Sunny writes:

Here is a link to the pointer I think Kathy was talking about in TWIV episode 494. It definitely is a great tool to have when presenting. Keep on TWIXing.

https://www.bestbuy.com/site/logitech-spotlight-presentation-remote-slate/5749105.p?skuId=5749105

Regards,

Sunny

Steve writes:

Hi —

Here’s an article:

http://cellculturedish.com/2015/02/developing-ebola-vaccine-securing-cold-chain-logistics-plan/

Dry ice is certainly the method of choice. It’s easy to handle safely, the procedures for air shipment are well defined, it’s easy to produce on demand from ambient temperature compressed gas with little machinery, it doesn’t require fragile or expensive containers (dewars), and it’s not too cold (as is liquid nitrogen). And it won’t carbonate vaccine (or bananas) that are in sealed containers. Those huge vaccine shipment boxes you see pictures of are probably mostly dry ice and (very important!) terrific insulation.

Not to minimize the many practical difficulties likely to be encountered in rural DRC, but maintaining the cold chain is just one among many.

Wink writes:

Dear TWIV Professors,

Thanks for the great Ebola update! As is customary, you began with a conversation about epidemiology. Missing from your discussion (and all other modern reviews of Ebola) is the originally reported mechanism for viral amplification in a human community (one reference below). This was by re-use of needles in medical facilities. This practice may be still going on; there is no data to the contrary. It may be that the subject doesn’t come up because it is too painful or embarrassing to think about, or (hopefully) the practice is merely historical. However, as this was Peter Piot’s initial finding, I think it at least deserves mention.

Wink Weinberg

Atlanta

[Bull World Health Organ. 1978;56(2):271-93. Ebola haemorrhagic fever in Zaire, 1976.]

Ted writes:

Hello TWIVeratti!

I wanted to follow up on last week’s discussion of the paper from Heinz Feldmann’s paper. I know Jeremy provided email input that you included in the show, but it seems he mixed up Mayinga and Makona in his response and I feel the need to clarify.

My main criticism of the paper has to do with the viral isolates that were used in the study, which were generated via tissue culture passage. Great work from the labs of John Connor (https://www.ncbi.nlm.nih.gov/pubmed/28539437)  and Jim Cunningham (https://www.ncbi.nlm.nih.gov/pubmed/28238624) has demonstrated that passage in culture often results in mutations in the fusion loop of Ebola GP. Perturbation of the fusion loop appears to have a similar biochemical consequence as the A82V: increased propensity for the GP to initiate the series of conformational changes leading to membrane fusion. Perturbing the fusion loop appears to provide a greater membrane fusion enhancement than A82V. This is important because all of the early (A82) isolates used in this study contain a mutation in their fusion loop. While it is unclear what the genotype of the other viral stocks are, if they match the NCBI accession # they harbor A82V but no fusion loop alterations. Thus, it is possible that the early isolates were observed to have enhanced virulence because their fusion loop mutations enhance fusion more than A82V does.

Overall, this raises an important point of using isolates to test the effects of specific mutations. Using this approach, it is difficult to control for the genetic backbone of the viruses being compared. Engineering the mutation directly into a relevant virus backbone using reverse genetics (and ensuring sequence fidelity of the recovered viral stocks) is a better approach for direct comparisons.

This paper also overlooks the epidemiological data. A82V was derived early during the epidemic, became widespread and nearly displaced A82, and was associated with increased mortality and higher viremia (the former statistically significant, the latter not). This could be due to genetic drift and founder effects, but all other published evidence (epidemiology, location of the mutation at the receptor binding interface, and biochemical changes to GP function) indicates selection. I should note that this evidence includes in vitro replication studies with recombinant Ebola virus (http://jvi.asm.org/content/early/2016/11/03/JVI.01913-16.abstract).

I also have a quibble with the animal models used. I believe that the mouse model is largely irrelevant for testing the effects of A82V, as no effect of A82V was observed in murine cells. Additionally, while the gold standard proxy for human infection, the macaque Ebola model is a classic example of “monkeys exaggerate”. This pathogenicity model results in 100% macaque fatality with a time to death of about 7 days. In contrast the case fatality rate in humans is 40-80% and the time to death is around 20 days. Because of these differences in comparison to human infection, it is possible that this pathogenesis model may not be suitable to detect subtle effects on virulence, but in my view this is all the more reason that very carefully controlled experimentation be done to address the question. Lastly, it is possible that mutations that arose during the Makona outbreak facilitate transmission, which was not assessed in this study (and may be prohibitively expensive to ever test).

This paper, however, does suggest that further investigation is required. We couldn’t agree more! We would like to see the isolates used in this paper should be compared using similar cell culture systems as performed by Stephan Becker’s group to see if the robust in vitro phenotype can be recapitulated. Not to beat a dead horse, but a reverse genetics approach with non-human primates or other relevant pathogenesis model is needed to directly assess the role of A82V in vivo.

Finally, I feel the need to feed Vincent’s pedantism. The title of the paper states that Makona mutations “do not alter pathogenicity”. Pathogenicity is a black and white characteristic. With a case fatality rate of ~40% it is clear that Ebola Makona is pathogenic. Virulence is rather what the authors were testing in the study.

Keep up the great work!

Ted Diehl
Sorry for the verbosity!

William Diehl

Postdoctoral Fellow

Program in Molecular Medicine

UMass Medical School

James writes:

Hi TWiV team!

On TWiV 451 you had a conversation regarding whether or not “all science is wrong”. During this discussion, Dr. Condit mentioned the Cochrane Collaboration which performs meta-analysis of published studies. This reminded me of a resource aimed at performing meta-analysis of RNA-seq data. It has a great name . . . “The Lair”. Here’s the link to the paper (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5131447/) and the website (http://pachterlab.github.io/lair/). I should mention that as a scientist myself, I agree that “not all science is wrong”, and I think meta-analysis tools like this empower the scientific community to “double check” findings, especially for “big data” that is hard to look at without proper training. If nothing else, the homepage of the website has a cute picture of a bear on it 🙂

On a more personal note, I wanted to say a quick “Hello” to Dr. Condit, who was on my thesis committee at the University of Florida for about two years before his retirement. I’ll never forget an AMAZING talk he gave to incoming graduate students on the “do’s/don’ts” of being a scientist. After that, I was desperate to soak up as much of his wisdom as possible and was thrilled Dr. Condit agreed to be on my committee despite being at the end of his tenure at UF.

Also, to Dr. Condit and the rest of the TWiV gang, I want to thank you for inspiring me to remain passionate about doing and loving science. I recently started my post-doc at the Fred Hutch in Seattle, and there have been quite a few growing pains (read: unfunded fellowship applications!!) which have occasionally put a damper on my belief of “I have what it takes”. That said, I recently picked TWiV back up after almost a year-long hiatus and it has been a lifesaver! Really, just hearing one of you say “Isn’t that cool?!?!” in response to some finding in a paper you discuss makes me believe that I never have to lose that same feeling of amazement. Anyways, just a big THANKS! to you all. I’m excited to catch back up with TWiV and haven’t been wasting any time (four episodes today alone!).

Thanks again to you all!

James Thomas

ps – Included in the growing pains mentioned above are all of this rain and lack of sun!! Phew, we’re not in Florida anymore!

Jennie writes:

Hi TWiVsters,

In episode 493, Rich shared his experience of an FDA Advisory Committee meeting. After mentioning the committee included a patient representative, Rich said he wondered about the process for selecting that rep. I participate in FDA’s Patient Representative Program, and can share some info.

FDA was an early adopter of incorporating patient experiences into decision making, largely as a result of HIV/AIDS activism. Today, the Program trains and provides representatives for FDA Advisory Committees and other review decision assignments. You can read more about the program here:

https://www.fda.gov/ForPatients/About/ucm412709.htm

Individuals can apply to the Program, and FDA looks for several qualifications (as detailed at the link above). Representatives are compensated for their time, and must complete regular training as well as financial disclosures. When preparing to serve on Advisory Committees, representatives (like other voting members of these committees) must undergo a detailed conflict of interest clearance process to ensure they have no financial conflicts.

Representatives are appointed for multi year terms, with no guarantee they will have the opportunity to serve on committees. Our experiences of specific diseases may never be relevant to drug or device reviews. Those with experience related to diseases that come up in reviews more frequently (some cancers come to mind) may have more opportunities.

In my experience, the FDA takes patient perspectives very seriously. The training is excellent, and so are FDA staff. Participating in the program has been a very positive experience for me personally.

Cheers,

Jennie

Chris writes:

Dear TWiV Team,

I was intrigued by some of your comments about the mode of action of Tecovirimat. It sounded to me that you thought that an inhibitor of ‘spreading’ of viruses from one host cell to another was unusual and wasn’t likely to be particularly effective, because it wouldn’t affect the production of virus particles within an infected cell. This is not so unusual however, after all, fusion inhibitors of HIV target the entry of virus to a new host cell, the protease inhibitors of HIV and HCV target the maturation of viruses, leading to the production of non-infectious virus, and the neuraminidase inhibitors of influenza viruses target the release of progeny viruses from infected cells. Hence, inhibitors of fusion, protease and neuraminidase inhibit the spread of virus to new host cells. Provided one stage of the replication cycle is targeted, that should inhibit overall production of virus. Targeting more than one stage of the replication cycle is likely to  have advantages of course, particularly in terms of inhibiting the development of drug resistance. I assume that, since poxviruses are double-stranded DNA viruses, mutations are likely to arise much less frequently than a virus such as HIV, where the use of multiple drugs, targeting multiple stages of the replication cycle is standard practice.

Thanks for your informative and entertaining podcasts.

Best wishes.

Chris

Christopher Ring MSc PhD FIBMS FHEA

Senior Lecturer in Microbiology.

First Year Tutor for BSc Biomedical Science.

Programme Leader for MSc Biomedical Science (Medical Microbiology) and the BSc Applied Biomedical Science programmes. Module Leader for specialist postgraduate Medical Microbiology and postgraduate Biomedical Science Department of Natural Sciences,

Faculty of Science & Technology,

Middlesex University,

The Burroughs,

Hendon,

London.