JoVE Journal > Immunology and Infection
Summary
Abstract
Introduction
Protocol
Resultados
Discussion
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Acknowledgements
Materials
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Inmunología y contagio

Tidlig Virale Adgangskrav Analyser for identifikation og evaluering af antivirale forbindelser

Published: October 29, 2015
doi:
10.3791/53124
, , , ,
1Department of Chinese Medicine,Taipei Medical University Hospital, 2Department of Obstetrics and Gynecology, School of Medicine, College of Medicine,Taipei Medical University, 3Department of Microbiology and Immunology, School of Medicine, College of Medicine,Taipei Medical University, 4Division of Hematology and Oncology, Department of Internal Medicine,Taipei Medical University Hospital, 5Department of Internal Medicine, School of Medicine, College of Medicine,Taipei Medical University, 6Graduate Institute of Medical Sciences, College of Medicine,Taipei Medical University

Summary

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

Abstract

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.

Introduction

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 ‘viral inactivation assay’ 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 °C, but is typically precluded at 4 °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.

Protocol

Bemærk: Sørg for, at alle procedurer, der involverer cellekultur og virusinfektion foregår på certificerede biosikkerhed emhætter, der er passende for biosikkerhed niveau af prøverne, der håndteres. Med henblik på at beskrive de protokoller, der er Gaussia luciferase reporter-mærket HCV bruges som model virus 32. I forbindelse med de repræsentative resultater, er forbindelserne chebulagic syre (chlA) og punicalagin (PUG), der anvendes som kandidat antivirale, der er målrettet virale glycoprotein interaktioner med celleoverflade glycosaminoglykaner under den tidlige virale post trin 31. Heparin, der vides at interferere med optagelse af mange vira 30,31,33,34, anvendes som en positiv kontrol behandling denne sammenhæng. For grundlæggende baggrund på virologiske teknikker, spredning af virus, bestemmelse af virustiter og ekspression af infektiøs dosis i plakdannende enheder (PFU), fokus-dannende enheder (FFU) eller infektionsmultiplicitet (MOI), læseren er reudskudt til at henvise 35. For tidligere eksempler og optimerede betingelser anvendt til virus vist i de repræsentative resultater, henvises læseren til referencer 30-32,36-39 samt oplysninger, der er anført i tabel 1, figur 1A og 2A. 1. Cell Culture, Compound Forberedelse og forbindelse cytotoksicitet Grow den respektive cellelinje for virusinfektion, der skal analyseres (tabel 1). For HCV, dyrke Huh-7,5-celler i Dulbeccos modificerede Eagles medium (DMEM) suppleret med 10% føtalt bovint serum (FBS), 200 U / ml penicillin G, 200 ug / ml streptomycin, og 0,5 ug / ml amphotericin B. Forbered testforbindelserne og kontrol ved hjælp af deres respektive opløsningsmidler: f.eks opløses chlA og PUG i dimethylsulfoxid (DMSO); forberede heparin i sterilt dobbeltdestilleret vand. For alle efterfølgende fortyndinger, brug kulturmedier. Bemærk: Den sidste concentration af DMSO i test sammensatte behandlinger er mindre end 1% i forsøgene; 1% DMSO er inkluderet som en negativ kontrol behandling i de assays til sammenligning. Bestem cytotoksicitet af testforbindelserne (f.eks chlA og Mops) på cellerne for virusinfektion ved hjælp af en bestemmelse af cellelevedygtighed reagens, såsom XTT (2,3-bis [2-methoxy-4-nitro-5-sulfophenyl] -5-phenylamino) carbonyl] -2H-tetrazolium hydroxide): For HCV, frø Huh-7,5-celler i en 96-brønds plade (1 x 10 4 celler pr brønd) og inkuberes ved 37 ° C i en 5% CO2-inkubator O / N for at opnå et monolag. Anvend DMSO kontrol (1%) eller stigende koncentrationer af testforbindelserne Chla og PUG (ex. 0, 10, 50, 100 og 500 uM) til dyrkningsbrøndene i tre eksemplarer. Der inkuberes ved 37 ° C i 72 timer, derefter kasseres mediet i pladen og vaske cellerne med 200 pi phosphatpufret saltvand (PBS) to gange. Tilføj 100 ul assaying opløsning fra XTT-baserede in vitro toksikologi analysekit til hver brønd og inkuberes pladerne ved 37 ° C i yderligere 3 timer for at tillade XTT formazanproduktion. Bestem absorbansen med en mikropladelæser ved en test på 492 nm og en referencebølgelængde på 690 nm. Beregn procentdelen af ​​overlevende celler ved hjælp af følgende formel: cellernes levedygtighed (%) = På / Som × 100%, hvor 'På "og" As' refererer til absorbansen af ​​forsøgsforbindelserne og opløsningsmidlet kontrol (tidl 1% DMSO. ) behandlinger, henholdsvis. Bestemme koncentrationen af 50% cellulær cytotoksicitet (CC 50) af testforbindelserne fra en analytisk software som GraphPad Prism ifølge producentens protokol. 2. Udlæsning af viral infektion Bemærk: udlæsning af virusinfektion afhænger af virusset, der anvendes, og kan involvere metoder såsom plakanalyser eller measuring reporter signaler fra reporter-mærkede virus. Metoden til påvisning af reporter-HCV-infektion baseret på luciferasereporteren aktivitet er beskrevet nedenfor. Saml supernatanterne fra de inficerede brønde og præcisere ved 17.000 xg i en mikrocentrifuge i 5 minutter ved 4 ° C. Bland 20 pi test supernatant til 50 pi luciferasesubstratet fra Gaussia luciferaseassaykit og gælder måle med et luminometer i overensstemmelse med producentens anvisninger. Hurtig HCV infektivitet som log 10 i relative lysenheder (RLU) for at bestemme viral inhibering (%) og beregne den 50% effektive koncentration (EC50) af testforbindelserne mod HCV-infektion ved hjælp af algoritmer fra GraphPad Prism software i overensstemmelse med producentens protokol. 3. virusinaktivering Assay Bemærk: Eksempler på inkubationstiden og viral dosis for forskellige vira enre angivet i figur 1A. Højere koncentrationer af virus kan også testes ved at øge MOI / PFU. Seed Huh-7,5-celler i en 96-brønds plade (1 x 10 4 celler pr brønd) og inkuberes ved 37 ° C i en 5% CO2-inkubator O / N for at opnå et monolag. Inkubér testforbindelserne eller kontroller (endelige koncentrationer er: Chla = 50 uM, PUG = 50 uM, heparin = 1.000 pg / ml; DMSO = 1%) med HCV-partikler ved 37 ° C (figur 1A, 'langsigtede' ) i en 1: 1-forhold. For eksempel kan en 100 pi virusinokulum indeholdende 1 x 10 4 FFU, tilsættes 100 pi af en 100 uM chlA brugsfortynding; dette giver Chla behandling ved en slutkoncentration på 50 uM. Fortynd virus-medikamentblandingen til "sub-terapeutisk" (ineffektive) koncentration af testforbindelserne. For eksempel den ineffektive koncentration af Chla og PUG mod HCV er 1 uM 31; derfordette kræver en 50-ganges fortynding af virus-drug blanding, som kan opnås med 9,8 ml basalt medium (celledyrkning medium med 2% FBS). Bemærk: Den fortynding til sub-terapeutisk koncentration forhindrer signifikant interaktion mellem testforbindelserne og værtscellen overflade og tillader undersøgelse af behandlingseffekt på cellefri virioner. Bemærk, at denne fortynding er afhængig af antivirale dosisrespons af testforbindelserne mod den særlige virusinfektion, og bestemmes før udførelse af denne særlige assay 31. Til sammenligning, bland virus med testforbindelserne og straks fortyndes (ingen inkubationstiden) til sub-terapeutisk koncentration forud for infektion (figur 1A, "Short-Term"). Tilsættes 100 pi af fortyndet HCV-drug blandingen på Huh-7,5 cellemonolaget (mængden af virus er nu på 1 x 10 2 FFU, endelige MOI = 0,01) og inkuberes i 3 timer ved 37 ° C for at tillade viraladsorption. Efter infektion fjernes den fortyndede podestoffer og forsigtigt vaske brøndene med 200 pi PBS to gange. Bemærk: Udfør vasker forsigtigt for at undgå at løfte cellerne. Påfør 100 pi basalt medium til hver brønd og inkuberes ved 37 ° C i 72 timer. Analyser af den resulterende infektion ved at analysere supernatanten for luciferaseaktivitet som beskrevet i "2. Udlæsning af viral infektion. 4. Viral Attachment Assay Bemærk: Eksempler på inkubationstid og viral dosis for forskellige vira er anført i figur 2A, "Attachment '. Højere koncentrationer af virus kan også testes ved at øge MOI / PFU. Seed Huh-7,5-celler i en 96-brønds plade (1 x 10 4 celler pr brønd) og inkuberes ved 37 ° C i en 5% CO2-inkubator O / N for at opnå et monolag. Pre-chill cellemonolagene i pladerne ved 4 ° C foR 1 time. Co-behandling af cellerne med HCV inoculum (MOI = 0,01) og testforbindelser eller kontrol (slutkoncentrationer er: chlA = 50 uM; PUG = 50 uM; heparin = 1.000 pg / ml; DMSO = 1%) ved 4 ° C for 3 timer. For eksempel kan en 90 pi virusinokulum indeholdende 1 x 10 2 FFU tilsættes 10 pi af en 500 uM chlA brugsfortynding; dette giver Chla behandling ved en slutkoncentration på 50 uM og en infektion med HCV ved MOI = 0,01 på cellemonolaget. Bemærk: Det er vigtigt at udføre forsøget ved 4 ° C, da det giver mulighed for virusbinding men hinder post, som mest effektivt finder sted ved 37 ° C. Udfør tilsætning af virus og testforbindelser på is og efterfølgende inkubation i en 4 ° C køleskab for at sikre, at temperaturen holdes på 4 ° C. Fjern supernatanten og forsigtigt vaske cellemonolaget med 200 pi iskold PBS to gange. Bemærk: Udfør vasker forsigtigt for at undgå at løfte cellerne <./ li> Påfør 100 pi basalt medium til hver brønd og inkuberes ved 37 ° C i 72 timer. Analyser af den resulterende infektion ved at analysere supernatanten for luciferaseaktivitet som beskrevet i "2. Udlæsning af viral infektion. 5. Viral opslag / Fusion Assay Bemærk: Eksempel på inkubationsperioder og viral dosis for forskellige vira er anført i figur 2A "adgangsbillet / Fusion". Højere koncentrationer af virus kan også testes ved at øge MOI / PFU. Seed Huh-7,5-celler i en 96-brønds plade (1 x 10 4 celler pr brønd) og inkuberes ved 37 ° C i en 5% CO2-inkubator O / N for at opnå et monolag. Pre-chill cellemonolagene i pladerne ved 4 ° C i 1 time. Inficere cellerne med HCV (MOI = 0,01) ved 4 ° C i 3 timer. For eksempel bruger en 100 pi virusinokulum indeholdende 1 x 10 2 FFU. Bemærk: Udfør suppletion af det virale inokulum på is og efterfølgende inkubation i en 4 ° C køleskab for at holde temperaturen ved 4 ° C, hvilket tillader viral binding, men ikke post. Fjern supernatanten og forsigtigt vaske cellemonolagene med 200 pi iskold PBS to gange. Bemærk: Udfør vasker forsigtigt for at undgå at løfte cellerne. Behandl brøndene med testforbindelserne eller kontroller (slutkoncentrationer er: chlA = 50 uM; PUG = 50 uM; heparin = 1.000 pg / ml; DMSO = 1%) og inkuberes ved 37 ° C i 3 timer. For eksempel, tilsættes 10 pi af en 500 uM Chla arbejder fortynding til 90 pi medier, mix, og behandle brøndene; dette giver Chla behandling ved en slutkoncentration på 50 uM. Bemærk: Skiftet fra 4 ° C til 37 ° C letter nu virusindtrængen / fusion begivenhed og derfor muligt at vurdere testforbindelser effekt på dette særlige trin. Aspirer lægemiddelholdige supernatant og fjerne ikke-internaliseresekstracellulære virus ved enten vask med 200 pi citratbuffer (50 mM natriumcitrat, 4 mM kaliumchlorid, pH 3,0) eller PBS. Påfør 100 pi det basale medium, før inkubering ved 37 ° C i 72 timer. Analyser af den resulterende infektion ved at analysere supernatanten for luciferaseaktivitet som beskrevet i "2. Udlæsning af viral infektion.

Representative Results

I figur 1 blev 'viral inaktivering assay' udført for at undersøge, om to specifikke naturlige forbindelser Chla og PUG kunne inaktivere de forskellige kappeklædte vira i celle-fri tilstand og forhindre efterfølgende infektion. Cytotoksicitet og antiviral dosis-respons af disse forbindelser er blevet bestemt før udførelse af mekanistisk forsøg 31. De vira blev forbehandlet med testforbindelserne og derefter virus-stofblandinger blev fortyndet til sub-terapeutiske koncentrationer før podning på respektive cellemonolaget for hver virus system. Som vist i figur 1, både Chla og PUG syntes at interagere med cellefrie virioner, hvilket resulterer i irreversible virkninger, beskyttede cellemonolaget fra efterfølgende infektion. De to testforbindelser opnåede en næsten 100% hæmning mod HCMV, HCV, og DENV-2, mens en 60-80% blok blev observeret mod MV og RSV. Disse resultater Suggest at Chla og PUG har direkte indflydelse på disse frie viruspartikler ved at inaktivere dem og neutralisere deres infektivitet. I figur 2, blev den vedhæftede fil og indrejse / fusion analyser udført for at udforske effekten af Chla og PUG mod disse tidlige virale entry-relaterede hændelser fra HCMV, HCV, DENV-2, MV, og RSV. Både Chla og PUG effektivt forhindret binding af de undersøgte vira onto respektive værtscelle som vist ved inhibering på den resulterende virusinfektion (figur 2, 'Attachment': lys grå søjler). Den inhiberende virkning på virus fastgørelse af begge forbindelser var ens over for HCMV (figur 2B), HCV (figur 2C), DENV-2 (figur 2D), og RSV (figur 2F), der spænder fra 90 – 100%. På den anden side, PUG syntes at være mere effektiv end Chla mod MV-binding (figur 2E), med inhiberingshastigheden fra two forbindelser varierer mellem 50 – 80%. Kontrolbehandlingen heparin, som er kendt for at blokere optagelse af mange vira, også hæmmede binding af HCMV, DENV-2, RSV, ad MV, men var mindre effektiv mod HCV. Den efterfølgende 'viral indgang / fusion assay' undersøgt, om Chla og PUG bevaret deres aktivitet under virus entry / fusion fase (Figur 2, "adgangsbillet / Fusion«: mørk grå søjler). Igen blev både Chla og PUG observeret for effektivt at forringe den virale trin af de undersøgte vira (figur 2B – F) post / fusion, hvilket gav en 50 – 90% beskyttende virkning på den respektive cellemonolaget. Heparin desuden effektivt inhiberede post / fusion i DENV-2 og RSV-infektioner, men var mindre effektivt over for HCMV, HCV og MV (<40% inhibering i gennemsnit). Virus Celletype HCMV <td> HEL HCV Huh-7.5 DENV-2 Vero MV CHO-SLAM RSV HEp-2 Tabel 1:. Host celletype for virusinfektion Den anvendte celletype for hver virusinfektion, der er beskrevet i de repræsentative resultater er angivet. Yderligere detaljer vedrørende de celler kan findes i reference 31. Figur 1. Inaktivering af virusinfektioner ved testforbindelserne Chla og PUG Forskellige vira blev behandlet med prøveforbindelserne i en lang periode. (Inkuberet i 1,5-3 timer før titrering, lys grå søjler) eller kort periode (umiddelbart fortyndet; mørkegrå søjler) ved 37 ° C før en fortynding til sub-terapeutisk koncention og efterfølgende analyse af infektion på de respektive værtsceller. (A) Skematisk af forsøget (vist til venstre) med den endelige koncentration af virus (PFU / brønd eller MOI), langsigtede virus-drug inkubationsperioden (i), og efterfølgende inkubationstid (ii) er angivet for hvert virus i tabellen til højre. Analyser for (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, og (F) RSV er indikeret på hver yderligere panel. Resultaterne er afbildet mod DMSO negativ behandling kontrol for virusinfektion og de viste data er middelværdier ± standardfejl af middelværdien (SEM) fra tre uafhængige forsøg. Dette tal har været ændret siden henvisning 31. Klik her for at se en større version af dette tal. Figur 2. Evaluering af antivirale aktiviteter af testforbindelserne Chla og PUG mod virus fastgørelse og indgang / fusion. (A) Den eksperimentelle procedure, viruskoncentration (PFU / brønd eller MOI), og tidspunktet for tilsætning og behandling med testforbindelserne (i, ii, iii) er præsenteret for hvert virus i Skema og de tilknyttede tabeller. I virus fastgørelse analyse (lys grå søjler), monolag af forskellige celletyper blev præ-kølet ved 4 ° C i 1 time, derefter co-behandlet med de respektive vira og testforbindelser ved 4 ° C (1,5 – 3 timer; i) før vask off Inokulater og testforbindelser til efterfølgende inkubation (37 ° C ii) og undersøgelse af virusinfektion. I virus entry / fusion analyse (mørkegrå søjler), podede cellemonolag blev præ-afkølet ved 4 ° C i 1 time og derefter udfordret med de respektive vira ved 4 ° C i 1,5 – 3 timer (i). Celler blev dereftervasket og behandlet med testforbindelserne i en yderligere inkubationsperiode (ii), hvorunder temperaturen blev overført til 37 ° C for at fremme virusindtrængen / fusion begivenhed. Ved slutningen af ​​inkubationen blev ekstracellulære vira fjernes ved enten citratbuffer (pH 3,0) eller PBS vaske og cellerne blev yderligere inkuberet (iii) til analyse af virusinfektion. Resultater for (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, og (F) RSV er indikeret på hver yderligere panel. Data er afbildet mod DMSO negativ kontrol behandling af virusinfektion og præsenteres som middelværdi ± SEM fra tre uafhængige eksperimenter. Dette tal har været ændret siden henvisning 31. Klik her for at se en større version af dette tal.

Discussion

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 ‘viral inactivation assay’, the dilution of the virus-drug mixture to sub-therapeutic concentration prevents significant interaction between the test compound and the host cell surface by ‘titrating out’ 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 °C (which allows for virus binding but not entry) and 37 °C (which facilitates virus entry/fusion) in the ‘viral attachment assay’ and ‘viral entry/fusion assay’ are crucial to determine the test compound’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 °C and by placing the sample directly in a 37 °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’ 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.

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

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).

Materials

DMEM GIBCO 11995-040
FBS GIBCO 26140-079
Penicillin-Streptomycin GIBCO 15070-063
Amphotericin B GIBCO 15290-018
DMSO Sigma D5879
In vitro toxicology assay kit, XTT-based Sigma TOX2
PBS pH 7.4  GIBCO 10010023
Microplate reader Bio-Tek Instrument, Inc. ELx800 
Microcentrifuge Thermo Scientific 75002420
BioLux Gaussia luciferase assay kit New England Biolabs E3300L   
Luminometer Promega GloMax-20/20
Sodium citrate, dihydrate Sigma 71402
Potassium chloride Sigma P5405
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Referencias

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Key Words:- Viral Entry Assays- Antiviral Compounds- Cell-based Systems- Viral Life Cycle- Viral Entry Stages- Viral Inactivation- Viral Attachment- Viral Entry/fusion- Hepatitis C Virus- Antiviral Assays- Antagonists- Inhibitors- Early Viral Entry- Drug Development

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Tai, C., Li, C., Tai, C., Wang, C., Lin, L. Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds. J. Vis. Exp. (104), e53124, doi:10.3791/53124 (2015).
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