Cultivation of virus in cell lines * The primary purpose of virus cultivation is. * To isolated and identify Viruses in clinical Samples * To research on viral structure, Replication, genetics and effects on host cell. Types of cell culture; * primary cell culture Diploid cell Culture * Heteroploid Culture virus are replicated within living cells. * Some viruses are resisticted in the kind of cells In which they replicated. * A few have not been cultivated at all under labortory conditions. * Most viruses are grown in cultured cells, embryo -nated hen's eggs (00) Labovõtry animals.. ~ cell lines of BHK BHK "Baby Hamster Kidney Fibroblasts" *BHK cell are in adherent cell line used in molecul -aru biology. 1 * The cells were derived in 1961 by I. A Macpherson and M.G.p stoker M * BHK cell lines is one of the most commonly used cell lines for the expression of "biopharm -aceuticals. ~ *BHK cell are originally isolated by polyoma transtormation of hamster cells. * These BHK cell is proved to be quite Succeful as host cell line for stable experssion of various recombinant proteins * The species Mesocricetus alratus, commonly Known as Syrian golden hamsters * These are most widely used line is a Subclone Infected diseases * In commercial manufacture of foot and mouth diseases Vaccines over a twenty year period. * Human adenovirus, Dreovirus, 3, vesicular Stomatitis vinus. * SBV CSchmallen berg virus) intected live Stock Such as cattle, sheep and goats. Cell lines of hela * Hela is an immortal cell line used in scienti -fic reseach. * It is the oldest and most commonly used in human cell line *The cell line is named after and derived from Cervical cancer cells * The cell line was found to be remarkably durable and prolific, which allows it to be Used extensively in scientific Study. * labortory studies, especially in reserch on viruses, cances, and human genetics * Hela cells are Cross contamination a common Source of of other cell lines and aspected cause of numerous of cell line misi dentification in stance * The Hela cell genome has also been shown to be highly unstable houing numesow genom -ic rearrangements Ex: abnormal no. of chromosomes (Chromothripsis) Countinuous cell lines. * continuous immortalized cell lines are comprised of a single cell type that can be serially propagated in culture either for a limited number of cell divisions. cell lines of afinite life are usually dipoloid and manitain some degree of differentiation. The standard Continous cell lines have been derived from human Cancer cell Such as Hela Derived from cervical cancer of a lady; hela by name, Hep2 and KB cells. production of important pharmaceituals like vaccines and hormones. They can be cloned Ext Measles virus, Rabies Virus. Cytopathic effects:- mainly consists of causation of morphological, changes in the host cells Sometimes these alterations may even lead to addeath. - There may be varfous types of such changes, death. upon the host viras system. For eg. we may cite the lytic effect of bacterioptages of cyanophages on the host bacterial and cyanorkyco cells, respectively. →The effects of some animal viruses on their host cells and are slightly different. Adenovirus infected cells roundup and aggregate in cluster before their eventful death. cellis affected by polioviras round ur and shrink, eventually getting totally destroyed The effects of some plant viruses on their host to partial (or) Cells are In host infected by plant viruses, CPE lead total chlorosis of the, chlorortyllous plants. - Eg: The virus mosaic diseases of cucurbitaceos and solana Ceocus plants chow such symptoms b) ancrocial grocuth of cell well leads to all sorts of Curvature of different organs particularly leaves. -Accompained by chlorotic symptoms." ()shrinkage and chostering of cells which beads to Cell death The viras which cause CPE are knocon as cytopathic viruses. CYTOPATHIC EFFETS OF SOME VIRAL INFECTIONS. Vird Host CPE Symptoms Bacteriophage Ta E.coli lysis due to release of hydrolytic enzyme Haque formation Influenza Non, alilse Secretion of to reins Causes cell necrosit ferer, body pains Babies Dog, cat Development of Prelosion bodies CNegribodies) Infection of CN leading to Corvolsion. Tobacco Mosaic virus Tobacco plant chlorophyll synthesis hampenned Cuncaval cell exull growth) chlorosis Mottling. vaccinia Virus Man Alternation of the cell membrane Aggregation of determination of leading to exter hal Viruses leads howing lirid membranes like myxoviruses, infection desintegraedfon of host cell membranes into fragments cohich are utilised in the formation of membrane coverings of the viras particles. The cells infected with hapes vires membranes of the interacting. contigous cells, dissolved resulting into the formation of giant cells. -Eventually these appear to develop as crystalline Structures upto a certain sife after cohich lysis occar resulting in the liberation of the particles. These structures could in the develop cytoplum in the case of cells infected with rabies vilces forming Negri bodies (or) with ming cwheel like inclusion bodres of poly are another example. bean mosaic Vivopin Virus Profections. - Sometimes the crystals may be formed in the nulleus e.z. adenovirus infection of mammalian has occasionally these crystals are Knocon to appear in both Nucleus. and the cytoplasm of the injected cells e.g. In meastos. - In certain eases conspicious bodies develop at the site of earlies viral vira infection that might have taken place e.g. Development of casinophilic bodies in cells infected cwith simplex viras. CHROMOSONAL ABBERATIONS cells in culture when infected by a variety of viruses may shoco chromosomal aberration like breaks and constructions. Such abbreations appear in the cell of organisms infected by herpes virus and by polyoma viscus. The breaks induced by hores vires occur only at Contain sites of tobo specific chromosomes, The progeny of Such abbreations the host cells contribute to show CELL TRANSFORMATION. -Some viral infection lead to alternation in the morphology of the infected cells. Transformation can also be recognised by a) Presence of intranudear t antigens b) loss contact inhibition. Ability host. of calle tumors. when inoculated into suitabl d) NECO surface antigers Trans for mation by retsovircu Production of infective particle. is associated with Due to this there on loss of control of the host cel cetkular metabolic functions Eg. cell caused by transformation of the virases. Rous saeroma Vircs (RSV) and sacromas ak simian virus 40 Gr40) + antigens plays an important role in cell transformation CELL FUSION: Some viruses like paramyxovirus, herpes viruses and pox viruses produce giant they are made to lab conditions. Cells cou) synciter when infect Cell monolayers under 2. primarily the host cell protein synthesis is shut down at the instance of viral proteins cohich may be part of the infective virion cor) to be synthesized using viral genes. Cellular DNA by RNA synthesis are affected only Secondarily the sheet doron is found to be particularly. rapid in cultured cells infected and herpes Viruses such as TMV by Picornaviruses. are knocon to inhibit cellular synthesis of RNA and proteins coithout affecting DNA synthesis by the host cells. -It has been discovered that rRNA synthesis is inversely related to the TMV RNA infected that cells. esis in the Synthesis - After the synthesis increases Normal level, though desring Production decreases. upto etc. TMV-RNA synthesis the - Another person reason. host cells is behind the adverse in-fluences on the found release of the hydrolytic enzyme (lysozymes) from the organelleds that contain them. This rescut in plant partial (or) total cou destruction Enclusion Bodies. Intracellular inclusion bodies are observed in quite large no. of maost of viron systems. Thes coubt duelop as a of virons • the aggregalin. Consecuence to the at a particular site in the cell line of their formation. at the In the case of plants infected with virus, various physiological disorders are known to take place a) Increase respiration b) Decreased photosynthetic activity. •) Decreased activity of groceoth regulators d) fecamellation of soluble Ne Compounds such as camides (e) Koncreased activity of polyphenyl oxidase and accumulation of oxidized phenols. activity Apart from these activities other metabolite processes such as photorespiration process such photorespiration, Nucleic acid the stress phenomenon infection. synthesis, lepid as Synthesis. are all effected by virds A high proteins is concentration of virions (or) of viral coat known to initiate cytopathic effects are Collectively called as toxic effects. A kind of effects also cecated cwith viral infection is not cytopathic bat leads to altesnations in the morphology of the host cells. 4) Development of inclusion bodies 2) Induction of chromosomal abbrevations 3) cell fusion 4) Metabolic inhibition 2) Bacteriophage, Cyanophage and some virus infected cells, depression of metabolic process occer. mammalian essential TMV tobacco Mosaic virus -Bt is a Helical symmetry Non Enveloped Rod shaped Capsid -capso meres It is 21600-2400 kange SSRNA It is genome organization. Single strandard Kila bosepal RNA organized helically of length 63-65 kho 3º sterminal is like tRNA and blend is methylate a.. The genome encodes for AD ORF's Copped- - Open Reading formes. 40RF'S codes for 4 proteins replicase -Helicase -RNA dependent DNA polymerase -Capsid proteins Helicase is an enzyme helps in unwindedin of genome. Reticase helps in replication. Replication sterlegies Assembly and maturation. lytic release Cross many veins necrolization Disease cycle/pathogenesis TMV enter through Hand damages-membrane Entry of infectious particle icle Into ↓ it kaks control of host viral proteins cytoplasm then machinery and synthesize genetic material. ↓ Assembly and makeration -lakes place the virus may selease by cell lysis or enters into a fresh cell includes through plasmodesmata Symptoms! 1) Tissue Necrosis 2) Mossaic appearance due to chlorophy !!. d pigmentation of 3) Mottling and leaf curling. (methy) growth of veind. 4 stunted growth of the plant Cintermodal elongation Control Measures coill not take place [Hindered] 1) Removing /uprouting of total plant. 2) virusidal agend, should be used. 3) The infected plants or virued plants should be incinerated. *20-30% bleach is used Cutting Detection, on tools to be used for PCR based drssay's.