In Vitro Antiviral Testing
The IAR routinely performs testing for antiviral efficacy in vitro. We have a myriad of viruses and models available, including BSL-3, BSL-3+, and select agents. Testing is done by neutral red assays, microscopic cytopathic effect (CPE) assays, plaque reduction assays, and/or virus yield reduction assays. We can also complete time-of-addition and mechanism-of-action studies. In addition, we have developed a high throughput assay where we screen up to 300 compounds at one time at a reduced cost. Please contact Michelle Mendenhall (firstname.lastname@example.org) or Dr. Brett Hurst (email@example.com) for details and pricing. Below is a description of the assays we have readily available for in vitro screening:
Antiviral Assay by CPE
This test is for initial screening of potentially antiviral compounds. The antiviral activity of the compound is evaluated based on the ability of the compound to prevent virus from causing viral CPE in mammalian cell culture. Eight dilutions of test compound are evaluated, and the effective antiviral concentration determined by regression analysis. The toxicity of the test compound is determined in parallel. An abbreviated test with four dilutions of compound may be employed to screen large numbers of compounds quickly and at a reduced cost. CPE is determined by microscopic observation of cell culture monolayers as well as uptake of neutral red dye.
Virus Yield Reduction Assay
This test evaluates the ability of the compound to inhibit virus production in mammalian cell culture. This is a two-step assay where virus is first produced in cultures containing the antiviral substance at varying dilutions, followed later by titration of the samples for virus titer by endpoint dilution in 96-well microplates. Eight dilutions of test compound are assayed, and the effective antiviral concentration determined by regression analysis. This test is normally requested as a follow-up for compounds that demonstrate activity in the CPE assay.
This assay determines if a test compound inactivates ("kills") virus outside of cells, or in other words, whether the compound inactivates the virus before infection of the cells. A virucidal compound may demonstrate activity in the antiviral assay, but virucidal compounds (e.g. alcohol, sodium hypochlorite, etc.) are normally not considered candidates for antiviral treatment. The assay is performed by incubating virus and compound for one hour (or a length of time requested by the sponsor), followed by determination of virus titer by endpoint dilution in 96-well microplates of cells.
Microneutralization or Virus Neutralization Assay
The microneutralization (MN) or virus neutralization (VN) assay is used to determine the presence of functional antibodies to prevent viral infection. In this assay, dilutions of antibody samples (either purified antibodies or animal serum samples) are prepared in test tubes. A fixed amount of virus is added to each dilution of antibody sample and the the resulting antibody/virus mixture is incubated for one hour (or a length of time requested by the sponsor). After the incubation period, the antibody/virus mixture is added to 96-well microplates containing cells susceptible to virus infection. The cell culture plate containing the antibody/virus mixture is incubated and CPE is determined by microscopic observation. The reciprocal of the lowest antibody dilution that is able to prevent CPE effect is calculated as the virus neutralizing (VN) titer.
Plaque Reduction Neutralization Test
The plaque reduction neutralization test determines the presence and concentration of neutralizing antibodies in a serum sample or antibody solution. In this assay, the sample is incubated with the virus and the mixture is then added to monolayers of cells. Agar or carboxymethyl cellulose is overlaid on the cell monolayer to prevent spreading of the virus which leads to plaque formation. After a certain number of days, the concentration of serum or antibody solution needed to reduce the number of plaques by 50% is calculated as the PRNT50. The number of plaques is determined by either visual observation or the use of dyes to stain the cell monolayer depending on which virus is used.
Hemagglutination Inhibition Assay
The hemagglutination inhibition (HAI) assay applies the process of hemagglutination of the sialic acid receptors of red blood cells (RBCs) by the hemagglutinin glycoprotein of influenza viruses. When RBCs are added to a round-bottom 96-well microplate, the RBCs will normally settle to the bottom of the plate forming a small "button" of RBCs in the bottom of the well. When an influenza virus is added to the RBC mixture, the influenza virus will hemagglutinate the RBCs and form a lattice-type structure that prevents the RBCs from settling to the bottom of the well. If a serum sample containing antibodies to the influenza virus is placed on the plate prior to adding the virus and then the RBCs, the antibodies present in the serum sample will bind the influenza virus and thus inhibit the hemagglutination process. Visual observation of the plate can determine an HAI titer (similar to a VN titer) that indicates the presence of influenza virus neutralizing antibodies.
NA-Star® Neuraminidase Assay
Using the NA-Star® neuraminidase inhibitor resistance kit from Invitrogen™, we can evaluate potential compounds for their ability to inhibit the neuraminidase enzyme of influenza viruses. Compounds can be evaluated against nearly all of our influenza viruses using this highly reliable assay. The signal for this assay relies on detection of luminescence using a microplate luminometer. Linear regression is used to calculate a 50% inhibitory concentration of compound against the influenza virus neuraminidase. More information about the NA-STAR® assay kit can be found at Thermofisher's website.
MatTek EpiAirway™ 3D Tissue Model
MatTek's 3D tissue models provide valuable insight into antiviral therapy efficacy by utilizing normal human-derived tracheal/bronchial epithelial cells. The cells are cultured at the air-liquid interface and are more human-relevant for evaluation of respiratory infections than traditional cell culture models. The 3D mucociliary model is beneficial for evaluating the effects of antiviral therapy upon normal human cells when infected with a number of respiratory pathogens. This model works well for the evaluation of compounds against influenza, rhinovirus, adenovirus, respiratory syncytial virus, SARS and MERS coronavirus, and enterovirus D68. More information about the 3D cell culture model can be obtained through MatTek's website.