability to detect norovirus in commercial ham, in that norovirus was successfully detected in artificially contaminated samples at a detection level as low as 1-10 RT-PCR unit/mL. Overall, such a detection limit suggests this protocol is both quick and efficient in terms of its potential use for detecting norovirus in meat products.

• 식물병연구
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• 제10권1호
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• pp.30-33
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• 2004

현재까지 벼 줄무의잎마름병(Rice stripe virus, RSV)은 남부지방에 국한되어 발생되어 왔다. 그러나 최근에는 RSV의 발생이 충청도, 경기도를 포함한 중부지방까지 확산되는 경향을 나타내고 있다. 이병의 병징은 육안으로는 생리적인 장해 현상과 구분하기가 힘들다 본 실험에서는 이병주 및 애멸구(Laodelphax striatellus)로 부터 viral RNA를 추출한 후 RNA복제효소 및 외피단백질유전자에 특이적인 primer를 제작하여 RT-PCR법에 의해 RSV를 검정하였다. 그결과 이병식물체 및 보독 애멸구로부터 RNA복제효소 유전자에 특이적인 band(1,023 bp) 및 외피단백질유전자에 특이적인 band(969 bp)가 관찰되었다.

• 한국수산과학회지
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• 제30권6호
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• pp.948-955
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• 1997

Immunomagnetic separation of virus coupled with .reverse transcription-polymerase chain reaction (IMS-PCR) was performed with infectious hematopoietic necrosis virus (IHNV). A DNA fragment of expected size was synthesized in the RT-PCR with total RNA extracted from IHNV inoculated CHSE-214. In a SDS-PAGE analysis, a protein band of over 70kDa was detected from non-infected cells and cells inoculated with IHNV and infectious pancreatic necrosis virus (IPNV). This protein was detected in the Western blot analysis probably because of non-specific reaction to monoclonal antibody against IHNV nucleocapsid protein. In the immunomagnetic separation, magnetic beads coated with monoclonal antibody against the IHNV nucleocapsid protein was incubated with supernatant from IHNV inoculated CHSE-214 cells. During this process, the non-specifically reacting protein could be removed by washing the magnetic bead with PBS in the presence of an external magnetic field, and viral proteins were detected from the remaining, cleaned magnetic beads. It was necessary to extract viral RNA from the captured virus particles before RT-PCR, and no DNA product was detected when the captured virus was only heated 5 min at $95^{\circ}C$. A PCR-product of expected size was synthesized from IMS-PCR with magnetic beads double coated either by goat anti-mouse IgG antibody -monoclonal antibody or streptavidin - biotin conjugated monoclonal antibody.

• 대한바이러스학회지
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• 제28권4호
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• pp.327-335
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• 1998

Hantaan (HTN) and Seoul (SEO) viruses, murid rodent-borne hantaviruses, are known to causes hemorrhagic fever with renal syndrome (HFRS) in Korea. To determine the genomic diversity and molecular phylogeny of HTN and SEO viruses found in Korea, we amplified for part of M and S genomic segments of hantaviruses from sera of HFRS patients and lung tissues of hantavirus seropositive striped-field mice. Both M and S segment of 16 HTN and 2 SEO viruses were amplified by nested reverse transcription-polymerase chain reaction. Based on 324 nucleotides in the M genomic segment, the HTN and SEO strains showed $93.8{\sim}100%$ and $99.1{\sim}99.4%$ homologies, respectively. Similarly, based on 230 nucleotides in the S genomic segment, HTN and SEO strains showed $90.9{\sim}100%$ and 100% homologies, respectively. Phylogenetic analysis of M and S segments indicated that HTN strains could be divided into at least two main groups in M and S trees and the sequence differences detected among the Sand M genomic segments of HTN viruses are consistent with reassortment having taken place between HTN virus strains.

• 미생물학회지
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• 제43권4호
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• pp.285-291
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• 2007

볼티모어의 분류체계에 의하면 바이러스는 복제 및 단백질합성 전략에 따라 6개의 집단으로나눌 수 있다. 몇 종류의 작은 DNA 바이러스를 제외한 대부분의 바이러스는 게놈 복제를 위한 자신의 핵산중합효소를 유전자로 암호화하고 있다. 바이러스 핵산중합효소에는 DNA-의존DNA 중합효수, RNA-의존RNA 중합효소, RNA-의존 DNA 중합효소 세 종류가 있으며, 이들은 모두 4개의 공통된 모티프(motif)를 가진다. 우리는 볼티모어의 분류체계와 바이러스의 핵산중합효소와의 관계를 아미노산 서열을 통해 분자 계통분류학적 분석을 통해 알아보고자 하였다. NCBI GenBank에서 얻은 바이러스 중합효소의 아미노산 서열을 CLUSTAL X 프로그램으로 다중서열하고, Neighbor-joining, Maximum-likelihood, Bayesian의 세 가지 방법으로 계통도를 그려보았다. 미세한 차이는 있었으나, 세 가지 방법 모두에서 볼티모어의 분류법과 일치하는 결과를 보였고, 특이하게도 두 가닥 RNA 바이러스는 숙주의 종류에 따라, (-)RNA 바이러스는 게놈의 절편화에 따라 각각2개의 소집단으로 나뉘어지는 것을 볼 수 있었다.

• 대한바이러스학회지
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• 제29권4호
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• pp.221-233
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• 1999

Prospect Hill Virus (PHV) is the well known serotype of hantavirus, a newly established genus in family Bunyaviridae. Extensive studies have upheld the original view of PHV genetics with three genes such as nucleocapsid (N) protein, envelope proteins (G1, G2) and RNA dependent RNA polymerase. In this study, we report the existence of additional gene that is encoded in an overlapping reading frame of the N protein gene within S genome segment of PHV. This gene is expected to encode a nonstructural small (NSs) protein and it seems to be only found in PHV infected cell. The presence and synthesis of NSs protein could be demonstrated in the cell infected with PHV using anti-peptide sera specific to the predicted amino acid sequence deduced from the second open reading frame. Ribosomal synthesis of this protein appears to occur at AUG codon at the 83rd base of S genome segment, downstream of N protein initiation codon. This protein is small in size (10.4 KDa) and highly basic in nature. The expression strategy of NSs protein appears that a signal mRNA is used to translate both N and NSs protein in PHV infected cell. 10 KDa protein in virus infected cell lysates can bind to mimic dsRNA. This fact strongly suggests that NSs protein may be involved in virus replication on late phase of viral life cycle.

• Journal of Veterinary Science
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• 제22권4호
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• pp.56.1-56.10
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• 2021

Background: Fluorescent antibody virus neutralization (FAVN) test is a standard assay for quantifying rabies virus-neutralizing antibody (VNA) in serum. However, a safer rabies virus (RABV) should be used in the FAVN assay. There is a need for a new method that is economical and time-saving by eliminating the immunostaining step. Objectives: We aimed to improve the traditional FAVN method by rescuing and characterizing a new recombinant RABV expressing green fluorescent protein (GFP). Methods: A new recombinant RABV expressing GFP designated as ERAGS-GFP was rescued using a reverse genetic system. Immuno-fluorescence assay, peroxidase-linked assay, electron microscopy and reverse transcription polymerase chain reaction were performed to confirm the recombinant ERAGS-GFP virus as a RABV expressing the GFP gene. The safety of ERAGS-GFP was evaluated in 4-week-old mice. The rabies VNA titers were measured and compared with conventional FAVN and FAVN-GFP tests using VERO cells. Results: The virus propagated in VERO cells was confirmed as RABV expressing GFP. The ERAGS-GFP showed the highest titer (10^8.0 TCID₅₀/mL) in VERO cells at 5 days post-inoculation, and GFP expression persisted until passage 30. The body weight of 4-week-old mice inoculated intracranially with ERAGS-GFP continued to increase and the survival rate was 100%. In 62 dog sera, the FAVN-GFP result was significantly correlated with that of conventional FAVN (r = 0.95). Conclusions: We constructed ERAGS-GFP, which could replace the challenge virus standard-11 strain used in FAVN test.

• 대한수의학회지
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• 제47권2호
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• pp.163-167
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• 2007

Bovine viral diarrhea (BVD) is very important disease in cattle industry with a worldwide distribution. Detection and elimination of persistently infected calves with bovine viral diarrhea virus (BVDV) was valuable strategy for BVD eradication because those calves were main source for transmission. We surveyed persistent infection with BVDV by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) using whole blood and skin. Five hundred thirty nine Korean native calves were tested. Four calves (0.7%) were positive for BVDV antigen for both tests. Those calves remained positive for follow-up by RT-PCR and IHC. Therefore they were identified as persistently infected with BVDV. We confirmed that immunohistochemistry using skin biopsy samples was very useful tool to detect persistently infected calves with BVDV. As far as we know, this would be first report on persistent infection with BVDV in Korea.