Diagnostic Virology

Question 1

What are the PCR based methods?

Answer 1

Standard PCR

qRT-PCR (quantitative real time) (more of a diagnostic tool)

Question 2

What is HTLV-1?

Answer 2

Human T cell leukaemia virus type 1

A virus that can cause cancer (ATL adult T cell leukaemia)

15-20 million people infected worldwide (but endemic in some regions, like japan and Africa)

About 5% of infected individuals will develop ATL

Transmitted via mother to infant, sexual contact, blood to blood contact

It also causes other diseases: adult T cell leukaemia, HTLV-1 associated myelopathy, tropical spastic paraparesis

Question 3

What type of virus is HTLV-1?

Answer 3

A single stranded enveloped RNA virus

Enveloped

ss RNA

The virus encodes many proteins and enzymes that help the virus to invade cells, and for assembly of the virus. It also contains reverse transcriptase.

As it can cancer, it encodes a viral protein called TAX, this has an important role in viral transcription and also ONCOGENESIS

Question 4

What is the replication cycle of HTLV-1?

Answer 4

Preferentially infects T cells

Enters T cells

ssRNA released into host cytosol

ssRNA reverse transcribed to ssDNA

ssDNA converted to dsDNA

dsDNA enters nucleus and integrates into host genome

Viral genome can replicate as part of the host chromosome

It is assumed that the number of infected T cells correlated with: disease severity, likelihood of transmitting the virus

Question 5

Give a brief overview of PCR?

Answer 5

The abbreviation PCR stands for polymerase chain reaction. A standard PCR reaction uses DNA as the starting material with the aim of amplifying a specific region. A typical PCR reaction will have 30 to 40 cycles of three main steps. The PCR product is visualised on an agarose gel using a stain that intercalates into the DNA and fluoreces under a specific light source

Question 6

What are the three steps in PCR?

Answer 6

Denaturing DNA (95 c)

Primer annealing (55 C)

Extending DNA strands (72 C)

Question 7

What is the serological method of diagnostics?

Answer 7

Western blot

Question 8

How does the western blot work?

Answer 8

When people are infected with HTLV-1 they produce antibodies against its viral proteins. WB tests for these

Question 9

What are the steps in Wedtern blot?

Answer 9

Separation: different viral proteins derived from in vitro propagated cultures are separated based on their size on pilyacrymide protein gel.

Transfer: proteins are transferred using an electric transfer system onto a PVDF membrane and the viral proteins will stick to this. So the protein bands will be on this membrane (but not yet visible)

Staining: membrane is incubated with human serum (primary AB). The membrane is washed. It is then incubated with a secondary AB that is linked to an enzyme. Then washed. Substrate is added for the enzyme

Visualisation: generation of a precipitate, or a luminescence signal

Question 10

What constitutes a positive result for HTLV-1 on a western blot?

Answer 10

Visualisation of:

Synthetic peptide: MTA-1

Viral core proteins: p53, p24, p19

Recombinant glycoprotein: gd21

Question 11

What are the steps of PCR?

Answer 11

Denaturaton - 1 minute 95C

Annealing - 45 seconds 54C. Allows the primers (forward and reverse) to anneal to the DNA

Extension - 2 mins 72C. Only dNTPs

Repeated 30-40 times

Generates millions of copies of specific DNA regions

Question 12

What are the primers designed to amplify the HTLV-1 tax gene?

Answer 12

Primer 1: HL43 (same sequence as the top strand)

Primer 2: HL44 (bottom strand)

This amplifies a region around 300 base pairs long around the tax gene

Primers must be written in the 5’ to 3’ direction

Question 13

What are the 5 key components in PCR?

Answer 13

1. DNA template
2. Primers
3. DNA polymerase (often Taq polymerase as it works at high temperatures)
4. dNTPs (deoxygenated nucleoside triphosphates)
5. Reaction buffer

Question 14

How is a PCR sample for HTLV-1 prepared?

Answer 14

Peripheral blood sample is taken

PBMCs (peripheral blood mononuclear cells) are isolated (T, B and NK)
This is done using a separation medium and centrifugation

Isolate DNA from PBMC

Question 15

How long does PCR take?

Answer 15

Around 2 hours

Question 16

How are the PCR results analysed?

Answer 16

DNA gel electrophoresis

DNA is -ve charged

Question 17

Why is qRT-PCR useful?

Answer 17

Provides information on the amount of viral DNA is a sample

Helpful in providing the severity and likelihood of transmission of the disease

Question 18

How are qRT-PCR results analysed?

Answer 18

There is a real-time increase in fluorescence of the sample, this coincides with the amount of Viral DNA

For a positive result, it must pass a threshold, it can be quantitate on how fast it reaches it

The two methods are fluorescence dye based (using SYBR green). And DNA probe based (TaqMan method)

Question 19

How does the TaqMan method work?

Answer 19

Along with the two primers, a TaqMan probe is added. This has a reporter dye at the 5’ end and a quencher at the 3’ end. At the beginning the reporter is too close to the quencher so is only lightly fluorescent

As the PCR progresses the probe is degraded and the reporter and quencher get further apart so the reporter fluorescence is brighter

This is proportional to the amount of desired DNA at a certain point in the reaction