Lesson 4d: Detection of replicating viruses

• Some viruses similar to widespread respiratory viruses (the influenza and parainfluenza viruses) and several other different viruses, mumps virus and Newcastle illness virus replicate in cell cultures however often don’t produce cytopathic results.
• Though the replication of those viruses could not produce cytopathic results, the presence of replicating virus may be detected by hemadsorption, virus Interference, virus neutralization take a look at (VNT). The later has already been covered under the topic of detection of antibodies.

Hemadsorption

• Hemadsorption is the ability of viral infected cells to bind red blood cells (RBCs)
• This occurs because certain viruses express on the surface of infected cells viral proteins that bind to erythrocyte membranes
  Paramyxoviruses, including influenza, parainfluenza, and mumps have this behaviour
• The binding is specific for the erythrocytes of certain species, such as guinea pig, rat, or monkey.

The final step in the maturation phase of these viruses is the budding of the virion through the plasma membrane of the cell. The insertion of viral proteins necessary for budding of virus from the cell membrane facilitates the binding of RBCs to the infected cells. This process
is referred to as hemadsorption, and the RBCs from several species (human O, chicken, or guinea pig) can be used in this method. This process is relatively simple. A suspension of RBCs is added to the cell monolayer, and the mixture is incubated at the required temperature and then observed microscopically for adsorption of RBCs to the cells in the monolayer.

Materials

• Guinea pig RBCs
• A 10% (vol/vol) suspension of RBCs in Alsever’s solution is used. This suspension is stable for 1 week when stored at 4 o C.
• Uninoculated cell culture controls
• Cell cultures inoculated with patient sample(s)
• Cell cultures inoculated with known hemadsorbing strains of virus

Method

1. Transfer 5 ml of the RBCs in Alsever’s solution to a graduated centrifuge tube, add 5 ml of phosphate-buffered saline (PBS), mix well, and centrifuge at 900 × g for 5 min.
2. Discard the supernatant, and resuspend the RBC pellet in 10 ml of PBS.
3. Repeat the pelleting and resuspension steps two more times.
4. After discarding the supernatant from the final centrifugation, determine the packed cell volume of the RBCs.
5. Resuspend the RBCs in PBS to a final concentration of 10% (vol/vol). The stock suspension of RBCs should not be stored for more than 1 week at 4°C.
6. On each day of testing, prepare a 0.4% (vol/vol) suspension of guinea pig RBCs in PBS from the 10% (vol/vol) stock solution of guinea pig RBCs.
7. Remove the cell culture fluid from each monolayer in a biological safety cabinet. The cell culture fluid can be saved in a sterile tube or discarded depending on the method used to identify hemadsorbing virus.
8. Add 0.2 ml of the 0.4% RBC solution to each cell culture. The guinea pig RBC suspension should cover the monolayer but not reach beyond the neck of the tube.
9. If mumps virus is in the healthcare provider’s request, incubate the cell cultures at 4 o C for 30 min.
10. Rock each tube gently, and then observe each monolayer microscopically.  However, as the cells in the monolayer age, they become sticky and can appear to have RBCs that are hemadsorbing. The cell cultures inoculated with a known hemadsorbing virus should have RBCs adsorbed to the cells (Figure below). The monolayers can show varying degrees of hemadsorption, and at times, the supernatant will also have hemagglutination of the RBCs.
11. If other hemadsorbing viruses are included in the test request, the laboratory will perform a second hemadsorption by incubating the tubes at room temperature for 30 min and observing them as described above. The room temperature incubation and observation may be the only hemadsorption assay performed if mumps virus isolation is not requested.
12. For hemadsorption-negative cultures, the RBC suspensioncan be removed, fresh cell culture media can be added, and the cultures can be reincubated.
13. The identifi cation of the hemadsorbing virus can be done with either the supernatant or the infected cells. The monolayer can be removed from the surface of the tube, and the cells are then attached to a glass slide and stained in either a direct or indirect immunofluorescent assay. The binding of virus-specific antibody is detected by direct observation of the stained cells with a fluorescent microscope. The supernatant can be tested by hemagglutination inhibition (HAI).