Diagnostic Virology

Question 1

What are the characteristics of HTLV-1?

Answer 1

• Causes ATL cases
• 1st virus linked to malignancy
• Enveloped virus
• ssRNA reverse transcripted to ssDNA in T cells
• has Tax gene
• linked with oncogenesis
• can cause ATL, HAM, dermatitis, HUS
• breast feeding, sexual contact and blood transmission

Question 2

Which proteins found by the western blot method would show a positive test for HTVL1?

Answer 2

Used to separate specific proteins :

Synthetic peptide:
MTA-1

Viral core proteins:
P53
P24
P19

Recombinant glycoprotein:
gd21

Question 3

Describe the western blot method? ( 4 steps )

Answer 3

1: separation
2: transfer
3: staining - incubate in human serum, wash, incubate with 2ndy AB linked to enzyme, wash, add substrate for enzyme linked to secondary AB
4: visualisation

Question 4

Describe the PCR method?

Answer 4

• Denature at 94 degrees 1 min
• Annealing at 54 degrees (forward and reverse primers) 45s
• Extension at 72 degrees (dNTPs) DNA polymerase
  buffer included. 2min (longer time for longer areas)

Question 5

How to prepare the sample of HTVL1 for PCR?

Answer 5

Require peripheral blood mononuclear cells

Isolate the PBMCs from the blood by centrifuging

Isolate DNA

Question 6

What components make up PBMCs?

Answer 6

Monocytes
Lymphocytes
T cells
B cells
NK- cells

Question 7

What is Sybr Safe dye used for?

Answer 7

Used in electropheresis, seen under blue light

Increases density of sample so goes deep into DNA well

Indicated how far DNA fragments have migrated

Question 8

Describe the Taqman probe method?

Quantitative real time PCR

Answer 8

Taqman dye has:

• Quencher dye
• Reporter dye ( fluorophore ) which becomes separated by TAQ polymerase

When that happens light emitted increases

Used to get primers as Taqman dye is at the tax gene region

Question 9

What is the Cycle threshold?

Answer 9

If the threshold is reached early ( low CT value)

= means high HTLV-1 amount

Plot on standard curve

calculate log10 of tax gene copy number

Question 10

What type of genetic material can be amplified by a standard PCR?

Answer 10

dsDNA

ssDNA

Question 11

HTLV-1 has a single strand RNA genome, but a standard PCR can still be used for diagnosis. What is the reason for this?

Answer 11

During virus replication, viral RNA is reverse transcribed to ssDNA and converted to dsDNA that integrates into the host-cell genome. Viral infection can thus be determined by standard PCR using host cell DNA as template

Question 12

How do you estimate the size of your PCR product on the agarose gel?

Answer 12

Run a DNA ladder alongside your PCR reactions

Question 13

What is the purpose of adding a loading buffer with dye to the PCR sample before loading on an agarose gel? (3)

Answer 13

• Visualise which wells contain samples
• Visualise how far the gel has migrated
• Makes samples sink into wells

Question 14

What is the typical number of repeat cycles for the three steps in a CR reaction?

Answer 14

30-40x

Question 15

HTLV replication cycle?

Answer 15

Infects T cells / T helper cells

HTLV enters T cell
ssRNA released into host cytosol
ssRNA reverse transcribed by RT-enzyme to ssDNA
ddDNA converted to dsDNA
dsDNA can enter nucleus and integrates into host genome
viral genome can replicate as part of the host chromosome

• number of T cells with HTLV-1 DNA can correlate with disease severity and likelihood of transmitting virus