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:

WORD->WORE->GORE->GONE->GENE

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 www.microbe.tv/twiv.

Why do viruses cause disease?

EvolutionVirulence, the capacity to cause disease, varies markedly among viruses. Some viruses cause lethal disease while others do not. For example, nearly all humans infected with rabies virus develop a disease of the central nervous system which ultimately leads to death. In contrast, most humans are infected with circoviruses with no apparent consequence. Is there a benefit for a virus to be virulent?

One explanation for viral virulence is that it facilitates transmission. However, a comparison of infections caused by two enteric viruses, poliovirus and norovirus, does not support this general view. Both viruses infect the gastrointestinal tract and are spread efficiently among humans by fecal contamination. However, norovirus infection causes vomiting and diarrhea, while poliovirus infection of the intestine is without symptoms (the rare invasion of the nervous system, and subsequent paralysis, is an accidental dead end). Both viruses have successfully colonized humans for many years, so why does only one of them cause gastrointestinal tract disease?

Two recent studies of bacterial virulence provide some clues about the evolution of virulence. In one a commensal strain of Escherichia coli was serially propagated in the presence of macrophages, which are cells of the immune system that take up and destroy the bacteria. After many such passages, bacterial clones were isolated that escape phagocytosis and killing by macrophages. These clones had also acquired increased pathogenicity in mice. In other words, the genetic changes that allowed the bacteria to evade the immune response also lead to increased virulence.

In another example of evolution to virulence, it was found the the bacterium Pseudomonas aeruginosa can sense the presence of competing gram-positive bacteria because the latter shed the cell wall component peptidoglycan. In response to this molecule, P. aeruginosa secretes proteins that kill the other bacteria. These secreted proteins also make the bacterium more virulent in a host – in their absence, the bacteria are less virulent. In other words, P. aeruginosa damages its host in an attempt to remove nearby bacterial competitors.

In both bacterial examples, virulence can be viewed as collateral damage: the consequence of evading the immune response, or killing off competitors. Being virulent was not the primary goal. This explanation for bacterial virulence is straightforward and compelling: virulence is not directly selected for during evolution but comes along for the ride. Can it be applied to viruses?

All eukaryotic viruses must encode at least one protein that antagonizes host immune responses, otherwise they would be eliminated. These immune evasion proteins are certainly virulence factors: in general, when they are deleted or altered, the capacity of the virus to cause disease in a host is reduced. Like bacterial virulence, viral virulence might be collateral damage incurred by having to evade immune responses. This hypothesis is attractive but seems overly simplistic. If the ubiquitous and benign circoviruses did not evade host responses, then they would be eliminated from the human population.

The reasons why some viruses are virulent and others are not remain elusive. It is possible to reduce viral virulence by mutation, but this type of experiment does not reveal why viruses cause disease. The inverse experiment would be more informative: to select from a population of avirulent virus those that can cause disease. The results of such an experiment would help to identify the selection pressures that allow viruses to evolve to virulence.

Changing influenza virus neuraminidase into a receptor binding protein

neuraminidaseThe hemagglutinin (HA) and neuraminidase (NA) glycoproteins of the influenza virus particle serve distinct functions during infection. The HA binds sialic acid-containing cellular receptors and mediates fusion of the viral and cell membranes, while the NA removes sialic acids from glycoproteins. Apparently this division of labor is not absolute: influenza viruses have been identified with NA molecules that serve as receptor binding proteins.

An influenza virus was created that could not bind sialic acid by introducing multiple mutations into the HA gene. This mutant virus was not expected to be infectious, but nevertheless did propagate to moderate titers in cell culture. A single amino acid change was identified in the NA protein of this virus: G147R, which is just above the active site of the enzyme (illustrated; active site marked with green spheres). Passage of the virus in cell culture produced a virus that multiplied to higher titers; improved growth was caused by a K62E change in the HA stalk. The results of site-directed mutagenesis showed that the G147R change allowed the NA protein to serve the receptor binding function normally provided by HA. It is not clear how the HA change leads to improved growth of the G147 virus.

Although the G147R NA can serve as receptor binding protein, the HA is still required for fusion: abolishing this activity by mutation or by treatment with a fusion-blocking antibody did not allow virus growth.

The influenza NA protein is an enzyme (sialidase) that cleaves sialic acids from cellular and viral proteins. The G147R NA is active as a sialidase, and this activity can be blocked by the antiviral compound oseltamivir, which is an NA inhibitor. Treatment of G147R-containing virus with oseltamivir also blocked virus binding to cells. Virus-like particles that contain G147R NA but not HA can attach to sialic acid-containing red blood cells. This attachment can be reversed by oseltamivir. After binding to red blood cells, these virus-like particles slowly fall off, a consequence of NA cleaving sialic acid receptors. These observations indicate that the G147R NA binds to sialic acids at the active site of the enzyme, and cleaves the same receptor that it binds.

Treatment of cells with a bacterial sialidase that removes a broad range of sialic acids only partially inhibits G147R NA-mediated binding to cells. In contrast, growth of wild type influenza virus is completely blocked by this treatment. Therefore the receptor recognized by G147R NA is not the same as that bound by wild type virus.

Changing the influenza virus NA to a receptor binding protein is not simply a laboratory curiosity: the G147R NA change was found in 31 of 19,528 NA protein sequences in the Influenza Virus Resource. They occur in seasonal H1N1 viruses that circulated before 2009, in the 2009 swine-origin pandemic H1N1 virus, and in avian H5N1 viruses. The presence of this change in phylogenetic clusters of seasonal H1N1 and chicken H5N1 sequences suggests that they are also found in circulating viruses, and are not simply sequence errors or the product of passage in the laboratory.

These observations emphasize the remarkable flexibility of the influenza viral glycoproteins in their ability to switch receptor binding function from HA to NA. They might also have implications for vaccines, whose effectiveness are thought to depend largely on the induction of antibodies that block the function of HA protein. The work underscores the importance of serendipity in science: the HA receptor binding mutant virus was originally produced as a negative control for a different experiment.

TWiV 242: I want my MMTV

On episode #242 of the science show This Week in Virology, the complete TWiV team talks about how two different viruses shape the evolution of an essential housekeeping protein.

You can find TWiV #242 at www.microbe.tv/twiv.

Dual virus-receptor duel

transferrin receptorViruses are obligate intracellular parasites: they must enter a cell to reproduce. To gain access to the cell interior, a virus must first bind to one or more specific receptor molecules on the cell surface. Cell receptors for viruses do not exist only to serve viruses: they also have cellular functions. An example is the transferrin receptor, which regulates iron uptake and assists in the entry of viruses from three different families. It might appear that such dual-use proteins cannot evolve to block virus entry because their cellular function would then be compromised. A study of two viruses that bind to the same cell surface receptor protein reveals how a cellular protein can change to prevent infection without affecting its role in the cell.

The virus-cell receptor interaction is one of the many arenas where the evolution of host-virus conflict can be studied. Because the virus-receptor interaction is essential for viral replication, host cells with a mutation in the receptor gene that prevents virus infection survive and eventually dominate the population. A virus could overcome this block with an amino acid change allowing binding to the altered receptor. Mutations that alter the interaction to favor the virus or the host are called ‘positively selected’ mutations. Such back-and-forth evolution between viruses and their host cells has been called host-virus arms races. Most have been identified by studying antiviral genes. This study is unusual in that it involves a housekeeping gene that has been usurped for viral attachment.

Evidence for positive selection of host genes can be detected by comparing gene sequences of phylogenetically related species. Nonsynonymous mutations lead to a change in the amino acid sequence, while synonymous mutations do not. The rate at which nonsynonymous mutations occur in the genome is typically much slower than synonymous mutations. The reason for this difference is that most mutations that change the amino acid sequence of a protein are lethal to the host. When genes have been subjected to positive selection by a virus, the ratio of nonsynonymous to synonymous mutations is higher, typically in host amino acids that interact with viral proteins. Computer programs have been designed to scan gene sequences and identify codons which are under positive selection by virtue of a high ratio of nonsynonymous to synonymous mutations.

To determine if the transferrin receptor (TfR1) has evolved to prevent virus attachment, sequences of the protein from seven different rodent species were compared. The analysis revealed that much of the protein is highly conserved, but a small part, comprising six amino acids, is evolving rapidly. Three of these amino acids  are located on the part of TfR1 that binds arenaviruses, and three are at the binding site for the retrovirus mouse mammary tumor virus (MMTV) (see illustration). Changing these three amino acids of TfR1 of the house mouse, which is susceptible to MMTV, to the sequence found in TfR1 of the MMTV-resistant vesper mouse, blocked entry of the virus into cells. In turn, changing these three amino acids of TfR1 of the MMTV-resistant short-tailed zygodont to the sequence of the house mouse enabled virus entry into cells. None of these changes had an effect on ferritin binding by TfR1.

Evidence for positive selection can also be detected in viral genes encoding proteins that interact with the host. The arenavirus glycoprotein, GP, is known to bind to TfR1. Ten GP amino acids were identified that are under positive selection, and four of these directly contact TfR1.

These findings demonstrate that there has been an arms race between TfR1 and both an arenavirus and retrovirus. An interesting question is whether human TfR1 will enter into an arms race with arenaviruses. As these viruses emerge into the human population, it is expected that humans with mutations that make them less susceptible to infection or severe disease will be positively selected. Amino acid 212 of human TfR1, which is near the positively selected resides in murine TfR1, varies in the human population. When this amino acid change (leucine to valine) is introduced into TfR1, it confers some protection against arenavirus entry. Curiously, this polymorphism has only been found in Asian populations, where arenaviruses that bind TfR1 are not found. The polymorphism is probably neutral with respect to TfR1 function, and if TfR1-binding arenaviruses are introduced into Asia, this change could be positively selected.

Because all viruses depend on many host proteins for replication, it will be interesting to use this approach to see how other highly conserved cell proteins balance cell function with the ability to resist virus infections. There are like to be many cell proteins that cannot change to evade viral use without destroying their cell function. Fortunately for cells there are exceptions.

TWiV 237: Paleovirology with Michael Emerman

Episode #237 of the science show This Week in Virology was recorded at the Fred Hutchinson Cancer Research Center in Seattle, WA, where Vincent and Rich met up with Michael to talk about his work on the molecular and evolutionary basis of HIV replication and pathogenesis.

You can find TWiV #237 at www.microbe.tv/twiv.