RESUMO
We explored the possibilities for purification of various ΔNS1 live, replication deficient influenza viruses on ion exchange methacrylate monoliths. Influenza A ΔNS1-H1N1, ΔNS1-H3N2, ΔNS1-H5N1 and ΔNS1-influenza B viruses were propagated in Vero cells and concentrated by tangential flow filtration. All four virus strains adsorbed well to CIM QA and CIM DEAE anion exchangers, with CIM QA producing higher recoveries than CIM DEAE. ΔNS1-influenza A viruses adsorbed well also to CIM SO3 cation exchanger at the same pH, while ΔNS1-influenza B virus adsorption to CIM SO3 was not complete. Dynamic binding capacity (DBC) for CIM QA, DEAE and SO3 methacrylate monoliths for influenza A ΔNS1-H1N1 virus were 1.9E+10 TCID50/ml, 1.0E+10 TCID50/ml and 8.9E+08 TCID50/ml, respectively. Purification of ΔNS1 viruses on CIM QA was scaled up and reproducibility was confirmed. Yields of infectious virus on CIM QA were between 70.8±32.3% and 87±30.8%. Total protein removal varied from 93.3±0.4% to 98.6±0.2% and host cell DNA removal efficiency was ranging from 76.4% to 99.9% and strongly depended on pretreatment with deoxyribonuclease.
Assuntos
Cromatografia por Troca Iônica/métodos , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Vírus da Influenza B/isolamento & purificação , Adsorção , Animais , Chlorocebus aethiops , Filtração , Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza A Subtipo H3N2/fisiologia , Virus da Influenza A Subtipo H5N1/fisiologia , Vírus da Influenza B/fisiologia , Células Vero , Cultura de Vírus , Replicação ViralRESUMO
Rotaviruses are the leading cause of gastroenteritis in children and they exist widely in water environments. Ingestion of 10-100 viral particles is enough to initiate disease, what calls for extremely sensitive detection methods. In this study we have confirmed the validity of a recently published method for rotavirus concentration and detection based on the combination of methacrylate monoliths and real-time reverse transcription-quantitative PCR (RT-qPCR). The method was used to concentrate rotaviruses from different tap water and environmental water samples collected in Slovenia within years 2007 and 2009. The performance of virus concentration using monolithic supports was improved in comparison to the one of tangential ultrafiltration upon application of both methods on a range of environmental samples. Several samples were successfully concentrated on-site after successful adaptation of the method to field requirements. In such on-site format, the combination of concentration using CIM and detection using RT-qPCR detected as low as 30 rotavirus particles/ml, spiked in an environmental water sample.
Assuntos
Metacrilatos/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Rotavirus/isolamento & purificação , Virologia/métodos , Microbiologia da Água , Cromatografia Líquida/métodos , Reprodutibilidade dos Testes , Rios/virologia , Cloreto de Sódio , Abastecimento de Água/análiseRESUMO
Human enteric viruses are detected frequently in various types of environmental water samples, such as irrigation water, wastewater, recreational water, ground or subsurface water and even drinking water, constituting a primary source of gastroenteritis or hepatitis outbreaks. Only a few, but still infective number of viral particles are normally present in water samples, therefore an efficient virus concentration procedure is essential prior to molecular detection of the viral nucleic acid. In this study, a novel chromatographic technology, Convective Interaction Media (CIM) monolithic supports, were optimized and applied to the concentration of hepatitis A virus (HAV) and feline calicivirus (FCV), a surrogate of norovirus (NoV), from water samples. Two-step real-time RT-qPCR was used for quantitation of the virus concentration in the chromatographic fractions. Positively charged CIM QA (quaternary amine) monolithic columns were used for binding of HAV and FCV present in previously inoculated 1.5 l bottled water samples. Column bound viruses were eluted from the monolith using 1M NaCl to a final volume of 15 ml. Elution volume was concentrated further by ultracentrifugation. When the CIM/ultracentrifugation method was compared with another concentration method employing positively charged membranes and ultrafiltration, the recovery of HAV was improved by approximately 20%.
Assuntos
Calicivirus Felino/isolamento & purificação , Cromatografia , Água Doce/virologia , Vírus da Hepatite A Humana/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ultracentrifugação , Aminas/metabolismo , Animais , Calicivirus Felino/genética , Calicivirus Felino/metabolismo , Gatos , Cromatografia/instrumentação , Cromatografia/métodos , Microbiologia de Alimentos , Vírus da Hepatite A Humana/genética , Vírus da Hepatite A Humana/metabolismo , Humanos , RNA Viral , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Microbiologia da ÁguaRESUMO
Rotaviruses are the leading cause of diarrhoea in infants around the globe and, under certain conditions they can be present in drinking water sources and systems. Ingestion of 10-100 viral particles is enough to cause disease, emphasizing the need for sensitive diagnostic methods. In this study we have optimized the concentration of rotavirus particles using methacrylate monolithic chromatographic supports. Different surface chemistries and mobile phases were tested. A strong anion exchanger and phosphate buffer (pH 7) resulted in the highest recoveries after elution of the bound virus with 1M NaCl. Using this approach, rotavirus particles spiked in 1l volumes of tap or river water were efficiently concentrated. The developed concentration method in combination with a real time quantitative polymerase chain reaction assay detected rotavirus concentrations as low as 100 rotavirus particles/ml.
