Laboratory Diagnosis of Viral Infections

Three Approaches1. Direct detection of virus• Electron Microscopy• Light Microscopy “Inclusion Bodies”• Antigen detection tests• Molecular Methods: PCR & Nucleic Acid Probes

2. Virus Isolation (Indirect detection)• Animal inoculation • Inoculation of eggs • In vitro Cell Culture

3. Serology (Detection of Antibodies)

Electron Microscopy (EM) &Immune Electron Microscopy• Magnification : 50,000 - 400,000• Mainly used for the diagnosis of viral gastroenteritis • The sensitivity and specificity of EM is enhanced by

immune electron microscopyDrawbacks of EM• Expensive • Poor sensitivity: at least 105 to 106 viruses per ml in the

sample required for visualization• Need highly skilled observer • Becoming less widely used due to availability of reliable

antigen detection and molecular methods.

Rotavirus Astroviruses

Adenovirus Norwalk-like viruses Transmission EM

Light Microscopy

• Detect inclusion bodies (IB)• IB are collections of replicating virus

particles in the nucleus or cytoplasm of infected cells.

• Seen in histological sections• May be characteristic or non-specific

e.g. Negri bodies in rabies

Antigen Detection Tests Viral antigens can be detected by a wide

range of serological techniques utilizing polyclonal or monoclonal antibodies.

The same techniques, utilizing purified viral antigens, can be used to detect specific antibodies to those viruses in the patient's serum.

Immunofluorescence • Rapid: result within a few hours• Technique is often tedious and time consuming• Result difficult to read and interpret• Poor sensitivity and specificity• The quality of the specimen obtained is of utmost

importance in order for the test to work properly

        

Molecular Methods

• In recent years extensively used for "non-cultivable" viruses

• Use in a routine diagnostic lab is increasing • The future of viral diagnosis • Have greatly improved the specificity of

virus diagnostic • Used for both protein and nucleic acid

• Polyacrylamide gel electrophoresis (PAGE) of protein fragments

• Western blotting• Polymerase Chain Reaction (PCR), to amplify

specific segments of viral nucleic acid • Southern blotting, and DNA hybridization

with labelled probes • Sequencing of portions of the viral genome • Restriction fragment Length Polymorphisms

(RFLP) of viral nucleic acid

Molecular Methods

Virus Isolation1. Inoculation of laboratory animals • Observing the animal for signs of disease. • One of the earliest ways of detecting a virus

2. Inoculation of fertile hens eggs Animals and eggs are difficult to handle and

are rarely used

3. "in vitro" cell cultures are still used

1. Primary cells - e.g. Monkey Kidney • These are normal cells obtained from

freshly killed adult animals. • These cells can only be passaged once or

twice o The best cell culture systems o Support the widest range of viruseso Are very expensive o Are difficult to obtain a reliable supply

Virus Isolation: Types of Cell Cultures

2. Semi-continuous cells - e.g. Human embryonic kidney and skin fibroblasts.

• Are taken from embryonic tissue• May be passaged up to 50 times. 3. Continuous cells - e.g. HeLa, Vero, Hep2 • These are immortalized cells i.e. tumor cell lines • May be passaged indefinitely• The most easy to handle but • The range of viruses supported limited

Virus Isolation: Types of Cell Cultures

• Cytopathic Effect (CPE) o Specific CPE: e.g. HSV and CMV o Non-specific CPE: e.g. enteroviruses

• Haemadsorption o Cells acquire the ability to stick to mammalian

RBCso Haemadsorption is mainly used for the

detection of influenza and parainfluenzaviruses.

Virus Isolation: Identification of growing virus

CPE

HSV RSV

Enterovirus 71

Problems with cell culture • Delayed results (up to 4 weeks) • Sensitivity is often poor• Susceptible to bacterial contamination and toxic

substances in the specimen. • Many viruses will not grow in cell culture at all e.g.

Hepatitis B and C, Diarrheal viruses, parvovirus The role of cell culture is declining due to rapid

methods like antigen detection & molecular methods

Virus Isolation: Identification of growing virus

The mainstay of viral diagnosis using polyclonal or monoclonal antibodies

Classical Techniques1. Complement fixation tests (CFT)2. Haemagglutination inhibition (HAI) tests3. Immunofluorescence techniques (IF)4. Neutralization tests

CFT and HAI, can only detect total antibody, which comprises mainly IgG

Are less sensitive

Serology

Newer Techniques1. Radioimmunoassay (RIA)2. Enzyme linked immunosorbent assay (EIA &

ELISA)3. Particle agglutination4. Western Blot (WB)5. Recombinant immunoblot assay (RIBA)

EIA and RIA, can detect specific IgM or IgG, EIAs and radioimmunoassays are the most

sensitive tests available

Serology

Primary Infection• IgM is the first antibody to appear; is

followed by a much higher titre of IgG.

Reinfection• The level of specific IgM either remains

the same or rises slightly• IgG shoots up rapidly and far more

earlier than in a primary infection

SerologyPrimary Infection