MB 558: Diagnostic Virology

1. Introduction to Diagnostic Virology

Viruses are obligate intracellular infectious agents and therefore differ fundamentally from bacteria and fungi in their laboratory diagnosis. Most viruses cannot multiply on ordinary bacteriological culture media. Their propagation requires living cells or other suitable biological systems. Consequently, diagnostic virology uses a combination of classical virological techniques, antigen-antibody reactions, molecular methods and microscopic methods.

The major objectives of laboratory diagnosis are to determine whether a patient is infected with a particular virus, identify the causative virus when possible, determine the stage of infection, assess the immune response, monitor viral load or treatment response, and support epidemiological and public-health investigations.

Major approaches in diagnostic virology

2. Cultivation and Purification of Viruses

2.1 General principle

Viruses are obligate intracellular parasites. They do not possess complete cellular machinery for independent energy production, protein synthesis and replication. Therefore, viruses must multiply inside living susceptible cells. This property forms the basis of virus cultivation in the laboratory.

The principal systems used for cultivation include cell cultures, embryonated chicken eggs and laboratory animals. The choice depends upon the virus, purpose of cultivation, availability of the system and diagnostic or research requirement.

2.2 Requirements for successful viral cultivation

• Susceptible cells or a suitable living host system.
• Presence of appropriate cellular receptors.
• Suitable temperature and pH.
• Adequate nutrients and physiological conditions.
• Appropriate incubation period.
• Suitable biosafety conditions.
• Proper specimen quality and adequate viral load.

2.3 Cell culture

Cell culture is the most widely used system for the propagation and study of many medically important viruses. Cells obtained from tissues are maintained under controlled conditions in suitable culture media.

Major types of cell culture

2.4 Primary cell culture

Primary cultures are prepared directly from freshly obtained tissues. The tissue is mechanically or enzymatically dissociated into individual cells and placed into an appropriate culture medium.

General steps

1. Collection of suitable tissue.
2. Removal of unwanted tissue and contamination.
3. Mechanical or enzymatic disaggregation.
4. Suspension of cells in culture medium.
5. Seeding into sterile culture vessels.
6. Incubation under controlled conditions.
7. Observation of cell attachment and growth.
8. Inoculation with the clinical specimen or virus preparation.
9. Observation for evidence of viral replication.

2.5 Monolayer cell culture

Many adherent cells grow as a single layer over the surface of a culture vessel. After reaching an appropriate degree of confluence, the cells may be inoculated with a clinical specimen containing virus.

2.6 Cytopathic effect

A cytopathic effect (CPE) is a visible structural or functional change produced in infected cells as a result of viral replication. CPE may be observed microscopically and can provide an important indication of viral growth.

Common cytopathic changes

• Cell rounding.
• Cell swelling.
• Cell shrinkage.
• Loss of adherence.
• Cell lysis.
• Formation of syncytia.
• Inclusion bodies.
• Alteration of cellular morphology.
• Detachment of infected cells.

Examples

2.7 Embryonated chicken egg

Embryonated chicken eggs were historically among the most important systems for virus cultivation and remain useful for selected viruses and vaccine production. The developing embryo provides several anatomical sites that support replication of different viruses.

Important sites of inoculation

• Chorioallantoic membrane.
• Allantoic cavity.
• Amniotic cavity.
• Yolk sac.

2.8 Laboratory animals

Some viruses can be cultivated or studied using susceptible laboratory animals. Animal inoculation has historically been important for viruses that do not grow well in routine cell cultures. It is now used less frequently because of the availability of cell culture and molecular methods, but it remains important in selected research and diagnostic applications.

2.9 Detection of viral growth

Viral multiplication may be demonstrated through direct or indirect indicators. These include:

• Cytopathic effect.
• Hemadsorption.
• Hemagglutination.
• Antigen detection.
• Immunofluorescence.
• Molecular detection of viral nucleic acid.
• Electron microscopic demonstration of particles.

2.10 Virus quantification

Quantification is important for determining the concentration or infectious activity of a virus preparation. Different methods measure different properties of the virus.

Major methods

2.11 Plaque assay

The plaque assay is a classical method for determining the concentration of infectious virus. A virus-containing sample is inoculated onto a susceptible cell monolayer. After adsorption, a semi-solid overlay restricts viral spread and allows localized infection. Each infectious unit may produce a visible area of cell destruction called a plaque.

Calculation

The result is expressed as plaque-forming units per millilitre (PFU/mL). Because a plaque may arise from more than one physical particle, PFU measures infectious units rather than total physical virus particles.

2.12 TCID₅₀

The tissue culture infectious dose 50% (TCID₅₀) is the amount of virus required to produce infection or a defined cytopathic effect in 50% of inoculated cell cultures.

Serial dilutions of virus are inoculated into replicate cell cultures. The presence or absence of infection is recorded for each dilution, and the dilution corresponding to 50% infection is calculated using an appropriate statistical method such as the Reed–Muench method.

2.13 Hemagglutination assay

Some viruses possess surface proteins capable of binding receptors on red blood cells and causing visible agglutination. This property can be used for detection and approximate quantification of hemagglutinating viruses.

3. Purification of Viruses

Virus purification refers to the separation of viral particles from cellular debris, proteins, nucleic acids and other unwanted components present in a virus-containing preparation.

Purification is important for research, vaccine development, structural studies, antigen preparation, antibody production and biochemical characterization.

3.1 General steps

1. Propagation of virus in a suitable host system.
2. Harvesting of infected cells or culture fluid.
3. Removal of large cellular debris.
4. Concentration of virus.
5. Purification using physical or biochemical methods.
6. Assessment of purity and viral infectivity.
7. Storage under appropriate conditions.

3.2 Differential centrifugation

Differential centrifugation separates particles according to differences in size and sedimentation characteristics. Lower-speed centrifugation can remove large cellular debris, whereas higher-speed centrifugation may sediment viral particles.

3.3 Density-gradient centrifugation

Density-gradient centrifugation separates particles according to their buoyant density or sedimentation behaviour. Sucrose and cesium chloride gradients have historically been used for purification of different viruses.

3.4 Chromatographic purification

Chromatography can separate viral particles or viral proteins according to physical and biochemical properties. Depending on the method, separation may be based on size, charge, affinity or hydrophobic interactions.

3.5 Filtration

Filtration can be used for clarification, removal of larger contaminants and sometimes concentration or purification of viral preparations. The choice of membrane depends upon particle size and the purpose of processing.

4. Principles and Applications of Serodiagnostic Methods

4.1 Introduction

Serodiagnosis is the diagnosis of infection through detection or measurement of antibodies or other immune responses in serum. It is particularly useful when the virus is difficult to isolate, when the patient presents late, or when evidence of previous exposure is required.

4.2 Important antibody classes

4.3 General principles of serological diagnosis

• Detection of virus-specific antibodies.
• Demonstration of seroconversion.
• Demonstration of a significant rise in antibody titre.
• Detection of virus-specific IgM.
• Measurement of neutralizing antibodies.
• Detection of antigen-antibody reactions using labelled reagents.

4.4 Haemagglutination Test

Haemagglutination is a reaction in which certain viruses bind receptors on red blood cells and produce visible agglutination. The test is mainly useful for viruses possessing hemagglutinating activity.

Principle

Serial dilutions of a virus-containing sample are mixed with a standardized suspension of red blood cells. If sufficient virus is present, the viral particles cross-link the cells and form a diffuse lattice. In the absence of hemagglutination, red cells settle as a compact button.

Applications

• Detection of hemagglutinating viruses.
• Approximate estimation of viral concentration.
• Standardization of viral antigen for HAI.
• Supportive laboratory diagnosis of selected viral infections.

4.5 Haemagglutination Inhibition Test

The haemagglutination inhibition (HAI) test detects antibodies that prevent a virus from agglutinating red blood cells.

Principle

Patient serum containing specific antibodies is mixed with a standardized virus preparation. The antibodies bind to the viral hemagglutinin and prevent the virus from attaching to red blood cells. Therefore, inhibition of hemagglutination indicates the presence of specific antibody.

4.6 Complement Fixation Test

The complement fixation test (CFT) is based on the ability of antigen-antibody complexes to fix complement. A second indicator system containing sensitized red blood cells is used to determine whether complement remains available.

Principle

If the patient's serum contains antibody specific for the test antigen, the antigen-antibody complex fixes complement. The complement is therefore not available to lyse the sensitized red blood cells in the indicator system. No hemolysis means a positive complement fixation reaction.

4.7 Neutralization Test

Neutralization tests detect antibodies capable of preventing the biological activity or infectivity of a virus. Neutralizing antibodies bind to viral surface structures and interfere with attachment, entry or another essential step of infection.

Basic procedure

1. Prepare serial dilutions of patient serum.
2. Mix serum with a standardized quantity of virus.
3. Allow antigen-antibody interaction.
4. Inoculate the mixture into susceptible cells or another appropriate system.
5. Observe for viral replication or cytopathic effect.
6. Determine the highest serum dilution that prevents infection.

The result is expressed as a neutralizing antibody titre.

4.8 Western Blot

Western blot is an immunoblotting technique used to detect specific proteins using antibodies. Viral proteins are first separated according to molecular size by electrophoresis, transferred onto a membrane and then detected using specific antibodies.

Major steps

1. Preparation of viral antigen/protein sample.
2. SDS-PAGE separation of proteins.
3. Transfer of proteins to a membrane.
4. Blocking of nonspecific binding sites.
5. Incubation with patient serum or primary antibody.
6. Washing.
7. Addition of labelled secondary antibody.
8. Substrate/reagent reaction.
9. Visualization of specific bands.
10. Interpretation according to the validated diagnostic criteria.

4.9 Radioimmunoprecipitation Assay (RIPA)

Radioimmunoprecipitation assay is an immunological technique in which radiolabelled viral proteins are used to identify antibodies capable of recognizing specific viral antigens.

In RIPA, antigen-antibody complexes are formed and precipitated. The associated radioactive proteins are then separated and detected using an appropriate radioactive detection system.

Applications

• Detection and characterization of virus-specific antibodies.
• Analysis of viral proteins recognized by patient sera.
• Research and specialized diagnostic applications.

Limitations

• Requires radioactive materials and specialized facilities.
• More technically demanding than many non-radioactive immunoassays.
• Requires strict radiation safety procedures.

4.10 Flow Cytometry

Flow cytometry is a technique that analyzes individual cells or particles as they pass through a laser beam in a fluid stream. Fluorescently labelled antibodies can be used to detect viral antigens or cellular markers associated with viral infection.

Major components

• Fluidics system.
• Laser light source.
• Optical filters and detectors.
• Signal-processing system.
• Computer for data acquisition and analysis.

Applications in virology

• Detection of viral antigen in infected cells.
• Identification of infected cell populations.
• Measurement of immune-cell responses.
• Quantification of selected cellular markers.
• Research on virus-host interactions.

4.11 Immunohistochemistry

Immunohistochemistry (IHC) detects specific antigens in tissue sections using antibodies. The antibody is linked directly or indirectly to an enzyme or other detectable label, allowing localization of viral antigen within infected tissues.

General procedure

1. Collection and fixation of tissue.
2. Preparation of tissue sections.
3. Antigen retrieval when required.
4. Blocking of nonspecific reactions.
5. Application of primary antibody.
6. Washing.
7. Application of labelled secondary antibody when required.
8. Addition of substrate/chromogen.
9. Microscopic examination.
10. Interpretation with appropriate controls.

5. Nucleic Acid-Based Diagnosis

Molecular diagnosis has become a central component of modern diagnostic virology because viral genomes can often be detected even when infectious virus cannot be cultured easily.

The major techniques included in the syllabus are nucleic-acid hybridization, polymerase chain reaction, microarray and nucleotide sequencing.

5.1 Nucleic Acid Hybridization

Nucleic-acid hybridization is based on complementary base pairing between single-stranded nucleic-acid sequences. A labelled nucleic-acid probe is designed to recognize a complementary sequence in the target viral genome.

Basic principle

1. Viral nucleic acid is extracted from the specimen.
2. The target nucleic acid is denatured into single strands.
3. A labelled complementary probe is added.
4. The probe hybridizes with its complementary target sequence.
5. Unbound probe is removed.
6. The hybridized probe is detected.

Applications

• Detection of specific viral sequences.
• Identification of viral genomes in clinical specimens.
• Research on viral gene expression.
• Localization of viral nucleic acid in tissues.

5.2 Polymerase Chain Reaction

PCR is an in-vitro nucleic-acid amplification technique that produces millions of copies of a specific DNA target from a small starting amount of template. It has become one of the most important molecular methods in diagnostic microbiology.

Basic components

• Template DNA.
• Forward primer.
• Reverse primer.
• Thermostable DNA polymerase.
• dNTPs.
• Reaction buffer.
• Magnesium ions.

Three fundamental steps

5.3 RT-PCR

RNA viruses cannot be amplified directly by conventional DNA PCR. In reverse-transcription PCR (RT-PCR), viral RNA is first converted into complementary DNA (cDNA) using reverse transcriptase. The cDNA is then amplified by PCR.

5.4 Real-Time PCR

Real-time PCR monitors amplification as it occurs using fluorescence. The amount of fluorescence generated during amplification is related to the amount of target nucleic acid present.

Advantages

• High sensitivity.
• High specificity.
• Rapid detection.
• Reduced post-amplification handling.
• Quantitative or semi-quantitative analysis is possible.
• Useful for viral load monitoring.

5.5 Multiplex PCR

Multiplex PCR uses more than one primer pair in a single reaction to detect multiple targets simultaneously. It can be useful when several viruses or several genetic targets need to be investigated from the same specimen.

5.6 Microarray

A nucleic-acid microarray contains a large number of immobilized probes arranged in a defined pattern. Labelled target nucleic acids hybridize to complementary probes, allowing simultaneous analysis of many genetic targets.

Applications

• Detection of multiple viral agents.
• Analysis of viral genes.
• Study of gene expression.
• Research on viral diversity.

5.7 Nucleotide Sequencing

Nucleotide sequencing determines the precise order of bases in a nucleic-acid molecule. It provides more detailed information than simple detection of a viral genome.

Applications in virology

• Identification of viral species or strains.
• Detection of mutations.
• Genotyping.
• Investigation of antiviral resistance-associated mutations.
• Molecular epidemiology.
• Outbreak investigation.
• Phylogenetic analysis.

5.8 Sanger sequencing

Sanger sequencing uses chain-terminating nucleotides to determine the sequence of a defined DNA region. It is particularly useful when a specific genomic region requires targeted sequencing.

5.9 Next-generation sequencing

Next-generation sequencing (NGS) allows parallel sequencing of a very large number of DNA fragments. In virology it can support broad pathogen detection, whole-genome sequencing, variant identification and molecular epidemiology.

6. Microscopic Techniques in Diagnostic Virology

Microscopy can be used to detect viral particles, viral antigens, infected cells or characteristic cellular changes. Because most viruses are below the resolution of conventional light microscopy, specialized microscopic methods are required for direct visualization of many virus particles.

6.1 Fluorescence Microscopy

Fluorescence microscopy uses fluorochrome-labelled molecules that emit light when excited by a suitable wavelength. In virology, fluorescent antibodies are commonly used to detect viral antigens in infected cells.

Direct immunofluorescence

A fluorescently labelled antibody directly binds the viral antigen in the clinical specimen or infected cell.

Indirect immunofluorescence

An unlabelled primary antibody binds the target antigen and a fluorescently labelled secondary antibody binds the primary antibody.

6.2 Confocal Microscopy

Confocal microscopy uses focused laser illumination and spatial filtering through a pinhole to obtain sharp optical sections of specimens. Multiple optical sections can be combined to produce three-dimensional information.

Advantages

• Improved optical sectioning.
• Reduced out-of-focus fluorescence.
• High-resolution imaging of labelled structures.
• Three-dimensional reconstruction is possible.
• Useful for studying virus-host interactions and intracellular localization.

6.3 Electron Microscopy

Electron microscopy uses a beam of electrons rather than visible light and therefore provides much higher resolving power. It can be used to visualize viral morphology and intracellular structures.

Transmission Electron Microscopy (TEM)

In TEM, electrons pass through an ultrathin specimen. The resulting image provides detailed information about internal and external structures. TEM is particularly useful for determining viral morphology and ultrastructure.

Scanning Electron Microscopy (SEM)

SEM scans the surface of a specimen with an electron beam and detects emitted signals to produce a detailed image of surface morphology.

7. Selection, Collection, Transportation and Processing of Specimens for Laboratory Diagnosis of Viral Infections

7.1 Importance of specimen collection

The accuracy of laboratory diagnosis begins with appropriate specimen selection and collection. Even a highly sensitive molecular or serological test may give an unreliable result if the specimen is collected from the wrong site, collected at the wrong time, inadequately transported or improperly stored.

7.2 General principles of specimen selection

• Select the specimen from the anatomical site where the virus is expected to be present.
• Collect specimens as early as possible during acute infection when viral load is often highest.
• Consider the clinical syndrome and suspected virus.
• Use appropriate sterile containers.
• Collect adequate volume or quantity.
• Avoid contamination.
• Use appropriate transport medium when required.
• Maintain the recommended temperature during transport.
• Provide complete clinical information to the laboratory.

7.3 Common clinical specimens in virology

7.4 Respiratory specimens

Respiratory specimens are important for diagnosis of viral infections involving the upper and lower respiratory tract. Nasopharyngeal swabs, nasopharyngeal aspirates, combined nasal/throat specimens and lower respiratory tract samples may be selected according to the clinical condition and test requirement.

Important considerations

• Collect before antiviral treatment whenever clinically appropriate.
• Use appropriate synthetic-fibre swabs and suitable transport systems according to laboratory protocol.
• Avoid unnecessary contamination.
• Transport promptly to the laboratory.

7.5 Blood and serum

Blood may be used for detection of viral nucleic acid, antigen or circulating virus, whereas serum is commonly used for serological diagnosis.

For serology, paired serum samples may sometimes be useful. An acute-phase sample is collected early and a convalescent-phase sample is collected later. A significant change in antibody titre may provide evidence of recent infection depending on the virus and assay.

7.6 Cerebrospinal fluid

CSF is an important specimen in suspected viral infections of the central nervous system, including viral meningitis and encephalitis.

Molecular detection, particularly nucleic-acid amplification, is frequently important because many neurotropic viruses are difficult to recover by routine culture.

7.7 Vesicular and lesion specimens

For vesicular diseases, material from a fresh lesion can be particularly useful. The specimen may include vesicle fluid, lesion swab or material from the lesion base depending on the suspected infection and laboratory protocol.

For herpesvirus infections, molecular detection from lesion material is an important diagnostic approach.

7.8 Stool specimens

Stool may be collected for diagnosis of selected enteric viral infections. Adequate specimen quantity should be collected in a clean, leak-proof container and transported promptly.

7.9 Tissue and biopsy specimens

Tissue specimens can be used for histopathology, immunohistochemistry, molecular detection or other specialized viral investigations.

The portion required for molecular or virological testing should be handled according to the laboratory protocol because inappropriate fixation may affect nucleic-acid or antigen detection.

7.10 Timing of specimen collection

The amount of virus or viral nucleic acid varies during infection. Therefore, specimen timing is critical. During the acute phase, the viral load may be sufficiently high for direct detection.

For serological diagnosis, antibodies may not be detectable during the earliest phase. Therefore, paired specimens or a later convalescent sample may sometimes be required.

7.11 Transportation of specimens

The purpose of transportation is to maintain the integrity and diagnostic value of the specimen from the point of collection to the laboratory.

Important principles

• Use leak-proof primary containers.
• Use appropriate secondary packaging.
• Clearly label the specimen.
• Include patient/sample identification according to laboratory policy.
• Maintain appropriate temperature.
• Prevent leakage and contamination.
• Use suitable transport medium where required.
• Minimize delay between collection and processing.

7.12 Cold chain

Many viral specimens require temperature-controlled transportation to preserve viral infectivity, antigenicity or nucleic-acid integrity. The appropriate temperature depends on the specimen, test and virus.

For molecular testing, preservation of nucleic acid is important. For virus isolation, maintaining infectivity is particularly important.

7.13 Viral Transport Medium

Viral transport systems are designed to maintain the integrity of viral specimens during transport while limiting bacterial and fungal contamination. The exact transport system and storage requirements depend on the diagnostic protocol and laboratory.

7.14 Specimen processing

After reaching the laboratory, specimens are checked, registered and processed according to the requested test.

General processing sequence

1. Verify patient and specimen identification.
2. Check specimen type and adequacy.
3. Assess transport and storage conditions.
4. Process the specimen using appropriate biosafety precautions.
5. Divide aliquots when required.
6. Perform the selected diagnostic test.
7. Store residual specimen appropriately when required.
8. Dispose of infectious material according to biosafety procedures.

7.15 Specimen rejection criteria

A specimen may be rejected or recollection requested when it is unsuitable for reliable testing.

• Incorrect or missing identification.
• Wrong specimen type.
• Insufficient quantity.
• Leaking container.
• Improper transport conditions.
• Excessive delay in transportation.
• Improper storage.
• Contamination where it affects the intended test.

7.16 Biosafety during specimen handling

Clinical specimens may contain infectious viruses. Therefore, all specimens should be handled using appropriate standard precautions and laboratory biosafety procedures.

• Use appropriate personal protective equipment.
• Perform procedures that may generate aerosols in appropriate containment.
• Use biological safety equipment when required.
• Disinfect work surfaces appropriately.
• Dispose of infectious waste safely.
• Follow institutional biosafety procedures.

8. Comparison of Major Diagnostic Approaches

9. Quality Assurance in Diagnostic Virology

Quality assurance is essential because viral diagnostic procedures are highly sensitive to specimen quality, reagent performance, contamination and interpretation.

Important components

• Proper specimen identification.
• Standardized specimen collection.
• Validated laboratory methods.
• Positive and negative controls.
• Internal controls in molecular assays.
• Equipment maintenance and calibration.
• Reagent quality control.
• Proper documentation.
• Staff competency.
• Participation in external quality assessment where available.

10. Important 2-Mark Questions

11. Important 5-Mark Questions

12. Important 10-Mark Long Questions

13. High-Yield Examination Points

14. Rapid Revision Table

15. Final Exam Focus