1. Virus Isolation and Cultivation
1.1. Animals
1.1.1. Used for routine cultivation of virus; they play an essential role in studies of viral pathogenesis.
1.1.2. Live animals such as monkeys, mice, rabbits, guinea pigs, ferrets are widely used for cultivating viruses.
1.1.3. After the animal is inoculated with the virus suspension, the animal is observed for signs of disease like visible lesions or killed so that infected tissues can be examined for virus.
1.1.4. Advantages A diagnostic procedure for identifying and isolating a virus from a clinical specimen. Mice provide a reliable model for studying viral replication. Gives unique insight into viral pathogenesis and host virus interactions. Study of immune responses, epidemiology and oncogenesis.
1.1.5. 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. It will lead to generation of escape mutants. Issues related to animal welfare systems
1.2. Cell Culture
1.2.1. Cell Cultures are most widely used for virus isolation in-vitro using monolayer cells cultures. However, some viruses cannot grow in-vitro e.g. Hepatitis C!
1.2.1.1. 1-Primary cells
1.2.1.1.1. e.g. Primary Rhesus Monkey Kidney (PMK) Cells
1.2.1.1.2. The cells in culture divide only a limited number of times, before their growth rate declines and they eventually die.
1.2.1.1.3. Prepared from cells obtained directly from the tissues or organs.
1.2.1.1.4. Viable cell suspensions may be obtained by dissociating tissues or organs, by enzymatic digestion or mechanical dispersion. e.g. human amnion, with trypsin, collagenase or other enzymes.
1.2.1.1.5. Primary cell lines are widely acknowledged as the best cell culture systems available since they support the widest range of viruses. However, they are very expensive and it is often difficult to obtain a reliable supply.
1.2.1.1.6. Advantages:
1.2.1.1.7. usually retain many different characteristics of the cell in-vivo.
1.2.1.1.8. Disadvantages:
1.2.1.1.9. Initially heterogeneous but later become dominated by fibroblasts.
1.2.1.1.10. The preparation of primary cultures is labor intensive.
1.2.1.1.11. It can be maintained in-vitro only for a limited period of time.
1.2.1.2. 2- Semi-continuous cells
1.2.1.2.1. e.g. Human embryonic kidney and skin fibroblasts
1.2.1.2.2. Semi-continuous Cell Cultures (Diploid cell lines or strains)
1.2.1.2.3. Those cell cultures are established with the successful subculture of primary cell monolayers.
1.2.1.2.4. These cultures consist mostly of spindle shaped fibroblast cells. E.g. Established from human embryonic tissue, or neonatal foreskin.
1.2.1.3. 3- Continuous cells
1.2.1.3.1. e.g. Human diploid fibroblast(HDF), Human Cervix Adenocarcinoma (HeLa), African Green Monkey Kidney(Vero), Human Epithelial Cells (Larynx carcinoma) (Hep2), Rhesus Monkey Kidney (LLC-MK2), Madin-Darby Canine Kidney (MDCK).
1.2.1.3.2. The following continuous cell lines are commonly used:
1.2.1.3.3. Characteristics of continuous cell lines:
1.2.1.3.4. The Cell Culture Experiment
1.2.1.3.5. Newer cell culture formats
1.3. Eggs Embryo
1.3.1. Good pasture in 1931 AD first used the embryonated hen’s egg. 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
1.3.1.1. An embryonated egg offers various sites for the cultivation of viruses
1.3.1.1.1. A-Chorioallantoic Membrane(CAM):
1.3.1.1.2. B- Allantoic Cavity:
1.3.1.1.3. C- Amniotic Cavity:
1.3.1.1.4. D- Yolk Sac:
1.3.1.2. Advantages Popular method for the isolation of virus and growth Ideal substrate for the viral growth and replication. Isolation and cultivation of many avian and few mammalian viruses. Cost effective and maintenance is much easier. Less labor is needed. The embryonated eggs are readily available. Sterile and wide range of tissues and fluids They are free from contaminating bacteria and many latent viruses. Specific and non specific factors of defense are not involved in embryonated eggs.
1.3.1.3. Disadvantages The site of inoculation for varies with different virus. That is, each virus have different sites for their growth and replication.
2. Viral Antigen Detection
2.1. Immunofluorescense (IF)
2.1.1. A Powerful technique that utilizes fluorescent-labeled antibodies to detect specific target antigens..
2.1.2. Fluorescein is a dye wich emits greenish fluorecence under UV light. it can bee tagged to immunoglobulin molecules.
2.1.3. This technique is sometimes used to make viral plaques more readily visible to human eye .
2.1.4. Labeled tissue sections are studied using a fluorescence microscope.
2.1.5. Technique allowing the visualization of a specific antigen in tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye (also called fluorophores or fluorochromes) such as fluorescein isothiocyante (FITC).
2.1.6. The specific antibodies are labelled with a compound (FITC) that makes the glow an apple-green colour when observed microscopically under ultraviolet light.
2.1.7. There are two ways of doing IF staining
2.1.7.1. Direct IF
2.1.7.1.1. • Antigen is fixed on the slide • Fluorescein labeled antibodies are layered over it • Slide is washed to remove unattached antibodies • Examined under UV light in a florescent microscope • The site where the antibodies attaches to its specific antigen will show apple green fluorescence • Use : direct detection of Pathogens or their antigen in tissues or in pathological samples
2.1.7.2. Indirect IF
2.1.7.2.1. • Indirect test is a double –layer technique • The unlabelled antibody is applied directly to the tissue substrate • Treated with a fluorochrome – conjugated anti- immunoglobulin serum.
2.1.8. Advantages Result available quickly, usually within a few hours.
2.1.9. Disadvantages Often very much reduced sensitivity compared to cell culture, can be as low as 20%. Specificity often poor as well. Requires good specimens. The procedures involved are often tedious and time- consuming and thus expensive in terms of laboratory time.
3. Serologic (Antibody Detection)
3.1. Enzyme Linked Immunosorbent Assay
3.1.1. Was Coined By Engvall and Pearlmann in 1971
3.1.2. Can be used to detect either antigen (as a direct test) or antibody (as a serology test).
3.1.3. Different Types
3.1.3.1. 1. Sandwich
3.1.3.2. 2. Indirect
3.1.3.3. 3. Competitive
3.2. Hemagglutination Inhibition Test (HAI)
3.3. Complement Fixation Test (CFT)
4. Nucleic Acid Amplification
4.1. Polymerase Chain Reaction PCR
4.1.1. In vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique.
4.1.2. It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest.
4.1.3. Further sensitivity and specificity may be obtained by the nested PCR.
4.1.4. Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis, hybridization with a specific oligonucleotide probe, restriction enzyme analysis, or DNA sequencing.
4.1.5. Advantages
4.1.5.1. Extremely high sensitivity, may detect down to one viral genome per sample volume
4.1.5.2. Easy to set up
4.1.5.3. Fast turnaround time
4.1.6. Disadvantages
4.1.6.1. Extremely liable to contamination
4.1.6.2. High degree of operator skill required
4.1.6.3. Not easy to set up a quantitative assay.
4.1.6.4. A positive result may be difficult to interpret, especially with latent viruses such as CMV, where any seropositive person will have virus present in their blood irrespective whether they have disease or not.
4.2. Real-time PCR
4.2.1. A specialized technique that allows a PCR reaction to be visualized “in real time” as the reaction progresses.
4.2.2. Allows us to measure minute amounts of DNA sequences in a sample!
4.2.3. Applications
4.2.3.1. quantitation of gene expression
4.2.3.2. drug therapy efficacy / drug monitoring
4.2.3.3. viral quantitation
4.2.3.4. pathogen detection
4.2.4. Advantages
4.2.4.1. amplification can be monitored real-time
4.2.4.2. high throughput, low contamination risk
4.2.4.3. most specific, sensitive and reproducible
4.2.5. Disadvantages
4.2.5.1. setting up requires high technical skill and support
4.2.5.2. high equipment cost
4.2.5.3. Runs are more expensive than conventional PCR
4.2.5.4. DNA contamination (in mRNA analysis)