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In Vitro Antiviral Testing

A key step in drug discovery is screening to evaluate antiviral activity. After determining the appropriate compounds and mechanisms, the next step is to perform cytotoxicity analysis of the compounds to ensure your efficacy data are meaningful and within a reasonable therapeutic window. Here is a short list of antiviral assays.

VSV-Pseudotyped Assays:  For this assay the Vesicular Stomatitis virus (VSV) glycoprotein (G) is substituted with Ebola, Sudan or Marburg Virus glycoprotein (GP) or SARS-CoV-2 spike (S) protein to create a recombinant virus that can be safely handled at biosafety level 2, BSL 2. The recombinant rVSV-SARS-CoV-2 Spike has been incorporated into a convenient luciferase-based neutralization assay for evaluating the efficacy of drug candidates that target spike-mediated infection.  Neutralization is determined relative to the untreated virus controls. In dose response experiments, the endpoint readout is the concentration of test article causing 50% neutralization (IC50). The rVSV-EBOV and rVSV-MARV  viruses are cytopathic. Infection efficiency can be measured by quantifying luciferase fluorescence or by plaque count.

Cytopathic effect (CPE) inhibition assay. CPE is morphological changes in cells caused by cytopathogenic virus infection. CPE assay is used to evaluate test articles’ ability to inhibit CPE. This is the most cost-effective and time-efficient assay we offer for high throughput screening of overall antiviral activity. For non-cytopathic viruses we offer cell-based enzyme-linked immunosorbent assay (ELISA) or quantitative real-time Polymerase Chain Reaction (PCR) assay.

Cell-based ELISA. Cell-based ELISA measures reduction of viral antigen in infected cells using anti-virus monoclonal antibody. The abundance of viral protein in infected cells treated with the test article compared to that of the untreated control is used as a measure of antiviral activity.

qPCR assay. qPCR assay uses oligonucleotide primers and a probe amplifying virus-specific target sequence to detect the presence of virus nucleic acids. Reduction of virus nucleic acid in infected cells is used an indicator of a test article’s antiviral efficacy.

Plaque reduction assay. Infectious virus particles multiply in cells and result in circular zones of infected regions, plaques. Plaque reduction assay measures the plaque forming efficiency of a virus in the presence of different concentrations of a test article. Plaque reduction neutralization test (PRNT), a variation of this assay, is considered the gold standard for detecting neutralizing antibodies to certain viruses (i.e., flavivirus).

Yield reduction assay. Yield reduction assay is a labor-intensive but powerful technique for evaluating a compound’s antiviral efficacy. The three-step assay involves: infecting cells in the presence of different concentrations of the test article; collecting the cells or cell culture supernatants after a cycle of virus replication; and determining virus titers by plaque assay, TCID50, or quantitative real-time PCR.

Antibody-dependent enhancement (ADE) assay. ADE occurs when non-neutralizing or sub-neutralizing antiviral proteins facilitate virus entry into host cells leading to enhanced infectivity. ADE, which has been observed in viruses such as Dengue and Influenza, poses a challenge in vaccine development. Using flow cytometry, plaque assay or qPCR, this assay evaluates the ADE effect of test articles on virus infection in Fc receptor bearing cells.

Hemagglutination-inhibition test (HAI). HAI assay tests the efficacy of influenza vaccine candidates in preventing virus-induced hemagglutination. We offer HAIs against H1, H7 and H10 subtypes of the Influenza virus.

Quantitative suspension test. This test is used to evaluate virucidal activity of chemical disinfectants within a given contact time in suspension. Generally, a 4 log10 reduction in virus titer (99.9% inactivation) is an indicator of a disinfectant’s virucidal properties detected under the test conditions.

IBT Bioservices offers a set of assays in key virus families. We can help you select the appropriate assays for your specific compound and mechanism of action, as outlined in the table on the following page. If you don’t see your pathogen we can provide additional validations upon request.  Contact us for a consultation and a no obligation quote.

Virus Family
Genus
Species
Strain
Details
Togaviridae Alphavirus

Chikungunya

Venezuelan Equine Encephalitis

181/25

TC-83


Flaviviridae Flavivirus

Dengue 1-4

Japanese Encephalitis

Yellow Fever

Zika 

Various

14-14-2


17D

FSS13025




Arenaviridae Arenavirus Junin Candid 1
Orthomyxoviridae Influenza A

Influenza B

H1N1

H3N2


Bunyaviridae Phlebovirus
Orthobunyavirus
Rift Valley Fever
La Crosse

MP12

 
Poxviridae Orthopoxvirus Vaccinia NYCHBH  
Paramyxoviridae Pneumovirus Human Respiratory Syncytial Virus
Herpesviridae Simplexvirus Herpes Simplex Virus
Picornaviridae Enterovirus

Enterovirus 70 (EV)

Coxsaekievirus

Echovirus

J670/71

DN-19

Gregory

 
Coronaviridae Betacoronavirus
SARS-CoV-2

(Pseudotyped-VSV)
Wuhan

Spike Variants

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Fig. 1 Survival after challenge with INFV H1N1 A/Pert/261/2009 (Tamiflu-resistant strain). Inoculum 1xLD90=1.0E+05 PFU/mouse
Survival after challenge with INFV H1N1 A/Pert/261/2009 (Tamiflu-resistant strain) 1.0E+05 PFU/mouse
Survival and weight change in BALB/c mice challenged with INFV A/ Texas/36/91 (H1N1) and treated with antiviral Osletamivir Phosphate (Tamiflu)
Lung viral load and Survival (30 % weight loss cut-off) in BALB/c mice challenged with INFV H3N2 A/HK/1/68.

Alpha (UK) – B. 1.1.7 / 501Y.V1

amino acid mutations: del69–70 HV, del144 Y, N501Y, A570D, D614G, P681H, T761I, S982A, D1118H

Beta (South Africa) – B.1.351

amino acid mutations: K417N, E484K, N501Y, D614G, A701V

Gamma (Brazil) – P.1

amino acid mutations: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I

Epsilon (Ca, USA) B.1.427

amino acid mutations: L452R, D614G

SARS-CoV-2 Parental Strain Wild Type (Wuhan)
SARS-CoV-2 D614G Variant

amino acid mutations: D614G

Epsilon (Ca, USA) B.1.429

amino acid mutations: S13I, W152C, L452R, D614G

SARS-CoV-2 Delta Variant

amino acid mutations: L452R, E484Q