Permissive vaccines and viral virulence

chicken farmA permissive vaccine prevents disease in the immunized host, but does not block virus infection. Would a permissive vaccine lead to the emergence of more virulent viruses?

This hypothesis is based on the notion that viruses which kill their hosts too quickly are not efficiently transmitted, and are therefore removed by selection. However a vaccine that prevents disease, but not viral replication in the host, would allow virulent viruses to be maintained in the host population. It has been suggested that in this scenario, viruses with increased virulence would be selected if such a property aids transmission between hosts.

On the surface this hypothesis seems reasonable, but in my opinion it is flawed. One problem is that increased transmission might not always be associated with increased virulence. The more serious flaw lies in making anthropomorphic assessments of what we think viruses require, such as concluding that increased viral transmission is a desired trait. Our assumptions fail to recognize the main goal of evolution: survival. Evolution does not move a virus along a trajectory aimed at perfection. Change comes about by eliminating those viruses that are not well adapted for the current conditions, not by building a virus that will fare better tomorrow. All the viruses on Earth today transmit well enough, or they would not be here; yet some kill their hosts clearly much faster than others. The fact is that humans have little understanding of what drives virus evolution in large populations. Our assumptions of what constitute the selective forces are usually tainted by anthropomorphism.

This long preamble is an introduction to a series of findings which are purported to support the idea that permissive vaccines (the authors call them ‘leaky’ and ‘imperfect’ vaccines but I dislike both names because they imply defects) can lead to the selection of more virulent viruses. The subject of the paper is Marek’s disease virus (MDV), a herpesvirus that infects chickens. MDV is shed from feather follicles of infected chickens and is spread to other birds when then inhale contaminated dust. Vaccines have been used to prevent MDV infection since the early 1970s. These vaccines prevent disease, but do not block viral replication, and vaccinated, infected birds can shed wild type virus. The virulence of MDV has been increasing since the 1950s, initially from a paralytic disease, to paralysis and death. The authors wonder if the use of permissive Marek’s vaccines has lead to the selection of more virulent viruses.

To address their hypothesis, the authors inoculate vaccinated or unvaccinated chickens with a series of MDV isolates that range from low to high virulence. Unvaccinated chickens inoculated with the most virulent MDV died within a week and shed little virus. In contrast, most vaccinated birds survived infection with virulent viruses, and shed virus for the length of the experiment, 56 days.

A transmission experiment was done to determine if shed virus could infect other birds. The authors infected vaccinated or unvaccinated birds and asked if sentinel, unvaccinated chickens became infected. Unvaccinated birds died within 10 days after infection with virulent MDV, and did not transmit infection. In contrast, vaccinated birds survived at least 30 days, and co-housed sentinel animals became infected and died.

The experiments are well done and the conclusions are clear: more virulent Marek’s disease viruses replicate longer in vaccinated than unvaccinated chickens, and can be readily transmitted to other chickens. But these results do not prove that more virulent MDV arose because of permissive vaccines. Nor do the results prove in general that leaky vaccines lead to selection of more virulent viruses. The results simply show that a vaccine that does not prevent replication will allow transmission of virulent viruses.

To prove that vaccinated chickens can allow the selection of more virulent viruses, vaccinated chickens could be infected with an avirulent virus, and the shed virus collected and used to infect additional, vaccinated birds. This process could be repeated to determine if more virulent viruses arise. While the results of this gain-of-function experiment would be informative, they would be done in a controlled laboratory setting which would not duplicate all the selective forces present on a poultry farm.

The authors note that most human vaccines do prevent replication of infecting virus. They do not mention the one important exception: the Salk poliovirus vaccines. People who are immunized with the Salk vaccine can be infected with poliovirus, which will then replicate in the intestines, be shed in the feces, and transmitted to others. This behavior has been well documented in human populations, yet the virulence of poliovirus has not increased for the 60 years during which the Salk vaccine has been used.

I do not feel that these experimental results have general implications for the use of any animal vaccine. It is unfortunate that the work has been covered in many news sources with the incorrect implication that vaccines may be responsible for the emergence of more virulent viruses.

TWiV 335: Ebola lite

On episode #335 of the science show This Week in Virology, the TWiVumvirate discusses a whole Ebolavirus vaccine that protects primates, the finding that Ebolavirus is not undergoing rapid evolution, and a proposal to increase the pool of life science researchers by cutting money and time from grants.

You can find TWiV #335 at

Describing a viral quasispecies

QuasispeciesVirus populations do not consist of a single member with a defined nucleic acid sequence, but are dynamic distributions of nonidentical but related members called a quasispecies (illustrated at left). While next-generation sequencing methods have the capability of describing a quasispecies, the errors associated with this technology have limited progress in our understanding of the genetic structure of virus populations. A new method called CirSeq reduces next-generation sequencing errors to allow an accurate description of viral quasispecies.

The key to eliminating sequencing errors is a clever approach based on the conversion of viral RNAs to circular molecules. When copied with reverse transcriptase, tandemly repeated cDNAs are produced (illustrated below). Mutations in the original viral RNA will be shared by all repeats derived from a circle, but not errors produced during copying or sequencing. The latter can be computationally subtracted, reducing sequencing error to a point that is much lower than the estimated mutation rate of an RNA virus.CirSeq

CirSeq was used to characterize poliovirus populations produced by seven serial passages in HeLa cells. The calculated mutation frequency, 2 X 10-4 mutations per nucleotide, was substantially lower compared with estimates determined by conventional sequence analysis. Over 200,000 sequence reads per nucleotide position were used to detect >16,500 variants per population per passage. This number represents ~74% of all possible alleles. Many mutations were detected at nearly all positions in the viral RNA. Most mutations occur at a frequency between 1 in 1000 to 1 in 100,000. The conclusion is that the virus population produced in HeLa cells consists mainly of genomes with the consensus sequence, and small amounts of many variant genomes. These variants are only those that give rise to viable viruses; lethal mutations are not observed.

CirSeq was also used to calculate the mutation rate of poliovirus. The rates vary according to type: transitions occurred at a rate of 2.5 X 10-5 to 2.6 X 10-4 substitutions per site, while transversions were observed at a rate of 1.2 X 10-6 to 1.5 X 10-5 substitutions per site. Nucleotide-specific differences in mutation rate were also observed: C to U and G to A transitions were 10 times more frequent than U to C and A to G. These rates are consistent with previously determined values using other methods.

This method can also be used to determine the fitness of each base at every position in the genome, according to changes observed during the seven passages in HeLa cells. This analysis allows determination of which bases are neutral, and which are selected, and when combined with analysis of protein structure, can provide new insights into viral functions.

By enabling a sequencing approach that gives an accurate description of virus populations at a single-nucleotide level, CirSeq can be used to provide an unprecedented view of how virus populations change during evolution.

TWiV 332: Vanderbilt virology

On episode #332 of the science show This Week in Virology, Vincent visits Vanderbilt University and meets up with Seth, Jim, and Mark to talk about their work on a virus of Wolbachia, anti-viral antibodies, and coronaviruses.

You can find TWiV #332 at

Viral genomes in 700 year old caribou scat

CaribouRecovering viral genomes from ancient specimens can provide information about viral evolution, but not many old nucleic acids have been identified. A study of 700 year old caribou feces reveals that viruses can be protected for long periods of time – under the right conditions.

The oldest virus recovered so far is the giant Pithovirus sibericum, which was isolated from 30,000 year old Siberian permafrost. Other attempts have yielded fragments of viral genomes. It was possible to reconstruct the 1918 influenza virus from small RNAs recovered from formalin fixed and frozen human tissues. However this feat was not achieved for viral RNA in 140,000 year old Greenland ice cores, 900 year old North African barley grains, or 7,000 year old Black Sea Sediments.

Caribou feces have been frozen for the past 5,000 years in ice patches in the Selwyn Mountains of the Canadian Northwest territories. To determine if viruses could be recovered from this material, the frozen feces were thawed, resuspended in buffer, filtered, and treated with nucleases to destroy any nucleic acids not contained within a viral capsid. Sequence analysis of the remaining nucleic acids revealed two different viruses.

Ancient caribou feces associated virus (aCFV) has a single stranded, circular DNA genome distantly related to plant-infecting geminiviruses and gemycircularviruses. The entire 2.2 kb genome of aCFV was amplified from the caribou feces specimen. This reconstructed viral DNA replicated upon introduction into tobacco plant leaves.

Sequences of an RNA virus distantly related to picornaviruses of insects (such as Drosophila C virus) were also identified in the caribou feces. These viral genomes exceed 7.4 kb, but it was only possible to recover a 1.8 kb fragment of this virus, ancient Northwest Territories cripavirus (aNCV).

Neither virus was isolated from contemporary Caribou feces collected from an animal living in the same region. The authors also went to great pains to demonstrate that the two 700 year old viral genomes were not contaminants. The isolation was repeated in a different laboratory, and was not to be a consequence of contamination from any laboratory reagent or apparatus used for purification of nucleic acids.

It is not likely that aCFV or aNCV infected a caribou 700 years ago. The viruses were probably acquired when a caribou ingested plant material infected with the plant virus; perhaps insects harboring aNCV were also present on these plants. The exact hosts for both viruses are unknown.

The fact that two relatively large fragments of viral DNA and RNA were identified suggests that intact capsids were present in the caribou feces. Their preservation is probably a consequence of the low temperature of the arctic ice, and the stable icosahedral capsids characteristic of members of geminiviruses, gemycircularviruses, and cripaviruses.

We already know that viruses have been around for a long time, more than hundreds of millions of years, so what is the value of this work? Studying ancient viruses can provide insight into viral diversity and evolution. However, the value of two viral genome sequences is limited, and additional work should be done to acquire additional specimens spanning a long period of time. Similar sampling of other environments would also be desirable, but it is unlikely that large fragments of viral genomes can be recovered from specimens that are not frozen. And as the ice caps melt away, we will lose our ability to decode this important viral record.

Image credit

What we are not afraid to say about Ebola virus

sneezeIn a recent New York Times OpEd entitled What We’re Afraid to Say About Ebola, Michael Osterholm wonders whether Ebola virus could go airborne:

You can now get Ebola only through direct contact with bodily fluids. If certain mutations occurred, it would mean that just breathing would put one at risk of contracting Ebola. Infections could spread quickly to every part of the globe, as the H1N1 influenza virus did in 2009, after its birth in Mexico.

Is there any truth to what Osterholm is saying?

Let’s start with his discussion of Ebola virus mutation:

But viruses like Ebola are notoriously sloppy in replicating, meaning the virus entering one person may be genetically different from the virus entering the next. The current Ebola virus’s hyper-evolution is unprecedented; there has been more human-to-human transmission in the past four months than most likely occurred in the last 500 to 1,000 years.

When viruses enter a cell, they make copies of their genetic information to assemble new virus particles. Viruses such as Ebola virus, which have genetic information in the form of RNA (not DNA as in other organisms), are notoriously bad at copying their genome. The viral enzyme that copies the RNA makes many errors, perhaps as many as one or two each time the viral genome is reproduced. There is no question that RNA viruses are the masters of mutation. This fact is in part why we need a new influenza virus vaccine every few years.

The more hosts infected by a virus, the more mutations will arise. Not all of these mutations will find their way into infectious virus particles because they cause lethal defects. But Osterholm’s statement that the evolution of Ebola virus is ‘unprecedented’ is simply not correct. It is only what we know. The virus was only discovered to infect humans in 1976, but it surely infected humans long before that. Furthermore, the virus has been replicating, probably for millions of years, in an animal reservoir, possibly bats. There has been ample opportunity for the virus to undergo mutation.

More problematic is Osterholm’s assumption that mutation of Ebola virus will give rise to viruses that can transmit via the airborne route:

If certain mutations occurred, it would mean that just breathing would put one at risk of contracting Ebola. Infections could spread quickly to every part of the globe, as the H1N1 influenza virus did in 2009, after its birth in Mexico.

The key phrase here is ‘certain mutations’. We simply don’t know how many mutations, in which viral genes, would be necessary to enable airborne transmission of Ebola virus, or if such mutations would even be compatible with the ability of the virus to propagate. What allows a virus to be transmitted through the air has until recently been unknown. We can’t simply compare viruses that do transmit via aerosols (e.g. influenza virus) with viruses that do not (e.g. HIV-1) because they are too different to allow meaningful conclusions.

One approach to this conundrum would be to take a virus that does not transmit among mammals by aerosols – such as avian influenza H5N1 virus – and endow it with that property. This experiment was done by Fouchier and Kawaoka several years ago, and revealed that multiple amino acid changes are required to allow airborne transmission of H5N1 virus among ferrets. These experiments were met with a storm of protest from individuals – among them Michael Osterholm – who thought they were too dangerous. Do you want us to think about airborne transmission, and do experiments to understand it – or not?

The other important message from the Fouchier-Kawaoka ferret experiments is that the H5N1 virus that could transmit through the air had lost its ability to kill. The message is clear: gain of function (airborne transmission) is accompanied by loss of function (virulence).

When it comes to viruses, it is always difficult to predict what they can or cannot do. It is instructive, however, to see what viruses have done in the past, and use that information to guide our thinking. Therefore we can ask: has any human virus ever changed its mode of transmission?

The answer is no. We have been studying viruses for over 100 years, and we’ve never seen a human virus change the way it is transmitted.

HIV-1 has infected millions of humans since the early 1900s. It is still transmitted among humans by introduction of the virus into the body by sex, contaminated needles, or during childbirth.

Hepatitis C virus has infected millions of humans since its discovery in the 1980s. It is still transmitted among humans by introduction of the virus into the body by contaminated needles, blood, and during birth.

There is no reason to believe that Ebola virus is any different from any of the viruses that infect humans and have not changed the way that they are spread.

I am fully aware that we can never rule out what a virus might or might not do. But the likelihood that Ebola virus will go airborne is so remote that we should not use it to frighten people. We need to focus on stopping the epidemic, which in itself is a huge job.

A WORD on the constraints of influenza virus evolution

NP evolutionEvolution proceeds by selection of mutants that arise by error-prone duplication of nucleic acid genomes. It is believed that mutations that are selected in a gene are dependent on those that have preceded them, an effect known as epistasis. Analysis of a sequence of changes in the influenza virus nucleoprotein provides clear evidence that stability explains the epistasis observed during evolution of a protein.

Evolutionary biologist John Maynard Smith used an analogy with a word game to explain how epistasis constrains the evolution of a protein. In this game, single letter changes are made to a four letter word to convert it to another valid word:


Although all the intermediates are valid words, the sequence of changes is important. For example, the G in GENE, if introduced into WORD would produce GORD which is not a word. D must be changed to E before W is changed to G. In a similar way mutations in a gene are likely to depend on the changes that have previously taken place.

Whether similar constraints affect protein evolution has been studied with the nucleoprotein (NP) of influenza virus. Between 1968 and 2007, 39 mutations appeared in the NP RNA of influenza virus H3N2. Because sequences of this viral RNA are available each year, it was possible to deduce the order in which these changes appeared in the viral genome (illustrated; figure credit). Plasmids encoding 39 different NP proteins were then constructed which represent viral NP sequences present from 1968 through 2007. All of the NP proteins were found to support similar levels of viral RNA synthesis.

The 39 mutations were then introduced singly into the NP RNA, and RNA synthesis was measured. Three of the altered proteins had large decreases in activity. Their presence also substantially reduced the growth of infectious viruses. However when these NP changes were combined with the amino acid changes that preceded it during evolution, replication was normal. The three NP changes that reduce viral RNA synthesis and replication also decrease the thermal stability of the protein.

These findings show that, from 1968-2007, three amino acid changes were fixed in the influenza virus NP protein whose deleterious effects on protein stability were compensated by previously accumulated changes in the protein. The three amino acids are located in a part of the protein that harbors sequences recognized by T cells. These changes likely allow the virus to escape the host immune response.

Protein stability clearly mediates the epistasis observed in the influenza virus NP protein. It will be important to determine which other protein properties determine the sequence of mutations that are fixed in a viral genome. Influenza viruses are ideal for this work because sequences of all of the viral RNAs are determined for multiple isolates on an annual basis. Studies of what regulates epistasis for other RNA and DNA viruses are also needed to provide an understanding of the constraints of viral evolution.

Virology question of the week: why a segmented viral genome?

influenza-reassortmentThis week’s virology question comes from Eric, who writes:

I’m working on an MPH and in one of my classes we are currently studying the influenza virus. I’d forgotten that the genome is in 8 separate parts. Curious, I’ve been searching but can’t find any information as to why that is?

What evolutionary advantage is conferred by having a segmented genome?

Terrific question! Here is my reply:

It’s always hard to have answers to ‘why’ questions such as yours. We answer these questions from a human-centric view of what viruses ‘need’. We might not be right. But I’d guess there are at least two important advantages of having a segmented RNA genome.

Mutation is an important source of RNA virus diversity that is made possible by the error-prone nature of RNA synthesis. Viruses with segmented genome have another mechanism for generating diversity: reassortment (illustrated).

An example of the evolutionary importance of reassortment is the exchange of RNA segments between mammalian and avian influenza viruses that give rise to pandemic influenza. The 2009 H1N1 pandemic strain is a reassortant of avian, human, and swine influenza viruses.

Having a segmented genome is another way to get around the limitation that eukaryotic mRNAs can only encode one protein. Viruses with segmented RNA genomes can produce at least one protein per segment, sometimes more. There are other ways to overcome this limitation – for example by encoding a polyprotein (picornaviruses), or producing subgenomic RNAs (paramyxoviruses).

Other segmented viral genomes include those of reoviruses, arenaviruses, and bunyaviruses.

There are various ways to achieve genetic variation and gene expression, and viruses explore all aspects of this space.

Virology question of the week

HIV binding CD4 and ccrOn the science show This Week in Virology we receive many questions and comments, which are read every week. I also get many questions here on virology blog, which I tend to answer by email. However I think that everyone could benefit from these questions, so I’ve decided to post one here each week along with my answer.

This week’s question is from Joseph, who wrote:

I’m relatively new to virology or anything biology-related. Hell, I’m studying computer science as an undergrad at the moment; however, there’s something about virology that fascinates me – the simplistic fact that we can’t cure viruses, which are less complex than bacterium (in which we can treat, and they’ll eventually pack their bags and leave).

I’ll get to my question … since most, if not all, cells in the body replicate and reproduce and none of them merge, why do our cells let virions in? You would think after years of viral/immune system encounters, our bodies would have adapted to repelling these viruses off. I understand it’s probably much more complicated than that, but I would love to hear your answer. Does it have anything to do with virions’ size being so small?

This is a great question. In fact, I had a similar question on a midterm examination in my virology course. I phrased it this way: Could cells evolve to not have receptors for binding viruses?

I sent this answer to Joseph:

Viruses get into cells by binding to proteins on the cell surface – viruses have evolved to do this: they are safecrackers.

You would think that the cells would evolve to change these proteins – and you would be right. Over thousands of years, the cell proteins change, so the viruses can’t bind anymore.

But guess what? The viruses change right back so that they can bind to the cell protein once more.

Now you might ask: why doesn’t the cell get rid of that surface protein? The answer there is that they are needed for the cell, so they can’t be removed.

There seems to be one exception to the last statement: about 4-16% of people of Northern European descent don’t make one of the receptors for HIV. They are resistant to infection. But this doesn’t happen for most other viruses.

Joseph wrote back:

Hmm. I thought by definition virions weren’t living organisms, yet they “adapt” to bind to living cells. Sounds like those emotional virions just can’t deal with rejection – that and our cells just aren’t as smart as we need them to be. I’m not sure if you are a Trekkie; however, it reminds me of the Borg and The Enterprise’s encounter – The Enterprise adapting to The Borg’s every frequency of their phasers, bypassing their bruteforce.

That does make sense that our cells do need that protein surface for energy; however, I never thought it would actually be the surface itself. Interesting.

I did read about that somewhere – because of the Bubonic Plague causing some genetic mutation, if I’m not mistaken.

To which I responded:

Virus particles are not alive – but once they infect a living cell they can evolve.

Both cells and viruses are smart – they both have managed to be around for a long time. We have great immune systems; virus infected cells can evolve very quickly. It’s an arms race.

Correct, one idea is that the mutation conferring resistance to HIV was acquired in the Plague, but that’s hard to prove.

The mutation we are discussing is of course ccr5delta32, which confers resistance to infection with HIV-1 (the illustration shows the HIV-1 glycoprotein binding CD4 and ccr, a chemokine receptor). You can read more about ccr5delta32 here or listen to us discuss it on TWiV #278. We also talked about virus-receptor arms races on TWiV #242, and I wrote about it here.

TWiV 275: Virocentricity with Eugene Koonin

On episode #275 of the science show This Week in Virology, Vincent and Rich meet up with Eugene Koonin to talk about the central role of viruses in the evolution of all life.

You can find TWiV #275 at