SLOWLY, INEXORABLY, Ebola virions are reproduced in the cell, and pass through the cell membrane, ready to infect new cells.
That does not sound like the Ebola we know. Surely Ebola ravages through the body at break-neck speed, devastating cells and tissue and organs as it makes it way through it typical route to the end-stage pathological crisis of coagulopathy.
But new evidence suggests the in the case of Ebola’s fitness, less may be more – and also that some mutations in Ebola may yet to be shown to have had an impact on the degree of virulence by influencing, as I also proposed was possible in “Ebola: An Evolving Story“- the rate of viral reproduction, which should then determine infectivity.
Readers will recall my missive against the widespread idea that high virulence and pathogenicity of an infectious agent are associated with increased mutation rates. This idea seems to have originated from the observation that non-synonymous substitution rates may be expected to higher than synonymous rates when positive Darwinian selection is occurring. Somehow, in the formation of public policy, however, the overall rate itself became a proxy measure of virulence – as if natural selection would act to increase the mutation rate rather than to be reflected by the increase in certain phenotypes encoded by from various genotypes that might emerge from substitutions that have occurred. Is worth pointing out that the least interest nucleotide substitutions nearly always have the highest rate (third positions in codons, introns in animal genomes), and thus the notion of the use of a high mutation rate as a sign of increase virulence or pathogenicity is not warranted based on evolutionary principles.
An alternative hypothesis worth consideration is whether Ebola (or any emerging infectious agent) that comes into the human species via what David Quammen has called “spillovers” adapts to us as a new host via a mechanism that involves, after a series of transmissions, an overall slowing of the rate of disease progression, because virion types (quasispecies) that stick around in a living host longer have a better shot at either being transmitted, or of having descendants that are transmitted, to another member of the same newly colonized species (in this case, Homo sapiens).
While the rest of the world focused on the low probability that Ebola may undergo a shift to become “airborne”, following the plot line of the movie “Outbreak”, the specific mechanisms that I envisioned by which Ebola might slow down, or speed up the disease progression rate included
- specific nucleotide substitutions that lead to increases or decreases in the rate at which the Ebola genome can be replicated;
- changes in the rate of the stuttering of the transcription of the GP gene, which leads to the production in sGP, the truncated version of the Ebola GP protein. The sGP protein is thought to be of benefit to Ebola by overwhelming the adaptive immune system with it large numbers;
- improving in the ability of the virus to undergo asymptomatic, or presymptomatic, transmission;
- change in the host (human patient) tissue virulence patterns;
- change in the primary mode of death.
Ebola from Guinea in 2013/2014 may have undergone any, or all of these types of shifts in phenotype.
I am not the first to conceive that specific mutations might also cause shifts in pathogenicity in Ebola. Take, for instance, this from a study in 2004:
” Likewise, GP shedding may play an important regulatory role in virus replication and pathogenesis” (Dolnik, 2004).
Public policy in the US was based primary on viewpoints from the CDC. I express in “Ebola” how shocked I was to hear Dr. Tom Freiden, Director of the CDC relay to the world, including the entire US medical community, that there we no mutations in Ebola from guinea. I also report my incredulity at the report to the scientific community, during a White House conference call, that Ebola from guinea was “well over 99.999% ” similar to Ebola zaire from 1995 (it’s not – it’s as much as 97.3% similar). The “99.999%” answer came from a CDC Senior Scientist in response to my question about what may, or may not be yet known, about the in the form of a question about the 396 mutations found to date (at the time) in Ebola from guinea. How many, and which mutations, is an important question for a virus as deadly as Ebola. Why the CDC consistently downplayed even the existence of potentially important mutations is a mystery – it has left much of the scientific community shaking their heads and wondering “Why“?
Studies of molecular phenotype is challenging science. Working with Ebola is even more challenging. So it is remarkable that a research team at Emory University were able to show convincingly that high amounts of the GP1,2 protein actually leads to lowered infectivity. Here is the abstract from their study (Mohan et al., 2015):
The Ebola virus (EBOV) surface glycoprotein (GP1,2) mediates host cell attachment and fusion and is the primary target for host neutralizing antibodies. Expression of GP1,2 at high levels disrupts normal cell physiology, and EBOV uses an RNA-editing mechanism to regulate expression of the GP gene. In this study, we demonstrate that high levels of GP1,2 expression impair production and release of EBOV virus-like particles (VLPs) as well as infectivity of GP1,2-pseudotyped viruses. We further show that this effect is mediated through two mechanisms. First, high levels of GP1,2 expression reduce synthesis of other proteins needed for virus assembly. Second, viruses containing high levels of GP1,2 are intrinsically less infectious, possibly due to impaired receptor binding or endosomal processing. Importantly, proteolysis can rescue the infectivity of high-GP1,2-containing viruses. Taken together, our findings indicate that GP1,2 expression levels have a profound effect on factors that contribute to virus fitness and that RNA editing may be an important mechanism employed by EBOV to regulate GP1,2 expression in order to optimize virus production and infectivity.
IMPORTANCE The Ebola virus (EBOV), as well as other members of the Filoviridae family, causes severe hemorrhagic fever that is highly lethal, with up to 90% mortality. The EBOV surface glycoprotein (GP1,2) plays important roles in virus infection and pathogenesis, and its expression is tightly regulated by an RNA-editing mechanism during virus replication. Our study demonstrates that the level of GP1,2 expression profoundly affects virus particle production and release and uncovers a new mechanism by which Ebola virus infectivity is regulated by the level of GP1,2 expression. These findings extend our understanding of EBOV infection and replication in adaptation of host environments, which will aid the development of countermeasures against EBOV infection.
A glimmer of light on the possible mechanisms involved in shifts in the rates of ebola virus disease progression in a patient appeared this month in The Journal of Infectious Disease. A study by Dolnik et al., (2015) reported that virions made to have specific mutations had a lower rate of ‘shedding’ out from infected cells than virions with the 1995 genotype [sentence edited 9/12/15]. The mutations in question are not from the Guinea isolates, but were studied based on their previously studied effects on specific pathways likely to influence pathogenicity.
Here’s the abstract from the Dolnik et al., (2015) study:
The surface glycoprotein (GP) is responsible for Ebola virus (EBOV) attachment and membrane fusion during virus entry. Surface expression of highly glycosylated GP causes marked cytotoxicity via masking of a wide range of cellular surface molecules, including integrins. Considerable amounts of surface GP are shed from virus-infected cells in a soluble truncated form by tumor necrosis factor α–converting enzyme. In this study, the role of GP shedding was investigated using a reverse genetics approach by comparing recombinant viruses possessing amino acid substitutions at the GP shedding site. Virus with an L635V substitution showed a substantial decrease in shedding, whereas a D637V substitution resulted in a striking increase in the release of shed GP. Variations in shedding efficacy correlated with observed differences in the amounts of shed GP in the medium, GP present in virus-infected cells, and GP present on virions. An increase in shedding appeared to be associated with a reduction in viral cytotoxicity, and, vice versa, the virus that shed less was more cytotoxic. An increase in shedding also resulted in a reduction in viral infectivity, whereas a decrease in shedding efficacy enhanced viral growth characteristics in vitro. Differences in shedding efficacy and, as a result, differences in the amount of mature GP available for incorporation into budding virions did not equate to differences in overall release of viral particles. Likewise, data suggest that the resulting differences in the amount of mature GP on the cell surface led to variations in the GP content of released particles and, as a consequence, in infectivity. In conclusion, fine-tuning of the levels of EBOV GP expressed at the surface of virus-infected cells via GP sheddin
g plays an important role in EBOV replication by orchestrating the balance between optimal virion GP content and cytotoxicity caused by GP.
Basically, this study also shows that the phenotype of interest – rate of progression – which in turn will influence overall infectivity – can be increased, or decreased, on the basis of individual nucleotide substitutions.
It is superb to see such excellence by researchers focusing on the right questions. Congratulations to Volchkov team at the Université de Lyon, Université Claude Bernard, and the Institut für Virologie, Philipps-Universität Marburg, and the team led by Yang at Emory University, and the Atlanta VA Medical Center (Mohan et al.).
Dolnik et al., 2004. Ectodomain shedding of the glycoprotein GP of Ebola virus. EMBO J. 23:2175-2184.
Dolnik O. et al., 2015. Shedding of Ebola virus surface glycoprotein is a mechanism of self-regulation of cellular cytotoxicity and has a direct effect on virus infectivity. J Infect Dis. pii: jiv268.
Mohan et al., 2015. Less Is More: Ebola virus surface glycoprotein expression levels regulate virus production and infectivity. J. Virology 89:1205-1217. jvi.asm.org/content/89/2/1205.full