Cultivation and Quantification of Animal Viruses

CULTIVATION AND QUANTIFICATION OF OPNIMAL VIRUSES Introduction:- Since the Viruses are obligate miracellular parasites, they cannot be grown on any inanimale culture metium. →Viruses can be cultivated within suitable hosts such Ge a living cell Animal virus viruses cause disrased in animals their Gerelic matrasal is either DNA of RNA, which Can be single or double stranded. Cultivation of Animal vhwes:- Animal viruses are generally cultivated by three methocla 4. Inoculation of virus into animals 2. Inoculation of Vious into embryonaled eggs 3. Tissue cullure 1. Inoculation of vious in Animals: Introduction:- → Laboratory animals play an essential rolt in studies of Viral pathogenesis → Live animals such as monkeys, mice, rabbits, guinea pigs ferrets are widely used for cultivating visus. →mice are the most widely employed animal' used in Virology. Condition:- Animal Should be free from contaminatian and disease free →free from bacteriological contamination Achieved by • Specific pathogen free animale [selection] •by beatment with antibiotics [Eg:- penicillin, streptongis process select animal [mitce] free from ciseases [hrothy mice] different routes of inoculation in mite dre • Inizacerebral Subcutaneous • Intraperitoneal • Intranasal After, the animal is inoculated with the vious suspension, the animal is observed for • Signs of disease • Visible lesions on • it is killed so that infected tissues can be examined for viaus • Abnormal behaviou's of the animal Advantages of Animal Inoculation:- →Diagnosis, pathogenesis and clinical symptoms ase cleterned. Ideal for studying viral replication → used for study of immune responses, epidemology and oncogenesis- Disadvantages of Animal inoculation: → Expensive and difficulties so moantenance of artimal →Difficully in Chemistry of animals for particular vious. → Issues related to animal welfare system. 2. Inoculation into embryonated 298: Introduction:- Good Pasture in 1931 first used the embryonated hen's egg for cultivation of vive The process of cultivation of 'ruses in embryonated egge depends on the type of egg which is wed. Embryonated egg does not support all animal viruses but most of avian Viruses grown Condition: the eggs should be healthy and free from disease and Contamination. → It is achieved by sterilization with Todide/thanol. process: →Selection of eggs Chealthy and free from microbes] →for inoculation, egg shell Surface it first Sterilized [disinfected with Todinel ethanol and penetrated with a Small Sterile dalll. → Inoculate virus through the small opening →After noculation, the opening vs sealed with gelatin fperaffin and incubated al 36°C for 2-3 days →After incubation, the egg is broken and visus isolated froon tissue of 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 typocks Leisions on the egg membrane. (1) →virusat can be cultivated in various parts of egg like choripallantoic membrane, allantoio cavity, amniotic sac and yolk sac. Shell Shell membrane Miumin. Amniotic cavity Allantoic York sac Chasianatlarlaic rembione meinbiane Chorioallantaic membrane [cam] This method has been widely und in vetensory virology →Many viruses grow readily on the Ang vioses Produce visible packs Lach infections visus particle forms are une pack viruses which can be grown Include Herpes viruses & pox riouses Amniotic Roure:- polmary isolation of influenza and mump viruses → Growth of virus detected by haemagglutination → viruses which can grow by this route includes Influenza Mumps Allantoic Route:- → most popular site of inoculation → Simple technique → most of avtan viruses can be grown by their route • Adenovirus • Mumps • Influenza • Newcastle disease vous Yolk sac:- It is also a simplest method for growth and multiplication of vizus. Mostly mammalian Viruses are isolated using this method- Herpes simplex virus. This method is also used for the cultivation of some bacteria like Chlamydia and Rickettsia Advantages of noculation into embryonated egg → Ideal substrate for the viral growth and replication. → used for many avion and few mammation Viruses cultivation 2016 Jabor is needed. →The embryonated eggs are readily avaliable → widely used method to grow visus for some vaccine Production. Disadvantages of inoculation into embryonated egg: →The site of inoculation for varies with different virus. That is each virus have different sites for their Growth and replication. 3. Tissue Culture:- Most widely used method for cultivation of viruses. →Again these are three types of tissue culture a, organ culture b. Explant Culture E. Cell culture a, organ culture Small bifs of organs can be maintained in vitro for days and weeks, preserving thers characteristics and function organ culture are useful for the isolation of some Viruses which appear to be highly specialised parasite of Certain organs. 29: The tracheal ing organ culture is employed for isolation of coronaviave, a respiratory pathogen. Explant Cultuar:- fragments of mixed tissus can be grown as explants embedded in plasma clots. They may also be cultivated in suspension Adenoid tissue explant culture were wird for the reviation of admoviruses. F. coll culture:- This is the type of culture vamosty employed for growing Viruses. Tissue are dissociated into Component Cell by the action of Proteolytic enzyme. The cells are wasted counted and suspended in a growth medium Media will enable most cell typer to multiply with a devition time of 24-48 hours The cell suspension to dispensed in bottles, Habes coupetostisbas The cell adbere to the glass surface and on incubation divide to form a confluent monolayer sheet of cells Covering the surface with about a week Based on theis ongin Chromosomal characters and the number of generation through which they can be maintained, cells cultures core Classified into 3 types They are:- 1. paimary cell cultuza in. Diploid cell culture Continuous cell culture primary cell culture- -Normal cell from fresh animals are oblained and being cullard 1 They are capable of only limited growth in culture, attachiment dependent Contact Inihilation [can't grow one oven stri Lg: monkey Kidney cell Culture Human embryonic Kidney Chick embayo cell culture →They are commonly employed for primary isolation of Viruset and in preparation of vaccine Riploid cell culture: → Dexived from Icell culture, contain the same number of Chromosome at the parent cells and are diploid. →These diplold cell strains can be subcultiome for limited number of times. -ie, possible upto 50 senal subcultures This culture employed for the production of viral vaccine. ig:- human embryonic ling cell strain (WI-38) Rhesus embryo cell strain [41-8] Continuove cell time:- →These are cells of Single type capable of infinite growth- In vitro →They are divided derived from cancer colle →They are termed continuous cell lines as they can be Sersally etthud cultivated Indefinitely. Eg:- Human cancer cell such as Hola cells, Hep-8, vera cells and Bик → Continuous cell lints-maintained either by serial Subcultore Stooing in deep freeze at-700 Hela crits derived from cervical cancer of a lady named →vero [vervet monkey kidney cell time] - BHK [Baby Hamster kidney cell line] Advantages of issue culture:- Growth can be detected easily. visuses can be grown in bulk → cells can be stored for longer period of lime Disadvantages of tissue cullore: Requirement of good laboratory facility mon costly compoored to the embryonated eggs Chances of presence of latent viruses in the Cultored cells QUANTIFICATION OF ANIMAL VIBUSES:- →vious quantification involved detecting the visvses [presence), counting the number of viruses in a specific Volume to determine the virus concentration. It is wed in both Rande as well as production Situations swhere the quality of virus steps in an Important variable →To count Idetect the viruses there are several methode 1 Microscopy- for lesions [Cytopathic effect) 2. Histopathology- examination of tissue for disease a serologically - for presence of viral antigen-Immunoflusse- Sence, EUSA, MA 4. Infectivity assay-plaque, pock Assay. 5. Molecular methods- PCB