Gostaríamos de agradecer a S. Werner para fornecer os corvos utilizados neste estudo e USDA, APHIS, WS, NWRC equipe de cuidados de animais para cuidar dos animais e monitoramento. Mencione ou utilização de um produto não implica o endosso do USDA. O financiamento para este estudo foi fornecido pelo USDA, APHIS, Serviços Veterinários.

Nosso protocolo foi adaptado de um nós publicado anteriormente 7. Todos os procedimentos envolvendo animais foram aprovados pelo Comitê do Departamento de Agricultura dos EUA (USDA), de animais e plantas Serviço de Inspeção de Saúde (APHIS), Wildlife Services (WS), Centro Nacional de Pesquisa da Vida Selvagem (NWRC) Institutional Animal Care e Use. 1. Corvo Gavaging <ol><li> Estimar o tempo de passagem do &#39;material cerebral pseudo&#39; através do trato digestivo de corvos americanos. <ol><li> Misture 5 ml de cozidos e ovos mexidos inteiro com corante azul e gavage um corvo com um 2 em agulha gavage (Figura 1). </li><li> Verifique corvo a cada 30 minutos até que azul / fezes verdes com manchas não estão mais sendo excretado. </li></ol></li><li> Obter RML (Chandler tensão) infectados C57BL / 6 cérebros não infectados e doentes terminais do mouse. </li><li> Gerar a 20% peso / volume normal e infectado homogeneizado mouse-cérebro em um homogeneizador de bala liquidificador com 1xsalina tamponada com fosfato (PBS) e esferas de vidro, em seguida, centrifugar a 3.000 g durante 1 min para remover as partículas maiores. Remover o sobrenadante e dilui-se com um volume igual de PBS 1x para gerar a 10% em peso / volume em PBS estéril. Congelar a -80 ° C até ser necessário. </li><li> 17 horas antes da gavage remover comida, mas não de água, a partir de penas de galinha. </li><li> Aleatoriamente alocar corvos para os grupos de tratamento e por via oral gavage cada corvo com 5 ml de homogeneizado normal ou infectado cérebro do rato usando um 2 em agulha gavage. </li><li> Transferência de corvos para gaiolas individuais e coletar e agrupar todas as fezes dentro de cada gaiola para 4 post hr gavage (Figura 2). </li><li> Homogeneizar fezes reunidas para cada corvo num homogeneizador bala misturador com esferas de vidro, até uma textura uniforme. * Fezes galinha são na maior parte de líquido e pode ser misturado facilmente. </li><li> Para cada corvo, diluir a 500 ul de homogeneizado fecal em 9,5 ml de 1x PBS para um volume total de 10 ml. </li><li> Centrifugar a homogena fecalte durante 15 minutos a 1400 xg e extrair o sobrenadante. </li><li> Para minimizar o risco de uma infecção microbiana secundária, tratar o sobrenadante com 1 ml de 100 unidades / ml de penicilina e 100 ug / ml de estreptomicina (Invitrogen, EUA) por cada 100 mL de homogeneizado, em seguida, colocar sob luz UV a temperatura ambiente durante 20 min para reduzir risco de contaminação viral e bacteriana. Após a exposição à radiação UV, as amostras sonicate em 3000 um Mp sonicador em configuração 70 para 30 segundos para romper a membrana de qualquer micróbio restantes. </li></ol> 2. Rato Inoculação <ol><li> Aleatoriamente alocar ratos para os seguintes grupos de tratamento (Tabela 1): <ol><li> Grupo 1 - camundongos positivos do tratamento inoculado por via intraperitoneal (IP) com 1 ml de homogeneizado fecal de corvos oralmente tratadas por gavage com 5ml de camundongo infectado cérebros. </li><li> Grupo 2 - Negativo camundongos inoculados tratamento IP com 1 ml de homogeneizado fecal de corvos oralmente tratadas por gavage com 5ml cérebros de camundongos normais. </li><li> Diluir infected e normal homogeneizado cérebro do rato para Grupos 3 e 4 a 1:100 w / v em 1x PBS. </li><li> Grupo 3 - murganhos de controlo positivo IP inoculados com 1 ml de homogeneizado de rato infectado-cérebro. </li><li> Grupo 4 - Negativo ratinhos de controlo inoculados IP com 1 ml de homogeneizado de cérebro de rato normal-. </li></ol></li></ol><table border="1"><tbody><tr><td></td><td> Grupo de Tratamento </td><td> Número de animais </td></tr><tr><td> Grupo 1 </td><td> Scrapie + Corvo Fezes </td><td> 100 </td></tr><tr><td> Grupo 2 </td><td> Scrapie - Corvo Fezes </td><td> 25 </td></tr><tr><td> Grupo 3 </td><td> Scrapie + Mouse Cérebro </td><td> 10 </td></tr><tr><td> Grupo 4 </td><td> Scrapie - Rato Cérebro </td><td> 5 </td></tr></tbody></table> Estatuto Tabela 1 Scrapie de inóculo (+ positivo, negativo -). Eo número de animais utilizados 7. <ol start="2"><li> Intraperitoneal inocular mouse: <ol><li> Scruff rato por dorsal pele do pescoço com o polegar eo dedo indicador e rode suavemente para expor o lado ventral. </li><li> Elevar extremidade posterior do mouse assim que a cabeça é um pouco menor. </li><li> Insira 25 G agulha através de um centímetro de pele, um centímetro lateral da linha média, e 1-2 cm anterior à articulação sacro-ilíaca, utilizando uma seringa de agulha de bloqueio. </li><li> Injectar 1 ml inocular lentamente rato cavidade do corpo. </li></ol></li></ol> 3. Rato de Monitoramento <ol><li> Monitorar os ratos diariamente até que eles expressam sinais clínicos de rato scrapie. Os sinais clínicos podem incluir: cifose, ataxia, rabo duro, falta de higiene, emagrecimento e letargia. </li><li> Pontuação ratos para cada um dos seis sinais clínicos, quando os sinais visíveis são evidentes, em que 0 = nenhum visível, 1 = moderado, 2 = e grave. </li><li> Eutanásia ratos quando escores totais diárias para cada signo atingir ≥ 8 para 1 dia, ≥ 6 continuamente por 3 dias, ou aos 365 dias após a inoculação (dpi). </li><li> Cérebros colheita imediatamente após euthanÁsia e armazenar a -70 ° C. </li><li> Para confirmar um diagnóstico de scrapie, digerir as amostras de cérebro com 3 mL de um 50 ug / ml de solução de proteinase K (PK) diluídos como se segue: 3.1. ul PK, 12,5 uL de EDTA 500 mM, pH 8, 109,39 ul de 1x PBS durante 30 min a 45 ° C, em seguida, desactivar o PK adicionando 8 ul de tampão de carga e incubando amostras a 95 ° C durante 5 min. Carregar as amostras num gel de SDS-PAGE a 12%, electroforese e transferência para PVDF Immobilon membrana e bloqueio com 5% de leite desnatado em 0,2% de Tween 20 em PBS durante 1 hora à temperatura ambiente. Em seguida sondar com Bar224 anticorpo monoclonal anti-PRP conjugado com peroxidase de rábano, diluído em Superblock, durante 1 hora à temperatura ambiente. Lavar membrana durante 1 hora com PBS-Tween 20 a 0,2%. Para visualizar, incubar Western blot 5 min com substrato quimioluminescente e imagem em um sistema de documentação de gel G-box. </li></ol>

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Nós demonstramos um procedimento para documentar a passagem de material de PrP Res através do sistema digestivo de corvos. Usamos métodos convencionais para determinar se os corvos têm a capacidade de translocação de material PrP Res para áreas geográficas livres da doença. Outros avaliaram a resistência de PrP Res para ruminantes 19-21 e roedores 22,23 fluidos digestivos, sendo que ambos não conseguiram eliminá-lo. A futura aplicação destas técnicas devem ser aplicadas a outros carnívoros 24 como eles também poderiam encontrar material PrP Res em carniça e transportar este material em toda a paisagem facilitando a propagação de doenças de príon. Nós adicionamos corante azul para o nosso ovos mexidos material &quot;pseudo cérebro &#39;, fornecendo uma maneira simples de avaliar o tempo aproximado de passagem cérebro homogeneizado do mouse através do trato digestivo dos corvos. Aconselhamos os outros, considerando que esta técnica primeiros insPect existente cor das fezes dos animais de estudo e, em seguida, usar corante alimentício que é mais contrastantes. Isto irá permitir a fácil identificação de cor alimento material manchado que passou por animais do estudo. Optamos por usar corante alimentar para estimar o tempo de passagem, mas outros autores utilizaram o pigmento fluorescente 25, óxido férrico 26, os marcadores de plástico colorido 27, e flocos metálicos coloridos (ie brilho) 28. Minimizar a ameaça de uma infecção microbiana secundário a partir de fezes de galinha e inoculando corretamente os ratos são pontos de partida fundamentais para estudos de mouse-prião que requerem longos períodos de incubação. Se qualquer uma dessas considerações são violados, mortalidade precoce do mouse é provável que resulte. Outra ferramenta possível para a investigação é a proteína de série misfolding amplificação cíclica ou sPMCA para avaliar amostras de fezes de galinha ao longo do tempo para estabelecer quanto tempo depois corvos sonda oral passar material infeccioso, coma necessidade de um bioensaio em ratos. Os recentes avanços na análise fecal PMCA por Pulford, et al. Indicam que a amplificação dos níveis de príons minutos pode ser detectada em mamíferos fezes 29, sugerindo PMCA também pode ser uma ferramenta útil para a avaliação de fezes de aves. O bioensaio e sPMCA abordagem poderia ser utilizado para avaliar a infecciosidade residual nas fezes dos corvos tratadas por gavage com materiais infectados com CWD. Uma linha de camundongo transgênico cervidized seria necessário e intracerebral contra inoculação intraperitoneal daria resultados mais rápidos. Catadores Avian, como corvos, abutres e águias, poderia desempenhar um papel na propagação de doenças TSE, ou seja, CWD na América do Norte. Estas espécies podem consumir tecido CWD-positivo a partir de carcaças doentes ou vísceras (no caso dos cervídeos matou-caçador) e translocar material infeccioso em suas fezes para áreas livres de CWD ou populações de cervídeos. Como a prática da alimentação de grãos para cervídeos, em natureza ou em cativeirodefinições, atrai corvos que pode defecar sobre a fonte de alimento e ser inadvertidamente consumidas por cervídeos, é, portanto, uma prática de alto risco (Vercauteren observação pessoal). Além disso, embora as chances de animais livres de CWD encontrando material de PrP Res através das fezes deposição aleatória pode ser baixo, áreas onde corvos e, portanto, suas fezes estão concentrados, como abaixo locais roosting comuns, poderia tornar-se de áreas de alto risco para a transmissão da doença desde prions são tão persistentes no ambiente 30,31.

As encefalopatias espongiformes transmissíveis (EET) são desordens neurodegenerativas fatais infecciosas que afetam a vida selvagem, os animais domésticos e seres humanos. O agente infeccioso de doenças TSE parece ser isoformas deformadas ou patogênicos (PrP Res) de uma proteína príon. EET animal incluem doença debilitante crônica (CWD) em veados (Odocoileus hemionus), veados de cauda branca (Odocoileus), alces (Cervus elaphus) e alce (Alces alces); scrapie em ovinos e caprinos; encefalopatia espongiforme bovina ( BSE) em gado doméstico; encefalopatia mink transmissível em mink viveiro; encefalopatia espongiforme felina em gatos; exótico encefalopatia espongiforme ungulados em exótico jardim zoológico rumina da família dos bovídeos, e encefalopatia espongiforme em primatas não humanos 8. A doença TSE humano único, a doença de Creutzfeldt-Jakob variante, é raro e pensado para ser adquirida através do consumo de PrP Res-contaminalimentos ciado 9. Da mesma forma, a EEB pode infectar os seres humanos se carne contaminada é consumida 10. De todas as doenças TSE, scrapie e CWD são os dois únicos com epidemias auto-sustentáveis ​​e fontes de infecção se presume ser de animal para animal 2,3,11, bem como a partir de fontes ambientais 4-6. A pesquisa sugere que a maioria das doenças TSE exigem períodos de incubação prolongados notáveis ​​de eventos de exposição naturais de PrP Res a manifestação de sinais clínicos 2-4,6,8 e barreiras de espécies aparentes minimizar, mas não eliminar o potencial de, a transmissão entre espécies 12-14 . Identificar mecanismos para a propagação do príon infeccioso (PrP Res) o material é extremamente importante para responder a perguntas sobre como as doenças TSE mover por toda a paisagem. Estudos experimentais têm sugerido que os insetos 15,16, aves e suínos 17, e os corvos-americanos (Corvus brachyrhynchos) 7,18 são portadores passivos ou dispersores de material PrP Res. A passagem de material de PrP Res através do sistema digestivo de corvos foi recentemente documentado, demonstrando o papel que podem desempenhar na dispersão de doenças TSE 7. Estes resultados tornam plausível que corvos, um limpador, poderia encontrar, consumir e transportar o material infeccioso via deposição de fezes, para áreas livres da doença. Os procedimentos que demonstram aqui foram usados ​​para documentar a passagem de material PrP Res através do sistema de digestão de corvos e vai facilitar muito a aplicação destes métodos para outro limpador e modelos específicos de espécies de carnívoros em futuras pesquisas relacionadas. Neste estudo foram utilizados os métodos convencionais para investigar um meio não-convencionais de tráfico de material PrP Res, o que poderia contribuir para a disseminação e carga global de material PrP Res.

<table border="0" cellpadding="0" cellspacing="0" width="545">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">RML Chandler strain mouse-adapted scrapie</td>
			<td style="width:112px;">Rocky Mountain Laboratories</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">RC57BL/6 mice</td>
			<td style="width:112px;">Hilltop Lab Animals</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">American crows</td>
			<td style="width:112px;">wild captured</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Pen/Strep</td>
			<td>Invitrogen</td>
			<td style="width:137px;">15140-122</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Phosphate buffered Saline</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">70011-044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Sonicator</td>
			<td>Misonix</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Proteinase-K solution</td>
			<td style="width:112px;">Roche</td>
			<td align="right" style="width:137px;">3115887001</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Loading buffer</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">NP0007 and 0009</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Bis-tris SDS PAGE 12% gel</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">NP0342</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Immobilon PVDF membrane</td>
			<td style="width:112px;">Millipore</td>
			<td style="width:137px;">1SEQ00010</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Tween 20</td>
			<td style="width:112px;">Sigma Aldrich</td>
			<td style="width:137px;">P2287</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Bullet blender homogenizer</td>
			<td style="width:112px;">Braintree Scientific</td>
			<td style="width:137px;">BBX24B</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">2.3 mm Zirconia/silica beads</td>
			<td style="width:112px;">BioSpec Products</td>
			<td style="width:137px;">11079125Z</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Bar224 anti-PrP monoclonal antibody</td>
			<td style="width:112px;">Cayman Chemical</td>
			<td align="right" style="width:137px;">10009035</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Superblock</td>
			<td style="width:112px;">Thermo Scientific</td>
			<td align="right" style="width:137px;">37517</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">chemiluminescent substrate</td>
			<td style="width:112px;">Millipore</td>
			<td style="width:137px;">WBKLS0500</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">G-box gel documentation system</td>
			<td style="width:112px;">Syngene</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

procedures for identifying infectious prions after passage through the digestive system of an avian species

Material de príon infeccioso (PrP Res) é provável que a causa da fatal, neurodegenerativa Encefalopatia Espongiforme Transmissível (EET) doenças 1. A transmissão de doenças TSE, tais como a doença debilitante crónica (CWD), presume-se ser de animal para animal de 2,3, bem como a partir de fontes ambientais 4-6. Catadores e carnívoros têm potencial para translocação de material PrP Res através do consumo e excreção de carniça contaminados com CWD. Trabalhos recentes tem documentado passagem de material PrP Res através do sistema digestivo dos corvos-americanos (Corvus brachyrhynchos), um limpador norte-americano comum 7. Nós descrevemos os procedimentos utilizados para documentar a passagem de material PrP Res através corvos americanos. Crows foram tratadas por gavage com RML-deformação scrapie adaptado do rato e suas fezes foram coletadas 4 post hr gavage. Fezes galinha foram em seguida reunidas e injectadas por via intraperitoneal emC57BL / 6. Ratos foram monitorados diariamente até que eles manifestaram sinais clínicos de rato scrapie e foram, posteriormente, submetidos à eutanásia. Camundongos assintomáticos foram monitoradas durante 365 dias após a inoculação. Análise de Western blot foi realizada para confirmar o estado da doença. Os resultados revelaram que os príons infecciosos permanecer depois de viajar através do sistema digestivo de corvos e estão presentes nas fezes, causando doença em ratos de teste.

Os procedimentos utilizados demonstram que o sistema digestivo do corvos não elimina PrP Res. infecciosidade 4 h após gavagem oral de homogeneizado de cérebro scrapie 7. Todos os vinte corvos que foram tratadas por gavage com material PrP Res posteriormente transmitidos material de PrP Res via fezes de ratos. Ratos doentes foram identificados pela manifestação dos sinais rato-scrapie clínicos e confirmação da doença foi completada por análise de Western blot. Investigação do tempo de retenção de material ingerido por corvos revelou tempo de passagem através do tracto digestivo de um corvo tratadas por gavage a ser de 4 h, com base na presença de corante nas fezes (Figura 1). Todas as fezes foram coletadas com pipetas descartáveis ​​e agrupados para cada corvo (Figura 2). Toxicidade aguda a partir de fezes de galinha não tratados ocorreu quando sobrenadante fecal-prima foi injetado em ratos IP de teste-piloto. Ao tratar inocular fecal com penicilina e streptomycin, luz UV, e sonificação que aliviou este problema. Todos os ratinhos inoculados com ou cérebro de ratinho do tremor epizoótico-positivo (9/9) ou a partir de fezes corvos inoculados com scrapie (66 * / 84) desenvolveram sinais clínicos e testaram positivo por análise de Western blot (Figura 3), que ilustra que o tremor epizoótico prontamente passada através do sistema digestivo de corvos e doença causada. * Dezoito ratos morreram no prazo de 3 dias da inoculação, provavelmente devido a toxicidade. Todos, exceto um (1/23) camundongos inoculados scrapie-negativos foram negativos pela análise de Western blot. Nossa hipótese é que este rato foi inadvertidamente inoculadas com fezes de galinha scrapie-positivas em vez de fezes de um corvo scrapie-negativo. <img alt="Figura 1" fo:content-width="6in" src="/files/ftp_upload/50853/50853fig1.jpg" width="500px" /> Figura 1. Corvo restringido manualmente e tratadas por gavage oral com 5 ml de ovo inteiro misturadas com corante azul (A) e blue / fezes verde manchada de 4 post hr gavage (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50853/50853fig1highres.jpg" target="_blank">Clique aqui para ver a imagem ampliada</a> . <img alt="Figura 2" fo:content-width="6in" src="/files/ftp_upload/50853/50853fig2.jpg" width="500px" /> Figura 2. Coleta de fezes Corvo com pipeta antes da homogeneização. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50853/50853fig2highres.jpg" target="_blank">Clique aqui para ver a imagem ampliada</a> . <img alt="Figura 3" fo:content-width="6in" src="/files/ftp_upload/50853/50853fig3.jpg" width="500px" /> Figura 3. . Representante SDS-página Western blot do cérebro do rato a partir de bioensaios homogeneizado NBH normal cérebro do rato, scrapie-negativos, TX gaiola 4 - cérebro de um camundongo inoculados com fezes de um corvo inoculados com scrapie. Com (+) e without (-). PK (proteinase K) digestão <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50853/50853fig3highres.jpg" target="_blank">Clique aqui para ver a imagem ampliada</a> .

Watch this Scientific Journal Video about Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species at JoVE.com

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Catadores têm potencial para translocam infecciosas transmissíveis prions encefalopatia espongiforme em suas fezes para áreas livres da doença. Nós métodos de detalhes usados ​​para determinar se prions de scrapie mouse adaptados permanecer infeccioso após a passagem que o trato digestivo dos corvos-americanos (Corvus brachyrhynchos), o consumidor comum de animais mortos.

Procedimentos para identificar Infecciosas príons após a passagem através do sistema digestivo de uma espécie de aves

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

procedures-for-identifying-infectious-prions-after-passage-through

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

11.0K visualizações.  USDA.  Catadores têm potencial para translocam infecciosas transmissíveis prions encefalopatia espongiforme em suas fezes para áreas livres da doença. Nós métodos de detalhes usados ​​para determinar se prions de scrapie mouse adaptados permanecer infeccioso após a passagem que o trato digestivo dos corvos-americanos (Corvus brachyrhynchos), o consumidor comum de animais mortos. 

Vídeo: Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of pleural effusion lymphomas (PEL) and some forms of multicentric Castleman's disease.3-5 Only 1-5% of cells in KS lesions actively support lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent associated with KS, and it is clear that cellular factors must interact with viral factors in the process of oncogenesis and tumor progression.6,7 Identifying novel host-factor determinants which contribute to KS pathology is essential for developing prognostic markers for tumor progression and metastasis as well as for developing novel therapeutics for the treatment of KS.8 The accompanying video details the methods we use to identify host cell gene expression programs altered in dermal microvascular endothelial cells (DMVEC) after KSHV infection and in KS tumor tissue.9 Once dysregulated genes are identified by microarray analysis, changes in protein expression are confirmed by immunoblot and dual labeled immunofluorescence. Changes in transcriptional expression of dysregulated genes are confirmed in vitro by quantitative real-time polymerase chain reaction (qRT-PCR). Validation of in vitro findings using archival KS tumor tissue is also performed by dual labeled immunochemistry and tissue microarrays.8,10 Our approach to identifying dysregulated genes in the KS tumor tissue microenvironment will allow the development of in vitro and subsequently in vivo model systems for discovery and evaluation of potential novel therapeutic for the treatment of KS.

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus KSHV

Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.

Identificação de genes desregulados Induzida pelo Herpesvirus sarcoma de Kaposi-associado (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

Imunologia e Infecção

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

Desenvolvimento de um marcador negativo selecionáveis ​​para Entamoeba histolytica</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

Visualizando Vírus da Dengue por meio Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

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

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

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

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

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

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor treatment options exist. The hallmarks of these laboratories are: custom-designed airtight doors, dedicated supply and exhaust airflow systems, a negative-pressure environment, and mandatory use of positive-pressure (&#8220;space&#8221;) suits. The risk for laboratory specialists working with highly pathogenic agents is minimized through rigorous training and adherence to stringent safety protocols and standard operating procedures. Researchers perform the majority of their work in BSL-2 laboratories and switch to BSL-4 suit laboratories when work with a high-consequence pathogen is required. Collaborators and scientists considering BSL-4 projects should be aware of the challenges associated with BSL-4 research both in terms of experimental technical limitations in BSL-4 laboratory space and the increased duration of such experiments. Tasks such as entering and exiting the BSL-4 suit laboratories are considerably more complex and time-consuming compared to BSL-2 and BSL-3 laboratories. The focus of this particular article is to address basic biosafety concerns and describe the entrance and exit procedures for the BSL-4 laboratory at the NIH/NIAID Integrated Research Facility at Fort Detrick. Such procedures include checking external systems that support the BSL-4 laboratory, and inspecting and donning positive-pressure suits, entering the laboratory, moving through air pressure-resistant doors, and connecting to air-supply hoses. We will also discuss moving within and exiting the BSL-4 suit laboratories, including using the chemical shower and removing and storing positive-pressure suits.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Although researchers are generally knowledgeable about procedures and safety precautions required for biosafety level 1 or 2 (BSL-1/2) experiments, they may not be familiar with experimental procedures in BSL-4 suit laboratories. This article provides a detailed visual demonstration of BSL-4 suit laboratory systems check, laboratory entry, movement, and exit procedures.

Precauções de segurança e Procedimentos Operacionais em um (A) BSL-4 laboratoriais: 1. Nível de Biossegurança 4 Suit Laboratório Suíte entrada e saída Procedimentos

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 2. General Practices

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory with non-high-consequence pathogens will take significantly longer in a BSL-4 setting. This increased time requirement is due to a multitude of factors that are aimed at protecting the researcher from laboratory-acquired infections, the work environment from potential contamination and the local community from possible release of high-consequence pathogens. Inside the laboratory, movement is restricted due to air hoses attached to the mandatory full-body safety suits. In addition, disinfection of every item that is removed from Class II biosafety cabinets (BSCs) is required. Laboratory specialists must be trained in the practices of the BSL-4 laboratory and must show high proficiency in the skills they are performing. The focus of this article is to outline proper procedures and techniques to ensure laboratory biosafety and experimental accuracy using a standard viral plaque assay as an example procedure. In particular, proper techniques to work safely in a BSL-4 environment when performing an experiment will be visually emphasized. These techniques include: setting up a Class II BSC for experiments, proper cleaning of the Class II BSC when finished working, waste management and safe disposal of waste generated inside a BSL-4 laboratory, and the removal of inactivated samples from inside a BSL-4 laboratory to the BSL-2 laboratory.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 2. General Practices

Performing viral assays in a BSL-4 laboratory is more involved compared to work in a BSL-2 laboratory due to required additional safety precautions. Here, we present an overview of practices and procedures used inside a BSL-4 laboratory illustrating proper Class II biosafety cabinet usage, waste management/disposal, and sample removal.

Precauções de segurança e Procedimentos Operacionais em um (A) de laboratório BSL-4: 2. Práticas Gerais

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 4. Medical Imaging Procedures

Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by high-consequence pathogens has not been possible. In 2014, the National Institutes of Health, National Institute of Allergy and Infectious Diseases, Integrated Research Facility at Fort Detrick opened an Animal Biosafety Level 4 (ABSL-4) facility to assess the clinical course and pathology of infectious diseases in experimentally infected animals. Multiple imaging modalities including computed tomography (CT), magnetic resonance imaging, positron emission tomography, and single photon emission computed tomography are available to researchers for these evaluations. The focus of this article is to describe the workflow for safely obtaining a CT image of a live guinea pig in an ABSL-4 facility. These procedures include animal handling, anesthesia, and preparing and monitoring the animal until recovery from sedation. We will also discuss preparing the imaging equipment, performing quality checks, communication methods from "hot side" (containing pathogens) to "cold side," and moving the animal from the holding room to the imaging suite.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 4. Medical Imaging Procedures

Here, we present an overview of the preparation and animal handling procedures required to safely perform medical imaging in an animal biosafety level 4 laboratory. Computed tomography of a mock-infected guinea pig illustrates these procedures that may be used to evaluate the disease caused by a high consequence pathogen.

Precauções de segurança e Procedimentos Operacionais em um (A) BSL-4 laboratoriais: 4. Procedimentos Médicos de Imagem

Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 3. Aerobiology

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol releases or unexpected natural aerosol transmission. Specific laboratories designed to perform these experiments require tremendous engineering controls to provide a safe and secure working environment and constant systems maintenance to sustain functionality. Class III biosafety cabinets, also referred to as gloveboxes, are gas-tight enclosures with non-opening windows. These cabinets are maintained under negative pressure by double high-efficiency-particulate-air (HEPA)-filtered exhaust systems and are the ideal primary containment for housing aerosolization equipment. A well planned workflow between staff members within high containment from, for instance, an animal biosafety level-4 (ABSL-4) suit laboratory to the ABSL-4 cabinet laboratory is a crucial component for successful experimentation. For smooth study execution, establishing a communication network, moving equipment and subjects, and setting up and placing equipment, requires staff members to meticulously plan procedures prior to study initiation. Here, we provide an overview and a visual representation of how aerobiology research is conducted at the National Institutes of Health, National Institute of Allergy and Infectious Diseases Integrated Research Facility at Fort Detrick, Maryland, USA, within an ABSL-4 environment.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 3. Aerobiology

As high-consequence pathogens can potentially infect subjects through airborne particles, aerobiology has been increasingly applied in pathogenesis research and medical countermeasure development. We present a detailed visual demonstration of aerobiology procedures during an aerosol challenge in nonhuman primates in an animal biosafety level 4 maximum containment environment.

Precauções de segurança e Procedimentos Operacionais em um (A) BSL-4 laboratoriais: 3. aerobiologia

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol ...

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

Virus Zika sistema de cultura celular Infecciosas e do<em&gt; In Vitro</em&gt; Efeito profilático de interferões

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

Isolando os linfócitos de rato pequeno Intestinal sistema imunológico

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...