Name: early viral entry assays for the identification and evaluation of antiviral compounds
Uploaded: 2015-10-29T14:00:00.000+00:00
Duration: 9 min 29 s
Description: Watch this Scientific Journal Video about Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds at JoVE.com

This study is supported by funding from Taipei Medical University Hospital (102TMU-TMUH-19) and the Ministry of Science and Technology of Taiwan (MOST103-2320-B-038-031-MY3).

참고 : 세포 배양 및 바이러스 감염과 관련된 모든 절차가 처리되는 샘플의 바이오 안전성 수준에 적합한 인증 바이오 안전성 후드에서 수행되었는지 확인합니다. 프로토콜을 설명하기 위해 사용 된 것으로, Gaussia 루시페라아제 리포터 - 태그 모델은 HCV 바이러스 (32)로서 사용된다. 대표적인 결과의 맥락에서, 화합물 chebulagic 산 (CHLA) 및 퓨니 칼라 (PUG)는 31 단계 초기 바이러스 진입 동안 세포 표면 글리코 사 미노 글리 칸 당 단백질과 바이러스의 상호 작용을 표적 후보 바이러스제로서 사용된다. 많은 바이러스 30,31,33,34의 진입을 방해하는 것으로 알려져 헤파린이 이러한 맥락에서 양성 대조군 처리로서 사용된다. 기본 바이러스학 기술에 대한 배경, 바이러스의 전파, 바이러스 역가를 결정하고, 플라크 형성 단위의 감염 량 (PFU)의 발현을 위해, 형성 단위 (FFU), 또는 감염 (MOI)의 다양성에 초점을, 독자는 다시는35를 참조 ferred. 종래 예 및 대표적인 결과에 도시 된 바이러스를 사용 최적화 조건, 리더를 표 1,도 1a 및도 2a에 나열된 참조 30-32,36-39뿐만 아니라 상세한 설명에 언급된다. 1. 세포 배양, 복합 준비, 복합 세포 독성 <ol><li> 바이러스 감염 시스템을위한 각 세포주의 성장은 (표 1)를 분석한다. HCV를 들면, 둘 베코 개질 된 이글 배지, 10 % 소 태아 혈청 (FBS)으로 보충 (DMEM), 200 U / ㎖ 페니실린 G, 200 μg의 / ㎖ 스트렙토 마이신에 응 - 7.5 세포 성장 및 0.5 μg의 / ml의 암포 테리 B. </li><li> 각각의 용매를 사용하여 시험 화합물을 제조 및 제어 예를 들어, CHLA 및 디메틸 설폭 시드에 퍼 (DMSO)에 용해; 멸균 두 번 증류수에 헤파린을 준비합니다. 모든 후속 희석을 위해, 문화 미디어를 사용합니다. 참고 : 최종 승낙을시험 화합물로 치료 DMSO의 비율은 실험에서 1 % 이하이고; 1 % DMSO는 비교 분석에서 음성 대조군 처리에 포함된다. </li><li> 이러한 XTT 같은 시약을 결정 세포 생존을 사용하여 바이러스 감염 세포에 대한 시험 화합물 (예를 들면, CHLA 및 PUG)의 세포 독성을 결정 (2,3- 비스 [2- 메 톡시 -4- 니트로 -5- 술포 페닐] -5- 페닐 아미노) 카르 보닐] -2H-수산화 테트라 졸륨) : <ol><li> HCV의 경우, 96 웰 플레이트에 응 - 7.5 세포 (웰 당 1 × 104 세포) 시드와 단층을 얻기 위해, 5 % CO 2 인큐베이터 O / N에서 37 ° C에서 배양한다. </li><li> 중으로의 배양 웰에 DMSO 제어 (1 %) 또는 시험 화합물 및 퍼 CHLA 증가 농도 (EX. 0, 10, 50, 100, 및 500 μM)을 적용한다. </li><li> 다음, 72 시간 동안 37 ° C에서 품어 접시에 매체를 버리고 두 번 인산염 완충 생리 식염수 200 μL (PBS)와 세포를 씻으십시오. </li><li> assayin 100 μl를 추가g 웰 각각 XTT 기반 관내 독성 분석 키트에서 용액 XTT 포르 마잔을 제조 할 수 있도록 또 다른 3 시간 동안 37 ℃에서 플레이트 배양한다. </li><li> 492 nm의 파장에서 테스트를 마이크로 플레이트 리더 및 690 nm의 기준 파장과 흡광도를 결정한다. </li><li> . 시험 화합물의 흡광도 및 용매 제어 참조 (예, 1 % DMSO &#39;로&#39;100 %, &#39;1&#39;× AS / 세포 생존율 (%) =로하고 다음 식을 이용하여 세포 생존 비율을 계산할 각각) 치료. 제조사의 프로토콜에 따라 이러한 그래프 패드 프리즘 소프트웨어로 분석에서 시험 화합물의 50 % 세포 독성 (CC 50)의 농도를 결정한다. </li></ol></li></ol> 바이러스 감염 2. 판독 참고 : 바이러스 감염의 판독이 사용 된 바이러스 체제에 따라 다릅니다 및 상패 분석​​ 또는 MEA 등의 방법을 포함 할 수있다기자 - 태그 바이러스에서 기자 신호를 가감하는. 루시퍼 라제 리포터 활동을 기반 리포터-HCV 감염을 검출하는 방법에 대하여 설명한다. <ol><li> 감염된 우물에서 상층 액을 수집하고 4 ℃에서 5 분 동안의 microcentrifuge에 17,000 XG에 명확히. </li><li> Gaussia 루시퍼 라제 분석 키트에서 루시페라아제 기질 50 ㎕의 상등액을 시험 20 μL를 혼합하고 직접적으로 제조자의 지시에 따라 조도계로 측정한다. </li><li> 제조 업체의 프로토콜에 따라 그래프 패드 프리즘 소프트웨어의 알고리즘을 사용하여 HCV 감염에 대한 시험 화합물의 50 % 유효 농도 (EC 50) 바이러스 억제 (%)을 결정하기위한 기준으로 빛 단위 (RLU)의 로그 10 HCV의 감염을 표현하고 계산한다. </li></ol> 3. 바이러스 불 활성화 분석 참고 : 여러 바이러스를 배양 기간의 예 바이러스 투여도 1a에 나열 재. 바이러스의 높은 농도는 MOI / PFU를 증가시킴으로써 테스트 될 수있다. <ol><li> 어 시드 - 7.5 96- 웰 플레이트에있는 세포 (웰 당 1 × 104 세포) 및 단층을 얻기 위해, 5 % CO 2 인큐베이터에서 37 ℃에서 O / N을 배양한다. </li><li> 시험 화합물 또는 컨트롤을 품어 (최종 농도는 다음과 같습니다 CHLA을 = 50 μm의; PUG = 50 μm의 헤파린 = 1,000 μg의 / ㎖, DMSO = 1 %) 37 ° C (그림 1A에서 HCV 입자와, &#39;장기&#39; ) 1 : 1의 비율. 예를 들어, 1 × 104를 함유 FFU 100 μL 바이러스 접종으로, 100 μM CHLA 작업 희석액 100 ㎕를 추가; 이것은 50 μM의 최종 농도 CHLA 처리를 산출한다. </li><li> 바이러스 약물 혼합물을 시험 화합물에 &quot;서브 치료&quot;(무효) 농도를 희석. 예를 들어, HCV에 대한 CHLA와 퍼그 효과 농도는 1 μm의 31에있다; 따라서이것은 (2 % FBS와 함께 세포 배양 배지) 기본 배지 9.8 ㎖로 수행 될 수 바이러스 약물의 혼합물을 50 배 희석을 요구한다. 주 : 서브 치료 농도에 희석 시험 화합물과 숙주 세포 표면 사이의 중요한 상호 작용을 방지하고, 무 세포 비리에 치료 효과를 조사 할 수있다. 이러한 희석은 특정 바이러스 감염에 대한 시험 화합물의 항 바이러스 투여 량 반응에 의존적이며, 특정 분석 (31)를 수행하기 전에 결정되는 것에주의. </li><li> 비교를 위해 시험 화합물과 바이러스를 혼합하고 즉시 서브 치료 농도 전에 감염 (도 1a &#39;단기&#39;)으로 (더 잠복기)를 희석하지 않는다. </li><li> 및 바이러스 있도록 37 ° C에서 3 시간 동안 배양; 응 - 7.5 세포 단층 상 희석 HCV 약물 혼합물 100 μl를 추가 (최종 MOI = 0.01 바이러스의 양이 1 × 2 FFU에서 지금)흡착. </li><li> 감염 후, 희석 접종을 제거하고 부드럽게 두 번 PBS 200 μL와 우물을 씻으십시오. 참고 : 세포를 해제하지 않도록 부드럽게 세척을 수행합니다. </li><li> 각 웰에 기초 배지 100 μl를 적용하고 72 시간 동안 37 ° C에서 품어. </li><li> 2 &#39;에 설명 된대로 루시 페라 제 활동에 대한 상층 액을 분석하여 그 결과 감염을 분석합니다. 바이러스 성 감염의 판독 &#39;. </li></ol> 4. 바이러스 첨부 파일 분석 참고 : 여러 바이러스를 배양 기간과 바이러스 투여의 예는,도 2a에 열거 된 &#39;첨부 파일&#39;됩니다. 바이러스의 높은 농도는 MOI / PFU를 증가시킴으로써 테스트 될 수있다. <ol><li> 어 시드 - 7.5 96- 웰 플레이트에있는 세포 (웰 당 1 × 104 세포) 및 단층을 얻기 위해, 5 % CO 2 인큐베이터에서 37 ℃에서 O / N을 배양한다. </li><li> 4 ° C FO에서 접시에 세포 단일 층을 사전 진정R 1 시간. </li><li> HCV 접종과 세포 (MOI가 0.01 =) 및 시험 화합물 또는 컨트롤 공동-치료 (최종 농도는 다음과 같습니다 CHLA을 = 50 μm의, DMSO = 1 % PUG = 50 μm의; 헤파린 = 1,000 μg의 / ㎖) 4 ° C에서 3 시간. 예를 들어, 1 × 2를 함유 FFU 90 μL 바이러스 접종으로, 500 μM CHLA 작업 희석액 10 μL를 추가; 이것은 CHLA 50 μM의 최종 농도로 처리하고 MOI에서 HCV에 의한 감염 = 0.01 세포 단층에를 산출한다. 참고 : 바인딩 바이러스 수 있지만 가장 효율적으로 37 ° C에서 발생하는 항목을 배제하기 때문에 그것은 4 ° C에서 실험을 수행하는 것이 중요합니다. 온도가 4 ℃로 유지되는 것을 보장하기 위해 얼음에 바이러스 및 시험 화합물의 추가와 4 ° C 형 냉장고에 계속되는 배양을 수행합니다. </li><li> 상층 액을 제거하고 부드럽게 두 번 얼음처럼 차가운 PBS 200 μL와 세포 단층을 씻는다. 참고 : 세포를 해제하지 않도록 부드럽게 세척을 수행 &lt;./ 리&gt; <li> 각 웰에 기초 배지 100 μl를 적용하고 72 시간 동안 37 ° C에서 품어. </li><li> 2 &#39;에 설명 된대로 루시 페라 제 활동에 대한 상층 액을 분석하여 그 결과 감염을 분석합니다. 바이러스 성 감염의 판독 &#39;. </li></ol> 5. 바이러스 성 항목 / 퓨전 분석 참고 : 배양 기간과 각종 바이러스에 대한 바이러스 투여의 예는 그림 2A &#39;입 / 퓨전&#39;에수록되어 있습니다. 바이러스의 높은 농도는 MOI / PFU를 증가시킴으로써 테스트 될 수있다. <ol><li> 어 시드 - 7.5 96- 웰 플레이트에있는 세포 (웰 당 1 × 104 세포) 및 단층을 얻기 위해, 5 % CO 2 인큐베이터에서 37 ℃에서 O / N을 배양한다. </li><li> 1 시간 동안 4 ℃에서 플레이트에서 세포 단층을 미리 차게. </li><li> 3 시간 동안 39 ° C에서 HCV와 셀 (MOI를 0.01 =) 감염. 예를 들어, 1 × 2 FFU 함유 100 μL 바이러스 접종 물을 사용한다. 참고 : ADDI를 수행얼음에 바이러스 접종 물과 4 ℃로 냉장고에 계속되는 인큐베이션 기 바이러스 결합이 아닌 엔트리를 허용하는 4 ℃에서 온도를 유지한다. </li><li> 상층 액을 제거하고 부드럽게 두 번 얼음처럼 차가운 PBS 200 μL와 세포 단일 층을 씻는다. 참고 : 세포를 해제하지 않도록 부드럽게 세척을 수행합니다. </li><li> (최종 농도는 다음과 같습니다; PUG = 50 μm의; 헤파린 = 1,000 μg의 / ㎖, CHLA = 50 μm의 DMSO = 1 %) 시험 화합물 또는 컨트롤 우물을 치료하고 3 시간 동안 37 ° C에서 품어. 예를 들어, 미디어의 90 μL에 500 μm의 CHLA 작업 희석의 10 μl를 추가 혼합하고, 우물을 치료; 이것은 50 μM의 최종 농도 CHLA 처리를 산출한다. 주 : 37 ° C에서 42 ° C에서 변화는 이제 바이러스 성 항목 / 퓨전 이벤트를 용이하게하고, 따라서이 특정 단계에 시험 화합물 &#39;효과의 평가를 할 수 있습니다. </li><li> 약물 함유 뜨는을 기음과 비 내면화 제거시트르산 완충액 (50 mM의 시트르산 나트륨, 4 mM의 염화칼륨, pH를 3.0) 또는 PBS 200 ㎕를 가진 하나 세척하여 세포 외 바이러스. 72 시간 동안 37 ℃에서 배양하기 전에 기초 배지 100 μl를 적용합니다. </li><li> 2 &#39;에 설명 된대로 루시 페라 제 활동에 대한 상층 액을 분석하여 그 결과 감염을 분석합니다. 바이러스 성 감염의 판독 &#39;. </li></ol>

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The authors declare that no competing interests exist.

In this report the methods to identify and evaluate antiviral compounds based on a mechanistic approach of dissecting the early viral entry events were described. Specifically, the assays allowed us to examine the effect of test compounds on free virus particles, viral attachment, and viral entry/fusion. Critical steps were implemented to distinctly evaluate the drug effect on the specific stage of early viral entry. For instance, in the &#8216;viral inactivation assay&#8217;, the dilution of the virus-drug mixture to sub-therapeutic concentration prevents significant interaction between the test compound and the host cell surface by &#8216;titrating out&#8217; the drug. This ensures that the inhibitory effect observed on the subsequent infection of the host cell is due to a direct interaction between the test compound and the cell-free virions, rather than an effect from the test compound on host cell membrane or membrane-associated molecules, including viral receptors30. Similarly, the shift in temperature between 4 &#176;C (which allows for virus binding but not entry) and 37 &#176;C (which facilitates virus entry/fusion) in the &#8216;viral attachment assay&#8217; and &#8216;viral entry/fusion assay&#8217; are crucial to determine the test compound&#8217;s effect on each of these specific events. This is feasible due to the temperature sensitivity of enveloped viruses during these steps in the infection24-29. It is therefore important that the assays are performed at the indicated temperature to ensure the accuracy of the results; for example, by carrying out the experiment on ice to maintain at 4 &#176;C and by placing the sample directly in a 37 &#176;C incubator for the temperature shift. In addition, the use of negative (ex. DMSO solvent for drug preparation) and positive (ex. heparin treatment) controls also help further establish the assays&#8217; accuracy. The utility and applicability of such methods have been demonstrated in many antiviral studies26,30,31,40,41. Note that while heparin is included as a control for all three assays in the context of the representative results, it typically blocks the initial virus binding rather than the ensuing fusion/entry step (as reflected by the data in Figure 2). Additional controls could also be used, such as neutralizing antibodies directed against the virus (for viral inactivation assay), antibodies that mask the cell surface receptors for the virus (for viral attachment assay), and membrane fusion inhibitors (for viral fusion/entry assay). 
The assays described in this report, which are specific to the early stages of the viral infection, are useful in terms of application as secondary tests to characterize the mechanism of action of candidate drugs from primary screens which typically target the viral infection more broadly. Alternatively, they could also be incorporated in primary screens if one is specifically looking for inhibitors of early viral entry, including virus inactivating agents, viral attachment antagonists, and inhibitors to viral entry/fusion. In this case, their use allows a more focused and precise screen analysis for the identification of mechanism-specific antiviral candidates, which, in turn, would expedite downstream drug development. 
The use of cell-based assays in identifying antiviral agents provides several important advantages compared to biochemical assays, including revealing potential off-target effects (such as cytotoxicity) and adding physiological relevance to the bioactivity of the test agents42. These issues are important considerations for deciding whether a candidate agent is of value for continuation in subsequent phases of drug development. Similarly, the early viral entry-specific assays described in this report allow examination of the drug effect on the distinct viral entry stage at the cellular level, and more specifically in the context of an authentic viral infection in vitro. The results obtained from such assays would therefore help better predict the antiviral efficacy of the test compounds and also identify potentially unwanted off-target effects against the host cell. One potential limitation though, is that an in vitro cell-based assay may not completely reflect the actual in vivo entry step in the context of a natural viral infection. Nonetheless, the assays presented in this protocol do serve as an analytical platform for mechanism-based identification and evaluation of novel antiviral agents.
The development of reporter viruses or reporter cell systems to quantitate the amount of viral infection has greatly facilitated cell-based screening and evaluation of antiviral compounds. Examples include the use of recombinant viruses carrying a reporter gene or by means of recombinant human cell lines containing a reporter gene driven by the specific virus promoter31,43. In this report, the infection from luciferase-tagged HCV can be easily monitored by quantitating the reporter signal, thus facilitating data analysis. By incorporating these useful reporter-based tools, the early viral entry assays described here can essentially be adapted into high-throughput format for mechanism-based screening of small molecule libraries.
In conclusion, a protocol was described for assays dissecting the early viral entry as a means of identifying and evaluating mechanism-specific antiviral compounds. Such assays would be useful for discovering novel antagonists/inhibitors to viral entry and help expand the scope of antiviral agents for development as prophylactic and/or therapeutic treatments.

Viral infections are a constant threat to the public health and a significant cause of epidemic diseases, morbidity, and deaths worldwide. Specific modes of control against viral infections include vaccine development and antiviral therapies. While vaccine efforts have proven successful in immunizing against several viruses, many viral pathogens remain without a protective vaccine including dengue virus (DENV), human cytomegalovirus (HCMV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and respiratory syncytial virus (RSV)1-5. Antivirals, on the other hand, play an important role for the management of these viral infections when prophylactic vaccines are unavailable. However, to date, only few licensed and cost-effective antiviral drugs are available compared to the number of viral pathogens that threatens the public health. More importantly, due to an increase in global travel and rapid urbanization, the situation is aggravated by risks of epidemic outbreaks from emerging/re-emerging viral infections that are being introduced into non-indigenous areas6. Recent outbreaks caused by severe acute respiratory syndrome (SARS) virus, influenza viruses (H1N1, H5N1, H3N2, and H7N9), DENV, West Nile virus (WNV), measles virus (MV), Middle East Respiratory Syndrome (MERS) virus, and Ebola virus6-12 are among the examples reflecting the need for antivirals development when immunization and/or therapeutics are unavailable. In addition, there is always a potential risk of generating drug-resistant infections with currently used antivirals. Thus, the continuous development and expansion of the scope of antivirals to these emerging/re-emerging viral infections are necessary to provide better management strategies and safeguard the public health.
Most antiviral therapies consist of direct acting antivirals (DAAs) which target a specific viral protein or cofactor that mediates important steps in the viral life cycle. For example, the nucleoside analogue Acyclovir inhibits herpesvirus DNA polymerase, protease inhibitors Boceprevir and Telaprevir antagonize the HCV NS3, and Oseltamivir and Zanamivir are neuraminidase inhibitors that block the release of influenza virus particles from infected cells13-15. There are however very few licensed viral entry inhibitors including Enfuvirtide, which targets HIV gp41 to prevent fusion, and Maraviroc, which blocks the HIV co-receptor CCR5, thereby preventing viral entry16. Exploring novel antagonists/inhibitors to viral entry could help provide additional therapeutics for prophylactic or therapeutic use, such as in combination with other antivirals with a different mechanism of action to better manage viral infections17-19. 
Identification of antivirals can involve structure-guided drug design and candidate drug screening-based strategy. Methods for assessing antiviral activity of test agents include biochemical assays of enzymatic activity and evaluation by cell-based systems20-23. In cell-based systems, the viral life cycle can be dissected into distinct stages of infection, such as entry events (attachment, fusion, uncoating), the replication phase (viral genome replication and protein translation), and virion egress (assembly, maturation, and release). Since the assays can be adapted to investigate each specific stage using various tools and methods, this approach allows identification/examination of potential candidate antivirals with a specific mechanism of action targeting the distinct stage analyzed. For instance, to analyze drug effect specifically on the free virus particles prior to binding to the host cell, a &#8216;viral inactivation assay&#8217; can be performed. In this assay, the virus is allowed to incubate with the test drug and then diluted to titrate out the drug before infecting a cell monolayer. Additional steps such as viral attachment and entry/fusion stages can also be analyzed individually, by shifting the temperature during the infection. For many enveloped viruses, viral entry/fusion at the host cell membrane is greatly facilitated at 37 &#176;C, but is typically precluded at 4 &#176;C which does not affect virus binding24-29. Finally, the use of reporter viruses or cell systems could facilitate these studies and permit a high-throughput analysis.
We previously employed the cell-based approach and dissected the early entry of various enveloped viruses for the examination of antiviral compounds that potentially antagonize viral entry30,31. Herein, the various methods used, including viral inactivation, viral attachment, and viral entry/fusion assays, are described.

<table><tbody><tr><td>DMEM</td><td>GIBCO</td><td>11995-040</td><td></td></tr><tr><td>FBS</td><td>GIBCO</td><td>26140-079</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>GIBCO</td><td>15070-063</td><td></td></tr><tr><td>Amphotericin B</td><td>GIBCO</td><td>15290-018</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D5879</td><td></td></tr><tr><td>In vitro toxicology assay kit, XTT-based</td><td>Sigma</td><td>TOX2</td><td></td></tr><tr><td>PBS pH 7.4&amp;nbsp;</td><td>GIBCO</td><td>10010023</td><td></td></tr><tr><td>Microplate reader</td><td>Thermo Scientific</td><td>89087-320</td><td></td></tr><tr><td>Microcentrifuge</td><td>Thermo Scientific</td><td>75002420</td><td></td></tr><tr><td>BioLux Gaussia luciferase assay kit</td><td>New England Biolabs</td><td>E3300L&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Luminometer</td><td>Promega</td><td>GloMax-20/20</td><td></td></tr><tr><td>Sodium citrate, dihydrate</td><td>Sigma</td><td>71402</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>P5405</td><td></td></tr></tbody></table>

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.

도 1에서, &#39;바이러스 불 활성화 분석은&#39;개의 특정의 천연 화합물이 PUG CHLA 및 무 세포 상태의 다른 외피 바이러스를 불활 화 및 후속 감염을 예방할 수 있는지 여부를 조사하기 위해 수행 하였다. 이들 화합물의 세포 독성 및 항 바이러스 투여 량 반응은 역학적 연구 (31)를 수행하기 전에 결정되어왔다. 바이러스는 시험 화합물로 전처리 한 후, 바이러스 - 약물 혼합물은 각 바이러스 시스템에 대한 각각의 세포 단층 상에 접종하기 전에 서브 치료 농도로 희석 하였다. 도 1에 도시 된 바와 같이, CHLA 및 PUG 모두 후속 감염으로부터 세포를 보호 단층 비가역 효과의 결과로, 무 세포와 상호 작용하는 비리 나타났다. 80 % 블록 MV 및 RSV에 대해 관찰 하였다 - 60 반면 두 시험 화합물, HCMV, HCV, 및 DENV-2에 대해 100 % 가까이 억제를 달성했다. 이러한 결과 suggEST CHLA와 퍼그을 비활성화하고 자신의 감염을 중화하여 이러한 무료 바이러스 입자에 직접 영향을 미칠 수있다. 그림 2에서, 첨부 파일 및 항목 / 융합 분석은 HCMV, HCV, DENV-2, MV, 그리고 RSV에서 이러한 초기의 바이러스 성 항목 관련 이벤트에 대해 CHLA와 퍼그 효과를 탐험하기 위해 수행되었다. CHLA 및 PUG 모두 효과적으로 얻어진 바이러스 감염 (: 밝은 회색 막대도 2 &#39;부착&#39;)에 의해 도시 된 바와 같이 저해 각 숙주 세포 상에 조사 바이러스의 결합을 억제. 두 화합물에 의한 바이러스 부착에 대한 억제 효과는 HCMV (도 2B), HCV (도 2c)에 대해 유사 하였다, DENV-2 (도 2D), 및 (90)에 이르기까지 RSV (도 2F), - 100 %. 한편, 퍼그 TW에서 저해율와 결합 MV (도 2e) CHLA 대해보다 효과적인 것으로 나타났다50-80% 사이 변화 O 화합물. 많은 바이러스의 침입, HCMV의 부착도 억제, DENV-2, RSV, MV 광고를 차단하는 것으로 알려져 있지만, HCV에 대해 덜 효율적이었다되는 제어 처리 헤파린. 계속되는 &#39;바이러스 성 항목 / 융합 분석은&#39;CHLA와 PUG 바이러스 항목 / 융합 단계에서 그들의 활동을 유지할지 여부를 검사 (그림 2, &#39;입 / 퓨전&#39;: 어두운 회색 바). 각각의 세포 단층에 90 % 보호 효과 - (50)를 산출, - 다시, CHLA과 PUG 모두 효과적으로 검사 바이러스 (F 그림 2B)의 바이러스 성 항목 / 융합 단계를 저해하는 것으로 관찰되었다. 헤파린은 강력하게 억제 항목 / DENV-2와 RSV 감염의 융합,하지만 HCMV, HCV, 및 MV (&lt;평균 40 % 억제)에 대한 덜 효과​​적이었다. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 바이러스 </td><td> 세포의 종류 </td></tr><tr><td> HCMV </td> &lt;TD&gt; HEL </td></tr><tr><td> HCV </td><td> 허 - 7.5 </td></tr><tr><td> DENV-2 </td><td> 베로 </td></tr><tr><td> MV </td><td> CHO-SLAM </td></tr><tr><td> RSV </td><td> HEP-2 </td></tr></tbody></table> 표 1. 바이러스 감염에 대한 숙주 세포 유형은 대표적인 결과에 기재된 각 시스템 바이러스 감염에 사용되는 세포 유형이 표시된다. 세포에 대한 추가 사항은 기준 (31)에서 찾을 수있다. <img alt="그림 1" src="/files/ftp_upload/53124/53124fig1.jpg" /> 그림 1. 시험 화합물 CHLA 및 PUG 의한 바이러스 감염의 불 활성화는 다른 바이러스가 장기간 동안 시험 화합물로 처리 하였다. (1.5 인큐베이션 - 적정 전에 3 시간; 밝은 회색 막대) 또는 단기간 (즉시 희석 어두운 회색 서브 치료 집중하고에 희석 전에 37 ° C에서 바)기 및 각각의 숙주 세포에 감염의 후속 분석. 최종 바이러스 농도 (왼쪽 그림) 실험 (A) 회로도 (PFU / 웰 또는 MOI), 장기 바이러스 약물 잠복기 (I) 및 (ii) 이후의 배양 시간은 오른쪽에 테이블의 각 바이러스에 대한 지시. (B) HCMV, (C) HCV에 대한 분석은, (D) DENV-2 (E) MV, 및 (F) RSV 각 추가 패널에 표시됩니다. 결과는 3 개의 독립적 인 실험에서 평균 (SEM)의 표준 오차 ± 수단은 바이러스 감염에 대한 DMSO 음성 대조군 처리 및 표시되는 데이터에 대해입니다 그려. 이 그림은 참조 (31)로부터 수정되었습니다. <a href="https://www.jove.com/files/ftp_upload/53124/53124fig1highres.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/53124/53124fig2.jpg" /> 도 2 바이러스 부착 및 입 / 융합에 대해 시험 화합물의 CHLA 및 퍼 바이러스 활동의 평가. (A) 실험 절차, 바이러스 농도 (PFU / 웰 또는 MOI) 및 시험 화합물과 함께 첨가하고 처리 시간 (난, II, III) 회로도 및 관련 테이블의 각 바이러스되게됩니다. 바이러스 부착 분석 (밝은 회색 막대)에서, 상이한 유형의 세포의 단층을 1 시간 동안 4 ℃에서 사전 냉각 가한 다음 4 ℃에서 각각의 바이러스 및 시험 화합물과 함께 공동 - 치료 (1.5-3 시간; I) 후속 인큐베이션 inoculates 및 시험 화합물을 씻어 전에 (37 ° C, II) 및 바이러스 감염의 진단. 바이러스 진입 / 융합 분석 (어두운 회색 막대)에서, 시딩 세포 단층을 1 시간 동안 4 ℃에서 사전 냉각 후 1.5이었다 4 ℃에서 각각의 바이러스 도전 - 3 시간 (I). 세포는 그 다음이었다세정 온도가 바이러스 진입 / 융합 이벤트를 용이하게하기 위해 37 ° C로 시프트하고 (ⅱ) 동안 추가 배양 기간 동안 시험 화합물로 처리 하였다. 인큐베이션의 끝에, 세포 외 바이러스는 어느 시트르산 완충액 (pH 3.0) 또는 PBS 세척에 의해 제거하고, 세포는 또한 바이러스 감염 분석 (III)을 배양 하였다. (B) HCMV, (C) HCV에 대한 결과, (D) DENV-2 (E) MV, 및 (F) RSV 각 추가 패널에 표시됩니다. 데이터는 바이러스 감염의 DMSO 음성 대조군의 치료에 대해 플롯과 3 개의 독립적 인 실험 ± SEM 의미로 표현됩니다. 이 그림은 참조 (31)로부터 수정되었습니다. <a href="https://www.jove.com/files/ftp_upload/53124/53124fig2highres.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds at JoVE.com

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

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 ...

early-viral-entry-assays-for-identification-evaluation-antiviral

Research

JoVE Journal

Immunology and Infection

30.1K 조회수.  Taipei Medical University Hospital. Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

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

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

모든 독감의 양적 분석 간단한 슬롯 얼룩 Assays에서 유니버설 항체를 사용하는 바이러스성 Hemagglutinins 및 Neuraminidases를 입력하십시오

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

연구

면역학 및 감염병학

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

Bluetongue 바이러스에 대한 항 바이러스제 소설의 식별에 대한 분석

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA 바이러스의 넓은 스펙트럼 화학 억제를위한 높은 처리량 검사

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

HIV 역전사 및 인테그라 제 억제제의 빠른 검사

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

Arenavirus 셀 첨부 파일의 측정을위한 고감도 분석

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral ...

Use of Viral Entry Assays and Molecular Docking Analysis for the Identification of Antiviral Candidates against Coxsackievirus A16

Antiviral assays that mechanistically examine viral entry are pertinent to discern at which step the evaluated agents are most effective, and allow for the identification of candidate viral entry inhibitors. Here, we present the experimental approaches for the identification of small molecules capable of blocking infection by the non-enveloped coxsackievirus A16 (CVA16) through targeting the virus particles or specific steps in early viral entry. Assays include the time-of-drug-addition analysis, flow cytometry-based viral binding assay, and viral inactivation assay. We also present a molecular docking protocol utilizing virus capsid proteins to predict potential residues targeted by the antiviral compounds. These assays should help in the identification of candidate antiviral agents that act on viral entry. Future directions can explore these possible inhibitors for further drug development.

The goal of the protocol is to illustrate the different assays relating to viral entry that can be used to identify candidate viral entry inhibitors.

Coxsackievirus A16에 대하여 항바이러스 후보의 확인을 위한 바이러스 성 입력 분석 및 분자 도킹 분석의 사용

Antiviral assays that mechanistically examine viral entry are pertinent to discern at which step the evaluated agents are most effective, and allow for ...

High-throughput Antiviral Assays to Screen for Inhibitors of Zika Virus Replication

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) formats. The flavivirus non-structural (NS) proteins are thought to co-translationally assemble on the endoplasmic reticulum (ER) membranes, forming the replication complex (RC). The NS3 and NS5 are the most studied enzymes of the RC and constitute the main targets for drug development due to their crucial roles in viral genome replication. NS3 protease domain, which requires NS2B as its cofactor, is responsible for the cleavage of the immature viral polyprotein into the mature NS proteins, whereas NS5 RdRp domain is responsible for the RNA replication. Herein, we describe in detail the protocols used in replicon-based screenings and enzymatic assays to test large compound libraries for inhibitors of the Zika virus (ZIKV) replication. Replicons are self-replicating subgenomic systems expressed in mammalian cells, in which the viral structural genes are replaced by a reporter gene. The inhibitory effects of compounds on viral RNA replication can be easily evaluated by measuring the reduction in the reporter protein activity. The replicon-based screenings were performed using a BHK-21 ZIKV replicon cell line expressing Renilla luciferase as a reporter gene. To characterize the specific targets of identified compounds, we established in-vitro fluorescence-based assays for recombinantly expressed NS3 protease and NS5 RdRp. The proteolytic activity of the viral protease was measured by using the fluorogenic peptide substrate Bz-nKRR-AMC, while the NS5 RdRp elongation activity was directly detected by the increase of the fluorescent signal of SYBR Green I during RNA elongation, using the synthetic biotinylated self-priming template 3&#8242;UTR-U30 (5&#39;-biotin-U30-ACUGGAGAUCGAUCUCCAGU-3&#39;).

In this work, we describe the protocols used in replicon-based and viral enzyme-based assays to screen for inhibitors of Zika virus replication in a high-throughput screening format.

지카 바이러스 복제억제제에 대한 화면에 높은 처리량 항 바이러스 적 소사

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) ...

Assessing the Effectiveness of an Antiviral Test Compound through a Viral Inactivation Assay

Source: Tai, C. et al., <a href="https://app.jove.com/v/53124">Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds.</a> J. Vis. Exp. (2015)
This video demonstrates the viral inactivation assay, showcasing the evaluation of the impact of antiviral agents on viral particles.

바이러스 불활성화 분석을 통한 항바이러스 테스트 화합물의 효과 평가

1. Viral Inactivation Assay
Note: Examples of incubation period and viral dose for various viruses are listed in Figure 1A. Higher concentrations of the ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

A Viral Attachment Assay for Screening of Antiviral Compounds

Source: Tai, C. et al., <a href="https://app.jove.com/v/53124">Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds.</a> J. Vis. Exp.&#160;(2015)
This video demonstrates a viral attachment assay for screening antiviral compounds. Using genetically modified hepatitis C virus and luciferase, it assesses inhibitory effects on viral attachment, as indicated by lower light intensity in co-treated wells compared to wells treated with the virus alone.

항바이러스 화합물 스크리닝을 위한 바이러스 부착 분석

NOTE: Ensure that all procedures involving cell culture and virus infection are conducted in certified biosafety hoods that are appropriate for the ...

An Assay to Evaluate the Effect of Test Compounds on Viral Entry and Fusion in Host Cells

Source: Tai, C., et al. <a href="https://app.jove.com/v/53124">Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds.</a> J. Vis. Exp. (2015).
This video demonstrates an assay to evaluate antiviral agents that target specific steps of the viral entry into host cells and fusion with the endosome. The cells are incubated at a low temperature and then infected with HCV. The low temperature permits viral binding to host cells but inhibits internalization via endocytosis. The test antiviral compound is added, and the cells are incubated at physiological temperature. This shift to a higher temperature facilitates viral entry and allows assessment of the impact of the test compound on viral envelope fusion with endosomes.

Note: Ensure that all procedures involving cell culture and virus infection are conducted in certified biosafety hoods that are appropriate for the ...