Professional Documents
Culture Documents
What
is
Virology
?
Virology
is
the
study
of
-
Viruses
and
virus-like
agents
-
Structure
-
Classica9on
-
Evolu9on
-
Reproduc9on,
the
diseases
they
cause
-
The
techniques
to
isolate
and
culture
them
-
Their
use
in
research
and
therapy.
Virological Methods
Direct
Examina9on
Direct examination methods are often also called rapid diagnostic methods because they can usually give a result either within the same or the next day
morphology of virus particles immune electron microscopy histological appearance hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
Indirect
Examina.on
Cell cultures, eggs, and animals may be used for isolation. However eggs and animals are difficult to handle and most viral diagnostic laboratories depend on cell culture only
1. Cell Culture
cytopathic effect (CPE) haemabsorption immunofluorescence pocks on CAM haemagglutination disease or death
2. Eggs
3. Animals
Serology
Direct Examina9on
Electron Microscopy
Virus particles are detected and identified on the basis of morphology.
Viruses may be detected in the following specimens. Faeces Rotavirus, Adenovirus Norwalk like viruses Astrovirus, Calicivirus HSV (Herpes simplex virus) VZV (Varicella zoster virus) Papillomavirus,
Vesicle Fluid
Skin scrapings
Rotavirus
Rabies Virus
Electronmicrographs
Expensive equipment Expensive maintenance Require experienced observer Sensitivity often low Limited use, good if virus shape and location are specific.
Immune electron microscopy (IEM) electron microscopy of specimens labeled with antibodies that have been conjugated with gold. The gold makes the antibody labels electron-dense.
Methods based on the detection of viral genome are also commonly known as molecular methods. It is often said that molecular methods is the future direction of viral diagnosis. However in practice, although the use of these methods is indeed increasing, the role played by molecular methods in a routine diagnostic virus laboratory is still small compared to conventional methods. It is certain though that the role of molecular methods will increase rapidly in the near future.
Dot-blot, Southern blot, in-situ hydridization are examples of classical techniques. They depend on the use of specific DNA/RNA probes for hybridization. The specificity of the reaction depends on the conditions used for hybridization. However, the sensitivity of these techniques is not better than conventional viral diagnostic methods. However, since they are usually more tedious and expensive than conventional techniques, they never found widespread acceptance.
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique. It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest. These oligonucleotides act as primers for the thermostable Taq polymerase. Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC), annealing of primers to their complementary sequences (50oC), and primer extension (72oC) result in the exponential production of the specific target fragment. Further sensitivity and specificity may be obtained by the nested PCR. 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.
Advantages of PCR:
Extremely high sensitivity, may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR
Extremely liable to contamination High degree of operator skill required 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.
Cytomegalovirus (CMV)
Schematic of PCR
Branched DNA is essentially a sensitive hydridization technique which involves linear amplification. Whereas exponential amplification occurs in PCR. Therefore, the sensitivity of bDNA lies between classical amplification techniques and PCR. Other Newer molecular techniques depend on some form of amplification. Commercial proprietary techniques such as LCR, NASBA, TMA depend on exponential amplification of the signal or the target. Therefore, these techniques are as susceptible to contamination as PCR and share the same advantages and disadvantages. PCR and related techniques are bound to play an increasingly important role in the diagnosis of viral infections. DNA chip is another promising technology where it would be possible to detect a large number of viruses, their pathogenic potential, and their drug sensitivity at the same time.
For propagation of influenza virus, pathogenfree eggs are used 11-12 days after fertilization.
The egg is placed in front of a light source to locate a non-veined area of the allantoic cavity just below the air sac.
After all the eggs have been nicked and drilled, they are inoculated with virus using a tuberculin syringe The virus is placed in the allantoic cavity, which is filled with allantoic fluid. The holes in the shell are sealed with melted paraffin, and the eggs are placed at 37 degrees C for 48 hours.
The shell membrane and chorioallantoic membrane are pierced with a pipette which is then used to remove the allantoic fluid about 10 ml per egg. Sufficient virus may be produced in one or two eggs (depending on the viral strain) to produce one 15 microgram dose of vaccine.
To study viruses which cannot be propagated in vitro, e.g. HBV To study the pathogenesis of virus infec.ons. E.g. Coxsackieviruses To test vaccine safety, e.g. oral Poliovirus vaccine.
Nevertheless, they are increasingly being discarded because: Breeding & maintenance of animals infected with viruses is expensive Whole animals are complex systems, in which it is some.mes dicult to interpret Results obtained are not always reproducible, due to host varia.on Unnecessary or wasteful use of experimental animals is morally repugnant They are rapidly being overtaken by cell culture & molecular biology
Virus
Isola.on
Cell Cultures are most widely used for virus isolation, there are 3 types of cell cultures:
1. Primary cells - Monkey Kidney These cells can only be passaged once or twice. 2. Semi-continuous cells - Human embryonic, kidney and skin fibroblasts, may be passaged up to 50 times 3. Continuous cells - HeLa, Immortalized cell line, may be passaged indefinitely. Primary cell culture 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. Continuous cells are the most easy to handle but the range of viruses supported is often limited.
Cytopathic Eect
Cytopathic effect of enterovirus 71 and HSV in cell culture: note the ballooning of cells.
Haemadsorp9on
Haemadsorption of red blood cells onto the surface of a cell sheet infected by mumps virus
Haemadsorp9on
Viral Particle
Cell Culture
Serum neutralization test 1. A specific amount of virus is added to progressively diluted serum probes. 2. Susceptible cells are added to the virus-antibody mixture. 3. The neutralization effect is made visible (cytopathic effect or immunostaining).
Long period (up to 4 weeks) required for result. Often very poor sensitivity, sensitivity depends on a large extent on the condition of the specimen. Susceptible to bacterial contamination. Susceptible to toxic substances which may be present in the specimen. Aged cells Requires equipment and operator skill. Ongoing costs even without doing any tests. Many viruses will not grow in cell culture e.g. Hepatitis B, Diarrhoeal viruses, parvovirus, papillomavirus.
The cell sheet is grown on individual cover slips in a plastic bottle. Following inoculation, the bottle then is spun at a low speed for one hour (to speed up the adsorption of the virus) and then incubated for 2 to 4 days. The cover slip is then taken out and examined for the presence of CMV early antigens by immunofluorescence.
Cytomegalovirus (CMV)
Serology
Serological
Antigen - antibody reactions studied under laboratory conditions are known as serological reactions, so named because they commonly involve serum from a patient. Serology (the testing for antibodies) is used to determine antibody positivity A large variety of serological tests are available eg. complement-fixation (CFT) haeagglutination-inhibition (HAI) enzyme-linked immunoassay (EIA) radioimmunoassay (RIA) particle agglutination immunofluorescence western blot
The complement fixation assay can be used to look for the presence of i) specific antibody or ii) specific antigen in a patient's serum. The test utilizes sheep red blood cells (SRBC), anti-SRBC antibody and complement, along with specific antigen (if looking for antibody in serum) or specific antibody (if looking for antigen in serum). If antibody (or antigen) is present in the patient's serum, then the complement is completely utilized and SRBC lysis is minimal. However, if the antibody (or antigen) is not present in the patient's serum, then the complement binds anti-SRBC antibody and lysis of the SRBCs ensues.
Principle of Immunoassays
ANTI-HUMAN IMMUNOGLOBULIN WITH DETECTOR
Western
Blot
HIV-1 Western Blot
Lane1: Positive Control Lane 2: Negative Control Sample A: Negative Sample B: Indeterminate Sample C: Positive
Western
Blot
Expensive
$
80
-
100
technically
more
dicult
visual
interpreta9on
lack
standardisa9on
How useful a serological result is depends on the individual virus. For example, for viruses such as rubella and hepatitis A, the onset of clinical symptoms coincide with the development of antibodies. The detection of IgM or rising titres of IgG in the serum of the patient would indicate active disease. However, many viruses often produce clinical disease before the appearance of antibodies such as respiratory. So in this case, any serological diagnosis would be retrospective and therefore will not be that useful. There are also viruses which produce clinical disease months or years after seroconversion e.g. HIV and rabies. In the case of these viruses, the mere presence of antibody is sufficient to make a definitive diagnosis.
Long period of time required for diagnosis for paired acute and convalescent sera. Extensive antigenic cross-reactivity between related viruses e.g. HSV and VZV, Japanese B encephalitis and Dengue, may lead to false positive results. immunocompromised patients often give a reduced or absent humoral immune response. Patients given blood or blood products may give a false positive result due to the transfer of antibody.