will allow a limit to the dissemination of the newborn viruses onto the tissue cell

culture and to have a transmission of the infection only to the closest cells. This way

of viral expansion led to the creation of lysis plaque within the tissue culture, which

ultimately will allow for counting the initial number of viruses applied in the

suspension. This is possible for a viral concentration, which allows for the visual or

microscopic counting of the lysis plaque.

The method assumes that only one virus infects one cell which might introduce a

bias. The case of several defective incomplete viruses entering the same cell and

self-complementing cannot be ruled out. Therefore, false positives are possible for

such assays.

TCID50- Tissue Culture Infectious Dose 50 uses a similar principle of ap-

plying a suspension of the virus at different concentrations onto a plated tissue

culture. In this case, the diffusion of the newborn viruses is not limited to inducing

several cycles or replications on the same plated culture. Ultimately the whole

culture should be infected and lysed. Therefore, the read-out of such assays is

evaluating the cytopathic effect of a virus suspension. Such an assay lasts between

4 to 7 days [4]. The test is commonly performed on 96-well plates with replicates to

evaluate the percent of tissue cultures that have been infected after several potential

replication cycles. Thus, the wells that are infected are counted, among the wells

where the tissue culture is healthy. A statistical analysis is performed to determine

Negative Stain Transmission Electron Microscopy (NSTEM)

Extracellular vesicles

Influenza viral particle

Influenza virus like particles

Clarified supernant

Sucrose cushion purified supernant

FIGURE 8.2 Negative Staining Transmission Electron Microscopy (NSTEM) observation

of viral preparations. A − Sucrose cushion and sucrose gradient purified preparation of

influenza virus, extracellular vesicles, or influenza virus like particles. B – Immunogold

labeling of sucrose cushion and sucrose gradient purified preparation of influenza virus.

C – Several purification steps of culture supernatant.

Analytics and virus production processes

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