• Journal of Plant Biology
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• 제38권2호
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• pp.129-136
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• 1995

Complementary DNA of the genomic RNA of odontoglossum ringspot virus Cymbidium strain (ORSV-Cy) was synthesized from polyadenylated viral RNA and cloned. Selected clones containing the viral RNA-dependent RNA polymerase gene of the virus has been sequenced by automated sequencing system. The complete nucleotide sequence of an open reading frame is 1377 base pairs in length, and encodes a protein of 458 amino acids about 52, 334 D. The 52 kD protein of ORSV shares four sequence motifs characteristic of viral RNA-dependent RNA polymerase. Comparison of the ORSV 52 kD protein sequence with that of other five viruses in tobamovirus group showed 76.0 to 60.7% homologies at the amino acid level and the conservation of the four motifs betwen the viruses.

• Bulletin of the Korean Chemical Society
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• 제26권2호
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• pp.285-291
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• 2005

The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is the viral RNA-dependent RNA polymerase (RdRp), which is the essential catalytic enzyme for the viral replication and is an appealing target for the development of new therapeutic agents against HCV infection. A small amount of serum from a single patient with hepatitis C was used to get the genome of a Korean HCV isolate. Sequence analysis of NS5B 1701 nucleotides showed the genotype of a Korean isolate to be subtype 1b. The soluble recombinant HCV NS5B polymerase lacking the C-terminal 24 amino acids was expressed and purified to homogeneity. With the highly purified NS5B protein, we established in vitro systems for RdRp activity to identify potential polymerase inhibitors. The rhodanine family compounds were found to be potent and specific inhibitors of NS5B from high throughput screening (HTS) assay utilizing the scintillation proximity assay (SPA) system. The binding mode of an inhibitor was analyzed by measuring various kinetic parameters. Lineweaver-Burk plots of the inhibitor suggested it binds not to the active site of NS5B polymerase, but to an allosteric site of the enzyme. The activity of NS5B in in vitro polymerase reactions with homopolymeric RNA requires interaction with multiple substrates that include a template/primer and ribonucleotide triphosphate. Steady-state kinetic parameter, such as Km, was determined for the ribonucleotide triphosphate. One of compounds found interacts directly with the viral polymerase and inhibits RNA synthesis in a manner noncompetitively with respect to UTP. Furthermore, we also investigated the ability of the compound to inhibit NS5B-directed viral RNA replication using the Huh7 cell-based HCV replicon system. The investigation is potentially very useful for the utility of such compounds as anti-hepatitic agents.

• 한국어병학회지
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• 제21권3호
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• pp.181-188
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• 2008

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was used to detect and identify four fish viruses, fish iridovirus, viral hemorrhagic septicaemia virus (VHSV), viral nervous necrosis virus (VNNV), hirame rhabdovirus (HRV). Four viruses were detected by PCR with each specific primers. Identification of iridovirus was achieved by digesting the PCR amplified fragment with a restriction enzyme ApaⅠ. It was possible to distinguish positive from false positive PCR amplicons of VHSV by RFLP of PstⅠ or HindⅢ restriction enzymes. VNNV was identified using RFLP of BamHⅠrestriction enzyme and HRV was identified by XbaⅠ restriction enzyme. This approach can be used for more rapid, simple and specific diagnosis of fish viral diseases.

• 대한수의학회지
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• 제57권1호
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• pp.37-42
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• 2017

Getah virus (GETV) infection causes sporadic outbreaks of mild febrile illness in horses and reproductive failure in pigs. In this study, we established a reverse transcription polymerase chain reaction (RT-PCR) method to detect GETV from suspected virus-infected samples. The reaction conditions were optimized and validated by using RNA extracted from GETV propagated in cell culture. A GETV-specific GED4 primer set was designed and used to amplify a 177 bp DNA fragment from a highly conserved region of the E1 glycoprotein gene in the GETV genome. RT-PCR performed with this primer set revealed high sensitivity and specificity. In the sensitivity test, the GED4 primer set detected GETV RNA at the level of $10^{2.0}\;TCID_{50}/mL$. In the specificity test, the GED4 primer set amplified only a single band of PCR product on the GETV RNA template, without non-specific amplification, and exhibited no cross-reactivity with other viral RNAs. These results suggest that this newly established RT-PCR method is useful for accurate identification of GETV infection in animals.

• 한국균학회지
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• 제49권3호
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• pp.285-294
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• 2021

In general, mycoviruses remain latent and rarely cause visible symptoms in fungal hosts; however, some viral infections have demonstrated abnormal mycelial growth and fruiting body development in commercial macrofungi, including Lentinula edodes. Compared to other cultivated mushrooms, L. edodes is more vulnerable to viral infections as it is still widely cultivated under near-natural conditions. In this study, we investigated whether Korean wild strains of L. edodes were infected by RNA mycoviruses that have previously been reported in other parts of the world (LeSV, LePV1, LeV-HKB, LeNSRV1, and LeNSRV2). Using specific primer sets that target the RNA-dependent RNA polymerase genes of each of the RNA mycovirus, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect viral infection. Viral infection was detected in about 90% of the 112 wild strains that were collected in Korea between 1983 and 2020. Moreover, multiple infections with RNA mycoviruses were detected in strains that had normal fruiting bodies. This work contributes to our understanding of the distribution of RNA mycoviruses in Korea and the impact of multiple viral infections in a single strain of L. edodes.

• Journal of Microbiology and Biotechnology
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• 제31권1호
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• pp.16-24
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• 2021

Hepatitis B virus (HBV) genome P-encoded protein HBV DNA polymerase (Pol) has long been known as a reverse transcriptase during HBV replication. In this study, we investigated the impact of HBV Pol on host cellular processes, mainly apoptosis, and the underlying mechanisms. We showed a marked reduction in apoptotic rates in the HBV Pol-expressed HepG2 cells compared to controls. Moreover, a series of assays, i.e., yeast two-hybrid, GST pull-down, co-immunoprecipitation, and confocal laser scanning microscopy, identified the host factor eEF1A2 to be associated with HBV Pol. Furthermore, knockdown of eEF1A2 gene by siRNA abrogated the HBV Pol-mediated anti-apoptotic effect with apoptosis induced by endoplasmatic reticulum (ER) stress-inducer thapsigargin (TG), thus suggesting that the host factor eEF1A2 is essential for HBV Pol's anti-apoptosis properties. Our findings have revealed a novel role for HBV Pol in its modulation of apoptosis through integrating with eEF1A2.

• 대한수의학회지
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• 제63권4호
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• pp.41.1-41.6
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• 2023

Many viruses can infect different types of birds, with poultry being the most susceptible. These viral diseases have a direct negative impact on the poultry industry, with significant economic losses. This study examined a group of the most important viruses that infect backyard chickens in 2 specific areas of Basrah Governorate, south of Iraq. The study analyzed avian influenza viruses (AIVs), Newcastle disease virus (NDV), and infectious bronchitis virus (IBV). Two hundred and ninety oropharyngeal swabs, 150 from Abu Al-Khasib and 140 from Shatt Al-Arab regions in the Basrah governorate, were obtained from backyard chickens with clear respiratory signs. The samples were subjected to viral RNA extraction, and the viral nucleic acids were detected using a reverse transcriptase polymerase chain reaction technique. The overall rate of viral infections was 74.8%, which varied depending on the type of virus: 15.8%, 31.3%, and 27.5% for AIV, NDV, and IBV, respectively. The NDV and IBV had much higher infection rates than that of AIV. In addition, the prevalence of AIV in the Shatt Al Arab district was significantly higher than in the Abul Khasib district. Moreover, there were no significant differences between the NDV and the IBV distributions in either of the targeted regions in this study.

• Journal of Crop Science and Biotechnology
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• 제11권2호
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• pp.127-134
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• 2008

Tobamoviruses are the major viral pathogens of tomato and bell pepper. The preliminary results showed that Acibenzolar-Smethyl(ASM; S-methylbenzo(1,2,3) thiadiazole-7-carbothiate) pre-treatment to tomato and tobacco plants reduces the concentration of Tomato mosaic tobamovirus(ToMV) and Tobacco mosaic tobamovirus(TMV) in tomato and bell pepper seedlings, respectively. Pre-treatment of the indicator plant(Nicotiana glutinosa) with the ASM followed by challenge inoculation with tobamoviruses produced a reduced number and size of local lesions(67 and 79% protection over control to TMV and ToMV inoculation, respectively). In order to understand the mechanism of resistance the gene expression profiles of antiviral genes was examined. RT-PCR products showed higher expression of two viral resistance genes viz., alternative oxidase(AOX) and RNA dependent RNA polymerase(RdRp) in the upper leaves of the ASM-treated tomato plants challenge inoculation with ToMV. Further, the viral concentration was also quantified in the upper leaves by reverse transcription PCR using specific primer for movement protein of ToMV, as well as ELISA by using antisera against tobamoviruses. The results provided additional evidence that ASM pre-treatment reduced the viral movement to upper leaves. The results suggest that expressions of viral resistance genes in the host are the key component in the resistance against ToMV in the inducer-treated tomato plants.

• 대한수의학회지
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• 제55권3호
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• pp.185-189
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• 2015

Bovine parainfluenza virus type 5 (bPIV5) was isolated from cattle with downer cow syndrome in 2012, and included both respiratory and neurotropic pathogens from a variety of animals. In the current study, we conducted serosurveillance using sera obtained from seven Korean farms and optimized a reverse transcription-polymerase chain reaction (RT-PCR) assay to detect bPIV5. The overall seropositive rate for Korean cattle was 21.4% (163/760). A farm located near the city of Milyang in Gyeoungnam province had a markedly elevated seropositive rate for bPIV5 compared to that of the other six farms. The regional seropositive rates were 4.2% (8/192) for Haman, 19.5% (18/55) for Hwasung, 73.9% (65/88) for Milyang, 26.0% (50/192) for Namwon, 1.0% (1/96) for Uljin, 13.5% (13/96) for Yeongju, and 32.7% (8/41) for Yongin. The sensitivity and specificity of three RT-PCR primer sets used to amplify the conserved fusion gene of bPIV5 were also evaluated. An RT-PCR assay using the bPIVFR3 primer set was 10-fold more sensitive than the assays using the two other primer sets and did not result in non-specific amplification. These results demonstrated that the bPIFR3 primer set can be used to detect bPIV5.

• BMB Reports
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• 제39권5호
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• pp.571-577
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• 2006

Dendrolimus punctatus tetravirus (DpTV) has been identified as a new member of the genus Omegatetravirus of the family Tetraviridae that may be related serologically to Nudaurelia capensis virus ($N{\omega}V$). To establish the function of DpTV RNA genome and to better understand the mechanism of viral replication, the putative RNA-dependent RNA polymerase (RdRp) domain has been cloned and expressed in Escherichia coli. The recombinant protein was purified on a Ni-chelating HisTrap affinity column and demonstrated to initiate viral RNA synthesis in a primer-independent manner but not by terminal nucleotidyle transferase activity in the presence of $Mg^{2+}$ and RNA template. Mutation of the GDD to GAA interferes with the residues at the polymerase active site and metal ions, and thus renders the polymerase inactive.