Season 4, Episode 9: Unbottled

A group of terrorists holds the NorBAC team hostage in their own lab, and forces them to each carry out part of an experiment in isolation from the others. By interpreting subtle signals from their coworkers, Rachel and David figure out that they are mutating the Edmonston Measles virus into a highly infectious and deadly strain. David instructs Bob to sabotage his part of the experiment to avoid adding a crucial - and very dangerous - mutation.

Image provided courtesy of Shaftesbury Films.

A terrorist prepares to test a new virus he forced NorBAC to make…on Rachel!

IS IT POSSIBLE TO MUTATE THE MEASLES VIRUS?

What is the Edmonston measles virus?

Measles is a contagious viral disease. The Edmonston strain of the virus was first isolated in 1954, and has since been used in most laboratory studies of the measles virus, including in the development of vaccines.

The measles virus is an RNA virus, which means that unlike most organisms its genetic material is not DNA (deoxyribonucleic acid, a two-stranded molecule) but RNA (ribonucleic acid, a single-stranded molecule). Cells that use DNA to store their genetic information (such as human cells) convert DNA to (messenger-)RNA before producing the corresponding proteins. In those cases, RNA is an intermediate product; however, in RNA viruses like the measles virus, it is the only genetic material, encoding, among other things, the instructions to build the virus' protein "shell". Like other viruses, measles viruses need a host cell to carry out these and other genetic instructions and to make more virus particles.

What are Vero cells?

Rachel is asked to grow Vero cells. These cells, originally isolated from a monkey kidney, are commonly used to screen toxins or to grow viruses. For example, they are used in measles research to develop vaccines. Vero cells can be made to produce measles virus either by infecting them with original virus, or by inserting measles-like DNA into the cells where it can then be transcribed to RNA that is similar to the viral RNA.

DNA copies of measles RNA can be made in the lab using an enzyme called reverse transcriptase. It's also possible to mutate this newly made DNA before introducing it to the cell, so that the cells will use the modified genetic instructions to produce an altered virus. That's what the NorBAC team is being forced to do in this episode.

How can PCR primers be used to mutate DNA?

PCR stands for polymerase chain reaction. It's a technique used to rapidly make many copies of a short fragment of DNA. PCR works by repeatedly heating and cooling a DNA sample. When it's heated, the two DNA strands separate. An enzyme called polymerase can then synthesize new strands of DNA on each individual strand, producing two new double stranded fragments. The cycle is repeated several more times, each time doubling the number of DNA fragments. To mark the beginning and end of the region that needs to be copied, a very short DNA fragment of about 20 nucleotides is needed as starting point for the enzyme. These short fragments are called "primers".

The primers need to match the region of the DNA at the start and end of the region to be copied, so they can bind at the correct spot. However, primers can still bind if one nucleotide is changed, or mutated. If you carry out PCR with primers containing a mutation, the resultant DNA copies will also contain this mutation. These copied and mutated fragments can then be pieced back into the full length DNA by other molecular biology techniques.

In this episode, Mayko was instructed to compare the sequence of normal measles with another strain, find the locations at which they were different, and design primer pairs that could be used to mutate regular measles into the dangerous strain. Bob was then given four sets of primer pairs and asked to make a fifth to make a total of five new mutations to change normal Edmonston measles into a form that would grow extra fast, would not be recognized by the immune system of people vaccinated for regular measles, and would be extra virulent to make it more likely to kill whoever got infected. David instructed Bob to secretly use one normal pair of primers (without mutations) to avoid introducing the most dangerous mutation, the one that would make the virus extra virulent.

How long would all this take?

Comparing two genetic sequences and designing the primers needed to mutate one into the other can be done quite fast with the right software. Mayko got 15 minutes to do this, and she could probably have finished it in that time.

Isolating RNA and producing DNA from it takes about 2 to 3 hours.

Vero cells grown in culture double in number about once per day. It would take several days to grow enough cells to work with. Rachel was given a protocol to speed up the growth rate, but such a protocol does not exist.

Synthesizing primers for PCR is usually outsourced to specialized companies or facilities, but even if Bob was able to do this himself in the lab it would take another few hours. Carrying out PCR to introduce one mutation takes about three hours, but cloning the mutated fragment back into a longer piece of DNA takes another day. Bob says it would take five days to make five mutations, and that's about right. There is currently no method known to introduce five specific mutations at the same time.

It takes another day or so to check the final DNA product and make sure all the mutations have been correctly introduced.
Finally, it would take 2 to 3 days for Vero cells to produce virus particles from the mutated DNA.

So?

Yes, it's possible to mutate viral genetic material and use that to instruct cells to produce a mutated virus, but this would take more than a week and can't be done in one night.

DID YOU KNOW?

At the beginning of the episode, David reveals that the monkeys they found in Roth's lab all had high levels of methoxytyramine. Methoxytyramine is a byproduct of dopamine, and when it's found in the blood in high levels it indicates that high amounts of dopamine were present. Dopamine is a neurotransmitter that has many different functions in the brain: For example, it influences motor control, mood, cognition, and learning. Excess dopamine is associated with psychotic disorders, which may explain the violent actions of the monkeys in Roth's lab.

-- Eva Amsen

Want to read and learn more?

Read about measles infection and symptoms:
http://www.phac-aspc.gc.ca/im/vpd-mev/measles-eng.php

Learn how PCR works through this interactive animation:
http://www.dnalc.org/ddnalc/resources/pcr.html

Learn more about dopamine:
http://thebrain.mcgill.ca/flash/d/d_03/d_03_m/d_03_m_que/d_03_m_que.html