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This is a microbiology lecture by Dr. Bijendra Raj Raghubanshi (Professor, Dept. of Microbiology) titled "Laboratory Diagnosis of Viral Infection". Here's a full breakdown:
Overview
The presentation covers how laboratories identify viral infections — from specimen collection all the way to molecular techniques like PCR.
1. Specimen Collection & Transport
- Specimens vary by infection type: nasal wash, throat swab, stool, CSF, urine, blood, etc.
- Must be collected early in the acute phase and processed immediately
- If delayed: store on ice (4°C) or freeze at −20°C or below
- Transported in Stuart's viral transport media
2. Five Main Laboratory Methods
I. Microscopy
a. Electron Microscopy (EM)
- Directly visualizes virus particles
- Mainly used for viral gastroenteritis: rotavirus, adenovirus, astrovirus, calicivirus, Norwalk-like viruses
- Immune EM uses virus-specific antibodies to agglutinate particles — but it's expensive and has poor sensitivity
b. Antigen Detection
- Immunofluorescence testing
- Rapid immunochromatographic tests (e.g., rapid antigen tests)
c. Cytopathology (Light Microscopy)
- Looks for cellular changes caused by virus replication:
- Syncytium formation, cell necrosis/lysis, cellular clumping
- Inclusion bodies — virus-specific intracellular masses (~20–25 µm) with distinct size, shape, location, and staining
- Intracytoplasmic: Negri bodies (rabies), Guarnieri bodies (smallpox), Bollinger bodies (fowl pox)
- Intranuclear: Cowdry A (herpes, yellow fever), Cowdry B (adenovirus, poliovirus)
II. Isolation & Identification
Three systems used to grow viruses:
a. Laboratory Animals
- White mice and chimpanzees
- Suckling mice (<48 hours old): susceptible to togaviruses and coxsackieviruses
b. Chick Embryo (7–12 days old)
- Chorioallantoic membrane (CAM): grows poxvirus → produces visible pocks
- Allantoic cavity: mainly for vaccine production
- Amniotic sac: isolation of influenza virus
- Yolk sac: some viruses + intracellular bacteria (Chlamydiae, Rickettsiae)
c. Tissue Culture
- Types: organ culture, explant culture, cell culture
- Cell culture subtypes:
- Primary (monkey kidney, human amniotic cells)
- Diploid (embryonic fibroblasts)
- Continuous (HeLa, Hep-2, KB cells)
- Viruses identified in culture by: electron microscopy, neutralization, haemadsorption/haemagglutination, cytopathic effects (CPE), immunofluorescence
III. Serology
Used to detect antibodies in patient serum. Paired sera (acute + convalescent) showing a 4-fold rise in antibody titre confirms recent infection.
| Test | What It Detects |
|---|---|
| Haemagglutination inhibition (HI) | Antibodies to viral haemagglutinin |
| Complement fixation test (CFT) | Complement-fixing antibodies |
| Immunofluorescence | Using fluorescein-conjugated antiserum |
| Neutralisation | Virus-neutralizing antibodies |
| ELISA | Antibodies via enzyme-labelled anti-human Ig |
| Western blot | Confirmatory test (historically for HIV) |
IV. Detection of Viral Particles
Mentioned as a category but covered under microscopy (EM above).
V. Molecular Methods
Nucleic Acid Probes / DNA Probes
- Useful for slowly replicating viruses
PCR (Polymerase Chain Reaction)
- Invented in 1983 by Kary B. Mullis
- Amplifies millions to billions of copies of a specific DNA segment
- Three steps: Denaturation → Primer Annealing → Extension (using heat-resistant Taq polymerase in a thermocycler)
- RT-PCR: RNA first converted to DNA (cDNA), then amplified — used for RNA viruses
- Real-time PCR (qPCR): quantifies viral load
Summary Table
| Method | Examples |
|---|---|
| EM | Rotavirus, Norwalk virus |
| Antigen detection | Flu rapid tests, immunofluorescence |
| Inclusion bodies | Negri (rabies), Guarnieri (smallpox) |
| Lab animals | Suckling mice (coxsackievirus) |
| Chick embryo | Influenza, poxvirus |
| Tissue culture | HeLa cells, CPE observation |
| Serology | ELISA, Western blot, HI test |
| PCR / RT-PCR / qPCR | HIV, influenza, most modern diagnostics |
The lecture flows logically from sample collection → direct detection → culture → antibody tests → molecular methods, covering both classical and modern virology diagnostics.
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