Firstly, we would like to thank you for the segment on the current debate as to the mechanism of how viruses can become attenuated through wholescale genome modification. We really appreciated the comments made during the discussion – and enjoyed listening to the viewpoints of those on the panel.
At one point during the discussion it was suggested RNA structure could play a role in the mechanism of attenuation. This had occurred to us, too, so we included a permuted control in our studies (Atkinson et al., 2014; http://www.ncbi.nlm.nih.gov/pu… and eLife paper). Here, the peptide sequence was maintained, but we randomised the order of codon usage – whilst preserving CpG/UpA dinucleotide frequencies. Permuting codon usage in this manner would destroy any undetected RNA structures within these regions of the viral sequence. The replication kinetics of this mutant virus were identical to wild-type, supporting the notion that in this case RNA secondary structure is not involved in virus attenuation.
A key point was made about the use of cell-free translation systems to analyse protein synthesis in the absence of any cellular innate immunity-based translational control mechanisms. We, and Burns et al.,(2006; http://www.ncbi.nlm.nih.gov/pu…, carried out such experiments and did not observe any differences in protein synthesis between wild-type and the different mutant viruses.
Whilst the mechanism of attenuation remains unknown, data generated by Atkinson et al., (2014) demonstrated that high and low CpG/UpA mutated genomes were not sensitive to the cellular IFN response but were, however, sensitive to the addition of the kinase inhibitor C16 (thought to be PKR specific; Jammi et al., 2003): the replication phenotype reversed following inhibitor treatment. With regards to the Dengue paper under discussion, (i) the results in BHK cells and (ii) the effects of the Jak inhibitor 1 upon the attenuated phenotype of both the hmin and NS3hmin viruses (in accord with the C16 Atkinson data), suggest attenuation occurs through an unknown component of a cellular stress response pathway – and does not support the hypothesis of attenuation arising from a defect in translation.
Again, thank you for highlighting and discussing this topic.
We think you all agreed it is certainly very interesting and we hope your listeners enjoy the podcast as much as we did.
Fiona Tulloch, Peter Simmonds, David Evans and Martin Ryan.
I can’t explain my excitement while listening at TWIV 351. Just yesterday I met for dinner a wonderful Physicist, Rudi Podgornik, who in collaboration with his former postdoc, Luca Tubiana, have come up with a remarkable idea. In the world of physical virology, i.e. physicists and chemists studying viruses, there is a very hot topic which is of great interest to myself; how to characterize the physical properties of viral ssRNA. This means to find metrics to characterize the overall size (3D size), the degree of branching, the number of stem loops, etc., of viral (genomic) RNAs and compare to compare them with other kinds of RNAS. This quest has led to the observation by a few theoretical physicists and physical chemists that viral RNA molecules of icosahedral viruses are more compact than “random” RNAs with the exact number of nucleotides and base composition. These random RNAs are nothing more than viral RNA sequences that have been shuffled enough. Aron Yoffe et al PNAS 2008 put this idea forward.
Since then, both experimental and theoretical, physical virologists have been trying to understand what this means. In the light of this quest Luca Tubiana and Rudolf (Rudi) Podgornik came with a beautiful paper in which they took Aron Yoffe’s observation and ask the following question: how many synonymous mutations are needed to make a viral RNA look like a “random” RNA? In short, what they did was to mutate the genome of a huge amount of viruses (all this work is theoretical) by following the next rules: all mutations are only in the ORF, they have to be synonymous, they cannot be done in splice donor or acceptor bases and they cannot change the overall dinucleotide composition of the genome. With these restrictions they found that mutating 5% of the genomic viral RNA is enough to completely disrupt the secondary structure of these RNAs, such that now they look like “random” RNAs. We and others have been trying to understand how the structure of the viral genomic RNA affects the packaging efficiency and selectivity of ssRNA viruses. Hence, this observation turned out to be an exciting one.
This paper, which I think is a beautiful one, opens the following question; is there a third layer of information encoded underneath the genomic code? I know that you might not be aware of the literature in packaging signals for ssRNA viruses but for sure there is a great debate on the nature of viral RNA packaging selectivity. It might be as well that for some viruses packaging signals are not the way to go to ensure packaging selectivity, but it might be that the secondary and tertiary structure of the viral RNA dictates the specific interactions needed for such selective packaging. This is part of my work as a postdoc at NCI.
The data of the papers you discussed in TWIV 351 go along with Luca Tubiana’s idea; I don’t want to get in the middle of the discussion between Simmonds and Wimmer but both experimental evidence are in agreement with Luca’s observations. In other words, Luca Tubiana proposed that by changing the third base in the codons without changing the amino acids they code for, one can completely disrupt the secondary and tertiary structure of the RNA. If by changing the dinucleotide frequencies you change the structure of the RNA and then the strength of the interactions between the viral RNA and its cognate proteins, then it is no surprise that you end up with an attenuated virus. We are still missing the link between both papers but it looks very promising.
All the best
Mauricio Comas-García, Ph.D.
HIV Dynamics and Replication Program
National Cancer Institute
Dear Vincent & Co-Hosts,
I really enjoyed the discussion on TWiV 351 and wanted to chime in on one topic that came up in your conversation.
We are most familiar with the flaviviruses that are transmitted by arthropod vectors to vertebrate hosts, especially dengue, West Nile, and yellow fever viruses which are transmitted by mosquitoes, as well as tick-borne flaviviruses. But there are also insect-specific flaviviruses that are transmitted vertically between insects, with no vertebrate host. Furthermore, there are also vertebrate-specific flaviviruses that have been isolated from mammals but never from an arthropod; many of these vertebrate-specific flaviviruses won’t even replicate in insect cells in culture. While flavivirus host range is largely reflected in the phylogeny of these viruses (i.e. mosquito-borne viruses group together, tick-borne viruses group together, insect-specific viruses group together), there are several notable exceptions to this pattern. One interpretation is that there may have been instances in which a previously insect-specific flavivirus gained the ability to infect a vertebrate host, and additional instances in which a previously vectored flavivirus lost the requirement for an arthropod vector for its transmission. Understanding the factors that control flavivirus host tropism is critical for recognizing potential new emerging infections.
Love the show, keep up the great work!
Helen Lazear, Ph.D.
Assistant Professor of Microbiology & Immunology
The University of North Carolina at Chapel Hill
Re twiv 351 and manipulation of dengue virus nucleotide sequences, there was a question as to whether DNA virus genomes had been studied similarly. I don’t know about de-optimization, but there is an extensive literature on optimization of human papillomavirus (HPV) gene codon usage. The objective is to study the less than optimal codon usage (for humans) of natural virus strains, presumably selected to attenuate replication and allow the virus to persist in its natural host (us). To me, that raises flags for anyone thinking about developing attenuated live HPV vaccines instead of the currently licensed “recombinant particle” (I’m not sure what else to call them) vaccines.
It’s sunny and an amazing 21° this afternoon in Hershey. Fall must be on the way.
Keep on twivvin’
David J. Spector, Ph. D
Professor Emeritus of Microbiology and Immunology
Penn State Hershey
Dear TWiV Gang,
I have a very short question concerning the dengue paper that you discussed recently. If I understood correctly Shen et al. showed that when infected as young mice with the recoded viruses they produced high levels of neutralising antibodies. Since the changes are only apparent on nucleotide level but should not alter the protein once it is translated, the antigen should in theory be the same. So does this suggest that the attenuated virus gives the immune system more time to react and produce antibodies?
Similarly I am confused about their conclusion that the attenuation of the viruses could be “related to the synthesis of the polyprotein, its processing, misfolding, and/or stability”. How can the folding of the protein differ when the amino acids are the same?
In addition I was wondering if you could explain why mice are injected with the virus intracranially?
After weeks of fluctuations between 15 and 40° Celsius today we have 19°C and 65% humidity in Heidelberg, Germany.
Since most groups here focus on HIV and you could get the impression that it is the only important and interesting virus I am extremely glad that you are very considerate of other viruses as well.
Thank you very much for everything
Thank you for reading my letter on TWIV.
I’ll be amazed if any sort of surveillance is in place. My assumption is that Health departments / animal control only act on complaints. And those reports will most likely be of superficial sanitation / cruelty issues.
Union City, NJ was — and might still be — the most densely populated city in the US. A significant demographic there travels to Central and South America. Hoboken also is densely populated. The upper income individuals there often work in New York and can be expected to visit various destinations about the US and around the globe. A sneeze here might mean that the world catches the flu.
Back in the early ’70s during the outbreak of Exotic Newcastle, my understanding was that commercial poultry facilities were all made bio-secure. The recent Midwest spread of Avian Flu proved that if that was ever true, it no longer is. Containment did not happen. Is there any reason to expect a better outcome if (when?) the next disease infects people?
Thank you for your time and energy that goes into TWIV!
My name is Kristine, and I have been a weekly listener after being introduced to the show by a professor at Brigham Young University-Idaho. I am now a Pathobiology PhD student at the University of Washington, with an almost exclusive interest in virology.
TWIV #350, being shorter than usual, allowed me to go back to the beginning and listen to TWIV #2 on Poliovirus. In this episode you referenced a paper entitled “Virus Attenuation By Genome-Scale Changes in Coding Pair Bias.” Just as a quick refresher, the crux of this paper was using the idea of “codon pair bias” (for example, the paper explained that the Ala codon GCC is used four times more frequently than the synonymous codon GCG) to synthesize the poliovirus capsid protein with over- and underrepresented synonymous codon pairs as a means of viral attenuation for vaccine development. This paper was published in 2008, and I was curious if you are aware of any progression in using this method either for polio or other viral vaccines? Also, do you know if the idea of over- and under-representation of synonymous codon pairs has been found to be influential in anything other than viral attenuation such as genetic diseases perhaps?
Thanks for doing the show each week. I love listening to your discussions and familiarizing myself with the weekly virology news.
Kristine wrote a few days later:
Earlier this week I sent you an email concerning some questions I had about TWIV #2 on polio viruses and the codon pair bias phenomenon. I had sent this email literally an hour before listening to this weeks episode, which of course discussed this exact topic. Being episode #351, I can’t help but think “what are the odds?” Anyways, sorry about that. Episode 351 was a great update and explanation.
Thanks again for continuing this great podcast.
Dear TWiV and TWiMers,
Your podcasts on all the mind-blowing science going on today are a weekly party for my mind. Thank you so much, love you guys!
Your recent TWiV episode with Michelle Banks made me wonder if you had ever heard of the Canadian conceptual poet Christian Bök and his Xenotext Experiment? A bit of background here: http://triplehelixblog.com/2014/01/the-xenotext-experiment/
Love to hear what you think!
Your devoted listener,
I found this:
Have you seen this? What are your thoughts?
82F and still “rainless” in Seattle.
Has this happened before? Does the mutated Polio virus put U.S. at risk? 92 degrees fahrenheit, sunny. A triple H day (Hazy, Hot and Humid) near D.C.
Two papers on cGAMP. I remember you followed this antiviral signaling molecule. They now show it can travel between cells using viruses! Thanks for the BPF (Best Podcast Forever J). Cheers, DJ
Viruses transfer the antiviral second messenger cGAMP between cells
Published online 30 July 2015 [DOI:10.1126/science.aab3632]
Transmission of innate immune signaling by packaging of cGAMP in viral particles
Published online 30 July 2015 [DOI:10.1126/science.aab3628]
Dirk Jochmans, PhD
Laboratory of Virology and Chemotherapy
Rega Institute for Medical Research | University of Leuven
Hey there folks,
I have copied and pasted some comments that were left on a Raw Story article about vaccination and some of these arguments are not ones I had read before so I thought I would send them to you folks and see if you have any comments I can use back to these folks. The truth is that I am not a scientific person and so I can’t argue a lot of this back. I just know that I believe vaccines are safe, but they do name someone who seems to be a good and knowledgeable person(?) so I hope you can deal with this.
I also wanted to make another quick comment, whoever it was that stopped you from using FU for follow up needs to move on and you guys should bring that back. I get tired of these holier than thou types ruining just about everything. There is no need to over censor your comments and shit. Thanks in advance for your help refuting this crap!
I have provided the link which you were unable to open above.
Have you heard of the Human Genome Project which was an attempt to map out the chromosomal locations of human genes. The technique for mapping genes fuses human and rat cells in tissue cultures. The cells have both rat and human chromosomes combined. How do they hybridize human and animal cells? They place both in a tissue culture and put through repeated cell cycle passage. The human chromosomes become “lost” and this allows scientists to mark protein expressing genes to individual Human chromosomes. So this can occur in a laboratory and it brings up the question whether vaccines which are contaminated and made with animal, human, non human cells/DNA can have the same effect in the human body.
Many vaccines use immortal cell lines and these are actually cancerous types of cells and they divide continuously. Most commonly used is the human diploid variety extracted from aborted fetal tissue. It is quite likely in the light of what we know about human-rodent somatic cells that these cells could hybridize with our own cells. There is also the concern that these cell lines may be contaminated with cancer causing pathogens.
“The swine flu narcolepsy saga is so important to the Great Vaccine Blitz because it so starkly contrasts the “no dark side” mantra from the Bill Gates crowd. And the narcolepsy findings are just one example in an emerging boatload of scientific evidence that vaccines induce autoimmune diseases.
That evidence was neatly rounded up in a medical textbook released last week called Vaccines and Autoimmunity (Wiley). It’s a collection of papers by more than 75 doctors and scientists and is edited by pre-eminent immunologist Yehuda Shoenfeld, founder of the Center for Autoimmune Diseases at the Sheba Medical Center in Tel Hashomer, Israel and a prolific research with more than 1,800 references to his name on PubMed and author of now 26 medical textbooks including foundations of clinical practice. Last year at the 9th International Congress on Autoimmunology (ICA) in France, which he founded, he was introduced to the 2,300 or so doctors in attendance as the “Godfather Autoimmunology”– the youngest branch of medicine and one of the fastest growing.
“One chapter calls adjuvants as “the ‘dirty little secret’ of immunologists” because, just like the natural infections they are used to deter, they carry a risk of triggering chronic disease. Scientists knew this at least as far back as 1956 when they induced arthritis in Wistar rats – known as “adjuvant’s disease” in the industry. It’s incredibly ironic that rather than removing them from vaccines immediately, they tailored this “right nuisance” and for six decades now have widely injected adjuvants into animals to create lab models of arthritis, allergies, asthma, lupus and more — so they can test drugs for the diseases that are increasingly plaguing humans injected with adjuvants
At the ICA last year, dozens of scientists reviewed a terrifying catalogue of recent findings about aluminum in vaccines: how it persists in the body long after injection and can migrate to brain, lymph, and spleen where it wreaks inflammatory havoc; how it was found in the nerve tissue of paralyzed Spanish sheep who wasted away and dropped dead in a post-vaccination plague that wiped out flocks, and how post-mortems revealed that it may have acted like a “Trojan Horse” carrying HPV-vaccine components into the brains of two young girls who died suddenly in their sleep following Gardasil shots.”
My concern is that vaccines are not tested for their ability to cause cancer – this is clearly stated on the package insert. Most vaccines are grown on animal tissue such as MDCK – dog kidney cells (Flucelvax) Human diploid tissue (rubella and polio) aborted fetal cell lines polio, MMR, chickenpox, Hep B, chick embryo – influenza, bovine serum MMR. No one knows the long term effect as these cells lines are immortal and the FDA has expressed concern over their ability to cause tumours. Vaccines are tested on mice, rats and hamsers etc but humans are different – it also depends on the laboratory doing the testing. Pharmaceutical companies do their own testing which in itself gives me no confidence due to Pharma’s track record of corruption.
“specific DNA segments either derived from virus derived from cells and in doing so this does not of course eliminate all the worry one might have. One of the issues is that there is the question of whether the specific DNA that when it is added actually has some sort of risk associated with it. Of course if this DNA is capable of causing the cells to grow forever in a culture it may have oncogenic potential and in fact as you have heard discussed by Andy there is some reason to think that in the case of the adeno early region that there is some oncogenic potential of that. So you would worry about carrying the DNA if you’re using a vaccine preparation with the vaccine material you’re going to use. So there is actually still a question of the degree to which this is a serious concern and you’ll hear more about this later.”
the incidence of childhood cancers has increased by 29% over the past 20 years.
Some other mechanism (e.g., oncogenic proteins, RNAs, or other factors that could induce heritable epigenetic changes) associated with immortalization or tumorigenicity could present a risk to the recipient of a vaccine manufactured in tumorigenic cells
- 70 per cent of all HPV infections resolve themselves without treatment in a year, and the number rises to well over 90 per cent in two years.
- All trials of the vaccines were done on children aged 15 and above, despite them currently being marketed for 9-year-olds.
- So far, 35,000 girls have reported adverse side effects from Gardasil alone to the Vaccine Adverse Event Reporting System (VAERS), and this number only reflects parents who underwent the hurdles required for reporting adverse reactions.
- To date, 200 girls are officially known to have died from these vaccines.
- The reported side effects of these vaccines include Guillian Barré Syndrome (paralysis lasting for years, or permanently — sometimes eventually causing suffocation), lupus, seizures, blood clots, and brain inflammation. Parents are usually not made aware of these risks.
Kia ora folks,
One of the topics that comes up routinely is on trying to place the level of conversation at a level that is not too high and not too low. As a software developer who is routinely talking to non technical clients I’ll offer a suggestion using a technique I use every day. Try to minimize the number of abbreviations or acronyms used during conversations. The theory I have on this is that abbreviations and acronyms are really useful to increase communication density within groups that know the domain. For people outside of such domains it can be very difficult to get an understanding when all you see is a wall of acronyms and abbreviations. Restricting such terms to only the most common like DNA, RNA, PCR, etc. and expanding the others can dramatically help with understanding. This helps improve understandability without really dumbing it down but rather reducing the data density.
Thanks for the awesome podcast,
I wanted to respond to Ralph’s letter in TWIV 348 about the possibility of viruses (and other microbes) escaping in the event of severe damage to a building. I feel like you responded well, but I had an additional, more general take. I don’t know what Ralph’s thinking is but the general public tends to think of viruses as nigh on invulnerable, They aren’t. Take flu for example (it’s the obvious choice since it’s the center of the gain of function controversy, although it’s not the hardiest of viruses). As best I could find from Google (correct me if I’m wrong) under good conditions on good surfaces (which building collapse and terrorist action are not), flu is transmissible for a matter of hours. Fomite transmission can be further reduced or eliminated if the first responders are wearing hazmat suits, which they should anyway. Aerosol transmission is even more limited. I don’t know that there are any studies of virus aerosolized by falling buildings, but it sounds like most of the virus particles settle out of the air within minutes after sneezing. Falling building, while it might aerosolize some virus, would also contain that virus as well, it seems to me. The trouble is that society at large has a tendency to think that once a virus is in the air, it’s going to float around until someone gets sick off it, which is patently not true. Nonetheless, for instance, one of the characters in Stephen King’s The Stand managed to catch the regular flu months after most of the human population had died off and long after the character had been anywhere near anyone had a case of flu to transmit. It seems to me that this kind of misunderstanding explains why people are so afraid of microbes getting out.
I ended up talking about this with another TWIV listener and we wondered if there have been any studies of lab leaks caused by damage to buildings. Undoubtedly there have been microbiology buildings damaged by earthquakes and hurricanes, and probably other natural disasters. Now, hopefully, all labs are up to code and not liable to crack open, but unfortunately that won’t be the case for some older buildings. As an extreme scenario, I could imagine that an earthquake could knock out of alignment filters protecting airflow out of a lab working with live anthrax. In such a case, you could create a incident like the one in Sverdlovsk where a Soviet bio-weapons program forgot to replace a filter and released large quantities of anthrax. Okay, my hypothetical wouldn’t be that much like the Sverdlovsk incident; volume of anthrax would be significantly lower, and a natural disaster would stop work, but it seems like enough anthrax might get out to cause some problem. So, has anyone studied what happens after such incidents? I don’t expect anything created in the lab got far, but it would be interesting to know if anything at all has ever been detected, or if anyone studied how far microbes managed to get. Are there published studies? The person I talked to suggested the risk management specialists might know more, but publish less, so it might be worth talking with one if you could get one on the podcast.
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