This work was supported in part by the Research Program on Hepatitis from Japan Agency for Medical Research and Development (AMED) and by Grants-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

리포터 단백질을 코딩하는 재조합 HBV 1. 제조 <ol><li> 인 HepG2 세포의 제조 <ol><li> 세포 배양 배지를 준비 (둘 베코 변형 이글 배지에 10 % 소 태아 혈청 (FBS로 보충 (DMEM)), 100 U / ㎖ 페니실린, 100 μg의 / ㎖ 스트렙토 마이신, 100 U / ㎖의 비 필수 아미노산). </li><li> 배지 10 ml의 형질 전날에 10 cm 콜라겐 코팅 접시 플레이트 4 × 10 6 인 HepG2 세포. 가습 된 5 % CO 2 인큐베이터에서 37 ℃ 인 HepG2 세포를 인큐베이션. 주 : 약 4 × 106 인 HepG2 세포 배양 배지 10 ㎖의 10cm 콜라겐 코팅 접시에 도금되는 경우, 그들은 70~90% 합류 다음날 것이다. </li></ol></li><li> 형질 <ol><li> pUC1.2HBV 델타 엡실론 (15)의 5 μg의 및 pUC1.2HBV / NL (15)의 5 μg의와 형질 인 HepG2 세포 형질을 사용하여제조업체의 지침에 따라 시약. </li><li> 다음날, 배지를 제거하고 신선한 배지를 가하여 10. </li><li> 1 주일 형질 감염 후, 50 ㎖ 튜브에 재조합 HBV를 포함하는 배양액을 옮기고 1.3.1 단계로 진행. 형질 감염된 세포를 함유하는 플레이트에 신선한 배지 10 ㎖를 추가한다. 참고 : 재조합 B 형 간염 바이러스의 생산 4 주 동안 유지된다. HBV 재조합 체를 함유하는 배지 1 개월 동안 4 ℃에서 저장 될 수있다. </li></ol></li><li> 재조합 B 형 간염 바이러스의 정제 <ol><li> 원심 분리하여 재조합 HBV (5 분 동안 2,300 XG)을 함유하는 배지에서 세포 파편을 제거한다. </li><li> 0.45 μm의 멤브레인 필터를 통해 뜨는을 전달합니다. </li><li> 문화 매체에 : 26 % PEG / 1.5 M의 NaCl (130g, 염화나트륨, 49g, 0.5 M EDTA 산도 8.0의 1 mL 및 1 M HEPES pH를 5 ml의 7.6 500 ml의 폴리에틸렌 글리콜 6000)의 동일한 볼륨을 추가 포함하는 재조합HBV 가볍게 섞는다. 4 ℃에서 밤새 품어. </li><li> 4 ℃에서 20 분 동안 2,300 XG에 원심 분리기. </li><li> 뜨는을 취소하고 TNE 0.5 ㎖ (10 mM 트리스, 50 밀리미터의 NaCl, 1 mM의 EDTA)에서 펠렛을 녹인다. </li><li> 5 분 동안 2,300 XG에서 원심 분리하여 이물질을 제거합니다. </li><li> TNE 0.8 ml의 20 % 자당 상에로드 재조합 B 형 간염 바이러스를 포함 TNE 0.5 ml의. </li><li> 15 ° C에서 3 시간 동안 100,000 × g에서 원심 분리기. </li><li> 가능한 상층 액만큼을 폐기하고, 펠렛을 저장합니다. 배지의 개시 40 mL의 무 혈청 DMEM 1 ㎖에 재현 탁을. </li><li> 4 ℃에서 밤새 품어. </li><li> 0.45 μm의 필터를 통해 필터링합니다. -80 ° C에서 0.5 ml의 분취 및 저장을 준비합니다. 주 : 원래 바이러스 시료의 리포터 단백질의 오염이 관찰되는 경우, 2 시간 동안 100,000 XG에 TNE에서 5~30% 자당 밀도 구배 초 원심 분리에 의해 바이러스를 정제 또는 아프로 CSCL 밀도 평형화 원심50 시간 동안 150,000 XG에 m 1.1-1.6 g / ㎖. </li></ol></li></ol> 재조합 B 형 간염 바이러스의 감염 (2) <ol><li> 10 % FBS, 100 U / ㎖ 페니실린, 100 μg의 / ㎖ 스트렙토 마이신, 250 μg의 / ㎖ G-418, 100 U / ㎖로 보충 된 DMEM에서 HBV 감염에 취약 안정적 NTCP (HepG2 세포 / NTCP) (15)를 발현하는 문화 인 HepG2 세포 가습 5 % CO2 인큐베이터에서 37 ℃에서 비 필수 아미노산. </li><li> 하루 감염 전에 플레이트 약 5 × 104 인 HepG2 / 배지 0.1 ㎖ 중의 96 웰 콜라겐 코팅 된 플레이트에 NTCP 셀 (14). </li><li> 37 ° C의 물을 욕조에 해동 재조합 B 형 간염 바이러스 유리 병에 남아있는 얼음의 작은 비트가있을 때까지. </li><li> 다음 결합하여 감염 배지를 준비 : 1X 인산 완충 식염수 (PBS), 디메틸 설폭 사이드 2 μL (DMSO)에 40 % PEG8000 10 μl를 재조합 HBV 10 ㎕의 신선한 배지 78 μL96- 웰 플레이트의 웰 당. </li><li> 96 웰 플레이트의 한 웰에 재조합 B 형 간염 용액 100 μl를 추가합니다. </li><li> 어느 날 감염 후, 바이러스 부분에서 오염 기자 단백질을 제거하는 웰 당 300 ㎕의 PBS에 감염된 세포를 3 회 반복한다. </li><li> 일주 이하 2 %의 DMSO를 함유하는 배지 200 μL에 감염된 세포를 인큐베이션. </li></ol> 3. 분석 <ol><li> 일주일 감염 후, PBS로 감염된 세포를 3 회 반복한다. </li><li> 감염된 세포에 용해 버퍼의 50 μl를 추가합니다. </li><li> 5 분간 배양 플레이트를 흔들어 후 5 분 동안 2000 × g으로 원심 분리기. </li><li> 루미 판에 기자 기판의 50 μl를 추가합니다. </li><li> 기자 기판을 포함하는 루미 판에 세포 용 해물 20 μl를 추가합니다. 잠시 텍싱하여 섞는다. </li><li> 루미에 접시를 놓고 15을 읽기 시작합니다. </li></ol>

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The authors declare that they have no competing financial interests.

B 형 간염 게놈 코어, 폴리머, 표면 및 X의 ORF (그림 1) 등 4 차 오픈 리딩 프레임 (ORF를)가 있습니다. 이 네 HBV 개의 ORF의 전사가 단단히 네 프로모터에 의해 조절된다 다음 precore / 핵심 프로모터 인핸서 II를 포함, S1 프로모터, S2 프로모터 및 X 프로모터 인핸서 (18) I를 포함. 3.5 킬로바이트 및 3.4 킬로바이트의 mRNA는 각각 PreCore 및 핵심 단백질로 번역된다. 큰 봉투 단백?...

B 형 간염 바이러스 (HBV) 만성 감염은 만성 간 질환 (1)의 주요 위험 인자이다. 현재의 치료 전략은 인터페론 - 자극 된 유전자의 기능이 관통 HBV의 POL 기능 및 / 또는 감염된 개인에서 면역 반응을 활성화 타입 I 인터페론의 투여뿐만 아니라 간접적으로 억제 HBV의 증식을 억제 뉴클레오티드 유사체에 기반하지만, 이러한 치료는 HBV를 제거 할 수없는 DNA 완전히 3. 또한, 안티 폴 화제에 내성 HBVs의 출현 문제 4이다. 직접 인간 면역 결핍 바이러스 (HIV) 또는 C 형 간염 바이러스 (HCV)의 라이프 사이클의 상이한 단계를 타겟팅 결합 항 바이러스제의 투여가 성공적으로 억제하거나 바이러스 (들)를 박멸 도시 하였다. 이 아이디어는 H의 상이한 단계에 직접 작용 항 HBV 제 (들)의 개발과 마찬가지로BV 라이프 사이클 미래 HBV 치료를 설정하는 것이 중요하다. 일반적으로, 대상 바이러스의 시험 관내 배양 시스템에서 간단한의 설립은 항 바이러스 제제의 개발을 용이하게한다. 그러나, 항 HBV 제를 선별 할 수있는 시험 관내 배양 시스템의 개발에 대한 적어도 두 개의 장벽이있다. 첫 번째는 HBV 감염 / 확산 편리한 생체 외 세포 배양 시스템의 부족이다. 확립 된 세포주에서 전파 예컨대 HIV와 같은 다른 HCV 바이러스, 달리 때문에 좁은 숙주 범위 제한을 포함하여 실험 관내에서 HBV 양성하기 어렵다. 이러한 HBV 감염, 5, 6에 취약 인간 간암 세포주 HepaRG, 특정 세포 배양 시스템의 사용은도 7에 이러한 문제를 극복하기 위해 개발되었다. 또한, 유로 키나아제 TY에서 분리 PXB 세포체육 플라스 미노 겐 활성제의 형질 전환 / 차 인간 간세포 (PHH) 접종 SCID 마우스는 HBV 감염과 복제 팔에 민감한 것으로 나타났다. 그러나 HepaRG에서 HBV 복제 수준은 HBV 감염 / 복제 수준의 일관성과 재생 불가능한 결과가 발생할 수 있습니다 배양 한 후 세포 분화 상태에 따라 달라집니다. PXB는 일반적으로 HBV 감염 실험에 사용되지만 가용성에 의해 제한된다. 테트라 사이클린 유도 HBV 발현 세포주 HepAD38도 널리 HBV 복제를 연구하는 데 사용했지만,이 시스템은 전사 후하지 HBV 감염 (9)의 입력 단계에서 평가를 허용한다. 최근에, HBV에 대한 기능적 수용체 나트륨 타우로 콜산의 cotransporting 폴리펩티드의 동정 (NTCP)는 가변 HBV 배양 시스템 (10)의 개발을 허용했다. 실제로, 이러한 Huh7 비 감수성 간암 세포에서 발현 NTCPHepG2 세포는 HBV 감염을 열 수있게하고, 따라서, HBV 감염 세포주의 선택은 실험 많은 한계를 해결 확장되었다. 두 번째 문제는 HBV 감염 및 복제를 평가하기위한 간단한 분석 시스템의 부족이다. HBV 감염의 평가는 일반적으로 HBV DNA, RNA 및 단백질을 분석함으로써 수행된다. 그러나 이러한 바이러스 마커의 정량은 종종 비용이 많이 들고 항상 간단하지가 시간 소비이다. 따라서, 간단한 분석 시스템의 개발은, 예컨대 리포터 유전자를 사용하여 같은 HBV 분석 시스템과 관련된 문제를 극복 할 수있다. 그러나, HBV 캡시드로 패키징 될 수있는 게놈 크기가 제한되기 때문에 - 미만 3.7 KB 11 - 리포터 유전자의 크기가 가능한 한 짧아야한다. 또한, 바이러스 복제에 필수적인 게놈 흩어져 여러 시스 엘리먼트의 존재에 대한 가능한 위치를 제한<html> 게놈에 리포터 유전자의 sertion. 몇몇 보고서는 HBV 게놈 11, 12, 13로, HIV-1 문신, 녹색 형광 단백질 및을 DsRed을 포함하여 외래 유전자를 삽입하려고했습니다. 그러나 이러한 재조합 HBVs는 HBV 감염 / 복제, 또는 HBV 감염 / 복제에 영향을 미치는 요인의 높은 처리량 검사 선별에 유용하지 않다. 이것은 주로 때문에 재조합 바이러스의 낮은 생산성과 비효율적 바이러스 생산에 의한 리포터 유전자 발현의 감소 된 강도이다. 이러한 문제를 극복하기 위해, 우리는 바이러스 생산의 고 수율을 가진 리포터 HBV를 구축 하였다. 이 바이러스는 전사 엔트리에 상기 HBV 복제 사이클의 초기 모니터링을위한 매우 민감하다. 이 작은 (171 개 아미노산)이 발광 기자를 설계하기 때문에이를 위해 NanoLuc (NL)는 마커 유전자로서 선택되었다클래스 = &quot;외부 참조&quot;&gt; 14. 또한, NL 반딧불 또는 레 닐라 루시퍼보다 밝은 150 배 정도이고, 상기 발광 반응을 잘못된 히트 율이 높은 처리량 스크리닝 낮을 것으로 제안 ATP 독립적이다. 재조합 HBV의 생산 효율은 약 1/5 모 HBV의 이전 HBV 재조합 바이러스보고 수준과 유사하다; 이 항 HBV 제제의 질량 검사에 사용될 수 있지만, NL의 밝기 바이러스 생산성 문제를 극복한다. 기본 간세포를 사용하여 안티 HBV 요원의 검사는 HepaRG, HepAD38 및 NTCP-형질 간세포는 종래의 방법 (들)에 의해 항 HBV 에이전트의 심사에 유용 할 수 있습니다. 그러나, 여기에 설명 된 시스템은 간단한 처리, 고감도 및 스크리닝 저비용 등의 다양한 이점이있다. 이러한 장점 개발 및 치료 목적 HBV 새로운 에이전트를 식별하기위한 고 처리량 분석에 적합하다.

<table><tbody><tr><td>Nano-Glo Luciferase Assay Regent</td><td>Promega</td><td>N1110</td><td></td></tr><tr><td>Penicillin-Streptomycin Mixed Solution</td><td>Nacalai tesque</td><td>09367-34&amp;nbsp;</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids Solution</td><td>Thermo Fisher Scientific</td><td>11140050</td><td></td></tr><tr><td>DMEM</td><td>Thermo Fisher Scientific</td><td>11995065</td><td></td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Thermo Fisher Scientific</td><td>31985070</td><td></td></tr><tr><td>100 mm/collagen-coated dish</td><td>Iwaki</td><td>4020-010</td><td></td></tr><tr><td>Lipofectamine 3000 Transfection Reagent</td><td>Thermo Fisher Scientific</td><td>L3000001&amp;nbsp;</td><td></td></tr><tr><td>Polyethylene glycol (PEG) 6000&amp;nbsp;</td><td>Sigma-Aldrich</td><td>81255</td><td></td></tr><tr><td>Polyethylene glycol (PEG) 8000&amp;nbsp;</td><td>Sigma-Aldrich</td><td>89510</td><td></td></tr><tr><td>NaCl</td><td>Nacalai tesque</td><td>31319-45&amp;nbsp;</td><td></td></tr><tr><td>0.5 mol/L EDTA Solution</td><td>Nacalai tesque</td><td>06894-14</td><td></td></tr><tr><td>Tris-HCl</td><td>Nacalai tesque</td><td>35434-21</td><td></td></tr><tr><td>Millex-HP, 0.45 &amp;mu;m, polyethethersulfone, filter</td><td>Merck Millipore</td><td>SLHP033RS</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>Collagen coated 96-well plate</td><td>Corning</td><td>NO3585</td><td></td></tr><tr><td>Passive Lysis 5x Buffer</td><td>Promega</td><td>E1941</td><td></td></tr><tr><td>GloMax 96 Microplate Luminometer</td><td>Promega</td><td>E6501</td><td></td></tr><tr><td>Sucrose</td><td>Nacalai tesque</td><td>30403-55</td><td></td></tr><tr><td>Luminometer plate</td><td>Greiner bio-one</td><td>655075</td><td></td></tr><tr><td>HepG2-NTCP1-myc-clone22</td><td>-</td><td>-</td><td>Reference 15</td></tr><tr><td>pUC1.2HBV delta epsilon</td><td>-</td><td>-</td><td>Reference 15</td></tr><tr><td>pUC1.2HBV/NL</td><td>-</td><td>-</td><td>Reference 15</td></tr><tr><td>50 ml tube</td><td>Violamo</td><td>1-3500-02</td><td></td></tr><tr><td>Anti-Myc antibody</td><td>Sigma-Aldrich</td><td>C3956</td><td></td></tr><tr><td>HBIG</td><td>Japan Blood Products Organization</td><td>-</td><td></td></tr><tr><td>IFN-&amp;beta;</td><td>Mochida Pharmaceutical</td><td>14987224005413</td><td></td></tr><tr><td>Heparin</td><td>Sigma-Aldrich</td><td>H3393</td><td></td></tr></tbody></table>

development of a hepatitis b virus reporter system to monitor the early stages of the replication cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

도 1은 게놈에서 HBV 게놈의 RNA 전사는 바이러스 단백질 및 코딩 영역의 개략도를 도시한다. 게놈의 리포터 유전자 및 그 위치도 표시됩니다. 도 2는 리포터 유전자를 포함하는 HBV 리포터 플라스미드 및 헬퍼 플라스미드를 나타낸다. pUC1.2HBV / NL 기자는 pUC1.2HBV (16)의 preCore의 mRNA의 전사 개시 사이트에서 염기 위치를 223-811을 삭제 ?...

Watch this Scientific Journal Video about Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle at JoVE.com

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

여기서 우리는 HBV의 생활주기의 초기 단계를 모니터링하는 새롭게 개발 된 B 형 간염 바이러스 (HBV) 리포터 시스템을 설명한다. 시험 관내 시스템에서 간략화 이것은 높은 처리량 전략을 사용 항 HBV 에이전트 스크리닝 도울 것이다.

B 형 간염 바이러스 리포터 시스템 개발은 복제주기의 초기 단계를 모니터링하기

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

development-hepatitis-b-virus-reporter-system-to-monitor-early-stages

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

13.1K 조회수.  National Center for Global Health and Medicine.  여기서 우리는 HBV의 생활주기의 초기 단계를 모니터링하는 새롭게 개발 된 B 형 간염 바이러스 (HBV) 리포터 시스템을 설명한다. 시험 관내 시스템에서 간략화 이것은 높은 처리량 전략을 사용 항 HBV 에이전트 스크리닝 도울 것이다. 

비디오: Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

C 형 간염 바이러스의 복제를 분석하기위한 프로토콜

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

연구

면역학 및 감염병학

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

유전자 발현의 모든 단계에서 바이러스 성 기능을 정량화 우두 바이러스 기자

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

초기 바이러스 성 항목 식별을위한 분석 실험 및 항 바이러스 화합물의 평가

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

A Competent Hepatocyte Model Examining Hepatitis B Virus Entry through Sodium Taurocholate Cotransporting Polypeptide as a Therapeutic Target

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load but not result in complete remission. An efficient hepatocyte model for HBV infection would offer a true-to-life viral life cycle that would be crucial for the screening of therapeutic agents. Most available anti-HBV agents target lifecycle stages post viral entry but not before viral entry. This protocol details the generation of a competent hepatocyte model capable of screening for therapeutic agents targeting pre-viral entry and post viral entry lifecycle stages. This includes the targeting of sodium taurocholate cotransporting polypeptide (NTCP) binding, cccDNA formation, transcription, and viral assembly based on imHC or HepaRG as host cells. Here, the HBV entry inhibition assay used curcumin to inhibit HBV binding and transporting functions via NTCP. The inhibitors were evaluated for binding affinity (KD) with NTCP using isothermal titration calorimetry (ITC)-a universal tool for HBV drug screening based on thermodynamic parameters.

We present a protocol to screen anti-hepatitis B virus (HBV) compounds targeting pre- and post-viral entry lifecycle stages, using isothermal titration calorimetry to measure binding affinity (KD) with host sodium taurocholate cotransporting polypeptide. Antiviral efficacy was determined through the suppression of viral lifecycle markers (cccDNA formation, transcription, and viral assembly).

치료 표적으로 폴리펩티드를 공동 수송하는 나트륨 타우로 콜레이트를 통한 B 형 간염 바이러스 진입을 조사하는 유능한 간세포 모델

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load ...

생화학

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1&#8211;2 copies) integration using inverse nested PCR.

낮은 복사의 번호 통합된 바이러스 성 DNA 생체 외에서 B 형 간염 감염에 의해 형성 된

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

The goal of this protocol is to provide a step-by-step guide to perform 3-D &#34;liver-on-a-chip&#34; infection experiments with the hepatitis B virus.

기본 Hepatocytes 및 B 형 간염 바이러스 감염에 대 한 Kupffer 세포의 "간-온-어-칩" 문화

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally ...

A Cell Culture Model for Producing High Titer Hepatitis E Virus Stocks

Hepatitis E virus is the leading cause of liver cirrhosis and liver failure with increasing prevalence worldwide. The single-stranded RNA virus is predominantly transmitted by blood transfusions, inadequate sanitary conditions and contaminated food products. To date the off-label drug ribavirin (RBV) is the treatment of choice for many patients. Nonetheless, a specific HEV treatment remains to be identified. So far, the knowledge about the HEV life cycle and pathogenesis has been severely hampered because of the lack of an efficient HEV cell culture system. A robust cell culture system is essential for the study of the viral life cycle which also includes the viral pathogenesis. With the method described here one can produce viral titers of up to 3 x 106 focus forming unit/mL (FFU/mL) of non-enveloped HEV and up to 5 x 104 FFU/mL of enveloped HEV. Using these particles, it is possible to infect a variety of cells of diverse origins including primary cells and human, as well as animal cell lines. The production of infectious HEV particles from plasmids poses an infinite source, which makes this protocol exceedingly efficient.

Described here is an effective method on how to produce high viral titer stocks of hepatitis E virus (HEV) to efficiently infect hepatoma cells. With the presented method, both non-enveloped, as well as enveloped viral particles can be harvested and used for inoculating various cell lines.

높은 티더 E 바이러스 주식을 생산하기위한 세포 배양 모델

Hepatitis E virus is the leading cause of liver cirrhosis and liver failure with increasing prevalence worldwide. The single-stranded RNA virus is ...

Modeling Hepatitis B Virus Infection in Non-Hepatic 293T-NE-3NRs Cells

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV models are limited to hepatoma cell lines (such as HepG2 and Huh7) overexpressing a functional HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP). Here, we used 293T-NE-3NRs (293T&#160;overexpressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and HepG2-NE (HepG2 overexpressing NTCP) as model cell lines.&#160;HBV infection in these cell lines was performed either by using concentrated HBV virus particles from HepG2.2.15 or co-culturing HepG2.2.15 with the target cell lines. HBcAg immunofluorescence for HBcAg was performed to confirm HBV infection. The two methods presented here will help us study HBV infection in non-hepatic cell lines.&#160;&#160;

This manuscript describes a detailed protocol for Hepatitis B virus (HBV) infection in novel engineered 293T cells (293T-NE-3NRs, expressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and traditional hepatic cells (HepG2-NE, expressing human NTCP).

비 간염 293T-NE-3NRs 세포에 있는 B형 간염 바이러스 감염을 모델링

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV ...

인간 혈청에서 B형 간염 바이러스 DNA의 검출 및 정량화

Real-Time Polymerase Chain Reaction-Based Detection and Quantification of Hepatitis B Virus DNA

Hepatitis B virus (HBV) is a significant cause of liver disease worldwide. It can lead to acute or chronic infections, making individuals highly susceptible to fatal cirrhosis and liver cancer. Accurate detection and quantification of HBV DNA in the blood are essential for diagnosing and monitoring HBV infection. The most common method for detecting HBV DNA is real-time PCR, which can be used to detect the virus and assess the viral load to monitor the response to antiviral therapy. Here, we describe a detailed protocol for the detection and quantification of HBV DNA in human serum or plasma using an IVD-marked real-time PCR-based kit. The kit uses primers and probes that target the highly conserved core region of the HBV genome and can accurately quantify all HBV genotypes (A, B, C, D, E, F, G, H, I, and J). The kit also includes an endogenous internal control to monitor possible PCR inhibition. This assay runs for 40 cycles, and its cutoff is 38 Ct. For the quantification of HBV DNA in clinical samples, a set of 5 quantification standards is provided with the kit. The standards contain known concentrations of HBV-specific DNA that are calibrated against the 4th WHO International Standard for HBV DNA for the nucleic acid test (NIBSC code 10/266). The standards are used to validate the functionality of the HBV-specific DNA amplification and to generate a standard curve, allowing the quantification of HBV DNA in a sample. HBV DNA as low as 2.5 IU/mL was detected using the PCR kit. The high sensitivity and reproducibility of the kit make it a powerful tool in clinical laboratories, aiding healthcare professionals in effectively diagnosing and managing HBV infections.

저자 스포트라이트: B형 간염 바이러스 검출의 발전과 과제 

Real-time polymerase chain reaction (PCR)-based detection and quantification of hepatitis B virus (HBV) DNA is a sensitive and accurate method for diagnosing and monitoring HBV infection. Here, we present a protocol for HBV DNA detection and the viral load measurement of a sample.