11.9 Diagnostic virology

Diagnostic Methods for viral infections

• Why diagnose viral infections?

  • Identify cause of illness

  • Determine stage of disease (e.g. early vs chronic HIV)

  • Monitor treatment response

  • Support epidemiological studies

  • Evaluate vaccination success

• Who uses these diagnostic methods?

  • Pathology laboratories

  • Research laboratories

Sample collection for virus diagnosis

• Depends on:

  • Clinical presentation: Site of infection and symptoms

  • Risk factors: Travel history, exposures

  • Test types: Is the goal to detect current or past infections

• Current infection tests:

  • Infectious virus particles

  • Viral nucleic acids (RNA/DNA)

  • Viral proteins (antigens)

• Past infection tests (serology):

  • Antibody response in blood

Sample collection by virus type

Enteric viruses

• Examples: Poliovirus, Norovirus, Rotavirus

• Mode of transmission: These viruses are typically spread via the faecal-oral route, meaning transmission occurs when contaminated food, water, or hands come into contact with the mouth. Poor transmission occurs when contaminated food, water, or hands come into contact with the mouth. Poor hygiene and sanitisation practices significantly contribute to the spread

• Site of replication: These viruses primarily replicate in the intestinal tract. Most are non-enveloped (naked icosahedral capsids), making them more stable in the environment, particularly in acidic and harsh gastrointestinal conditions

• Sample types:

  • Faeces or vomitus: These are the primary samples used to detect the presence of viral particles, genomes (RNA or DNA), or viral proteins (antigens)

  • Blood (serum): Used to detect antibodies against the virus, which can help identify current or previous exposure through serological methods

Respiratory viruses

• Examples: Influenza A virus

• Mode of transmission: Spread occurs primarily through aerolised droplets and inhalation of airborne virus particles, especially via coughing, sneezing, or close contact with infected individuals.

• Site of replication: These viruses replicate in the epithelium of the respiratory tract, often causing symptoms such as runny nose, sore throat, coughing, and sneezing

• Sample types:

  • Respiratory tract samples (e.g. nasal swabs or throat swabs): These are tested for the presence of virus particles, genetic material (RNA or DNA), or viral antigens using PCR or rapid antigen tests

  • Blood (serum): Collected for serological testing to detect the presence of antibodies indicating past or current infections

Blood-borne viruses

• Example: Hepatitis B Virus (HBV), Hepatitis C virus (HCV)

• Mode of transmission: These viruses are primarily transmitted through exposure to infected infected blood, often via contaminated needles, blood transfusions, or unprotected sexual contact

• Site of replication: Both HBV and HCV replicate in liver cells and are released into the bloodstream, resulting in viraemia (virus present in blood

• Sample types:

  • Blood samples: Used to detect viral particles, genetic material, antigens, and antibodies. This is the most common and convenient sample type

  • Liver biopsy samples: Sometimes necessary to assess the extent of liver damage and detect viral genomes or antigens within liver tissue. Histopathological evaluation helps determine disease prgression and liver pathology.

Human Immunodeficiency Virus (HIV)

• Mode of transmission: HIV is a blood-borne virus transmitted through exposure to infected blood or body fluids, such as through unprotected sex, shared needles, or from mother to child during birth or breastfeeding.

• Target cells: HIV infects CD4+ T lymphocytes, a key cell type in the immune system, leading to progressive immune deficiency

• Sample types:

  • Blood samples: Used to detect infectious virus particles, viral RNA, viral antigens (e.g. p24), and HIV-specific antibodies through various methds such as ELISA and PCR

  • Heparinised blood: Used to quantify CD4+ T cell counts, which helps assess immune function and monitor HIV disease progression and treatment efficacy

Sexually transmitted viruses

• Example: Herpes simplex virus (HSV)

• Mode of Transmission: These viruses are transmitted via unprotected sexual contact, through exposure to infected blood, mucous membranes, or bodily secretions.
  Clinical Presentation: HSV typically presents with vesicular lesions on the genitals, mouth, or other mucosal surfaces.
  Sample Types:

  • Vesicle fluid: Collected directly from active lesions to detect virus particles, genetic material, or viral proteins using PCR or antigen tests.

  • Blood (serum): Used for serological testing to identify HSV-specific antibodies, which helps confirm prior exposure or differentiate between HSV-1 and HSV-2 infections

Specimen transport and handling

• Maintains virus viability for culture or genome detection

• Use cold chain: 4°C, -80°C, dry ice, or liquid nitrogen

• Avoid: -20°C and repeated freeze-thaw cycles

• Enveloped viruses are more fragile

Diagnostic methods

In vitro culture (cell lines)

• Amplifies virus for easier detection

• Requires cells with appropriate receptors

• Look for cytopathic effects (CPE):

  • Cell damage, plagues, inclusion bodies

• Can be confirmed with:

  • Immunostaining

  • PCR/hybridisation

• Examples:

  • A549 – Lung (HSV, Adenovirus)

  • MDCK – Canine kidney (Influenza)

  • VERO – Monkey kidney (Polio, Flu)

  • HEL – Human embryonic lung (HSV)

Electron microscopy

• Direct visualisation of virus particles

• Advantages:

  • Rapid

  • Useful as “catch-all” for unknown agents

• Disadvantages:

  • Low sensitivity (needs >10⁸ virus/mL)

  • Cannot differentiate species

• Examples:

  • Poliovirus: 20–25 nm

  • Adenovirus: ~70 nm

  • Influenza: 80–120 nm

  • Herpesvirus: ~150 nm

Antigen detection (virus proteins)

• Immunostaining

  • Uses antibodies tagged with enzymes (HRP) or fluorescent dyes (FITC)

  • Detects viral proteins in cells/tissues

  • High specificity but labour-intensive

• Rapid Antigen Tests

  • Used for public health (e.g. COVID-19 RATs)

  • Detect viral proteins from swabs or saliva

  • Quick (<30 mins), portable, but ~78% accuracy

  • No amplification → lower sensitivity than PCR

Genome detection (PCR/RT-PCR)

• Principle:

  • Amplifies viral DNA/RNA to detectable levels

  • Very sensitive – can detect single genome copies

• Steps:

  1. Collect sample

  2. Extract nucleic acid

  3. Design specific primers (20 nt upstream/downstream)

  4. Amplify and detect using thermal cycler

• Types:

  • RT-PCR: for RNA viruses

    • PCR: for DNA viruses

• Challenges:

  • Risk of contamination with amplicons

    • Requires intact nucleic acid

• Validation:

  • Use melting curve analysis to distinguish products (e.g. HSV-1 vs HSV-2)

Antibody detection (serology)

• Uses:

  • Epidemiology studies

  • Immunity assessment

  • Vaccine monitoring

  • Diagnosing past or current infections

• Technique: ELISA

  • Uses enzyme-linked antibodies to detect patient antibodies

  • Objective and scalable

• Results Interpretation:

  • Single serum with IgM → Current/recent infection

  • Paired sera with 4x rise → Confirmed acute infection

  • IgG indicates past exposure or immunity

Summary table: Diagnostic approaches