Immuno - Diagnostic Virology Flashcards
(43 cards)
What does HTLV-I stand for?
Human T-cell leukaemia virus type I
What family is the virus part of?
Delta retrovirus family
What is HTLV-I the causative agent of?
Tropical Spastic Paraparesis (TSP)
HTLV-I associated myelopathy (HAM)
adult T-cell leukaemia/lymphoma (ATLL)
other inflammatory diseases such as:
- HTLV-I associated infectious dermatitis
- HTLV-I-associated uveitis (HAU)
What kind of gene does the virus not harbour?
a viral homologue of a cellular proto-oncogene
What protein is the transforming entity ascribed to?
Viral Tax protein
What is the Tax protein and what does it affect?
It is a nuclear phosphoprotein and it can affect cell-cycle progression, cyclic AMP and nuclear factor kB (NF-kB)
Modes of Infection with HTLV-I virus
Cell to cell contact primarily:
- Mother-infant (mainly through breast feeding)
- Sexual contact
- Parenteral transmission (other than the digestive system)
What is the dominant contributor to increase in number of HTLV-I?
Not de novo infection of cells but rather clonal proliferation
Describe the HTLV-I genetic material?
Monopartite, linear, dimeric, ssRNA(+) with a 5’-cap and a poly-A tail
Function of the 5’ cap
Added to the first nucleotide during transcription and it’s a modified Guanine nucleotide. It protects the transcript from being broken down and it also facilitates the attachment of the mRNA to the ribosome.
Role of the poly-A tail
A chain of adenine nucleotides added to mRNA in order to increase its stability and prevent it from being broken down.
What sample is needed for the detection of HTLV-I in patients?
Blood from which peripheral blood mononuclear cells (PBMCs) are isolated using Ficoll gradient centrifugation.
What is the first step of the PCR
Primers are designed to amplify the pol or tax gene of HTLV-I
What does a PCR tube contain?
DNA template containing target, primers, free deoxynucleotides and DNA polymerase
Describe the steps of heating and cooling of the mixture
- Heated to 95 degrees to separate the DNA strands
- Cooled to 55 degrees so primers anneal to ssDNA
- Heated back up to 72 degrees so DNA polymerase starts to synthesise new strands of DNA starting from primers
what are the Tax and Rex proteins in HTLV-I used for?
Rex - positive post-transcriptional regulation
Tax - Viral transcription and oncogenesis
What does the number of infected cells correlate to?
Disease severity
Likelihood of transmission
What is the separation method behind the Western blot?
Separated based on size on polyacrylamide protein gel so the smaller the DNA chain is the further it will go
PVDF membrane used
How do you transfer the bands onto a PVDF membrane from the gel in Western blot?
Using electricity
What is a positive HTLV-I test?
Bands for:
- synthetic peptide - MTA-1
- viral core proteins - p53, p24, p19
- recombinant glycoprotein - gd21
Needs to have all of them otherwise the sample is inconclusive and requires further PCR analyses
What key feature must the primer include?
A free 3’ hydroxyl group because the DNA polymerase extends strand in 5’ to 3’ direction
What needs to be adjusted if the DNA fragment is larger/
Incubation time with temperature changes
How are primer sequences written and what needs to be done to transform the reverse primer?
From 5’ to 3’ end so the reverse primer sequence needs to be written in reverse
What sort of genetic material does PCR require
dsDNA or ssDNA
