Where did our smallpox vaccine come from?

Edward Jenner's smallpox vaccination

Bring to mind the now famous 'first scientific exploration of vaccination', when, in the late 1700's, Edward Jenner - an English physician - first came up with the idea of using a non-pathogenic cowpox virus to vaccinate people against its deadly relative, smallpox (variola virus). 

Well, this virus and others like it, such as vaccinia virus (and its own viral derivatives, like the highly attenuated modified vaccinia virus Ankara) have been used worldwide to protect human populations from contracting smallpox (See dryvax and it's recombinant clone ACAM2000), which resulted in the eradication of variola virus during the second half of last century. These viruses are thought to all trace there ancestry back to cowpox isolates from around Jenner's time in the late 1700's, yet we don't really know for 100% where they originated.

Despite its renowned success, showing society the power of vaccination, the origins of cowpox have so far remained elusive. The story goes that Jenner's original cowpox isolate, through generations and generations has somehow become what we know of as vaccinia virus but how is anyone's guess. Maybe recombination with other poxviruses out there or maybe it is the last living representative of an extinct virus is the reason why.

What human cowpox looks like

Now, an international team of researchers (see paper here) has shed light on it's origins by sequencing and studying whole genomes - contrary to the single-gene-centric studies in the past - of multiple currently circulating isolates of cowpox from around the world in order to uncover the secrets of poxvirus evolution in general.

Viruses are a haven of genetic diversity - even DNA viruses, which have been largely ignored on that front in favor of their more mutation-prone RNA cousins; this fact is no more apparent than in the case of poxviruses. These viruses, including smallpox and the re-emerging human pathogen monkeypox represent an immense amount of genotypic and phenotypic variation, which is in itself medically and evolutionarily important. Just think of the devastation that smallpox caused to the human population and have a look at what monkeypox has been up to.

False-color electron micrograph of vaccinia virus particle

This group compared the DNA of  the cowpox strains to other closely related pox viruses, such as: smallpox itself, monkeypox, camelpox and tatera pox (viruses which themselves have a difficult to trace past) as well as current vaccine vaccinia strains. They thus generated large phylogenetic trees based on the compared sequence and then mapped these onto a map of Europe to see if they could uncover some geographical pattern of cowpox evolution.

This analysis found an as yet unappreciated diversity hidden under what we called 'cowpox viruses' through identification of a number of well-defined monophyletic groups that should in their own rights be designated separate species. Interestingly, it also appears that our vaccine strain has jumped species to horses and buffalo as viruses isolated from these species have close relatives in vaccinia-like strains.

Cowpox viruses were found to cluster in two major groups - cowpox like and vaccinia virus like suggesting that our smallpox 'vaccinia' vaccine potentially originated as a cowpox virus (as we thought) yet it was endemic to mainland Europe, something that goes against the tale of Jenner's isolation of cowpox from the UK.

The authors suggest that further sampling of more isolates from within the UK and across Europe may clear up any taxonomic uncertainties here. What this work does highlight is the oft under appreciated diversity of large DNA viruses, especially the medically important pox viruses and the difficulties of doing evolutionary analysis on viruses which such large genomes who like to recombine with each other.

Carroll, D., Emerson, G., Li, Y., Sammons, S., Olson, V., Frace, M., Nakazawa, Y., Czerny, C., Tryland, M., Kolodziejek, J., Nowotny, N., Olsen-Rasmussen, M., Khristova, M., Govil, D., Karem, K., Damon, I., & Meyer, H. (2011). Chasing Jenner's Vaccine: Revisiting Cowpox Virus Classification PLoS ONE, 6 (8) DOI: 10.1371/journal.pone.0023086

Can we target antivirals to the host cell?

Two papers published recently explore the idea of targeting antiviral compounds to the host cell using high-throughput organic synthesis and an in vitro screen. Both groups identify a single novel inhibitor of virus replication and both are host specific.  One paper goes further and identifies the target and also the in vivo potential of this compound. These papers highlight the hopes and pitfalls of targeting the host cell against viruses while shedding light on the basic biology of these viruses.

   We can protect ourselves from the harmful effects of viral infection through the use of vaccination, antivirals or indeed behavioral changes. Yet not all viruses have proved to be amenable to these and some, like measles and mumps, are quite easily combated with mass immunization campaigns while the likes of HIV have shown to be quite sensitive to antiviral compounds.

Respiratory infections are a problem! hastings.gov.uk

   For a majority of viruses though, we have neither effective vaccines nor antivirals. We need these now more than ever. And, even those which we do have vaccines and/or antivirals (Influenza A), resistance is quickly built up over time rendering them clinically useless. How then are we able to generate new, more effective antiviral molecules against a whole range of viral pathogens, especially given the cost of taking a single drug to patients?; we want more bang for our buck with antivirals. One way maybe through the targeting not of a viral gene or protein but a cellular one. The virus life cycle is so intimately connected to that of the host cell that removal of cellular pathways may have a dramatic effect on virus functioning and applications like these have the beauty of possibly targeting both established and emerging pathogens.

   Krumm et al, from the Emory University School of Medicine, Atlanta, U.S.A, demonstrate the feasibility of this approach using viruses from the paramyxo- and the orthomyxoviruses (generally called the 'myxoviruses') as their model of choice. Read the paper here. Using this, they discover a novel compound that demonstrates host-cell specificity while greatly inhibiting virus replication. Even at nanomolar concentrations (potent!).

   This work falls on the back of a series of experiments they reported back in 2008 where they screened a 137,500 compound library against in vitro infection with the related measles, canine distemper and human parainfluenza 3 viruses. This was to determine if they could uncover broad range, potent inhibitors of a large number of viruses. Moving away from the 'one-bug, one-drug' paradigm, this work identified a class of 11 compounds that did just that: they significantly inhibited replication and cytopathic effect of all the three viruses. They use the data from this series of experiments to find a host-targeted compound.

They state that their 'ideal' inhibitor would be:

In search of candidates with a host-directed antiviral profile, we anticipated three distinct features of desirable compounds: a) potent inhibition of virus replication at the screening concentration (3.3 µM); b) a primary screening score, representative of the selectivity index (CC₅₀/EC₅₀), close to the cut-off value for hit candidates due to some anticipated host-cell interference ( = 1.9); and c) a broadened viral target spectrum in counter screening assays that extends to other pathogens of the myxovirus families

initial compound A) and B), the synthetically optimised JMN3-003

   A single compound was taken forward and then synthetically optimized (high-throughput synthetic chemistry anybody?) to achieve even better inhibition. No longer do we have to rely on the natural diversity of compounds out there. This new and improved compound (JMN3-003) could inhibit not just myxo-virus replication but also that of clinically relevant RNA (Sindbis virus) and to a lesser extent DNA viruses, such as vaccinia virus. The broad activity of the molecule suggests that it may target a more general aspect of viral replication than others had previously. The activity of this drug was also host-cell specific, suggesting that it did not target a virus structure but rather one found within the host cell, whose activity would change depending on the species assayed.

JMN3-003 antiviral activity

   This molecule had little or no effect on the metabolic activity of the immortalized cell lines used or of other primary cell cultures when administered at >7,000X the active dose, possibly hinting at the safety profile of such a drug. And, under liver cell extract stability tests, the compound showed a desirable extrapolated half-life of around 200 minutes. Host transcription and translation weren't affected by drug administration.To be safe, the authors put forward that this compound may only be preferentially used in acute respiratory infections where the treatment time would be substantially shorter. This group did not carry out any in vivo work so the real safety of this compound cannot really be assessed. See below for how this should be done.

Activity changes depending on the cell used. Human PBMCs or primate Veros

   So, we have demonstrated that the compound inhibits virus replication. But just how exactly does it do this? At what stage of the virus replication cycle does it act? Entry? Transcription? replication? assembly? exit? The group addressed these points through the use of assays that measure virus envelope fusion with the cell membrane (virus entry) and virus gene expression and replication (post entry). This work showed that fusion was not inhibited when the drug was added suggesting a post entry step was being targeted. When compared to a known inhibitor of virus polymerase activity, JMN3-003 looked very similar. Other experiments, assessing levels of mRNA and genomes produced from virus replication showed a significant inhibition in one or both processes during measles or influenza virus infection. Some component associated with virus polymerase reactions is being targeted. What it is, sadly this paper did not address, yet further experiments I am sure will uncover the mechanism of action of this compound and maybe through this identify an important host-virus association that has been previously overlooked. Recently, evidence has suggested that these viruses require the activity of host kinases for proper, regulated replication. Could it be these?

Little risk of resistance?

   Their experiments also suggest that it is extremely difficult for these viruses to adapt to the inhibition from JMN3-003. After 90 days of replication on cells treated with the compound, little resistance had emerged. Again, the reasons for this were not addressed. When compared to previous problems of drug resistance seen in Influenza A and HIV-1, this activity looks very promising.

RSV - the biggest cause of bronchiolotis in young children. http://www.empowher.com/

   Interestingly, this approach has been used for another medically important virus (Respiratory syncitial virus, RSV - also a paramyxovirus) and just published recently in the journal PNAS (Bonavia et al 2011). This group used effectively the same strategy as described above, except using a lot more compounds (1.7 million). They picked up a number of molecules that also targeted a post-entry step in virus replication, possibly virus replication ( RdRp activity); it also shows broad spectrum activity against a range of RNA viruses. And again, no resistant viruses emerged in the experiments. 

   This work went a little further than that I mentioned earlier and Bonavia et al were able to pull down the drug target using a type of affinity purification with immobilized samples of the compound (iTRAQ quantitative chemical proteomics). The protein binding most strongly to this may mostly likely be the target. In this case, the two compounds tested both targeted seperated enzymes in the same pathway, the de novo pyrimidine biosynthesis pathway, that is the nucleotides A, T and U (A and U being used extensively by RNA viruses). To assess the safety of targeting this important cellular pathway, the group looked at both in vitro and in vivo rodent models of disease. Rapidly-dividing cells showed high sensitivity to the compound while significant histopathological changes were observed in lymphoid and gut tissues in the cotton rat. This is not good for a potential clinical drug that may be administered to very young babies. 

Pyramidine biosynthesis - a good antiviral target? http://upload.wikimedia.org/

   Sadly, these compounds exhibited no antiviral activity in vivo and rather one increased RSV virus replication. This is possibly down to the potential immunosupressive nature of the target (inhibits rapidly dividing cells - T and B cells) or the ability of circulating pyramidines in the animal's body to rescue the inhibition of their synthesis inside the cell.

   I wonder how the first compound, JMN3-003 would fare in the many animal models of myxovirus infection. And, what would the target be when the chemical pull-down was applied? would it be the same pathway? Both these papers highlight the ability of this approach to identify readily targeted host cell components to reduce virus replication in vitro and potentially overcome the problems of narrow virus targeting and resistance while the second paper goes further and shows us how the results may not be pleasing. The targeting of host factors has obvious implications for patient health and this cotton rat model displays what can go wrong. We should learn from this and expand any potential drug candidates into in vivo modeling quickly and at the start of any experiments, not like what was done with JMN3-003, although that's not to say the same will happen with that compound.



Bonavia, A., Franti, M., Pusateri Keaney, E., Kuhen, K., Seepersaud, M., Radetich, B., Shao, J., Honda, A., Dewhurst, J., Balabanis, K., Monroe, J., Wolff, K., Osborne, C., Lanieri, L., Hoffmaster, K., Amin, J., Markovits, J., Broome, M., Skuba, E., Cornella-Taracido, I., Joberty, G., Bouwmeester, T., Hamann, L., Tallarico, J., Tommasi, R., Compton, T., & Bushell, S. (2011). Organic Synthesis Toward Small-Molecule Probes and Drugs Special Feature: Identification of broad-spectrum antiviral compounds and assessment of the druggability of their target for efficacy against respiratory syncytial virus (RSV) Proceedings of the National Academy of Sciences, 108 (17), 6739-6744 DOI: 10.1073/pnas.1017142108

Krumm, S., Ndungu, J., Yoon, J., Dochow, M., Sun, A., Natchus, M., Snyder, J., & Plemper, R. (2011). Potent Host-Directed Small-Molecule Inhibitors of Myxovirus RNA-Dependent RNA-Polymerases PLoS ONE, 6 (5) DOI: 10.1371/journal.pone.0020069

Yoon, J., Chawla, D., Paal, T., Ndungu, M., Du, Y., Kurtkaya, S., Sun, A., Snyder, J., & Plemper, R. (2008). High-Throughput Screening--Based Identification of Paramyxovirus Inhibitors Journal of Biomolecular Screening, 13 (7), 591-608 DOI: 10.1177/1087057108321089

The ecology of virus emergence - the role of rodents and biodiversity

   The inter-species transmission of viruses and other pathogens (see data below) poses a serious threat to public health, the global economy as well our environment and biodiversity. Just look at Ebola; SARS and Hendra viruses. With the numbers of emerging viruses increasing year on year, how best are we to deal with this incoming threat? As they say, the most effective bioterrorist is nature herself; so how can we stop her?

Increasing numbers of emerging infectious diseases. Jones et al (2008)

First of all, we can identify a number of possible routes that may be followed if we are to successfully combat or at least limit human infection with other animal viruses. These would include:

• Identifying what viruses may make (or are in the process of making) the jump into humans, i.e. what viruses are out there?

• What are the molecular mechanisms behind inter-species transmission and adaptation?

• Can we identify temporal and geographical (or cultural) 'hotspots' that correlate with increased risk of virus emergence?

• How can we develop potential vaccines/antivirals to further protect vulnerable local/global populations?

And, perhaps most importantly - and the most difficult aspect:

• How are we going to fund this and what are the most cost-effective measures of doing this, i.e can we identify the best places to protect ourselves and place our resources there?

  

Hantavirus - a deadly group of re-emerging viruses. http://virology-online.com/

Deer mouse

   

Orrock et al, publishing recently in the journal American Naturalist, give their contribution to this complex virus protection scheme by identifying key ecological regulators of the potential emergence of a fatal human virus from its rodent species reservoir. Knowledge of this may allow us to pin-point potential danger areas in terms of countries/regions or seasons (by applying their principles to other viruses and ecosystems), which would increase the risk of human infection. By placing emphasis on these areas we could develop a safer, more cost effective strategy to protect at-risk populations.

  

   The model system the group used was that of the rodent-borne hantvirus, Sin Nombre virus (SNV) infecting its host, the deer mouse, Peromyscus maniculatus on the Californian Channel islands. This virus was only relatively recently found to be present among Channel islands deer mice. Hantaviruses are a group of trisegmented negative sense RNA viruses that naturally infect rodent species around the world. Two groups are recognised: one found throughout the new world and the other, throughout the old. When a human is infected by one of the new world viruses (Sin nombre virus, for example), they may develop what is known as hantavirus cardiopulmonary syndrome (HCPS), a life-threatening disease (with up to 50% mortality) caused by leakage of fluids into the lungs. Humans get infected through coming into contact with infected rodents through their aerosolised urine, feces or saliva. In the U.S, the deer mouse (Peromyscus maniculatus) is the primary reservoir of this viruses. Environmental determinants of rodent density are thought to play a significant role in the risk of rodent-human disease through increasing the chances of human/deer mouse contact.

   

Specifically, they obtained SNV-specific antibody data relating to infected mice across all islands, giving them an accurate estimate as to the prevalence of SNV in these mouse populations. The group also compiled data corresponding to a number of environmental factors of these islands, including: area, perimeter, elevation, annual precipitation ( a good correlate with island productivity) and finally the number of deer mouse predators found across the islands. These numbers allowed Orrock et al to determine which ecological factor correlated well with SNV prevalence individually or in combination with others; data that would allow for the prediction of at-risk areas across the islands.

   A number of factors were identified, for example: SNV prevalence correlated well with annual precipitation on the islands as well as island area, which I guess may be expected given that these factors influence the food sources that the mice eat as well as potential space to leave and breed.

   Predator richness negatively correlates with SNV prevalence, suggesting that if we were to artificially remove top predator species from this ecosystem, rodent population density would increase, leading to more and more SNV-infected mice with a greater chance of infecting both themselves and humans. The authors state that the protection of both biodiversity and individual predators within ecosystems would serve to protect human populations from rodent-human virus transmission through the better regulation of host density. Also, environmental increases in primary productivity within the isalnd may also increase the risk of emergence.

   So, Orrock et al have demonstrated the key role of a number of ecological regulators of virus prevalence using a unique island-rodent virus model system. They specifically focused on a potentially fatal virus that can infect humans and hence their work has medical significance within the island system. They have also identified possible situations (bigger islands/more rainfall/low predator richness) that favor an increase in SNV prevalence although, they have not determined exactly why each occurs. This adds to the debate on the relevance of biodiversity to general protection from zoonotic disease.


   This work supplies important evidence for the potential prediction of 'at-risk' regions - not just within the Californian Channel islands - that pose a threat of virus emergence. Seasons with increased rainfall that increase primary production and hence possibly increase rodent densitie. Predictions such as these have recently been used within China and South Korea to identify areas for targeted control of hantavirus infections.

Orrock, J., Allan, B., & Drost, C. (2011). Biogeographic and Ecological Regulation of Disease: Prevalence of Sin Nombre Virus in Island Mice Is Related to Island Area, Precipitation, and Predator Richness The American Naturalist, 177 (5), 691-697 DOI: 10.1086/659632

Can we prevent gorilla extinction with vaccination?

Western Lowland gorilla

Gorillas, Gorilla spp. are found only throughout central African rainforest where there are in total over 200,000 individuals living in the wild. Two gorilla species are recognised, split between east and west Africa with at least two sub-species recognized in both. Their numbers are rapidly decreasing with problems such as habitat loss, poaching and human war contributing  greatly to a rapid reduction in their numbers. Sadly, one other factor on top of these in which these large primates must worry about is that of the transfer of infectious agents arising from humans and other animals. 

Viruses are constantly being transferred between populations of animals but may not establish infection all that easily in a non-host species. Although occasionally infection will result in virus replication and significant disease and this is known as a zoonotic infection -  when non-human viruses are transmitted to humans (see HIV, Influenza A and SARS-coronaviruses) and 'reverse zoonosis' when human viruses infect other animals. This pathogen transfer may be especially important when occurring in a critically endangered species such as gorillas; one recent example is that of human metapneumovirus.



EBOV effects not just humans http://turbo.indyposted.com/



We have been able to detect the effect of a number of viruses on gorilla populations including enteroviruses, adenoviruses and parvoviruses for example, although a small number are known to cause disease. In the last decade thousands of gorillas, chimpanzees and other mammals were killed through infection with the Zaire strain of ebola virus (EBOV) in the rainforests throughout the Congo basin area of central Africa. EBOV is a highly infectious and deadly RNA filovirus which causes a nearly always fatal hemorrhagic fever. The reservoir species for EBOV has been linked to central African fruit bat populations and ebola has caused hundreds of human deaths since its first recorded emergence in the 1970s.  Significantly more non-human primates have been victim to ebola than humans. We can implement a number of control measures for example limiting human-ape contact especially when ill to prevent this virus transfer but this may be more difficult when humans are not involved as in the case of EBOV. There are also a number of therapeutic options available although in the case of ape infection, would be logistically impossible. One strategy we therefore must consider is that of potentially protecting these Gorilla populations through vaccination against a number of potential viral pathogens.



The group VaccinApe is attempting to do just that. A volunteer consortium lead by the charity group the World Wildlife Fund, a vaccine developer Integrated Biotherapuetics and two academic institutions, the Max Plank institute for Evolutionary Anthropology and Kansas state University, VaccinApe is trying to develop an easy and effective method of vaccinating Gorilla populations in the wild. Currently in a 'proof of concept' phase, the group will lead the development of non-human primate vaccinology in order to generate a safe and reliable Ebola virus vaccine to be used through darting of individual gorillas. A large scale vaccination program may therefore afford protection of critically endangered gorilla populations against future EBOV emergences.

In light of the clinical severity of EBOV infection in humans a number of potential vaccine candidates have been developed which rely upon the generation of a protective immune response specifically to EBOV. One main candidate the group are interested in is the EBOV virus-like particles (VLPs). These are effectively non-infectious viruses lacking a viral genome but retaining virus antigenic proteins. Therefore EBOV-specific immunity will be generated against whatever EBOV proteins are found within the VLP. In early trials in macaques, this VLP strategy protected individuals against EBOV challenge although whether this could safely be transferred to gorillas isn't known.

EBOV VLP. Looks and acts antigenically like 'live' ebola. http://www.integratedbiotherapeutics.com/

Endangered wild gorilla and chimpanzee populations are at a great risk from a number of emerging viruses with the most important being EBOV. The difficulties in preventing direct transmission/therapeutic intervention have led people to consider the development of anti-EBOV vaccines. A number of candidates have already been tested and proved safe and effective in non-human primate models possibly allowing these vaccines to be transferred to gorilla population testing. It is hardly surprising that the work required to carry out such large-scale and difficult vaccination campaigns in wild gorillas in the African rain forest will be extremely difficult. The main problems include safety/efficacy testing in gorillas, physical vaccination methods and tracking anti-EBOV immunity non-invasively. Despite these difficulties, only time will tell whether the work of VaccinApe and their partners is to be supported as a worthwhile investment to save these animals.

Le Gouar PJ, Vallet D, David L, Bermejo M, Gatti S, Levréro F, Petit EJ, & Ménard N (2009). How Ebola impacts genetics of Western lowland gorilla populations. PloS one, 4 (12) PMID: 20020045


Richardson JS, Dekker JD, Croyle MA, & Kobinger GP (2010). Recent advances in Ebolavirus vaccine development. Human vaccines, 6 (6), 439-49 PMID: 20671437

Warfield KL, Swenson DL, Olinger GG, Kalina WV, Aman MJ, & Bavari S (2007). Ebola virus-like particle-based vaccine protects nonhuman primates against lethal Ebola virus challenge. The Journal of infectious diseases, 196 Suppl 2 PMID: 17940980

On the origins of smallpox - where and when did variola virus emerge?

2011 may be the year where the last known officially acknowledged stocks of the deadly smallpox virus, variola are destroyed - a virus that claimed over 500 million lives in the 20th century alone. The extensive collection of 'live' virus and DNA stocks totalling over 500 isolates/strains, which are held between the US Centres for Disease Control and the Russian State Research Centre of Virology and Biotechnology may be ordered to be eliminated following World Health Organisation (WHO) recommendations soon to be announced.

Although the impending fate of this pathogen has been covered elsewhere by Vincent Racaniello and Steven Salzberg I have been led to ponder its beginnings, at least in humans: where and when, over the course of human history did variola virus emerge  and have we always suffered from it? What confuses the matter further is that there are two clinical forms of smallpox - major (30% mortality) and minor, including both African and Alastrim minor (<1% mortality)- do these viruses have the same evolutionary history and if so, when and where did they diverge? Luckily, we can now study the origins of infectious diseases through both molecular and historical records.

A depiction of Shapona the west-African Yoruba god of smallpox. Courtesy James Gathany (photo), CDC/ Global Health Odyssey.

Conflicting historical records

It has been very confusing trying to make sense of the historical records of suspected smallpox cases as there are significant gaps in documentation and many conflicting reports. Smallpox-like skin lesions have been observed on Egyptian mummies dating from as far back as 1580 B.C yet there is no mention of the disease at all in the Old or New testaments nor even the Hippocratic texts. There was some mention of a smallpox-like disease in China and India as early as 1500 B.C but the only unmistakable description can be found from the 4th century A.D in China.  Interestingly there was no mention of smallpox in the American continents nor in sub-Saharan Africa prior to European exploration. But as shown in the picture above, smallpox has shaped west-African culture. So, did smallpox originate in Asia and spread to Egypt around 1,500 B.C? Or, is smallpox a relatively recent human disease, emerging around the 4th century A.D in Asia?

Molecular data shed light on variola evolution

A 2007 study using genomic data from the CDC's variola collections - the same ones that may soon be destroyed, added a phylogenetic perspective to the origins of smallpox and how it spread worldwide. Through studying single nucleotide polymorphisms (SNPs) from 47 variola genome isolates from geographically distant areas and collected between the 1940s and '60s they examined the genetic relatedness between isolates and were able to estimate the time since they shared a last common ancestor. They combined this DNA evidence with the above historical records to generate an idea as to where, when and how smallpox originated and spread throughout human populations.

Variola genome phylogeny

Abstract: Human disease likely attributable to variola virus (VARV), the etiologic agent of smallpox, has been reported in human populations for >2,000 years. VARV is unique among orthopoxviruses in that it is an exclusively human pathogen. Because VARV has a large, slowly evolving DNA genome, we were able to construct a robust phylogeny of VARV by analyzing concatenated single nucleotide polymorphisms (SNPs) from genome sequences of 47 VARV isolates with broad geographic distributions. Our results show two primary VARV clades, which likely diverged from an ancestral African rodent-borne variola-like virus either ≈16,000 or ≈68,000 years before present (YBP), depending on which historical records (East Asian or African) are used to calibrate the molecular clock. One primary clade was represented by the Asian VARV major strains, the more clinically severe form of smallpox, which spread from Asia either 400 or 1,600 YBP. Another primary clade included both alastrim minor, a phenotypically mild smallpox described from the American continents, and isolates from West Africa. This clade diverged from an ancestral VARV either 1,400 or 6,300 YBP, and then further diverged into two subclades at least 800 YBP. All of these analyses indicate that the divergence of alastrim and variola major occurred earlier than previously believed.

Hypothesised spread of variola worldwide

When analysed, variola fell into two large monophyletic clades signifying a historical divide in their genetic relatedness. The earliest representative - or most basal - of the variola major smallpox viruses are the Asian isolates. This suggests that major may have originated in Asia followed by geographic radiation across the Old world and into Africa. Using historical records as a means to calibrate variola evolutionary history, their results indicated that smallpox spread from Asia as much as 1,600 years ago which neatly backed up the historical records of 4th Century China. By the time smallpox reached out of East-Asia, the ancient Greek and Roman civilisations were no more - hinting that the reason they didn't observe smallpox was because at that time, in the Mediterranean region there wasn't any variola virus transmission. Despite this, analysis of the second major clade suggested a split 6,300 years ago placing variola well into ancient history. So, is smallpox a very old or relatively recent human pathogen? And, if so, where did it occur? The molecular data also showed that the clinically 'minor' forms of smallpox - African minor and Alastrim minor are very much related to the major viruses; evolutionarily speaking these viruses are thus very smilier.

A rodent origin of smallpox?

We can investigate the origin of smallpox through the molecular characterisation of other poxviruses. Variolataterapox virus) and camelpox viruses and they all are more related to each other than to other poxviruses, such as monkeypox. When their genomes were compared to that of variola, a time since divergence was estimated at between 16,000 and 68,000 years ago. As taterapox and camelpox are primarily found throughout Africa and Asia this suggests a possible origin of variola and the other poxviruses from ancient endemic poxviruses in Africa, possibly from rodents. Upon human infection this virus may have followed us out of Africa entering Asia and spreading across the globe. Or possibly, the virus emerged in rodent populations only to pop up again in Asia thousands of years later.

Although this period is quite a bit before the development of human agriculture and increased population density as is possibly required for such a highly infectious and lethal virus like smallpox to persist, the ancient variola virus might have behaved very differently from the one we know and fear. Sadly, we cannot say for sure exactly how and where variola emerged because we simply do not know a lot about the natural diversity of poxviruses in rodents or other mammal species and until we do, we will not have an accurate answer.

Future Pox?

Monkeypox from bushmeat? www.lynnjohnsonphoto.com/

What does this history of smallpox say about its impending eradication and the threat of a future virus emergence? Well, sometime in our ancient past variola virus emerged into prehistoric human populations and the data indicate that this may well have occurred within the African continent or nearby in Asia and it is likely that this same ancestral virus emerged into other mammalian species, such as Camels and Rodents. Smallpox then might have followed us through our journey out of Africa and became endemic in large population centres across the Eurasian landmass.

Although we have now effectively eradicated variola from the human population - and soon seek to destroy its last remaining stocks - might another poxvirus emerge just like it did before? We are now in a situation where the human population has very little immunity to variola and other related poxviruses, a situation which last would have existed prior to the initial emergence of smallpox. This provides ample breeding ground for novel poxviruses to emerge and fill the niche emptied by waning population immunity. Alarmingly, the rate of monkeypox infections - another rodent poxvirus - has been increasing (20X)  in the last 3 decades following cessation of smallpox vaccination. May this, or another related poxvirus, be the new smallpox? Could existing smallpox stocks not be used to study poxvirus/human interactions? May it be premature to destroy them in light of possible future pandemics? Only the WHO can decide later ths year.

Gubser C, & Smith GL (2002). The sequence of camelpox virus shows it is most closely related to variola virus, the cause of smallpox. The Journal of general virology, 83 (Pt 4), 855-72 PMID: 11907336

Li, Y., Carroll, D., Gardner, S., Walsh, M., Vitalis, E., & Damon, I. (2007). From the Cover: On the origin of smallpox: Correlating variola phylogenics with historical smallpox records Proceedings of the National Academy of Sciences, 104 (40), 15787-15792 DOI: 10.1073/pnas.0609268104

Raymond S. Weinstein (2011). Should Remaining Stockpiles of Smallpox Virus (Variola) Be Destroyed? Emerg Infect Dis, 17 (Apr) : 10.3201/eid1704.101865

Rimoin AW, Mulembakani PM, Johnston SC, Lloyd Smith JO, Kisalu NK, Kinkela TL, Blumberg S, Thomassen HA, Pike BL, Fair JN, Wolfe ND, Shongo RL, Graham BS, Formenty P, Okitolonda E, Hensley LE, Meyer H, Wright LL, & Muyembe JJ (2010). Major increase in human monkeypox incidence 30 years after smallpox vaccination campaigns cease in the Democratic Republic of Congo. Proceedings of the National Academy of Sciences of the United States of America, 107 (37), 16262-7 PMID: 20805472

“The virus is ‘dead’. Long ‘live’ the virus.” – what does history say about how virology should develop in the future?

In the modern world, we are continuously challenged by viral disease; well established pathogens such as the measles and mumps viruses alongside recently (re)-emerging viruses such as ebola-virus and even those viruses which we currently know little about (XMRV?) all represent a continuous threat to human health and well-being. Yet how can this be true when we have been developing anti-viral vaccines for half a decade – surely we should be good at it by now? And, is this idea that we can easily eradicate viruses hollow, considering how relatively easy it may be to recreate long extinct pathogens from their nucleic acid sequence alone? These investigations will require years of research and billions of dollars in funding but how should it achieved?

NEIDL building in Boston

In a recent article (UK Society for General Microbiology publication 'Microbiology Today', can be found here), Paul Duprex and  Elke Mühlberger, both virologists from the Boston UniversitySchool of Medicine and associated National Emerging Infectious Disease Laboratories (NEIDL) put forward their view on how best virology may be able to face up to this global challenge and outline how it may be achieved. Through a firm grasp of its historical context, combined with recent developments in molecular biology, future scientists will better be able to understand the intertwined relationship between viral pathogenesis and its rational attenuation. If we understand how viruses cause disease at the molecular level, altering this through well-established DNA technologies we may be able to mitigate pathogenesis and develop improved or novel vaccines – and antivirals - on a rational level.

In the early days of virology (see site on the history of vaccines), bent on developing vaccines under the paradigm of “isolate, attenuate and vaccinate”, scientists barely understood the mechanisms behind the production of live-attenuated vaccines, such as those for measles and smallpox. They didn’t need to; they worked superbly and were of course highly effective allowing for the eradication of one of the worst diseases of mankind. But this golden age didn’t last long, with countless viruses proving somewhat more resistant to this ‘black box’ method of vaccinology; HIV-1, SARS and Ebola had not yet been observed by scientists and nothing was known about them. This was an age concentrated on investigating viral pathogenesis and how best to change it but with the developments of recombinant DNA methodology (two important papers concerning virus cloning and synthetic virology: 1 and 2) this agenda shifted in favour of the virus genome and it is hard to even outline the tremendous impact this molecular understanding of viruses has had on both basic and applied virology. Yet bear in mind that it is this same technology that could facilitate the resurrection and recreation of ‘eradicated’ virues.

Knowledge of the molecular biology of viruses (in this case measles virus) will go a long way in developing much needed novel, rational vaccines

Despite this word of caution, Duprex and Mühlberger argue that virology has – or at least should – come back full circle, back to understanding basic pathogenesis with the aim in mind of developing more effective therapies and vaccines; this, they say, is needed now more so than ever. This generation, and the next, of molecular virologists should take heed of the long historical roots their discipline has and highlight the importance of understanding disease and attenuation as two sides of the same coin. This of course, would allow for a better grasp of the basic biology of these long established pathogens; those viruses which are now extinct but which may resurface, or even those viruses which are constantly in our minds as agents of natures bioterrorism.  They conclude that “a long overdue renaissance in vaccinology has commenced and it is with anticipation and excitement that we wait to see progress in the next decade”.

Mahalingam S, Damon IK, & Lidbury BA (2004). 25 years since the eradication of smallpox: why poxvirus research is still relevant. Trends in immunology, 25 (12), 636-9 PMID: 15530831

Mueller S, Coleman JR, Papamichail D, Ward CB, Nimnual A, Futcher B, Skiena S, & Wimmer E (2010). Live attenuated influenza virus vaccines by computer-aided rational design. Nature biotechnology, 28 (7), 723-6 PMID: 20543832

Racaniello VR, & Baltimore D (1981). Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome. Proceedings of the National Academy of Sciences of the United States of America, 78 (8), 4887-91 PMID: 6272282

Wimmer E, Mueller S, Tumpey TM, & Taubenberger JK (2009). Synthetic viruses: a new opportunity to understand and prevent viral disease. Nature biotechnology, 27 (12), 1163-72 PMID: 20010599

Seeing the big picture of RNA virus evolution

From both a medical and a scientific viewpoint, the evolution of viruses is extremely important to us;  viral adaptation to their ever changing environment is responsible for major morbidity and mortality worldwide so maybe studying this  may allow us to predict virus evolution in the future and may help prevent pandemics occuring?

We kind of know a lot about how viruses evolve at the small-scale; we know how viruses generally create genetic diversity - mutations - and how processes such as natural selection and genetic drift act on these genetic changes and influence the way viral genomes change over time. What we don't know however, is how viruses change at the larger-scale - how these above processes influence viral genomes over thousands of years, including: how and why viruses speciate, how their genome structure evolves and how and when do new viruses originate.

[caption id="" align="aligncenter" width="375" caption="An example phylogenetic tree - The paramyxoviridae containing a number of important human and animal pathogens. Notice the host/viral species distribution."][/caption]

A recent study, investigating the evolution of a number of RNA viruses has sought to reconcile this lack of understanding by attempting to assess virus 'macroevolution'- specifically viral speciation. By generating large and highly robust phylogenetic trees (using significantly highly conserved amino-acid sequences of a single viral protein) for 5 genera of RNA virus including: the Alphaviruses, Caliciviruses, Paramyxoviruses, Rhabdoviruses and the Flaviviruses, the team were able to map the host species of each virus species onto the trees and this allowed them to infer the mode of speciation of each virus genus.

More specifically they asked: Do closely related viral species infect the same host and are therefore believed to have speciated in that host or do they infect completely different hosts which are believed to have speciated following host jumping?

What is a virus species?

[caption id="" align="aligncenter" width="300" caption="General modes of speciation - we may think of viruses speciating by either allopatric (host shift) or sympatric (intra-host divergence)."][/caption]

The concept of the viral species has been a hard one to determine becuase viruses don't reproduce sexually. It is generally thought to be rather a arbitrary classification, however, most virus 'species' tend to be phylogenetically and often phenotypically stable genetic lineages and hence may be thought of as 'biological relevant'. We may think of viral speciation much like we think of speciation in the classic sense: allopatric or 'geographical speciation' (virus adaptation to a new host species) and sympatric - that not requiring the forces of georgaphic isolation (generation of viral speciation within a single host). Virus sympatric speciation requires the adapation to a new infectious niche within a host, for example a new lineage may infect new cell types within that host. Virus allopatric speciation requires host-jumping or adaptation to a new host altogether but may result from co-divergence follwoing host speciation. Both processes may result in two or more 'stable, phylogenetic and phenotypic genetic lineages. But what does the data say about it - how do viruses evolve in the real world?

What the data says

The results were split - at leats 50% were found to have 'speciated' via sympatric-like processes and half from allopatric-like processes. The group stress, however, that a major caveat of this study is that it highlights our limited understanding of what specific host speces particular viruses infect; in this study most hosts were classified as 'birds' or 'plants' or 'Carnivores' which limits the resolution of phylogenetic studies and leads to the overestimation of sympatric speciation events which would otherwise not exist if exact hosts were known. This leads us to put little confidence on our earlier 50/50 estimate and most likely the role of sympatric speciation would be a lot less important than allopatric modes of speciation in reality.

Why do RNA viruses evolve this way - What controls viral speciation?

So, we may say that most RNA virus speciation is caused by allopatric modes - or host jumping, but this may seem counterintuitive as there are some major barriers to viral emergence. The group argue, however, that it may take a lot more - genetically speaking - for a virus to speciate within a host than it does for a virus to jump species - eg. replicate in a new cell type/alter antigenic epitopes. The apparant preference for allopatric speciation may be controlled by intrinsic biological factors of these RNA viruses, namely: their extremely small genome size which effectively constrains evolutionary innovation. Those changes required for host jumping (change in receptor binding sites for instance) may be relatively minor when compared to those and the more closely related the host species are then the more easily host-jumping will occur - which is what we see here.

This study highlights the key role that viral 'allopatric' speciation or host - jumping plays in the evolution of RNA viruses yet further emphasizes the need to better study and understand viral biodiversity and host range in the wild - not only focusing on those medically important human viruses. Further research may be carried out on the molecular barriers to both cellular and host switching for these RNA viruses. This study will act as a model system that may be applied to other viral lineages - what about the RNA viruses with segmented genomes? What about the DNA viruses? Retroviruses?


Kitchen A, Shackelton LA, & Holmes EC (2011). Family level phylogenies reveal modes of macroevolution in RNA viruses. Proceedings of the National Academy of Sciences of the United States of America, 108 (1), 238-43 PMID: 21173251