ELISA and Viral Detection Challenges

Questions and Answers

What is the primary purpose of isolating and cultivating viruses in a clinical context?

To enhance virus stability outside the host

To permanently eliminate the virus from the environment

To genetically modify the virus

For diagnosis and treatment options (correct)

Which of the following methods best describes how Western blot assays function?

Viruses are visualized directly under an electron microscope

Proteins are separated using a sodium dodecyl sulfate (SDS) process (correct)

Antibodies are used to neutralize virus particles

Cultured viruses are treated with UV light to enhance visibility

Which characteristic of naked viruses contributes to their ability to survive in the environment?

They replicate faster inside host cells

They are dependent on host cellular machinery for survival

They are resistant to physical and chemical inactivation (correct)

Their lipid envelopes protect them from drying

What type of sample is typically collected for diagnosing viral meningitis or encephalitis?

Cerebrospinal fluid (CSF) Signup and view all the answers

In the context of viral assays, what is the primary role of antigen capture assays?

To detect and quantify specific viral antigens Signup and view all the answers

What factor significantly affects the preservation of virus infectivity during cultivation?

Temperature and freezing conditions Signup and view all the answers

Which assay is known for its high specificity in confirming the presence of antiviral antibodies?

Western blot assay Signup and view all the answers

What is a common characteristic of enveloped viruses that affects their survival outside a host?

They do not survive well outside host cells Signup and view all the answers

What is a primary disadvantage of using ELISA for viral detection?

It can produce a high number of false positives. Signup and view all the answers

What characteristic makes PCR a preferable method for viral nucleic acid detection?

It is cheap and highly sensitive. Signup and view all the answers

How does Reverse Transcriptase PCR (RT-PCR) differ from regular PCR?

RT-PCR converts RNA into DNA before amplification. Signup and view all the answers

What is the purpose of a Southern blot in viral detection?

To detect viral nucleic acids. Signup and view all the answers

What is a significant limitation of using culture methods for studying viruses?

Only a portion of assembled particles are infective. Signup and view all the answers

What is the main advantage of using ViroChip for viral detection?

It can analyze viral strains quickly and efficiently. Signup and view all the answers

Why is automated PCR considered beneficial in viral detection?

It reduces the chance of human error. Signup and view all the answers

What distinguishes gel electrophoresis from other viral detection methods?

It separates nucleic acids based on size and charge. Signup and view all the answers

What is the main difference between rate zonal density gradient centrifugation and equilibrium (isopycnic) centrifugation?

Rate zonal centrifugation separates based on size, while equilibrium separates based on density. Signup and view all the answers

Which purification method is most similar to bacterial culture for isolating viruses?

Plaque purification Signup and view all the answers

What type of gel electrophoresis is used to disrupt virions and analyze proteins?

SDS gel electrophoresis Signup and view all the answers

In the context of virus purification, what does the term 'pellet' refer to?

The mass of viral particles that are collected after ultracentrifugation. Signup and view all the answers

What method is commonly employed to convert RNA into free nucleotides during identification processes?

Ribonuclease (RNase) treatment Signup and view all the answers

What is the purpose of using SDS in gel electrophoresis for viral analysis?

To bind to proteins and impart a negative charge for migration Signup and view all the answers

What characteristic feature distinguishes density gradient ultracentrifugation in viral particle purification?

It uses a unique combination of density and the sedimentation coefficient for separation. Signup and view all the answers

Which statement about limiting dilution of the original sample in virus purification is true?

It allows for isolation of a single viral particle from a complex mixture. Signup and view all the answers

Which of the following best describes the process of obtaining biologically pure viruses?

Using a combination of plaque purification, limiting dilution, and molecular cloning. Signup and view all the answers

Study Notes

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.

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Description

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.