CULTIVATION OF VIRUSES

As the viruses do not reproduce independent of living host cells, they cannot be cultured in the same as bacteria and eukaryotic microorganisms. However, the cultivation of viruses can be discussed under following heads: (i) cultivation of animal viruses, (ii) cultivation of plant viruses, and (iii) cultivation of bacteriophages.

Cultivation of Animal Viruses

(i) In Animal Cells

Suitable living mammals (such as sheep or calves or rabbits) are selected for cultivation of viruses. The selected animals should be healthy and free from any communicable diseases. The specific virus is introduced into the healthy animals. The site of administration varies according to the type of virus is allowed to grow in the living animal. At the end of incubation period, the animals are slaughtered and washed thoroughly and viruses are obtained from them.

(ii) In Chick-Embryo

The animal viruses can be successfully cultivated using chick-embryo technique. In this method fertile hen eggs are selected. Eggs must not be more then 12 days old. To prepare the egg for virus cultivation, the shell surface is first disinfected with iodine and penetrated with a small sterile drill. After inoculation, the drill hole is sealed with gelatin and the egg is then incubated. Viruses may be able to region. For convenience, the myxoma virus grows well on the chorioallantoic membrane, whereas the mumps virus prefers the allantoic cavity. The infection may produce a local tissue lesion known as pock, whose appearance often is characteristic of the virus.

(iii) In Vitro Culture (Tissue Culture Technique)

More recently developed in vitro cultivation of animal viruses has eliminated the need to kill the animals. This technique has become possible by the development of growth media for animal cells and by the availability of antibiotics which prevent bacterial and fungal contaminations in cultures. Cultivating animal viruses using tissue culture technique involves following three main steps:

Cultured cells

• Primary

– Heterogeneous – many cell types

– Closest to animal

– Technical hassle

• Diploid cell strain

– Relatively homogeneous – fewer cell types

– Further from animal

– Technically less hassle

• Continuous cell line

– Immortal

– Most homogeneous

– Genetically weird – furthest from animal

– Hassle free

–Suspension or monolayerMonolayer Preparation. Live tissues of vital organs (e.g., heart or kidney) are taken and the cells are separated from the tissue by digesting the intracellular cement substance with dispersing agents such as trypsins or collagenase or ethylenediaminetetraacetic acid (EDTA). The cell suspension is passed through screen filters so that the coarse particles are removed from the separated cells. The cells are washed free of dispersing agents. The cells are centrifuged if required and resuspended in nutrient medium contained in glass or plastic vessels. The composition of medium and other conditions of incubation depends on the type of cells used. Upon incubation the cells quickly settle and attach firmly to the bottom of the flask. If undisturbed, these cells grow and spread to form monolayers.

Clonal Cell Line Preparation

The monolayer cells are first removed and washed with saline solution devoid of calcium and magnesium ions and then added to the dilute solution of EDTA (1: 3000) to chelate intracellular magnesium or calcium ions. After sometime, the loosened cells are shaken and resuspended in growth medium in fresh culture vessels and incubated. The cells are cultivated under 5% CO2 condition. The cultures of cell obtained so are called diploid cell strain. It is extremely difficult to distinguish primary cell and the diploid cell strain. On repeated subculturing, each cell starts multiplying to form separate colony. If each colony is removed and cultivated separately, it forms pure culture. These bunches of cells from single cell is called clonal cell lines.

Infection with Virus

The clonal cell lines suspended in suitable media are infected with any desired virus which replicates inside the multiplying cells. If the virus is virulent, they cause lysis of cells and virus particles are released in the surrounding medium. These newly produced virus particles (virions) infect the adjacent cells. As a result localized areas of cellular destruction and lysis (called plaques) often are formed. Plaques may be detected if stained with dyes, such as neutral red or trypan blue that can distinguish living from dead cells. Viral growth does not always result in the lysis of cells to form a plaque. Animal viruses, in particular, can cause microscopic or macroscopic degenerative changes or abnormalities in host cells and in tissues called cytopathic effects, cytopathic effects may be lethal, but plaque formation from cell lysis does not always occur.

Cultivation of Plant Viruses

There are several methods of cultivation of viruses such as plant tissue cultures, cultures of separated cells, or cultures of protoplasts, etc. Viruses also can be grown in whole plants.

Leaves are mechanically inoculated by rubbing with a mixture of viruses and an abrasive such as carborundum. When the cell wall is broken by the abrasive, the viruses directly contact the plasma membrane and infect the exposed host cells. (The role of the abrasive is frequently filled by insects that suck of crush plant leaves and thus transmit viruses.) A localized necrotic lesion often develops due to the rapid death of cells in the infected area. Even when lesions do not arise, the infected plant may show symptoms such as change in pigmentation or leaf shape. Some plant viruses can be transmitted only if a diseased part is grafted onto a healthy plant.

Cultivation of Bacteriophages

Bacterial viruses of bacteriophages (phages for short) are cultivated in either broth or agar cultures of young, actively growing bacterial cells. Several host cells are destroyed that turbid bacterial cultures may clear rapidly because of cell lysis:

Cultures are prepared by mixing the bacteriophage sample with cool, liquid agar and a suitable bacterial culture medium. The mixture is quickly poured into a petri dish containing a bottom layer of sterile agar. After hardening, bacteria in the layer of top agar grow and reproduce, forming a continuous, opaque layer or lawn. Wherever a virion comes to rest in the top agar, the virus infects an adjacent cell and reproduces. Finally, bacterial lysis generates a plaque or clearing in the lawn.

What is Plaques?

Plaques represent clearing the areas of bacterial growth due to lysis by a bacteriophage. If lytic bacteriophage is absent bacteria grow luxuriantly on culture medium. But if they are present, formation of clear zones or plaques can be seen here and there on the bacterial lawn. Each plaque corresponds to the site where a single bacteriophage particle acted as an infectious unit and initiated its lytic reproductive cycle. The spread of infectious bacteriophage from the initially infected bacterial cell to the surrounding cells results in the lysis of many of the bacterial cell and, therefore, a clear zone develops.