Ebola virus mutations do not affect pathogenicity

EbolavirusSeveral mutations that arose during the 2013-2016 outbreak of Ebola virus in West Africa were previously found to increase infectivity for human cells. However, a study in two animal models show no effect of these mutations on disease.

Among the many mutations identified among the hundreds of genome sequences obtained during the 2013-2016 Ebola virus epidemic, a change from alanine to valine at position 82 (A82V) that arose early in the outbreak was found to increase infectivity in human cells of HIV particles with the Ebola virus glycoprotein. The authors suggested that this change might have been in part responsible for the extent and severity of the outbreak. [click to continue…]

Good viruses visiting bad neighborhoods

Marco VignuzziWhat would happen to an RNA virus if its genome were placed in a bad neighborhood? The answer is that fitness plummets.

RNA virus populations are not composed of a single defined nucleic acid sequence, but are dynamic distributions of many nonidentical but related members. In the past I have referred to these populations as quasispecies but that is no longer the preferred term: mutant swarms or heterogeneous virus populations should be used instead.

The term for all possible combinations of a viral genome sequence is sequence space; for a 10,000 nucleotide genome this would be theoretically 410,000 different genomes – a huge number, more than the atoms in the universe. Any RNA virus population occupies only a fraction of this sequence space, in part because many mutations are deleterious. Studies have shown that viral genomes occupy specific parts of sequence space, called neighborhoods, and movement to different neighborhoods is important for viability. If the viral genome is placed in a bad neighborhood – one that is detrimental for virus fitness – the ability to explore sequence space is restricted.

An example of the effect of changing viral sequence space is shown by a study in which hundreds of synonymous mutations (they did not change the amino acid sequence) were introduced in the capsid region of poliovirus (link to paper). Such rewiring, which placed the virus in a different sequence space, reduced viral fitness and attenuated pathogenicity in a mouse model. In other words, the viral genome was placed in a bad neighborhood, from where it could not move to other neighborhoods needed for optimal replication and pathogenesis. While the genome rewiring did not affect the protein sequence, it might have had deleterious effects on RNA structures or codon or dinucleotide frequency. For example, introduction of codon pairs that are under-represented in the human genome can produce less fit viruses.

A recent study avoids these potential issues by introducing changes in the viral genome that do not affect protein coding, RNA structures or codon or dinucleotide frequency, yet place the viral genome in a different sequence space (link to paper). All 117 serine/leucine codons in the capsid region of Coxsackievirus B3 were changed so that a single nucleotide mutation would lead to a stop codon, terminating protein synthesis and virus replication (this virus is called 1-to-Stop). The serine codons were changed to UUA or UUG; one mutation changes these to the terminators UAA, UGA, or UAG. Another virus was made in which two mutations were needed to produce a stop codon (NoStop virus).

1-to-Stop viruses replicated normally, but when mutagenized, they had significantly lower fitness than wild type or NoStop viruses. Extensive passage of the virus in cells, which would be expected to cause accumulation of mutations, had the same effect on fitness. When a high fidelity RNA polymerase was introduced into 1-to-Stop virus, it replicated like wild type virus. Similar results were obtained with an influenza virus when one of its 8 genome segments was rewired to produce 1-to-Stop and NoStop counterparts.

The 1-to-Stop Coxsackieviruses were attenuated in a mouse model of infection. Furthermore, mice infected with 1-to-Stop virus were protected against replication and disease after challenge with wild type virus. These observations suggest that rewiring viral RNA genomes could be used to design vaccines.

These findings show that recoding a viral genome places it an different sequence space than wild type virus, in which single mutations can lead to inactivation of viral replication. This new neighborhood is unfavorable (‘bad’) because the virus cannot readily move to other neighborhoods to accommodate the effects of mutation.

For more discussion of viral sequence space and rewiring viral genomes, listen to the podcast This Week in Evolution #24: our guest is Marco Vignuzzi (pictured), senior author on the second paper discussed here.

TWiV 431: Niemann-Pick of the weak

The TWiVirions reveal bacteriophage genes that control eukaryotic reproduction, and the biochemical basis for increased Ebolavirus glycoprotein activity during the recent outbreak.

You can find TWiV #431 at microbe.tv/twiv, or listen below.

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TWiV 415: Ebola pipettors and the philosopher’s clone

Jeremy Luban, Aaron Lin, and Ted Diehl join the TWiV team to discuss their work on identifying a single amino acid change in the Ebola virus glycoprotein from the West African outbreak that increases infectivity in human cells.

You can find TWiV #415 at microbe.tv/twiv, or listen below.

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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.