Virology Lab

Question 1

What can we detect?

Answer 1

Infectious virus – virus isolation and EM

Protein components – antigens of the virus

Genetic components – DNA or RNA

Host response – antibody or cell responses

Question 2

What are methods of diagnosing?

Answer 2

• Cell culture and electron microscopy – not used as commonly now, as replaced by PCR.
• PCR – Polymerase chain reaction – PCR detects specific sequences of DNA.
• Antibody detection – serology (EIA = Enzyme Immunoassay).
• Antigen detection – serology (EIA, IF = Immunofluorescence).
• Quantification of antibody or antigen.
• Serotyping – i.e. in HIV.
• Quantification of genomes – viral load.
  > This is essential for diagnosis and monitoring of HIV, HBV, HCV and CMV/EBV in the immunocompromised.
• Genome sequencing – genotyping and antiviral resistance testing

Question 3

What are the limitations of the tests?

Answer 3

False negatives results – SENSITIVITY – the tests ability to correctly identify positive samples

False positive results – SPECIFICITY – the tests ability to correctly identify negative samples

Question 4

What samples can be tested?

Answer 4

Throat swab, nasopharyngeal aspirate (NPA), Bronchoalveolar lavage (BAL), ET (Endotracheal tube) secretions – PCR

Stools – rotavirus, adenovirus, norovirus, antigen detection (EIA) or PCR

Urine – BK virus and adenovirus – PCR

CSF – herpes viruses and enteroviruses – PCR

Blood (clotted) – serology (antibody detection)

Blood (EDTA) – PCR / viral load testing.

Saliva – serology and/or PCR (e.g. measles)

Question 5

When is IgM produced and when is IgG produced?

Answer 5

IgM is a marker of RECENT INFECTION whereas IgG is created later in the host response and lasts longer

Both IgG and IgM are created in the acute phase of disease but the IgG levels only get higher and plateau whilst the IgM levels peak early and drop after a few weeks.
E.G. Positive IgG with absent IgM is consistent with past infection or immunisation

Question 6

What is antibody avidity testing?

Answer 6

This confirms a positive IgM result

Avidity = strength with which antibodies bind to a specific antigen

Early on in infection avidity is low but it gets better over a period of months as the antibodies mature

Virus isolation in cell culture is rarely used now as its slow and time-consuming (expensive) but it is still useful for phenotypic antiretroviral susceptibility testing

Electron microscopy is also rarely used

Question 7

What happens during HIV serology testing?

Answer 7

• We are on the 4th generation EIA for HIV – antibody and p24 antigen detection.

A positive result goes onto confirmatory testing in a second assay to reduce false positives.

Confirmed positives then undergo typing (HIV 1 vs. 2).

Blood samples are repeated and EDTA blood for the HIV viral load – for genotyping and baseline resistance testing.

Point-of-care testing also exists outside the lab (small equipment)

Question 8

When is immunofluorescence used?

Answer 8

Useful for direct detection of viral antigens in clinical samples

Can also be used for typing and cell culture conformation

It is rapid and inexpensive but produces very subjective results as is very dependent upon skill of technician and quality of sample

Question 9

What samples would you look at for a respiratory disease?

Answer 9

throat swab +/- nose swab
nasopharyngeal swab
nasopharyngeal aspirate (NPA)
bronchoalveolar lavage (BAL)
endotracheal tube (ET) secretions

*Multiplex PCR – rather than use a single test tube for each virus, use several viruses in one tube – only do 3 or 4 at once

Question 10

What samples would you take for a CNS disease (meninges/ encephalitis)?

Answer 10

CSF for PCR (HSV,VZV, enterovirus)
stool and throat swab (enterovirus detection)
blood for serology and/or PCR for west nile an dother arboviruses

Question 11

How does a clinical history help identify viruses?

Answer 11

Meningitis or encephalitis – HSV, VZV and enterovirus

Young child with febrile fits – Add HHV-6 + parechovirus

Immunocompromised (HIV) – Add CMV, EBV, JC virus

Recent exotic travel – Japanese encephalitis, West Nile virus, equine encephalitides, tick borne encephalitis

Outbreaks – mumps

Question 12

What happens in PCR?

Answer 12

To amplify specific RNA (RT-PCR) or DNA

Carry out 30 cycles usually

Starting block is dsDNA (so if the initial virus has RNA, use RT-PCR to form dsDNA)

Denaturing the dsDNA into strands is achieved by heating

Taq polymerase elongates the chain

Cycles of this induce exponential expansion

“Real-time” PCR can produce a read-out of results