Lecture 4 - Diagnostic Virology: Isolation and Titration Flashcards
How are viral diseases diagnosed in a clinical and laboratory setting?
Clinical Signs – Assessed by the clinician.
Sample Collection – From live animals or post-mortem (PM).
Laboratory Diagnosis:
Direct Detection (identifies virus or components):
* Antigen detection
* Virus cultivation (cell culture)
* Electron microscopy
* Detection of viral genome (e.g., PCR)
Indirect Detection (evidence of immune response):
* Serology (antibody detection)
* Cellular response tests (e.g., intradermal TB test)
What laboratory assays are used to detect viruses, and how do they work?
Virus Culture – Growth of live virus in host cells (requires viable virus).
Antigen Detection – Does not require live virus:
* Direct staining in infected cells
* Use of specific antibodies to detect viral antigens
* Detection via antigen-antibody complexes (e.g., ELISA, immunofluorescence)
Serology (Antibody Detection) – Detects immune response to virus (indirect):
* Uses known viral antigens to test for antibodies
* Detection via antigen-antibody complexes (ELISA, immunofluorescence)
Nucleic Acid Detection – Identifies viral DNA or RNA (e.g., PCR)
What methods are used for virus culture and visualization in the lab?
Virus Culture Methods:
Laboratory Animals
* Different species/routes used for different viruses.
* Allows study of disease progression and viral replication in vivo.
Embryonated Chicken Eggs
* Common for viruses like influenza.
* Multiple injection routes (e.g., allantoic, amniotic) target fast-dividing cells.
Cell Cultures
* Growth of animal/human cells in vitro.
* Supports virus replication and allows observation of cytopathic effects.
How are cell cultures used in viral diagnosis, and what are key considerations?
Cell Cultures for Viral Diagnosis:
Specific Cell Lines – Some viruses grow only in certain cells (e.g., available from ATCC).
Culture Conditions – Require controlled temperature, media, and pH to mimic the physiological environment.
Source of Cells – Can be from foetal tissue or organ biopsies.
Process:
* Inoculate virus into prepared cell culture.
* Observe cytopathic effects (CPE) caused by viral replication.
Timing – Samples must be collected early in infection (during viraemia) for best results.
What cytopathic effects (CPE) do viruses cause in host cells?
Cytopathic Effects (CPE) of Viruses:
Cell Damage/Death:
* Necrosis – uncontrolled cell death
* Apoptosis – programmed cell death
* Vacuolisation – formation of vacuoles in cytoplasm
* Crystal formation – viral protein aggregates
Syncytial Formation:
* Fusion of infected cell membranes → giant multinucleated cells
Inclusion Bodies:
* Sites of viral replication or assembly
* RNA viruses → usually intra-cytoplasmic inclusions
* DNA viruses → usually intra-nuclear inclusions
(General rule with exceptions)
How are antisera prepared for use in diagnostic tests like ELISA?
Preparation of Antisera (Antibody Reagents):
Antisera – Antibodies produced against a specific viral antigen.
Animal Models – Rabbits commonly used to produce immunoglobulins (e.g., IgG, IgM).
Use in Tests – Antisera are key reagents in ELISA and other immunological assays for detecting viral antigens or antibodies.
What is indirect immunofluorescence staining and how does it work?
Indirect Immunofluorescence Staining (IIF):
Purpose – Detects specific antigens or antibodies in a sample.
Process:
* Primary antibody binds to the target antigen.
* A fluorescently labeled secondary antibody binds to the primary antibody.
Benefits:
* Amplifies signal (multiple secondary antibodies bind one primary).
* Highly sensitive and used in viral diagnostics, autoimmunity, and research.
How does the Immunofluorescent Antibody Test (IFAT) work in viral diagnostics?
Immunofluorescence Staining (IFAT):
Principle – Detects viral antigens in infected cells using antibodies conjugated to fluorochromes (e.g., FITC – Fluorescein isothiocyanate).
Application – Used on smears or cell cultures fixed onto glass slides (commonly with acetone).
Detection:
* Specimen is illuminated with blue or UV light.
* FITC fluoresces in the yellow-green spectrum.
* Observed using interference filters matched to the emission wavelength.
What is Immunoperoxidase Staining (IIP) and how is it used in viral diagnostics?
Application – Used on cell cultures fixed with reagents that do not dissolve plastic.
Process:
* Antibody is conjugated to an enzyme (e.g., peroxidase).
* Substrate/chromogen solution is added.
* A coloured precipitate forms at the site of viral antigen.
Counterstaining – Enhances contrast to distinguish specific staining from background.
How does immunochromatography work in rapid diagnostic tests?
Immunochromatography (Lateral Flow Test):
Structure:
* A nitrocellulose strip is mounted on an absorbent card.
* Reagent lines are pre-placed on the nitrocellulose during manufacturing.
Mechanism:
* Sample and reagents migrate along the strip by capillary action (chromatography).
* As they move, they react with fixed reagents, forming visible lines.
Controlled Flow:
* Absorbent pads regulate reagent movement and maintain flow direction.
Use:
* Quick, easy detection of antigens or antibodies (e.g., pregnancy tests, COVID-19 tests).
