CULTIVATION AND IDENTICATION OF VIRUSES.pptx

viruses are intracellular in nature, so they can not be grown in ordinary culture media they need living cells for growth. they are very small in size can not be seen by light microscope they need Electron microscope for morphology of viruses. there are different methods for cultivation of virus, egg inoculation, animal inoculation and tissue culture.

1. CULTIVATION OF VI R USES

2. Introduction to Vir uses A virus is an obligate intracellular parasite containing genetic material surrounded by protein . Virus particles can only be observed by an electron microscope ( range in sizes from 20 – 250 nm). Unlike  bacteria, many of which can be grown on an artificial nutrient medium, viruses require a living host cell for  growth. . 2

3. Cultivation of Viruses Virus cultivation refers to the process of growing and propagating viruses in a laboratory setting. This process is important for studying the properties and behavior of different viruses, as well as for developing  vaccines   and treatments for viral infections. Viruses can be grown:   in vivo (within a whole living organism, plant, or animal)   in vitro (outside a living organism in cells in an artificial environment, such as a test tube, cell culture flask, or agar plate)

4. There are three methods employed for the cultivation of viruses. Animal inoculation Embryonated eggs or chick embryo method. Tissue culture or cell culture.

5. Animal Inoculation Susceptible experimental animals like Mice, Monkey, Rabbits, Guinea Pigs etc. are used for the cultivation of viruses. The animals should be free of  communicable diseases   and in good health. It is important to select specific host animal for particular viruses. Age and immunity of laboratory animal also affect the growth of viruses in the host. V iruses to be cultivated can be administered to laboratory animals via intraperitoneal, subcutaneous, intracerebral, and intranasal routes .

6. Mice are the most widely employed animals in virology. After the animal is incubated with the virus, the animal is: Observed for signs of the disease Visible lesions or is sacrificed so, that infected tissue can be examined for virus. Animal inoculation Method of virus Cultivation

7. Advantages: Mimics human infection:  Animal inoculation can provide a more accurate representation of human infection than cell culture methods, as it involves the virus interacting with a living organism and its immune system. Produces larger quantities of virus:  Animal inoculation can produce larger quantities of virus compared to cell culture, making it useful for producing viral vaccines or developing diagnostic tests. Enables study of disease progression:  In vivo virus cultivation allows for the study of the entire disease process, including how the virus interacts with the host immune system and how the disease progresses over time. Facilitates the development of treatments : In vivo virus cultivation can be used to test the effectiveness of treatments and vaccines in animals before they are tested in humans.

8. Disadvantages: Expensive and difficulties in maintenance of animals. Difficulty in choosing of animals for particular virus. Some human viruses cannot be grown in animals, or can be grown but do not cause disease. Mice do not provide models for vaccine development. Issues related to animal welfare systems.

9. Inoculation of Virus into Embryonated eggs Good pasture (1931) was the first who used hen’s embryonated egg for the cultivation of viruses. Embryonated egg provides several sites for the cultivation of viruses. Chorio - allantoic membrane Allantoic cavity Amniotic cavity Yolk sac Embryo

10. Different site is used for growth of different viruses. Chorio -allantoic membrance is used for the cultivation of pox virus. Allantoic cavity is employed for the Influenza virus, paramyxovirus. The amniotic sac is employed inoculated for primary isolation of influenza A virus and the mumps virus. Yolk Sac is also used for the cultivation of some bacteria like Chlamydiae and Rickettsiae.

11. The procedure for inoculating viruses into embryonated eggs involves the following steps: Prepare the eggs : Use an egg candler to check the viability of the eggs and mark the inoculation site. The egg should be sterile and have an intact shell.  Disinfect : Use 70% alcohol to swab the end of the egg and the eggshell punch.  Drill a hole : Drill a small hole in the shell at the marked location. The location depends on the type of inoculation:  Allantoic cavity : Drill on the side of the egg, just above the bottom of the air sac.  Yolk sac : Drill at the top of the egg, above the yolk sac.  Inject the virus : Use a needle attached to a syringe to inject the virus into the egg. The needle should penetrate about 16 mm into the egg to reach the allantoic cavity.  Seal the hole : Use melted wax or stationery tape to seal the hole in the shell.  Incubate : Place the inoculated eggs in an incubator and observe daily.

12. Viral growth and multiplication in the egg embryo is indicated by: The death of the embro embryo cell damage The formation of typical pocks or lesions on the egg membranes

13. Cell Culture (Tissue Culture) Steinhardt and colleagues (1913), was the first who used bits of tissue or organ for the cultivation of viruses. Now advance techniques are develop in Tissue culture. There are three types of tissue culture: Organ cultures Small bits of organs are used for the cultivation of virus. e.g., tracheal ring culture is done for isolation of coronavirus. Explant culture In this small fragment of tissue is extracted from human or animal and used for virus culture and is rarely done. Cell culture is mostly used for identification and cultivation of viruses. Cell culture is the process by which cells are grown under controlled conditions. Cells are grown in vitro on glass or a treated plastic surface in a suitable growth medium.

14. Method of Virus Cultivation This is the most frequently employed method. Labs routinely use cell line culture for virus culture, isolation, and identification. First, in cell line culture, a balanced salt concentration, all essential  amino acids , glucose, buffering agents, antibiotics, serum, etc. are included in the preparation of the growth medium. The fragment of tissue is then trypsinized to separate the cells. The dissociated cells are rinsed, suspended in culture media in a  test tube  or  petri dish , and incubated for the appropriate amount of time. Upon incubation, the cells divide and form a confluent monolayer on the glass surface, which is now used for virus culture. 

15. On the basis of their origin, chromosomal characteristics, and the number of generations through which they can be maintained, there are three categories of cell line cultures. Primary cell line Semi-continuous ( diploid ) cell line Continuous cell line

16. Primary cell culture: These are normal cells derived from animal or human cells. 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. Examples: Monkey kidney cell culture, Human amnion cell culture

17. Diploid cell culture (Semi-continuous cell lines): They are diploid and contain the same number of chromosomes 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. Examples: Human embryonic lung strain, Rhesus embryo cell strain

18. Continuous cell lines: These are the cells of single type, usually derived from cancer cells. They can be serially cultured indefinitely so named as continuous cell lines They can be maintained either by serial subculture or by storing in deep freeze at -70°c. Due to derivation from cancer cells, they are not useful for vaccine production. Examples: HeLa (Human Carcinoma of cervix cell line), HEP-2 (Human Epithelioma of larynx cell line), Vero (Vervet monkey) kidney cell lines, BHK-21 (Baby Hamster Kidney cell line).

19. DETECTION OF VIRAL GROWTH Viral growth in cell culture can be detected by the following methods Cytopathic effect (CPE) Due to the viral growth, morphology of cultured cell change, these changes can be readily observed under microscope. These morphological changers in cell culture is called Cytopathic effect (CPE) and viruses causing CPE are called cytopathogenic virus.’ Eg. Adeno virus cause large granular clumps in cell culture. Fluorescence Antibody Technique (FAT):- In this technique, cell from virus infected cultures can be stained by fluorescent conjugated antigen. Fluorescent dye such as fluorescein isothioacynate and rhodamine are generally used to tag with antibodies. FAT is very useful in testing for rabies virus in clinical specimen within few hours with 100% accuracy. Haemagglutination :- Haemagglutination is the phenomenon of clumping of RBC s . Viruses such as mumps, measals and influenza can able to agglutinate the RBC s . their presence can be indicated by addition of guinea pig erythrocytes to the culture. If the viruses are cultivated in the cell culture, the erythrocytes will adsorb onto the cell surface also called ‘haemadsorption.’

20. Cytopathic effect (CPE) and FAT

21. Heamagglutination method for virus detection

22. Inclusion bodies Intracytoplasmic inclusion bodies Negri bodies – seen in Rabies virus Paschen body- seen in Variola virus Guarnieri bodies - seen in Vaccinia virus ­ Bollinger bodies - seen in Fowl pox virus Molluscum bodies - seen in Molluscum contagiosum virus Perinuclear cytoplasmic body- seen in Reovirus Intranuclear inclusion bodies A) Cowdry type A inclusions Torres body- seen in Yellow fever Lipschultz body - seen in Herpes simplex B) Cowdry type B inclusions - seen in Poliovirus Adenovirus Intracytoplasmic & intranuclear inclusion bodies Owl’s eye appearance- seen in Cytomegalovirus Measles

23. Purpose of virus cultivation Understanding virus behavior : to study their properties and behavior. This can help them understand how viruses cause diseases, how they replicate, and how they interact with host cells. Developing vaccines : for the development of vaccines against viral diseases. By growing and propagating the virus, researchers can develop weakened or inactivated forms of the virus that can be used as vaccines to stimulate the immune system. Developing antiviral drugs :  useful for developing drugs to treat viral infections. Researchers can test different compounds and drugs to see if they can stop the replication of the virus or prevent it from infecting host cells. Studying viral evolution : to study viral evolution over time. By comparing different strains of the virus and how they evolve, scientists can gain insights into how viruses adapt and mutate to new environments.