It was recently discovered in an Australian wild animal population that a rare f

Question

It was recently discovered in an Australian wild animal population that a rare form of oral/facial cancer was spreading through the population. This contagious cancer has been reducing the animal population. Design an experiment to determine if this contagious cancer is due to a virus or the result of a non-viral mutation. (You must consider that this could be a novel virus that ONLY grows in the wild animal tissues indicated (or of course, it might NOT be a virus at all. That is what you need to find out). And, you must consider that this is a situation where the tumor is contagious. Something is being passed from one animal to another. Also, think about early studies on viruses and cancer.)

Explanation / Answer

Answer:

Transmissible cancers are rare and are spread through somatic cell lineages between individuals of a population, mediated by living cancer cells.

Inorder to identify the causative agent (mutation or virus) of the facial cancer in the these Australian wild animals, we can perform the following experiments:

Experiments for non-viral mutations:

a. Study of tissue histology:

Tissue biopsies and tumor fine needle aspirates sholud be collected either post mortem, or from live animals showing signs of the disease to study the specific features the tumor cells exhibit. These results can be used to compare with previously known cases of transmissible cancers.

b. Cytogenetic study:

Karyotype analysis of the isolated tumor cells can reveal possible chromosomal alterations (aneuploidy) when compared to the wild type karyotype.

c. Microsatellite genotyping:

Microsatellites are repetitive DNA regions that occur throughout the genome, and variations within microsatellites can affect cellular function through mechanisms including promoting alternative splicing, altering protein sequence, and affecting gene regulation

Genomic instability at microsatellite loci is a hallmark of many cancers. Microsatellite-based genotyping pipeline helps to identify microsatellite loci that can distinguish between the germline of individuals diagnosed with facial cancer and healthy animals.

Experiments to identify if its a virus:

Virus isolation in cell cultures, immunofluorescence based assay and molecular techniques to determine nucleic acid, have all been used successfully to detect and identify the viruses.

a. Indirect methods:

The virus (if any) need to be isolated from intact infected cells from diseased animals and studied for CPE (Cytopathic effects). Other methods include studying nucleic acids and antigens for detection.

b. Direct detection:

Isolated viruses from infected cells can be observed directly under the EM (electron microscope) followe by negative staining with uranyl acetate/formate or through Cryo-EM (time consuming) to get the atomic level structure of the virus.

c. Immunofluorescence assay:

The IFA (also known as fluorescent foci assay FFA) is a rapid method (antibody based staining methods) of virus titration that allows the quantification of virus in cell lines, which does not support plaque formation or do not exhibit detectable CPE.

d. Immunoprecipitation: (Requires specific antibody which should be raised in a different animal)

Immunoprecipitation method utilizes specific antigen-antibody interaction and binding of protein A/G to FC fragment of antibody. As most IP performed, protein A/G is first allow to bind to agarose beads, that reacts with mixture of sample solution (antigen) and antibody.

Thus protein A/G on the beads adsorbs the target antigen which can be easily isolated from mixture of proteins in sample solution by low-speed centrifuge. The samples are then analyzed by SDS-PAGE to determine their quantity and molecular weights of proteins, study protein-protein interaction or to monitor protein post-translational modifications.

e. Isolation of viral nucleic acid and sequencing:

The nucleic acid isolated from the viruses can be used for sequencing and data base comparison to identify whether its a novel virus that infected the animal.

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