Influenza Virus Testing

The Schultz-Cherry laboratory is uniquely suited to undertake highly infectious influenza product testing, given that it is one of the few laboratories in the country with the high containment facilities, trained personnel, and animal model systems to test novel compounds.

Companies provide compounds they want tested for efficacy against influenza viruses, including the highly pathogenic avian H5N1 influenza strains (“bird flu”).  The Schultz-Cherry lab completes the test(s) ordered, and provides results to the company.  Analysis of results is not included as part of the service provided.  If the assay does not work due to unforeseen circumstances in the laboratory, it will be repeated without charge to the customer.  If the doses supplied by the customer fail to produce the wanted effect, the customer will be responsible for trouble-shooting and requesting additional assays (for additional fees).

Description of Assays

To request any of the following tests, please fill out the request for service form.

Cytotoxicity Assay (1 compound – 4 doses and controls)

Method

• To determine if the test compound has any associated toxicity, Madin Darby canine kidney (MDCK) cells will be plated at 1 x 10⁴ cells per well in a 96-well tissue culture plate and incubated until confluent (overnight).
• Cells will be washed to remove sera and up to 4 doses of the test compound (provided by customer) will be added to the cells and incubated for 24 hours.
• Cytotoxicity will be determined by adding the tetrazolium salt MTT to the wells and measuring the production of the colored product formazan.
• Results will be measured photometrically at 570 nm and presented as percentage viability (Y axis) versus concentration.  Cells alone will be considered 100% viable.
• The result from this assay is required prior to performing further cell-based experiments.

 

Fluorescent Focus Assay (1 compound – 4 doses and controls)

Method

• To determine whether the test compound inhibits viral replication, MDCK cells will be plated in 24 well tissue culture plates on glass coverslips and grown until confluent.
• Cells will be washed to remove sera and treated with media alone (negative control) or increasing by four times the concentrations of the test compound provided by the customer.  Cells will then be infected with influenza virus at a standard concentration and incubated for 6 hours.
• Cells will then be washed and fixed with paraformaldehyde prior to staining for the viral nucleoprotein using a specific antibody.
• Viral antigen will be detected by immunofluorescent microscopy and the number of fluorescent cells counted.
• Results will be presented as the number of infected cells (Y-axis) versus virus alone or in the presence of test compound (X-axis).

 

Virus Neutralization Assay (1 compound or sera – 4 doses and controls)

Method

• To determine if the test compound inhibits viral replication, MDCK cells will be plated in 6 wells tissue culture plates and incubated until confluent.
• Cells will be washed to remove sera and a standard concentration of virus will be incubated with up to 4 doses of the test compound (provided by customer) and used to infect cells. Alternatively, the cells may be pre-treated with the compound prior to infection (again – this information must be provided by customer).
• 24 to 48 hr after infection, media will be removed from cells and 10-fold dilutions prepared.
• MDCK cells previously plated in a 96-well tissue culture plate will be washed to remove sera and the above dilutions added in duplicate and incubated for 72 hours.
• The viral titer will be determined based on the last dilution demonstrating viral-induced cell death.  This information will be provided as the Viral titer in log/ml (Y-axis) versus virus alone or with test compounds (X-axis)

 

Hemagglutination Inhibition (HI with serum) (1 compound or sera – 4 doses and controls)

Method

• To determine whether the test compound or sera inhibits the agglutination of chicken red blood cells by a standardized concentration of influenza virus, test samples (up to 4 different dilutions as provided by the customer) are incubated with virus prior to the addition of red blood cells.
• Agglutination is visually determined and results are provided as YES/NO inhibits agglutination; if YES – we will tell the dose of compound that inhibited.

 

Neuraminidase Inhibition (NI with serum) (1 compound or sera – 4 doses and controls)

Method

• To determine whether the test compound or sera inhibits influenza neuraminidase activity, test samples (up to 4 different dilutions as provided by the customer) are incubated with a standardized concentration of virus and neuraminidase activity determined by MUNANA assay (fluorometric substrate cleaved by influenza neuraminidase).
• The results are quantitated on a fluorescent plate reader and will be provided as the fluorescence activity (Y-axis) versus the virus alone (positive control) with or without test compound.

 

Animal Experiments

You must consult with Dr. Schultz-Cherry (slschul2@wisc.edu) before test price can be determined and a request for service can be initiated. It requires prior in vitro testing prior to initiation. Mice may require treatment with the compound prior to infection.  This information and the dose must be provided by the customer and will not be optimized by Dr. Schultz-Cherry

Method

• Five mice per group (groups including control uninfected mice; mice infected with virus alone; and test compounds of interest) will be weighed and intranasally inoculated with saline or a standard concentration of virus in 25 ul saline.
• Mice will be monitored daily for general health and weighed every 48 hours post-infection for up to 10 days.
• It may be necessary to treat with compound or measure other health parameters including clinical score, temperature, or lung function.  This will need to be agreed upon prior to initiation of experiments.
• All mice will be euthanized at termination of the experiment.
• Results are dependent on parameters requested and could be presented as:
  
  
  1. Percentage weight loss
  2. Clinical score
  3. Lethality
  4. Temperature
  5. Lung function

 

Viral Titers (pooled lungs from one group of mice at one time point)

You must consult with Dr. Schultz-Cherry (slschul2@wisc.edu) before test price can be determined and a request for service can be initiated.

Method

• To determine viral titers in lungs of infected mice, lungs will be collected from one group of mice at one time point, pooled, and homogenized in antibiotics.
• Lung homogenates will be prepared as 10-fold dilutions.
• MDCK cells previously plated in a 96-well tissue culture plate will be washed to remove sera and the above dilutions added in duplicate and incubated for 72 hours.
• The viral titer will be determined based on the last dilution demonstrating viral-induced cell death.  This information will be provided as the Viral titer in log/ml (Y-axis) versus virus alone or with test compounds (X-axis).