CULTIVATION OF VIRUS

•  Viruses are obligate intracellular parasites that depend on a host for survival and replication.
• They require living cells for their replication process.
• The primary purpose of cultivating viruses is to isolate and identify the diseases. 
• Also, to understand the viral structure, replication, genetics, and effects on the host cell.
• To prepare the virus for vaccine production.

Mostly virus is cultivated based on

1. Experimental animals.
2. Embryonated eggs.
3. Tissue culture.

Animal Inoculation:

• A mouse is frequently used for the isolation of a virus by animal inoculation.
• Rabbits, hamsters, and newborn rodents are also used.
• This inoculation method is used rarely but plays an essential role in the study of pathogenesis.
• The introduction path will be by intracerebral, subcutaneous, intraperitoneal, or intranasal path.
• After inoculation, the animal is checked for any infections or disease.
• Mice are used for the isolation of the rabies virus and Coxsackie virus.

Embryonated eggs:

• It was used first for the cultivation of the virus by Goodpasture in 1931.
• The method was developed by Burnet and used on different types of embryonated eggs at different sites.
• Prominently, 8-11-day eggs are used for this process.
• Viruses are isolated from the yolk sac, amniotic cavity, allantoic cavity, and chorioallantoic membrane.

Yolk sac: Mainly used for the cultivation of Japanese encephalitis and West Nile virus.

Amniotic cavity: cultivation of Influenza virus.

Allantoic cavity: cultivation of the Influenza virus, yellow strain, and rabies virus.

Chorioallantoic membrane: Cultivation of pox virus-like variola or vaccinia.

 

Tissue culture:

• The most used technique or method for the cultivation of the virus and the assay of the virus.
•  Tissue culture was first applied in diagnostic virology by Steinhardt and his colleagues in 1913.
• Maitland 1928 used cut tissues and kept them in nutrient media for the cultivation of the virus.
• Enders, Weller, and Robins 1949 were the first to culture polio virus.

Different types of tissue culture are used to grow viruses like

1. Organ culture.
2. Explant culture.
3. Cell culture.

Organ culture:

• In this method, small pieces of organs are maintained in vitro conditions for days or weeks, preserving their original morphology and functions.
• For example, coronavirus, a respiratory pathogen, was isolated in tracheal ring organ culture.

Explant culture:

• The components of minced tissues are grown as explants embedded in plasma clot.
• For example, adenoid tissue explants are used to cultivate the adenovirus.

Cell culture: Tissues are dissociated into component cells by treatment with proteolytic enzymes (trypsin or collagenase) followed by mechanical shaking.

• The cells are then washed, counted, and suspended in a growth medium containing essential amino acids and vitamins, salts, glucose, and a buffering system. This medium is supplemented by up to 5% of fetal calf serum and antibiotics.
• The cell suspension is dispensed in glass or plastic bottles, tables, or Petri dishes.
• On incubation, the cells adhere to the glass surfaces and divide to form a confluent monolayer sheet of cells covering the surface within a week.
• The cell culture may be incubated either as a stationery culture or as a roller drum culture. The latter is useful for growth of some fastidious viruses due to better aeration by rolling of theculture bottle in special roller drums.
• The cell cultures are classified into three different types based on their origin, chromosomal characters, and number of generations for which they can be maintained.

Primary cell culture:

• These cell cultures can be established from whole animal embryo or from selected tissues from adult, newborn, or embryos.
• These cells have the normal diploid chromosomal number and are capable of only limited growth (5–10 divisions) in culture.
• Monkey kidney cell culture, human embryonic kidney cell culture, and chick embryo cell culture are the common examples of primary cell culture.

Diploid cell strains:

• Diploid cell strains are of a single cell type that retains their original diploid chromosome number and karyotype. However, they have specific characteristics and compositions and are usually composed of one basic cell type.
• They are usually fibroblasts and can be cultured for maximum 50 serial passages before they undergo senescence (die off) or undergo a significant change in their characteristics.
• They are also used for production of vaccines; for example, WI-38 human embryonic, lung cell stem is used for the cultivation of fixed rabies virus, and human fetal diploid cells for isolation of adenovirus, picornaviruses, HSV, CMV, and VZV.

Continuous cell lines:

• The cells are usually derived from diploid cell lines or from malignant tissues and have altered and irregular number of chromosomes.
•  Hep-2, HeLa, and KB derived from human carcinoma cervix, human epithelioma of larynx, and human carcinoma of nasopharynx and other cell lines are excellent for recovery of a large number of viruses.
• Most of the viruses can be isolated by using one of these cell lines.