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).

Nota: Certifique-se de que todos os procedimentos que envolvem cultura celular e infecção pelo vírus são realizados em capuzes de biossegurança certificados que são apropriadas para o nível de biossegurança das amostras sendo manipulado. Para a finalidade de descrever os protocolos, Gaussia luciferase de VHC marcada com repórter é utilizado como um vírus modelo 32. No contexto dos resultados representativos, o ácido compostos chebulagic (CHL) e punicalagina (PUG) são utilizados como medicamentos antivirais candidatos que têm como alvo as interacções de glicoproteínas virais com os glicosaminoglicanos da superfície celular durante a entrada viral precoce passos 31. A heparina, que é conhecida por interferir com a entrada de muitos vírus 30,31,33,34, é utilizada como um tratamento de controlo positivo, em tal contexto. Para o fundo de base em técnicas de virologia, propagação de vírus, a determinação do título do vírus e a expressão de dose infecciosa em unidades formadoras de placas (PFU), concentrar unidades formadoras de FFU (), ou multiplicidade de infecção (MOI), o leitor é rerido para fazer referência a 35. Para exemplos anteriores e condições otimizadas utilizados para vírus mostrados nos resultados representativos, o leitor é remetido para referências 30-32,36-39, bem como detalhes listados na Tabela 1, Figura 1A e 2A. 1. Cultura celular, Composto de preparação, e o Composto Citotoxicidade <ol><li> Crescer a linha celular correspondente para o sistema de infecção por vírus a ser analisado (Tabela 1). Para o HCV, crescer as células Huh-7.5 em meio de Eagle modificado por Dulbecco (DMEM) suplementado com soro a 10% de bovino fetal (FBS), 200 U / ml de penicilina G, 200 ug / ml de estreptomicina, e 0,5 ug / ml de anfotericina B. </li><li> Preparar os compostos de teste e controlos utilizando os seus respectivos solventes: por exemplo, dissolver CHL e PUG em sulfóxido de dimetilo (DMSO); preparar heparina em água bidestilada estéril. Para todas as diluições subseqüentes, utilize meios de cultura. Nota: O último concentração de DMSO nos tratamentos composto do teste é inferior a 1% nas experiências; 1% de DMSO é incluído como um tratamento de controlo negativo nos ensaios para comparação. </li><li> Determinar a citotoxicidade dos compostos de teste (por exemplo, Chla PUG) e sobre as células para a infecção virai utilizando uma determinação de viabilidade celular, tais como reagente de XTT (2,3- bis [2-metoxi-4-nitro-5-sulfofenil] -5-fenilamino) -carbonil] -2H-tetrazólio): <ol><li> Para o HCV, sementes células Huh-7.5 numa placa de 96 poços (1 x 10 4 culas por po) e incuba-se a 37 ° C numa atmosfera de 5% de CO 2 incubadora O / N para se obter uma monocamada. </li><li> Aplicar controlo de DMSO (1%) ou concentrações crescentes de compostos de teste a CHL e PUG (ex. 0, 10, 50, 100, e 500 ^ M) aos poços de cultura em triplicado. </li><li> Incubar a 37 ° C durante 72 h, em seguida, descartar o meio na placa e lavar as células com 200 ul de solução salina tamponada com fosfato (PBS) duas vezes. </li><li> Adicionar 100 ul de assaying de solução a partir do kit de ensaio de toxicologia in vitro na base de XTT-se a cada poço e incubar as placas a 37 ° C durante mais 3 horas para permitir a produção de formazano XTT. </li><li> Determinar a absorvência com um leitor de microplacas a um comprimento de onda de teste de 492 nm e um comprimento de onda de referência de 690 nm. </li><li> Calcula-se a percentagem de células sobreviventes com a seguinte fórmula: viabilidade celular (%) = A / Como × 100%, em que &#39;A&#39; e &#39;As&#39; referem-se a absorvância dos compostos de teste e o controlo de solvente (1% de DMSO ex. ) tratamentos, respectivamente. Determinar a concentração de 50% de citotoxicidade celular (CC50) dos compostos de teste a partir de um software de análise, tais como o GraphPad Prism de acordo com o protocolo do fabricante. </li></ol></li></ol> 2. Leitura de infecção viral Nota: A leitura da infecção virai depende do sistema de vírus utilizado e pode envolver métodos tais como ensaios de placa ou MEAsuring sinais repórter de vírus marcado-repórter. O método para detectar a infecção por HCV-repórter com base na actividade repórter da luciferase é descrito abaixo. <ol><li> Recolhe-se os sobrenadantes dos poços infectados e clarificar a 17000 xg numa microcentrífuga durante 5 min a 4 ° C. </li><li> Misturar 20 ul de sobrenadante de teste a 50 ul de substrato de luciferase a partir do kit de ensaio de luciferase e Gaussia medir directamente com um luminómetro de acordo com as instruções do fabricante. </li><li> Expresso de infecciosidade de HCV como log 10 de unidades relativas de luz (RLU) para a determinação da inibição virai (%) e calcular a concentração 50% eficaz (CE 50) dos compostos de ensaio contra a infecção pelo HCV usando algoritmos de software GraphPad Prism de acordo com o protocolo do fabricante. </li></ol> 3. Inactivação Viral Assay Nota: Os exemplos de período de incubação e a dose virai de vários vírus deestá listada na Figura 1A. Concentrações mais elevadas do vírus também pode ser testado através do aumento da MOI / UFP. <ol><li> Semente células Huh-7.5 numa placa de 96 poços (1 x 10 4 culas por po) e incuba-se a 37 ° C numa atmosfera de 5% de CO 2 incubadora O / N para se obter uma monocamada. </li><li> Incubar os compostos de teste ou controles (concentrações finais são: CHLA = 50 mM; PUG = 50 mM; heparina = 1,000 g / ml; DMSO = 1%) com as partículas de HCV a 37 ° C (Figura 1A, &quot;a longo prazo&quot; ) em uma proporção de 1: 1. Por exemplo, para um inoculo de vírus de 100 ul contendo 1 x 10 4 FFU, adicionar 100 ul de uma diluição de trabalho 100 uM CHL; este tratamento proporciona CHLA a uma concentração final de 50 uM. </li><li> Dilui-se a mistura de vírus-droga (ineficaz) concentração de &quot;sub-terapêutica&quot; dos compostos de teste. Por exemplo, a concentração da CHL ineficaz e PUG contra o HCV é a 1 uM 31; portantoisto requer uma diluição de 50 vezes da mistura de vírus-droga que pode ser realizada com 9,8 ml de meio basal (meio de cultura de células com 2% de FBS). Nota: A diluição para a concentração sub-terapêutica impede interacção significativa entre os compostos de teste e da superfície da célula o hospedeiro e permite o exame do efeito do tratamento sobre os viriões livres de células. Note-se que esta diluição é dependente da dose resposta antiviral dos compostos de ensaio contra a infecção virai em particular, e é determinada antes de se efectuar este ensaio particular 31. </li><li> Para comparação, o vírus misturar com os compostos de ensaio e imediatamente diluídas (sem período de incubação) a concentração sub-terapêutica antes da infecção (Figura 1A, &quot;Short-Term&quot;). </li><li> Adicionar 100 ul da mistura de HCV-droga diluída para o Huh-7,5 monocamada de células (a quantidade de vírus é agora a 1 x 10 2 FFU; finais MOI = 0,01) e incubar durante 3 horas a 37 ° C para permitir viraladsorção. </li><li> Após a infecção, remover os inóculos diluído e lava-se suavemente os poços com 200 ul de PBS, duas vezes. Nota: Execute as lavagens com cuidado para evitar levantar as células. </li><li> Adicionar 100 ul de meio basal de cada poço e incuba-se a 37 ° C durante 72 h. </li><li> Analisar a infecção resultante por doseamento do sobrenadante para a actividade de luciferase tal como descrito em &#39;2. Leitura de infecção viral &#39;. </li></ol> 4. Acessório viral Assay Nota: Os exemplos de período de incubação e a dose viral para vários vírus são listados na Figura 2A, &quot;Anexo&quot;. Concentrações mais elevadas do vírus também pode ser testado através do aumento da MOI / UFP. <ol><li> Semente células Huh-7.5 numa placa de 96 poços (1 x 10 4 culas por po) e incuba-se a 37 ° C numa atmosfera de 5% de CO 2 incubadora O / N para se obter uma monocamada. </li><li> Pré-relaxar as monocamadas de células em placas a 4 ° C FoR 1 h. </li><li> Co-tratar as células com HCV inoculo (MOI = 0,01) e os compostos de teste ou os controlos (concentrações finais são: CHL = 50 | iM; PUG = 50 | iM; heparina = 1000 ug / ml; DMSO = 1%) a 4 ° C durante 3 hr. Por exemplo, para um inoculo de vírus de 90 ul contendo 1 x 10 2 FFU, adicionar 10 ul de uma diluição de trabalho de 500? M CHL; este tratamento proporciona CHLA a uma concentração final de 50 uM e uma infecção por HCV a MOI = 0,01 na monocamada de células. Nota: É importante realizar a experiência a 4 ° C uma vez que permite a ligação do vírus, mas impede a entrada que ocorre mais eficientemente a 37 ° C. Realizar a adição de compostos de vírus e de ensaio em gelo e o que se seguiu incubação num frigorífico a 4 ° C para assegurar que a temperatura é mantida a 4 ° C. </li><li> Remover o sobrenadante e lavar cuidadosamente a monocamada de células com 200 ul de PBS gelado duas vezes. Nota: Execute as lavagens com cuidado para evitar levantar as células &lt;./ li&gt; <li> Adicionar 100 ul de meio basal de cada poço e incuba-se a 37 ° C durante 72 h. </li><li> Analisar a infecção resultante por doseamento do sobrenadante para a actividade de luciferase tal como descrito em &#39;2. Leitura de infecção viral &#39;. </li></ol> 5. Viral Entrada / ensaio de fusão Nota: Exemplo de períodos de incubação ea dose viral para vários vírus são listados na &#39;Entry / Fusão&#39; Figura 2A. Concentrações mais elevadas do vírus também pode ser testado através do aumento da MOI / UFP. <ol><li> Semente células Huh-7.5 numa placa de 96 poços (1 x 10 4 culas por po) e incuba-se a 37 ° C numa atmosfera de 5% de CO 2 incubadora O / N para se obter uma monocamada. </li><li> Pré-relaxar as monocamadas de células em placas a 4 ° C durante 1 h. </li><li> Infectar as células com VHC (MOI = 0,01), a 4 ° C durante 3 h. Por exemplo, usar um inoculo de vírus de 100 ul contendo 1 x 10 2 UFF. Nota: Execute o addição do inoculo virai em gelo e o que se seguiu incubação num frigorífico a 4 ° C para manter a temperatura a 4 ° C, o que permite a entrada de ligação mas não virai. </li><li> Remover o sobrenadante e lavar suavemente as monocamadas de células com 200 ul de PBS gelado duas vezes. Nota: Execute as lavagens com cuidado para evitar levantar as células. </li><li> Tratar os poços com os compostos de teste ou os controlos (concentrações finais são: CHL = 50 | iM; PUG = 50 | iM; heparina = 1000 ug / ml; DMSO = 1%) e incuba-se a 37 ° C durante 3 h. Por exemplo, adicionar 10 ul de uma diluição de trabalho 500 mm CHLA para 90 l de mídia, misture, e tratar os poços; este tratamento proporciona CHLA a uma concentração final de 50 uM. Nota: A passagem de 4 ° C a 37 ° C agora facilita o evento de entrada / fusão viral e, portanto, permite a avaliação do efeito de compostos de teste &#39;sobre este passo particular. </li><li> Aspirar o sobrenadante que contém o fármaco e remover não-internalizadovírus extracelulares através de lavagem com 200 ul de tampão citrato (citrato de sódio 50 mM, cloreto de potássio a 4 mM, pH 3,0) ou PBS. Adicionar 100 ul do meio de base antes da incubação a 37 ° C durante 72 h. </li><li> Analisar a infecção resultante por doseamento do sobrenadante para a actividade de luciferase tal como descrito em &#39;2. Leitura de infecção viral &#39;. </li></ol>

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broad-spectrum+antiviral+activity+of+chebulagic+acid+and+punicalagin+against+viruses+that+use+glycosaminoglycans+for+entry.">Broad-spectrum antiviral activity of chebulagic acid and punicalagin against viruses that use glycosaminoglycans for entry.</a> BMC Microbiol. 13, 187 (2013).</li><li>Marukian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture-produced+hepatitis+C+virus+does+not+infect+peripheral+blood+mononuclear+cells.">Cell culture-produced hepatitis C virus does not infect peripheral blood mononuclear cells.</a> Hepatology. 48, 1843-1850 (2008).</li><li>Baba, M., Snoeck, R., Pauwels, R., de Clercq, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulfated+polysaccharides+are+potent+and+selective+inhibitors+of+various+enveloped+viruses,+including+herpes+simplex+virus,+cytomegalovirus,+vesicular+stomatitis+virus,+and+human+immunodeficiency+virus.">Sulfated polysaccharides are potent and selective inhibitors of various enveloped viruses, including herpes simplex virus, cytomegalovirus, vesicular stomatitis virus, and human immunodeficiency virus.</a> Antimicrob Agents ChemotheR. 32, 1742-1745 (1988).</li><li>Barth, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+binding+of+hepatitis+C+virus+envelope+glycoprotein+E2+requires+cell+surface+heparan+sulfate.">Cellular binding of hepatitis C virus envelope glycoprotein E2 requires cell surface heparan sulfate.</a> J Biol CheM. 278, 41003-41012 (2003).</li><li>Flint, S. J., Enquist, L. W., Racaniello, V. R., Skalka, A. 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> Principles of Virology. , (2008).</li><li>Brown, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dramatic+caspase-dependent+apoptosis+in+antibody-enhanced+dengue+virus+infection+of+human+mast+cells.">Dramatic caspase-dependent apoptosis in antibody-enhanced dengue virus infection of human mast cells.</a> J Leukoc Biol. 85, 71-80 (2009).</li><li>Huang, Y., Cyr, S. L., Burt, D. S., Anderson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+host+responses+to+respiratory+syncytial+virus+(RSV)+following+intranasal+administration+of+a+Protollin-adjuvanted,+epitope-enhanced+recombinant+G+protein+vaccine.">Murine host responses to respiratory syncytial virus (RSV) following intranasal administration of a Protollin-adjuvanted, epitope-enhanced recombinant G protein vaccine.</a> J Clin Virol. 44, 287-291 (2009).</li><li>Isaacson, M. K., Compton, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cytomegalovirus+glycoprotein+B+is+required+for+virus+entry+and+cell-to-cell+spread+but+not+for+virion+attachment,+assembly,+or+egress.">Human cytomegalovirus glycoprotein B is required for virus entry and cell-to-cell spread but not for virion attachment, assembly, or egress.</a> J Virol. 83, 3891-3903 (2009).</li><li>Leonard, V. H., 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=Measles+virus+blind+to+its+epithelial+cell+receptor+remains+virulent+in+rhesus+monkeys+but+cannot+cross+the+airway+epithelium+and+is+not+shed.">Measles virus blind to its epithelial cell receptor remains virulent in rhesus monkeys but cannot cross the airway epithelium and is not shed.</a> J Clin Invest. 118, 2448-2458 (2009).</li><li>Ciesek, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+green+tea+polyphenol,+epigallocatechin-3-gallate,+inhibits+hepatitis+C+virus+entry.">The green tea polyphenol, epigallocatechin-3-gallate, inhibits hepatitis C virus entry.</a> Hepatology. 54, 1947-1955 (2011).</li><li>Lin, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saikosaponin+b2+is+a+naturally+occurring+terpenoid+that+efficiently+inhibits+hepatitis+C+virus+entry.">Saikosaponin b2 is a naturally occurring terpenoid that efficiently inhibits hepatitis C virus entry.</a> J Hepatol. 62, 541-548 (2015).</li><li>Atkins, C., Evans, C. W., White, E. L., Noah, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+methods+for+influenza+antiviral+drug+discovery.">Screening methods for influenza antiviral drug discovery.</a> Expert Opin Drug Discov. 7, 429-438 (2012).</li><li>Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+novel+virus+inhibitors+by+influenza+A+virus+specific+reporter+cell+based+screening.">Identification of novel virus inhibitors by influenza A virus specific reporter cell based screening.</a> Antiviral Res. 93, 48-54 (2012).</li></ol>

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 border="0" cellpadding="0" cellspacing="0">
	<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&#160;</td>
			<td>GIBCO</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Bio-Tek Instrument, Inc.</td>
			<td>ELx800&#160;</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&#160;&#160;&#160;</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.

Na Figura 1, foi realizado o &quot;ensaio de inactivação virai&quot; para examinar se dois compostos específicos e CHLA PUG natural poderia inactivar os vírus com envelope diferentes em estado isento de células e prevenir a infecção subsequente. A citotoxicidade e de resposta à dose antiviral destes compostos foram determinados antes de se efectuar o estudo mecanicista 31. Os vírus foram pré-tratadas com os compostos de teste e, em seguida, as misturas de vírus-drogas foram diluídas para concentrações sub-terapêuticos antes da inoculação para a respectiva monocamada de células para cada sistema vírus. Como mostrado na Figura 1, tanto CHL e PUG apareceu para interagir com os viriões livres de células, resultando em efeitos irreversíveis que a monocamada de células protegidas da infecção subsequente. Os dois compostos de teste alcançou uma inibição quase 100% contra HCMV, HCV e DENV-2, enquanto que um 60 - bloco de 80% foi observada contra MV e RSV. Estes resultados suggest que CHLA e PUG têm impacto direto sobre estas partículas virais livres, desativando-os e neutralizando sua infectividade. Na Figura 2, o anexo e entrada / ensaios de fusão foram realizados para explorar o efeito da CHLA e PUG contra esses eventos relacionados à entrada virais início de HCMV, HCV, DENV-2, MV, e RSV. Ambos CHL e PUG prevenida eficazmente a ligação dos vírus investigados para a respectiva célula hospedeira, como mostrado pela inibição da infecção viral resultante (Figura 2, &#39;anexo&#39;: barras cinzentas claras). O efeito inibitório sobre a fixação do vírus por ambos os compostos foi semelhante contra HCMV (Figura 2B), HCV (Figura 2C), DENV-2 (Figura 2D), e RSV (Figura 2F), variando de 90 - 100%. Por outro lado, PUG pareceu ser mais eficaz do que CHLA contra MV ligação (Figura 2E), com a taxa de inibição do TWS compostos que variam entre 50 - 80%. O tratamento de controlo de heparina, que é conhecido por bloquear a entrada de muitos vírus, também inibiu a fixação de HCMV, DENV-2, RSV, anúncio MV, mas foi menos eficaz contra o HCV. O &#39;ensaio de entrada / fusão viral &quot;que se seguiu examinou se CHLA e PUG mantido a sua actividade durante a fase de entrada do vírus / fusão (Figura 2,&#39; Entry / Fusão &#39;: barras cinza escuro). Novamente, tanto CHL e PUG foram observados para prejudicar a eficácia do passo de entrada / fusão viral dos vírus examinados (Figura 2B - F), obtendo-se um 50 - efeito protector de 90% no respectivo monocamada de células. A heparina também inibiu potencialmente entrada / fusão em infecções de RSV e DENV-2, mas foi menos eficaz contra CMVH, VHC, e MV (&lt;40% de inibição, em média). <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Vírus </td><td> Tipo celular </td></tr><tr><td> HCMV </td> &lt;td&gt; HEL </td></tr><tr><td> HCV </td><td> Huh-7.5 </td></tr><tr><td> DENV-2 </td><td> Vero </td></tr><tr><td> MV </td><td> CHO-SLAM </td></tr><tr><td> RSV </td><td> HEp-2 </td></tr></tbody></table> Tabela 1:. Tipo de célula hospedeira por infecção viral O tipo de célula utilizado para cada sistema de infecção virai descrito nos resultados representativos está indicado. Detalhes adicionais referentes as células podem ser encontradas na referência 31. <img alt="figura 1" src="/files/ftp_upload/53124/53124fig1.jpg" /> Figura 1. A inactivação de infecções virais por o CHLA compostos de teste e PUG vírus diferentes foram tratadas com os compostos de teste durante um longo período. (Incubadas durante 1,5 - 3 horas antes da titulação; barras cinzentas claras) ou curto prazo (imediatamente diluída; cinzento escuro bares) a 37 ° C antes de uma diluição à Concentra sub-terapêuticação e subsequente análise de infecção sobre as células respectivo hospedeiras. (A) Esquema da experiência (mostrada à esquerda) com a concentração de vírus final (PFU / poço ou MOI), período de incubação de vírus-drogas de longo prazo (i), e tempo de incubação posterior (ii) indicada para cada vírus no quadro à direita. Análise para (B) de HCMV, (C) de HCV, (D) DENV-2, (E) MV, e (F) de RSV são indicados em cada painel adicional. Os resultados são representados graficamente contra o tratamento de DMSO de controlo negativo para a infecção pelo vírus e os dados apresentados são as médias ± erros padrão da média (EPM) de três experiências independentes. Esta figura foi modificado a partir da referência 31. <a href="https://www.jove.com/files/ftp_upload/53124/53124fig1highres.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53124/53124fig2.jpg" /> Figura 2. A avaliação das actividades anti-virais dos compostos de teste a CHL e PUG fixação contra vírus e entrada / fusão. (A) O procedimento experimental, a concentração de vírus (pfu / poço ou MOI), e o tempo de adição e o tratamento com os compostos de ensaio (I, II, III) são apresentados para cada virus nos esquemas e as tabelas associadas. Na análise acessório do vírus (barras cinzentas claras), as monocamadas de diferentes tipos de células foram pré-arrefecidas a 4 ° C durante 1 h, em seguida, co-tratadas com os respectivos vírus e os compostos de teste a 4 ° C (1,5 - 3 h; i) antes de lavar os inóculos e compostos de teste para a subsequente incubação (37 ° C, ii) e exame da infecção pelo vírus. Na entrada do vírus / análise de fusão (barras cinza escuro), as monocamadas de células semeadas foram pré-refrigerada a 4 ° C durante 1 hr e depois desafiado com os respectivos vírus a 4 ° C durante 1,5 - 3 h (i). As células foram entãolavados e tratados com os compostos de teste durante um período de incubação adicional (ii) durante o qual a temperatura foi mudada para 37 ° C para facilitar o evento de entrada / fusão viral. No final da incubação, os vírus extracelulares foram removidos por qualquer tampão de citrato (pH 3,0) ou de lavagens com PBS e as células foram incubadas adicionalmente (iii) para a análise da infecção pelo vírus. Os resultados para (B) de HCMV, (C) de HCV, (D) DENV-2, (E) MV, e (F) de RSV são indicados em cada painel adicional. Os dados são representados graficamente contra o controlo negativo DMSO tratamento da infecção pelo vírus e são apresentados como médias ± SEM de três experiências independentes. Esta figura foi modificado a partir da referência 31. <a href="https://www.jove.com/files/ftp_upload/53124/53124fig2highres.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

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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.

No início virais Ensaios de entrada para a identificação e avaliação de compostos antivirais

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

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30.0K visualizações.  Taipei Medical University Hospital. Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

Vídeo: 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.

Análise quantitativa de todos Influenza Tipo A Hemaglutininas Virais e neuraminidases usando anticorpos Universal em Ensaios Blot simples slot

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

Imunologia e Infecção

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

Amplificar e Quantificação do HIV-1 RNA no indivíduos infectados pelo HIV com carga viral abaixo do limite de detecção pela Standard Ensaios Clínicos

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

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.

Ensaios para a identificação de novos antivirais contra a febre catarral ovina vírus

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

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional&#160;overlays for certain heat labile viruses.

Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

Concentração Determinação Viral Através de placa Ensaios: Usando tradicionais e novos Sistemas de sobreposição

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

De alto rendimento quantitativo em tempo real de RT-PCR Ensaio para Determinar Perfis de Expressão do tipo I e III do interferão subtipos

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Qualitativos e quantitativos Os ensaios para a detecção e caracterização de antimicrobianos Proteína

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

O Desenvolvimento da liofilizado Loop-mediadas isotérmicas de amplificação Reagentes para a detecção de<em&gt; Burnetii Coxiella</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

Swab método de amostragem para a detecção do norovírus Humano em Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

Purificação de DNA Viral para a identificação de proteínas Viral e celulares associadas

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

Avaliação das respostas de células T Zika vírus específicos em órgãos Immunoprivileged de infectados Ifnar1- / - ratos

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...