A zero time point is essential

The recent release of grant materials from EcoHealth Alliance pertaining to their research with the Wuhan Institute of Virology has been used to demonstrate that gain of function research was funded by the NIH. This conclusion is correct, although not all the experiments done would fall into this category.

One of the figures from the released grant materials shows the results of two experiments using recombinant SARS-like coronaviruses that were constructed in the laboratory. As I described previously, after the SARS-CoV pandemic of 2003, wildlife sampling efforts in China revealed many SARS-like coronaviruses in bats. To assess the potential of these viruses for infecting humans, their spike protein encoding genes were substituted into the SARS-like CoV WIV1.

The reproduction of these recombinant viruses were assessed in two experimental systems. ACE2 transgenic mice were inoculated with the recombinant viruses to assess their pathogenic potential, by measuring weight loss. The results (figure below, left panel) show that only one recombinant virus, carrying the spike gene from SHC014, caused more rapid weight loss than did WIV-1. Hence, SCH014 has a gain of function. However the recombinant virus 4231 caused the same rate of weight loss as WIV-1 and therefore does not have a gain of function. Recombinant virus WIV-16 caused no weight loss – its curve resembles that of uninfected mice – and therefore this virus has a loss of function.

The second experiment is not, in my opinion, proof that any of these recombinant viruses have gained a new function. The experiment was to infect ACE2 transgenic mice with WIV1 and the three recombinant viruses, and then remove lung tissue at different times after infection and determine viral RNA loads. The first problem with this experiment is that RT-PCR was used to measure viral RNA loads, which does not directly measure viral infectivity. Therefore it cannot be concluded that any of the viruses reproduced to higher levels than WIV1. More problematic is that there is no time zero – the measurement of lung tissues begins at day 2. Consequently, one cannot reliably compare viral RNA loads of any of the recombinant viruses with WIV1. When doing kinetic experiments, a time zero is ALWAYS needed, so you can see how much virus was actually instilled in the mice. It gives you a baseline from which to compare subsequent time points. Even though the authors state the amount of virus added to the mice, they do not know how much was actually added. Operator error, binding to tissues, and a host of other factors can interfere early in the infection. For example, it is possible that the viral RNA loads on day 0 were the same as in subsequent time points, which showed little change over the course of the experiment. One cannot conclude from this experiment anything about the viral RNA loads without a time zero. No viruses gained any function in this experiment.

Unfortunately, many expert virologists do not have a problem with the absence of time zero. When I spoke to a reporter about these results, and told her my interpretation, she told me other ‘experts’ did not agree with me. You will find many examples of published time courses without a time zero – they are worthless.

Why the difference between virology experts? I was trained to always have a time zero; the others were not. They continue to get away with it so they believe it is correct. It is not proper research practice and no amount of arguing will make it right.

3 thoughts on “A zero time point is essential”

  1. Brian Hanley, PhD

    The primary point about monitoring and gain of function research is that this is how we know what is going on, and what is likely to emerge. The people screaming about it, are essentially saying, “Noooooo! Don’t look over there! It’s scaring me!”

    The people amplifying that head-in-the-sand fear are doing it for political gains. They are repeating it because they think that “their side” will get elected. That is why Mike Pompeo, Fox News, and the other usual suspects are yelling about this. It’s a wedge that can get them votes – they think. But the reality of what they are doing is making the USA less safe.

    Well, technically, it is almost certainly paid internet publicity consultants who are doing this. I hired one of those firms a few years ago for a book. It was a disaster because their idea was to create the greatest noise, regardless of content. So they played on fears, and contributed to the problem I was opposing in my book. Politics, which I have some experience in from working on a friend’s congressional campaign, is filled with such people. They don’t look at the world and see what is true or right. They look at the world and see, “What triggers can I exploit?”

  2. All studies need a baseline/t0. That’s basic science. I’m horrified at any scientist that shrugs that off. Sloppy science.

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