Later this month (May 2014) the World Health Assembly will decide whether to destroy the remaining stocks of variola virus – the agent of smallpox – or to allow continued research on the virus at WHO-approved laboratories.
After the eradication of smallpox in 1980, the World Health Organization called for destruction of known remaining stocks of variola virus. The known remaining stocks of the virus are closely guarded in the United States and Russia. These consist not of a single vial of the virus, but of hundreds of different strains, many of which have not been fully characterized, nor has their genome sequence been determined.
It can be argued that there still remains a good deal of work to be done on variola virus, including development of newer diagnostic tests, and identification of additional countermeasures (antivirals and vaccines have been stockpiled in the US). Damon, Damaso, and McFadden have written a summary of the research on variola virus that should be done. We also discussed whether the remaining variola virus stocks should be destroyed on episode #284 of This Week in Virology.
We are interested in what readers of this blog think about this issue – please fill out the poll below.
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Might as well kill the wild stocks left. We have the genome of the thing. If we need one to play with in the future we can build a new one.
As discussed in the latest TWIV, it may not be trivial to build the genome as the makeup of the hairpin structure at the end of the genome are difficult to sequence, so a reliable copy of the genome may not be available. Not to mention that to develop new antivirals, it may be beneficial to have stocks available now for research.
I voted to not destroy these, as regardless of what the US or Russian governments do or say, they will keep their own stocks for military purpose. If the purpose of destroying the stocks is to eliminate risk, we would in fact be increasing risk by lowering our ability to research and access the virus to allow us to develop new technologies to protect ourselves from harm.
Sure. However, as discussed on Virology I && II and in my current gig Epigenetics and Gene Expression, there never is a “standard” genome. I should think that we have sequenced variola enough times by now as to have a pretty good consensus picture of the beast.
If we removed a few decent computers away from these “climate change” nutjobs we could really throw some CPU at the task! 😉
Antivirals can be pretty much developed in-silico if CPU and budgets are not a problem.
I agree that the question is somewhat moot if the virus is going to be kept regardless of a vote.
You wrote: “I should think that we have sequenced variola enough times by now as to have a pretty good consensus picture of the beast.”
This is not the case, which was what Rich was made a point of.
Rich said on TWiV 284, starting from 45:20 (Paraphrasing): There are two repositories, one in Moscow, the other in the CDC. Hundreds of vials, representing hundreds of isolates (some duplicates). Potentially multiple strains, from many localities. Of around 500 samples, only ~50 have been sequenced.
Then later on, at 1:00:00 Dixon asked, paraphrased: “What has stopped us from sequencing them all to at least have a permanent record of whats out there”… “Isn’t that an easy thing to do?”
Rich’s answer, paraphrased: “It’s in progress, but it’s not done.”
Later on at 1:08:14: Rich goes on to say, paraphrased: “The sequence information is limited, the hairpin ends are really hard to sequence, and typically that is not done. It is not known how the hairpin ends differ from one sample to another, and the effect that would have on the properties of the virus”
I’m sure that you will appreciate, having completed Virology I, the importance of the hairpin structure for the contiguous replication of the genome. But as shown with other loop structures, these can have serve many functions within a cell.
Sure. Doesn’t take away from the fact that any virus is a “quasi-species”. Arguing against destruction because every single one hasn’t been sequenced is pointless. As soon as you warm the things up they will surely mutate yes? ALL sequencing is re-sequencing by consensus pattern for anything larger that a protein! If there are 500 samples in the world and we have sequenced 10% of them then that 10% is plenty for a consensus pattern.
I’m a bit of a newbie at the molecular biology stuff, only around six years or so. I have been a software engineer for thirty five years and I know a LOT about statistics and pattern matching!
Argue for keeping the stocks by all means. Just don’t use this particular argument as it makes little to no sense.
You wrote: “If there are 500 samples in the world and we have sequenced 10% of them then that 10% is plenty for a consensus pattern.”
Yes, even with very few genomes, you could map a genome on a consensus basis by law of average. As has been done with the Human Genome Project with as little as 4 (2 male, 2 female from a random selection out of 20 of each), or 5 in the case of Celera Genomics.
However, for anti-virals it’s important to target conserved genes. In order to measure single-nucleotide polymorphism, the sample size needs to me much larger. In the case of the human equivalent, the HapMap project, the sample size was raised to 270 genomes.
Also, the point which I keep making is that of the 10%, hardly any are a complete genome. Very important.
Rich said, paraphrased: “The hairpin ends are really hard to sequence, and typically that is not done.
“Variola virus genomics
J.J. Esposito, S. Sammons, M. Frace, Y. Li, M. Olsen-Rasmussen, M. Zhang, J. Osborne, M. Laker, R. Kline, I.K. Damon, J. LeDuc, R.M. Wohlhueter
Centers for Disease Control and Prevention, Atlanta, United States
Please direct all queries to the authors at the addresses given.
The genomes of 8 variola strains, chosen by systematically by reducing the approximately 400 variola virus specimens in the repository at CDC to a sample size of 45 strains, mainly on epidemiological criteria, were selected by correlating the 45 strains by restriction fragment length polymorphism (RFLP) cluster analysis. The RFLP assays, which should be useful for comparatively screening for differences in entire genomes, and the sequencing templates essentially used sets of 20 PCR amplicons designed to overlap to virtually span each selected viral DNA genome.
The sequences showed that the coding regions of the 8 strains were rather alike, containing about 200 predicted genes or gene vestiges of which under half were identical and the majority encoded for proteins whose counterparts had at least amino acid difference. The similarity of predicted proteins of the newly sequenced variola viruses and two prior published strains suggest that a diverse battery of test probes and antivirals should be unnecessary to verify a clinical diagnosis and treat smallpox if it reappears, and antigenic variation should not be a major issue for future vaccine design. More, the sequences and variations observed should help improve diagnostic and forensic tests, targeting antivirals, and understanding pathogenesis mechanisms and putative animal models.”
http://www.who.int/csr/disease/smallpox/virusgenomics/en/
I haven’t looked before, I assumed people have been squireling away at this task for a few decades at least.
Sounds like the boys and girls at CDC are not having a lot of difficulty.
I’d be surprised if the Rooskies had not done this.
From the dim dark past….
“Virology. 1994 Jun;201(2):215-40.
Analysis of the complete genome of smallpox variola major virus strain Bangladesh-1975.
Massung RF1, Liu LI, Qi J, Knight JC, Yuran TE, Kerlavage AR, Parsons JM, Venter JC, Esposito JJ.
Author information
Abstract
We analyzed the 186,102 base pairs (bp) that constitute the entire DNA genome of a highly virulent variola virus isolated from Bangladesh in 1975. The linear, double-stranded molecule has relatively small (725 bp) inverted terminal repeat (ITR) sequences containing three 69-bp direct repeat elements, a 54-bp partial repeat element, and a 105-base telomeric end-loop that can be maximally base-paired to contain 17 mismatches. Proximal to the right-end ITR sequences are another seven 69-bp elements and a 53- and a 27-bp partial element. Sequence analysis showed 187 closely spaced open reading frames specifying putative major proteins containing > or = 65 amino acids. Most of the virus proteins correspond to proteins in current databases, including 150 proteins that have > 90% identity to major gene products encoded by vaccinia virus, the smallpox vaccine. Variola virus has a group of proteins that are truncated compared with vaccinia virus counterparts and a smaller group of proteins that are elongated. The terminal regions encode several novel proteins and variants of other poxvirus proteins that potentially augment variola virus transmissibility and virulence for its only natural host, humans.
PMID: 8184534 [PubMed – indexed for MEDLINE]”
VR is the Pox man. What say boss?
PS. Virology i && II really taught me a LOT! Thanks so much!
Augmenting that recent knowledge with Epigenetics and Gene Expression, Melbourne U.
I am starting to think for the first time I am beggining to understand some of this stuff!
Cheers.
@addinall “We have the genome of the thing”: which one? Which of the many, many strains referred to in the piece above have been sequenced?
Precious few, in answer. We would be throwing away all sorts of solutions to the biological puzzle which is “variola virus” and its pathogenicity – and we CANNOT explore that sequence space without knowing what it is.
Any virus is NOT a quasispecies – unless it has a high mutation rate, like Vincent’s favourite picornaviruses or like flu and HIV. dsDNA viruses tend NOT to mutate like ssDNA or RNA viruses do – so you will NOT re-establish the “same” population present in any of those mixed stocks by multiplying one.
Like I said, my degrees seem to be mathematics, computer science and engineering. It is the only last half decade I have done a few courses in molecular biology, virology, genomics, epidemiology, epigenetics, molecular systematics etc. SOMEHOW I managed a distinction on a few of those, dunno how, I am a bit of a cloth ear most of the time, and I seem to remember it came up… Guess who said this…
“Any biological population is a mix of sequences and therefore a quasispecies. RNA viruses get all the attention because they cannot correct errors and their sheer numbers makes their quasispecies more extensive than any other.”
Variola may not mutate as quickly as influenza, however I am betting it mutate more quickly than Crocodylus porosus.
I pay attention when bright people tell me stuff!
I stand by my assertion. If we have sequenced 10% of all the variola left on the planet then we have enough information for in-silico representation.
Cheers.
“Any biological population is a mix of sequences and therefore a quasispecies. RNA viruses get all the attention…” yeeeeeaaaaahhhh. True in the strict sense, not in the sense of “how many distinct variola genomes are there?” Because you will find that any one person’s infection is essentially clonal – as it is for the much smaller HPV, for example – and there may be important differences between clonal isolates that you will miss if you throw them away.
Like what makes one isolate kill 60% of people it infects, and another 40%.
Hang about!
This argument has gone circular! First I was given the suggestion that we need to keep live virus intact because it is NOT a mutator to now, we need to keep the same virus because it IS (highly) mutative!
I don’t really mind which is correct as it has no bearing on my assertion nor argument.
You seem to be arguing against the development of broad-spectrum anti-vaccines, even within the same species. Using this as a base argument, then all antiviral research is pointless because the vaccine stock and the virus recovered from a ‘current’ infection MAY have nucleotide point variations.
Not only that, but the host species (humans) each possess an individual genome which includes an individual intrinsic and adaptive immune response.
Clearly you would not argue that, as it makes Virology rather pointless.
If the epigenetic structure of the viral species is similar enough to be CALLED a species group, then point mutations that increase or decrease virulence is an academic curiosity.
I suppose different people approach viruses in different fashions. Many, many, many moons ago I was a regular Army MEDIC living and working in tropical environments. I had the pleasure of being invited to work in the feild with Lassa. Then I thought a virus was a king of super sneaky bug with a BAD attitude.
Now I regard a virus as a really well built molecular machine who’s actions and functions can be described at a molecular level, and with the knowledge of the virus/host interaction, we can pretty well describe most of the past ‘mysteries’ at an electro-static level. Epigenetics has started that road to discovery. Modern computers allow us to engineer and visualise these interactions in-silico.
The morbidity/mortality rate of a viral infection may well indeed be changed my genome mutations, however there are a LOT of other factors that need to be considered. A case in point is Lassa. It turned out that Epidemiology solved the morbidity/mortality mortality questions without sophisticated molecular biology resources. African miners “hot swapping” beds with each other and a Multimammate Rat of the genus Mastomys.
The in-silico power we can bring to a problem today is truly astounding over that of four decades ago. Now in the field of “Genomics and Precision Medicine” clinicians can order whole Exome screening (WES) or whole Genome screening (WGS) to identify Mendelian disorders including autosomal recessive disorders.
My assertion still stands. If we have sequenced 10% of all the variola on the planet, we have enough information to carry on research in-silico. If we can prise some decent computing infrastructure away from the “global warming will kill us all” nutjobs.
Cheers.
PS. The “Any biological population is a mix of sequences and therefore a quasispecies” quote was from VR. THAT made me even MORE convinced that we should be designing broad spectrum anti-viral agents at a molecular level.
Destroying the stocks is like destroying a key to a locked room for fear of getting stuck inside…
So, one position could be: “We can destroy it all when we know how to regenerate all the remaining virus from their gene sequences”. So in a sense the destruction would only make sense if it in some other sense didn’t make much sense 🙂
Nonsense. The pathogenicity of variola is quite simple to look up in the 21st century. Even I managed it as homework on virology i && II.
“Pathogenicity: There are two slightly different virions of variola that are morphologically distinct, called the intracellular mature virion (IMV) and the extracellular enveloped virus (EEV). These virions are surrounded by different numbers of membranes, they have different proteins on their surface, and they bind to different cell receptors, which causes some confusion on how exactly the variola virus enters the host cell. The majority of IMV remain intracellular until cell lysis, but some become wrapped by additional membranes during a complex morphogenesis pathway to form intracellular enveloped virus (IEV). IEV are transported on microtubules to the cell surface, where they fuse with the plasma membrane to expose a virion on the cell surface. This virion may be retained on the cell surface as cell-associated enveloped virus or released as EEV.
The binding proteins and their functions for variola virus are unknown, however it has been determined that only the IMV virion has the proteins needed for fusion to the cell. In this case, it is deduced that the outer layer of an EEV is discarded before fusion to the host cell. The specific entry path of the virion depends on the virus strain or type; the virus may enter the cell through either fusion at the plasma membrane or through the endosomal pathway. Finally, immediately before entering the cell the outer membrane of the virion is shed, which leaves only the dumbbell-shaped core to enter the host cell.
Once the virus enters into the cell, a host RNA polymerase is activated. Along with other accessory enzymes, this polymerase begins to be translated by the host cell. This leads to the uncoating of the virus. The dumbbell-shaped core of the virus is revealed by the early mRNA. The nucleoproteins pass through the core wall making it disappear. The uncoating is a process completed by RNA and protein synthesis. The DNA within the variola virion is cut in half by the enzyme helicase. DNA polymerase then matches the strand with each unique pair, therefore creating a new strand of identical DNA for the progeny virion. Once all the essential proteins are transcribe and translated, progeny virions are assembled. Infection of the host cell typically results in about 10000 copies of the genome per cell.”
Riedel, S. (2005). Smallpox and biological warfare: a disease revisited. Baylor University Medical Center Proceedings, 18: 13-20.
Given the fact that the smallpox vaccinations DO work, and have done since the efforts of the remarkable Edward Jenner, leads one to believe that the “hundreds” of strains are structurally alike does it not?
You can probably recognize me as a human without knowing how much change I have in my pocket.
I have read “Are We There Yet? The Smallpox Research Agenda Using Variola Virus” of course. I rarely study anything without a purpose and usually to some depth.
It states:
“In 1999, following an independent report on variola research by the Institute of Medicine (IOM), a decision by WHO was made to increase the amount of research that utilized live variola virus. The WHO Advisory Committee on Variola Virus Research (ACVVR) was subsequently formed to oversee the research, and research began to generate additional virus genomic information, characterize new antivirals and therapeutics, evaluate newer generations of vaccines and biologics, develop diagnostics, better define disease pathogenesis, and generate animal models of smallpox disease. This work was further refined by the World Health Assembly (WHA) to focus on “essential public health research†in 2005 and was subsequently comprehensively reviewed by the IOM in 2009 [1], and the WHO ACVVR and its assembled external advisory group (called AGIES) in 2010 and 2013. Each of the last two comprehensive reviews was prepared to summarize research advances and to recommend whether additional research with live virus would be required in order to fulfill the original WHO-mandated agenda, in advance of a WHA-wide discussion about the fate of the remaining variola virus materials stored at the two WHO Collaborating Centers. These reviews are available online (http://apps.who.int/iris/bitstream/10665​/97033/1/WHO_HSE_PED_CED_2013.2_eng.pdf; http://apps.who.int/iris/bitstream/10665​/97034/1/WHO_HSE_PED_CED_2013.3_eng.pdf). Because of the advances made in the acquisition of knowledge to support diagnostics, antiviral, and vaccine research and development through to the regulatory review process, the majority opinions of those in these groups are now, in 2014, more supportive of discontinuing the use of live variola virus for future research studies.”
I don’t much care either way. However I am of the opinion that we should be concentrating on developing broad spectrum anti-viral drugs along the lines of CMX001, a DNA polymerase inhibitor active against multiple DNA viruses. And we can do this in-silico REMOVING the chance of some virus being tipped into an air conditioning duct, AGAIN.
Cheers.
Damon IK, Damaso CR, McFadden G (2014) Are We There Yet? The Smallpox Research Agenda Using Variola Virus. PLoS Pathog 10(5): e1004108. doi:10.1371/journal.ppat.1004108
If there is ever another outbreak of smallpox, the only strain that will matter is the one that is circulating then, and there will be plenty of it for research. One dead body will suffice. The technology is such that it will be sequenced in a day. I say cook every last vial, and use our limited resources to study the viruses that are killing people now.
Yes; destroy the stocks immediately.
Our current risk management methodology is decisively inferior. Our closely interconnected systems are collapsing at an increasingly rapid rate; consider the two recent examples of Fukushima and the Great Financial Crash.
The top mathematicians in the world assured us that neither of those two events would happen more frequently than once every several million years, i.e. they were 12 sigma events.
But that was false and it was stupid. But most of the brightest risk management folks don’t know why. That portends danger for retaining such stocks.
One key point is that these models that say we have a 12 sigma chance (or thereabouts) of a catastrophic accident from a virus laboratory are making the same mistake made in both Fukushima and Goldman Sachs risk modelling.
To wit, proper risk assessement must take into account that these are complex systems of interconnected and layered probabilities. Additionally, any estimate of a very small risk contains a margin of error. That margin of error is often larger than the actual risk being estimated. When you layer those massive uncertainties, their products must be multiplied for an accurate risk assessment to be made.
These clowns are off by orders of magnitude. They do not employ what is known as metaprobability analyses.
Simply stated, we are too stupid as a species at the present time to continue research with smallpox.
The foregoing analysis is informed by pages 91-98 of a seminal new text on risk management to be published next year.
That text, entitled “Silent Risk” is available free of charge below. Virologists need to understand this so we don’t have a viral “Fukushima”.
http://www.fooledbyrandomness.com/FatTails.html
I think they should be destroyed with the exception of one. I don’t agree with the twiv hosts when they say that we are going to lose the information in there. As long as one survives we can use error prone polymerases and reconstruct a genomic space that far surpasses the one that is stored in those samples.
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The recent discovery of viable variola samples, maintained for decades by the NIH FDA, indicates that we never know how or where this virus may emerge. I am not in favor of destroying the remaining stocks, as long as we are completely certain that they are properly stored and protected.
lol
Sorry about that but i think it should be destroyed in the future because due to the fact that we can all lie to other countries that we destroyed small pox but make vaccines of small pox and secretly make it we can hide anything