The virulence of a virus – its capacity to cause disease – is determined by both viral and host factors. Even among healthy individuals, infection with a particular virus may have different outcomes ranging from benign to lethal. The study of influenza viruses that cause mild or fatal outcomes reveals that defective viral genomes play a role in determining viral virulence.
Not 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.
On episode #348 of the science show This Week in Virology, Vincent and Rich discuss fruit fly viruses, one year without polio in Nigeria, and a permissive Marek’s disease viral vaccine that allows transmission of virulent viruses.
You can find TWiV #348 at www.microbe.tv/twiv.
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.
On episode #284 of the science show This Week in Virology, the TWiV team discusses how skin scarification promotes a nonspecific immune response, and whether remaining stocks of smallpox virus should be destroyed.
You can find TWiV #284 at www.microbe.tv/twiv.
Virulence, 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.
On episode #262 of the science show This Week in Virology, Vincent returns to the University of Wisconsin – Madison to speak with Ann Palmenberg about her career in virology.
You can find TWiV #262 at www.microbe.tv/twiv.
On episode #233 of the science show This Week in Virology, Vincent, Rich, Alan and Kathy review aerosol transmission studies of influenza H1N1 x H5N1 reassortants, H7N9 infections in China, and the MERS coronavirus.
You can find TWiV #233 at www.microbe.tv/twiv.
Vincent, Michael, and Stanley recorded episode #8 of the podcast This Week in Microbiology live at the 2011 ASM General Meeting in New Orleans, with guests Andreas Baümler, Nicole Dubilier, and Paul Rainey. They spoke about how pathogens benefit from disease, symbioses between chemosynthetic bacteria and marine invertebrates, and repetitive sequences in bacteria.
Click the arrow above to play, or right click to download TWiM #8 (60 MB, .mp3, 87 minutes).
Links for this episode:
- Salmonella invasion from the gut lumen into tissues (PLoS Pathogens)
- Symbiotic diversity in marine animals (Nature Rev Micro)
- REPINs, a new family of mobile DNA in bacteria (PLoS Genetics)
- Letters read on TWiM #8
- Video of TWiM #8 – view below
Send your microbiology questions and comments (email or mp3 file) to firstname.lastname@example.org, or call them in to 908-312-0760. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twim.
The World Health Organization has declared the end of the pandemic caused by H1N1 influenza virus. According to Director-General Margaret Chan,
The world is no longer in phase 6 of influenza pandemic alert. We are now moving into the post-pandemic period. The new H1N1 virus has largely run its course.
As we enter the post-pandemic period, this does not mean that the H1N1 virus has gone away. Based on experience with past pandemics, we expect the H1N1 virus to take on the behaviour of a seasonal influenza virus and continue to circulate for some years to come.
According to the Director-General, levels and patterns of H1N1 transmission are now different from those observed during the pandemic. Out-of-season outbreaks are no longer being reported, and their intensity is similar to that seen during seasonal epidemics. In addition, multiple influenza viruses are being isolated in many countries, a pattern typical of many recent seasonal epidemics.
I take particular interest in what the Director-General believes did not happen:
This time around, we have been aided by pure good luck. The virus did not mutate during the pandemic to a more lethal form. Widespread resistance to oseltamivir did not develop. The vaccine proved to be a good match with circulating viruses and showed an excellent safety profile.
I continue to wonder why the Director-General, and many others, feel that influenza virus must change to a more lethal form. Although the four previous influenza pandemics occurred in multiple waves of increasing lethality, there is no evidence that they are a consequence of viral mutation. For example, the only virus available from the 1918 pandemic was rescued from an Alaskan influenza victim who was buried in permafrost in November of that year, when higher mortality was already evident. This makes it impossible to correlate any genetic changes in the virus with increased virulence. Viruses are available from different stages of the pandemics of 1957 and 1968, which also occurred in waves of increasing lethality, but to my knowledge the virulence studies have not been done.
I believe that a major selective force for viral evolution is the need to maintain efficient transmission among hosts. This may be achieved by any number of phenotypic changes, such as increases in stability and virion production. Changes in lethality might also lead to more effective transmission – for example, by inducing more severe coughing, the virus could be better transmitted among humans. But there is no genetic evidence that such changes have occurred during influenza virus pandemics.
How has the idea that influenza virus mutates to greater lethality permeated our popular culture? I don’t know the answer, but John Barry’s The Great Influenza is a prime suspect.