Plaque virus assay

As a result, clear plaques can be distinguished from the purple monolayer. For a comparison of the two protocols, see Commentary. During this incubation, avoid movement of the plates, which can disrupt the liquid overlay and cause plaques to smear.

Incubate at room temperature for 1 hr. Staining : Aspirate fixative from wells and discard into a waste bottle containing the appropriate amount of Formalex. The negative control shows an intact monolayer stained purple. Use Formula 7 see Basic Protocol to calculate the average number of plaques at this dilution.

Liquid waste containing disinfectant should be incubated at room temperature overnight prior to disposal. The accurate quantification of infectious virus specimens is crucial for a multitude of applications in virology. Noble agar, the solid matrix used in the overlay in the Basic Protocol, is considered a traditional overlay matrix for plaque assays.

However, it comes with disadvantages. The secondary overlay in this protocol uses neutral red to stain the monolayer and enhance the visualization of plaques. As a vital stain, neutral red has the advantage of being applied during incubation, enabling live monitoring of plaque formation. However, the contrast between plaques and viable cells stained with neutral red is not as distinct as that produced by crystal violet and fixation in the Alternate Protocol, and requires a light box to further enhance visualization.

However, the disadvantage of the liquid overlay matrix is that disruption of the liquid overlays can result in smeared plaques, and thus great care must be taken to prevent movement of plates during the incubation period. The fixation and subsequent staining with crystal violet used to enhance plaque visualization in the protocol has several advantages, including increased contrast between plaques from the purple monolayers, as well as the inactivation of the virus.

In addition, fixation enables plates to be stored and plaques to be enumerated at a later time. However, some disadvantages of this method includes the generation of formaldehyde waste and the additional need to properly inactivate that chemical. A comparison conducted in our laboratory demonstrated that there were no significant differences in viral titers calculated using either method.

The monolayer is susceptible to damage from forceful pipetting, which can then be mistaken for plaques. Instead of adding medium directly on top of the monolayer, slowly add it by aiming for the walls of each well. A representative plaque assay plate exhibiting commonly encountered problems.

However, if starting Vero E6 cells from cryostocks, it would be necessary to passage the cell line at least three times prior to use in either assay to remove excess cryopreservative and allow cells to return to normal growth, extending the duration by a few days; this is not taken into account in the protocols. If plates are seeded as described in the protocols on a Monday, the plaque assay can begin on Tuesday and end with plaque enumeration on Friday.

Using this schedule, the secondary overlay would be applied on Thursday for the Basic Protocol. Development of this protocol was supported by the Public Health Agency of Canada. We thank Dr.

Darwyn Kobasa, Dr. Yohannes Berhane Canadian Food Inspection Agency for their constructive commentary regarding the optimization of the protocols. Mendoza, E. Current Protocols in Microbiology , 57 , e National Center for Biotechnology Information , U. Curr Protoc Microbiol.

Published online May Emelissa J. Author information Copyright and License information Disclaimer. Michael Drebot, Email: ac. Corresponding author. This article has been cited by other articles in PMC. Open in a separate window. Figure 1. Cell culture Day 0 1. Figure 2. Secondary overlay and staining Day 3, 2 dpi 7.

Enumeration of plaques and titer calculation Day 4, 3 dpi 9. Figure 3. Cells can be maintained in vitro outside of their original body by this […]. Last updated on June 1st, Shell vial cell culture, a centrifuge enhanced tissue culture assay, is a modification of conventional cell culture for the rapid detection of viruses in vitro. Contents1 Principle1.

Last updated on May 22nd, Embryonated chicken eggs are used for the cultivation of some viruses. The viruses grow in the cells of the embryo and membranes. Specimens are inoculated into pathogen-free fertilized eggs […]. News Ticker. Viral Culture Technique. About Acharya Tankeshwar Articles. Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. Virus Genes — DOI Cook et al. Coates M et al. Reich S et al.

Hanson QM et al. Le Rouzic E et al. Retrovirology volume 10, Article number: Rusanov et al. Scientific Reports volume 8, Article number: July 16, Dr Andrea Krumm. What is a virus? The replicative life cycle of viruses Outside infected cells, viruses exist as independent particles called virions. Although the viral life cycle differs greatly between species, six basic stages are essential for viral replication: 1. Viral structure Virions consist of nucleic acid and a protein coat called capsid.

Virus genome — Baltimore classification The most common classification is based on the viral nucleic acid and its replication. Virus types differ in nucleic acid type and its replication. Example: Rotavirus: infects small intestine cells, leading to diarrhoea and gastroenteritis in children. The disease often presents mild symptoms, such as a scratching throat, dry cough and fever but can also escalate to pneumonia, multiple organ failure or even death, the mechanism of which is not fully understood.

This virus has spread rapidly and was declared a pandemic in early by the WHO. No vaccine is currently available, only symptomatic treatments to suppress the virus and reduce transmission.

Example: Hepatitis B: may be asymptomatic or cause severe liver inflammation. A vaccination for long term immunity is available. Virus transmission Viruses spread using living organisms as vectors. Treatment of viral infections Modern medicine combats viral infections mainly through vaccination and drug treatments.

Diagnostic virus assays Diagnostic methods accurately identify viral infections in patients. Long-established virus assays for diagnosis Traditional laboratory identification methods of viral infections focus mainly on optical detection of virions by microscopy techniques or by indirect serologic evidence.

Phase-contrast microscopy under green light. Source: Y tambe, Wikimedia Commons. Infected cells expressing viral antigens can be directly or indirectly recognised by virus-specific antibodies.

Modern techniques for the rapid diagnosis of viral infections Whilst still employed, traditional virus assays have been or are being replaced by more rapid methods allowing qualitative testing for screening and surveillance, as well as confirmation of diagnosis. A fluorescent probe binds the amplified target and emits a detectable signal.

Once fluorescence intensity rises above background, the detectable level proportionally corresponds to the initial number of template DNA molecules in the sample.

Unlike PCR, it does not rely on temperature cycling, and can be hence measured in real-time in a microplate reader with periodic shaking. The assay can be colorimetric or fluorescent. Compared to PCR, this approach has speed and cost advantages. In addition, it is highly automatable, making it one of the best virus assays for rapid diagnosis These methods can provide important information on novel viruses to assist in the production of vaccines and treatments against them.

The plaque assay represents the gold standard and most used quantitative virus assay. In plaque assays, a confluent monolayer of cells is infected with unknown concentrations of a lytic virus at varying dilutions. Infected monolayers are then covered with an immobilizing overlay medium to prevent viral spread. Zones of cell death plaques will begin to develop as result of constrained infection and replication.

Infected cells will propagate the replication-lysis-infection cycle, resulting in increasingly distinct and discrete plaques fig. Visible plaque formation can take 2—14 days, depending on the virus and host cells used. Plaques are typically counterstained by neutral red or crystal violet to be counted manually.

Serial dilutions of the viral samples are applied to plates with a confluent monolayer of host cells. Different numbers of plaques will develop as a result of viral dilution.

Focus Forming Assays FFAs are modified plaque assays that utilise an antibody-based staining method to detect infected cells. Unlike plaque assay, FFAs detect lytic and non-lytic viruses with increased sensitivity and decreased incubation times FFAs are limited in their need for appropriate antibodies and their ability to only probe viral protein subunits and not infectious virions.

It is an endpoint dilution assay used to quantify viruses that do not form plaques. In brief, serial dilutions of the virus are added to host cells. After incubation, the percentage of cell death is measured from a cell viability assay using a plate reader. Alternatively, microscopy can be used. Recombinant viruses can be engineered to express a fluorescent or luminescent marker.

This virus assay detects virions faster than plaque assays or TCID50 and is ideally suited to screen for antivirals. Additionally, it can be run on a microplate reader allowing automation. Samples containing unknown quantities of luciferase-expressing or fluorescence-expressing viruses are transferred on to a plaquing cell line in parallel with the standard dilution curve. The luminescence or fluorescence is read after incubation and viral titres are calculated based on the standard curve.

Hemagglutination assay and Hemagglutination Inhibition Assay HIA Haemagglutinin is a protein on the envelope of arboviruses, influenza and parainfluenza virus subtypes which binds red blood cells RBCs to form a lattice of agglutinated cells. In the hemagglutination assay, serial dilutions of the virus are added to RBCs. Samples are then screened for agglutinated cells. A variation of this virus assay is the hemagglutination inhibition assay used to measure specific antibodies in serum. If antibodies are present in the serum at a sufficient concentration, they will interfere with the viral attachment to RBCs, resulting in inhibition of hemagglutination.

Both virus assays provide a relative virus quantitation 3, 13, The images across a plate typically progress from agglutinated wells with a diffuse reddish appearance to wells with low agglutination, containing a red pellet in the well center. As antibodies persist in the bloodstream even for many years after infection, a positive test is not an indicator of active infection but determines patient immunity resulting from exposure to the virus, reinfection, or a reactivation state.

ELISAs are invaluable for studying the epidemiology of disease as they study present and past disease prevalence in different populations over time. To screen for viral antibodies a viral antigen is immobilised on the bottom of a microplate well direct and indirect ELISA.

To screen for viral antigens, antibodies against the virus are immobilised direct and indirect sandwich ELISA. Unlike ELISAs, removal of the unbound components from the well is not required to reduce the background and detect the bound complex.

Hence, homogeneous assays do not necessitate in-between separation or washing steps and can be executed with a simple add-and-read protocol. This minimises handling steps, operation time and makes them particularly suited for automation-supported screening. Due to their nature and chemistry, homogeneous immunoassays do not require washing steps to remove unbound components from the well. They can be immediately measured upon incubation.

Functional virus assays: research and high throughput methods There are different types of functional assays to study how a virus works, such as how it infects cells and which mechanisms and enzymes are important for the virus. Methods in basic research : in basic research, functional virus assays help to find immunogenic components for vaccines and potential drug targets for therapy.

Microplate-based tests employed for virus characterization , mainly use plates up to 96 wells. As soon as a virus is understood and potential drug targets are identified, higher throughput virus assays search for molecules interfering with pathogenic processes.

High throughput assays : the development of antivirals requires to screen compound libraries for various aspects and relies on high throughput compatible virus assays. These are performed in plate formats of wells or wells. In addition to the plate density, high throughput virus assays are automatically prepared with the use of automated liquid handlers and industrial robots.

Neuraminidase assay Neuraminidase is one of two key glycoproteins found on the surface of influenza viruses the other being hemagglutinin, fig. Figure Structure of the influenza virus.

Viral neutralization assays The effectiveness of an antibody, vaccine treatment or infection-related immunity is tested with a neutralization assay. In a luminescence-based neutralization assay, virus pseudotypes are pre-incubated with neutralizing samples such as sera of immunized organisms or antibodies.

Subsequently, cells are added and incubated with the virus samples to allow infection. Upon addition of the luciferase substrate, luminescence is measured on a microplate reader.

Neutralized viruses are not capable of infecting cells and display low luminescence while infectious pathogens show high luminescence. Virus infectivity assays to study antiviral activity Methods used to test the antiviral effect of compounds overlap with assays used to diagnose infections.

Binding assays for compound screens In the context of virus research and antiviral development, binding assays screen for molecules that interact with target structures essential for the virus lifecycle. A donor molecule e. The acceptor molecule e. As energy transfer occurs only in proximity of donor and acceptor, the acceptor emission intensity directly reports on a binding event.