Virological Diagnostics Overview

Study Notes

Learning Outcomes: Students will be able to apply viral structural analysis, analyze virally infected cells, and apply molecular virological diagnostics for detecting virus infections.

Structural Investigation Tools: Electron microscopy is used to investigate viral structure and virus-infected cells.
- Samples are stained with potassium phosphotungstate or ammonium molybdate.
- This allows estimation of viral shape and size.
- Electron microscopy uses an electron beam.
X-ray Crystallography: Virions are condensed and dehydrated into crystals, allowing study of their molecules and atoms using X-rays.
Cryo-electron Microscopy: Three-dimensional (3D) images of frozen virus structures are analyzed using computer software.

Animal Cell Culture: Animal cell culture (e.g., Vero, MDCK, HepG2, Calu-3, A549) is used to cultivate viruses from samples.
Cytopathic Effects (CPEs): Inverted light microscopy observes effects of virus infections in host cells, including cytopathic effects (e.g., cell shrinking and rounding up).
Multinucleated Giant Cells (Syncytia): Some viruses cause multinucleated giant cells (e.g., Nipah, measles, parainfluenza virus).
Cellular Functions: CPEs are signs of cell damage and loss of normal cellular functions.

Confocal microscopy uses fluorescently tagged antibodies that bind to virus antigens in infected cells (nucleus, cytoplasm, or membranes).

Laboratory Animals: Nonhuman primates and mice are used as virus infection models.
Embryonated Eggs: Embryonated eggs are used for virus cultivation, particularly for vaccine production (e.g., herpes, varicella-zoster, influenza).
Tissue Culture: Viruses can be cultivated into tissue culture.

Plaque Assay: Plaques form in areas where cells are infected and killed by viruses.
Virus Isolation: Virus particles are isolated from plaque centers.
Plaque Purification: Isolated virus particles are inoculated onto monolayer cells for amplification.
Laboratory Strain Development: Viruses that replicate poorly in a lab can be 'passaged' (cultured) to increase replication in lab environments.

A. Detection of Virus Nucleic Acids:
- Hybridization (Southern or Northern blotting) uses specific nucleic acid probes that bind to complementary virus genes using labeled probes.
B. Detection of Virus Antigens: -Western blotting detects virus-specific antibodies or antigens with labeled antibodies.

Amplifies specific virus genes using oligonucleotide primers.
Converts viral RNA to cDNA using reverse transcriptase for RNA viruses, then amplified using PCR.
Amplified genes are separated by agarose gel electrophoresis and stained for analysis.

Real-time PCR quantifies viral gene copies in a sample using fluorescent dyes (e.g., SyberGreen).
Monitors gene amplification in real-time.
Higher viral load correlates to faster fluorescent signal.

Sanger and Next-Generation Sequencing: Amplified viral genes/whole genomes are sequenced using these methods.
Sequence similarity is compared to databases (e.g., BLAST).
Virus strains, open reading frames, promoters, and enhancers are identified.
Phylogenetic trees can be constructed.