Virological Diagnostics Overview

Virological Diagnostics

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

Part 1: Observation of Viral Structures

• 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.

Cultivation and Isolation of Viruses

• 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

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

Virus Infection Models

• 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.

Isolation and Purification of Viruses

• 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.

Part 2: Molecular Diagnostics for Virus Infections

• 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.

Polymerase Chain Reaction (PCR)

• 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

• 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.

Genome Sequencing

• 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.

Enzyme-linked Immunosorbent Assay (ELISA)

• Detects virus particles, proteins, or antigens using microplates, virus-specific antibodies, and high sensitivity.