An outbreak of respiratory disease in Ugandan chimpanzees provides insight into how virus infection can shape the genome and lead to differences in the cell receptor gene that regulate susceptibility to infection.
Vincent speaks with Sandy Weller about her career and her work on the mechanisms of synthesis, maturation, cleavage and packaging of viral DNA genomes.
You can find TWiV 398 at microbe.tv/twiv, or listen/watch below.
Become a patron of TWiV!
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.
Today it is well known that viruses may contain DNA (poxvirus, mimivirus) or RNA (influenza virus, Zika virus), but for many years it was thought that genomes were only made of DNA. The surprise at finding only RNA in a virus is plainly evident in a 1953 letter from Harriett Ephrussi-Taylor to James D. Watson (pictured, Cold Spring Harbor Archives Repository*).
While DNA was discovered in the late 1800s, its role as genetic material was not proven until the famous experiments of McLeod, Avery, and McCarty, published in 1944. They showed that DNA from a strain of Pnemococcus bacteria that formed smooth colonies, when added to a rough colony former, produced smooth colonies.
By this time many viruses had been identified, and it was assumed that their genetic information was DNA. The ‘kitchen blender’ experiments of Hershey and Chase in 1952 proved that the genetic information of bacteriophage T2 is DNA. Watson and Crick proposed the double-helical structure of DNA in 1953, and a few years later published the Central Dogma, which suggested that information flowed in biological systems from DNA to RNA to protein.
Amidst all these experimental findings, which gave rise to the field of molecular biology, comes the note in 1953 from Ephrussi-Taylor to Watson. Under the heading TOP SECRET she writes:
Burnet swears, from work in his lab, that flu virus has principally, if not exclusively RNA. Suspects the same for polioviruses. ??
During her career, Dr. Ephrussi-Taylor carried out work on bacterial transformation by DNA and was knowledgeable about its history as genetic material. Frank Macfarlane Burnet was an Australian immunologist who worked on influenza virus early in his career.
By the 1950s many viruses had been isolated which we now know have genomes of DNA (bacteriophage, poxvirus) or RNA (yellow fever virus, poliovirus, influenza virus). But it was the first virus discovered – tobacco mosaic virus, in the 1890s – that lead the way to establishing RNA as genetic material. Wendell Stanley produced crystals of TMV in 1935 and found that they contained 5% RNA. But Stanley and others thought TMV was a protein, and that the RNA was either a contaminant, or played a structural role.
A structural role for RNA was reinforced as late as 1955 when Heinz Fraenkel-Conrat separately purified TMV protein and RNA. When he mixed the two components together, they formed infectious, 300 nm rods. When the RNA was omitted, noninfectious aggregates formed. This finding reinforced the belief that RNA helped form virus particles.
This view changed when Fraenke-Conrat gave his wife, Beatrice Singer, the task of purifying TMV RNA until it had lost all infectivity. To everyone’s surprise she found that TMV RNA itself was infectious, proving in 1957 that it was the viral genetic material. However, RNA also has a structural role in TMV virus particles, as it organizes the capsid protein (yellow in illustration at left) into regularly repeated subunits.
Demonstration of infectivity of RNA from animal viruses soon followed, for mengovirus, a picornavirus, in 1957 and for poliovirus in 1958 (the latter done at my own institution, the College of Physicians and Surgeons of Columbia University!).
By the early 1950s the idea that RNA could be viral genetic material was clearly in the minds of virologists, hence Ephrussi-Taylor’s amusing letter on influenza virus and poliovirus.
*Thanks to @infectiousdose for finding this amazing letter.
On episode #376 of the science show This Week in Virology, the TWiV team discusses the latest data on Zika virus, including ocular defects in infants with microcephaly, and isolation of the entire viral genome from fetal brain tissue.
You can find TWiV #376 at microbe.tv/twiv.
About eight percent of human DNA is viral – remnants of ancestral infections with retroviruses. These endogenous retroviral sequences do not produce infectious viruses, and most are considered to be junk DNA. But some of them provide important functions. The protein called syncytin, which is essential for formation of the placenta, originally came to the genome of our ancestors, and those of other mammals, via a retrovirus infection. Another amazing role of endogenous retroviruses is that they regulate the stem cells that are the precursors of all the cells in our body.
The genetic material of retroviruses is RNA, but during infection it is converted to DNA which then integrates into the chromosome of the cell. If the infected cell happens to be a germ cell, then the viral DNA, now called called an endogenous retrovirus, becomes a permanent part of the animal and its offspring. One of our endogenous retroviruses, called HERV-H, infected human ancestors about 25 million years ago. HERV-H has been found to be important for the properties of human embryonic stem cells.
Embryonic stem cells (ES cells), which are derived from the inner cell mass of a blastocyst (which forms 4-5 days after implantation), are pluripotent – they can differentiate into every cell type in the human body. Being pluripotent means expressing a very different set of genes compared with somatic cells – the cells of skin, muscle, organs, to name a few. The genes that are expressed in ES cells are controlled by a small number of key proteins that regulate mRNA synthesis. If these proteins – just four – are produced in a differentiated cell, it will turn into an ES cell – an induced, pluripotent embryonic stem cell, or iPSC. This observation garnered Shinya Yamanaka the Nobel Prize in 2012.
The first clue that HERV-H might be important for the pluripotency of ES cells was the finding that this DNA is preferentially expressed in human ES cells (the figure [credit] shows the expression of HERV-H in ES and two other cell types). When the levels of HERV-H RNAs are reduced (by RNA interference) in ES cells, the morphology of the cells changes – they become fibroblast-like, a sign of differentiation. In contrast, when fibroblasts are reprogrammed to become iPSCs, the levels of HERV-H RNAs rise. These findings suggest that HERV-H is essential for keeping ES cells pluripotent, and for making somatic cells pluripotent.
The HERV-H DNA in our genome is flanked by viral sequences called long terminal repeats, or LTRs. These provide initiation sites for the synthesis of viral mRNAs. In human ES cells the HERV-H LTRs appear to be enhancing the transcription of nearby human genes that are important for maintaing pluripotency. In an interesting twist, the HERV-H viral RNA is important for this activity: it appears to bind proteins involved in the regulation of mRNAs important for pluripotency. This observation explains why reducing HERV-H viral RNA leads to loss of pluripotency.
The HERV-H RNA made in human ES cells is not translated into protein because it contains many mutations that have accumulated over the past 25 million years. Therefore HERV-H is a long, non-coding RNA (lncRNA), a relatively recently discovered class of regulatory RNAs. There are about 35,000 lncRNAs in human cells that are involved in controlling a variety of processes such as splicing, translation and epigenetic modifications. Now we know that endogenous retroviruses can also produce lncRNAs.
Without endogenous retroviruses, humans might not be recognizable as the Homo sapiens that today walk the Earth. They might also be egg-layers – but the eggs would be white. Viruses don’t just make us sick.
On episode #268 of the science show This Week in Virology, Vincent, Alan, Kathy, and Ashlee discuss fomites in physicians offices, plant virus factories involved in aphid transmission, and clues from the bat genome about flight and immunity.
You can find TWiV #268 at www.microbe.tv/twiv.
On episode #267 of the science show This Week in Virology, Vincent, Alan, Rich and Kathy review a protease essential for influenza pathogenesis in mice, and directionality of rhinovirus RNA exit from the capsid.
You can find TWiV #267 at www.microbe.tv/twiv.
On episode #246 of the science show This Week in Virology, Vincent, Alan, Rich, and Kathy discuss the huge Pandoravirus, virologists planning H7N9 gain of function experiments, and limited access to the HeLa cell genome sequence.
You can find TWiV #246 at www.microbe.tv/twiv.
We recorded this episode of TWiV as a Google hangout on air. Consequently the audio is not the same quality as you might be used to. But the tradeoff is that you can see each of us on video.