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CULTIVATION OF VIRUS.pptx

The document discusses methods for cultivating viruses, highlighting animal inoculation, embryonated eggs, and tissue culture as the primary techniques. It outlines the purpose of virus cultivation, including isolation for research and vaccine production, and describes processes involved in each cultivation method. Additionally, it details monitoring techniques for viral growth and identification in cell cultures through various biochemical and morphological assessments.

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VIVA MARIYA K V MSC.BIOTECHNOLOGY ST.MARY’S COLLEGE THRISSUR
Viruses are obligate intracellular parasites which means they can only grow or reproduce inside a host cell. The primary purpose of virus cultivation:  To isolate and identify viruses in clinical samples. To do research on viral structure , replication , genetics and effects on host cell. To prepare viruses of vaccine production. Isolation of virus is always considered as a gold standard for establishing viral origin of disease
CULTIVATION OF VIRUSES Animal inoculation Embryonated eggs  CAM Allantoic cavity  Aminotic cavity  Yolk sac Tissue culture Organ culture Explant culture Cell culture  Primary cell culture  diploid cell culture  Continues cell lines
Mainly 3 methods are employed 1. Animal inoculation 2. Embryonated eggs 3. Tissue culture CULTIVATION OF VIRUS
 Earliest method of cultivation of viruses causing human viral disease was inoculation into human volunteers. • Mouse is most frequently used for isolation of viruses by animal inoculation. • After inoculation, the animals are observed for signs of disease or death. • The infected animals are then sacrificed and infected tissues are examined for the presence of viruses by various tests, and also for inclusion bodies in infected tissues. • Furthermore, infant (suckling) mice are used for isolation of coxsackie virus and rabies virus.
Guinea pigs Rabbits Ferrets
Inoculation routes in mice  Intranasal  Intracerebral  Subcutaneous Inoculation routes in mice
 Animal inoculation may be used as a diagnostic procedure for identifying and isolating a virus for a clinical specimen.  Mice provides a reliable model for studying viral replication.  Gives unique insight into viral pathogenesis and host virus replication.  Expensive and difficulties in maintenance.  Mice don’t provide models for vaccine production.  It will lead to generation of escape mutants.  Issues related to animal welfare systems.
 1931 Good Pasture first used the embryonated hen’s egg for cultivation of virus.  The egg used for the cultivation must be sterile and the shell should be intact and healthy.  A hole is drilled in the shell of the embryonated egg, and a viral suspension or suspected virus- containing tissue is injected into the fluid of the egg.  Viral growth and multiplication in the egg embryo is indicated by the death of the embryo, by embryo cell damage, or by the formation of typical pocks or lesions on the egg membranes.
Routes of Egg Inoculation
Chorioallantoic membrane (CAM) Allantoic Cavity Aminotic Cavity Yolk Sac Virus growth and replication in the CAM is indicated by visible lesions (pocks); grey white area in transparent CAM.. Allantoic cavity Is the most popular and simple method for viral inoculation. The amniotic sac is employed inoculated for primary isolation of influenza a virus and the mumps virus. It is also a simplest method for growth and multiplication of virus. Each pock is derived from a single virion. The morphology of the pocks may vary depending on the nature of the virus. Allantoic inoculation is employed for the growth and replication of the influenza virus for vaccine production. Other allantoic vaccines include Yellow fever and rabies vaccines. Growth and replication of virus in egg embryo can be detected by haemagglutination assay. Immune interference mechanism can be detected in most of avian viruses. This method is also used for the cultivation of some bacteria like Chlamydiae and Rickettsiae  Under optimal conditions, each infectious virus particle can form one pock. Hence this method is suitable for plaque studies. Mostly mammalian viruses are isolated using this method.
 Widely used method for the isolation of virus and growth.  Ideal substrate for the viral growth and replication.  Isolation and cultivation of many avian and few mammalian viruses.  Cost effective and maintenance is much easier.  Less labor is needed.  The embryonated eggs are readily available.  Sterile and wide range of tissues and fluids.  Widely used method to grow virus for some vaccine production.  The site of inoculation for varies with different virus. That is, each virus have different sites for their growth and replication.
Organ culture Explant culture Cell culture •Cell culture is most widely used in diagnostic virology for cultivation and assays of viruses. •Different types of tissue cultures are used to grow viruses. Tissue culture can be of three different types as follows:
• This was used earlier for the isolation of some viruses, which appear to show affinity for certain tissue organs. For example, coronavirus, a respiratory pathogen, was isolated in the tracheal ring organ culture. • In this method, small bits of the organs are maintained in vitro for days and weeks preserving their original morphology and function. • Nowadays, organ culture is not used.
• In this method, components of minced tissue are grown as explants embedded in plasma clots. • Earlier, adenoid tissue explant cultures were used for isolation of adenoviruses. • This method is now seldom used in virology
• Cell culture is now routinely used for growing viruses. • In this method, 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 the culture bottle in special roller drums.
Primary culture Diploid cell structure Continuous cell lines • 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 are normal cells freshly taken from animal or human body. •They are able to grow only for limited time and cannot be maintained in serial culture. •They are used for the primary isolation of viruses and production of vaccine. •Example : Monkey kidney cell culture, Human embryonic kidney and Chick embryo cell culture. Diploid cell strains: • Also known as semi-continuous cell lines. • They are diploid and contain the same number of chromosome as the parent cells. • They can be sub-cultured up to 50 times by serial transfer following senescence and the cell strain is lost. • They are used for the isolation of some fastidious viruses and production of viral vaccines. • Example :Human embryonic lung strain, Rhesus embryo cell strain.
Continuous cell lines: • They are derived from cancer cells . • They can be seriously cultured so called as continues cell lines. • They can be maintained either by serial subculture or by storing in deep freeze at - 70 degree Celsius. • Due to derivation from cancer cell they are not useful for vaccine production. • Example : HeLa-Human carcinoma of cervix cell HEP2-Human epithelioma of larynx cell lines BHK-21 –Baby hamster kidney cell lines.
Recognition of Viral Growth in Cell Culture  The growth of viruses in cell culture can be monitored by a number of biochemical procedures indicative of the intracellular increase in viral macromolecules and virions .  In addition, there are simpler methods that are more commonly used for diagnostic work Cytopathic Effects Hemadsorption and Hemagglutination. Immunofluorescence Interference.
Cytopathic Effects:  Many viruses can be detected and initially identified by observation of the morphological changes in the cultured cells in which they replicate.  The CPE produced by different types of viruses are characteristic and help in the initial identification of virus isolates.  Nuclear shrinking, vacuoles in the cytoplasm, syncytia formation, rounding up, and detachment are the examples of alteration of morphology of the cells.  Most CPEs can be demonstrated in unfixed and unstained monolayer of cells under low power of microscope.  For example, adenoviruses produce large granular changes resembling bunches of grapes, SV-14 produces well-defined cytoplasmic vacuolation, measles virus produces syncytium formation, herpes virus produces discrete focal degeneration, and enteroviruses cause crenation of cells and degeneration of the entire cell sheet.
Hemadsorption and Hemagglutination  Cultured cells infected with orthomyxoviruses, paramyxoviruses, or togaviruses, all of which bud from cytoplasmic membranes, acquire the ability to adsorb erythrocytes. This phenomenon, know as hemadsorption, is due to the incorporation into the plasma membrane of newly synthesized viral protein that binds red blood cells .  Hemadsorption can be used to demonstrate infection with non- cytopathogenic as well as cytocidal viruses, and can be demonstrated very early, e.g., after 24 hours, when only a small number of cells in the culture are infected.  Hemagglutination is a different, though related phenomenon, in which erythrocytes are agglutinated by free virus .  Virions or hemagglutinin may thus be demonstrated in the supernatant fluid of an infected culture.
Immunofluorescence Newly synthesized intracellular viral antigen can be detected by staining the fixed cell monolayer with specific antiviral antibody which has been labeled with a fluorescent dye, or with an enzyme such as peroxidase. Interference  The replication of one virus in a cell usually inhibits the replication of another virus .  The viruses of rubella and of the common cold were first discovered by showing that infected cell cultures, which showed no cytopathic effect, were nevertheless resistant to challenge with an unrelated enterovirus.  Cell lines have now become available in which these viruses produce cytopathic effects, but interference is still used for the diagnosis of bovine virus diarrhea virus.
CULTIVATION OF VIRUS.pptx