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    Hi,

    I have been given the following question as an assignment but I am not sure what exactly the results mean, can anyone help or offer ideas?:

    You have been given brain biopsies from 5 patients with suspected measles inclusion body encephalitis (MIBE).

    cDNA samples have been prepared from these. You wish to determine whether the samples are positive for measles virus and also whether mutations may have occurred in either the fusion (F) or matrix (M) genes from the virus.

    Primers designed to the mRNA sequence of β actin (across two exons), to the virus nucelocapsid (N) gene, wild type measles virus F gene (cytoplasmic tail region) and the wild type M gene sequence (known region of hypermutation) have been supplied. You carry out single PCR reactions for each set of primers. Results shown below.

    Q1. What are your conclusions with regard to these patients having MIBE and whether mutations have occurred? How would you confirm if mutations have occurred?

    Results:
    Patient 1
    · Nucleocapsid gene: +
    · Fusion gene: +
    · Matrix gene: +
    · Beta-actin: +
    I'm guessing this patient has MIBE with no mutations in the M or F genes.

    Patient 2

    · Nucleocapsid gene: +
    · Fusion gene: +
    · Matrix gene: -
    · Beta-actin: +
    This patient has MIBE with a mutation in the M gene?

    Patient 3

    · Nucleocapsid gene: -
    · Fusion gene: -
    · Matrix gene: -
    · Beta-actin: -
    The beta-actin is being used as a control for RNA extraction right? Does this mean this extraction failed?

    Patient 4

    · Nucleocapsid gene: +
    · Fusion gene: -
    · Matrix gene: +
    · Beta-actin: +
    This patient has MIBE with a mutation in the F gene?

    Patient 5

    · Nucleocapsid gene: -
    · Fusion gene: -
    · Matrix gene: -
    · Beta-actin: +
    This patient has no MIBE?

    Q2. How do I confirm if mutations have occurred? – Any ideas? Really struggling with this.


    -----------------------------------
    In addition, PCR was carried out on the cDNA derived from each brain sample for a number of other viruses. A product was amplified from one of the brain samples and required confirmation by sequencing. The following sequence was obtained:-

    cactccacgc aatttcgcgg tatacccgcc gccattggat cgagtggggc cctaaagaag
    ccctacacgt cctcatcgac ccaagcccgg gcctgctccg cgaggtcgct cgcgttgagc

    Q3. What is the virus and which brain is this likely to have been found in and why?
    I ran this sequence through BLAST and it showed it as a Rubella virus strain, but I'm not sure which brain this is likely to have been found in – any ideas?
    (I was thinking that if you cannot have measles and rubella infection at the same time then it must be Patient 5 but I can’t find anything to back this up. Also I thought that maybe Rubella degraded Beta actin and it is patient 3 but there's no info about this.)

    Thanks for your help!
    Sandy
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    You've got the first question right but Patient 3 has Rubella so the extraction hasn't failed. Suggest using another housekeeping gene like GAPDH or 18S ribosomal RNA (look them up) for this sample.

    Rubella does disrupt actin filament formation. Check out this reference and scroll down to cytoskeletal changes.

    To confirm mutations you'd probably have to sequence the genes.
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    Thanks for your reply.

    I had thought that Patient 3 may have Rubella initially but there is very little information to support that the Rubella virus disrupts actin filament formation. There are however, a lot of studies published were they have used beta-actin as a loading control in detection of the Rubella virus which is why I chose patient 5 to possibly have the Rubella virus as MIBE and progressive rubella panencephalitis have similar symptoms??
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    If you look up the reference I gave you there is some evidence that rubella virus disrupts actin filament formation. That should lead you to other references.

    Yes beta actin is used as a loading control but there are other genes you can use. They're called housekeeping genes which I've also talked about in my post.


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