We thank F. Chisari for providing Huh-7.5.1 cell line. We would like to thank Justine Ho for editing the manuscript. This work was supported by Cedars-Sinai Medical Center Institutional Programmatic Research Award and National Center for Advancing Translational Sciences, Grant UL1TR000124 to V.A.

프로토콜의 일반적인 개요는도 1에 도시되어있다. 1. 셀 <ol><li> 15 % 소 태아 혈청 (FBS), 10 mM의 비 필수 아미노산, 10mM의 헤 페스, 페니실린 (100 단위 / ml), 스트렙토 마이신 (100 ㎎ / ㎖), 및 2 mM의 L-글루타민을 포함 완전 성장 배지를 준비한다. </li><li> C 형 간염 바이러스의 복제주기의 체외 분석을 위해 위의 보충제를 포함하는 완전한 성장 매체 허-7.5.1 셀 (13)을 유지한다. </li><li> 5 % CO 2와 37 ° C에서 지정한 보충 성장 매체 허-7.5.1 세포 배양 바이러스의 변종. </li></ol> 2. 바이러스와 플라스미드 구문 <ol><li> HCV RNA의 상보 적 DNA (cDNA를) 형태로 생성 및 유전자 조작의 용이성을 위해 플라스미드 벡터로 복제. 참고 : 일본의 전격 성 간염 1의 발견은, JFH-1 (유전자형 2A의 HCV), HCV 연구에 대한 비판적이고있다 격리주로 HCV의 복제 사이클 11-13를 연구하는 데 사용. JFH-1 HCV에 따라 간 및 intragenotypic 키메라 바이러스는 일반적으로 연구 14-18에서 사용된다. 합성 intragenotype의 2A 키메라 바이러스, FNX-HCV, 그리고 monocistronic 키메라 기자 균주의 건설은 이미 19 건설 자세한 내용은이 프로토콜의 범위를 벗어 설명했다. </li><li> 그것은 (뉴클레오티드 1-2878) J6CF 부모의 변형 (NCBI의 기탁 아니오. AF177036)의, 같은 5&#39;NTR, 구조 지역과 P7 및 NS2 비 구조 영역의 일부를 포함로 pFNX-HCV intragenotype의 2A 바이러스를 사용 잘 JFH-1 바이러스 균주 (NCBI의 기탁 없습니다. AB047639)의 비 구조적인 구성 요소로. 참고 : FNX-HCV가 JC1의 intragenotype의 2A 키메라 HCV의 순서에 따라 합성 하였다 이전에 (15) 보도했다. </li><li> R을 삽입하여 (단계 2.2 이상) pFNX-HCV의 변형을 기반으로 monocistronic 키메라 기자 바이러스 구조, pFNX-Rluc를 생성5 &#39;NTR과 (NT 388과 389 사이의) 핵심 유전자 사이 enilla 루시퍼 라제 유전자. 이어서, 절단 신호로서 작동 할 구제역 바이러스 2A (F2A) 펩티드 서열을 사용하여 루시 페라 제 유전자 및이 구조의 핵심 유전자를 연결한다. </li><li> pFNX-HCV 또는 pFNX-Rluc 바이러스 배경을 사용하여 RNA 중합 효소 널 (폴) 바이러스 구조 엔지니어. , AAG의 아미노산 잔기 이러한 바이러스 백본 중 하나의, NS5B 효소 촉매 잔류 물, GDD (8615-8623을 NT AA 2,759에서 2,761 사이)를 교체합니다. </li></ol> 3. 체외 HCV의 RNA의 전사 <ol><li> intragenotype의 2A 키메라 바이러스 FNX-HCV와 FNX-HCV의 유류 널 (null) 바이러스 복제 (그림 2A)의 평가를위한 (폴) HCV를 사용합니다. </li><li> 를 XbaI 제한 효소로 바이러스 플라스미드를 선형화 한 다음 평활 말단을 생성하는 녹두 클레아로 취급한다. 음이온 교환 크로마토 그래피에 의해 소화 플라스미드를 정화. O를 무결성을 확인F 겔 전기 영동 (그림 2B)를 아가 로스의 DNA를 쓰는하여 플라스미드를 선형화. </li><li> -T7 RNA 중합 효소를 이용하여 선형화 된 바이러스 플라스미드를 표기. </li><li> RNA 정제 키트를 사용하여 새로 합성 된 DNA 분해 효소 처리 RNA를 정화. </li><li> 아가로 오스 겔 전기 영동 (그림 2C)에 의해 RNA의 생산을 확인합니다. </li><li> 분광 광도법에 의한 RNA의 양을. </li><li> 10 μg 작업 분량 씩 -80 ° C에서 생성 된 RNA를 저장합니다. 참고 : 같은 시간에 각각의 바이러스 샘플 및 제어를위한 모든 RNA를 해독하여 각 실험 설계 내에서 RNA의 품질의 변화를 최소화하기 위해. </li></ol> 4. HCV RNA 형질 및 시료 채취 <ol><li> 트립신을 사용하는 허-7.5.1 세포를 수확. </li><li> 세포 원심 분리기를 씻어 차가운 낮은 혈청 미디어로 두 번 정학을 재현 탁합니다. ML 당 1 × 10 7 세포에서 낮은 혈청 배지에서 세포를 Resuspend. </li><li> 10 &amp; # 총 혼합956, 0.4-CM의 일렉트로 큐벳에 재현 탁 세포 (4 × 10 6 세포)의 400 μL와 전사 바이러스 성 RNA의 g. 270 V에서 전기 충격을 통해 100 Ω, 950 μF을 세포를 형질. </li><li> 15 % FBS와 10 ㎖ 완전한 성장 미디어 일렉트로 세포를 Resuspend. 참고 : 허-7.5.1 세포는 15 % 소 태아 혈청에서 배양 할 때 일렉트로 세포의 증가 생존을 볼 수 있습니다. </li><li> 4, 48 및 96 시간 : 두 T-25 플라스크 (플라스크 당 ~ 1.2 × 10 6 세포) 및 48 - 웰 플레이트 다음과 같은 시점마다 (물론 당 1 × 10 4 세포)에서 세포를 접시. </li><li> 배양 플라스크와 접시에서 죽은 세포 파편을 제거하기 위해 10 % FBS와 신선한 보충 성장 매체와 4-8 시간 후 - 형질 전환에 용지를 교체하십시오. </li><li> 48, 96 시간의 시점에서, 4에서 10 분 동안 1,500 rpm에서 원심 분리하여 세포를 수집 한 시료에서 세포 파편을 제거 수확 세포 배양 상청액 º C. </li><li> -80 º C에서 세포가없는 상층 액을 저장 를 Lyse 웨스턴 블롯에 의해 단백질과 RNA 분석 및 지정된 시점에서 전사 정량적 PCR 역 세포. </li></ol> 5. HCV 게놈 복사를 평가하기위한 전사 정량 PCR (RT-qPCR에)를 역방향 <ol><li> HCV 게놈 RNA 카피 수를 결정하는 두 단계 RT-qPCR에를 수행합니다. </li><li> 역 쓰다 1 역전사 효소 및 5&#39;NTR (JFH RTQ R : 5&#39;CCTATCAGGCAGTACCACA-3 &#39;)에 결합 HCV의 가닥 특정 프라이머 사용하여 총 세포 RN​​A ㎍의 또는 하우스 키핑 유전자 peptidylprolyl 이성화 효소 G (삑 R 특정 프라이머를 : 5&#39;-GTCTCTCCTCCTTCTCCTCCTATCTTT-3 &#39;). 또한 HCV의 가닥 프라이머를 사용하여 알려진 게놈 사본 (10 1 9 표준 10)의 (3 단계에서 생성 된) FNX-HCV RNA를 표기 역. </li><li> (JFH RT HCV 특정 프라이머를 이용하여 생성 된 cDNA의 전사 50 NG를 사용하여 qPCR에를 수행QF : 5&#39;CTGGGTCCTTTCTTGGATAA-3; JFH RTQ R : 5&#39;CCTATCAGGCAGTACCACA-3 &#39;)와 DNA는 qPCR에 슈퍼 믹스를 포함하는 녹색 염료를 결합. 또한 하우스 키핑 유전자 삑위한 qPCR에 수행 (프라이머 삑 F : 5&#39;-GAAGAGTGCGATCAAGAACCCATGAC-3 &#39;, 삑 R : 5&#39;-GTCTCTCCTCCTTCTCCTCCTATCTTT-3&#39;) 참고 : 예제를 통해 정확한 세포 내 HCV의 RNA 수준을 계산하는 경우, 휴대 하우스 키핑 유전자 삑를 사용 정규화에 대한 발현 수준 (CT주기). 카피 수 결정을위한 표준으로 FNX-HCV 게놈의 카피 수를 사용합니다. </li><li> 15 초 동안 95 º C ~ 60 : qPCR에를 실행하는 HCV의 RNA 사본의 수를 결정하는 경우 다음과 같은 조건을 사용하여 &#160; 실시간 PCR 시스템을 사용하여 30 초 (40 회)에 대해 C를 º. </li><li> 게놈의 복제 결과를도 3a 및도 3b를 참조하십시오. </li></ol> 6. 웨스턴 블로 팅 분석 HCV 단백질 발현 (그림 3C)를 검출 <ol><li> 세포 용 해물의 FR을 사용하여톰 바이러스 성 RNA는 단백질 웨스턴 블롯 분석을위한 96 시간 포스트 형질에 형질 전환. </li><li> SDS-PAGE 및 폴리 비닐 리덴 디 플루오 라이드 (PVDF) 멤브레인으로 이동을 사용하여 세포 용 해물을 해결. </li><li> 5 % 탈지 분유, 인산염 완충 식염수 (PBS)에서 0.2 % 트윈 20을 함유하는 차단 용액을 사용하여 멤브레인을 차단. </li><li> 기본 마우스 단일 클론 항체 NS3와 막 (1 천에 희석) 및 베타 - 액틴 (1 5,000 희석)을 품어. </li><li> 고추 냉이 퍼 옥시 다제 (1 5,000 희석)에 접합 된 염소 항 - 마우스 IgG의 추가 및 화학 발광에 의해 감지. </li></ol> 7. 면역 형광 분석 (IFA) <ol><li> 면역 형광 분석 -20 º C에서 30 분 동안 메탄올을 사용하여 HCV 감염과 형질 전환 세포를 고정합니다. </li><li> PBS로 3 회 세포를 세척하고 IFA 버퍼를 차단으로 차단합니다. </li><li> 토끼 폴리 클로 날 또는 마우스 단일 클론 항-DSR (친절 박사 다스 굽타, UCLA에서 제공) 항 NS5A 주로 항체를 사용하여NA 항체 1:200 (1 ㎍ / ㎖)의 희석 J2와 5 시간에 4 박에서 알을 품다 º C. </li><li> 세 번 차 항체 후 PBS로 세포를 씻으십시오. </li><li> 염소 항 - 토끼 IgG의-488 클론 차 항체 또는 1:1,000 희석 (1 ㎍ / ㎖)에 염소 항 - 마우스 IgG-594 클론 차 항체를 추가하고 실온에서 1 시간 동안 배양한다. </li><li> PBS 형광 현미경 (그림 3D)를 사용하여 훽스트 염료 및 뷰를 사용하여 3 회 얼룩 핵으로 세포를 씻으십시오. </li></ol> 8. 측정 바이러스 역가 <ol><li> 판 순진 허-7.5.1 세포 약 3 × 10 3 세포 / 잘 96 - 웰 플레이트를 사용하여. 다음 날, 성장 미디어를 사용하여 HCV의 RNA 형질 세포에서 수확 한 무 세포 배양 상층 액의 10 배 시리얼 희석을 수행하고 허-7.5.1 세포에 배수로 접종. </li><li> -20에서 30 분 동안 메탄올 (사용하여 72 시간 게시물 감염에서 세포를 수정 </suC)과 7 장에서 언급 한 바와 같이 HCV의 NS5A 단백질에 대한 immunostain º P&gt;. </li><li> NS5A 긍정적 인 초점을위한 카운트 밀리리터 당 포커스 형성 단위의 평균 수 (FFU)를 계산하는 가장 높은 희석을 사용합니다. HCV 역가 그림 4를 참조하십시오. </li></ol> 바이러스 게놈 복제 및 감염성 9. 닐라 루시퍼 라제 리포터 분석 <ol><li> 바이러스 게놈 복제, 48 - 웰 플레이트에 세중 판 HCV의 RNA-일렉트로 허-7.5.1 세포 (1 × 104 세포 / 웰) 평가하십시오. </li><li> 를 Lyse 6 시간에서 수동 용해 완충액 75 μL, 48 시간, 96 시간 후 - 형질을 가진 세포. </li><li> -80 º C에서 실내 온도와 매장에서 15 분 동안 부드럽게 플레이트 록 </li><li> , 레 닐라 루시퍼 라 아제의 효소 활성을 측정하고, 실온으로 단백질 및 파쇄물 레 닐라 루시퍼 라제 분석 시약을 해동. </li><li> 96 - 아, 잘 당 단백질의 용해 액의 20 μl를 추가합니다<html> L 화이트 발광 판과 루미에 접시를 놓습니다. </li><li> 레 닐라 루시퍼 라제 분석 시약 (엘렌 테라 기판 + 버퍼) 잘 당 2 초 미리 읽기 지연 시간 후 100 μl를 분배; 10 초 동안 방출되는 빛을 통합 할 수 있습니다. </li><li> 생물학적 삼중의 루시퍼 라제 값으로부터 각 샘플에 대한 평균 및 표준 편차를 계산 및 통계 분석 데이터 (도 5) 주체. </li><li> 감염을 평가하기위한 48 - 웰 플레이트에 순진 허-7.5.1 세포에 세중 표시된 시점 (48 시간 및 96 시간)에 대한 HCV의 RNA 형질 세포에서 얻은 세포가없는 상층 액 500 μl를 접종. </li><li> 물론 후 6 시간 게시물 감염 당 신선한 매체의 500 μL와 바이러스 접종을 교체합니다. </li><li> 를 Lyse 8.2-8.7 (그림 5) 단계에 설명 된대로 레 닐라 루시퍼 라제 분석에 48 시간의 후 감염과 피사체에 세포. </li></ol>itle &quot;&gt; 10. 통계 분석 <ol><li> 그래프에 오차 막대는 표준 편차 (SD)를 나타냅니다. P 값은 짝 t 테스트에 의해 산출 하였다. </li></ol>

<ol>
	<li>Alter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=J.+Epidemiology+of+hepatitis+C.">J. Epidemiology of hepatitis C.</a> Hepatology.. 26(3 Suppl 1), (1997).</li><li>Alter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=J.+Epidemiology+of+hepatitis+C+virus+infection.">J. Epidemiology of hepatitis C virus infection.</a> World J Gastroenterol. 13 (17), 2436-2441 (2007).</li><li>Lavanchy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+global+burden+of+hepatitis+C.">The global burden of hepatitis C.</a> Liver Int. 29 (Suppl 1), 74-81 (2009).</li><li>Lindenbach, B. D., Thiel, H. J., Rice, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Flaviviridae: viruses and their replication in Fields Virology. , 1101-1152 (2007).</li><li>Ciesek, S., Manns, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+in+2010:+the+dawn+of+a+new+era+in+HCV+therapy.">Hepatitis in 2010: the dawn of a new era in HCV therapy.</a> Nat Rev Gastroenterol Hepatol. 8 (2), 69-71 (2011).</li><li>Ghany, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+update+on+treatment+of+genotype+1+chronic+hepatitis+C+virus+infection:+2011+practice+guideline+by+the+American+Association+for+the+Study+of+Liver+Diseases.">An update on treatment of genotype 1 chronic hepatitis C virus infection: 2011 practice guideline by the American Association for the Study of Liver Diseases.</a> Hepatology. 54 (4), 1433-1444 (2011).</li><li>Choo, Q. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+cDNA+clone+derived+from+a+blood-borne+non-A,+non-B+viral+hepatitis+genome.">Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome.</a> Science. 244 (4902), 359-362 (1989).</li><li>Kolykhalov, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+of+hepatitis+C+by+intrahepatic+inoculation+with+transcribed+RNA.">Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA.</a> Science. 277 (5325), 570-574 (1997).</li><li>Lohmann, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+subgenomic+hepatitis+C+virus+RNAs+in+a+hepatoma+cell+line.">Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line.</a> Science. 285 (5424), 110-113 (1999).</li><li>Blight, K. J., Kolykhalov, A. A., Rice, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+initiation+of+HCV+RNA+replication+in+cell+culture.">Efficient initiation of HCV RNA replication in cell culture.</a> Science. 290 (5498), 1972-1974 (2000).</li><li>Lindenbach, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+replication+of+hepatitis+C+virus+in+cell+culture.">Complete replication of hepatitis C virus in cell culture.</a> Science. 309 (5734), 623-626 (2005).</li><li>Wakita, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+infectious+hepatitis+C+virus+in+tissue+culture+from+a+cloned+viral+genome.">Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.</a> Nat Med. 11 (7), 791-796 (2005).</li><li>Zhong, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+hepatitis+C+virus+infection+in+vitro.">Robust hepatitis C virus infection in vitro.</a> Proc Natl Acad Sci U S A. 102 (26), 9294-9299 (2005).</li><li>Yi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compensatory+mutations+in+E1,+p7,+NS2,+and+NS3+enhance+yields+of+cell+culture-infectious+intergenotypic+chimeric+hepatitis+C+virus.">Compensatory mutations in E1, p7, NS2, and NS3 enhance yields of cell culture-infectious intergenotypic chimeric hepatitis C virus.</a> J Virol. 81 (2), 629-638 (2007).</li><li>Pietschmann, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+characterization+of+infectious+intragenotypic+and+intergenotypic+hepatitis+C+virus+chimeras.">Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras.</a> Proc Natl Acad Sci U S A. 103 (19), 7408-7413 (2006).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+full-length+hepatitis+C+virus+genotype+1+(strain+TN)+infectious+culture+system.">Highly efficient full-length hepatitis C virus genotype 1 (strain TN) infectious culture system.</a> Proc Natl Acad Sci U S A. 109 (48), 19757-19762 (2012).</li><li>Yi, M., Lemon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotype+1a+HCV+(H77S)+infection+system.">Genotype 1a HCV (H77S) infection system.</a> Methods Mol Biol. 510, 337-346 (2009).</li><li>Gottwein, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+application+of+hepatitis+C+reporter+viruses+with+genotype+1+to+7+core-nonstructural+protein+2+(NS2)+expressing+fluorescent+proteins+or+luciferase+in+modified+JFH1+NS5A.">Development and application of hepatitis C reporter viruses with genotype 1 to 7 core-nonstructural protein 2 (NS2) expressing fluorescent proteins or luciferase in modified JFH1 NS5A.</a> J Virol. 85 (17), 8913-8928 (2011).</li><li>Arumugaswami, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+functional+profiling+of+hepatitis+C+virus+genome.">High-resolution functional profiling of hepatitis C virus genome.</a> PLoS Pathog. 4 (10), (2008).</li><li>Chu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+analysis+of+enhancer+and+critical+cis-acting+RNA+elements+in+the+protein-encoding+region+of+the+hepatitis+C+virus+genome.">Systematic analysis of enhancer and critical cis-acting RNA elements in the protein-encoding region of the hepatitis C virus genome.</a> J Virol. 87 (10), 5678-5696 (2013).</li><li>Salloum, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab18+binds+to+hepatitis+C+virus+NS5A+and+promotes+interaction+between+sites+of+viral+replication+and+lipid+droplets.">Rab18 binds to hepatitis C virus NS5A and promotes interaction between sites of viral replication and lipid droplets.</a> PLoS Pathog. 9 (8), (2013).</li><li>Gonzalez, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+an+optimal+RNA+transfection+reagent+and+comparison+to+electroporation+for+the+delivery+of+viral+RNA.">Selection of an optimal RNA transfection reagent and comparison to electroporation for the delivery of viral RNA.</a> J Virol Methods. 145 (1), 14-21 (2007).</li><li>Phan, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+C+virus+NS2+protein+contributes+to+virus+particle+assembly+via+opposing+epistatic+interactions+with+the+E1-E2+glycoprotein+and+NS3-NS4A+enzyme+complexes.">Hepatitis C virus NS2 protein contributes to virus particle assembly via opposing epistatic interactions with the E1-E2 glycoprotein and NS3-NS4A enzyme complexes.</a> J Virol. 83 (17), 8379-8395 (2009).</li><li>Schaller, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+hepatitis+C+virus+superinfection+exclusion+by+using+novel+fluorochrome+gene-tagged+viral+genomes.">Analysis of hepatitis C virus superinfection exclusion by using novel fluorochrome gene-tagged viral genomes.</a> J Virol. 81 (9), 4591-4603 (2007).</li><li>Li, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+hepatitis+C+virus+lacking+the+envelope-encoding+genes+for+single-cycle+infection+by+providing+homologous+envelope+proteins+or+vesicular+stomatitis+virus+glycoproteins+in+trans.">Production of hepatitis C virus lacking the envelope-encoding genes for single-cycle infection by providing homologous envelope proteins or vesicular stomatitis virus glycoproteins in trans.</a> J Virol. 85 (5), 2138-2147 (2011).</li><li>Jones, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+C+virus+p7+and+NS2+proteins+are+essential+for+production+of+infectious+virus.">Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virus.</a> J Virol. 81 (16), 8374-8383 (2007).</li><li>Steinmann, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+C+virus+p7+protein+is+crucial+for+assembly+and+release+of+infectious+virions.">Hepatitis C virus p7 protein is crucial for assembly and release of infectious virions.</a> PLoS Pathog. (7), (2007).</li></ol>

The authors have nothing to disclose.

이 그림은 C 형 간염 바이러스의 복제 사이클을 분석하는 방법을 설명한다. HCV는 인간의 병원체이며, 소정의 바이오 안전성 의정서는 엄격하게 준수해야하는 것입니다. HCV 감염 세포 배양 시스템은 이전 11-13,16,17 설명되었다. 예시 된 프로토콜을 다음과 같은 때 우리가 구현하는 몇 가지 중요한 포인트가 있습니다. 첫째, 다운 스트림 연구에 그대로 전체 길이 바이러스 게놈 RNA의 좋은 품?...

C 형 간염 바이러스 (HCV)는 간경변과 간암의 원인이됩니다. 그것은 350,000의 사람들이 매년 1-3 죽어과 170 만명이 전 세계적으로 영향을 미칩니다. HCV는 9.6 KB의 게놈의 크기와 양의 가닥 RNA 바이러스이다. HCV 게놈은 단백질 가수 분해 10 폴리펩티드에 다양한 세포 및 바이러스 프로테아제에 의해 절단된다 ~ 3000 개의 아미노산의 단일 폴리 단백질로 번역된다. HCV는 속 Hepacivirus에 프로토 타입 바이러스와 가족 플라 비 바이러스과 4에 속한다. 노출되면, HCV는 개인의 80 %에 만성 감염을 설정합니다. 감염이 진단은 간 저하를 방지하기 위해 치료 적 개입을 허용 할 수 있습니다 대부분 증상이 적시에 있습니다. 현재 치료는 차선이고 백신은 5,6 사용할 수 없습니다. C 형 간염의 원인은 1989 7 설명했다. HCV 복제를 공부하는 것은 C 형 간염 백신과 치료 연구를위한 중요하지만이 있었다긴 효율적인 바이러스 배양 시스템의 부족에 의해 방해. HCV의 분자 클론은 간내 접종 8시 침팬지에 감염 될 것으로 나타났다. 이어서, HCV 서브 게놈 플리은 세포 배양 시스템 9,10에서 바이러스 게놈 복제 단계를 절개 할 수있는 기술되었다. 유전자형 2A의 HCV의 발견은 JFH-1 (일본어 전격 성 간염 1) 분리, 세포 배양을 감염시킬 수는 HCV의 복제 연구를 11 ~ 13를위한 새로운 길을 열었습니다. 유전자형 2A 변형 JFH-1 간 및 내 유전자형 키메라 바이러스와 유전자형 1 HCV 기반 기반 감염성 문화 시스템뿐만 아니라 14 ~ 18 사용할 수 있습니다. 우리는 성공적으로 단백질 도메인 및 시스 - 작용하는 RNA 요소 (19, 20)의 고해상도 기능 프로파일의지도를 구하는 것이 JFH-1 균주 및 HCV의 intragenotype의 2A 키메라 바이러스를 사용했습니다. 이 기사에 따르면, 우리가 할 수 일상적으로 사용하는 효과적인 문화 시스템을 설명HCV의 복제주기 및 호스트 병원체 상호 작용의 다양한 단계를 공부. 우리는 바이러스 게놈 복제 및 intragenotype 2a 개의 HCV와 레 닐라 루시퍼 기반 기자 HCV의 새로이 감염을 평가 한 바이러스 분석을 제시한다.

<table border="0" cellpadding="0" cellspacing="0" width="1121">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Name of Material/ Equipment</td>
			<td style="width:267px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td style="width:267px;">Fisher Scientific</td>
			<td style="width:119px;">10-017-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Non essential amino acid</td>
			<td style="width:267px;">Fisher Scientific</td>
			<td style="width:119px;">MT25025CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">HEPES</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">15630080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Glutamax</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Opti-MEM Reduced Serum Medium,no Phenol Red</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">11058-021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Huh-7.5.1</td>
			<td style="width:267px;">The Scripps Research Institute</td>
			<td style="width:119px;"></td>
			<td>The cell line was kindly provided by Dr. Francis Chisari to Dr. Arumugaswami under executed MTA between The Scripps Research Institute and Cedars-Sinai Medical Center</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:569px;">Plasmids (pFNX-HCV, pFNX-HCV Pol null, pFNX-Rluc, and pFNX-Rluc Pol null)&#160;</td>
			<td style="width:267px;">Cedars-Sinai Medical Center</td>
			<td style="width:119px;"></td>
			<td>The HCV plasmids were synthesized by Dr. Arumugaswami using overlapping oligo-nucleotides.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">XbaI</td>
			<td>New England Biolabs Inc.</td>
			<td style="width:119px;">R0145S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Mung Bean Nuclease</td>
			<td>New England Biolabs Inc.</td>
			<td style="width:119px;">M0250S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">T7 RiboMAX Express Large Scale RNA Production System</td>
			<td style="width:267px;">Promega</td>
			<td style="width:119px;">P1320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Rneasy Mini Kit</td>
			<td style="width:267px;">Qiagen</td>
			<td style="width:119px;">74104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Nanodrop 2000</td>
			<td style="width:267px;">Thermo Scientific</td>
			<td style="width:119px;">Nanodrop 2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Electroporation Cuvette (4 mm)</td>
			<td style="width:267px;">Bioexpress</td>
			<td>E-5010-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Gene Pulser Xcell Total System</td>
			<td style="width:267px;">Bio-Rad</td>
			<td style="width:119px;">165-2660</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse monoclonal anti-dsRNA antibody J2&#160;</td>
			<td>English &#38; Scientific Consulting Kft.</td>
			<td style="width:119px;">10010200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Goat anti-rabbit IgG Alexa Fluor 488</td>
			<td style="width:267px;">Life Technologies</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Goat anti-rabbit IgG Alexa Fluor 594</td>
			<td style="width:267px;">Life Technologies</td>
			<td>A11020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PVDF membrane package</td>
			<td style="width:267px;">Bio-Rad</td>
			<td style="width:119px;">162-0263</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Blotting Grade Blocker Non Fat Dry Milk</td>
			<td style="width:267px;">Bio-Rad</td>
			<td style="width:119px;">170-6404XTU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Tween-20</td>
			<td style="width:267px;">Bio-Rad</td>
			<td style="width:119px;">170-6531XTU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Hepatitis C Virus NS3 antibody [8 G-2]</td>
			<td style="width:267px;">Abcam</td>
			<td style="width:119px;">ab65407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Hepatitis C Virus NS3 antibody [H23]</td>
			<td style="width:267px;">Abcam</td>
			<td style="width:119px;">ab13830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-mouse IgG conjugated with horseradish peroxidase (HRP)&#160;</td>
			<td>Jackson ImmunoResearch Laboratories Inc.</td>
			<td style="width:119px;">115-035-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amersham ECL Prime Western Blotting Detection Reagents&#160;</td>
			<td style="width:267px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">RPN2236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">SUPERSCRIPT III RT&#160;</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">18080085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">SYBR QPCR SUPERMIX W/ROX</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">11744500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ViiA 7 real-time PCR system</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Renilla Luciferase Assay System kit</td>
			<td style="width:267px;">Promega</td>
			<td style="width:119px;">E2810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">RNase-Free DNase</td>
			<td style="width:267px;">Promega</td>
			<td style="width:119px;">M6101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">GloMax-Multi Detection System (Luminometer)</td>
			<td style="width:267px;">Promega</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol for analyzing hepatitis c virus replication

C 형 간염 바이러스 (HCV)는 세계 인구의 3 %에 영향을 미치는 만성 간염, 간경변 및 간암 등의 심각한 간 질환의 원인이됩니다. HCV는 가족 플라 비 바이러스과에 속하는 포락선 RNA 바이러스이다. 현재의 치료는 완전히 효과가없고, 부작용이 발생합니다. 가능한 HCV 백신이 없습니다. 따라서, 지속적인 노력은 백신과 더 나은 치료를 개발하기 위해 필요합니다. HCV 세포 배양 시스템은 바이러스 진입, 게놈 복제, 포장, 및 송신을 포함하여 HCV 성장의 각종 단계 연구에 중요하다. 제시된 현재 프로 시저에서, 우리는 야생형 intragenotype의 2A 키메라 바이러스, FNX-HCV, 그리고 바이러스 복제를 연구하는 레 닐라 루시퍼 라제 리포터 유전자를 운반하는 재조합 FNX-Rluc 바이러스를 사용했습니다. 인간 간암 세포주 (허-7을 기준으로)의 시험 관내 형질 도입에 사용 된은 HCV 게놈 RNA를 전사를. 무 세포 배양 상층 액, 단백질 해물과 t전 체 RNA는 HCV의 성장을 평가하는 사후 형질 포인트는 다양한 시간에 수확. HCV 게놈의 복제 상태 정량적 RT-PCR 및 HCV의 존재를 시각화하는 이중 가닥 RNA에 의해 평가 하였다. HCV 단백질 발현은 HCV NS3 및 NS5A 단백질에 대한 특이 항체를 이용하여 웨스턴 블롯 및 면역 형광 분석에 의해 확인되었다. 배양 상청 및 바이러스 역가에 감염성 입자를 방출 HCV의 RNA 형질 감염 세포를 측정 하였다. 루시페라아제 리포터 분석법은 HCV의 복제 수준 및 감염성을 평가하기 위해 사용되었다. 결론적으로, 우리는 HCV의 복제주기의 다른 단계를 특성화하기위한 다양한 바이러스 적 분석을 제시한다.

C 형 간염 바이러스는 RNA 바이러스이다. 따라서, 유전자 조작 목적, HCV 게놈 cDNA를 박테리아 플라스미드 벡터에 클로닝되었다. T7 RNA 폴리머 라제 프로모터 서열은 HCV 게놈의 5 &#39;말단 바로 전에 도입되었다. HCV 분석 워크 플로우에 대한 일반적인 개요를 그림 1에 제시되어있다. 정확한 3 &#39;끝 HCV 게놈 RNA를 생성하려면, 플라스미드를 포함하는 HCV 게놈은 XBA I 제한 효소 생성 된 ...

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A Protocol for Analyzing Hepatitis C Virus Replication

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 ...

a-protocol-for-analyzing-hepatitis-c-virus-replication

Research

JoVE Journal

Immunology and Infection

23.9K 조회수.  Cedars-Sinai Medical Center. 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. 

비디오: A Protocol for Analyzing Hepatitis C Virus Replication

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

사이 Synergism을 맡은 이후로 발광 생물 이미징을 사용하여 연쇄상 구균 pneumoniae</em유아 마우스에서>와 인플루엔자 바이러스

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

연구

면역학 및 감염병학

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

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

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 Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

헤르페스의 연구를위한 예비 신경 세포 문화 시스템은 바이러스 지연 및 재가입을 단순

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

표현형 높은 처리량 분석<em&gt; 살모넬라 엔테</em&gt; 티피 뮤 리움 협회, 침략, 그리고 대 식세포에서의 복제

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

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

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

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 Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

인플루엔자 바이러스 Hemagglutinins의 결합력 및 특이도를 평가하기위한 소형화 글리 칸 마이크로 어레이 분석

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

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.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

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), ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

새로운 세계 Zika 바이러스 전염성 클론으로부터 재조합 바이러스의 구조 및 특성 규명

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

"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.

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

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 ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

코딩 및 비 코딩 RNA 클래스의 식별 표시 돼지에서 전 혈

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...