Indirect methods (virus isolation)
1- Inoculation of lab animal
For observing the animal for signs of diseases One the earliest ways of detection a virus
2- Inoculation of fertile hens eggs
Both animal and egg are difficult to handle and are rarely to used
3- In vitro cell culture
a- Primary cells
o Use normal cells derived from animal or human cell such Monkey
kidney and human amnion cell culture
o Monkey kidney cells are obtained from freshly killed and these cell can only be passaged one or twice
➤ The best cell culture system
➤ Support the widest range of viruses
➤ Are very expensive
➤ Use for viruses vaccine
b- Semi- continuous cells
Cells taken from embryonic tissue such as, human embryonic kidney and skin fibroblast and may be passaged up to 50 times, used for virus
isolation and viral vaccines
c- Continuous cells
Immortalized cells for example, HeLa cells (tumor cells) May be passaged indefinitely
Easy to handle but the range of viruses supported is limited
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Identification of growing virus
The presence of growing virus is usually detected by:
* Cytopathic effect (CPE)
Change in morphology of infected cells such as, cell degeneration, rounding and shrinkage Specific e.g. herpes simplex (HSV) and cytomegalovirus (CMV) Non-specific e.g. enteroviruses
* Haemadsorption
Cells acquire the ability to stick to mammalian Red Blood Cells (RBCS) Mainly used for detection of influenza and parainfluenza virus
Problem with cell culture
1- Delay results up to 4 weeks
2- Sensitivity is often poor depand on many factors such as, the condition of the specimen and the condition of the cell sheet
3- Susceptible to bacterial contamination and toxic substances in the specimen
4- Some viruses will not grow in cell culture at all e.g. hepatitis B and C
5- The role of cell culture is declining due to rapid methods like antigen detection and molecular methods
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Cultivation of Virus
- Since the viruses are obligate intracellular parasites, they cannot grown on any culture medium
- Viruses can be cultivated within suitable hosts, such as a living cell
• The primary purposes of viral cultivation are
1. To isolate and identify viruses in clinical specimens
2. To prepare viruses for vaccines
3. And to do detailed research on viral structure, multiplication cycles, genetics, and effects on host cells
Viruses not only need living cells to grow in but also they are specific about the type of cell they infect and grow in
There is no universal cell that will support all viruses
Viruses tend to be host specific; therefore:
➤ Human viruses grow best in cells of human origin,
➤ Bovine viruses in bovine cells,
➤ Canine viruses in canine cells,
➤ While some viruses will not grow in vitro at all
Therefore in the laboratory the suspected virus must be grown in a culture method known to support its growth
Methods for Cultivation of Virus
- Generally three methods are employed for the virus cultivation
1. Inoculation of virus into animals
2. Inoculation of virus into embryonated eggs
3. Tissue culture
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- Animals are used for studying viruses which do not grow in cell cultures or eggs, and for testing vaccines
- Eggs support a fairly wide range of animal and human viruses - hence their importance in the diagnostic service
- Cell cultures; different types of cell lines will support different types of viruses
Inoculation of Virus in Animals
Laboratory animals are widely used for routine cultivation of virus; they play an essential role in studies of viral pathogenesis. Live animals such as monkeys, mice, rabbits, guinea pigs, are widely used for cultivating virus. Monkeys were used for the isolation of Poliovirus. But due to their risk to handlers, monkeys find only limited applications in Virology. Mice are the most widely employed animals in virology. The different routes of inoculation in mice are intracerebral, subcutaneous, intraperitoneal or intranasal. After the animal is inoculated with the virus suspension, the animal is observed for signs of disease, visible lesions or is killed so that infected tissues can be examined for virus.
الفيديو يشرح طرية تلقيح الفئران في intraprtitoneal
Advantages:
1. Animal inoculation may be used as diagnostic procedure for identifying and isolating a virus from a clinical specimen.
2. Mice provide a reliable model for studying viral replication.
3. Gives unique insight into viral pathogenesis and host virus relation.
4. Used for the study of immune responses, epidemiology and oncogenesis.
Disadvantages:
1- Expensive and difficulties in maintenance of animals.
2- Difficulty in choosing of animals for particular virus.
3- Some human viruses cannot be grown in animals, or can be grown but do not cause disease.
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4- Mice do not provide models for vaccine development.
5- It will lead to generation of escape mutants.
6- Issues related to animal welfare systems.
Inoculation of Virus into Embryonated eggs
Prior to the advent of cell culture, animal viruses could be propagated only on whole animals or embryonated chicken eggs. Good pasture in 1931 first used the embryonated hen's egg for the cultivation of virus. The process of cultivation of viruses in embryonated eggs depends on the type of egg which is used. The egg used for 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. An embryonated egg offers various sites for the cultivation of viruses.
The different sites of viral inoculation in embryonated eggs are:
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