PROPAGATION OF VIRUSES IN THE LABORATORY

Introduction Viruses are important agents of many human diseases, ranging from the trivial (e.g. common colds) to the lethal (e. g . rabies) Viruses also play roles in the development of several types of cancer. As well as causing individuals to suffer, virus diseases can also affect the well-being of societies. Smallpox had a great impact in the past and AIDS is having a great impact today.

Purpose of Growing Viruses in the Laboratory Diagnosis of infection Research Production of antigens for vaccines and serological reagents

Important Characteristics of Viruses Related to Propagation Viruses can not grow outside a living cell. The range of cell types in which they will replicate is limited. A few viruses can not be grown at all (neither in vivo nor in vitro) Viruses can be propagated in: Animals Chick embryos Cell Culture

1. Propagation in Animals The first virus vaccines, smallpox and rabies, were prepared in animals. Smallpox on the skin of calves, sheep or water-buffaloes. Rabies in the spinal cords of rabbits, sheep or goats. Mice or other small animals are sometimes used in research laboratory.

2. Propagation in chick embryos The time from fertilization of hen’s eggs to hatching is 21 days. Before inoculation the eggs are incubated at 37˚C, usually for 10-14 days. There are different inoculation sites in the embryonated eggs

2. Propagation in chick embryos Amniotic inoculation of 10-11 days-old embryos used to isolate influenza viruses. Influenza, may require repeated amniotic passages before becoming adapted to the egg and grown in the allantoic cavity Used for vaccine production. Chorioallantoic membrane Pox and herpes simplex viruses produce lesions (pocks) 1-3 mm in diameter, each of which is a focus of cell proliferation.

3. Propagation in Cell and Tissue Cultures Cell culture is a collection of dispersed living cells, either in suspension or as continuous (confluent) sheets adhering to glass or plastic surfaces. Such sheets, one cell thick, are called monolayers. It is the most commonly used culture method

3. Propagation in Cell and Tissue Cultures Culture media These are nutrient rich media and usually contain salts at physiological concentrations, glucose, amino acids, essential vitamins, and antibiotics. All ingredients are buffered at pH 7.2-7.4. Fetal calf serum (10-20%) is added to provide essential growth factors. Cells in culture media are incubated at 37oC in CO2 atmosphere.

Effects of Viral Infections on Host Cells Cytopathic Effects When viruses are added to the cell culture, cells undergo changes, which are referred to as “cytopathic effects”. Examples for these effects include: Intracytoplasmic inclusion bodies, e.g., rabies virus Intranuclear inclusions, e.g., herpesvirus Cytoplasmic and nuclear inclusions, e.g., measles virus Lysis of cells (many viruses) Syncytium formation, e.g., respiratory syncytial virus Transformation of cells (oncogenic viruses)

Effects of Viral Infections on Host Cells (A) uninfected cells, (B) nuclear inclusions & cell enlargement, (C) cell degeneration (i.e., lysis), (D) a focus of infected cells in a cell monolayer (i.e., a plaque)

Effects of Viral Infections on Host Cells Cell Transformation Cell transformation is an alteration in growth properties of virus-infected cells. Such alterations are manifested in the formation of a tumor if the virus is used to infect an animal. In infected cell cultures, morphology, growth properties and cellular metabolism are altered. These changes are referred to as cellular transformation and are a direct result of expression of one or more viral genes. These changes are passed on to the infected cell progeny. Viral DNA must be integrated into host cell genome.

Effects of Viral Infections on Host Cells Plaque Formation Cells infected with some viruses are destroyed showing clear areas within the monolayer. These areas are referred to as plaques. When lawns of bacteria are infected with bacteriophages, after incubation, clear areas appear which represent sites of lysed bacteria (plaques). Plaque formation can be used to quantitate virus titers by counting plaques produced in cell monolayers.

Virus Purification After a virus has been propagated it is usually necessary to remove host cells debris and other contaminants before the virus particles can be: used for laboratory studies, for incorporation into a vaccine, or for some other purpose

Partial purification of virions by differential centrifugation Virus Purification Differential centrifugation Involves alternating cycles of low-speed centrifugation, after which most of the virus is still in the supernatant, and high-speed centrifugation, after which the virus is in the pellet Partial purification of virions by differential centrifugation

Purification of virions by density gradient centrifugation Virus Purification Density gradient centrifugation Involves centrifuging particles (such as virions) in a solution of increasing concentration (cesium chloride(CsCl), potassium tartrate, potassium citrate, or sucrose), and therefore density Purification of virions by density gradient centrifugation he complete, infective form of a virus outside a host cell, with a core of RNA or DNA and a capsid.

Reaction to Physical and Chemical Agents Heat and Cold Virus infectivity is generally destroyed by heating at 50-60oC for 30 minutes. Some exceptions may include Hepatitis B virus and Prions. Viruses can be preserved by storage at subfreezing temperatures and some may withstand lyophilization and then preserved at 4oC or even at room temperature. Enveloped viruses tend to lose infectivity after prolonged storage even at-90oC and are sensitive to repeated freezing and thawing. These viruses are effectively stabilized in the presence of 5% dimethyl sulfoxide

Stabilization of Viruses By Salts Many viruses can be stabilized by molar concentrations of salts and cannot be inactivated even by heating at 50oC for 1 hour. The stability of viruses is important in the preparation of vaccines. The nonstabilized polio vaccine must be stored at freezing temperatures to preserve its potency. However, with the addition of molar MgCl2, the potency can be maintained for weeks at room temperature, even in the high temperatures of the tropics

Stabilization of Viruses By pH Viruses are usually stable between pH values of 5.0 and 9.0. Because of the electrostatic forces in the hemagglutination reactions, variations of few tenths of a pH unit may be the deciding factor in obtaining positive or negative results in this test.

Inactivation of Viruses By Radiation Ultraviolet, X-ray, and high energy particles inactivate viruses. Vital Dyes Viruses are penetrable to a varying degree by vital dyes such as toluidine blue, neutral red, and proflavine. These dyes unite with nucleic acid, and the virus then becomes susceptible to inactivation by visible light. Photodynamic inactivation can be used for treating herpetic lesions.

Inactivation of Viruses Ether Susceptibility Enveloped viruses are inavtivated by ether while naked viruses are resistant to ether inactivation. Disinfectants High concentrations of chlorine are required to destroy viruses while formalin destroys resistant viruses