Make me a virus: goodbye MTAs

Material transfer agreements - MTAs - can be infuriating. They will almost certainly exist forever, in some form or other. They say knowledge is power, so it's no surprise that people/institutions want to hold on to anything which may offer them a competitive advantage. It clearly makes sense. I've always been curious though about the amount of metaphorical wheel re-inventing which goes on across the world purely due to the fact that MTAs get in the way; considering the time and money it seems crazy.

Problems arise in the form of time delays and restrictions on what can be done with the materials, whatever they may be. Swapping the simplest of items can take what seems like forever to change hands, and even when it does eventually happen there seems to be a lot of hand tying involved.

But, the concept of virus rescue/reverse genetics means that, for a lot of viruses, the days of the MTA may be limited. Classically, cDNA clones of virus genomes were generated by cloning bits of a virus you already have. This could be extremely laborious and time consuming and meant that you were required to already possess the virus. Now though, gene synthesis is becoming cheaper and cheaper and that means, assuming the sequence is known, that infectious clones can be ordered online by labs with smaller and smaller budgets. Our lab did this recently with Schmallenberg virus. In order to get working as quickly as possible, and allow us to work on whatever aspect we wanted, we ordered the clones online and received them a few weeks later. Not long after, we had Schmallenberg virus, with no restrictions on what it could be used for. 

The virus rescue strategy for Schmallenberg virus; the pUC plasmids were simply ordered online, from Varela et al 2013

For some groups of viruses, rescue systems haven't been established, for example the rotaviruses and some other members of the Reoviridae. For the majority though, there are established systems and clones, including many viruses which are under strict restrictions in laboratories. Accession number AF086833 is the full genome sequence of Ebola Zaire 1976, the original strain associated with horrifying levels of mortality. Similarly, accession number AJ539141 is the sequence of the Foot and Mouth Disease Virus from the UK in 2001 which was estimated to cost the UK government £8bn ($16bn). I'm not aware of questions being asked when ordering such sequences to be synthesised. I thought about experimenting and doing some sort of dummy orders of sequences such as these online to see whether there were any blocks in the way. In theory there should be questions from the company regarding what it is and where it's being sent once it's made. All I know for now is that we were essentially able to order a newly emerged virus online. But arguably the most positive aspect of this, is that it hasn't taken months of paperwork to formulate a restrictive MTA!

Man flu or real flu? - DIY Diagostics

Someone in the lab emailed me today saying that she wouldn't be coming in as she was ill, and that she hoped it was a cold instead of flu. If you're in the same situation, wouldn't it be good if you could test yourself to find out? Well, you can, using a simple dip-stick style test which you can buy online.

That's great and it's easy to envisage how useful this can be but, for me, the interesting page is the one describing the test specifications - how good is the test? The most notable values are the positive (PPV) and negative (NPV) predictive values, which represent the accuracy of a result; PPV represents the proportion of positive results that are truly positive and likewise the NPV is the proportion of test negatives that are truly negative. A PPV of 62% for a nasal swab doesn't seem to be particularly high. As these values very much rely upon seroprevalence it's hard to really tell how useful this is. 

These assays do work, and simple lateral flow devices worked really well during the foot and mouth outbreak in 2007 in the UK. These assays currently use antibodies and, without a good antibody, assay sensitivity can be poor. In lab settings, molecular assays based upon detecting viral nucleic acid are generally more sensitive, in particular real-time RT-PCR. Sadly, molecular methods also require much more expensive equipment, making it more difficult to convert a lab procedure into a 'point-of-care' test. Perhaps unsurprisingly, the initial major steps were taken by the military as a response to the threat of biowarfare. Now however, more commercial machines have made their way to the market, for example machines made by Smiths detection.

Smiths detection PCR machine: essentially a robot for nucleic acid extraction and a real-time PCR machine packaged in a (very heavy and expensive) briefcase.

Field based diagnosis is as close now as it has ever been. Ultimately though, the problems of hardware still exist. The machines are expensive; essentially they are the same machines that a lab has packaged into a briefcase. I think molecular assays in the field will only really take off once isothermal assays, such as loop-mediated isothermal amplification (LAMP), are more widespread. As their name suggests, isothermal assays are performed at a single temperature, so all that is required is a simple waterbath set to a particular temperature, as opposed to a block of metal heating up and cooling down. 

Loop mediated isothermal amplification: modified primers loop back to prime the alternative strand. A strand displacing enzyme abrogates the need for variable temperatures.

Detection is the other problem; fluorescence as a read-out is expensive to a) achieve and b) detect, therefore the gold standard for field-based detection of amplification (PCR, LAMP etc.) is likely to be dipsticks/lateral flow devices as these are easy, more foolproof and provide a clear answer. For now, extraction of the nucleic acid probably remains the biggest obstacle. Another benefit of assays based around isothermal amplification and simple methods of detection is that it allows the use of molecular assays with minimal equipment, meaning that such assays can then be used in resource poor countries. 

It is more than likely that the future will be much more sophisticated with much greater scope for what can be detected, although the requirements for simplicity are unlikely to change much. For now, imagine settings such as clinics where patients suspected of hepatitis C can get an instant result. LAMP assays are already available for many of the pathogens that would be on a list of desirable tests; I suspect it won't be long before point of care testing becomes even more widespread than it already is.