Cultivation of viruses

animal viruses

1. Cultivation of Viruses By DR.S.Dhiva Assistant Professor, Department of Microbiology, Sree Narayana College, Alathur , Palakkad, Kerala.678682

2. General Characteristics Viruses are too small to be seen with a light Microscope viruses are obligate intracellular parasites, require a living host cell for its replication Viruses have few or no enzymes of their own for metabolism Viruses takes host metabolic machinery for its multiplication It Cannot be cultured out side the living host cell Viruses may infect Bacteria, plants and Animals

3. Cultivation of Viruses Unlike bacteria, Viruses cant be grown on an artificial nutrient medium Viruses can be grown   in vivo  (within a whole living organism, plant, or animal) or   in vitro  (outside a living organism in cells in an artificial environment, such as a test tube, cell culture flask, or agar plate).

4. Bacteriophage viruses which infects bacteria are called Bacteriophages   It can be grown on bacterial lawn (dense layer of bacteria) 0.7 % soft agar is used to grow Bacteriophages in a Petri dish . soft agar allows the bacteriophages to diffuse easily through the medium. So that lysed bacterial cells can be observed as a clear zone which is called as   plaque . Based on the lysis of bacteria in the lawn, many plaques are observed. Concentrations of viral suspensions is measured by the number of plagues are called as plaque- forming units ( pfu ) E.coli , C ornyebacterium sp , etc., can be used as a host for Bacteriophage cultivation

5. Need of virus cultivation Virus cultivation is needed for various reasons such as 1. Diagnosis of Plant diseases 2. for the production of viral vectors 3. Diagnosis of viral infections in clinical specimens, 4. Vaccine production and 5. other research studies .

6. Methods of cultivation In the Laboratory, three methods are commonly used for culturing animal viruses. These methods involve: Embryonated eggs Living Animals Cell cultures

7. Goodpasture in 1931 . first discovered that Embryonated chick eggs can be used for the cultivation of animal viruses Inoculation at different sites of the embryonated egg was developed by Burnet Fertilized eggs of birds such as (e.g., chicken, turkey) incubated about 6-8 days after laying is used for inoculation Before inoculation the shell surface is first disinfected with iodine and a hole is drilled in the shell of the embryonated egg Through the hole Virus is injected into various target sites of the egg such Chorioallantoic membrane, amniotic cavity, allantoic membrane and Yolk sac Inoculation on Embryonated Egg

8. Pock formation After inoculation, the drilled hole is sealed with gelatin and the egg is incubated Virus growth begins with the death of the embryo, by embryo cell damage or by the formation of typical pocks or lesions on the egg membranes This method is still used to produce some vaccines Persons allergic to egg will not be injected with this vaccine because it may carry egg proteins during vaccine production and leads to allergic reaction.

9. Animal Inoculation Mice , rabbits, hamsters, newborn or suckling rodents are also used . Mouse is most frequently used for viral cultivation and replication Animal inoculation is mainly used for diagnostic purposes. Some human viruses cannot be grown in animals or it can be grown and do not cause disease

10. After inoculation the animal is observe for the development of signs and symptoms of the disease, or it is killed for the tissue examination for the development of inclusion bodies But due to ethical issues Experimental animals are rarely used for cultivation of viruses Animals can be inoculated through Intracerebral , subcutaneous, intraperitoneal , or nasal instillation routes

11. Cell culture Cell culture has replaced embryonated eggs for the cultivation and assays of viruses. Steinhardt and colleagues in 1913 first used tissue culture for diagnostic pur p ose Because these cultures are generally rather homogenous collections of cells and can be propagated and handled much like bacterial cultures They are more convenient to work with than whole animals or embryonated eggs

12. Cell culture Cell culture can be divided into three types; 1. Fragment cultures, 2. Cell cultures and 3. Organ culture

13. Fragment culture/ explant culture Maitland in 1928 introduced cut tissues to the nutrient media for cultivation of viruses It is a simplest form of cell culture It consists of fragments of tissue or minced tissues suspended in a fluid medium. Eg . plasma clot cultures Here the cell remain viable for several days to allow the sufficient growth of virus Draw back behind this culture is fragments does not multiply.

14. Cell line preparation Cell lines are prepared by treating a slice of animal tissues with a proteolytic enzyme usually trypsin or with the chelating agent versene (EDTA, 171 ethylenediamine tetra-acetic acid, sequenstrene ) that separate the individual cells. These cells are suspended in a solution that provides the osmotic pressure, nutrients and growth factors needed for the cells to grow. Normal cells tend to adhere to the glass or plastic container and reproduce to form a monolayer. Viruses infecting such a monolayer cause the cells to deteriorate as they multiply This cell deterioration is called cytopathic effect (CPE ), it can be detected and counted as plaques in bacteriophage assay

15. Cell Culture Cell culture can be divided into three types: Primary cell lines Diploid cell lines Continuous cell lines Primary cell lines: Are derived from tissue slices, tend to die out after only a few generations.

16. Diploid cell lines : They are developed from human embryos, can be maintained for about 100 generations and are widely used for culturing viruses that require a human host. Cell lines developed from embryonic human cells are used to culture rabies virus for a rabies vaccine production called human diploid culture vaccine Genetically , diploid cell lines differ from continuous cell lines and appears as similar to normal cells. Rhinoviruses and the cytomegalovirus can be easily cultivated using Diploid cell lines

17. Continous cell lines : It is used Routinely for the cultivation of viruses They are cancerous (transformed) cells that can be maintained through an indefinite number of generations, and they are sometimes called immortal cell lines. After years of laboratory cultivation many such cell lines have lost almost all the original characteristics of the cell such as contact inhibition, etc., However these changes facilitate the viral propagation, so that enough number cell lines can synthesized for viral cultivation Eg . HeLa cell line which was isolated from a human cervical caocinoma , and the HEp-2 cell derived from a carcinoma of Larnyx

18. Organ Culture S mall bits of the organs are used for organ culture To preserve its original morphology and function they are maintained in vitro for few days. Nowadays, organ culture is not used .

19. References www.researchgate.com www1.mans.edu.eg www.biologyreader.com www.courses.lumenlearning.com www.virusabc.weebly.com www.slideshare.net Tortora , Funke , Case, Microbiology An introduction, 8 th edition, pg 411-414 Lansing M.Prescott , John P.Harley and Donald A. Klein, Microbiology 4 th edition pg.. 337-338

20. Thank you