Laboratory Diagnosis of Viral Infections Flashcards
(a) What is cell culture?
(b) What is the primary application of cell culture in viral diagnosis?
(a) The process of growing cells under controlled conditions, typically outside their natural environment.
(b) Virus isolation from patient samples to increase their quantity for further testing.
List three types of cell cultures.
(a) Primary cell cultures
(b) Secondary cell cultures [aka. diploid cell cultures, aka. semi-continuous]
(c) Cell lines [aka. heteroploid cultures]
What is a primary cell culture?
These are cells taken directly from tissues and grown until they occupy the available substrate. Once the cells get attached to the vessel surface, they undergo mitosis until a confluent monolayer of cells covers the surface. These cells are capable of only limited growth in culture and cannot be maintained in serial culture.
They are commonly employed for primary isolation of viruses and in preparation of vaccine.
What is a secondary cell culture?
These cell cultures comprise a single type of cells, usually fibroblasts, and can be subcultured for a limited number of times. There is a rapid growth rate and after about 50 serial subcultures, they undergo senescence and the cell strain is lost.
What is a cell line culture?
Cells that have been transformed to proliferate indefinitely.
Further notes:
The standard continuous cell lines have been derived from human cancers, such as HeLa (derived from cervical cancer of a lady, Hela by name), Hep2 and KB cells.
These are not used for preparation of viral vaccines, as vaccines prepared in cancer cells are considered unsafe for human use.
What are some common cytopathic effects caused by viruses that can be observed under light microscopy?
(1) syncytium formation
(2) cell necrosis and lysis
(3) cellular clumping
(4) inclusion bodies
(5) discrete focal degenration
(6) transformation
What are some problems that may be encountered when cell cultures are used for virus isolation?
(a) long period (up to 4 weeks) required for result
(b) susceptible to bacterial contamination
(c) susceptible to toxic substances which may be present in the specimen
(d) many viruses will not grow in cell culture e.g. hepatitis B, diarrheal viruses, parvovirus, papillomavirus
(e) safety concerns
Embryonated Egg method of Virus Culture
Fertilized chicken eggs, typically 10-12 days old, are used because ____________.
the provide a variety of cell types that support the replication of different viruses.
Embryonated Egg method of Virus Culture
List four inoculation sites and one virus for each.
(1) Allantoic sac: influenza virus, mumps virus, Newcastle disease virus [The virus replicates in the cells lining the allantoic cavity.]
(2) Amniotic sac: influenza virus, mumps virus. [The virus grows in the amniotic fluid and the cells of the amniotic membrane.]
(3) Chorioallantoic Membrane: herpes simplex, poxvirus, Rous sarcoma virus [The virus forms visible lesions or pocks on the membrane.]
(4) Yolk sac: Used for some herpes viruses. The virus replicates in the cells of the yolk sac.
Suckling mice are often used in virology for the isolation and identification of viruses. Why suckling mice?
Newborn mice, typically 1-3 days old, are used because their immune systems are not fully developed, making them more susceptible to viral infections.
Further notes:
A virus-containing sample is prepared and injected into the suckling mice, usually via intracerebral, intraperitoneal, or subcutaenous routes. The inoculated mice are monitored for signs of infection, such as lethargy, paralysis or death.
Electron microscopy enables visualization of a virus, and this useful in assigning it under a correct family. What are three techniques that can be used to enhance the sensitivity and specificity of electron microscopy?
(a) Negative stain electron microscopy
(b) Thin section electron microscopy
(c) Immune electron microscopy
Further notes:
Negative stain electron microscopy involves applying a heavy metal stain to a biological sample, which enhances contrast by staining the background rather than the specimen itself. This technique allows for the visualization of small structures like viruses, bacteria and proteins by making them appear lighter against a dark background.
Thin section electron microscopy involves cutting specimens into ultra-thin slices, typically less than 100nm thick, using an ultramicrome. These slices are then examined under a transmission electron microscope (TEM), where electrons pass through the specimen to form highly detailed images of its internal structure.
Immune electron microscopy is a technique that combines immunology and electron microscopy to identify and localize specific proteins within cells. It uses antibodies tagged with electron-dense markers, such as gold particles, to bind to target proteins, making them visible under an electron microscope. This allows for high-resolution visualization of the protein’s location and distribution within the cell.
What are some disadvantages of electron microscopy in laboratory diagnosis of viral infections?
☑ expensive equipment
☑ expensive maintenance
☑ require experienced observer
☑ sensitivity often low
Explain how Immunochromatography [aka. lateral flow immunoassay] is used to detect viral infections in the lab.
Don’t worry about the details. Understand the basic principle and rate yourself a five!
(1) A biological sample (e.g. blood, urine, saliva) is applied to the sample pad of a test strip.
(2) The sample moves along the strip by capillary action, carrying any target substances (like viral antigens or antibodies) with it.
(3) The strip contains a conjugate pad with colored particles (such as gold nanoparticles) coated with specific antibodies or antigens. These particles bind to the target substances in the sample
(4) As the sample continues to migrate, it reaches the test line, which has immobilized antibodies or antigens specific to the target. If the target substance is present, it binds to these immobilized antibodies or antigens.
(5) The binding reaction at the test line causes a visible colored line to form, indicating a positive result.
[2-minute video]
Explain how immunofluorescence is used in the laboratory diagnosis of viral infections.
Don’t worry about the details. Understand the basic principle and rate yourself a five!
(1) A tissue sample is obtained from the patient.
(2) The collected sample is fixed onto a microscope slide to preserve the structure and integrity of the cells.
(3) A primary antibody that specifically binds to the viral antigen present in the infected cells is applied.
(4) A secondary antibody that binds to the primary antibody is applied. The secondary antibody is conjugated with a fluorescent dye.
(5) Incubation…
(6) Washing of the slide to remove unbound antibodies, reducing background noise.
(7) Visualization: The slide is examined under a fluorescence microscope. The fluorescent dye emits light when exposed to a specific wavelength, highlighting the presence of a virus.
