Agradecemos Markus Thali, Nathan Roy, Christopher Ziegler, Emily Bruce, e Benjamim Rei para discussões úteis e os Institutos Nacionais de Saúde para apoio a estes estudos por meio de doações T32 AI055402 (JK), R21 AI088059 (JB), e P20RR021905 (JB) .

Nota: É crítico que o protocolo descrito ser realizada utilizando rigorosa técnica de pré-PCR (ver 28 para uma excelente visão geral sobre como configurar um laboratório de pré-PCR e como conduzir experimentos usando boa técnica de pré-PCR). É imperativo que todos os reagentes e equipamentos são livres de DNA amplificado contendo a sequência alvo qPCR e que os controles apropriados estão no local para detectar a contaminação. Uma visão geral do protocolo é demonstrado na Figura 1. 1. Prepare Mídia e células de sementes em placas de 48 poços <ol><li> Preparar 500 ml de completa de Dulbecco Modified Eagle Médium (DMEM) contendo soro de bovino fetal a 10%, 1% de penicilina-estreptomicina, e 1% de HEPES (ácido N-2-hidroxietilpiperazina-N-2-etano sulfónico) solução tampão através da adição de 50 ml de calor de soro de bovino fetal inactivado e 5 mL de cada vez de uma solução tampão de 100x de penicilina-estreptomicina e tampão HEPES 100x para um frasco de 500 ml de DMEM. Este reagente pode ser armazenada a 476; C durante meses. </li><li> Semente 2,5 x 10 4 células Vero E6 por poço numa placa de cultura de tecido de 48 poços num volume final de 500 ul de DMEM completo. Incubar a placa a 37 ° C numa incubadora humidificada contendo 5% de CO2 durante 10 a 18 h. <ol><li> Semente da placa no final do dia de trabalho. Teste cada amostra de vírus tanto em duplicado ou triplicado poços. Nota: Embora uma plataforma de poços com base em 48 para este ensaio é descrito, deve ser possível dimensionar o ensaio para baixo para utilização em placas de 96 ou de placas de 384 poços. </li></ol></li></ol> 2. Executar Anexo de células-Virus Nota: O vírus utilizados no presente protocolo, estirpe JUNV Espontânea # 1 (C # 1), foi utilizado com sucesso como uma vacina viva atenuada para a prevenção da doença JUNV 29. JUNV C # 1 foi derivada a partir da estirpe XJ JUNV virulento através de passagens em série tanto in vivo como in vitro e difere da estirpe parental XJ por 12 aminoácidos 30. because JUNV C # 1 é um nível de biossegurança (BSL) patógeno -2-classificado, o trabalho descrito para esta seção devem ser realizados utilizando apropriadas BSL-2 práticas 31. Isso inclui o uso de equipamento de protecção individual (por exemplo, protecção para os olhos, bata de laboratório, luvas duplas), uma cabine de segurança biológica classe II, e garantir que todo o pessoal tenha recebido a formação institucional e aprovações necessárias para lidar com segurança com este organismo. <ol><li> Dilui-se a JUNV C # 1 da amostra (s) a ser testado para o PFUs desejado ou se concentrar em unidades formadoras gelada DMEM completo e armazenar em gelo até estar pronto para adicionar a cada poço. Observação: Como uma alternativa para a realização dos estudos de ligação com vírus diluída em DMEM completo, usar um meio mínimo contendo PBS com uma concentração reduzida de soro (2%) e um tampão apropriado para determinar se o soro pode causar interferência com o acessório do vírus. Similar protocolos de ligação para os vírus alternativas têm relatado o uso de RPMI 1640 sem bicarbonate contendo 0,2% de albumina de soro bovino, 10 mM de (2- (morfolino) etanossulfónico), e 10 mM de HEPES, pH 6,8 32. Este os meios de ligação podem ser superior a evitar alterações de pH que podem ocorrer quando o material é removido a partir do CO 2 -Ambiente da incubadora. <ol><li> Inocular poços com PFU que variam de 200 a 2000 para limitar eventos de fixação não produtivas. Como mostrado na Figura 2, este ensaio é capaz de detectar anexo usando tão poucos como 20 PFU (ou tantos quanto 2 x 10 6 pfu). </li><li> Criar uma mistura mestre do vírus para uma inoculação em células. Cada amostra de vírus será inoculado em poços duplicados num volume de 50 ul por cavidade. Portanto, se a adição de 2.000 PFU de virus por poço é desejado, dilua 4.800 PFU em um volume total de 120 ul de DMEM completo gelada. <ol><li> Para controlar a especificidade do ensaio, usar um par correspondente de linhas de células de ovário de hamster chinês que não expressam quer TfR1 (TRVb) ou express hTfR1 (TRVb-1) como anexo JUNV a estas células deveria ser reduzida ou não, respectivamente 13,33. Alternativamente, o tratamento de células com uma protease, tal como proteinase K para impedir a fixação arenavírus 34 ou entrada 35. Solúvel hTfR1 ou o anticorpo ch128.1 monocolonal, que é específico para hTfR1, também poderia ser considerado como eles são conhecidos por bloquear a entrada de JUNV em células 13,36. Além disso, o pré-tratamento de células com manose livre 14 ou incubação de partículas com anticorpos neutralizantes específicos de JUNV 37 pode inibir a entrada do vírus. </li><li> Por favor note, no entanto, que estes últimos reagentes e abordagens, apesar de bloquear a entrada JUNV, não foram confirmados para bloquear a adesão, embora esta seja uma explicação provável. O protocolo aqui descrito pode ser usado para determinar se se trata formalmente várias abordagens ligação de viriões de impacto. </li></ol></li></ol></li><li> Retirar as placas da incubadora a 37 ° C elugar quer num 4 ° C frigorífico ou em gelo durante 15 minutos para inibir a capacidade de as células plaqueadas para efectuar a endocitose. </li><li> Em seguida, aspirar o DMEM completo a partir de cada cavidade e lavar rapidamente cada poço duas vezes com 100 ul de PBS gelado (soro fisiológico tamponado com fosfato) pH 7,4. Em seguida, adicionar 50 ul das amostras de vírus pré-diluído (preparado no passo 2.1) aos poços apropriados. </li><li> Enrole a placa em filme plástico e coloque imediatamente, quer de volta no gelo ou em um 4 ° C frigorífico durante 1 a 1,5 horas para permitir a ligação do vírus a ocorrer. Manter a placa, tampão de lavagem, e as amostras de vírus frio a 4 ° C ao longo deste passo. </li><li> Após a conclusão da incubação de 4 ° C, aspirar o inoculo de vírus e lavar cada poço 3 vezes com 200 ul de PBS gelado. </li></ol> 3. Extracção de ARN viral <ol><li> Purifica-se ARN viral a partir de JUNV C # 1 partículas aderidas às monocamadas usando o kit comercial (por exemplo, RNeasy Mini Kit) acording o protocolo do fabricante. Especificamente, siga a &quot;purificação de RNA total a partir de células animais utilizando protocolo de tecnologia de spin&quot; nas páginas 23-28 no RNeasy Mini Handbook (4ª edição, Abril de 2006), com as modificações enumeradas abaixo. <ol><li> Lisar as células, conforme indicado no passo 1B por adição de 350 ul de tampão de lise directamente RLT a cada poço. Misture o tampão várias vezes para assegurar a lise das células. Note-se que as células lisam facilmente neste tampão de lise completa e pode ser verificada por meio de visualização poços sob um microscópio invertido. </li><li> Transferir o lisado de células resultante para o kit de coluna (passo 3a) e seguir o resto do protocolo, tal como descrito. Neste ponto, o vírus foi neutralizada pelo tampão de lise de modo que os passos restantes do protocolo pode ser feito fora da cabine de segurança biológica de classe II, desde que os tubos kit não foram contaminadas com vírus infeccioso. </li><li> Incluir o passo opcional de 9 o protocolo do fabricante, que é um additional centrifugação da coluna de centrifugação para eliminar qualquer possibilidade de reporte de Tampão RPE. </li><li> No passo final o protocolo do fabricante (passo 10), elui-se o ARN purificado a partir da coluna de spin com 30 ul de DDH livre de nuclease 2 O. ARN armazenar a -80 ° C neste ponto ou usar directamente no ensaio de qRT-PCR. Para evitar a degradação das amostras de RNA viral purificado por nucleases, utilizar reagentes e equipamento livre de nuclease e manter as amostras de ARN descongeladas em gelo. Nota: Os tampões RLT e RW1 contêm sal de guanidina e não deve ser misturado com água sanitária. Tampão RW1 contém etanol. </li></ol></li></ol> 4. qRT-PCR Medição de genoma viral <ol><li> Para converter JUNV C # 1 de ARN em ADNc segmento S para qPCR subsequente, sujeitar o ARN viral (gerado no passo 3.1.4) à temperatura ambiente, num volume total de 50 ul. <ol><li> Para configurar a reacção RT, primeiro criar uma mistura principal contendo cada um dos seguintes reagentes por reacção: 6,75 mL de nucleaselivre de ddH2O, 5 ul de estoque de um iniciador de RT o S 2 uM para 2098- 2075- (5&#39;-AAGGGTTTAAAAATGGTAGCAGAC-3 &#39;), que é complementar à região de NP do ARN genómico segmento S, 5 ul de tampão de PCR 10X II, 11 ul de uma solução de MgCl 2 25 mM, 1,25 mL (62,5 unidades) da enzima RT, 1 ul (0,4 unidades) de inibidor de RNase, e 10 ul de um stock de dNTPs 500 jiM. Nota: Durante o curso da replicação arenavírus, 4 espécies de ARN segmento S virais são gerados: um ARN de comprimento completo genómico (ARNv), um comprimento total de ARN antigenómico (vcRNA) (que é o complemento reverso do ARNv), e dois mRNAs subgenómicos codifica os genes NP e GP, respectivamente. O iniciador de RT listados aqui é específica apenas para o segmento S ARNv, mas não o vcRNA, ARNm de NP, ou ARNm de GP. </li><li> Misture suavemente a mistura principal RT e depois se alíquota de 40 mL em 0,2 mL tubos de PCR individuais. Adicionar 10 ul de ARN viral para cada tubo. Incluir i) um tubo nenhum controle modelo (por exemplo,. Adicione 10 ml de 2 O a 40 mL livre de nuclease DDH da mistura principal) e ii) um controlo sem enzima RT (por exemplo, adicionar 10 ul de uma amostra de RNA viral conhecido positiva para 40 l de mistura principal que não receberam qualquer enzima RT) para o rastreio de contaminação dos reagentes temperatura ambiente com ADN amplificado contendo a sequência alvo viral. Incluem ARN extraído de células infectadas com a mistura principal completa de RT para controlar a especificidade do ensaio na presença de RNA celular. </li><li> Além disso, incluem uma amostra de ARN viral conhecido-positivo para a mistura principal completo para verificar a qualidade dos reagentes RT. Note-se que o volume da reacção RT pode ser reduzida para 25 ul, se necessário. </li></ol></li><li> Sujeitar os tubos de RT para as seguintes condições de ciclo: 25 ° C durante 10 min, 48 ° C durante 30 min, e 95 ° C durante 5 min. Note-se que as amostras podem ser armazenadas a -20 ° C neste ponto ou deixada durante a noite no termociclador pela adição de um passo de 4 ° C. </li><li> Perform qPCR em um volume total de 25 ul. <ol><li> Adicione uma mistura mestre qPCR, combinando o seguinte: 2,5 ul (de um estoque 9 uM) de cada iniciador directo de PCR para S1937 + 1958+ (5&#39;-CATGGAGGTCAAACAACTTCCT-3 &#39;) e iniciador de PCR inverso 2001- S para 1982- (5 &#39;-GCCTCCAGACATGGTTGTGA-3&#39;), 2,5 ul (de um estoque 2 mM) do qPCR sonda S 1960+ a 1975+ (5&#39;-6FAM-ATGTCATCGGATCCTT-MGBNFQ-3 &#39;), e 12,5 ul de 2X o mestre universal qPCR misturar. Note-se que o iniciador directo aqui relatado, que é específico para JUNV C # 1, difere por 1 nt a partir da sequência do iniciador originalmente relatado 38, que tem como alvo a estirpe virulenta JUNV Romero. </li><li> Misture suavemente a solução e, em seguida, adicionar 20 ul a cada qPCR bem. Adicionam-se 5 ul de cada reacção de RT para os poços apropriados qPCR. Realizar qPCR em triplicado para cada amostra testada. Note-se que o volume da reacção de PCR pode ser reduzida para tão pouco quanto 12,5 ul, se necessário. Nota: O software associadoa máquina qPCR irá gerar números de cópias absolutos de JUNV C # 1 RNA genómico segmento S, comparando o valor de cada amostra desconhecida a uma série de diluições padrão de plasmídeo que codifica o gene NP JUNV C # 1, que é o alvo do iniciador qPCR e sonda. O ensaio qPCR só pode proporcionar a quantificação dos valores que caem na gama da curva padrão. Por esta razão, incluem as seguintes quantidades de plasmídeo (em poços em triplicado): 5, 50, 5 x 10 3, 5 x 10 5 e 5 x 10 7 cópias por poço. </li></ol></li><li> Carregue a placa qPCR no termociclador e executar as seguintes condições de reacção: 95 ° C durante 10 min e 40 ciclos de 95 ° C durante 15 seg e 60 ° C durante 1 min. <ol><li> Recolher e analisar os dados para determinar a quantidade de ARN virai presente em cada amostra de acordo com o protocolo do fabricante. </li></ol></li><li> Para estabelecer uma curva padrão, utilizando uma amostra recentemente purificado do plasmídeo contendo o alcatrão qPCRsequência obter, em primeiro lugar determinar o número de cópias do plasmídeo nesta solução. <ol><li> Calcule o peso molecular do plasmídeo. Se a sequência de todo o plasmídeo é conhecido, calcular esta com a seguinte fórmula: MW = ([A * 312,2] + [L * 328,2] + [C * 288,2] + [T * 303,2] - 61,13). Lembre-se de incluir o número total de um nucleotídeo especial encontrada em cada mecha do plasmídeo de cadeia dupla. </li><li> Se o tamanho do plasmídeo é conhecido, mas a sua sequência exacta não é, calcular o MW sob a suposição de que cada par de bases dsDNA tem um PM de 600. Nota: O plasmídeo pCAGGS NP-JUNV C # 1 aqui utilizado tem um peso molecular de 4,2 x 10 6. </li><li> Uma vez que o PM do plasmídeo foi determinada, calcular o número de cópias por ul estão presentes na solução estoque do plasmídeo. </li><li> Em primeiro lugar calcular a ug total de plasmídeo na solução, em seguida, convertê-lo em moles de plasmídeo (por exemplo, se 69 ug de plasmídeo estão contidas em 300 ul de água, seguida de 6,9 x 10 -5 g de plasmídeo * 1 mole / 4,2 x 10 6 g de plasmídeo = 1,6 x 10 -11 moles de plasmídeo), que pode ser convertido para o número de cópias (por exemplo, 1,6 x 10 -11 moles de plasmídeo * 6,022 x 10 23 moléculas / mole = 9,8 x 10 12 moléculas (ou cópias)). </li><li> Separe as cópias de plasmídeo totais na solução de plasmídeo por o volume da referida solução para obter cópias por ul (por exemplo, 9,8 x 10 12 300 cópias / ul = 3,28 x 10 10 cópias). </li><li> Dilui-se esta solução de forma apropriada para que os volumes de 5 ul pode ser carregado para a placa de PCR para proporcionar a gama de números de cópias necessárias para estabelecer a curva padrão. </li></ol></li></ol>

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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+rapid+and+efficient+method+for+studies+of+virus+interaction+at+the+host+cell+surface+using+enteroviruses+and+real-time+PCR.">A rapid and efficient method for studies of virus interaction at the host cell surface using enteroviruses and real-time PCR.</a> Virol J. 6, 217 (2009).</li><li>Jiang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+C+virus+attachment+mediated+by+apolipoprotein+E+binding+to+cell+surface+heparan+sulfate.">Hepatitis C virus attachment mediated by apolipoprotein E binding to cell surface heparan sulfate.</a> J. Virol. 86, 7256-7267 (2012).</li><li>Shannon-Lowe, C., 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=Epstein-Barr+virus-induced+B-cell+transformation:+quantitating+events+from+virus+binding+to+cell+outgrowth.">Epstein-Barr virus-induced B-cell transformation: quantitating events from virus binding to cell outgrowth.</a> J. Gen. Virol. 86, 3009-3019 (2005).</li><li>Mifflin, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Setting+up+a+PCR+laboratory.">Setting up a PCR laboratory.</a> CSH Protoc. 2007, pdb top14 (2007).</li><li>Maiztegui, J. I., 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=Protective+efficacy+of+a+live+attenuated+vaccine+against+Argentine+hemorrhagic+fever.+AHF+Study+Group.">Protective efficacy of a live attenuated vaccine against Argentine hemorrhagic fever. AHF Study Group.</a> J. Infect. Dis. 177, 277-283 (1998).</li><li>Goni, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+features+of+attenuated+Junin+virus+vaccine+strain+candidate.">Genomic features of attenuated Junin virus vaccine strain candidate.</a> Virus Genes. 32, 37-41 (2006).</li><li>Chosewood, L. C., Wilson, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biosafety in microbiological and biomedical laboratories. , (2009).</li><li>White, J., Matlin, K., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+fusion+by+Semliki+Forest,+influenza,+and+vesicular+stomatitis+viruses.">Cell fusion by Semliki Forest, influenza, and vesicular stomatitis viruses.</a> J. Cell Biol. 89, 674-679 (1981).</li><li>McGraw, T. E., Greenfield, L., Maxfield, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+expression+of+the+human+transferrin+receptor+cDNA+in+Chinese+hamster+ovary+cells+deficient+in+endogenous+transferrin+receptor.">Functional expression of the human transferrin receptor cDNA in Chinese hamster ovary cells deficient in endogenous transferrin receptor.</a> J. Cell Biol. 105, 207-214 (1987).</li><li>Borrow, P., Oldstone, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+lymphocytic+choriomeningitis+virus-binding+protein(s):+a+candidate+cellular+receptor+for+the+virus.">Characterization of lymphocytic choriomeningitis virus-binding protein(s): a candidate cellular receptor for the virus.</a> J. Virol. 66, 7270-7281 (1992).</li><li>Rojek, J. M., Spiropoulou, C. F., Kunz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+cellular+receptors+for+the+South+American+hemorrhagic+fever+viruses+Junin,+Guanarito,+and+Machupo.">Characterization of the cellular receptors for the South American hemorrhagic fever viruses Junin, Guanarito, and Machupo.</a> 346. , 476-491 (2006).</li><li>Helguera, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+antibody+recognizing+the+apical+domain+of+human+transferrin+receptor+1+efficiently+inhibits+the+entry+of+all+new+world+hemorrhagic+Fever+arenaviruses.">An antibody recognizing the apical domain of human transferrin receptor 1 efficiently inhibits the entry of all new world hemorrhagic Fever arenaviruses.</a> J. Virol. 86, 4024-4028 (2012).</li><li>Sanchez, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Junin+virus+monoclonal+antibodies:+characterization+and+cross-reactivity+with+other+arenaviruses.">Junin virus monoclonal antibodies: characterization and cross-reactivity with other arenaviruses.</a> J. Gen. Virol. 70, 1125-1132 (1989).</li><li>Trombley, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+panel+of+real-time+TaqMan+polymerase+chain+reaction+assays+for+detection+and+absolute+quantification+of+filoviruses,+arenaviruses,+and+New+World+hantaviruses.">Comprehensive panel of real-time TaqMan polymerase chain reaction assays for detection and absolute quantification of filoviruses, arenaviruses, and New World hantaviruses.</a> Am. J. Trop. Med. Hyg. 82, 954-960 (2010).</li><li>Helenius, A., Marsh, M., White, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Semliki+forest+virus+penetration+by+lysosomotropic+weak+bases.">Inhibition of Semliki forest virus penetration by lysosomotropic weak bases.</a> J. Gen. Virol. 58 (Pt 1), 47-61 (1982).</li><li>Nour, A. M., Li, Y., Wolenski, J., Modis, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+membrane+fusion+and+nucleocapsid+delivery+into+the+cytoplasm+are+distinct+events+in+some+flaviviruses.">Viral membrane fusion and nucleocapsid delivery into the cytoplasm are distinct events in some flaviviruses.</a> PLoS pathogens. 9, e1003585 (2013).</li></ol>

Os autores declaram que não têm interesses financeiros concorrentes.

O protocolo que descrevemos é simples e robusta desde que as seguintes considerações críticas são observadas. Em primeiro lugar, rigorosa técnica de pré-PCR deve ser empregado (ver 28 para uma excelente revisão). É imperativo que todos os reagentes e equipamentos são livres de DNA amplificado contendo a sequência alvo qPCR e que os controles apropriados estão no local para detectar a contaminação. Em segundo lugar, para a parte de fixação de células com vírus do protocolo, o vírus, células...

Arenavírus são uma família de vírus com envelope, de ARN de cadeia simples que são mantidas essencialmente em roedores na natureza 1-3. Embora estes vírus tipicamente estabelecer uma infecção assintomática, persistente em reservatórios de roedores 1,2, que pode causar doença grave em humanos 3. Espécies patogênicas importantes incluem o vírus Novo Mundo Junin (JUNV) 4 e o vírus Old Lassa Mundial 5, que são os agentes etiológicos da febre hemorrágica argentina na América do Sul e da febre de Lassa na África, respectivamente. Vírus da coriomeningite linfocítica (LCMV), o vírus protótipo da família 6, tem uma distribuição mundial e é responsável por uma variedade de doenças, incluindo meningite asséptica em indivíduos imunocompetentes 6, defeitos congênitos graves no feto em desenvolvimento 7, ou de alta letalidade em imunossuprimidas indivíduos na sequência de órgãos sólidos transplante 8,9. A falta de Estados UnidosFood and Drug Administration (FDA) -aprovado vacinas ou medicamentos antivirais eficazes para combater estes vírus destaca a necessidade crítica de desenvolver novas estratégias para atingir terapeuticamente esses patógenos emergentes / re-emergente. O genoma arenavírus consiste em dois segmentos de ARN de cadeia simples, a grande (L) (~ 7,2 kb) e pequena (s) (~ 3,6 kb) segmentos 10. O segmento S codifica a nucleoproteína viral (NP) e a glicoproteína do envelope (GP), enquanto o segmento L codifica a polimerase viral (L) e proteína de matriz (Z). O L e segmentos de ARN genómico S (ARNv) são empacotados em viriões 10-12. O passo inicial de infecção arenavírus é a ligação de partículas virais às células hospedeiras através de uma interacção entre o GP viral e os seus receptores de células hospedeiras correspondentes que, por JUNV, inclui o receptor de transferrina humana 1 (hTfR1) 13,14. Após a ligação, as partículas JUNV são levados para dentro da célula através de endocitose mediada por clatrina 15.Partículas de tráfego, em seguida, para o final do endossoma onde a baixo pH deste compartimento desencadeia a activação da GP 16,17. Uma vez activadas, as inserções de péptidos de fusão codificada-GP na membrana endossomal, iniciando a fusão entre as membranas viral e endossomais. resultados da fusão de sucesso na liberação dos vRNAs embalados-virion no citoplasma, onde servem como modelos para replicação genômica e transcrição. Quando uma quantidade suficiente de proteínas estruturais virais e ARNv são gerados, novas partículas de montar e botão da célula infectada para completar o ciclo de vida de 18 anos. Para facilitar a descoberta de novas estratégias para atingir terapeuticamente os arenavírus patogênicos, o nosso grupo tem-se centrado na identificação de factores do hospedeiro que são críticas para a propagação de vírus, mas dispensável para o anfitrião. Neste contexto, a definição do estádio preciso (s) do ciclo de vida viral controlada por esses factores do hospedeiro é necessária para guiar odesenvolvimento de estratégias antivirais. Aqui, nós descrevemos um (Q) ensaio quantitativo com base em RT-PCR que podem ser utilizados para medir a ligação de células arenavírus para o propósito de identificar se as proteínas hospedeiras seleccionadas e / ou moléculas antivirais influenciar esta fase inicial da entrada viral 19. Abordagens alternativas para medir a fixação de células arenavírus têm caracterizado viriões marcados com biotina 20, corantes fluorescentes 21, ou isótopos radioactivos 22. Inconvenientes para estes métodos podem incluir o requisito de grandes quantidades de vírus de entrada (por exemplo, elevadas multiplicidades de infecção (MOI)), a utilização de radioactividade, e / ou passos adicionais de manuseamento para a preparação do vírus de entrada (por exemplo, concentração e / ou a rotulagem de vírus) . Em particular, o uso de alta MOI torna difícil distinguir produtivo contra eventos de fixação não produtivas 15,23. O ensaio à base de qRT-PCR descrito aqui pode detectar eventos de ligação utilizando tão pouco quanto 20 por placaunidades Ming (PFU) de vírus, que se traduz em um MOI de 0,0004 para uma placa de 48 poços. Portanto, a forte sensibilidade do ensaio supera a necessidade de alta MOI assim como quaisquer passos de rotulagem ou de vírus de concentração. Além disso, o ensaio pode ser i) adaptado para medir virião endocitose, bem como libertação de genoma do vírus no citoplasma da seguinte fusão e ii) adaptado para utilização com outros vírus através do desenvolvimento de reagentes de qRT-PCR específica para o vírus (para exemplos, ver 24-27).

<table border="0" cellpadding="0" cellspacing="0" width="730">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">DMEM</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">11965-118</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Penicillin-Streptomycin</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">15140-163</td>
			<td style="width:160px;">100x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">HEPES Buffer Solution</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">15630-130</td>
			<td style="width:160px;">100x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">PBS pH 7.4</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">10010-023</td>
			<td style="width:160px;">1x</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Vero E6 cells</td>
			<td style="width:131px;">American Type Culture Collection</td>
			<td style="width:141px;">CRL-1586</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Costar 48-well plate</td>
			<td style="width:131px;">Corning</td>
			<td style="width:141px;">3548</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">RNeasy mini kit</td>
			<td style="width:131px;">Qiagen</td>
			<td style="width:141px;">74106</td>
			<td style="width:160px;">do not mix buffers RLT or&#160; RW1 with bleach</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">QIAshredder homogenizer</td>
			<td style="width:131px;">Qiagen</td>
			<td style="width:141px;">79654</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Taqman Universal PCR Master Mix</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">4326614</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Multiscribe Reverse Transcriptase (50U/&#181;l)</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">4311235</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">dNTP Mixture (10 mM; 2.5 mM each dNTP)</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">N808-0260</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">GeneAmp 10X PCR Buffer II and 25 mM MgCl2 solution</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">N808-0010</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">RNase Inhibitor (5000U/100 &#181;l)</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">N808-0119</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">RT primer 5&#8217;-AAGGGTTTAAAAATGGTAGCAGAC-3&#8217;</td>
			<td style="width:131px;">Integrated DNA Technologies</td>
			<td style="width:141px;">250 nmol DNA oligo</td>
			<td style="width:160px;">standard desalting</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Forward qPCR primer&#160; 5&#8217;-CATGGAGGTCAAACAACTTCCT-3&#8217;</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">4304971</td>
			<td style="width:160px;">standard desalting</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Reverse qPCR primer 5&#8217;-GCCTCCAGACATGGTTGTGA-3&#8217;</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">4304971</td>
			<td style="width:160px;">standard desalting</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">TaqMan Probe 5&#8217;-6FAM-ATGTCATCGGATCCTT-MGBNFQ-3&#8217;</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">4316033</td>
			<td style="width:160px;">HPLC purified</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">MicroAmp Fast Optical 96-well reaction plate (0.1 mL)</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">4346906</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">StepOnePlus Real-Time PCR System</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">4376598</td>
			<td style="width:160px;">or other qPCR instrument</td>
		</tr>
	</tbody>
</table>

highly sensitive assay for measurement of arenavirus-cell attachment

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

Para demonstrar a sensibilidade e gama dinâmica do ensaio qPCR, quantidades conhecidas de um plasmídeo que codifica o gene NP de JUNV C # 1 (intervalo 5-5 x 10 7 cópias) foram submetidos a qPCR como descrito na Seção 4 do protocolo. O ensaio é sensível e pode detectar de forma fiável tão poucos como 5 cópias do ácido nucleico alvo (Figura 2). Além disso, a gama dinâmica do ensaio é grande e, como mostrado na Figura 2, pode cobrir...

Watch this Scientific Journal Video about Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment at JoVE.com

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

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

Ensaio altamente sensível para a medição do Anexo de células Arenavirus

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

highly-sensitive-assay-for-measurement-of-arenavirus-cell-attachment

Research

JoVE Journal

Immunology and Infection

9.5K visualizações.  University of Vermont. The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

Vídeo: Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with causing food- and water-borne disease outbreaks. The definitive host is the feline species where sexual replication occurs resulting in the development of the highly infectious and environmentally resistant oocyst. Infection occurs via ingestion of tissue cysts from contaminated meat or oocysts from soil or water. Infection is typically asymptomatic in healthy individuals, but results in a life-long latent infection that can reactivate causing toxoplasmic encephalitis and death if the individual becomes immunocompromised. Meat contaminated with T. gondii cysts have been the primary source of infection in Europe and the United States, but recent changes in animal management and husbandry practices and improved food handling and processing procedures have significantly reduced the prevalence of T. gondii cysts in meat1, 2. Nonetheless, seroprevalence in humans remains relatively high suggesting that exposure from oocyst contaminated soil or water is likely. Indeed, waterborne outbreaks of toxoplasmosis have been reported worldwide supporting the theory exposure to the environmental oocyst form poses a significant health risk3-5. To date, research on understanding the prevalence of T. gondii oocysts in the water and environment are limited due to the lack of tools to detect oocysts in the environment 5, 6. This is primarily due to the lack of efficient purification protocols for obtaining large numbers of highly purified T gondii oocysts from infected cats for research purposes. This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation7.

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation

Obtenção altamente purificada Toxoplasma gondii Oocistos por um gradiente de cloreto de césio descontínuos

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with ...

Imunologia e Infecção

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase 1, 2. Manual FV assays have been described 3, 4, but they are time consuming and subjective. Automated FV assays have been reported 5-7, but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput 8, 9. Microplate assays have been reported for clot lysis 10, platelet aggregation 11, and coagulation Factors 12, but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma.
This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405nm during fibrin formation in human plasma (Figure 1) 13. The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only &mu;l quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity).
Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections 14. DIC is associated with a poor prognosis and increases mortality above the pre-existing pathology 15. The assay was used to show that in 9 patients with DIC, the FV 1-stage, 2-stage, and total activities were decreased, on average, by 54%, 44%, and 42%, respectively, compared with normal pooled human reference plasma (NHP).
The FV microplate assay is easily adaptable to measure the activity of any coagulation factor. This assay will increase our understanding of FV biochemistry through a more accurate and complete measurement of its activity in research and clinical settings. This information will positively impact healthcare environments through earlier diagnosis and development of more effective treatments for coagulation disorders, such as DIC.

This study describes a novel microplate assay that measures FV coagulation activity during fibrin clot formation in human plasma which has not been reported previously. The method uses a kinetic microplate reader to continuously measure the change in absorbance at 405nm during fibrin clot formation in human plasma.

Medição da actividade de factor V no plasma humano, utilizando um ensaio de coagulação de Microplacas

 In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin ...

Plaque Assay for Murine Norovirus

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3.
Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4.
In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.

Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV&#x2019;s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.

Ensaio de placas para Norovirus Murine

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

Medição quantitativa da resposta imune e do sono em<em&gt; Drosophila</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

Altamente Resolvido Microscopia intravital Striped-iluminação dos centros germinativos

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

A transfecção altamente eficiente de THP-1 por macrófagos humanos Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 &#176;C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.

We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.

Medindo Anexo e Internalização do Vírus da Influenza A em células A549 por citometria de fluxo

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

Desenvolvimento de uma mais sensível e específica Cromogênico Agar para a detecção de<em&gt; Vibrio parahaemolyticus</em&gt; E outro<em&gt; Vibrio</em&gt; Espécies

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

Medição de<em&gt; In Vitro</em&gt; Actividade integração do HIV-1 de pré-integração Complexos

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.