ELISA and Viral Detection Challenges

ELISA and Viral Detection Methods

• ELISA is generally slow; detection of related viruses is challenging due to cross-reactivity among flaviviruses.
• High sensitivity but low specificity may lead to numerous false positives in antibody detection assays.
• Viral nucleic acid detection via PCR (Polymerase Chain Reaction) is cheap, fast, highly sensitive, and specific, yet can be contaminated.
• Reverse Transcriptase PCR (RT-PCR) is used for RNA detection; Southern and Northern blotting techniques for DNA and RNA respectively; sequencing provides detailed viral identification.

ViroChip Features

• ViroChip contains 36,000 sequences representing all known viral sequences as of December 2019.
• Each microarray element is converted into stripes organized by viral family.
• Stripes' hybridization intensity is represented in a linear scale; very specific for viral identification.

Comparison of Assays

• Titre values can vary significantly based on the assay used; only a small fraction of viral particles may actually replicate.
• In vivo, 1 in 100 assembled particles in a cell may be infective.

Virus Cultivation and Importance

• Cultivation is essential for virology research and production of reagents such as antivirals and antibodies.
• Certain viruses lack culture systems, requiring alternative methods for diagnosis and public health measures.
• Virus stability varies; enveloped viruses are sensitive to environmental conditions, while naked viruses like Norovirus can survive for years.

Clinical Sample Collection Methods

• Samples vary by infection type: throat washings for respiratory, fecal for enteric, CSF for meningitis, and blood for systemic infections.
• Samples should be refrigerated or frozen in buffered media for transport.

Viral Detection Techniques

• Direct visualization using electron microscopy allows family identification and confirmation via antibody specificity.
• Western blotting uses detergents to solubilize proteins; high specificity for antiviral antibodies.

Traditional and Modern Cultivation Techniques

• Chicken egg cultivation is dated but still used for some flu vaccines; inoculations can produce visible pocks.
• Mammalian cell cultures are preferred, utilizing primary cells, diploid, and continuous cell lines, including HeLa cells.
• Safety protocols require class II biohazard cabinets for handling viral cultures; class IV isolation for highly pathogenic viruses.

Organ-on-a-Chip Technology

• Organoids, mini organs using stem cells, assist in growing various viruses and studying host responses.

SARS Outbreak Case Study

• SARS outbreak began in November 2002, with a peak reported in April 2003, resulting in 5,327 cases and 349 deaths.
• Control measures included strict public health cooperation and monitoring, including identification of bats as a natural reservoir.

Viral Structure and Composition

• Definitions:
  • Virion = virus particle
  • Capsid = protein shell of genome
  • Nucleocapsid = nucleic acid-protein assembly
  • Envelope = lipid bilayer from host.

Energy Requirements for Viral Stability

• Viruses must balance stability to protect nucleic acid with the ability to release genetic material when targeting host cells.

Purification of Viral Particles

• Purification strategies include plaque purification, dilution, and generation from molecular clones.
• Cell disruption methods are crucial for isolating viral particles, often employing freeze-thaw cycles or homogenization techniques.

Centrifugation Techniques in Viral Research

• Various centrifugation methods are used to separate nucleic acids and proteins; key processes include density gradient ultracentrifugation for purifying viruses.

Identifying Viral Components

• SDS-PAGE allows for protein identification; mass spectrometry reveals protein abundance and modifications.
• Distinguishing DNA from RNA involves specific labeling techniques.

Icosahedral Structure in Viruses

• Icosahedral capsids use identical protein arrangements for maximum stability, containing 20 triangular faces.
• Examples include adenovirus and poliovirus, both having distinct structural arrangements that facilitate host interaction.

Triangulation in Virus Structure

• The triangulation number (T) helps describe the arrangement of proteins in icosahedral viruses, with varying sizes impacting assembly and structural integrity.

This quiz explores the complexities of the ELISA test, particularly in the context of detecting flavivirus-related diseases. It covers issues such as cross-reactivity and the balance between sensitivity and specificity in viral protein assays. Test your understanding of these important diagnostic challenges.