• 대한의생명과학회지
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• 제22권3호
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• pp.83-97
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• 2016

The measurement of viral load in HIV-1 infected patients is essential for the establishment of a therapeutic strategy. Several commercial assays have shown shortcomings in quantifying rare genotypes of HIV-1 such as minor groups of N and O. In this study, the HIV-1 RT-qPCR assay was developed. The primers and probe of HIV-1 were designed to target the pol gene and to increase the detection efficiency of various subtypes including group N and O. The HIV-1 quantitative RT-qPCR assay was assessed for its analytical performance and clinical evaluation. The LoD was determined to 33.9 IU/ml. The LoD of several subtypes including A, C, D, CRF_01AE, F, CRF_02AG, G and H, were determined to less than 40 IU/ml. The HIV-1 quantitative RT-qPCR assay was evaluated using the China National Reference Panel of HIV-1 RNA to determine the analytical performance. The results were all within the acceptable range. The clinical evaluation was performed at Hunan CDC in China. The clinical evaluation results were compared with those of the China domestic commercial kit. A significant correlation (fresh samples; $R^2=0.84$, P<0.001, frozen samples; $R^2=0.76$, P<0.001) between the two systems was observed for 64 fresh samples and 76 frozen samples with viral loads, and the Bland-Altman plot showed good agreement (98.4%, 96.1%, respectively). In conclusion, the HIV-1 quantitative RT-qPCR assay had comparable analytical performance with several commercial kits. The study provides basic data for the research of HIV-1 diagnosis and the development of P < HIV-1 molecular diagnostic assay.

• 한국동물위생학회지
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• 제27권4호
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• pp.355-369
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• 2004

The equine herpesvirus type 1 (EHV-1) immediate-early (IE) protein is a potent transactivator responsible for the activation of both early and late genes during the course of infection and is comprised of discrete functional domains that mediate its many functions. Interaction between trans activators such as the IE protein and various components of the RNA polymerase II transcription initiation machinery has been demonstrated to be critical for transactivation. In the present report, it is addressed the hypothesis that the IE protein interacts with various components of transcription machinery to mediate transactivation of target viral genes. In these studies, it is demonstrated that in vitro transcribed and translated IE protein interacts with TFIIB-agarose conjugate but not with TFIID-agarose conjugate. Additional immunoprecipitation studies using nuclear extracts derived from EHV-1 infected RK-13 cells confirmed that the IE protein interacts strongly with TFIIB, but fails to interact with TFIID. IR2, a truncated form of the IE protein lacking the potent transactivation domain and involved in the down-regulation of the IE gene, also interacted with TFIIB but not with TFIID. Studies were also performed to ascertain if particular TBP-associated factors (TAFs) could mediate IE or IR2 binding to TFIID. In vitro transcribed and translated TAF250 added to nuclear extracts generated from EHV-1 infected cells also failed to mediate an interaction between the IE protein or the IR2 protein and TFIID. This study demonstrated that the IE protein mediates transactivation of target viral genes by a mechanism that involves TFIIB. This is in contrast to mechanisms that have been proposed for both the herpes simplex virus ICP4 and VP16 protein which have been proposed to transactivate viral genes through interactions involving both TFIIB and TFIID. This study also intimates that IR2 mediate its repressive effects during the course of EHV-1 infection by a mechanism that involves sequestration of various transcription factors.

• Genomics & Informatics
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• 제18권4호
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• pp.43.1-43.13
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• 2020

The coronavirus disease 2019 is a contagious disease and had caused havoc throughout the world by creating widespread mortality and morbidity. The unavailability of vaccines and proper antiviral drugs encourages the researchers to identify potential antiviral drugs to be used against the virus. The presence of RNA binding domain in the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could be a potential drug target, which serves multiple critical functions during the viral life cycle, especially the viral replication. Since vaccine development might take some time, the identification of a drug compound targeting viral replication might offer a solution for treatment. The study analyzed the phylogenetic relationship of N protein sequence divergence with other 49 coronavirus species and also identified the conserved regions according to protein families through conserved domain search. Good structural binding affinities of a few natural and/or synthetic phytocompounds or drugs against N protein were determined using the molecular docking approaches. The analyzed compounds presented the higher numbers of hydrogen bonds of selected chemicals supporting the drug-ability of these compounds. Among them, the established antiviral drug glycyrrhizic acid and the phytochemical theaflavin can be considered as possible drug compounds against target N protein of SARS-CoV-2 as they showed lower binding affinities. The findings of this study might lead to the development of a drug for the SARS-CoV-2 mediated disease and offer solution to treatment of SARS-CoV-2 infection.

• The Plant Pathology Journal
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• 제40권4호
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• pp.390-398
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• 2024

The Chinese artichoke (Stachys affinis syn. S. sieboldii) is a widely cultivated crop, and its rhizome is used as a medicinal vegetable. To investigate the causes of viral diseases in Chinese artichokes, the infection rates of four virus species infecting Chinese artichoke were investigated. Since the Chinese artichoke propagates through its tuber, this study aimed to determine whether viral transmission to the progeny is possible through the tuber, by identifying the virus present in the tuber and investigating its accumulation. First, reverse transcription polymerase chain reaction analysis was performed to detect viruses using total RNA extracted from the flowers, leaves, and tubers of Chinese artichoke plants. Alfalfa mosaic virus (AMV) and Chinese artichoke mosaic virus (ChAMV) had high infectivity in Chinese artichoke and most plants were simultaneously infected with AMV and ChAMV. These viruses were present in all tissues, but their detection frequency and accumulation rates varied across different tissues of the Chinese artichoke. Also, we sequenced the coat protein (CP) genes of AMV and ChAMV to investigate genetic variations of virus between the leaf and tuber. It provides information on CP gene sequences and genetic diversity of isolates identified from new hosts of AMV and ChAMV. This study offers valuable insights into the distribution and spread of the ChAMV and AMV within Chinese artichoke plants, which have implications for the management and control of viral infections in crops.

• Applied Biological Chemistry
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• 제37권3호
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• pp.148-153
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• 1994

우리나라에서 발생하는 주요 벼 바이러스로써 저항성 유전자원이 없어 현재까지 저항성 품종이 육성되지 못하고 있는 흑조위축병(Rice Black-Streaked Dwarf Virus, RBSDV)에 대한 유전정보에 대하여 연구하였다. 매개충인 보독 애멸구를 이용하여 이병주를 생산한 후 바이러스 입자를 순수 분리하여 전기영동한 결과 10개의 band를 확인하였다. RBSDV RNA로부터 역전사 효소를 이용 cDNA를 합성한 후 ${\lambda}gt11$에 삽입하여 cDNA library를 만들었다. 이 library에서 6개의 단편을 선발하였으며 그중 한 개의 clone(pRV3)은 hybridization을 통해 RBSDV 게놈 조각 3번 유래인 것을 확인하였다. pRV3의 염기서열을 결정한 결과 2개의 ORF의 일부분들을 갖고 있었으며 이것은 바이러스 저항성 작물개발에 이용될 수 있을 것으로 생각된다.

• IMMUNE NETWORK
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• 제8권1호
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• pp.21-28
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• 2008

Background: A co-inhibitory molecule, B7-H4, is believed to negatively regulate T cell immunity by suppressing T cell proliferation and inhibiting cytokine production. However, the mechanism behind B7-H4-mediated tolerance remains unclear. Methods: Balb/c $(H-2^d)$ mice were fed with dendritic cell line, DC2.4 $(H-2^d)$ every day for 10 days. Meantime, mice were hydrodynamically injected with recombinant plasmid expressing B7-H4 fusion protein (B7-H4.hFc) or hFc via tail vein. One day after last feeding, mice were immunized with allogeneic B6 spleen cells. 14 days following immunization, mice were challenged with B6 spleen cells to ear back and the ear swelling was determined the next day. Subsequently, a mixed lymphocyte reaction (MLR) was also performed and cytokines profiles from the reaction were examined by sandwich ELISA. Frequency of immunosuppressive cell population was assayed with flow cytometry and mRNA for FoxP3 was determined by RT-PCR. Results: Tolerant mice given plasmid expressing B7-H4.hFc showed a significant reduction in ear swelling compared to control mice. In addition, T cells from mice given B7-H4.hFc plasmid revealed a significant hyporesponsiveness of T cells against allogeneic spleen cells and showed a significant decrease in Th1 and Th2 cytokines such as IFN-${\gamma}$, IL-5, and TNF-${\alpha}$. Interestingly, flow cytometric analysis showed that the frequency of CD4+CD25+FoxP3+ Tregs in spleen was increased in tolerant mice given recombinant B7-H4.hFc plasmid compared to control group. Conclusion: Our results demonstrate that B7-H4 synergistically potentiates oral tolerance induced by allogeneic cells by increasing the frequency of FoxP3+ CD4+CD25+ Treg and reducing Th1 and Th2 cytokine production.

• Journal of Microbiology and Biotechnology
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• 제28권12호
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• pp.2133-2140
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• 2018

Norovirus is the most common cause of acute gastroenteritis. Its pathogenesis is poorly understood owing to the difficulty of establishing viral infection in animal models. Here, post-weaning gnotobiotic pigs were infected with human norovirus genogroup II genotype 4 (HuNoV GII.4) to investigate the pathogenesis and replication of the virus. Three groups of four pigs were infected with $1{\times}10^5$, $1{\times}10^6$, or $1{\times}10^7$ genomic equivalent (GE) copies of HuNoV GII.4. Four pigs were used as negative controls. Blood and rectal swab samples were collected after viral infection, and gross legions were examined after necropsy. Diarrhea was induced in 25% and 75% of pigs infected with $1{\times}10^6$ and $1{\times}10^7$ GE copies, respectively. Viral shedding was detected in 50%, 75%, and 50% of pigs infected with $1{\times}10^5$, $1{\times}10^6$, and $1{\times}10^7$ GE copies, respectively. Viremia was detected in 25% of pigs infected with either $1{\times}10^6$ or $1{\times}10^7$ GE copies. When gross lesions of gastroenteritis were investigated, the ileum walls of the infected pigs were thinner than those of the controls. Villi atrophy and inflammatory cell infiltration were identified in the ileum of each infected pig. Viral capsid was identified in the jejunum, ileum, colon, spleen, and mesenteric lymph node. Virus replication was newly verified in the spleen and mesenteric lymph nodes by detection of negative-sense viral RNA. In conclusion, HuNoV GII.4 could induce acute gastroenteritis and replicate in the extra-intestinal lymphoid tissues in post-weaning gnotobiotic pigs. Therefore, such pigs would be a suitable animal model for studying the pathogenesis and replication of HuNoV.

• Biomolecules & Therapeutics
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• 제31권3호
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• pp.350-358
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• 2023

Hand-foot-and-mouth disease (HFMD) is a viral infectious disease that occurs in children under 5 years of age. Its main causes are coxsackievirus (CV) and enterovirus (EV). Since there are no efficient therapeutics for HFMD, vaccines are effective in preventing the disease. To develop broad coverage against CV and EV, the development of a bivalent vaccine form is needed. The Mongolian gerbil is an efficient and suitable animal model of EV71 C4a and CVA16 infection used to investigate vaccine efficacy following direct immunization. In this study, Mongolian gerbils were immunized with a bivalent inactivated EV71 C4a and inactivated CVA16 vaccine to test their effectiveness against viral infection. Bivalent vaccine immunization resulted in increased Ag-specific IgG antibody production; specifically, EV71 C4a-specific IgG was increased with medium and high doses and CVA16-specific IgG was increased with all doses of immunization. When gene expression of T cell-biased cytokines was analysed, Th1, Th2, and Th17 responses were found to be highly activated in the high-dose immunization group. Moreover, bivalent vaccine immunization mitigated paralytic signs and increased the survival rate following lethal viral challenges. When the viral RNA content was determined from various organs, all three doses of bivalent vaccine immunization were found to significantly decrease viral amplification. Upon histologic examination, EV71 C4a and CVA16 induced tissue damage to the heart and muscle. However, bivalent vaccine immunization alleviated this in a dose-dependent manner. These results suggest that the bivalent inactivated EV71 C4a/CVA16 vaccine could be a safe and effective candidate HFMD vaccine.

• 대한수의학회지
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• 제34권2호
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• pp.321-326
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• 1994

돼지 유행성 설사 바이러스(KPEDV-9)주를 이용하여 세포내 증식성을 비롯한 혈청학적 역학조사를 수행하였던 바 다음의 결과를 얻었다. 돼지 유행성 설사바이러스 국내 분리주는 Vero 세포에 연속계대시 증식성이 증가되었으며 90대 계대시 $10^{5.5}TCID_{50}/ml$의 역가를 나타내었다. 조직배양 순화주를 이용하여 간염세포내에서 20Kb 이상의 RNA가 존재함을 확인할수 있었으며 전자현미경 검사시 5~10nm의 외피항원 및 80~300nm크기의 coronavirus특징을 나타내었다. 설사증상을 나타내는 돼지의 장가검재료를 이용하여 유행성 설사 바이러스의 감염실태를 조사하였던 바 18%에 상당하는 감염 양성율을 확인하였으며 ELISA법에 의한 항체검사결과 전국적으로 약 45%의 항체 양성율을 나타내었다.

• Molecules and Cells
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• 제21권1호
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• pp.63-75
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• 2006

The 5′-region of Potato virus X (PVX) RNA, which contains an AC-rich, single-stranded region and stem-loop structure 1 (SL1), affects RNA replication and assembly. Using Systemic Evolution of Ligands by EXponential enrichment (SELEX) and the electrophoretic mobility shift assay, we demonstrate that SL1 interacts specifically with tobacco protoplast protein extracts (S100). The 36 nucleotides that correspond to the top region of SL1, which comprises stem C, loop C, stem D, and the tetra loop (TL), were randomized and bound to the S100. Remarkably, the wild-type (wt) sequence was selected in the second round, and the number of wt sequences increased as selection proceeded. All of the selected clones from the fifth round contained the wt sequence. Secondary structure predictions (mFOLD) of the recovered sequences revealed relatively stable stem-loop structures that resembled SL1, although the nucleotide sequences therein were different. Moreover, many of the clones selected in the fourth round conserved the TL and C-C mismatch, which suggests the importance of these elements in host protein binding. The SELEX clone that closely resembled the wt SL1 structure with the TL and C-C mismatch was able to replicate and cause systemic symptoms in plants, while most of the other winners replicated poorly only on inoculated leaves. The RNA replication level on protoplasts was also similarly affected. Taken together, these results indicate that the SL1 of PVX interacts with host protein(s) that play important roles related to virus replication.