Name: a small volume procedure for viral concentration from water
Uploaded: 2015-02-03T13:00:00.000+00:00
Duration: 7 min 28 s
Description: Watch this Scientific Journal Video about A Small Volume Procedure for Viral Concentration from Water at JoVE.com

We would like to thank Dr. Nicholas J. Ashbolt and Dr. G. Shay Fout for their review of the manuscript.

1. Preparação de material de vidro e Carcaças de filtro <ol style="margin-left: 40px;"><li> Salvo disposição em contrário, esterilizar todos os copos, caixas de filtro e soluções a 121 ° C por 15 min. Para garantir a esterilidade, cobrir todas as aberturas ou as superfícies expostas, quer com a folha de alumínio ou papel fixada com fita antes da esterilização. </li><li> Montar aparelho de filtração, anexando carcaça do filtro (47 mm de diâmetro) para 1 L Erlenmeyer braço lateral. Recolha filtros necessários: 47 milímetros de diâmetro electropositivos / electronegative filtros de disco. </li><li> Prepare 1 L de 1,5% de extracto de carne de bovino (floculação; produz partículas de flocos quando o pH da solução é reduzido para 3,5, ou não-floculação, o que não agregar a pH 3,5), com 0,05 M de glicina, por dissolução de 15 g de extracto de carne em 1 L de água desionizada e adição de 3,75 g de glicina. NOTA: extractos de carne não-floculantes irá requerer a adição de celite antes da concentração terciária. </li><li> Adicionar eluição polyph aditivo de sódioosphate de extracto de carne a uma concentração de 0,1%. </li><li> Adicionar uma solução de ácido clorídrico M, gota a gota, a mistura de extrato de carne, o pH da solução de pH desejado. Extracto de carne autoclave durante 15-30 min a 121 ° C. </li><li> Meça 0,1 g de pó fino, médio ou grande celite de partículas (para uso com o não-floculação apenas extrato de carne). </li><li> Prepare vácuo / aspiração, anexando tubo compatível para Erlenmeyer de braço lateral e para a saída de vácuo. </li></ol> 2. Preparação de Soluções e vírus Stock <ol style="margin-left: 40px;"><li> Prepare 1 litro de 1 M de HCl. </li><li> Prepare 1 L de NaOH 1M. </li><li> Preparar a solução de PBS 1x (NaCl 137 mM, KCl 2,7 mM, 1,47 mM de K 2 PO 4, e 4,3 mM de NaH 2 PO 4). <ol><li> Ajustar o pH de 1x PBS a 9,0 usando NaOH 1 M. </li></ol></li><li> Prepare e diluir vírus estoque para 10 4 -10 5 número mais provável (NMP) ml -1 misturando 1 ml de vírus estoque com 9 ml de PBS (pH7), como anteriormente descrito 8,13. Armazenar a -80 ° C em alíquotas de 1 ml. <ol><li> Esterilizar estoque de vírus por meio de filtragem seringa (0,22 tamanho dos poros) para remover contaminantes potenciais. </li></ol></li><li> Água da torneira sem cloro <ol><li> Medida 1 L de água da torneira esterilizada utilizando um cilindro graduado estéril e despeje em um copo de 2 L contendo uma barra magnética. </li><li> Adicionar 0,7 g de tiossulfato de sódio e misturar até dissolver grânulos. Ajustar o pH da água da torneira para 7-7,5, se necessário usando 1 M HCl ou solução de NaOH 1 M. </li><li> Spike vírus Thaw (1 ml) e adicionar volume inteiro de pico para 1 L de água sem cloro torneira esterilizada. Mistura-se durante 10 min. </li><li> Preparar um método em branco (sem água declorada adenovírus) com cada experimento e processo da mesma maneira como a amostra enriquecida. </li></ol></li></ol> 3. Preparação de filtro Millipore Aparelho <ol style="margin-left: 40px;"><li> Retirar cobertura estéril sobre caixa do filtro. Garantirbom ajuste entre a caixa do filtro bacia superior e inferior da tela do filtro para evitar a fuga de amostra durante a filtração. </li><li> Retirar cobertura estéril a partir de 1 frasco L Erlenmeyer. Assegure-se que a cortiça da unidade de filtro se encaixa perfeitamente na abertura superior do balão de Erlenmeyer, criar uma boa vedação. </li><li> Remover bacia superior do filtro do filtro de tela menor e coloque 47 milímetros disco filtro electropositive diretamente sobre a tela com uma pinça estéril. Anexar caixa do filtro tigela para a tela de fundo e bloquear no lugar por torção anti-horário. </li></ol> 4. Toque em Filtragem de água (Concentração Primário) <ol style="margin-left: 40px;"><li> Medir 100 ml de vírus cravado água da torneira usando estéril proveta graduada. </li><li> Despeje 100 ml de vírus cravado a água da torneira em carcaça de filtro contendo um filtro de 47 milímetros de vidro plissado / celulose ou de um filtro de disco de fibra nano-alumina / vidro. </li><li> Deixe a água passar lentamente pelo filtro. Aplicar vácuo suave para remover os resíduos restantes oágua da torneira f da superfície do filtro. </li><li> Se utilizando tratamento com tripsina, adicionar 5 ml de tripsina a 0,25% para filtrar superfície e incubar durante 5 min. </li><li> Lavar tripsina da superfície do filtro com 50 ml de água desionizada estéril. </li></ol> 5. Vírus de eluição Filtrar <ol style="margin-left: 40px;"><li> Remover caixa do filtro de 2 balão L Erlenmeyer, e coloque-frasco de 250 ml de braço lateral. </li><li> Medir 100 ml de extrato de carne usando proveta graduada. Juntar cuidadosamente 100 ml de extrato de carne em carcaça de filtro. </li><li> Permitir extrato de carne bovina para derramar através do filtro, captando extrato de carne em um pequeno (250 ml) Erlenmeyer. Aplicar vácuo para puxar através de extrato de carne residual de filtro. </li></ol> 6. Celite Concentração Secundário <ol style="margin-left: 40px;"><li> Frascos de transferência contendo os vírus eluídas a uma placa de agitação. Adicionar estéril barra magnética e lugar na placa de agitação, mistura para criar ligeiro vórtice. </li><li> Adicionar 0,1 g de celiteem pó a 100 ml de extracto de carne e permitir celite para dispersar. Coloque sonda pH estéril em extrato de carne. </li><li> Adicionar gota a gota 1 M HCl de extrato de carne bovina para atingir pH 4.0. Permitir que a mistura lentamente, durante 10 min. </li><li> Colocar 47 milímetros pré-filtro no alojamento do filtro (como descrito anteriormente) utilizando um balão de Erlenmeyer de 2 L. </li><li> Despeje 100 ml de extrato de carne / mistura celite sobre pré-filtro. Permitir extrato de carne bovina para derramar through. Aplique uma leve vácuo para puxar através de extrato de carne residual de filtro. </li><li> Coloque clipe de tubo de metal no final do bico caixa do filtro. Anexar estéril 15 ml tubo de coleta de polipropileno para o clipe tubo de metal. Coloque caixa do filtro de volta para Erlenmeyer. </li><li> Adicionar 5 ml de tampão 1x PBS, pH 9.0 sobre celite recolhido em pré-filtro. </li><li> Permitir PBS a gotejar através de pré-filtro. Aplique uma leve vácuo para puxar através PBS residual em 15 ml tubo de coleta. </li></ol> 7. Floculação Organic Concentração Secundário <ol style="margin-left: 40px;"><li>Transfira os copos contendo o vírus eluída a uma placa de agitação. Adicionar estéril barra magnética a 100 ml de extrato de carne e misture para criar ligeiro vórtice. </li><li> Coloque sonda pH estéril em extrato de carne. </li><li> Adicione 1 HCl M gota a gota a extrato de carne e ajustar o pH para 3,5. Mistura-se durante 30 min. </li><li> Verter o eluído em 250 ml de tubos de centrífuga cónicos e centrifugar a 2500 xg durante 15 min a 4 ºC. </li><li> Deitar fora sobrenadante tomando cuidado para não para não perturbar o sedimento. Ressuspender o sedimento em 5 ml de 1x PBS, pH 9. </li><li> Centrifugar a suspensão a 4000 xg durante 10 min a 4 ºC. Retirar o sobrenadante e descartar o pellet. </li><li> Ajustar o pH a 7-7,5, filtro de esterilização através de filtro de seringa de 0,22 um e congelamento a -80 ° C. </li></ol> 8. Viral Extraction Nucleic Acid <ol style="margin-left: 40px;"><li> Extrai-se os ácidos nucleicos virais utilizando um kit comercial adequado de acordo com as instruções do fabricante. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 9. AdV 40/41 qPCR <ol style="margin-left: 40px;"><li> Use iniciadores e sondas, bem como mistura de reacção e perfis de ciclos térmicos como publicado 8,13. NOTA: O limite de detecção estimado para o ensaio é de cerca de 5 unidades de qPCR por reacção. </li><li> Adicionar componentes de misturas de amplificação por PCR em concentrações como se segue: Tris 10 mM (pH 8,3), KCl 50 mM, 4,5 mM de MgCl 2, 10 mM dNTPs, 10 uM iniciadores e sonda de 1 uM e 0,5 jil de polimerase de modo a que os volumes finais de 45 ul pode ser adicionado ao número adequado de poços no prazo de uma placa de 96 poços de reacção de PCR. </li><li> Carga 45 ul de mistura de amplificação de PCR a cada poço adequado de uma de 96 poços, de reacção rápida placa de PCR. </li><li> Adiciona-se 5 ul de amostra de ADN extraído a poços apropriados da placa de PCR. </li><li> Espelho com vedação da tampa resistente ao calor, girar a placa para mover as gotas para o fundo da PCR poços da placa. </li><li> Definir ciclos de amplificação de PCR como Follows: 95 ° C durante 10 min, seguido de 40 ciclos de 95 ° C durante 15 seg e 60 ° C durante 1 min. </li></ol> 10. Análise de Dados <ol style="margin-left: 40px;"><li> Analisar as medições em triplicado de diluições em série de 5 vezes das amostras distribuidas 1: 5 a 1: 625 de diluição intervalo, a fim de determinar o número mais provável (MPN) das partículas virais, como anteriormente descrito 8,13. </li><li> Analise diluições em série de 5 vezes de picos experimentais adenovirus abrangendo 1: 5 a 1: 15.625 gama de diluição, a fim de determinar o número mais provável (MPN) das partículas virais. </li><li> Usando a calculadora EPA MPN (http://www.epa.gov/nerlcwww/online.html), ajustar a calculadora para contabilizar tipo de diluição (1: 5 ou 1:10), o número de diluições realizadas e número de duplicados de amostras em séries de diluição. </li><li> Realizar log 10 de transformação dos dados, seguido por normalização para uma unidade de volume (por exemplo, 1 ml, 100 ml, etc. </em&gt;). Calcular as recuperações para dada experiência por divisão do número de partículas virais presentes na amostra concentra com o número de partículas virais presentes em pontos experimentais. </li><li> Avaliar o efeito das diferentes variáveis ​​experimentais (por exemplo, diferentes tipos de Celite, gamas de pH, os diferentes tipos de filtros e diferentes técnicas de concentração secundários) sobre recuperações por AdV realizar testes estatísticos paramétricos ou não-paramétricos (por exemplo, teste-t emparelhado, um ou dois -way ANOVA), dependendo da distribuição dos dados e do desenho experimental. </li></ol>

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Filtros electropositiva são úteis na concentração vírus de água; no entanto, estes filtros podem diferir na sua estrutura e composição, que por sua vez poderia alterar a sua eficácia. Para agravar este problema, as estruturas do capsídeo e encargos variam entre cepas de vírus que requerem ser adaptados técnicas de concentração para garantir a recuperação óptima 15. Através de modificações simples das técnicas de concentração existentes (por exemplo, filtros electropositivos, ex...

Vírus entéricos humanos são importantes causadoras doenças transmitidas pela água 1-3, mas são geralmente presentes em números baixos nas águas ambientais contaminados, tornando difícil a sua detecção, sem concentração. Os procedimentos usados ​​para concentrar os vírus incluem, tipicamente, uma etapa de filtração, seguido por eluição do filtro, e concentração secundária do eluato filtro. Um procedimento de filtração comum se baseia em uso de membranas carregadas, tais como filtros electropositivos (recentemente revisados ​​em 4,5). Estes filtros dependem de captura de vírus em suspensão em água usando interacções electrostáticas entre a superfície de filtro (carregado positivamente) e partículas de vírus segmentados (carregada negativamente). Dois filtros electropositivos que estão disponíveis comercialmente confiam nesta tecnologia, os filtros de vidro / celulose e nano-alumina / fibra de vidro. Os custos de filtro de vidro / celulose são até 10 vezes maior do que a nano-alumina / fibra de vidro, o que limita o uso do vidro / cefiltros llulose para monitoramento de vírus de rotina. Estudos recentes concluíram que as diferenças são nominais entre estes dois filtros em recuperação de enterovírus de 6,7 água do ambiente, o que justifica o uso de um filtro alternativa mais barata. Outras opções de filtros, como filtros electronegativos e de fibra de vidro foram estudadas, no entanto, que quer impor ao pré-tratamento da água de nascente (filtros electronegativos) ou não estão disponíveis comercialmente (filtros de fibra de vidro). O desenvolvimento de processos de concentração de vírus tenha focado principalmente na optimização das técnicas de concentração primária (filtros), a fim de melhorar a recuperação de vírus a partir de água. No entanto, os procedimentos de concentração secundários, os quais reduzem o volume de eluente, tipicamente de 1 L para mililitro volumes, também pode ter um impacto significativo sobre a recuperação de vírus 8. Concentração Secundária de vírus entéricos normalmente se baseia em um agente de floculação, como alguns tipos de extrato de carne bovina (floccu orgânicamento) ou floculação leite desnatado 9-12 para remover partículas do vírus a partir de superfícies de filtro. Recentemente, um outro processo de concentração secundária utilizando extracto de carne juntamente com a adição de Celite (partícula fina) mostrou-se promissor para a recuperação de adenovírus, enterovírus, e norovírus 8,13,14. Obras de concentração de celite sob princípios semelhantes aos do método de floculação orgânico em que as partículas de vírus para anexar e são libertados de partículas (ou flocos de celite), alterando o pH da solução de suspensão. As comparações entre estas duas técnicas de concentração secundários foram avaliadas em recuperação de adenovírus cravado (Adv) tipos 40 e 41 8. Este estudo concluiu que as duas técnicas de concentração secundários foram estatisticamente semelhantes em recuperação de adenovírus. No entanto, o método de floculação biológica requer um 30 min. de incubação a pH 3,5, enquanto que a técnica de celite requer uma incubação mais curto (10 minutos) a pH 4,0. O flocculat orgânicaião também requer a utilização de equipamento de laboratório caro (centrífugas), para recolher as partículas de flocos durante a concentração terciária, a técnica de celite em contraste utiliza apenas equipamentos básicos de laboratório (filtração no vácuo) para separar as partículas de celite suspensão. Determinadas combinações de filtros e técnicas de eluição secundárias pode também afectar as recuperações de vírus. Um estudo concluiu que certas combinações de (filtros) electropositivos primárias e secundárias técnicas de concentração (Celite ou floculação orgânica) teve um impacto significativo de recuperação de adenovirus 13. Estes resultados sugerem que a optimização é necessária, a fim de recuperar o vírus alvo de forma óptima a partir de uma dada matriz de água quando se utiliza estas técnicas. Optimization é um demorado, árduo processo muitos pesquisadores evitam ativamente desde inúmeras variáveis ​​serão avaliadas (tipo de filtro / marca, solução de eluição pH, Celite / floculação orgânica). Para este study, foi desenvolvido um processo para identificar as condições óptimas para a concentração de vírus a partir da água utilizando adenovírus humano cravado estirpes 40 e 41. Presumivelmente, uma vez que cada tipo de vírus exibe uma morfologia única e capsídeo cápside carga específica, protocolos de concentração pode necessitar de ser optimizado para cada vírus alvo, a fim de alcançar a recuperação viral ideal. Este estudo fornece uma abordagem para AdV 40 e 41 concentração por: 1) avaliar as recuperações de vírus na água da torneira usando discos de filtro electropositivos seguidos de 2) avaliação de um método de floculação orgânica estabelecida versus a técnica de celite como uma concentração secundária, e 3) avaliação da tampões de eluição para a concentração terciária.

<table><tbody><tr><td>Adenovirus 40 stock</td><td>ATCC</td><td>VR-931</td><td></td></tr><tr><td>Adenovirus 41 stock</td><td>ATCC</td><td>VR-930</td><td></td></tr><tr><td>Sodium Thiosulfate</td><td>Fluka Chemical Co.</td><td>72051</td><td></td></tr><tr><td>Celites #577</td><td>Fluka Chemical Co.</td><td>22142</td><td></td></tr><tr><td>NanoCeram 47 mm</td><td>Argonide</td><td>N/A</td><td></td></tr><tr><td>1MDS 47 mm</td><td>3M</td><td>6408502</td><td></td></tr><tr><td>AP-20 Prefilter 47 mm</td><td>Millipore Corp.</td><td>AP2004700</td><td></td></tr><tr><td>Glycine&amp;nbsp;</td><td>Sigma</td><td>50046-1KG</td><td></td></tr><tr><td>Sodium Polyphosphate</td><td>Acros Organics</td><td>390930010</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>25200</td><td></td></tr><tr><td>PBS</td><td>Sigma</td><td>P5368</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Fisher</td><td>A481-212</td><td></td></tr><tr><td>BBL Beef Extract</td><td>BD Biosciences</td><td>212303</td><td></td></tr><tr><td>Difco Beef Extract</td><td>BD Biosciences</td><td>211520</td><td></td></tr><tr><td>ABI 7900 Real-time PCR system</td><td>ABI</td><td>N/A</td><td></td></tr><tr><td>Stainless Steel Filter Housing</td><td>Millipore Corp.</td><td>XX2004720</td><td></td></tr><tr><td>Blood DNA Extraction Kit</td><td>Qiagen&amp;nbsp;</td><td>51104</td><td></td></tr><tr><td>EPA MPN Calculator</td><td></td><td></td><td>http://www.epa.gov/nerlcwww/online.html</td></tr></tbody></table>

a small volume procedure for viral concentration from water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.

<html> Seleção Celite Três tipos diferentes de celite foram testados antes da selecção da variante com melhor desempenho. Celites com multa de partículas de tamanho médio produzido as maiores recuperações de adenovírus. Uso de celites maiores percentagens de recuperação mais baixas para ambos AdV40 e 41 (gama de 32% -100%) (Figura 1). A recuperação média de adenovirus 40 eram de 144% ± 52% (multa), 115% ± 28% (média) e 82% ± 53% celites (grande) de partícu...

Watch this Scientific Journal Video about A Small Volume Procedure for Viral Concentration from Water at JoVE.com

A Small Volume Procedure for Viral Concentration from Water

An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation.

Um Procedimento volume pequeno para Concentração Viral da Água

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to ...

a-small-volume-procedure-for-viral-concentration-from-water

Research

JoVE Journal

Environment

10.0K visualizações.  U.S. EPA. An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation. 

Vídeo: A Small Volume Procedure for Viral Concentration from Water

Estimating Virus Production Rates in Aquatic Systems

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative estimates of this process is critical as we can then develop better models of microbial community structure and function as well as advance our understanding of how viruses work to alter aquatic biogeochemical cycles. The virus reduction technique allows researchers to estimate the rate at which virus particles are released from the endemic microbial community. In brief, the abundance of free (extracellular) viruses is reduced in a sample while the microbial community is maintained at near ambient concentration. The microbial community is then incubated in the absence of free viruses and the rate at which viruses reoccur in the sample (through the lysis of already infected members of the community) can be quantified by epifluorescence microscopy or, in the case of specific viruses, quantitative PCR. These rates can then be used to estimate the rate of microbial mortality due to virus-mediated cell lysis.

The turnover rate of viruses in marine and freshwater systems can be estimated by a reduction and reoccurrence technique. The data allow researchers to infer rates of virus-mediated microbial mortality in aquatic systems.

Estimar as taxas de produção de vírus em Sistemas Aquáticos

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative ...

Imunologia e Infecção

Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.

Here, we present a comprehensive protocol to assess the organic and inorganic nutrient availability and the abundance and structure of microbial and viral communities in remote marine environments.

Desvendando os jogadores invisíveis no Oceano - Um Guia de Campo para Química da Água e Microbiologia Marinha

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling ...

Ambiente

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. The U.S Environmental Protection Agency&#8217;s (USEPA) Method 1615 was developed with the goal of providing such a standard for measuring Enterovirus and Norovirus in these waters. Virus is concentrated from water using an electropositive filter, eluted from the filter surface with beef extract, and then concentrated further using organic flocculation. Herein we present the protocol from Method 1615 for filter elution, secondary concentration, and measurement of total culturable viruses. A portion of the concentrated eluate from each sample is inoculated onto ten replicate flasks of Buffalo Green Monkey kidney cells. The number of flasks demonstrating cytopathic effects is used to quantify the most probable number (MPN) of infectious units per liter. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. Laboratories must meet defined performance standards. Method 1615 was evaluated by examining virus recovery from reagent-grade and ground waters seeded with Sabin poliovirus type 3. Mean poliovirus recoveries with the total culturable assay were 111% in reagent grade water and 58% in groundwaters.

Here we present a procedure to measure total culturable viruses using the Buffalo Green Monkey kidney cell line. The procedure provides a standardized tool for measuring the occurrence of infectious viruses in environmental and drinking waters.

EPA Método 1615. A medição do Enterovirus e Norovirus Ocorrência na água por cultura e RT-qPCR. II. De ensaio total de Vírus cultiváveis

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

EPA Método 1615. Medição de Enterovirus e Norovirus Ocorrência na água por cultura e RT-qPCR. Parte III. Detecção de vírus por RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

Relação custo-benefício Método de Rastreamento Fonte microbiana, utilizando específicas Vírus Humano e Animal

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

Preparação de DNA viral a partir nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens

The key first step in evaluating pathogen levels in suspected contaminated water is concentration. Concentration methods tend to be specific for a particular pathogen group, for example US Environmental Protection Agency Method 1623 for Giardia and Cryptosporidium1, which means multiple methods are required if the sampling program is targeting more than one pathogen group. Another drawback of current methods is the equipment can be complicated and expensive, for example the VIRADEL method with the 1MDS cartridge filter for concentrating viruses2. In this article we describe how to construct glass wool filters for concentrating waterborne pathogens. After filter elution, the concentrate is amenable to a second concentration step, such as centrifugation, followed by pathogen detection and enumeration by cultural or molecular methods. The filters have several advantages. Construction is easy and the filters can be built to any size for meeting specific sampling requirements. The filter parts are inexpensive, making it possible to collect a large number of samples without severely impacting a project budget. Large sample volumes (100s to 1,000s L) can be concentrated depending on the rate of clogging from sample turbidity. The filters are highly portable and with minimal equipment, such as a pump and flow meter, they can be implemented in the field for sampling finished drinking water, surface water, groundwater, and agricultural runoff. Lastly, glass wool filtration is effective for concentrating a variety of pathogen types so only one method is necessary. Here we report on filter effectiveness in concentrating waterborne human enterovirus, Salmonella enterica, Cryptosporidium parvum, and avian influenza virus.

Glass wool filters have been used to concentrate waterborne viruses by a number of research groups around the world. Here we show a simple approach for constructing glass wool filters and demonstrate the filters are also effective in concentrating waterborne viral, bacterial and protozoan pathogens.

Vidro Filtros de lã para a concentração de vírus e transmitidas pela água Agrícolas patógenos zoonóticos

The key first step in evaluating pathogen levels in suspected contaminated water is concentration.  Concentration methods tend to be specific for a ...

Concentration of Virus Particles from Environmental Water and Wastewater Samples Using Skimmed Milk Flocculation and Ultrafiltration

Water and wastewater-based epidemiology have emerged as alternative methods to monitor and predict the course of outbreaks in communities.&#160;The recovery of microbial fractions, including viruses, bacteria, and microeukaryotes from wastewater and environmental water samples&#160;is one of the challenging steps in these approaches. In this study, we focused on the recovery efficiency of sequential ultrafiltration and skimmed milk flocculation (SMF) methods using Armored RNA as a test virus, which is also used as a control by some other studies. Prefiltration with 0.45 &#181;m and 0.2 &#181;m membrane disc filters were applied to eliminate solid particles before ultrafiltration to prevent the clogging of ultrafiltration devices. Test samples, processed with the sequential ultrafiltration method, were centrifuged at two different speeds. An increased speed resulted in lower recovery and positivity rates of Armored RNA.&#160;On the other hand, SMF resulted in relatively consistent recovery and positivity rates of Armored RNA. Additional tests conducted with environmental water samples demonstrated the utility of SMF to concentrate other microbial fractions. The partitioning of viruses into solid particles might have an impact on the overall recovery rates, considering the prefiltration step applied before the ultrafiltration of wastewater samples. SMF with prefiltration performed better when applied to environmental water samples&#160;due to lower solid concentrations in the samples and thus lower partitioning rates to solids. In the present study, the idea of using a sequential ultrafiltration method arose from the necessity to decrease the final volume of the viral concentrates during the COVID-19 pandemic, when the supply of the commonly used ultrafiltration devices was limited, and there was a need for the development of alternative viral concentration methods.

Virus concentration from environmental water and wastewater samples is a challenging task, carried out primarily for the identification and quantification of viruses. While several virus concentration methods have been developed and tested, we demonstrate here the effectiveness of ultrafiltration and skimmed milk flocculation for RNA viruses with different sample types.

Concentração de Partículas de Vírus de Amostras Ambientais de Água e Efluentes Usando Floculação e Ultrafiltração de Leite Desnatado

Water and wastewater-based epidemiology have emerged as alternative methods to monitor and predict the course of outbreaks in communities.&#160;The ...

Lentivirus Production

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are silenced at the post-transcriptional state. Short hairpin RNAs, precursors to siRNA, can be expressed using lentivirus, allowing for RNAi in a variety of cell types. Lentiviruses, such as the Human Immunodeficiency Virus, are capable to infecting both dividing and non-dividing cells. We will describe a procedure which to package lentiviruses. Packaging refers to the preparation of competent virus from DNA vectors. Lentiviral vector production systems are based on a 'split' system, where the natural viral genome has been split into individual helper plasmid constructs. This splitting of the different viral elements into four separate vectors diminishes the risk of creating a replication-capable virus by adventitious recombination of the lentiviral genome. Here, a vector containing the shRNA of interest and three packaging vectors (p-VSVG, pRSV, pMDL) are transiently transfected into human 293 cells. After at least a 48-hour incubation period, the virus containing supernatant is harvested and concentrated. Finally, virus titer is determined by reporter (fluorescent) expression with a flow cytometer.

To make lentiviruses, DNA vectors are transfected into human 293 cells. After harvest and concentrating the supernatant, virus titer is determined by fluorescence expression with a flow cytometer.

Produção lentivírus

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are ...

Biologia

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

Uma estratégia rápida para o isolamento de novos Faustoviruses de amostras ambientais Usando<em&gt; vermiformis Vermamoeba</em

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...