KGI: Diagnosis of Viral Diseases – Lecture 3

Flashcards covering Koch’s postulates, specimen types, ELISA and PCR principles, nucleic acid detection, Western blot, and general diagnostic approaches for viral diseases as presented in the lecture notes.

What is the purpose of Koch’s postulates in virology?

To establish causation between a virus and the disease it causes.

List the six steps of the modified Koch's postulates for viruses in order.

1) Isolate the virus from a diseased host; 2) Cultivate the virus; 3) Filter out bacteria; 4) Reproduce the disease; 5) Re-isolate the virus; 6) Detect a specific immune response.

What percentage of infectious disease cases are due to viral infections?

Over 60%.

Name the specimen types commonly used for diagnosing respiratory viral infections.

Nasal/pharyngeal aspirates, throat swabs, sputum (bronchoalveolar lavage for lower respiratory tract infections).

Which viruses are commonly associated with skin rashes and what specimens are collected?

Herpes simplex viruses, measles virus, rubella, varicella zoster virus, human herpesviruses 6 and 8, monkeypox; specimens: swab or aspirate from vesicle and scrape cells at the base of the lesion.

What specimens are used for central nervous system viral infections and which viruses are commonly tested?

Cerebrospinal fluid (CSF) or brain tissue; blood for arboviruses and arenaviruses; viruses include HSV, CMV, coxsackievirus, enteroviruses, rabies, VZV, arboviruses, arenaviruses.

What specimen is typically used for hepatitis virus diagnostics?

Serum.

What specimens are used for congenital infections diagnostics?

Serum; urine (CMV); amniotic fluid.

Which viruses are associated with infectious mononucleosis and what specimens are used?

Epstein-Barr virus (EBV) and CMV; blood, plasma, and peripheral blood lymphocytes.

What viruses cause eye infections and what specimens are collected?

Herpes simplex virus (HSV) and adenovirus; corneal scraping, dermal swab, throat/eye swab.

Which viruses are tested in immunodeficiency (HIV) diagnostics and what specimens are used?

HIV-1 and HIV-2; blood and plasma.

What are the five general approaches for laboratory diagnosis of viral infections?

Microscopy; detection of viral antigens; detection of viruses by culturing; detection of viral nucleic acids; detection of viral antibodies.

What is the typical resolution of microscopy mentioned in the notes?

0.1 micrometers (0.1 μm).

In antigen detection, what do B-cells (or antibodies) recognize on antigens?

Specific epitopes (epitope determinants) on the antigen.

What are the basic steps of ELISA for detecting viral antigens in a clinical sample?

Add viral antigen to wells; add antibodies specific for the virus; add enzyme substrate; wash; add secondary enzyme‑labeled antibodies; wash; observe color change.

How does antibody-detection ELISA (serology) work?

Plate is coated with antigen; patient antibodies bind to the antigen; enzyme‑labeled anti-human IgG binds the patient antibodies; substrate yields color.

How does antigen-detection ELISA differ from antibody-detection ELISA?

Antigen-detection uses antigen present in the sample captured by plate-bound antibodies; antibody-detection uses patient antibodies captured by plate-bound antigen and detected with labeled anti-human immunoglobulin.

What are the main steps of PCR and their typical temperatures?

Denature at ~95°C; anneal at ~50–70°C; extension at ~72°C; repeat for ~35 cycles.

What is RT-PCR and when is reverse transcriptase used?

Reverse transcription PCR; used for RNA viruses to convert RNA to cDNA before amplification.

What does nucleic acid detection aim to show in viral diagnostics?

Presence of viral nucleic acids by amplification and detection.

What is the general workflow of virus culture and visualization in shell vial culture?

Inoculate specimen onto tissue culture monolayer, centrifuge to enhance infection, incubate, stain with antiviral fluorescent monoclonal antibodies, mount and read under fluorescence microscope.

What is the principle of Western blot and its advantage over ELISA?

Proteins are separated by gel electrophoresis, transferred to a membrane, probed with patient antibodies to detect specific viral proteins; higher specificity and often confirmatory compared with ELISA.

Which diagnostic method is described as rapid (~1 hour) and easily automated?

Antigen detection by ELISA.

What are some advantages and limitations of nucleic acid detection tests?

Advantages: highly sensitive and automatable; can be rapid. Limitations: may be oversensitive and expensive.

What is a major limitation of culture-based viral diagnostics?

Time-consuming (days to weeks) and requires highly skilled personnel.

What are some challenges in serology for viral diagnosis?

Antibody responses may take up to 10 days; IgM indicates recent infection; interpretation can be difficult and not applicable to all viruses.

How do you prove if a virus exists?

Isolate virus from diseased hosts

Cultivate virus in host cells
Prove it passes through a filter that stops non-viral pathogens

Produce a comparable disease when the cultivated virus is used to infect experimental animals

Re-isolate the same virus from infected experimental animal

Look for a specific antibody immune response to the virus

What are the five general approaches for lab diagnosis of viral infections

Microscopy, Detection of viral antigens, Detection of viruses by culturing detection of viral nucleic acids and detection of viral antibodies

Immunofluorescence

is a technique used to visualize the presence of viral antigens in infected cells using fluorescently labeled antibodies under a fluorescence microscope.

Electron Microscopy

is a powerful imaging technique that allows for the visualization of viruses by providing high-resolution images through the use of electron beams instead of light. useful for viruses that cant be grown in cell cultures

ELISA

Rapid inexpensive and technically easy to perform

When viruses are cultured and grown in media what happens next

Fluorescent monoclonal antibodies are used to see virus

PCR

is a molecular technique used to amplify specific DNA sequences, enabling detection of viral genetic material even in low quantities. It is essential for diagnosing viral infections.

When do we use RT-PCR?

for RNA viruses

What are the three steps for nucleic acid detection

1.) use PCR for amplification 2.). hybridize with probe DNA with or without fluro dye

3.) detect double stranded DNA with without Fluro dye use agarose gel to detect specific size

Western Bolt

is a laboratory method used to detect specific proteins in a sample. It involves the separation of proteins by gel electrophoresis, followed by transfer to a membrane and probing with antibodies.