RESUMEN
Plaque assay method enables the quantification of infectious baculovirus when defined as plaque forming units (PFU). It allows to determine the amount of infectious virus needed to infect the cells at a specific multiplicity of infection (MOI). Serial dilutions of baculovirus stock are added to the Sf9 cells monolayer followed by addition of 5% Agarose overlay. Six days after infection clear infection halos are observed using a neutral red solution. Here we describe the quantification of recombinant baculovirus expression vector (rBEV) carrying a transgene in an rAAV expression cassette. Reproducible quantification of PFU is obtained with this method.
Asunto(s)
Baculoviridae , Vectores Genéticos , Ensayo de Placa Viral , Baculoviridae/genética , Células Sf9 , Ensayo de Placa Viral/métodos , Animales , Vectores Genéticos/genética , Transgenes , Virión/genética , Dependovirus/genética , Spodoptera/virologíaRESUMEN
The plaque reduction neutralization test (PRNT) and the enzyme-linked immunosorbent assay (ELISA) are both widely used to assess immunity to infectious diseases such as measles, but they use two different measurement principles: ELISA measures the ability of antibodies to bind to virus components, while the PRNT detects the aptitude of antibodies to prevent the infection of a susceptible cell. As a result, detection of measles virus (MV) neutralizing antibodies is the gold standard for assessing immunity to measles. However, the assay is laborious and requires experience and excellent technical skills. In addition, the result is only available after several days. Therefore, the classical PRNT is not suitable for high-throughput testing. By using an immunocolorimetric assay (ICA) to detect MV-infected cells, the standard PRNT has been developed into a focus reduction neutralization test (FRNT). This assay is faster and has improved specificity. The FRNT described here is extremely useful when immunity to measles virus needs to be assessed in patients with a specific medical condition, such as immunocompromised individuals in whom presumed residual immunity needs to be assessed. The FRNT is not generally recommended for use with large numbers of specimens, such as in a seroprevalence study.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , Virus del Sarampión , Sarampión , Pruebas de Neutralización , Pruebas de Neutralización/métodos , Virus del Sarampión/inmunología , Sarampión/inmunología , Sarampión/diagnóstico , Sarampión/virología , Humanos , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/inmunología , Chlorocebus aethiops , Animales , Células Vero , Ensayo de Placa Viral/métodos , Ensayo de Inmunoadsorción Enzimática/métodosRESUMEN
The endpoint dilution assay's output, the 50% infectious dose (ID50), is calculated using the Reed-Muench or Spearman-Kärber mathematical approximations, which are biased and often miscalculated. We introduce a replacement for the ID50 that we call Specific INfection (SIN) along with a free and open-source web-application, midSIN (https://midsin.physics.ryerson.ca) to calculate it. midSIN computes a virus sample's SIN concentration using Bayesian inference based on the results of a standard endpoint dilution assay, and requires no changes to current experimental protocols. We analyzed influenza and respiratory syncytial virus samples using midSIN and demonstrated that the SIN/mL reliably corresponds to the number of infections a sample will cause per mL. It can therefore be used directly to achieve a desired multiplicity of infection, similarly to how plaque or focus forming units (PFU, FFU) are used. midSIN's estimates are shown to be more accurate and robust than the Reed-Muench and Spearman-Kärber approximations. The impact of endpoint dilution plate design choices (dilution factor, replicates per dilution) on measurement accuracy is also explored. The simplicity of SIN as a measure and the greater accuracy provided by midSIN make them an easy and superior replacement for the TCID50 and other in vitro culture ID50 measures. We hope to see their universal adoption to measure the infectivity of virus samples.
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Bioensayo/métodos , Biología Computacional/métodos , Ensayo de Placa Viral/métodos , Virosis/virología , Teorema de BayesRESUMEN
For a cytopathic virus such as severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), the neutralization capacity of serum from convalescent or vaccinated persons or of therapeutic antibodies can be tested on adherent cell cultures. Here, a simple and tissue culture infectious dose-derived protocol for assessment of neutralization of SARS-CoV-2 is described. Compared with the often applied plaque-forming unit assay, the working load is lower, and fewer manipulations of the infected cultures are required. Hence, the method is safer for the personnel.
Asunto(s)
Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , COVID-19/inmunología , Pruebas de Neutralización/métodos , SARS-CoV-2/inmunología , Ensayo de Placa Viral/métodos , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/sangre , COVID-19/terapia , Chlorocebus aethiops , Ensayo de Inmunoadsorción Enzimática , Humanos , Células VeroRESUMEN
Conventional plaque assays rely on the use of overlays to restrict viral infection allowing the formation of distinct foci that grow in time as the replication cycle continues leading to countable plaques that are visualized with standard techniques such as crystal violet, neutral red, or immunolabeling. This classical approach takes several days until large enough plaques can be visualized and counted with some variation due to subjectivity in plaque recognition. Since plaques are clonal lesions produced by virus-induced cytopathic effect, we applied DNA fluorescent dyes with differential cell permeability to visualize them by live-cell imaging. We could observe different stages of that cytopathic effect corresponding to an early wave of cells with chromatin-condensation followed by a wave of dead cells with membrane permeabilization within plaques generated by different animal viruses. This approach enables an automated plaque identification using image analysis to increase single plaque resolution compared to crystal violet counterstaining and allows its application to plaque tracking and plaque reduction assays to test compounds for both antiviral and cytotoxic activities. This fluorescent real-time plaque assay sums to those next-generation technologies by combining this robust classical method with modern fluorescence microscopy and image analysis approaches for future applications in virology.
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Imagen Óptica/instrumentación , Imagen Óptica/métodos , Análisis de la Célula Individual/métodos , Ensayo de Placa Viral/métodos , Automatización de Laboratorios , Línea Celular , Efecto Citopatogénico Viral , Análisis de la Célula Individual/instrumentación , Ensayo de Placa Viral/instrumentaciónRESUMEN
Isolating single phages using plaque assays is a laborious and time-consuming process. Whether single isolated phages are the most lyse-effective, the most abundant in viromes, or those with the highest ability to make plaques in solid media is not well known. With the increasing accessibility of high-throughput sequencing, metaviromics is often used to describe viruses in environmental samples. By extracting and sequencing metaviromes from organic waste with and without exposure to a host-of-interest, we show a host-related phage community's shift, as well as identify the most enriched phages. Moreover, we isolated plaque-forming single phages using the same virome-host matrix to observe how enrichments in liquid media correspond to the metaviromic data. In this study, we observed a significant shift (p = 0.015) of the 47 identified putative Pseudomonas phages with a minimum twofold change above zero in read abundance when adding a Pseudomonas syringae DC3000 host. Surprisingly, it appears that only two out of five plaque-forming phages from the same organic waste sample, targeting the Pseudomonas strain, were highly abundant in the metavirome, while the other three were almost absent despite host exposure. Lastly, our sequencing results highlight how long reads from Oxford Nanopore elevates the assembly quality of metaviromes, compared to short reads alone.
Asunto(s)
Metagenoma , Metagenómica , Fagos Pseudomonas/fisiología , Pseudomonas/virología , Ensayo de Placa Viral , Viroma , Biología Computacional , Especificidad del Huésped , Metagenómica/métodos , Fagos Pseudomonas/clasificación , Ensayo de Placa Viral/métodosRESUMEN
Bacteriophage plaque size measurement is essential for phage characterisation, but manual size estimation requires a considerable amount of time and effort. In order to ease the work of phage researchers, we have developed an automated command-line application called Plaque Size Tool (PST) that can detect plaques of different morphology on the images of Petri dishes and measure plaque area and diameter. Plaque size measurements using PST showed no difference to those obtained with manual plaque size measurement in Fiji, indicating future results using PST are backwards compatible with prior measurements in the literature. PST can be applied to a range of lytic bacteriophages producing oval-shaped plaques, including bull's-eye and turbid morphology. The application can also be used for titer calculation if most of the plaques are stand-alone. As laboratory automation becomes more commonplace, standardised and flexible open-source analytical tools like PST will be important parts of biofoundry and cloud lab bacteriophage workflows.
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Bacteriófago phi X 174/crecimiento & desarrollo , Bacteriófagos/crecimiento & desarrollo , Ensayo de Placa Viral/métodos , Automatización de Laboratorios , Bacteriófago phi X 174/ultraestructura , Bacteriófagos/ultraestructura , Procesamiento de Imagen Asistido por Computador , Reproducibilidad de los Resultados , Programas InformáticosRESUMEN
Enteric viruses, such as poliovirus, are a leading cause of gastroenteritis, which causes 2-3 million deaths annually. Environmental surveillance of wastewater supplements clinical surveillance for monitoring enteric virus circulation. However, while many environmental surveillance methods require liquid samples, some at-risk locations utilize pit latrines with waste characterized by high solids content. This study's objective was to develop and evaluate enteric virus concentration protocols for high solids content samples. Two existing protocols were modified and tested using poliovirus type 1 (PV1) seeded into primary sludge. Method 1 (M1) utilized acid adsorption, followed by 2 or 3 elutions (glycine/sodium chloride and/or threonine/sodium chloride), and skimmed milk flocculation. Method 2 (M2) began with centrifugation. The liquid fraction was filtered through a ViroCap filter and eluted (beef extract/glycine). The solid fraction was eluted (beef extract/disodium hydrogen phosphate/citric acid) and concentrated by skimmed milk flocculation. Recovery was enumerated by plaque assay. M1 yielded higher PV1 recovery than M2, though this result was not statistically significant (26.1% and 15.9%, respectively). M1 was further optimized, resulting in significantly greater PV1 recovery when compared to the original protocol (p < 0.05). This method can be used to improve understanding of enteric virus presence in communities without liquid waste streams.
Asunto(s)
Monitoreo del Ambiente/métodos , Poliovirus/aislamiento & purificación , Aguas del Alcantarillado/virología , Residuos Sólidos/análisis , Carga Viral/métodos , Infecciones por Enterovirus/prevención & control , Floculación , Gastroenteritis/prevención & control , Gastroenteritis/virología , Humanos , Poliomielitis/prevención & control , Ensayo de Placa Viral/métodos , Microbiología del AguaRESUMEN
The potential zoonotic coronaviruses (SARS-CoV, MERS-CoV, and SARS-CoV-2) are of global health concerns. Early diagnosis is the milestone in their mitigation, control, and eradication. Many diagnostic techniques are showing great success and have many advantages, such as the rapid turnover of the results, high accuracy, and high specificity and sensitivity. However, some of these techniques have several pitfalls if samples were not collected, processed, and transported in the standard ways and if these techniques were not practiced with extreme caution and precision. This may lead to false-negative/positive results. This may affect the downstream management of the affected cases. These techniques require regular fine-tuning, upgrading, and optimization. The continuous evolution of new strains and viruses belong to the coronaviruses is hampering the success of many classical techniques. There are urgent needs for next generations of coronaviruses diagnostic assays that overcome these pitfalls. This new generation of diagnostic tests should be able to do simultaneous, multiplex, and high-throughput detection of various coronavirus in one reaction. Furthermore, the development of novel assays and techniques that enable the in situ detection of the virus on the environmental samples, especially air, water, and surfaces, should be given considerable attention in the future. These approaches will have a substantial positive impact on the mitigation and eradication of coronaviruses, including the current SARS-CoV-2 pandemic.
Asunto(s)
COVID-19/diagnóstico , Ensayos Analíticos de Alto Rendimiento/métodos , Síndrome Respiratorio Agudo Grave/diagnóstico , Ensayo de Inmunoadsorción Enzimática/métodos , Técnica del Anticuerpo Fluorescente/métodos , Genoma Viral/genética , Humanos , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus/genética , Ensayo de Placa Viral/métodosRESUMEN
There remains an urgent need for assays to quantify humoral protective immunity to SARS-CoV-2 to understand the immune responses of COVID-19 patients, evaluate efficacy of vaccine candidates in clinical trials, and conduct large-scale epidemiological studies. The plaque-reduction neutralization test (PRNT) is the reference-standard for quantifying antibodies capable of neutralizing SARS-CoV-2. However, the PRNT is logistically demanding, time-consuming, and requires containment level-3 facilities to safely work with live virus. In contrast, a surrogate virus neutralization test (sVNT) manufactured by Genscript is a quick and simple assay that detects antibodies that inhibit the RBD-ACE2 interaction, crucial for virus entry into host cells. In this study, we evaluate the sensitivity, specificity, and cross-reactivity of the sVNT compared with the PRNT using both 50% and 90% SARS-CoV-2 neutralization as a reference-standard. We found that the sVNT provides a high-throughput screening tool prior to confirmatory PRNT testing for the evaluation of SARS-CoV-2 neutralizing antibodies.
Asunto(s)
Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , SARS-CoV-2/inmunología , Ensayo de Placa Viral/métodos , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/diagnóstico , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Pruebas de Neutralización/métodosRESUMEN
As new tests and technologies advance our understanding and diagnostic capabilities of the severe acute respiratory syndrome coronavirus 2 and the coronavirus disease 2019, they must be appropriately validated to make sure test performance is following manufacturer claims. In this study, we evaluated the Vazyme 2019-nCoV IgG/IgM Detection Kit, which is a lateral flow assay (LFA), by the plaque reduction neutralization test (PRNT) using 100 patient plasma/serum samples. As compared to the PRNT results, the Vazyme LFA had 95.9% sensitivity and 96.1% specificity. Along with the increased need for rapid, effective, and affordable point of care tests to help provide meaningful epidemiological data, we demonstrated that the Vazyme LFA performed well on IgG detection but cannot be judged on the performance of IgM detection using PRNT alone. However, our observation of the low IgM-positive rate supported the poor performance of IgM detection of this LFA which led to the disapproval of its Emergency Use Authorization recently.
Asunto(s)
Anticuerpos Antivirales/sangre , COVID-19/diagnóstico , Pruebas de Neutralización/métodos , SARS-CoV-2/inmunología , Ensayo de Placa Viral/métodos , Humanos , Inmunoensayo/métodos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Pruebas en el Punto de AtenciónRESUMEN
A major limitation hindering the widespread use of synthetic phages in medical and industrial settings is the lack of an efficient phage-engineering platform. Classical T4 phage engineering and several newly proposed methods are often inefficient and time consuming and consequently, only able to produce an inconsistent range of genomic editing rates between 0.03-3%. Here, we review and present new understandings of the CRISPR/Cas9 assisted genome engineering technique that significantly improves the genomic editing rate of T4 phages. Our results indicate that crRNAs selection is a major rate limiting factor in T4 phage engineering via CRISPR/Cas9. We were able to achieve an editing rate of > 99% for multiple genes that functionalizes the phages for further applications. We envision that this improved phage-engineering platform will accelerate the fields of individualized phage therapy, biocontrol, and rapid diagnostics.
Asunto(s)
Bacterias/virología , Bacteriófago T4/genética , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Ingeniería Genética/normas , Ensayo de Placa Viral/métodos , Bacterias/metabolismo , Bacteriófago T4/metabolismo , Edición Génica/normas , Ingeniería Genética/métodosRESUMEN
In this invited article, we explain technical aspects of the lymphocytic choriomeningitis virus (LCMV) system, providing an update of a prior contribution by Matthias von Herrath and J. Lindsay Whitton. We provide an explanation of the LCMV infection models, highlighting the importance of selecting an appropriate route and viral strain. We also describe how to quantify virus-specific immune responses, followed by an explanation of useful transgenic systems. Specifically, our article will focus on the following protocols. © 2020 Wiley Periodicals LLC. Basic Protocol 1: LCMV infection routes in mice Support Protocol 1: Preparation of LCMV stocks ASSAYS TO MEASURE LCMV TITERS Support Protocol 2: Plaque assay Support Protocol 3: Immunofluorescence focus assay (IFA) to measure LCMV titer MEASUREMENT OF T CELL AND B CELL RESPONSES TO LCMV INFECTION Basic Protocol 2: Triple tetramer staining for detection of LCMV-specific CD8 T cells Basic Protocol 3: Intracellular cytokine staining (ICS) for detection of LCMV-specific T cells Basic Protocol 4: Enumeration of direct ex vivo LCMV-specific antibody-secreting cells (ASC) Basic Protocol 5: Limiting dilution assay (LDA) for detection of LCMV-specific memory B cells Basic Protocol 6: ELISA for quantification of LCMV-specific IgG antibody Support Protocol 4: Preparation of splenic lymphocytes Support Protocol 5: Making BHK21-LCMV lysate Basic Protocol 7: Challenge models TRANSGENIC MODELS Basic Protocol 8: Transfer of P14 cells to interrogate the role of IFN-I on CD8 T cell responses Basic Protocol 9: Comparing the expansion of naïve versus memory CD4 T cells following chronic viral challenge.
Asunto(s)
Inmunidad Adaptativa , Interacciones Huésped-Patógeno/inmunología , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/inmunología , Animales , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Técnicas de Cultivo de Célula , Línea Celular , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Técnica del Anticuerpo Fluorescente/métodos , Memoria Inmunológica , Depleción Linfocítica , Coriomeningitis Linfocítica/transmisión , Ratones , Especificidad del Receptor de Antígeno de Linfocitos T , Linfocitos T/inmunología , Linfocitos T/metabolismo , Carga Viral/métodos , Ensayo de Placa Viral/métodosRESUMEN
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is commonly diagnosed by reverse transcription polymerase chain reaction (RT-PCR) to detect viral RNA in patient samples, but RNA extraction constitutes a major bottleneck in current testing. Methodological simplification could increase diagnostic availability and efficiency, benefitting patient care and infection control. Here, we describe methods circumventing RNA extraction in COVID-19 testing by performing RT-PCR directly on heat-inactivated or lysed samples. Our data, including benchmarking using 597 clinical patient samples and a standardised diagnostic system, demonstrate that direct RT-PCR is viable option to extraction-based tests. Using controlled amounts of active SARS-CoV-2, we confirm effectiveness of heat inactivation by plaque assay and evaluate various generic buffers as transport medium for direct RT-PCR. Significant savings in time and cost are achieved through RNA-extraction-free protocols that are directly compatible with established PCR-based testing pipelines. This could aid expansion of COVID-19 testing.
Asunto(s)
Betacoronavirus/genética , Betacoronavirus/aislamiento & purificación , Técnicas de Laboratorio Clínico/métodos , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/virología , Neumonía Viral/diagnóstico , Neumonía Viral/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Benchmarking , COVID-19 , Prueba de COVID-19 , Técnicas de Laboratorio Clínico/normas , Técnicas de Laboratorio Clínico/estadística & datos numéricos , Infecciones por Coronavirus/epidemiología , Cartilla de ADN/genética , Calor , Humanos , Pandemias , Neumonía Viral/epidemiología , ARN Viral/genética , ARN Viral/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/estadística & datos numéricos , SARS-CoV-2 , Sensibilidad y Especificidad , Suecia/epidemiología , Ensayo de Placa Viral/métodosRESUMEN
Vesicular stomatitis virus (VSV) is the prototypical member of the Rhabdoviridae family of negative-sense single-stranded RNA viruses. This virus has been used as a powerful model system for decades and is currently being used as a vaccine platform and an oncolytic agent. Here, we present methods to propagate, quantitate, and store VSV. We also review the proper safety protocol for the handling of VSV, which is classified as a Biosafety Level 2 pathogen by the United States Centers for Disease Control and Prevention. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation, purification, and storage of vesicular stomatitis virus stocks Basic Protocol 2: Quantification of vesicular stomatitis virus by plaque assay Support Protocol: Propagation of Vero cells.
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Preservación Biológica/métodos , Manejo de Especímenes/métodos , Virus de la Estomatitis Vesicular Indiana/crecimiento & desarrollo , Virus de la Estomatitis Vesicular Indiana/aislamiento & purificación , Ensayo de Placa Viral/métodos , Cultivo de Virus/métodos , Animales , Línea Celular , Chlorocebus aethiops , Humanos , Células Vero , Estomatitis Vesicular/virologíaRESUMEN
Several techniques are currently available to quickly and accurately quantify the number of virus particles in a sample, taking advantage of advanced technologies improving old techniques or generating new ones, generally relying on partial detection methods or structural analysis. Therefore, characterization of virus infectivity in a sample is often essential, and classical virological methods are extremely powerful in providing accurate results even in an old-fashioned way. In this chapter, we describe in detail the techniques routinely used to estimate the number of viable infectious coronavirus particles in a given sample. All these techniques are serial dilution assays, also known as titrations or end-point dilution assays (EPDA).
Asunto(s)
Coronavirus/patogenicidad , Ensayo de Placa Viral/métodos , Animales , Células Cultivadas , Coronavirus/crecimiento & desarrollo , Virus de la Bronquitis Infecciosa/crecimiento & desarrollo , Virus de la Bronquitis Infecciosa/patogenicidad , Tráquea/citologíaRESUMEN
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been identified as the causal agent of COronaVIrus Disease-19 (COVID-19), an atypical pneumonia-like syndrome that emerged in December 2019. While SARS-CoV-2 titers can be measured by detection of viral nucleic acid, this method is unable to quantitate infectious virions. Measurement of infectious SARS-CoV-2 can be achieved by tissue culture infectious dose-50 (TCID50 ), which detects the presence or absence of cytopathic effect in cells infected with serial dilutions of a virus specimen. However, this method only provides a qualitative infectious virus titer. Plaque assays are a quantitative method of measuring infectious SARS-CoV-2 by quantifying the plaques formed in cell culture upon infection with serial dilutions of a virus specimen. As such, plaque assays remain the gold standard in quantifying concentrations of replication-competent lytic virions. Here, we describe two detailed plaque assay protocols to quantify infectious SARS-CoV-2 using different overlay and staining methods. Both methods have several advantages and disadvantages, which can be considered when choosing the procedure best suited for each laboratory. These assays can be used for several research purposes, including titration of virus stocks produced from infected cell supernatant and, with further optimization, quantification of SARS-CoV-2 in specimens collected from infected animals. © 2019 The Authors. Basic Protocol: SARS-CoV-2 plaque assay using a solid double overlay method Alternate Protocol: SARS-CoV-2 plaque assay using a liquid overlay and fixation-staining method.
Asunto(s)
Betacoronavirus/aislamiento & purificación , Protocolos Clínicos , Ensayo de Placa Viral/métodos , Animales , Chlorocebus aethiops , Humanos , SARS-CoV-2 , Coloración y Etiquetado , Células VeroRESUMEN
In late 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, the capital of the Chinese province Hubei. Since then, SARS-CoV-2 has been responsible for a worldwide pandemic resulting in over 4 million infections and over 250,000 deaths. The pandemic has instigated widespread research related to SARS-CoV-2 and the disease that it causes, COVID-19. Research into this new virus will be facilitated by the availability of clearly described and effective procedures that enable the propagation and quantification of infectious virus. As work with the virus is recommended to be performed at biosafety level 3, validated methods to effectively inactivate the virus to enable the safe study of RNA, DNA, and protein from infected cells are also needed. Here, we report methods used to grow SARS-CoV-2 in multiple cell lines and to measure virus infectivity by plaque assay using either agarose or microcrystalline cellulose as an overlay as well as a SARS-CoV-2 specific focus forming assay. We also demonstrate effective inactivation by TRIzol, 10% neutral buffered formalin, beta propiolactone, and heat.
Asunto(s)
Betacoronavirus/fisiología , Infecciones por Coronavirus/virología , Neumonía Viral/virología , Ensayo de Placa Viral/métodos , Inactivación de Virus , Animales , Betacoronavirus/efectos de los fármacos , Betacoronavirus/crecimiento & desarrollo , Betacoronavirus/patogenicidad , COVID-19 , Celulosa , Chlorocebus aethiops , Medios de Cultivo/química , Formaldehído , Guanidinas/farmacología , Células HEK293 , Humanos , Pandemias , Fenoles/farmacología , Propiolactona/farmacología , SARS-CoV-2 , Sefarosa , Células VeroRESUMEN
Zika virus (ZIKV) is an important pathogen transmitted to humans by the mosquito vector Aedes aegypti. ZIKV is able to infect several tissues and organs and, importantly, has been associated with microcephaly and central nervous system abnormalities in fetuses and newborn babies of mothers exposed to ZIKV during pregnancy, as well as neurological diseases such as Guillain-Barré syndrome in adults. There is currently no vaccine or drug licensed to prevent or treat ZIKV infections. The use of ZIKV isolation in disease diagnosis has been largely replaced by new techniques. However, virus isolation is still considered as a gold standard for the detection of ZIKV and is usually performed in research and reference laboratories for characterization, sequencing, and a variety of research experiments including pathogenesis, drug susceptibility, and vaccine efficacy. The experimental procedures presented here describe the most common techniques used for ZIKV isolation, propagation, purification, and quantification.
Asunto(s)
Volumetría/métodos , Virología/métodos , Infección por el Virus Zika/virología , Virus Zika/crecimiento & desarrollo , Virus Zika/aislamiento & purificación , Animales , Chlorocebus aethiops , Células Vero , Ensayo de Placa Viral/métodos , Infección por el Virus Zika/patologíaRESUMEN
Neutralizing antibodies against Zika virus (ZIKV) are a robust indicator of exposure to ZIKV. Here we describe the plaque reduction neutralization test (PRNT), which quantifies ZIKV-neutralizing antibodies (nAbs) by incubating infectious ZIKV with different dilutions of sera and applying the mixture to a susceptible Vero cell monolayer. After 4 days of incubation, neutral red dye is added to optimize the visualization of plaques caused by ZIKV cytopathic effect. Compared with controls, sera containing nAbs against ZIKV will demonstrate ≥90% reduction in the number of plaques observed. This protocol can be utilized for research applications and serological diagnostic testing with the use of additional controls.