Name: development of a hepatitis b virus reporter system to monitor the early stages of the replication cycle
Uploaded: 2017-02-01T13:00:00
Description: 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

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 3...

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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.

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			<td height="20" style="height:20px;width:332px;">Nano-Glo Luciferase Assay Regent</td>
			<td style="width:201px;">Promega</td>
			<td style="width:160px;">N1110</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin Mixed Solution</td>
			<td>Nacalai tesque</td>
			<td>09367-34&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM Non-Essential Amino Acids Solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>11995065</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Opti-MEM I Reduced Serum Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>31985070</td>
			<td></td>
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			<td height="18" style="height:18px;width:332px;">100 mm/collagen-coated dish</td>
			<td>Iwaki</td>
			<td>4020-010</td>
			<td></td>
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			<td height="20" style="height:20px;">Lipofectamine 3000 Transfection Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>L3000001&#160;</td>
			<td></td>
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			<td height="20" style="height:20px;">Polyethylene glycol (PEG) 6000&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>81255</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyethylene glycol (PEG) 8000&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>89510</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Nacalai tesque</td>
			<td>31319-45&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5 mol/l-EDTA Solution</td>
			<td>Nacalai tesque</td>
			<td>06894-14&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris-HCl</td>
			<td>Nacalai tesque</td>
			<td>35434-21</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Millex-HP, 0.45 &#956;m, polyethethersulfone, filter</td>
			<td>Merck Millipore</td>
			<td>SLHP033RS</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagen coated 96-well plate</td>
			<td>Corning</td>
			<td>NO3585</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Passive Lysis 5xBuffer</td>
			<td>Promega</td>
			<td>E1941</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GloMax 96 Microplate Luminometer</td>
			<td>Promega</td>
			<td>E6501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose</td>
			<td>Nacalai tesque</td>
			<td>30403-55</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Luminometer plate</td>
			<td>Greiner bio-one</td>
			<td>655075</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HepG2-NTCP1-myc-clone22</td>
			<td>-</td>
			<td>-</td>
			<td>Reference 15</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pUC1.2HBV delta epsilon</td>
			<td>-</td>
			<td>-</td>
			<td>Reference 15</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pUC1.2HBV/NL</td>
			<td>-</td>
			<td>-</td>
			<td>Reference 15</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 ml tube</td>
			<td>Violamo</td>
			<td>1-3500-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-Myc antibody</td>
			<td>Sigma-Aldrich</td>
			<td>C3956</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBIG</td>
			<td>Japan Blood Products Organization</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IFN-&#946;</td>
			<td>Mochida Pharmaceutical</td>
			<td>14987224005413</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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), ...

The overall goal of this recombinant HBV reporter system is to make it easy to conduct mass screening for anti-Hepatitis B virus, or HBV agents. This method can help answer key questions in the HBV field, such as screening for anti-HBV agents and identification HBV factors. The main advantage of this technique is that the HBV reporter system can monitor the earlier stages of the HBV replication cycle.After preparing cell culture medium, the day before transvection, place four times 10 to the six HepG2 cells in medium, on a 10 cm collagen coated dish. Incubate the cells at 37 degrees C in a humidifed 5%CO2 incubator. Using a transvectionary agent according to the manufacturer's instructions, transvect the cells with 5 mcg of pUC1 2HBV delta epsilon, and 5 mcg of pUC1 2HBV/NL.The next day, remove the culture medium and add 10 mL of fresh culture medium. Then, one week after transvection, transfer the culture medium containing the recombinant HBV to a 50 mL tube. Next, add 10 mL of fresh culture medium to the plate containing transvected cells.Then collect and centrifuge the culture medium at 2, 300 x g for five minutes to remove the cell debris containing the recombinant HBV. Pass the supernatant through a 45 mcm membrane filter. Then add an equal volume of 25%PEG, 1.5 molar NaCl, to the filtrate, and gently mix.Incubate the sample overnight at four degrees C.The following day, spin the solution at 2, 300 x g and four degrees C, for 20 minutes. Discard the supernatant and dissolve the pellet in 5 mL of TNE buffer. Now centrifuge the sample again to remove cell debris, then load 5 mL of the TNE-containing recombinant HBV onto 8 mL of 20%sucrose in TNE.Centrifuge the two at 100, 000 x g and 15 degrees celsius for three hours. Discard as much of the supernatant as possible and use 1 mL of serum-free DMEM per 40 mL of starting culture to re-suspend the pellet. Incubate the suspension at four degrees celsius overnight.The following morning, filter the suspension through a 45 mcm filter. Then prepare 5 mL aliquots and store the tubes at minus 80 degrees celsius. Plate HepG2 cells, stably expressing sodium taurocholate cotransporting polypeptide, or NTCP, that are susceptible to HBV infection, and incubate at 37 degrees celsius and 5%CO2.One day before infection, plate approximately five times 10 to the fourth HepG2 NTCP cells into the wells of a 96-well collagen coated plate, in 1 mL of culture medium. Thaw recombinant HBV in a 37 degree celsius water bath, until there is a small bit of ice remaining in the vial. Prepare medium for infection by combining 78 mcL of fresh culture medium, 2 mcL of DMSO, 10 mcL of 40%PEG 8000, in 1x PBS, and 10 mcL of recombinant HPV.Add 100 mcL of recombinant HBV solution to each well of a 96-well plate. One day after infection, use 300 mcL of PBS to wash the cell three times to remove the contaminating reporter protein from the virus fraction. Add 200 mcL of culture medium, containing 2%DMSO, to the infected cells, and incubate for one week or less.To carry out analysis of the HBV infection, one week after infection, use PBS to wash the infected cells three times. Add 50 mcL of Lysis Buffer to the infected cells. Rock the culture plate for five minutes then centrifuge at 2, 000 x g for five minutes.Add 50 mcL of the reporter substrate to a Luminometer plate. Then add 20 mcL of the cell lysate to the plate and mix by briefly shaking. Finally, place the plate in the Luminometer and initiate reading.This gel shows digestion of the constructed pUC1 2HBVNL reporter plasmid, and the pUC1 2HBV delta helper plasmid with HindII and EcoRI. Both samples produce the expected band sizes. In this western blot, lysates from HepG2 cells, or HepG2 cells expressing recombinant myc-tagged NTCP and infected with recombinant HBV, were probed with an anti-myc antibody that recognizes the 34 kDa unglycosylated and the 65 kDa glycosolated recombinant proteins.The graphs shown here illustrate the kinetics of the reporter gene. RNA and DNA levels in recombinant HBV infected HepG2 NTCP myc-clone 22, or human primary hepatocyte cells. The levels of HBV RNA in the reporter activity were elevated at three days after infection, in both the infected cells and the primary hepatocytes.In contrast, there was no change in DNA levels at nine days post-infection, because recombinant HBV is deficient for functional core and pole, that play important roles in the intracellular DNA replication pathway. In this figure, inhibition of recombinant HBV by Hepatitis B Immune Globulin, or HBIG, Heparin, and IFN-beta, are illustrated. HBIG and Heparin strongly suppressed the reporter activity to less than 10%at 75 U/mL, and 150 U/mL, respectively.IFN-beta on the other hand, suppressed reporter activity to less than 50%at 1000 U/mL. Once mastered, this technique can be done in one week for preparation of recombinant HBV, in six days for infection recombinant HBV, and in summary, means for measurement of the reporter protein, if it is performed properly. While attempting this procedure, it's important to remember the washing step that removes the contaminating reporter protein from the bias fraction.Following this procedure, other results like infection with wide HBV can be performed in order to answer additional question to that the recombinant HBV did not produce first positive results. After its development, this technique paved the way for researchers in the field of HBV to explore the development of anti-HBV agent in a cell culture model. After watching this video, you should have a good understanding of how to purify and infect cells with recombinant HBV.Don't forget that working with recombinant HBV can be extremely hazardous and precautions such as wearing gloves should always be taken while performing this procedure.

Research

JoVE Journal

Immunology and Infection

Development of a Negative Selectable Marker for Entamoeba histolytica

Development of a Negative Selectable Marker for Entamoeba histolytica

에 대한 부정적인 선택 마커의 개발 Entamoeba histolytica</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

연구

면역학 및 감염병학

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

모델 Herpesvirus의 Lytic 복제의 해부 호스트 바이러스 상호 작용

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

A Protocol for Analyzing Hepatitis C Virus Replication

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 ...

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

마우스 태아 간 문화 시스템은 터미널 적혈구의 초기와 후기 단계에서 대상 유전자의 기능을 해부하기

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

웨스트 나일 바이러스 돌연변이 균주의 감염에에서 MyD88 매개 세포 면역 반응의 생체 외 분석

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

C 형 간염 바이러스에 대한 인간 락토페린의 세포 흡수 및 관련 바이러스 활동을 모니터링하는 면역 형광

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

소설 급지대 무료 시스템 대량 생산을 위한 생체 외에서 Murine 자연 킬러 세포의

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Assembly and Purification of Prototype Foamy Virus Intasomes

조립 및 프로토 타입 거품 바이러스 Intasomes의 정화

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

전체 혈액 샘플에서 바이러스의 검출, 안전, 신속 하 고 간단한 수동 침대 핵 산 추출

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

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

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 ...