Diagnostic Virology

Question 1

What is HTLV and when was it first identified?

Answer 1

Human T-Cell Leukaemia Type 1. Mid-late 1970s in Japan. Associated with adult T-cell leukaemia cases. First virus associated with human malignancy.

Question 2

What is the prevalence of HTLV?

Answer 2

15-20 million people infected. Worldwide distribution but endemic is some places like Japan, Carribean, Iran, Romania and approx. 5% will develop ATL.

Question 3

What diseases can HTLV cause?

Answer 3

• Adult T-cell leukaemia (ATL)
• Adult T-cell leukamia/lymphoma (ATLL)
• HTLV-1 associated myelopathy (HAM)
• Tropical spastic paraparesis (TSP)
• HTLV-1 associated infectious dermatitis
• HTLV-1 associated uveitis (HAU)

Question 4

How is HTLV transmitted?

Answer 4

From mother to infant (breastfeeding or during birth), Through sexual contact, Through blood (blood transfusion)

Question 5

Describe HTLV’s genetic makeup and the functions of its viral proteins

Answer 5

HTLV-1 is a single-strand enveloped RNA virus with ssRNA genome but during time of infection, genetic information also present in the form of double-stranded DNA
Gag+ Pro+ Pol: Virion assembly and maturation
Env: Viral entry and transformation tropism
p13: Viral persistence
p12: Viral infectivity and persistence
Rex: Positive post-transcriptional regulation
Tax: Viral transcription and oncogenesis – found specifically in HTLV-1
p30: Transcriptional and post-transcriptional regulation

Question 6

Describe HTLV-1’s replication cycle

Answer 6

1. HTLV-1 enters T-cell
2. ssRNA released into host cell cytosol
3. ssRNA reverse transcribed into ssDNA
4. ssDNA converted to dsDNA
5. dsDNA enters nucleus and integrates into host genome
6. Viral genome can replicate as part of the host chromosome

Question 7

What does the number of cells containing HTLV-1 DNA tell us?

Answer 7

Disease severity and likelihood of transmission

Question 8

What is the Western blot used for and what are its steps?

Answer 8

Used to assess if patients have antibodies specific to HTLV-1 proteins. Steps: Taking blood, separation, transfer, staining and visualisation.

Question 9

Describe process of separation and transfer in the Western blot

Answer 9

Separation: viral proteins derived from in vitro propagated viral cultures separated based on size on a polyacrylamide protein gel. Smaller proteins migrate faster.
Transfer: proteins transferred using electro transfer system onto PVDF membrane and viral proteins stick to membrane creating image of gel and each viral protein forms distinct bands but not visible yet.

Question 10

Describe process of staining in the Western blot

Answer 10

The viral proteins immobilised on the membrane are used to determine if patients have antibodies to the diff proteins present.
• First membrane incubated with patient serum
• If antibodies present, they bind to it at specific spot
• Membrane washed to remove antibody and plate incubated with a secondary antibody
• Secondary antibody recognises Fc region of antibodies that could be present in patient serum
• Secondary antibody conjugfated to an enzyme so once incubation over, membrane washed to remove unbound secondary antibody and then substrate specific to the enzyme added
• If enzyme present, reaction occurs and output signal received

Question 11

Describe visualisation in the Western blot

Answer 11

Colour change occurs or generation of a precipitate which can be seen by eye or a luminescent signal detected with special camera

Question 12

What constitutes a positive test for HTLV-1?

Answer 12

If a patient is truly infected with a HTLV-1 virus, antibodies will be present against many different viral proteins. Positive result is when: Synthetic peptide MTA-1 present, Viral core proteins p53 + p24 + p19 present, Recombinant glycoprotein gd21 present. Esp in tropical regions, indeterminate results obtained where they only have a few HTLV-1 viral proteins and hence samples must be analysed with additional tests such as PCR

Question 13

Why is PCR used for HTLV-1 and what is a prerequisite for PCR?

Answer 13

HTLV-1 TAX gene amplified through PCR. DNA usually found as double strand, where top strand runs 5’ to 3’ while bottom runs 3’ to 5’. DNA polymerase adds bases going from 5’ to 3’ but to do this need a primer with a free 3’ hydroxyl group.

Question 14

What are the 3 steps in the PCR reaction?

Answer 14

1. Denaturation: Sample heated to 95 degrees to denature the double stranded DNA and produce a single strand of DNA (hydrogen bond breaking)
2. Annealing: Sample cooled to 50-60 degrees, allowing primer to anneal to DNA with a complementary sequence. For standard PCR, need a forward and a reverse primer.
3. Extension: Sample heated to 70-72 degrees which is optimal temp for a thermostable DNA polymerase to extend the DNA and produce a 2nd strand. Exact time taken for step depends on length of final PCR fragment.

Question 15

How many times is PCR done?

Answer 15

Steps repeated 30-40 times so sufficient DNA produced – subsequently separated on an agarose gel and can be visualised with stains.

Question 16

How are the primers designed?

Answer 16

Forward primer called HL43 has the same sequence as the top strand and reverse primer is named HL44 which has sequence of bottom gene – 300 bp region of tax gene is amplified. Primers must be written in 5’ to 3’ direction.

Question 17

What are the 5 key reagents of PCR?

Answer 17

1. DNA Template – DNA isolated from T cells with the integrated virus DNA
2. 2 tax specific primers
3. DNA polymerase (Taq polymerase)
4. Nucleotides (dNTPs)
5. Buffer component which provides appropriate salt + pH for polymerase

Question 18

What are the 3 steps of sample preparation for PCR?

Answer 18

1. Peripheral blood taken from patients and peripheral blood mononuclear cells (PBMCs) isolated – these are a mix of lymphocytes and monocytes
2. Peripheral blood sits atop the separation medium and is then centrifuged so the plasma collects at the top, then PBMCs, then granulocytes and finally RBCs
3. PBMC fraction taken and subjected to protein degradation, capture and cleaning of DNA until DNA isolated and obtained.

Question 19

What must also be done and how are PCR results analysed?

Answer 19

Must also set up a positive control and a negative control in addition to the patient samples. DNA Gel electrophoresis used to analyse results. Agarose electrophoresis separates DNA based on size. DNA fragments negatively charged and hence will migrate towards positive anode but smaller fragments will move faster. To see this DNA, must add a specific DNA stain to gel which intercalates into DNA – if UV light being used, ethidium bromide and if blue light being used, Sybr Safe. To load DNA sample onto agarose gel, may need to add a DNA loading dye to samples. Size of PCR product determined by DNA marker/ladder which contains guide on how far different DNA sizes run – used as reference to find size of our DNA product.

Question 20

Why does a DNA loading dye need to be added to samples?

Answer 20

1. PCR solution made heavier.
2. Allows sample to sink to bottom.
3. DNA loading dye also contains colour dye making it visual so can see which well already contains a sample.
4. Estimate how far DNA has migrated in the gel during run

Question 21

What concentration of agarose is used in the gel and why?

Answer 21

Prepped in conc between 0.7% to 2.0% depending on size of bands that need to be separated. Agarose percentage determines size of pores in the gel matrix
Short DNA = Higher agarose concentration = Greater resolution
Long DNA = Lower agarose concentration = Larger pores so easier movement

Question 22

What buffer is used and why?

Answer 22

TAE buffer/TBE buffer used – TBE less prone to overheating so better for longer electrophoresis runs. Agarose dissolved into this buffer. Gel box must be filled with same gel used to prepare the buffer.

Question 23

What is done to the prepared agarose gel before DNA is loaded?

Answer 23

Microwave gel but not too much as some will evaporate affecting concentrations. Once flask cool enough to touch with just gloved hand (or cooled to 55 degrees C), it is ready to pour. Add ethidium bromide to the prepared agarose gel. Place comb in plate with needed number of wells and pour the gel in, moving bubbles away with pipette. Allow to cool for an hour and then pour the buffer that was used to prepare the gel until wells filled.

Question 24

What occurs once DNA is loaded?

Answer 24

Load DNA ladders next to samples and fill samples into well. Connect to power and run between 80-100V – too high a power causes fast migration but gel will melt. Run for hour and a half. Visualise bands using ultraviolet light. 100-150 nanograms of DNA loaded and run electrophoresis for 1.5 hours.

Question 25

How can the visualisation of results be improved on agarose gel?

Answer 25

To increase resolution of DNA bands: Use gel comb with wider/thinner teeth, load less DNA or run gel for a longer time at lower voltage.

Question 26

What is qqRT-PCR and what is its purpose?

Answer 26

Quantitative real time PCR provides information on the amount of viral DNA present in the sample rather than a yes/no answer obtained from standard PCR. Helps to predict severity of disease and predict transmission likelihood. Aim of method is to amplify a specific piece of DNA (tax gene).

Question 27

How does qqRT-PCR work?

Answer 27

Instead of gel electrophoresis, amount of PCR product monitored throughout reaction cycle and readout is increase in fluorescent signal where signal is proportional to the amount of DNA in the sample (Fluorescence dye-based method). If readout crosses a threshold called CT, person is infected. In negative patient, threshold would never be reached. How quickly this threshold is reached depends on amount of HTLV-1 DNA present.

Question 28

Describe how the fluorescence dye method of qqRT-PCR works?

Answer 28

Dye added to reaction mixture which is non-fluorescent in solution but fluorescent once it binds to DNA so fluorescence increases in each cycle since DNA amount increases.

Question 29

Describe how the DNA probe method (TaqMan method) of qqRT-PCR works?

Answer 29

3rd oligo probe also added to mixture which binds to the specific gene that needs to be amplified. Binds to a region between DNA region that is to be amplified by forward and reverse primer. Oligo contains fluorophore and quencher molecule at 5’ and 3’ ends – hence when oligo intact, they are quite close in space. Thus even if fluorophore excited, will not fluorescence. Fluorescence in sample will increase as the fluorophore and quencher are physically separated which occurs during PCR when oligo degraded. When separated, fluorescence increases. Fluorescence increases in every PCR cycle and increases proportional to how many molecules of HTLV-1 present.

Question 30

What is a drawback of the TaqMan method and why?

Answer 30

TaqMan more expensive as requires oligo probe but because oligo probe binds to that specific DNA fragment which is amplified, makes assay more specific and hence used in diagnostics

Question 31

What does each primer end link to?

Answer 31

5’ end must be linked to fluorophore (FAM – 6-carboxyfluorescein)
3’ end must be linked to quencher (BHQ1 – Black Hole Quencher 1)

Question 32

How is the exact number of tax gene copies in the sample determined from TaqMan method?

Answer 32

Standard curve must be obtained plotting log10 input copy number on x-axis and cycle threshold on y-axis. Straight line with negative correlation obtained. This can then be used to estimate number of copies of tax gene in DNA present in patient sample based on CT values obtained.