Biochemical and structural comparisons of non-nucleoside reverse transcriptase inhibitors against feline and human immunodeficiency viruses

Siriluk Rattanabunyong,Khuanjarat Choengpanya,Chonticha Suwattanasophon,Duangnapa Kiriwan,Peter Wolschann,Thomanai Lamtha,Abdul Rajjak Shaikh,Jatuporn Rattanasrisomporn,Kiattawee Choowongkomon 대한수의학회 2023 Journal of Veterinary Science Vol.24 No.5

Background: Feline immunodeficiency virus (FIV) causes an acquired immunodeficiency-like syndrome in cats. FIV is latent. No effective treatment has been developed for treatment the infected cats. The first and second generations non-nucleoside reverse transcriptase inhibitors (NNRTIs) for HIV treatment, nevirapine (NVP) and efavirenz (EFV), and rilpivirine (RPV), were used to investigate the potential of NNRTIs for treatment of FIV infection. Objective: This study aims to use experimental and in silico approaches to investigate the potential of NNRTIs, NVP, EFV, and RPV, for inhibition of FIV reverse transcriptase (FIV-RT). Methods: The FIV-RT and human immunodeficiency virus reverse transcriptase (HIV-RT) were expressed and purified using chromatography approaches. The purified proteins were used to determine the IC50 values with NVP, EFV, and RPV. Surface plasmon resonance (SPR) analysis was used to calculate the binding affinities of NNRTIs to HIV-RT and FIV-RT. The molecular docking and molecular dynamic simulations were used to demonstrate the mechanism of FIV-RT and HIV-RT with first and second generation NNRTI complexes. Results: The IC50 values of NNRTIs NVP, EFV, and RPV against FIV-RT were in comparable ranges to HIV-RT. The SPR analysis showed that NVP, EFV, and RPV could bind to both enzymes. Computational calculation also supports that these NNRTIs can bind with both FIV-RT and HIV-RT. Conclusions: Our results suggest the first and second generation NNRTIs (NVP, EFV, and RPV) could inhibit both FIV-RT and HIV-RT.

FIV-Tet-On Vector System을 이용한 hG-CSF 유전자의 효율적인 발현 조절

권모선,구본철,김태완 한국동물생명공학회(구 한국동물번식학회) 2007 Reproductive & developmental biology Vol.31 No.3

본 연구에서는 hG-CSF의 발현을 유도적으로 조절하기 위한 FIV-Tet-On lentivirus vector system을 구축하고자 하였다. hG-CSF는 호중성구 계열 세포의 증식과 분화, 생존에 영향을 미치는 물질로서, 이 유전자의 발현을 증가시키기 위하여 FIV-Tet-On vector 상의 hG-CSF나 rtTA2SM2 서열의 3' 위치에 WPRE 서열을 도입하였다. 구축된 각각의 vector는 293FT 세포에 일시적으로 transfection하여 virus를 생산하였으며, 이 virus를 일차 배양 세포인 CEF와 PFF에 감염시켰다. 각 세포에 전이된 hG-CSF의 발현 양상을 관찰하기 위하여 doxycycline을 첨가하거나 첨가하지 않은 배지에서 배양한 후 quantitative real-time PCR, Western blot과 ELISA를 이용하여 hG-CSF 유전자의 발현 정도를 비교 측정한 결과, CEF에서는 WPRE가 hG-CSF의 3' 위치에 도입된 경우에 발현량과 유도율이 가장 높은 것으로 나타났고, PFF에서는 rtTA 서열의 3'위치에 도입된 경우에 발현량과 유도율이 가장 큰 것으로 확인되었다. 이 FIV-Tet-On vector system은 형질 전환 동물의 생산이나 유전자 치료에서 문제시되는 외래 유전자의 지속적인 과다 발현에 의한 개체의 생리적인 부작용을 최소화하기 위한 해결 방법으로 제시될 수 있을 것이다. In this study, using FIV-based lentivirus vector system, we tried to express hG-CSF in tetracycline-controllable manner. hG-CSF influences the proliferation, differentiation, and survival of cells in the neutrophil lineage. To enhance stability and translation of hG-CSF transcript, WPRE sequence was also introduced into FIV-Tet-On vector at downstream region of either the hG-CSF gene or the sequence encoding rtTA. Primary culture cells (CEF, chicken embryonic fibroblast; PFF, porcine fetal fibroblast) infected with the recombinant FIV were cultured in the medium supplemented with or without doxycycline for 48 hours, and induction efficiency was measured by comparing the hG-CSF gene expression level using quantitative real-time PCR, Western blot and ELISA. Higher hG-CSF expression and tighter expression control were observed from the vector in which the WPRE sequence was placed at downstream of the hG-CSF (in CEF) or rtTA (in PFF) gene. This FIV-Tet-On vector system may be helpful in solving serious physiological disturbance problems which has continuously hampered successful production of transgenic animals and gene therapy.

FIV Vector System을 이용하여 생산한 hEPO 형질전환 닭에서의 유전자 결손

구본철,권모선,김도향,남유화,김태완 한국동물생명공학회(구 한국동물번식학회) 2017 발생공학 국제심포지엄 및 학술대회 Vol.2017 No.10

형질전환 닭을 생산하는데 있어서 필수적인 유전자 전이 방법은 retrovirus 또는 lentivirus vector system을 이용하여 X기의 배반엽 세포에 외래 유전자를 전이시키는 방법이 가장 널리 이용되고 있다. 그러나 최근의 형질전환 닭의 생산에 관한 몇몇의 연구 보고 에서 도입된 virus vector 내의 일부 서열이 결손된 것으로 확인되었으며, 결손된 부위의 유전자 서열은 포유류를 대상으로 한 연구에서 보고된 이소성의 splicing donor 부분과는 상관관계가 없는 다른 염기 서열이 해당되는 것으로 보고되었다. 본 연구에서는 고양이 면역결핍 바이러스(feline immunodeficiency virus, FIV) 유래의 FIV-Ov23p-hEPOW lentivirus vector를 이용하여 난관 조직 특이적으로 hEPO 유전자가 발현되는 형질전환 닭을 생산하였으며, 이로부터 유래한 G1 세대 형질전환 개체에서 닭의 genome에 도입된 lentivirus vector 서열의 결손을 발견하였다. Virus vector 서열의 결손은 Southern blotting, genomic DNA PCR 분석 및 DNA sequencing을 실시하여 확인하였다. 그 결과, 도입된 lentivirus 서열 중 2.3 kb 크기의 ovalbumin promoter 내의 estrogen response element (ERE) 부분에서 443 bp가 결손된 것을 확인할 수 있었으며, 앞서 보고된 여러 연구 결 과와 마찬가지로 특정 서열과는 상관관계가 없는 부분이 소실된 것으로 확인되었다. 그 러나 promoter 부분의 결손에서 불구하고 G1 형질전환 닭이 산란한 계란에는 최대 371 IU/mL (3.1 μg/mL) hEPO 단백질이 생산되었으며, 혈액에서는 22.5 mIU/mL (0.19 ng/ mL)의 hEPO가 포함되어 있었다. 즉, enhancer로 알려진 ERE의 일부 결손이 난관 조직 을 통한 단백질의 발현량을 크게 증가시키진 못하였으나 난관 조직 특이적 발현에는 영 향이 없는 것으로 나타났다. 이상의 연구 결과는 FIV 유래의 lentivirus vector system을 이용하여 생산한 형질전환 닭에서 도입되는 vector 서열의 결손에 대한 실례를 제공하는 것으로, 결손이 일어나지 않는 새로운 형태의 virus vector system의 개발을 모색할 필요 성을 제시하는 데에 의의가 있다.

Azidothymidine and recombinant human interferon-alpha therapy in a cat with feline immunodeficiency virus

장혜진,Yen-Kang Ho,강민희,김승곤,박우정,최인수,김대영,박희명 충북대학교 동물의학연구소 2014 Journal of Biomedical and Translational Research Vol.15 No.2

A 7-year-old, spayed female, domestic short hair cat showed signs of a 2-week history of chronic anorexia, de- pression, and severe weight loss. Upon physical examination, pyrexia, mild gingivitis, and pale mucus membranes were noted. Laboratory analysis revealed normocytic normo- chromic non-regenerative anemia, severe thrombocytope- nia, and hypergammaglobulinemia. Serum protein electro- phoresis revealed the presence of elevated alpha-2 fraction within the globulin concentration. Based on history, clinical signs, and laboratory results, systemic viral infection was strongly suspected. Reverse transcriptase polymerase chain reaction identified the presence of feline immunodeficiency virus (FIV) in the serum. Furthermore, gene sequencing revealed the virus as FIV subtype A. Treatment with anti- retroviral agents, including azidothymidine (AZT) and recombinant human interferon-alpha, was continued for 4 weeks. However, the patient’s clinical condition deteriorat- ed, resulting in death 1 month after initiation of treatment due to progressive renal failure. Necropsy and histopathol- ogy revealed hepatic and renal necrosis with hyper-cellular bone marrow mainly comprised of myeloid precursor cells. This case report is the first to describe phylogenetic sub- typing, anti-retroviral combination treatment, and clinical outcomes in an FIV-infected cat in Korea. In addition, this report suggests that treatment should be initiated during the early phase of infection that could be effective for the virus.

Azidothymidine and recombinant human interferon-alpha therapy in a cat with feline immunodeficiency virus

Hye-Jin Jang,Yen-Kang Ho,Min-Hee Kang,Seung-Gon Kim,Woo-Jung Park,In-Soo Choi,Dae-Young Kim,Hee-Myung Park 충북대학교 동물의학연구소 2014 Journal of Biomedical and Translational Research Vol.15 No.2

A 7-year-old, spayed female, domestic short hair cat showed signs of a 2-week history of chronic anorexia, depression, and severe weight loss. Upon physical examination, pyrexia, mild gingivitis, and pale mucus membranes were noted. Laboratory analysis revealed normocytic normochromic non-regenerative anemia, severe thrombocytopenia, and hypergammaglobulinemia. Serum protein electrophoresis revealed the presence of elevated alpha-2 fraction within the globulin concentration. Based on history, clinical signs, and laboratory results, systemic viral infection was strongly suspected. Reverse transcriptase polymerase chain reaction identified the presence of feline immunodeficiency virus (FIV) in the serum. Furthermore, gene sequencing revealed the virus as FIV subtype A. Treatment with anti-retroviral agents, including azidothymidine (AZT) and recombinant human interferon-alpha, was continued for 4 weeks. However, the patient’s clinical condition deteriorated, resulting in death 1 month after initiation of treatment due to progressive renal failure. Necropsy and histopathology revealed hepatic and renal necrosis with hyper-cellular bone marrow mainly comprised of myeloid precursor cells. This case report is the first to describe phylogenetic subtyping, anti-retroviral combination treatment, and clinical outcomes in an FIV-infected cat in Korea. In addition, this report suggests that treatment should be initiated during the early phase of infection that could be effective for the virus.

The Analysis of Flow-Induced Vibration and Design Improvement in KSNP Steam Generators of UCN #5, 6

Kim, Sang-Nyung,Cho, Yeon-Sik The Korean Society of Mechanical Engineers 2004 JOURNAL OF MECHANICAL SCIENCE AND TECHNOLOGY Vol.18 No.1

The KSNP Steam Generators (Youngkwang Unit 3 and 4, Ulchin Unit 3 and 4) have a problem of U-tube fretting wear due to Flow Induced Vibration (FIV). In particular, the wear is localized and concentrated in a small area of upper part of U-bend in the Central Cavity region. The region has some conditions susceptible to the FIV, which are high flow velocity, high void fraction, and long unsupported span. Even though the FIV could be occurred by many mechanisms, the main mechanism would be fluid-elastic instability, or turbulent excitation. To remedy the problem, Eggcrate Flow Distribution Plate (EFDP) was installed in the Central Cavity region or Ulchin Unit 5 and 6 steam generators, so that it reduces the flow velocity in the region to a certain level. However, the cause of the FIV and the effectiveness of the EFDP was not thoroughly studied and checked. In this study, therefore the Stability Ratio (SR), which is the ratio of the actual velocity to the critical velocity, was compared between the value before the installation of EFDP and that after. Also the possibility of fluid-elastic instability of KSNP steam generator and the effectiveness of EFDP were checked based on the ATHOS3 code calculation and the Pettigrew's experimental results. The calculated results were plotted in a fluid-elastic instability criteria-diagram (Pettigrew, 1998, Fig. 9). The plotted result showed that KSNP steam generator with EFDP had the margin of Fluid-Elastic Instability by almost 25%.

시험용 핵연료집합체의 유동유발진동에 대한 실험적 연구

이강희(Kang-Hee Lee),윤경호(Kyung-Ho Yoon),송기남(Kee-Nam Song),김재용(Jae-Yong Kim),박미연(Mi-Yeon Park) 한국유체기계학회 2006 유체기계 연구개발 발표회 논문집 Vol.- No.-

The Flow-Induced Vibration (FIV) test for the test fuel assembly was carried out to investigate FIV characteristics using the KAERI-devised hydraulic test loop. The motivation of this study was to evaluate the effect of Spacer Grid (SG)s design on the vibration of the fuel bundle and to understand the flow-induced dynamic characteristics of the test bundle. A test bundle consists of 2 guide tubes, 23 dummy fuel rods (embedded with Pb pellets), and 5 SGs with the mixing vane. The fuel bundle was mounted by clamping ends of the guide tubes on the test section. The test flow velocity range was about 2 m/s ~ 10 m/s of cold water (25℃) such that the test condition covered the operating flow range of 4.5 ㎧ to 5.5 ㎧. Two fuel rods were instrumented with embedded accelerometers, permitting measurement of vibration in two mutually perpendicular planes of motion. A non-contact vibrometer were also used to measure oscillating motion of the bundle. The FIV characteristics of the test bundle with the flow variation were p esented in terms of temporal and spectral functions. The test results will be used to develop theoretical model for FIV of nuclear fuel and help to determine the final design of the SGs for the commercial usage.

Expression of Recombinant hEPO in the Egg White of Transgenic Chickens

Bon Chul Koo,Mo Sun Kwon,Dohyang Kim,Yuhwa Nam,Teoan Kim 한국동물생명공학회(구 한국동물번식학회) 2017 Reproductive & Developmental Biology(Supplement) Vol.41 No.2

In this study, we report successful expression of recombinant human erythropoietin (hEPO) in the egg white of transgenic hens using a feline immunodeficiency virus (FIV)- based lentiviral vector as an exogenous gene deliverer. hEPO is a glycoprotein hormone that controls erythropoiesis, or red blood cell production. FIV vectors permit high levels of transgene expression in quails and chickens. We constructed a FIV vector containing a hEPO cDNA sequence driven by an oviduct-specific ovalbumin promoter, and microinjected into the subgerminal cavity of stage X chick embryos to generate transgenic chicken. Out of 208 injected eggs, 10 chicks were hatched after 21 days of incubation, and one of the G0 hatched chicken expressed the vector-encoded hEPO gene in sperm. Successful germline transmission of the transgene was also confirmed in G1 transgenic chicks produced from crossing G0 transgenic roosters with non-transgenic hens. One rooster was mated to wild-type hens to produce 518 G1 progeny. PCR analysis of blood samples from these progeny revealed that there were four G1 transgenic offspring, corresponding to a 0.77% germline transmission rate. Subsequently, Southern blot analysis of the genomic DNA from four G1 transgenic chickens was carried out to verify the stable genomic integration and copy number of the transgene in the genome. Quantitative analyses of the blood and egg white samples taken from G1 transgenic chickens resulted in 4,810~6,600 IU/mL (40.1~55.0 ㎍/mL) of hEPO in egg white, whereas 18~25 mIU/mL (0.15~0.2 ng/mL) in serum. The biological activity of the recombinant hEPO in egg white was comparable to its commercially available counterpart. We conclude that successful expression of recombinant hEPO in the egg white of transgenic chickens implies an important step towards efficient production of human cytokine from the transgenic animal bioreactor.

유체유발진동 시험용 유동루프의 자유진동해석

이강희(K-H Lee),강흥석(H-S Kang),송기남(K-N Song),윤경호(K-H Yoon),최명환(M-H Choi) 대한기계학회 2004 대한기계학회 춘추학술대회 Vol.2004 No.4

Vibration characteristics of the FIV test loop for the Flow-Induced Vibration(FIV) study of a PWR partial(5x5) fuel assembly are investigated by the Finite Element(FE) analysis and the modal test. For the FE analysis, 3-D beam element is used for the pipes and the test section and mass element used for the valves and flanges. The 'U' restrainer stiffness determined by numerical simulation is used for the FE model. The result of the FE analysis is compared with that of the modal test. The higher mode similarity between the test and analysis is observed in a few low modes. After that, the mode similarity reduce as the mode goes high. It is concluded that the first to the third vibration modes are observed at the lower parts of the 6 inches restoring line, followed by a local mode at the test section, and the natural frequencies of the modes are 22.4 Hz, 26.0 Hz, 27.5 Hz and 31.4 Hz.

Expression of Active Human Interferon Alpha 2b gene nder the Control of Ovalbumin Promoter in Transgenic Chickens

Bon Chul Koo,Mo Sun Kwon,Dohyang Kim,Sanga Kim,Museog Choe,Yuhwa Nam,Teoan Kim 한국수정란이식학회 2016 한국수정란이식학회 학술대회 Vol.2016 No.10

Human interferon alpha 2b (hIFNα-2b) is an important immune regulator widely used in clinic, for the treatment of chronic hepatitis, hairy cell leukemia, chronic myelogenous leukemia and multiple myeloma, etc. The clinically used hIFNα-2b is generally produced by E. Coli, which lacks the post-translational O-glycosylation of naturally synthesized protein, and has a short serum half-life. In this study, we report the successful generation of transgenic chickens that produce hIFNα-2b in the egg white using a feline immunodeficiency virus (FIV)-based lentiviral vector. In preliminary in vitro study, the hIFNα-2b gene under the control of CMV promoter expressed as much as 2,650 ng/㎖ in CEF-LNC-hIFNα-2bW cell. A FIV vector packaged with vesicular stomatitis virus G glycoprotein (VSV-G) was injected underneath the blastoderm of freshly laid chicken eggs (stage X) to produce a hIFNα -2b transgenic chicken. Out of 187 injected eggs, 55 chicks were hatched after 21 days of incubation, and 27 of the G0 hatched chicks expressed the vector-encoded hIFNα-2b gene. The expression of recombinant hIFNα-2b in transgenic chickens constitutes an important step towards low-cost and full biological activity production of this protein drug in bioreactor. This work was supported by the Bio-industry Technology Development Program, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea, and by a grant from the Next-Generation BioGreen 21 Program (No. PJ011178), Rural Development Administration, Republic of Korea.