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.
During breastfeeding, mothers provide the infant with nutrients, beneficial bacteria, and immune protection. Fluids from the infant may also enter the mammary gland through retrograde flux of the nipple. Studies in a ferret model reveal that influenza virus replicates in the mammary gland, is shed in breast milk and transmitted to the infant. Virus may also travel in the opposite direction, from infant to mother.
The role of the mammary gland in influenza virus transmission was studied using a ferret model comprising lactating mothers and nursing infants. Intranasal inoculation of nursing mother ferrets with the 2009 H1N1 influenza virus lead to viral replication and development of influenza in both mother and infant. When the study design was reversed, and 4 week old nursing ferrets were inoculated intranasally with the same virus, viral replication and disease ensued first in the infants, and then in the mothers. Infectious virus was recovered both in the mammary glands and in the nipples at day 4 post infant inoculation, and in mother’s milk from 3-5 days post infant inoculation. Histopathological examination of sections of mammary glands from infected mothers revealed destruction of the mammary architecture.
These results show that nursing infants may pass influenza virus to mothers. It seems clear that influenza virus replicates in the mammary gland and that infectious virus is present in milk. How does this virus infect the mother? One possibility is that infection is transmitted by respiratory contact with virus-containing milk, or by inhalation of aerosols produced by nursing. How influenza virus in the mammary gland would reach the mother’s lung via the blood to cause respiratory disease is more difficult to envision and seems unlikely.
When influenza virus was inoculated into the mammary gland of lactating mothers via the lactiferous ducts, both mother and breast feeding infant developed serious influenza. Infectious virus was detected first in the nasal wash of infants, then later in the nasal wash of mothers. Breast milk contained infectious virus starting on day 2 after inoculation. Histopathological examination of sections from infected mammary glands revealed destruction of glandular architecture and cessation of milk production. This observation is consistent with the results of gene expression analysis of RNA from virus infected mammary glands, which revealed reduction in transcripts of genes associated with milk production.
To determine if human breast cells can be infected with influenza virus, three different human epithelial breast cell lines were infected with the 2009 H1N1 virus strain. Virus-induced cell killing was observed and infectious virus was produced.
Even if we assume that influenza virus can replicate in the human breast, the implications for influenza transmission and disease severity are not clear. Transmission of HIV-1 from mother to infant by breast milk has been well documented. In contrast to influenza virus, HIV-1 is present in the blood from where it spreads to the breast. Most human influenza virus strains do not enter the blood so it seems unlikely that virus would spread to the breast of a mother infected via the respiratory route. However, viral RNA has been detected in the blood of humans infected with the 2009 H1N1 strain, the virus used in these ferret studies. Therefore we cannot rule out the possibility that some strains of influenza virus spread from lung via the blood to the breast, allowing infection of a nursing infant. Some answers might be provided by determining if influenza virus can be detected in the breast milk of humans with influenza.
What would be the implication of a nursing infant infecting the mother’s breast with influenza virus? As I mentioned above, it seems unlikely that this virus would enter the blood, and even if it could, how would the virus infect the apical side of the respiratory epithelium? What does seem clear is that viral replication in the breast could lead to a decrease in milk production which could be detrimental to the infant. If the mother had multiple births, then influenza virus might be transmitted to siblings nursing on the infected mother.
Are you wondering how an infant drinking influenza virus-laded breast milk acquires a respiratory infection? Recently it has been shown that influenza virus replicates in the soft palate of ferrets. The soft palate has mucosal surfaces that face both the oral cavity and the nasopharynx. Ingested virus could first replicate in the soft palate, then spread to the nasopharynx and the lung. A simpler explanation is that nursing produces virus-containing aerosols which are inhaled by the infant.
On episode #303 of the science show This Week in Virology, the TWiV team discusses transmission of Ebola virus, and inhibition of Borna disease virus replication by viral DNA in the ground squirrel genome.
You can find TWiV #303 at www.microbe.tv/twiv.
Vincent, Rich, and Kathy and their guests Clodagh and Ron recorded episode #291 of the science show This Week in Virology at the 33rd annual meeting of the American Society for Virology at Colorado State University in Ft. Collins, Colorado.
You can find TWiV #291 at www.microbe.tv/twiv.
On episode #287 of the science show This Week in Virology, Matt Frieman updates the TWiV team on MERS-coronavirus, and joins in a discussion of whether we should further regulate research on potentially pandemic pathogens.
You can find TWiV #287 at www.microbe.tv/twiv.
It is well known that aquatic birds are a major reservoir of influenza A viruses, and that pandemic human influenza virus strains of the past century derive viral genes from this pool. The recent discovery of two new influenza A viruses in bats suggests that this species may constitute another reservoir with even greater genetic diversity.
A new influenza virus had previously been isolated from little yellow-shouldered bats (Sturnira lilium) in Guatemala. Three of 316 rectal swabs were positive when tested by a pan-influenza polymerase chain reaction assay. Viral sequences were also detected in liver, intestine, lung, and kidney tissues, suggestive of viral replication and not passage of ingested material through the intestinal tract. Analysis of the viral genome sequence revealed that A/little yellow-shouldered bat/Guatemala/164/2009 (H17N10) is significantly diverged from all known influenza viruses.
When the same PCR approach was used to screen 114 rectal swabs from 18 different species of bats captured in Peru, a single flat-faced fruit bat (Artibeus planirostris) was positive. Viral sequences were also detected in liver, intestine, and spleen tissues from the same bat. Comparison of the sequences of all 8 genome RNA segments with those of the H17N10 Guatemalan isolate revealed sufficient divergence to justify naming it a new HA and NA subtype, A/flat-faced bat/Peru/033/2010 (H18N11).
Comparison of the nucleotide sequences of bat influenza A viruses from Peru and Guatemala with other influenza viruses leads to two amazing conclusions. First, 7 of the 8 viral RNAs of the bat influenza A viruses group separately from the RNAs of all other known influenza viruses. Second, the RNA sequences encoding four proteins, PB2, PB1, PA and NA, display greater genetic diversity than in all non-bat influenza virus sequences combined. The implication is that New World bats harbor a diverse pool of influenza viruses.
The H17 and H18 HA RNA sequences are, in contrast, far more related to known influenza virus HA and NA sequences. The implication of this observation is clear: some time after the bat and non-bat influenza A viruses diverged, a reassortment event occurred that introduced the HA of a non-bat influenza A virus into the genome of a bat influenza A virus.
Serological studies have revealed widespread circulation of these two new influenza viruses in bats. Sera from 55 of 110 (50%) Peruvian bats representing 13 different species were positive for antibodies against the viral HA or NA proteins. Twenty-one of these samples were positive for antibodies against both viral glycoproteins, while 30 were positive only for anti-HA18 antibodies and 4 were positive for only anti-N11 antibodies. These observations suggest that some bats are infected with reassortant viruses carrying the H18 or N11 genes. A study of sera from 8 different species of Guatemalan bats revealed antibodies to the H17 HA protein in 86 of 228 sera (38%).
A number of human viruses, such as SARS-coronavirus and Nipah and Hendra viruses, are known to have originated in bats. Can bat influenza A viruses infect humans and serve as a source of future pandemic strains? The answer to this question is not known, but the two new bat viruses cannot infect human cell lines in culture. However, it is possible that acquisition of other (e.g. avian or swine) influenza virus genes by reassortment could produce a virus with bat influenza virus genes that is capable of infection humans. The pathogenic and pandemic potential of such viruses is unknown. A first step to answering this question would be to determine if human populations with contact with bats have antibodies to the two new bat influenza A viruses.
The cell receptor for all known influenza A viruses is the carbohydrate molecule known as sialic acid The cell receptor for the two new bat influenza A viruses is not known, but it is clearly not sialic acid, a conclusion reached by studying the crystal structures and binding properties of the H17 (paper one and two) and H18 HA (illustrated) molecules. Furthermore, the crystal structures of the N10 (paper one and two) and N11 proteins reveal that their substrate cannot be sialic acid (the function of the influenza A virus NA is to remove sialic acids from the cell surface, allowing newly synthesized virions to move away from the cell). For this reason the N10 and N11 proteins are called ‘NA-like’.
Bats also harbor many other kinds of viruses, including hepatitis B viruses, Marburg virus, hepaciviruses, pegiviruses, paramyxoviruses, coronaviruses, and many more. They also contain parasites – specifically, malaria parasites. For more information, listen to these podcast episodes:
Hedging our bats (TWiV 258)
More bats out of hell (TWiP 62)
Hepaciviruses and pegiviruses in bats and rodents (TWiV 231)
Bats out of hell (TWiV 183)
Going to bat for flu research (TWiV 173)
Matt’s bats (TWiV 65)
Avian influenza H7N9 virus has caused over 130 human infections in China with 43 fatalities. The source of the virus is not known but is suspected to be wet market poultry. No human to human transmission have been detected, and the outbreak seems to be under control. According to the authors of the letter, the virus could re-emerge this winter, and therefore additional work is needed to assess the risk of human infection.
The research that the virologists propose involve gain-of-function experiments which provide the H7N9 virus with new properties. The isolation of avian influenza H5N1 viruses that can transmit by aerosol among ferrets is an example of a gain-of-function experiment.
The proposed gain-of-function experiments fall into five general categories:
- Determine whether viruses with altered virulence, host range, or transmissibility have changes in antigenicity, or the ability of the virus to react with antibodies. The results of these studies would suggest whether, for example, acquisition of human to human transmissibility would have an impact on protection conferred by a vaccine produced with the current H7N9 virus strain.
- Determine if the H7N9 virus could be adapted to mammals and whether it could produce reassortants with other influenza viruses. The results of this work would provide information on how likely it is that the H7N9 virus would become better adapted to infect humans.
- Isolate mutants of H7N9 virus that are resistant to antiviral drugs. The purpose of these experiments is to identify how drug resistance arises (the mutations can then be monitored in clinical isolates), determine the stability of drug resistant mutants, and whether they confer other properties to the virus.
- Determine the genetic changes that accompany selection of H7N9 viruses that can transmit by aerosol among mammals such as guinea pigs and ferrets. As I have written before, the point of these experiments, in my view, is not to simply identify specific changes that lead to aerosol transmission. Such work provides information on the mechanisms by which viruses can become adapted to aerosol transmission, still an elusive goal.
- Identify changes in H7N9 virus that allow it to become more pathogenic. The results of these experiments provide information on the mechanism of increased pathogenicity and whether it is accompanied by other changes in properties of the virus.
I believe that the proposed gain-of-function experiments are all worth doing. I do not share the concerns of others about the potential dangers associated with gain-of-function experiments: for example the possibility that a virus selected for higher virulence could escape the laboratory and cause a lethal pandemic. Gain-of-function is almost always accompanied by a loss-of-function. For example, the H5N1 viruses that gained the ability to transmit by aerosol among ferrets lost their virulence by this route of infection. When these experiments are done under the proper containment, the likelihood that accidents will happen is extremely small.
All the proposed experiments that would use US funds will have to be reviewed and approved by the Department of Health and Human Services:
The HHS review will consider the acceptability of these experiments in light of potential scientific and public-health benefits as well as biosafety and biosecurity risks, and will identify any additional risk-mitigation measures needed.
While I understand that the authors wish to promote a dialogue on laboratory safety and dual-use research, I question the ultimate value of the communication. Because the letter has been published in two scientific journals, I assume that the target audience of the letter is the scientific community. However, the letter will clearly have coverage in the popular press and I am certain that it will be misunderstood by the general public. I can see the headlines now: “Scientists inform the public that they will continue to make deadly flu viruses”. The controversy about the H5N1 influenza virus transmission studies in ferrets all began with a discussion of the results before the scientific papers had been published. I wonder if the publication of these letters will spark another controversy about gain-of-function research.
In my view, science is best served by the traditional process known to be highly productive: a grant is written to secure funding for proposes experiments, the grant proposal is subject to scientific review by peers, and based on the review the work may or may not be supported. The experiments are done and the results are published. I do not understand why it is necessary to trigger outrage and debate by announcing the intent to do certain types of experiments.
I am curious to know what the many readers of virology blog – scientists and non-scientists – feel about the publication of this letter. Please use the comment field below to express your views on this topic.
Influenza virus initiates infection by attaching to the cell surface, a process mediated by binding of the viral hemagglutinin protein (HA) to sialic acid. This sugar is found on glycoproteins, which are polypeptide chains decorated with chains of sugars. The way that sialic acid is linked to the next sugar molecule determines what kind of influenza viruses will bind. Human influenza viruses prefer to attach to sialic acids linked to the second sugar molecule via alpha-2,6 linkages, while avian influenza viruses prefer to bind to alpha-2,3 linked sialic acids. (In the image, influenza HA is shown in blue on the virion (left) and as a single polypeptide at right. Alpha-2,3 linked sialic acid is shown at top).
Adaptation of avian influenza viruses to efficiently infect humans requires that the viral HA quantitatively switches to human receptor binding – defined as high relative binding affinity to human versus avian receptors. Such a switch is caused by amino acid changes in the receptor binding site of the HA protein. The HA of the H1N1, H2N2, and H3N2 pandemic viruses are all derived from avian influenza viruses that underwent such a quantitative switch in binding from avian to human sialic acid receptors.
Avian H5N1 influenza viruses have not undergone a quantitative switch to human receptor binding, which is one of the reasons why these viruses do not undergo sustained human-to-human transmission. It has been possible to introduce specific amino acid changes in the H5 HA protein that enable these viruses to recognize human sialic acid receptors. Such changes were required to select variants of influenza H5N1 virus that transmit via aerosol among ferrets. However none of these viruses have quantitatively switched to human receptor specificity.
In the H5N1 paper, the authors compared the structure of an H5 HA bound to alpha-2,3 linked sialic acid with the structure of an H2 HA (its closest phylogenetic neighbor) bound to alpha-2,6 linked sialic acid, revealing substantial differences in the receptor binding site. To predict what residues could be changed in the H5 HA to overcome these differences, the authors developed a metric to identify amino acids within the receptor binding site that either contact the receptor or might influence the interaction. They examined these amino acids in different H5 HAs, and identified residues which might change the H5 HA to human receptor specificity. As a starting point they picked two H5 viruses that have already undergone amino acid changes believed to be important for human receptor binding. The changes were introduced into the HA of a currently circulating H5 HA by mutagenesis and then binding of the HAs to purified sialic acids and human tracheal and alveolar tissues was determined.
The HA receptor binding site amino acid changes required for aerosol transmission of H5N1 viruses in ferrets did not quantitatively switch receptor binding of a currently circulating H5 HA from avian to human (the ferret studies were done using H5N1 viruses that circulated in 2004/05). The authors note that “These residues alone cannot be used as reference points to analyze the switch in receptor specificity of currently circulating and evolving H5N1 strains”.
However introducing other amino acid changes which the authors predicted would be important did switch the H5 HA completely to human receptor binding. Only one or two amino acids changes are required for this switch in recently circulating H5 HAs.
This work is important because it defines structural features in the receptor binding site of H5 HA that are critical for quantitative switching from avian to human receptor binding, a necessary step in the acquisition of human to human transmissibility. These specific residues can be monitored in circulating H5N1 strains as indicators of a quantitative switch to human receptor specificity.
Remember that switching of H5 HA to human receptor specificity is not sufficient to gain human to human transmissibility; what other changes are needed, in which genes and how many, is anyone’s guess.
These authors have also published (in the same issue of Cell) a similar analysis of the recent avian influenza H7N9 virus which has emerged in China to infect humans for the first time. They model the binding of sialic acid in the H7 HA receptor binding site, and predict that the HA would have lower binding to human receptors compared with human-adapted H3 HAs (its closest phylogenetic neighbor). This prediction was validated by studies of the binding of the H7N9 virus to sections of human trachea: they find that staining of these tissues is less intense and extensive than of viruses with human-adapted HAs. They predict and demonstrate that a single amino acid change in the H7 HA (G228S) increases binding to human sialic acid receptors. This virus stains tracheal sections better than the H7 parental virus.
These results mean that the H7N9 virus circulating in China might be one amino acid change away from acquiring higher binding to human alpha-2,6 sialic acid receptors. I wonder why a virus with this mutation has not yet been isolated. Perhaps the one amino acid change in the viral HA exerts a fitness cost that prevents it from infecting birds or humans. Of course, as discussed above, a switch in receptor specificity is likely not sufficient for human to human transmission; changes in other genes are certainly needed. In other words, the failure of influenza H7N9 virus to transmit among humans can be partly, but not completely, explained by its binding properties to human receptors.
At the 2013 ASM meeting in Denver, Colorado, Stanley Maloy discussed human infections with avian influenza H7N9 virus with Ronald Atlas, Ph.D., University of Louisville, KY; Robert Webster; St. Jude’s Research Hospital, Memphis, TN; Albert Osterhaus; Erasmus Medical Center, Rotterdam, The Netherlands; and Carole Heilman, NIAID, NIH, Bethesda, MD.