Zika virus, like all other viruses, is mutating

Zika virusNot long after the appearance of an outbreak of viral disease, first scientists, and then newswriters, blame it all on mutation of the virus. It happened during the Ebolavirus outbreak in West Africa, and now it’s happening with Zika virus.

The latest example is by parasitologist Peter Hotez, who writes in the New York Times:

There are many theories for Zika’s rapid rise, but the most plausible is that the virus mutated from an African to a pandemic strain a decade or more ago and then spread east across the Pacific from Micronesia and French Polynesia, until it struck Brazil.

After its discovery in 1947 in Uganda, Zika virus caused few human infections until the 2007 outbreak on Yap Island. The virus responsible for this and subsequent outbreaks in Pacific Islands is distinct from the African genotype, but there is no experimental evidence to suggest that sequence differences in the Asian genotype were responsible for the spread of the virus. For this reason I disagree with Dr. Hotez’ conclusion that mutation of the virus is the ‘most plausible’ explanation for its global spread. It is just as likely that the virus was in the right place at the right time to spark an outbreak in the Pacific.

We will never have experimental evidence that emergence of the Asian genotype allowed pandemic spread of Zika virus, because we cannot test the effect of individual mutations on spread of the virus in humans. Consider this experiment: infect a room of humans (and mosquitoes) with either the African or Asian genotype of Zika virus, then measure virus replication and transmission. If there is a difference between the two viruses, engineer specific mutations into the virus, reinfect another batch of humans, and continue until the responsible mutations are identified. Obviously we cannot do such an experiment! We could instead use animal models, but these have limitations in extrapolating results to humans. For this reason we have never identified any specific mutation that allows an animal virus to replicate more efficiently in humans.

The same experimental limitations do not apply to animals. An example is Chikungunya virus, spread by Aedes ageyptii mosquitoes. Before 2004, outbreaks of infection were largely confined to developing countries in Africa and Asia. The virus subsequently spread globally, due to a single amino acid change in the envelope glycoprotein which allows efficient replication in Aedes albopictus, a mosquito with a greater range than A. ageyptii. It was possible to prove this point by assessing the effects of changing this single amino acid on virus replication in mosquitoes. The same experiment cannot be done in humans.

There is no evidence that the Asian genotype of Zika virus is any more competent to replicate in mosquitoes than the African strain. Results of a study of replication of Asian genotypes of Zika virus revealed that Aedes aegypti and Aedes albopictus are not very good vectors for transmitting ZIKV. The authors smartly suggest that “other factors such as the large naïve population for ZIKV and the high densities of human-biting mosquitoes contribute to the rapid spread of ZIKV during the current outbreak.” In other words, don’t blame the Zika virus genome for the expanded range of the virus.

The Zika virus that has been spreading in Brazil, and which has been associated with microcephaly, shares a common ancestor with the Asian genotype. In a recent study of the genomes of 7 Brazilian isolates, there was no evidence that specific mutations are associated with microcephaly. Those authors conclude (also smartly):

Factors other than viral genetic differences may be important for the proposed pathogenesis of ZIKV; hypothesized factors include co-infection with Chikungunya virus, previous infection with Dengue virus, or differences in human genetic predisposition to disease.

It’s easy to blame mutations in the viral genome for novel patterns of transmission or pathogenesis. Viral mutations arise during every replication cycle, due to errors made by viral enzymes as they copy nucleic acids. RNA viruses are the masters of mutation, because, unlike the polymerases of DNA viruses, RNA polymerases cannot correct any errors that arise. As viruses spread globally through different human populations, it is not surprising that different genotypes are selected. These may reflect adaptation to various selective pressures, including different humans, vectors, climate, or geography. There is no reason to assume that such changes influence virulence, disease patterns, or transmission in humans. Whether they do so can never be tested in humans.

Blaming the viral genome is nothing new. At the onset of the 2014 Ebolavirus outbreak in West Africa there were many claims that the unprecedented size of the outbreak was a consequence of mutations in the viral genome. Genomic analysis of isolates early in the epidemic suggested that the large number of infections was leading to rates of mutation not previously observed. This work lead to dubious claims of  “Ebolavirus mutating rapidly as it spreads” and Ebolavirus is mutating (Time Magazine). Richard Preston, in the New Yorker article Ebola Wars quoted scientist Lisa Hensley:

In the lab in Liberia, Lisa Hensley and her colleagues had noticed something eerie in some of the blood samples they were testing. In those samples, Ebola particles were growing to a concentration much greater than had been seen in samples of human blood from previous outbreaks. Some blood samples seemed to be supercharged with Ebola. This, too, would benefit the virus, by enhancing its odds of reaching the next victim. “Is it getting better at replicating as it goes from person to person?” Hensley said.

And let’s not forget the absurd speculation, fueled by these data, that Ebolavirus would go airborne.

Within a year all this nonsense was proven wrong. Ebolavirus had not sustained mutations any faster than in previous outbreaks. Furthermore, the observed mtuations  did not change the virus into a more dangerous strain.

Go back to any viral outbreak – MERS-coronavirus, SARS-coronavirus, influenza virus, HIV-1 – and you will find the same story line. Mutation of the virus is leading to more virulence, transmission, spread. But in no case has cause and effect been proven.

Let’s stop blaming viral mutation rates for altered patterns of virus spread and pathogenesis. More likely determinants include susceptibility of human populations, immune status, vector availability, and globalization, to name just a few. Not as spectacular as ‘THE VIRUS IS MUTATING!’, but nearer to the truth.

TWiV 360: From Southeastern Michigan

On episode #360 of the science show This Week in Virology, Vincent visits the University of Michigan where he and Kathy speak with Michael, Adam, and Akira about polyomaviruses, virus evolution, and virus assembly, on the occasion of naming the department of Microbiology & Immunology a Milestones in Microbiology site.

You can find TWiV #360 at www.microbe.tv/twiv. Or you can watch the video below.

TWiV 340: No shift, measles

On episode #340 of the science show This Week in Virology, the TWiV teams reviews a MERS-coronavirus serosurvey and an outbreak in South Korea, and constraints on measles virus antigenic variation.

You can find TWiV #340 at www.microbe.tv/twiv.

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 www.microbe.tv/twiv.

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 www.microbe.tv/twiv.

Ebolavirus will not become a respiratory pathogen

sneezeAn otherwise balanced review of selected aspects of Ebolavirus transmission falls apart when the authors hypothesize that ‘Ebola viruses have the potential to be respiratory pathogens with primary respiratory spread.’

The idea that Ebolavirus might become transmitted by the respiratory route was suggested last year by Michael Osterholm in a Times OpEd. That idea was widely criticized by many virologists, including this writer.  Now he has recruited 20 other authors, including Ebola virologists, in an attempt to lend legitimacy to his hypothesis. Unfortunately the new article adds no new evidence to support this view.

In the last section of the review article the authors admit that they have no evidence for respiratory transmission of Ebolavirus:

It is very likely that at least some degree of Ebola virus transmission currently occurs via infectious aerosols generated from the gastrointestinal tract, the respiratory tract, or medical procedures, although this has been difficult to definitively demonstrate or rule out, since those exposed to infectious aerosols also are most likely to be in close proximity to and in direct contact with an infected case.

It is possible that some short-distance transmission of Ebolavirus occurs through the air. But claiming that it is ‘very likely’ to be taking place is an overstatement considering the lack of evidence. As might be expected, ‘very likely’ is exactly the phrase picked up by the Washington Post.

I find the lack of critical thinking in the following paragraph even more disturbing:

To date, investigators have not identified respiratory spread (either via large droplets or small-particle aerosols) of Ebola viruses among humans. This could be because such transmission does not occur or because such transmission has not been recognized, since the number of studies that have carefully examined transmission patterns is small. Despite the lack of supportive epidemiological data, a key additional question to ask is whether primary pulmonary infections and respiratory transmission of Ebola viruses could be a potential scenario for the future.

Why is the possibility of respiratory transmission of Ebolaviruses a ‘key additional question’ when there has been no evidence for it to date? To make matters worse, the authors have now moved from short-range transmission of the virus by droplets, to full-blown respiratory aerosol transmission.

The authors present a list of reasons why they think Ebolavirus could go airborne, including: isolation of Ebolaviruses from saliva; presence of viral particles in pulmonary alveoli on human autopsies; and cough, which can generate aerosols, can be a symptom of Ebolavirus disease. The authors conclude that because of these properties, the virus would not have to change very much to be transmitted by aerosols.

I would conclude the opposite from this list of what Ebolavirus can do: there is clearly a substantial block to respiratory transmission that the virus cannot overcome. Perhaps the virus is not stable enough in respiratory aerosols, or there are not enough infectious viruses in aerosols to transmit infection from human to human. Overcoming these blocks might simply not be biologically possible for Ebolavirus. A thoughtful discussion of these issues is glaringly absent in the review.

The conclusion that Ebolavirus is  ‘close’ to becoming a full-blown respiratory pathogen reveals how little we understand about the genetic requirements for virus transmission. In fact the authors cannot have any idea how ‘close’ Ebolavirus is to spreading long distances through the air.

It is always difficult to predict what viruses will or will not do. Instead, virologists observe what viruses have done in the past, and use that information to guide their thinking. If we ask the simple question, 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. There is no evidence to believe that Ebolavirus is any different.

Viruses are masters of evolution, but apparently one item lacking from their repertoire is the ability to change the way that they are transmitted.

Such unfounded speculation would largely be ignored if the paper were read only by microbiologists. But Ebolavirus is always news and even speculation does not go unnoticed. The Washington Post seems to think that this review article is a big deal. Here is their headline: Limited airborne transmission of Ebola is ‘very likely’ new analysis says.

Gary Kobinger, one of the authors, told the Washington Post that ‘we hope that this review will stimulate interest and motivate more support and more scientists to join in and help address gaps in our knowledge on transmission of Ebola’. Such hope is unrealistic, because few can work on this virus, which requires the highest levels of biological containment, a BSL-4 laboratory.

I wonder if Osterholm endorses Kobinger’s hopes. After all, he opposed studies of influenza virus transmission in ferrets, claiming that they are too dangerous. And the current moratorium on research that would help us understand aerosol transmission of influenza viruses is a direct result of objections by Osterholm and his colleagues about this type of work. The genetic experiments that are clearly needed to understand the limitations of Ebolavirus transmission would never be permitted, at least not with United States research dollars.

The gaps in our understanding of virus transmission are considerable. If virologists are not able to carry out the necessary experiments to fill these gaps, all we will have is rampant and unproductive speculation.

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.

TWiV 302: The sky is falling

On episode #302 of the science show This Week in Virology, the TWiVers discuss the growing Ebola virus outbreak in West Africa, and an epidemic of respiratory disease in the US caused by enterovirus D68.

You can find TWiV #302 at www.microbe.tv/twiv.

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.