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

Um esboço geral do protocolo é ilustrada na Figura 1. 1. Células <ol><li> Preparar o meio de crescimento completo, que contém 10-15% de soro fetal bovino (FBS), 10 mM de aminoácidos não essenciais, 10 mM de Hepes, penicilina (100 unidades / ml), estreptomicina (100 mg / ml), e 2 mM de L-glutamina. </li><li> Manter Huh-7.5.1 células 13 em meios de cultura completos contendo os suplementos acima para análise in vitro do ciclo de replicação viral da hepatite C. </li><li> Culturas estirpes virais em células Huh-7.5.1 com o meio de crescimento suplementado especificado a 37 ° C com 5% de CO 2. </li></ol> 2. Vírus e plasmídeos As construções <ol><li> Gerar a forma complementar de ADN (ADNc) do ARN de HCV e cloná-lo num vector de plasmídeo para a facilidade da manipulação genética. Nota: A descoberta da hepatite fulminante japonês 1, JFH-1 (genótipo 2a HCV), isolar tem sido fundamental para a pesquisa de HCV e éusado principalmente para estudar o ciclo de replicação do HCV 11-13. Vírus quiméricos inter e intragenotypic baseados em JFH-1 HCV são comumente usados ​​em pesquisas 14-18. Construção de intragenotype 2a sintético vírus quimérico, FNX-HCV, e uma cepa repórter quimérico monocistrónicas foi descrito anteriormente 19 e construção detalhes estão fora do âmbito do presente protocolo. </li><li> Use o pFNX-HCV vírus intragenotype 2a vez que contém um 5 &#39;NTR, regiões e p7 estruturais e parte das regiões não-estruturais NS2 (nucleotídeos 1-2878) da J6CF cepa parental (NCBI adesão não. AF177036), como bem como os componentes não estruturais do vírus da estirpe JFH-1 (n ° de acesso NCBI. AB047639). Nota: FNX-HCV foi sintetizado com base na seqüência de Jc1 intragenotype 2a HCV quimérico relatado anteriormente 15. </li><li> Gerar um construto quimérico monocistrónicas repórter viral, o pFNX-Rluc, com base na estirpe pFNX-HCV (anteriormente no passo 2.2) através da inserção de um Rdo gene da luciferase enilla entre a NTR 5 &#39;e o gene núcleo (entre 388 e 389 nt). Posteriormente, ligar o gene da luciferase e gene núcleo dessa construção usando uma 2A vírus da febre aftosa (F2A) sequência peptídica, que funcionaria como um sinal de clivagem. </li><li> Engenheiro da construção viral RNA polimerase-null (Pol-) usando o pFNX-HCV ou o fundo viral pFNX-Rluc. Substitua os resíduos catalíticos NS5B polimerase, GDD (aa 2759-2761; nt 8615-8623), de qualquer um desses backbones virais com resíduos de ácido amino AAG. </li></ol> 3. In Vitro HCV RNA Transcrição <ol><li> Use uma intragenotype 2a vírus quimérico FNX-VHC e FNX-HCV Pol nula (Pol-) HCV para avaliação da replicação viral (Figura 2A). </li><li> Linearizar os plasmídeos virais com a enzima de restrição Xbal e, em seguida, tratar com nuclease de feijão mung para produzir extremidades cegas. Purificar os plasmídeos digeridos por cromatografia de troca aniônica. Verifique a integridade of o plasmídeo linearizado submetendo ADN de electroforese em gel de agarose (Figura 2B). </li><li> Transcrever os plasmídeos linearizados virais usando a polimerase T7 RNA. </li><li> Purifica-se o ARN tratado com ADNase recém-sintetizado utilizando um kit de purificação de RNA. </li><li> Verificar a produção de RNA por electroforese em gel de agarose (Figura 2C). </li><li> Quantificar o RNA por espectrofotometria. </li><li> Armazenar o ARN gerado em -80 ° C em aliquotas de 10 ug de trabalho. Nota: Para minimizar a variação da qualidade de ARN em cada desenho experimental transcrevendo todos RNA para cada amostra e de controlo virai, ao mesmo tempo. </li></ol> 4. HCV RNA transfecção e Coleta de Amostras <ol><li> Colher as células Huh-7.5.1 utilizando tripsina. </li><li> Para lavar as células, centrífuga e ressuspender a suspensão duas vezes com a mídia frio baixo soro. Ressuspender as células em meios de baixa no soro a 1 x 10 7 células por ml. </li><li> Misturar um total de 10 &amp; #956; g de ARN viral transcrito com 400 ul de células ressuspensas (4 x 10 6 células) numa cuvete de electroporação de 0,4 cm. Transfectar as células através de electroporação a 270 V, 100 Ω, e 950 uF. </li><li> Ressuspender as células electroporadas em 10 ml de meio de crescimento completo com 15% de FBS. Nota: O aumento da capacidade de sobrevivência das células electroporadas é visto quando as células Huh-7.5.1 foram cultivadas em 15% de soro fetal bovino. </li><li> Placa as células em ambos os frascos T-25 (1,2 x 10 ~ 6 células por frasco) e as placas de 48 poços (1 x 10 4 culas por po) para cada um dos seguintes pontos de tempo: 4, 48 e 96 horas. </li><li> Substitua a mídia a 4-8 horas após a transfecção com a mídia de crescimento suplementado fresco com 10% de FBS para remover restos de células mortas dos frascos de cultura e placas. </li><li> Os sobrenadantes de cultura de células da colheita nos 48 pontos de tempo, 96 h e remover os detritos celulares a partir de amostras recolhidas por centrifugação das células a 1500 rpm durante 10 min a 4 º C. </li><li> Armazenar os sobrenadantes isentos de células a -80 º C. Lisar as células de proteína e análise por Western blot de ARN e a transcrição reversa-PCR quantitativa em pontos de tempo indicados. </li></ol> 5. Transcrição reversa-PCR quantitativo (RT-qPCR) para a Avaliação Cópias HCV Genoma <ol><li> Realize uma de duas etapas RT-qPCR para determinar o número de cópias do genoma do HCV RNA. </li><li> Transcrever Reversa 1 ug de RNA celular total, utilizando a transcriptase reversa e um iniciador específico para a cadeia de sentido directo do HCV que se liga a 5 &#39;NTR (JFH RTQ R: 5&#39;CCTATCAGGCAGTACCACA-3&#39;) ou um primer específico para o gene doméstico peptidylprolyl isomerase de L (R PPIG : 5&#39;-GTCTCTCCTCCTTCTCCTCCTATCTTT-3 &#39;). Também a transcrição reversa do RNA de HCV-FNX (gerado na Etapa 3) de cópias do genoma conhecidos (10 janeiro - 10 setembro padrão) usando sentido HCV cordão iniciador. </li><li> Realizar qPCR utilizando 50 ng do cDNA transcrito resultante usando primers específicos do HCV (JFH RTQF: 5&#39;CTGGGTCCTTTCTTGGATAA-3; JFH RTQ R: 5&#39;CCTATCAGGCAGTACCACA-3 &#39;) e DNA de ligação corante verde contendo qPCR de super mix. Realize qPCR para PPIG gene housekeeping, bem como (primers PPIG F: 5&#39;-GAAGAGTGCGATCAAGAACCCATGAC-3 &#39;; PPIG R: 5&#39;-GTCTCTCCTCCTTCTCCTCCTATCTTT-3&#39;) Nota: Para o cálculo nível RNA HCV intracelular preciso através de amostras, use gene housekeeping celular, PPIG , o nível de expressão (ciclo Ct) para normalização. Utilizar o número de cópias do genoma FNX-HCV, como o padrão para a determinação do número de cópias. </li><li> Use as seguintes condições durante a execução de qPCR para determinar o número de cópias de RNA do HCV: 95 º C por 15 segundos e 60 &#160; º C por 30 segundos (40 ciclos), utilizando sistema em tempo real PCR. </li><li> Ver Figuras 3A e 3B para resultados de replicação do genoma. </li></ol> 6. Ocidental análise de hibridação para detecção de expressão da proteína do VHC (figura 3C) <ol><li> Use lisatos celulares frRNA viral om transfectadas em 96 horas pós transfecção para a proteína análise Western blot. </li><li> Resolver o ligado celular utilizando SDS-PAGE e transferência de difluoreto de polivinilideno (PVDF) da membrana. </li><li> Bloquear a membrana com uma solução de bloqueio contendo 5% de leite desnatado, 0,2% de Tween 20 em tampão fosfato salino (PBS). </li><li> Incubar a membrana com anticorpo primário monoclonal NS3 (1 em 1000 de diluição) e da beta-actina (diluição de 1 em 5.000). </li><li> Adicionar cabra anti-IgG de ratinho conjugado com peroxidase de rábano (diluição de 1 em 5000) e detectadas por quimiluminescência. </li></ol> 7. Reação de Imunofluorescência Indireta (RIFI) <ol><li> Fixar as células transfectadas e infectadas por HCV utilizando metanol durante 30 min a -20 º C para o ensaio de imunofluorescência. </li><li> Lava-se a células com PBS três vezes, e bloquear com IFA tampão de bloqueio. </li><li> Use policlonal de coelho anti-NS5A principalmente anticorpo (gentilmente cedido pelo Dr. Dasgupta, UCLA) ou anticorpo monoclonal anti-DSRNA J2 anticorpo a uma diluição de 1:200 (1 ug / ml) e incubar durante 5 horas a durante a noite a 4 º C. </li><li> Lavar as células com PBS três vezes, após o anticorpo primário. </li><li> Adicionar cabra anti-IgG de coelho-488 de anticorpo secundário ou policlonal de cabra anti-IgG de ratinho-594 de anticorpo policlonal secundário numa diluição de 1:1000 (1 ug / ml) e incubar durante 1 hora à temperatura ambiente. </li><li> Lave as células com PBS três vezes e núcleos mancha usando corante Hoechst e vista usando microscópio de fluorescência (Figura 3D). </li></ol> 8. Titer medição Vírus <ol><li> Placa naive Huh-7.5.1 células em cerca de 3 x 3 10 células / poço usando uma placa de 96 poços. No dia seguinte, realizar diluições em série de 10 vezes de livre de células do sobrenadante da cultura colhida a partir de RNA de HCV transfectadas células usando a mídia de crescimento e inocular em triplicado para Huh-7.5.1 células. </li><li> Fix células em 72 horas pós-infecção, utilizando metanol (durante 30 min a -20 </sup&gt; º C) e immunostain para a proteína NS5A do HCV como indicado na secção 7. </li><li> Utilizar a diluição mais elevada para contar para os focos positivo NS5A e calcular o número médio de unidades formadoras de foco (UFF) por mililitro. Ver Figura 4 para o HCV título. </li></ol> 9. Renilla luciferase Ensaio para Viral Genome replicação e infectividade <ol><li> Para avaliar a replicação do genoma viral, a placa de HCV RNA electroporado-Huh-7.5.1 células em triplicado em placas de 48 poços (1 x 10 4 células / cavidade). </li><li> Lisar as células com 75 ul de tampão de lise passivo no 6 h, 48 h e 96 h pós-transfecção. </li><li> Rocha as placas suavemente durante 15 minutos à temperatura ambiente e armazenar a -80 ° C. </li><li> Para medir a actividade enzimática de luciferase de Renilla, descongelar o lisado de proteína e luciferase de Renilla reagentes de ensaio a temperatura ambiente. </li><li> Adicionar 20 ul de lisado de proteína por cavidade de uma 96-wel<html> l placa luminescência branca e coloque a placa em um luminómetro. </li><li> Dispensar 100 l de Renilla luciferase reagente de ensaio (substrato celenterazina + buffer) por poço e depois de um atraso de 2 segundos pré-leitura; integrar a luz emitida durante 10 segundos. </li><li> Calcula-se a média eo desvio padrão para cada amostra a partir dos valores da luciferase de triplicados biológicos e submeter os dados para análise estatística (Figura 5). </li><li> Para avaliar a infectividade, inocular 500 mL de sobrenadante isento de células obtidas a partir de células transfectadas com ARN de HCV para os pontos de tempo indicados (48 hr e 96 hr), em triplicado, para naive Huh-7.5.1 células em placas de 48 poços. </li><li> Substituir o inoculo virai com 500 ul de meio fresco por po, depois de 6 horas pós-infecção. </li><li> Lise das células às 48 horas pós-infecção e sujeitas a ensaio de luciferase de Renilla como descritos nos passos 8,2-8,7 (Figura 5). </li></ol>itle &quot;&gt; 10 Análise Estatística. <ol><li> As barras de erro nos gráficos indicam os desvios padrão (SD). Os valores de p foram calculados pelo teste t não pareado. </li></ol>

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The authors have nothing to disclose.

Esta ilustração descreve um método para a análise do ciclo de replicação do vírus da hepatite C. HCV é um patógeno humano eo protocolo de biossegurança prescrito terá que ser rigorosamente seguidas. Sistemas de cultura de células de HCV infecciosas foram descritos anteriormente 11-13,16,17. Existem alguns pontos cruciais que implementamos quando seguindo o protocolo ilustrado. Primeiro, é de grande importância para uma boa qualidade de comprimento RNA genômico viral completo intacto para os est...

Vírus da hepatite C (HCV) causa cirrose e câncer de fígado. Ela afeta 170 milhões de pessoas em todo o mundo, com 350.000 pessoas morrem anualmente 1-3. O HCV é um vírus de cadeia positiva de ARN com um tamanho de genoma de 9,6 kb. O genoma do HCV é traduzido como uma única poliproteína de ~ 3000 resíduos de aminoácidos que é clivada proteoliticamente por diversas proteases celulares e virais em 10 polipéptidos. O HCV é o vírus protótipo do género Hepacivirus e pertence à família Flaviviridae 4. Após a exposição, o HCV estabelece a infecção crónica em 80% dos indivíduos. A infecção é geralmente assintomática e oportuna diagnóstico pode permitir a intervenção terapêutica para evitar a deterioração do fígado. O tratamento atual é de qualidade inferior e nenhuma vacina está disponível 5,6. A etiologia da hepatite C foi descrita pela primeira vez em 1989 7. Estudar a replicação do HCV é importante para a vacina contra a hepatite C e pesquisa de tratamento, mas que tinha sidolongo dificultada pela falta de um sistema de cultura de vírus eficiente. Um clone molecular de HCV foi demonstrado ser infeccioso em chimpanzés mediante inoculação intra-8. Subsequentemente, os replicões de HCV sub-genómicos foram descritas, o que permitiu a dissecar a fase de replicação do genoma viral em um sistema de cultura de células de 9,10. Descoberta de um VHC genótipo 2a isolar JFH-1 (Japanese fulminante da hepatite-1), capaz de infectar cultura de células abriu novos caminhos para a pesquisa a replicação do HCV 11-13. Genótipo 2a tensão JFH-1 baseada vírus quiméricos inter-e intra-genotípicas e do genótipo 1 do HCV sistemas de cultura infecciosas base também estão disponíveis 14-18. Temos utilizado com sucesso JFH-1 tensão e HCV intragenotype 2a vírus quimérico para obter o mapa de alta resolução de perfil funcional de domínios de proteínas e cis-atuantes elementos de RNA 19,20. De acordo com isto, aqui descrevemos um sistema de cultura eficaz utilizado rotineiramente que permiteestudando várias fases do ciclo de replicação do HCV e interação patógeno-hospedeiro. Apresentamos ensaios virológicos para avaliar replicação do genoma viral e infectividade de novo de intragenotype 2a HCV e HCV repórter baseado Renilla luciferase.

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			<td>The cell line was kindly provided by Dr. Francis Chisari to Dr. Arumugaswami under executed MTA between The Scripps Research Institute and Cedars-Sinai Medical Center</td>
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			<td style="width:267px;">Cedars-Sinai Medical Center</td>
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			<td>The HCV plasmids were synthesized by Dr. Arumugaswami using overlapping oligo-nucleotides.&#160;</td>
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a protocol for analyzing hepatitis c virus replication

Vírus da Hepatite C (HCV) afeta 3% da população mundial e causa graves doenças do fígado, incluindo hepatite crônica, cirrose e carcinoma hepatocelular. O HCV é um vírus de ARN encapsulado pertencente à família Flaviviridae. O tratamento atual não é totalmente eficaz e causa efeitos colaterais adversos. Não há vacina HCV disponível. Assim, é necessário um esforço continuado para o desenvolvimento de uma vacina e terapia melhor. Um sistema de cultura de células de VHC é fundamental para o estudo de vários estágios de crescimento de HCV, incluindo a entrada do vírus, a replicação do genoma, as embalagens, e de saída. No processo agora apresentado, foi utilizado um vírus de tipo selvagem intragenotype 2a quimérico, FNX-HCV, e um vírus FNX-Rluc recombinante portador de um gene repórter de luciferase de Renilla para estudar a replicação do vírus. Uma linha de células de hepatoma humano (Huh-7 com base) foi utilizado para a transfecção in vitro do transcrito ARN genómico de HCV. Os sobrenadantes de cultura isentos de células, lisados ​​de proteína e tRNA otal foram colhidas em vários pontos de tempo pós-transfecção para avaliar o crescimento HCV. Estado de replicação do genoma do HCV foi avaliada por RT-PCR quantitativo e visualizar a presença de ARN de HCV de cadeia dupla. A expressão da proteína do VHC foi verificada por imunofluorescência blot e ensaios de Western utilizando anticorpos específicos para as proteínas do HCV NS3 e NS5A. RNA de HCV de células transfectadas divulgados partículas infecciosas em cultura sobrenadante e o título virai foi medido. Os ensaios de luciferase, foram utilizados para avaliar o nível de replicação e infecciosidade de HCV repórter. Em conclusão, nós apresentamos vários ensaios virológicos para caracterizar diferentes estágios do ciclo de replicação do HCV.

O vírus da hepatite C é um vírus de RNA. Assim, para fins de manipulação genética, o ADNc genómico do HCV foi clonado num vector de plasmídeo bacteriano. Uma sequência de promotor de polimerase de ARN T7 foi introduzida imediatamente antes da extremidade 5 &#39;do genoma do VHC. Um esquema geral de fluxo de trabalho de análise do HCV é apresentada na Figura 1. Para gerar o ARN genómico de HCV com precisão a extremidade 3 &#39;, o genoma do VHC que contém o plasmídeo é cortado com a enzim...

Watch this Scientific Journal Video about A Protocol for Analyzing Hepatitis C Virus Replication at JoVE.com

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

Um protocolo para hepatite C Analisando replicação de vírus

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

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

Research

JoVE Journal

Immunology and Infection

23.9K visualizações.  Cedars-Sinai Medical Center. Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle. 

Vídeo: A Protocol for Analyzing Hepatitis C Virus Replication

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

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

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

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

Usando imagens Bioluminescent para Investigar sinergismo entre Streptococcus pneumoniae E Vírus da Influenza A em Ratos infantil

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

Imunologia e Infecção

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

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

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

Dissecando interação hospedeiro-vírus em replicação lítica de um herpesvírus modelo

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

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

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

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

Um sistema de cultura primária Neuron para o Estudo da Herpes Simplex Virus latência e reativação

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

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

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

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

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

De alta capacidade: Ensaio para Fenótipo<em&gt; Salmonella enterica</em&gt; Typhimurium Association, invasão e replicação em macrófagos

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

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

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

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

Imunofluorescência para monitorar a Incorporação celular de lactoferrina humana e sua Associated atividade antiviral contra o vírus da hepatite C

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

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

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

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

A Miniaturized Glycan Microarray Ensaio para a Avaliação de avidez e especificidade do Hemaglutininas vírus da gripe A

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

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

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

Desenvolvimento de um Sistema Reporter vírus da hepatite B de acompanhar as fases iniciais do ciclo de replicação

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

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

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

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

Salvamento e Caracterização do Vírus Recombinante de um Clone Infeccioso do Vírus do Novo Mundo Zika

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

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

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

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

The goal of this protocol is to provide a step-by-step guide to perform 3-D &#34;liver-on-a-chip&#34; infection experiments with the hepatitis B virus.

"Fígado-on-a-Chip" culturas de hepatócitos primários e células de Kupffer para infecção pelo vírus da hepatite B

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally ...

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

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

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

Identificação de codificação e Classes de RNA não-codificante expressa em suínos, sangue total

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