Virology

Question 1

Describe the electron micrograph of a herpes virus

Answer 1

• Nucleocapsid surrounds viral DNA and an outer envelope

- Symmetry= icosahedral

Question 2

What is viral antigen detection?

Answer 2

• Frequent test used for virus detection

- Blood screened for surface antigens in immunoassay test

Question 3

Describe the HBV test

Answer 3

• HB antigens present in serum of chronic hepatitis B carrier
• Excess protein assembles into small spheres or tubules, which are not infectious
• Material aggregated by specific antibody to HBs antigen (immune EM)

Question 4

Why is there HBV carriers?

Answer 4

Excess protein assembles into small spheres or tubules, which are not infectious- a liver cell infected by hepatitis B virus manufactures a huge excess of HBsAg which enters the blood making this a highly sensitive marker of hepatitis B infection

Question 5

What test is better than electron microscopy?

Answer 5

ELISA= Enzyme-Linked Immuno Assay

• means of detecting antigen (or antibody) that is widely used in diagnostic virology and other work.
• EIA is carried out on liquid samples so blood serum or plasma can be used directly in tests, or (for other viruses) extracts may be prepared from respiratory secretions or faeces for testing

Question 6

Describe the stages of an ELISA test

Answer 6

1. Antibody= specific antibody is require to react with the antigen of interest in the sample cup- can be used as coating to trap antigen from solution
2. Enzyme-Labelled Antibody= labelled antibody against antigen or first antibody will be bound to the antigen hence enzyme-label can be detected
3. Enzyme substrate= chromogen that changes colour/ light-emitting reagent.

Question 7

How are the results from the ELISA calculated/ automated?

Answer 7

• testing large numbers of samples efficient.
• Many laboratories use closed system Random Access analysers, identical to those in biochemistry, for enzyme-linked immunoassays for antigen or for antibody.
• These assays are now sensitive enough to detect 0.5 international units of HBs antigen in 1 ml plasma/serum - important for excluding infected blood or tissue donations

Question 8

How may immunofluorescence be used?

Answer 8

• In some centres immunofluorescence is still used to reveal viral antigens in cell samples.
• This can easily be done for individual samples or small numbers at a time, by hand. This is useful in small labs which don’t have the sample throughput to carry out molecular tests for viruses
• For instance epithelial cells fixed on a glass slide can be stained with fluorescein (FITC)-labelled antibody to a specific virus, and viewed in a special light microscope with UV illumination to detect the fluorescence emitted.

Question 9

When may immunofluorescence be used?

Answer 9

-Direct immunofluorescence can be applied to cells from epithelial or mucosal sites, and is used for rapid direct detection of any respiratory virus, such as influenza, or of herpes simplex or varicella-zoster in cells from base of skin blister.
-Getting an accurate diagnosis can influence specific antiviral treatment, management of contacts, and infection control decisions.
-Immunofluorescence tests have the advantage of being rapid to carry out on a small number of samples but may be less sensitive than molecular tests, which are now tending to replImmunofluorescence is also used for rapid identification of viruses causing cytopathic effects in cell cultures, by fixing some cells from the tube culture on a slide. Type-specific monoclonal antibodies are also used, commonly to differentiate HSV type1 from HSV type 2 in cells. (The type-specific antigens are on surface glycoproteins)ace them.
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Question 10

What is Respiratory syncytial virus (RSV)?

Answer 10

RSV is the most important respiratory pathogen in infancy. One in every hundred babies born is admitted to hospital in the first year of life with serious RSV infection, usually bronchiolitis.
As RSV spreads it may induce adjacent cells to fuse into a syncytium (hence the name of this virus). Human metapneumovirus (hMPV) is another paramyxovirus like RSV that causes respiratory infections that are more severe in young children, the elderly and the immunosuppressed. Its importance has become more widely understood with the introduction of molecular diagnostic tests

Question 11

What is sensitivity and specificity?

Answer 11

Sensitivity= how many of the true positives are found
no. true positives/ no. true positives + false negatives
Specificity= how many of the true negatives are identified
no. true negative/ no. true negative + false positive

Question 12

Describe nucleic acid detection

Answer 12

• When sufficient nucleic acid is present, separation of fragments by size on gel electrophoresis is possible.
• Polymerase chain reaction (PCR)

Question 13

When can gel electrophoresis be used?

Answer 13

Children with gastroenteritis due to rotavirus infection shed large numbers of virus particles in stools, as seen in the EM image earlier. Rotavirus has 11 RNA genes, and these are easily extracted and detected by electrophoresis in a polyacrylamide gel (PAGE). Note that because so much rotavirus was shed in these stools there has been no need to amplify a fragment of its genome by PCR in this case - the entire genome has simply been extracted and detected.

Question 14

What does PCR require?

Answer 14

• A known (usually short) DNA sequence of interest
• synthetic oligonucleotide sequences (“primers”)
• deoxyribonucleotide triphosphates (dNTPs)
• heat-stable DNA polymerase (eg “Taq”)
• buffer with MgCl, etc
• sample with DNA to be tested
• positive and negative control material
• a thermal cycler

Question 15

How does PCR work?

Answer 15

The microprocessor-controlled thermal cycler repeats a chosen number of cycles of denaturation, hybridisation, and DNA synthesis (eg 30 to 40 cycles).
Eventually, a vastly-amplified amount of double-stranded DNA copied from the original sample is present in the small reaction tube.
The chosen stretch of DNA that has been amplified must then be detected by electrophoresis, or by a hybridisation probe, or an EIA for a label incorporated into the new DNA.

Question 16

What is the process of PCR

Answer 16

1. ds DNA
2. at 94 degrees strands denature and separate
3. at 50 degrees, primers hybridise, annealing to homologous nucleotide sequences
4. at 68 degrees DNA synthesis begins

Question 17

How is PCR used?

Answer 17

PCR is used in research, but is also now an important tool in diagnostic virology. The remarkable sensitivity creates a problem - amplified DNA can contaminate reactions if separate laboratory areas and other precautions are not used in the separate stages. PCR is particularly useful for viruses that are difficult, dangerous or impossible to grow in cell cultures, such as HIV or hepatitis C virus. These are both RNA viruses; the HIV provirus (DNA) may be amplified directly by PCR, but RNA has to be copied to DNA first.
Patients with HIV, or hepatitis C disease, or at risk of cytomegalovirus disease (a herpesvirus infection) after transplant surgery, are monitored with PCR for the respective viral load in their plasma. This information guides the clinician to start or change treatment

Question 18

What is Virus transport medium?

Answer 18

Virus transport medium (VTM): a buffered salt solution, with added protein for stabilising viruses, and antibiotics to control bacterial contaminants.
A pH indicator dye shows if medium becomes too acid (yellow) or alkaline (purple) for use. Bottles are marked with an expiry date, and should be kept refrigerated at 4°C.

Question 19

Why should samples be sent for virus detection?

Answer 19

• to confirm a clinical diagnosis
• to make or eliminate a diagnosis of virus infection
• to support infection control measures
• to guide specific therapy
• for epidemiological surveillance and public health

Question 20

What are the guidelines for success in virus isolation?

Answer 20

1. take sample early in infection
2. obtain some cells from site of infection
3. use sterile container
4. swabs should be placed in virus transport medium
5. keep samples cold (not frozen)
6. send promptly to laboratory
7. tell the laboratory what signs and symptoms the patient has

Question 21

Describe the Herpes virus

Answer 21

Viruses belonging to the Herpesvirus family have many properties in common: they are all enveloped DNA viruses and can establish lifelong latency in the host after infection, reactivating when the host becomes immune suppressed. Herpes simplex virus, varicella zoster virus and CMV are all members of this family. This makes them important in transplant patients.

Question 22

Describe epidemiological surveillance of infleunzae A epidemic

Answer 22

PCR is now used to detect influenza A and B viruses in viral throat swabs.
In earlier times detection of outbreaks of “flu-like illness” depended more on clinical symptoms, but other respiratory viruses (such as RSV and hMPV) don’t respond to the same antiviral drugs as influenza A and B and are not covered by the influenza vaccine. PCR data may nowadays be useful to define influenza outbreaks in real time.