1. Virus Isolation in tissue culture & Virus Quantitation Lab 5

2. 2 Cell Culture Isolation of most viruses is carried out in cell culture. To establish cell cultures, tissue must be obtained originally from an animal. Common sources are embryonic tissues, tissues such as kidney, or tumour tissues. All the manipulations in cell culture are carried out in aseptic conditions to avoid contamination of the cells by bacteria or fungi. All materials used are sterilised. The suspension is washed and the cells are then counted and distributed into flasks in appropriate concentrations in medium. Cell culture medium is chemically defined and consists of an isotonic, buffered salt solution containing all the necessary amino acids (including glutamine) and vitamins for growth with glucose as a source of energy. Usually serum, often foetal calf serum, is added to a concentration of 10% as a source of growth factors that are necessary for cell growth. There are many varieties of cell culture media with different formulae depending on the requirements of specific cells.

3. 3 Cell Culture Many cells grown in culture are adherent; that is, they attach to the flat surface of a plastic flask and grow until the surface is covered with cells. Since the culture is one cell thick, it is called a monolayer. This type of culture is often used for isolation of viruses. Once the cells cover the whole surface of the flask, they are said to be confluent. They can then be removed from the surface of the flask by treating with trypsin and redistributed among several new flasks. This is called passaging or sub-culturing the cells. Some tissues, particularly embryonic tissues, yield cells that will grow for many passages before they stop (reach the senescence).

4. 4 Virus Isolation An appropriate sample is taken from the animal and placed in transport medium for transfer to the laboratory. Transport medium is cell culture medium with additional high concentrations of antibiotics to prevent bacterial contamination. In the laboratory the sample is treated to release any virus it may contain. This may involve simple washing of an oropharyngeal swab or to homogenise a piece of tissue. The medium is then centrifuged to remove large cellular debris and the fluid is inoculated (or may be first passed through a 0.45μm filter) onto a previously prepared cell culture. The inoculum is left on the cells for a period of about one hour to allow any virus to infect the cells and is then removed. The cells are washed to remove any debris or contaminants and fresh medium is added to the cell cultures which are then incubated to allow the virus to grow. This usually takes several days, depending on the growth rate of a particular virus. Virus growth is recognised by the development of a CPE which may be characteristic. Confirmation of the identity of the virus is by subjecting the culture fluid or the cells to a serological test that is specific for the virus.

5. 5 Assays of Infectious Virus The cytopathic effect (CPE) produced by the growth of viruses can be used to determine the presence and the amount of infectious virus in a suspension. The capacity to titrate, or assay, viruses in this way is crucial to experimental and diagnostic virology. There are two main methods of assay of viruses:  Quantal, or end-point assays  Quantitative assays (plaque assays) The plaque assay is to be preferred since it is more precise but not all viruses form plaques.

6. 6 Quantal assay of virus infectivity The suspension of virus to be titrated is diluted in, for example, ten-fold steps. A standard volume of each dilution is added to a cell culture. The cultures are incubated for a few days to allow any virus present to grow, spread and kill all the cells. The cultures are then observed for CPE. At low dilutions of virus all the cultures are killed. With further dilution, a dilution will be reached which contained no virus and the cells will have survived. The highest dilution containing virus that killed the cells is called the end-point and is said to contain one tissue culture infectious dose (TCID) of virus. For example, if the end-point is at a dilution of 1:10 6, the original virus suspension is said to contain 10 6 TCID. In practice, several replicate cultures are used for each dilution of virus and the end-point is taken as that dilution of virus that kills half of the cultures. The titre of the virus is then expressed as cell culture infectious dose 50%, or TCID 50. The use of replicates increases the precision of the assay.

7. 7 Quantitative assays (plaque assays) The plaque assay is more precise than an end-point assay and is the method of choice for infectivity assays. A plaque is a localised area of CPE in a cell culture produced by the growth of the progeny of a single virus particle (i.e., it contains one clone of virus). The steps in a plaque assay are: 1. Dilutions of virus are made (often 5 or 10-fold). 2. A volume of each virus dilution is inoculated into a cell culture (often a plate). 3. The virus is allowed to infect the cells for about one hour. 4. The inoculum is then replaced by medium containing 1% agar or agarose. 5. The cultures are incubated for a few days (depending on the virus). 6. The agar overlay is removed and the cells are fixed and stained. 7. The plaques on each plate are counted.

8. 8 Quantitative assays (plaque assays) The agar overlay is necessary to restrict progeny virus to the local plaque. Otherwise the virus would spread throughout the culture via the medium and generalised CPE but no plaques would be produced. A plaque is produced when a virus particle infects a cell, replicates, and then kills that cell. Surrounding cells are infected by the newly replicated virus and they too are killed. This process may repeat several times. The cells are then stained with a dye which stains only living cells. The dead cells in the plaque do not stain and appear as unstained areas on a colored background. Each plaque is the result of infection of one cell by one virus followed by replication and spreading of that virus. However, viruses that do not kill cells may not produce plaques. Since each plaque arises from a single virus particle, the amount of virus in the original virus suspension can be calculated by multiplying the number of plaques on a plate with the dilution factor of the virus that was inoculated on to that plate. This gives the titre of the virus and is expressed in plaque forming units (PFU). For example, if a culture inoculated with a virus dilution of 1:10 5 contained 50 plaques, the titre of the virus would be 50x10 5 (or 5x10 6 ) PFU.

9. 9 plaque assay. Serial dilutions of virus have been plated on confluent monolayer cultures of cells. The cells are stained after a period of time in which a single virus infects a cell, produces new virus particles and infects surrounding cells. The white areas show areas of the culture in which the cells have been killed. Each "plaque" is the result of the presence of one original infectious virus particle.

10. 10 Cytopathic changes Any detectable changes (i.e. visible by light microscopy) in the host cell due to infection are known as a cytopathic effect. Such changes include swelling or shrinkage of cells, the formation of multinucleated giant cells (syncytia), and the production of inclusions (visible by staining) in the nucleus or cytoplasm of the infected cell. The most efficient way to demonstrate cellular changes is by staining with chromatic dyes. Cell monolayers are fixed and then exposed to basic and acidic dyes that accentuate the nature and location of the changes. Haemotoxylin (basic dye) and eosin (acidic dye) is often used.

11. 11 Cytopathic changes The gross appearance of the cellular changes, and the location and nature of the "inclusions" - i.e. basophilic or eosinophilic - can in many instances be used as a diagnostic criterion to identify the causative virus. These will be illustrated for some of the viruses commonly isolated in cell culture

12. 12 Syncytia Many enveloped viruses possess a fusion protein in their envelopes. This confers the ability of the virion to fuse with the host cell membrane and thus allow entry of the infectious genomic material into the cell cytoplasm. During replication of the virus, expression of the fusion protein at the cell membrane can result in the fusion of neighboring cells, and the formation of multi-nucleate cells or syncytia. Example: Herpes and measles

13. 13 Cytoplasmic inclusion bodies Replication of reovirus particles occurs in the cytoplasm of the cell, and in the final stages of assembly the virus particles bud through the endoplasmic reticulum membrane. Cytoplasmic sites of accumulated viral protein are stained with eosin (deep pink).

14. 14 intranuclear inclusion bodies Herpesviruses replicate in the nucleus of the cell. An accumulation of viral proteins results in the formation of characteristic eosinophilic intranuclear inclusions. Minor differences in CPE allow distinction between the different types of herpesvirus