Honeysuckle Targets Viruses.. Researchers call it a ‘Virological penicillin’ MIR2911 ( + the Original Ebola discussion )

* We are Posting our videos at request, from our clinicalnews.org site..

Honeysuckle, clinical tests may of just confirmed it is a powerful virus killer. MIR2911
– In a new study, Chen-Yu Zhang’s group at Nanjing University present an extremely novel finding that a plant microRNA, MIR2911, which is enriched in honeysuckle, directly targets influenza A viruses (IAV) including H1N1, H5N1 and H7N9. Drinking of honeysuckle soup can prevent IAV infection and reduce H5N1-induced mice death.
– Furthermore, one of their ongoing studies shows that MIR2911 also directly targets Ebola virus, which is pandemic in West Africa and is becoming a crisis of public health. Thus, MIR2911 is able to serve as the “virological penicillin” to directly target various viruses.
* Cell Research advance online publication 7 October 2014; doi: 10.1038/cr.2014.130 Honeysuckle-encoded atypical microRNA2911 directly targets influenza A viruses Continue reading “Honeysuckle Targets Viruses.. Researchers call it a ‘Virological penicillin’ MIR2911 ( + the Original Ebola discussion )”

Scientists creating viruses deadlier to humans

Sunday, 22 December 2013

Some of the world’s most eminent scientists have severely criticised the arguments used by some influenza researchers who are trying to make the H5N1 bird-flu virus more dangerous to humans by repeatedly infecting laboratory ferrets.

More than 50 senior scientists from 14 countries, including three Nobel laureates and several fellows of the Royal Society, have written to the European Commission denouncing claims that the ferret experiments are necessary for the development of new flu vaccines and anti-viral drugs. Continue reading “Scientists creating viruses deadlier to humans”

Scientists to make mutant forms of new bird flu to assess risk

Source: Reuters – Wed, 7 Aug 2013 05:00 PM

Author: Reuters


* Controversial research sparked previous security fears

* Flu experts say it is critical to prepare for threat

* New H7N9 bird flu strain has killed 43 people so far

* Outbreak currently controlled, may return in autumn

By Kate Kelland, Health and Science Correspondent

LONDON, Aug 7 (Reuters) – Scientists are to create mutant forms of the H7N9 bird flu virus that has emerged in China so they can gauge the risk of it becoming a lethal human pandemic.

The genetic modification work will to result in highly transmissible and deadly forms of H7N9 being made in several high security laboratories around the world, but it is vital to prepare for the threat, the scientists say.

The new bird flu virus, which was unknown in humans until February, has already infected at least 133 people in China and Taiwan, killing 43 of them, according to the latest World Health Organization (WHO) data.

Announcing plans to start the controversial experiments, leading virologists Ron Fouchier and Yoshihiro Kawaoka said H7N9’s pandemic risk would rise “exponentially” if it gained the ability to spread easily among people.

And the only way to find out how likely that is, and how many genetic changes would need to take place before it could happen, is to engineer those mutations in laboratory conditions  and test the virus’s potential using animal models, they said.

“It’s clear this H7N9 virus has some hallmarks of pandemic viruses, and it’s also clear it is still missing at least one or two of the hallmarks we’ve seen in the pandemic viruses of the last century,” Fouchier told Reuters in a telephone interview.

“So the most logical step forward is to put in those (missing) mutations first.”

Writing in the journals Nature and Science on behalf of 22 scientists who will carry out various aspects of the H7N9 work, Fouchier said because the risk of a pandemic caused by a bird flu virus exists in nature, it was critical for risk-mitigation plans to study the likely mutations that could make that happen.

This kind of science is known as “gain of function” (GOF) research. It aims to identify combinations of genetic mutations that allow an animal virus to jump to humans and spread easily.

By finding the mutations needed, researchers and health authorities can better assess how likely it is that a new virus could become dangerous and if so, how soon they should begin developing drugs, vaccines and other scientific defences.

Yet such work is highly controversial. It has fuelled an international row in the past two years after it was carried out on another threatening bird flu virus called H5N1.


When Fouchier, of the Erasmus Medical Centre in Rotterdam, The Netherlands, and Kawaoka, at the University of Wisconsin in the United States, announced in late 2011 they had found how to make H5N1 into a form that could spread between mammals, the U.S. National Science Advisory Board for Biosecurity (NSABB) was so alarmed that it took the unprecedented step of trying to censor publication of the studies.

The NSABB said it feared details of the work could fall into the wrong hands and be used for bioterrorism. A year-long moratorium on such research followed while the World Health Organisation, U.S. security advisers and international flu researchers sought ways to ensure the highest safety controls.

The laboratory Fouchier will be working in is known as a  BSL3 Enhanced lab (Bio-Safety Level 3), the highest level of biosecurity that can be achieved in academic research.

“Nature is the biggest threat to us, not what we do in the lab. What we do in the lab is under very intense biosecurity measures,” he said. “There are layers upon layers of layers of biosafety measures such that if one layer might break there are additional layers to prevent this virus ever coming out.”

Fouchier conceded that GOF research has been “under fire” recently. “One of the accusations against the flu community was that we were not transparent enough about what experiments were being done, and why and how they were being done,” he said. “We’re trying to pre-empt such accusations this time.”

The H7N9 bird flu outbreak currently appears under control with only 3 new human cases in May after 87 in April and 30 in March. Experts say this is largely thanks to the closure of many live poultry markets and because of warmer weather.

Yet as winter approaches in China, many experts believe H7N9 could re-emerge, meaning the threat of a pandemic looms if it mutates to become easily transmissible between people.

The first scientific analysis of probable human-to-human transmission of H7N9 raised concern about its pandemic potential and prompted scientists James Rudge and Richard Coker of the London School of Hygiene and Tropical Medicine to warn: “The threat posed by H7N9 has by no means passed.”

Fouchier and colleagues said they hope to unravel the molecular processes behind H7N9 by manipulating its genetic material to increase virulence or induce drug resistance.

Wendy Barclay, an Imperial College London flu expert, said it would be ludicrous to shy away from such studies. “This type of work is like fitting glasses for someone who can’t see well,” she said. “Without the glasses the vision is blurred and uncertain, with them you can focus on the world and deal with it a lot more easily.”   (Reporting by Kate Kelland; editing by David Evans)



Scientists create hybrid flu that can go airborne : Mixed Genes from H5N1 & H1N1

H5N1 virus with genes from H1N1 can spread through the air between mammals.

02 May 2013

Researchers have crossed two strains of avian flu virus to create one that can be transmitted through the air — and possibly settle on the cilia of lung cells as in this conceptual image.


As the world is transfixed by a new H7N9 bird flu virus spreading through China, a study reminds us that a different avian influenza — H5N1 — still poses a pandemic threat.

A team of scientists in China has created hybrid viruses by mixing genes from H5N1 and the H1N1 strain behind the 2009 swine flu pandemic, and showed that some of the hybrids can spread through the air between guinea pigs. The results are published in Science1.

Flu hybrids can arise naturally when two viral strains infect the same cell and exchange genes. This process, known as reassortment, produced the strains responsible for at least three past flu pandemics, including the one in 2009.

There is no evidence that H5N1 and H1N1 have reassorted naturally yet, but they have many opportunities to do so. The viruses overlap both in their geographical range and in the species they infect, and although H5N1 tends mostly to swap genes in its own lineage, the pandemic H1N1 strain seems to be particularly prone to reassortment.

“If these mammalian-transmissible H5N1 viruses are generated in nature, a pandemic will be highly likely,” says Hualan Chen, a virologist at the Harbin Veterinary Research Institute of the Chinese Academy of Sciences, who led the study.

“It’s remarkable work and clearly shows how the continued circulation of H5N1 strains in Asia and Egypt continues to pose a very real threat for human and animal health,” says Jeremy Farrar, director of the Oxford University Clinical Research Unit in Ho Chi Minh City, Vietnam.

Flu fears

Chen’s results are likely to reignite the controversy that plagued the flu community last year, when two groups found that H5N1 could go airborne if it carried certain mutations in a gene that produced a protein called haemagglutinin (HA)2, 3. Following heated debate over biosecurity issues raised by the work, the flu community instigated a voluntary year-long moratorium on research that would produce further transmissible strains. Chen’s experiments were all finished before the hiatus came into effect, but more work of this nature can be expected now that the moratorium has been lifted.

“I do believe such research is critical to our understanding of influenza,” says Farrar. “But such work, anywhere in the world, needs to be tightly regulated and conducted in the most secure facilities, which are registered and certified to a common international standard.”

Virologists have created H5N1 reassortants before. One study found that H5N1 did not produce transmissible hybrids when it reassorts with a flu strain called H3N24. But in 2011, Stacey Schultz-Cherry, a virologist at St. Jude Children’s Research Hospital in Memphis, Tennessee, showed that pandemic H1N1 becomes more virulent if it carries the HA gene from H5N15.

Chen’s team mixed and matched seven gene segments from H5N1 and H1N1 in every possible combination, to create 127 reassortant viruses, all with H5N1’s HA gene. Some of these hybrids could spread through the air between guinea pigs in adjacent cages, as long as they carried either or both of two genes from H1N1 called PA and NS. Two further genes from H1N1, NA and M, promoted airborne transmission to a lesser extent, and another, the NP gene, did so in combination with PA.

“It’s a very extensive paper,” says Schultz-Cherry. “It really shows that it’s more than just the HA. The other proteins are just as important and can drive transmission.” Chen says that health organisations should monitor wild viruses for the gene combinations that her team identified in the latest study. “If those kinds of reassortants are found, we’d need to pay high attention.”

Knowledge gap

It is unclear how the results apply to humans. Guinea pigs have bird-like receptor proteins in their upper airways in addition to mammalian ones, so reassortant viruses might bind in them more easily than they would in humans.

And scientists do not know whether the hybrid viruses are as deadly as the parent H5N1. The hybrids did not kill any of the guinea pigs they spread to, but Chen says that these rodents are not good models for pathogenicity in humans.

There is also a chance that worldwide exposure that already occurred to the pandemic H1N1 strain might actually mitigate the risk of a future pandemic by providing people with some immunity against reassortants with H5N1. In an earlier study, Chen and her colleagues showed that a vaccine made from pandemic H1N1 provided some protection against H5N1 infections in mice6.

“If you take [antibodies] from people who have been vaccinated or naturally infected, will they cross-react with these viruses?” asks Schultz-Cherry. “That’s an important study that would need to be done.”

Ironically, Chen’s team is now too busy reacting to the emerging threat of a different bird flu — H7N9. Research on H5N1 will have to wait.

Journal name:

‘Appalling irresponsibility’: Senior scientists attack Chinese researchers for creating new strains of influenza virus in veterinary laboratory

Experts warn of danger that the new viral strains created by mixing bird-flu virus with human influenza could escape from the laboratory to cause a global pandemic killing millions of people.

Steve Connor

Thursday, 2 May 2013

Senior scientists have criticised the “appalling irresponsibility” of researchers in China who have deliberately created new strains of influenza virus in a veterinary laboratory.

They warned there is a danger that the new viral strains created by mixing bird-flu virus with human influenza could escape from the laboratory to cause a global pandemic killing millions of people.

Lord May of Oxford, a former government chief scientist and past president of the Royal Society, denounced the study published today in the journal Science as doing nothing to further the understanding and prevention of flu pandemics.

“They claim they are doing this to help develop vaccines and such like. In fact the real reason is that they are driven by blind ambition with no common sense whatsoever,” Lord May told The Independent.

“The record of containment in labs like this is not reassuring. They are taking it upon themselves to create human-to-human transmission of very dangerous viruses. It’s appallingly irresponsible,” he said.

The controversial study into viral mixing was carried out by a team led by Professor Hualan Chen, director of China’s National Avian Influenza Reference Laboratory at Harbin Veterinary Research Institute.

Professor Chen and her colleagues deliberately mixed the H5N1 bird-flu virus, which is highly lethal but not easily transmitted between people, with a 2009 strain of H1N1 flu virus, which is very infectious to humans.

When flu viruses come together by infecting the same cell they can swap genetic material and produce “hybrids” through the re-assortment of genes. The researchers were trying to emulate what happens in nature when animals such as pigs are co-infected with two different strains of virus, Professor Chen said.

“The studies demonstrated that H5N1 viruses have the potential to acquire mammalian transmissibility by re-assortment with the human influenza viruses,” Professor Chen said in an email.

“This tells us that high attention should be paid to monitor the emergence of such mammalian-transmissible virus in nature to prevent a possible pandemic caused by H5N1 virus,” she said.

“It is difficult to say how easy this will happen, but since the H5N1 and 2009/H1N1 viruses are widely existing in nature, they may have a chance to re-assort,” she added.

The study, which was carried out in a laboratory with the second highest security level to prevent accidental escape, resulted in 127 different viral hybrids between H5N1 and H1N1, five of which were able to pass by airborne transmission between laboratory guinea pigs.

Professor Simon Wain-Hobson, an eminent virologist at the Pasteur Institute in Paris, said it is very likely that some or all of these hybrids could pass easily between humans and possess some or all of the highly lethal characteristics of H5N1 bird-flu.

“Nobody can extrapolate to humans except to conclude that the five viruses would probably transmit reasonable well between humans,” Professor Wain-Hobson said.

“We don’t know the pathogenicity [lethality] in man and hopefully we will never know. But if the case fatality rate was between 0.1 and 20 per cent, and a pandemic affected 500 million people, you could estimate anything between 500,000 and 100 million deaths,” he said.

“It’s a fabulous piece of virology by the Chinese group and it’s very impressive, but they haven’t been thinking clearly about what they are doing. It’s very worrying,” Professor Wain-Hobson said.

“The virological basis of this work is not strong. It is of no use for vaccine development and the benefit in terms of surveillance for new flu viruses is oversold,” he added.

An increasing number of scientists outside the influenza field have expressed concern over attempts to deliberately increase the human transmissibility of the H5N1 bird-flu virus. This is done by mutating the virus so that it can pass by airborne droplets between laboratory ferrets, the standard “animal model” of human influenza.

Two previous studies, by Ron Fouchier of Erasmus Medical Centre in Rotterdam and Yoshihiro Kawaoka of the University of Wisconsin, Madison, caused uproar in 2011 when it emerged that they had created airborne versions of H5N1 that could be passed between ferrets.

The criticism led to researchers to impose a voluntary moratorium on their H5N1 research, banning transmission studies using ferrets. However they decided to lift the ban earlier this year, arguing that they have now consulted widely with health organisations and the public over safety concerns.

However, other scientists have criticised the decision to lift the moratorium.

Scientists described small genetic changes that enable the H5N1 bird flu virus to replicate more easily in the noses of mammals


Bird flu mutation study offers vaccine clue

by  Sam Wong   08 April 2013                   

main image


shadow Scientists have described small genetic changes that enable the H5N1 bird flu virus to replicate more easily in the noses of mammals.

So far there have only been isolated cases of bird flu in humans, and no widespread transmission as the H5N1 virus can’t replicate efficiently in the nose. The new study, using weakened viruses in the lab, supports the conclusions of controversial research published in 2012 which demonstrated that just a few genetic mutations could enable bird flu to spread between ferrets, which are used to model flu infection in humans.

Researchers say the new findings could help to develop more effective vaccines against new strains of bird flu that can spread between humans.

“Knowing why bird flu struggles to replicate in the nose and understanding the genetic mutations that would enable it to happen are vital for monitoring viruses circulating in birds and preparing for an outbreak in humans,” said Professor Wendy Barclay, from the Department of Medicine at Imperial College London, who led the study.

“The studies published last year pointed to a mechanism that restricts replication of H5N1 viruses in the nose. We’ve engineered a different mutation with the same effect into one of the virus proteins and achieved a similar outcome. This suggests that there is a common mechanism by which bird flu could evolve to spread between humans, but that a number of different specific mutations might mediate that.”

Bird flu only rarely infects humans because the human nose has different receptors to those of birds and is also more acidic. The Imperial team studied mutations in the gene for haemagglutinin, a protein on the surface of the virus that enables it to get into host cells. They carried out their experiments in a laboratory strain of flu with the same proteins on its surface as bird flu, but engineered so that it cannot cause serious illness.

The more we understand about the kinds of mutations that will enable them to transmit between humans, the better we can prepare for a possible pandemic.– Professor Wendy Barclay

Department of Medicine

The research found that mutations in the H5 haemagglutinin enabled the protein to tolerate higher levels of acidity. Viruses with these mutations and others that enabled them to bind to different receptors were able to replicate more efficiently in ferrets and spread from one animal to another.

The results have important implications for designing vaccines against potential pandemic strains of bird flu. Live attenuated flu vaccines (LAIV) might be used in a pandemic situation because it is possible to manufacture many more doses of this type of vaccine than of the killed virus vaccines used to protect against seasonal flu. LAIV are based on weakened viruses that don’t cause illness, but they still have to replicate in order to elicit a strong immune response. Viruses with modified haemagglutinin proteins induced strong antibody responses in ferrets in this study, suggesting that vaccines with similar modifications might prove more effective than those tested previously.

“We can’t predict how bird flu viruses will evolve in the wild, but the more we understand about the kinds of mutations that will enable them to transmit between humans, the better we can prepare for a possible pandemic,” said Professor Barclay.

The research was funded by the Medical Research Council and the Wellcome Trust and published in the Journal of General Virology.


H Shelton et al. ‘Mutations in hemagglutinin that affect receptor binding and pH stability increase replication of a PR8 influenza virus with H5 HA in the upper respiratory tract of ferrets and may contribute to transmissibility.’ Journal of General Virology (2013) doi:10.1099/vir.0.050526-0



Mutation altering stability of surface molecule in acid enables H5N1 infection of mammals

Contact: Jim Sliwa jsliwa@asmusa.org 202-942-9297 American Society for Microbiology

A single mutation in the H5N1 avian influenza virus that affects the pH at which the hemagglutinin surface protein is activated simultaneously reduces its capacity to infect ducks and enhances its capacity to grow in mice according to research published ahead of print today in the Journal of Virology.

“Knowing the factors and markers that govern the efficient growth of a virus in one host species, tissue, or cell culture versus another is of fundamental importance in viral infectious disease,” says Charles J. Russell of St. Jude Children’s Research Hospital, Memphis, TN, an author on the study. “It is essential for us to identify influenza viruses that have increased potential to jump species, to help us make decisions to cull animals, or quarantine humans.” The same knowledge “will help us identify targets to make new drugs that stop the virus… [and] engineer vaccines.”

Various influenza viruses are spreading around the globe among wild birds, but fortunately, few gain the ability to jump to humans. However, those that do, and are able to then spread efficiently from person to person, cause global epidemics, such as the infamous pandemic of 1918, which infected one fifth and killed an estimated 2.7 percent of the world’s population. Occasionally, one of these viruses is exceptionally lethal. For example, H5N1 has killed more than half of the humans it has infected. The specter of such a virus becoming easily transmissible among humans truly frightens public health officials. But understanding the mechanisms of transmission could help microbiologists find ways to mitigate major epidemics.

When influenza viruses infect birds, the hemagglutinin surface protein of the virus is activated by acid in the entry pathway inside the host cell, enabling it to invade that cell. In earlier work, Russell and collaborators showed that a mutant version of the influenza H5N1 virus called K58I that resists acid activation, loses its capacity to infect ducks. Noting that the upper airways of mammals are more acidic than infected tissues of birds, they hypothesized, correctly, that a mutation rendering the hemagglutinin protein resistant to acid might render the virus more infective in mammals.

In this study the investigators found that K58I grows 100-fold better than the wild-type in the nasal cavities of mice, and is 50 percent more lethal. Conversely, the mutant K58I virus failed completely to kill ducks the investigators infected, while the wild-type killed 66 percent of ducks, says Russell. “A single mutation that eliminates H5N1 growth in ducks simultaneously enhances the capacity of H5N1 to grow in mice. We conclude that enhanced resistance to acid inactivation helps adapt H5N1 influenza virus from an avian to a mammalian host.”

“These data contribute new information about viral determinants of influenza virus virulence and provide additional evidence to support the idea that H5N1 influenza virus pathogenesis in birds and mammals is linked to the pH of [hemagglutinin] activation in an opposing fashion,” Terence S. Dermody of Vanderbilt University et al. write in an editorial in the journal accompanying the paper. “A higher pH optimum of [hemagglutinin] activation favors virulence in birds, whereas a lower pH optimum… favors virulence in mammals.”

Based on this and another study, “…surveillance should include phenotypic assessment of the [hemagglutinin] activation pH in addition to sequence analysis,” Dermody writes.

The journal carefully considered whether to publish the paper, because it raised issues of “dual use research of concern” (DURC), writes Dermody. DURC is defined as “Life sciences research that, based on current understanding, can be reasonably anticipated to provide knowledge, information, products, or technologies that could be directly misapplied to pose a significant threat with broad potential consequences to public health and safety, agricultural crops and other plants, animals, the environment, materiel, or national security,” according to a US government policy document. However, both the National Institute of Allergy and Infectious Diseases and the St. Jude Institutional Biosafety Committee concluded that the study failed to meet the definition of DURC. Clinching the case, “the addition of the key mutation in the Russell paper to other previously reported mutations would not result in an even more virulent H5N1 influenza virus,” says Dermody.


A copy of the research manuscript can be found online at http://bit.ly/asmpr0213b.  The manuscript of the accompanying editorial can be accessed at http://bit.ly/asmpr0213c. Both are scheduled to be formally published in the May 2013 issue of the Journal of Virology.

(H. Zaraket, O.A. Bridges, and C.J. Russell, 2013. The pH of activation of the hemagglutinin protein regulates H5N1 influenza virus replication and pathogenesis in mice. J. Virol. online ahead of print February 28, 2013, doi:10.1128/JVI.03110-12.)

The Journal of Virology is a publication of the American Society for Microbiology (ASM).  The ASM is the largest single life science society, composed of over 39,000 scientists and health professionals. Its mission is to advance the microbiological sciences as a vehicle for understanding life processes and to apply and communicate this knowledge for the improvement of health and environmental and economic well-being worldwide.

Man’s best friend: Common canine virus may lead to new vaccines for deadly human diseases

Public Affairs News Service

Tuesday, Nov. 27, 2012

Writer: James  E.  Hataway, 706/542-5222, jhataway@uga.edu Contact: Biao He, 706/542-2855, bhe@uga.edu

Athens, Ga. – Researchers at the University of Georgia have discovered that a virus commonly found in dogs may serve as the foundation for the next great breakthrough in human vaccine development.

Although harmless in humans, parainfluenza virus 5, or PIV5, is thought to contribute to upper respiratory infections in dogs, and it is a common target for canine vaccines designed to prevent kennel cough. In a paper published recently in PLOS ONE, researchers describe how this virus could be used in humans to protect against diseases that have eluded vaccine efforts for decades.

“We can use this virus as a vector for all kinds of pathogens that are difficult to vaccinate against,” said Biao He, the study’s principal investigator and professor of infectious diseases in UGA’s College of Veterinary Medicine. “We have developed a very strong H5N1 flu vaccine with this technique, but we are also working on vaccines for HIV, tuberculosis and malaria.”

PIV5 does not cause disease in humans, as our immune system is able to recognize and destroy it. By placing antigens from other viruses or parasites inside PIV5, it effectively becomes a delivery vehicle that exposes the human immune system to important pathogens and allows it to create the antibodies that will protect against future infection.

This approach not only ensures full exposure to the vaccine but also is much safer because it does not require the use of attenuated, or weakened, pathogens. For example, an HIV vaccine delivered by PIV5 would contain only those parts of the HIV virus necessary to create immunity, making it impossible to contract the disease from the vaccine.

“Safety is always our number one concern,” said He, who is also a Georgia Research Alliance distinguished investigator and member of the Faculty of Infectious Diseases. “PIV5 makes it much easier to vaccinate without having to use live pathogens.”

Using viruses as a delivery mechanism for vaccines is not a new technique, but previous efforts have been fraught with difficulty. If humans or animals already possess a strong immunity to the virus used for delivery, the vaccine is unlikely to work, as it will be destroyed by the immune system too quickly.

“Pre-existing immunity to viruses is the main reason most of these vaccines fail,” He said.

But in this latest study, He and his colleagues demonstrate that immunity to PIV5 does not limit its effectiveness as a vaccine delivery mechanism, even though many animals-including humans- already carry antibodies against it.

In their experiments, the researchers found that a single dose inoculation using PIV5 protected mice from the influenza strain that causes seasonal flu. Another single dose experimental vaccine also protected mice from the highly pathogenic and deadly H5N1 virus commonly known as bird flu.

This recent work is a culmination of more than fifteen years of research and experimentation with the PIV5 virus, and He has confidence that it will serve as an excellent foundation for vaccines to treat diseases in both animals and humans.

“I believe we have the best H5N1 vaccine candidate in existence,” He said. “But we have also opened up a big field for a host of new vaccines.”

UGA Faculty of Infectious Diseases The University of Georgia Faculty of Infectious Diseases was created in 2007 to address existing and emerging infectious disease threats more effectively by integrating multidisciplinary research in animal, human and ecosystem health. Researchers from across the university focus on epidemiology, host-pathogen interactions, the evolution of infectious diseases, disease surveillance and predictors and the development of countermeasures such as vaccines, therapeutics and diagnostics. For more information about the Faculty of Infectious Diseases, see fid.ovpr.uga.edu.

UGA College of Veterinary Medicine The UGA College of Veterinary Medicine, founded in 1946, is dedicated to training future veterinarians, to conducting research related to animal and human diseases, and to providing veterinary services for animals and their owners. Research efforts are aimed at enhancing the quality of life for animals and people, improving the productivity of poultry and livestock, and preserving a healthy interface between wildlife and people in the environment they share. The college enrolls 102 students each fall out of more than 800 who apply.

CDC Wants Safety Threat Information on Goose Flu

WASHINGTON (CN) – The Centers for Disease Control and Prevention request information and comments to questions on a highly contagious “goose” variant of avian influenza H5N1 viruses.

The viruses contain a hemagglutinin from the Goose/Guangdong/1/96 lineage. The CDC, among other questions, asks about “their potential to pose a severe threat to public health and safety.”

The CDC notes on its website that ferrets can transmit this variant, and it has been associated with infections in humans.

Comments will be accepted until Dec. 17.

For more information, click the document icon for this regulation and others.


Human nose too cold for bird flu, says new study ( H5N1 )

2009 study posted for filing

Contact: Lucy Goodchild
Imperial College London

Avian influenza viruses do not thrive in humans because the temperature inside a person’s nose is too low, according to research published today in the journal PLoS Pathogens. The authors of the study, from Imperial College London and the University of North Carolina, say this may be one of the reasons why bird flu viruses do not cause pandemics in humans easily.

There are 16 subtypes of avian influenza and some can mutate into forms that can infect humans, by swapping proteins on their surface with proteins from human influenza viruses.

Today’s study shows that normal avian influenza viruses do not spread extensively in cells at 32 degrees Celsius, the temperature inside the human nose. The researchers say this is probably because the viruses usually infect the guts of birds, which are warmer, at 40 degrees Celsius. This means that avian flu viruses that have not mutated are less likely to infect people, because the first site of infection in humans is usually the nose. If a normal avian flu virus infected a human nose, the virus would not be able to grow and spread between cells, so it would be less likely to damage cells and cause respiratory illness.

The researchers also found that when they created a mutated human influenza virus by adding a protein from the surface of an avian influenza virus, this mutated virus struggled to thrive at 32 degrees Celsius. This suggests that if a new human influenza strain evolved by adopting proteins from an avian influenza virus, this would need to undergo further changes in order to adapt to the conditions in the human body.

The researchers reached their conclusions by growing cells from the human airway and infecting them with different human and avian influenza viruses, including H5N1, to see how well the viruses grew and spread. The human influenza viruses grew equally well in the cells whether they were maintained at 37 degrees Celsius, our core body temperature, or at 32 degrees Celsius, the temperature of the nose. In contrast, the four avian influenza viruses tested grew well at 37 degrees Celsius but grew very slowly at 32 degrees Celsius.

When the researchers added proteins from an avian influenza virus to a human influenza virus, the human influenza virus also grew slowly and struggled to replicate at 32 degrees Celsius.

As viruses kill the cells they infect, the researchers also measured the extent of cell death in the model. This showed that at 32 degrees Celsius, far fewer cells died as a result of infection with avian influenza compared with human influenza, supporting the idea that the avian virus could not thrive at that temperature.

Professor Wendy Barclay, one of the authors of the study from the Division of Investigative Science at Imperial College London, said: “Bird viruses are out there all the time but they can only cause pandemics when they undergo certain changes. Our study gives vital clues about what kinds of changes would be needed in order for them to mutate and infect humans, potentially helping us to identify which viruses could lead to a pandemic.

“It would be impossible to develop vaccines against all 16 subtypes of avian flu, so we need to prioritise. By studying a range of different viruses in systems like this one we can look for warnings that they are already beginning to make the kinds of genetic changes in nature that mean they could be poised to jump into humans; animal viruses that spread well at low temperatures in these cultures could be more likely to cause the next pandemic than those which are restricted,” added Professor Barclay.




The research was funded by the Medical Research Council in the UK and by the NIH in the USA.

St. Jude develops vaccine against potential pandemic influenza virus H5N1 using reverse genetics (Using H1N1, requested repost 2003)

Contact: Bonnie Cameron bonnie.cameron@stjude.org 901-495-4815 St. Jude Children’s Research Hospital

Special modification of reverse genetics created at St. Jude allowed vaccine to be custom-made within weeks of emergence of virus

(MEMPHIS, TENN.–April 2, 2003) Scientists at St. Jude Children’s Research Hospital announced today the development of a vaccine against H5N1, a new lethal influenza virus that triggered the World Health Organization (WHO) to declare a pandemic alert in February 2003.

The virus appeared in birds in Hong Kong late last year and subsequently killed one of two infected people with rapidly progressive pneumonia in the past month.  St. Jude developed the vaccine in only four weeks from the time it received the H5N1 sample from colleagues in Hong Kong.

The announcement comes at a time when a second, as-yet-unidentified virus, has taken several lives around the world. The unknown virus, which causes severe acute respiratory syndrome (SARS), appears to have originated at the same time and in the same place as the new “flu.”

The development of the initial (“seed”) batch of H5N1 vaccine is significant because humans do not have a natural immunity to the virus, according to Robert Webster, Ph.D., a member of the Department of Infectious Diseases at St. Jude. Rather, humans appear to become infected through contact with chickens and other birds. In the past the virus killed only the chickens it infected. But the new variant of H5N1 also killed many kinds of wild birds, which is unusual.

If H5N1 acquires the ability to pass from human to human, there would be the potential for concern similar to that for SARS, according to Webster.

“It’s likely there were two things that prevented the 1997 poultry influenza outbreak in Hong Kong from becoming more deadly–its inability to spread from human to human and the slaughter of more than 1.5 million chickens and other birds in the open-air markets of Hong Kong, which eliminated the source of the virus,” Webster said. “In fact, the sudden appearance of SARS in the same region of the world is just another warning that the large populations of people and poultry in this region are a potential source of viruses.”

Webster is the director of the WHO’s U.S. Collaborating Center at St. Jude that studies animal influenza viruses. It is the only WHO laboratory that focuses on the transmission of animal viruses to humans.

Webster’s laboratory has sent the seed H5N1 vaccine to the Centers for Disease Control in Atlanta and the World Influenza Center in London for further testing, in preparation for initial Phase I and Phase II trials in humans.  “It’s important to move right along with these trials in case the virus begins spreading from person to person,” Webster says. Led by Richard Webby, Ph.D., and Daniel Perez, Ph.D., the St. Jude laboratory team successfully modified a technique called reverse genetics to permit them to develop the H5N1 vaccine so quickly. Using the samples of H5N1 obtained from Hong Kong, Webby mixed two genes from H5N1 with six genes from a second virus (A/PR8/34)[H1N1]). H1N1 is a rapidly growing “master” strain of virus commonly used to make vaccines.

The genes from flu viruses produce proteins called HA and NA, which are on the surface of the virus, in full “view” of the immune system. Webby took the modified gene for HA and the NA from H5N1 and mixed them inside a cell with six genes from H1N1. The HA gene was modified to abolish its ability to cause disease and therefore made it safer to use in the vaccine.

The genes mixed together, and the resulting vaccine virus produced in the cell thus carried HA and NA from H5N1. But because of the alterations to the HA, and the rest of the genes being derived from H1N1, the new virus vaccine cannot cause disease. Rather, it can only stimulate the immune system to respond to H5N1.

“The St. Jude vaccine is like a gun without ammunition,” said Elaine Tuomanen, M.D., director of the St. Jude Department of Infectious Diseases. “The vaccine looks deadly enough for its HA and NA proteins to alert the immune system. But in reality, it’s carrying blanks that can’t cause disease.”

Key to the quick success in developing the vaccine was the on-campus availability of GMP (Good Manufacturing Practices) facilities, which are equivalent in quality to those used by pharmaceutical companies to make biological agents such as vaccines. In addition, the centralization of genetic analysis and other molecular biology work, performed in the Hartwell Center for Bioinformatics and Biotechnology at St. Jude, greatly accelerated the process of building the vaccine components.

“We’ve been lucky twice with H5N1–once in 1997 and once so far during this current outbreak–in not experiencing human-to-human transmission,” Webster says. “But the mixing bowl in Hong Kong is still stirring up new variations of familiar viruses. Although we just made a vaccine against one of that mixing bowl’s nasty viral brews, SARS shows us there’s always another threat down the road.”


St. Jude Children’s Research Hospital St. Jude Children’s Research Hospital, in Memphis, Tennessee, was founded by the late entertainer Danny Thomas. The hospital is an internationally recognized biomedical research center dedicated to finding cures for catastrophic diseases of childhood. The hospital’s work is supported through funds raised by ALSAC. ALSAC covers all costs not covered by insurance for medical treatment rendered at St. Jude Children’s Research Hospital. Families without insurance are never asked to pay. For more information, please visit http://www.stjude.org.

A small genetic change makes flu virus deadly ( H5N1 2001 Requested Repost)

Contact: Jeff Minerd jminerd@niaid.nih.gov 301-402-1663 NIH/National Institute of Allergy and Infectious Diseases

A small genetic change makes flu virus deadly

A tiny change in one of the influenza virus’s 10 genes is key to making certain strains of the virus especially virulent to humans, scientists report in the Sept. 7 issue of Science. This discovery helps explain why an influenza outbreak four years ago in Hong Kong killed an unusually high proportion of the people it infected – six out of 18, says lead researcher Yoshihiro Kawaoka, D.V.M., Ph.D., of the University of Wisconsin-Madison.

“We have found that a limited number of very tiny genetic changes in a specific gene, one called PB2, can have a big effect on how potent the influenza virus is,” says Dr. Kawaoka, a grantee of the National Institute of Allergy and Infectious Diseases (NIAID).  “Because the influenza virus constantly mutates, and because only a few changes can make a non-pathogenic virus highly pathogenic, we should assume that an outbreak of any new strain or subtype is potentially dangerous to humans.”

“To prepare for future influenza pandemics, NIAID has supported efforts to understand how new virus strains potentially harmful to humans appear,” says Anthony S. Fauci, M.D., NIAID director. “This study is an elegant example of research that provides insight into the emergence of virulent viruses and can help us develop better strategies for detecting future outbreaks.”

Wild waterfowl are natural reservoirs for the influenza virus; these birds transmit the virus to pigs or chickens, which then pass it on to people. The deadly outbreak of influenza virus subtype H5N1 in Hong Kong in 1997 was the first documented case of an influenza virus jumping directly from chickens to people.  Public health authorities responded by ordering the slaughter of more than 1 million live poultry to prevent further spread of the virus to humans.

Dr. Kawaoka and colleagues obtained samples of the H5N1 viruses that had infected Hong Kong residents during the 1997 outbreak. Testing these viruses in laboratory mice, the researchers found good correlation between how sick certain H5N1 strains made mice and how sick they had made humans.  The researchers divided the H5N1 strains into two groups: one that caused systemic lethal infection in the mice and one that was relatively benign.  Mice are a good model for studying H5N1, Dr. Kawaoka says, because this virus affects mice and humans similarly.

Next, Dr. Kawaoka used a technology that allows him to genetically engineer “designer” influenza viruses from scratch.  By systematically swapping the genes from the harmful and benign viruses, then testing how those engineered viruses affected mice, he discovered that the PB2 gene from the harmful group gives the virus its potency.  Then, through testing viruses that contained variations of this PB2 gene, he further identified a tiny change within the gene – a change of just one unit of RNA – that appears to be key to the virus’s virulence.

The function of the PB2 gene is not completely understood, but scientists believe it codes for an enzyme that helps force the host cell’s molecular machinery to make more viruses, Dr. Kawaoka explains.  “We don’t know if the mutation we studied is involved in that process, but our next step will be to find out,” he says.

Just over 10 years ago, researchers developed the ability to genetically engineer influenza viruses, a process known as reverse genetics.  In 1999, Dr. Kawaoka, with support from NIAID, streamlined this technology, making it much more efficient.  Without the ability to engineer influenza viruses through the reverse genetics system, it would not have been possible to create and study variations of the H5N1 virus, Dr. Kawaoka says.  “Just a few years ago, this discovery would not have been possible,” says Carole Heilman, Ph.D., director of NIAID’s Division of Microbiology and Infectious Diseases. “We believe this is the first of many more important discoveries that will arise from this technology.”


For more information on Dr. Kawaoka’s work in this field, other NIAID-supported influenza research, and background on the virus itself, visit Focus on the Flu on the NIAID Web site at http://www.niaid.nih.gov/newsroom/focuson/flu00.  Focus on the Flu also contains information on NIAID-sponsored efforts to prepare for future influenza pandemics.  Such efforts include helping to fund ongoing monitoring of influenza virus strains circulating through live poultry markets in Hong Kong, a project that could nip future outbreaks in the bud.  Other NIAID-supported researchers are examining the history of influenza virus evolution for clues about which new strains might emerge next.

NIAID is a component of the National Institutes of Health (NIH).  NIAID supports basic and applied research to prevent, diagnose, and treat infectious and immune-mediated illnesses, including HIV/AIDS and other sexually transmitted diseases, tuberculosis, malaria, autoimmune disorders, asthma and allergies.


Hatta M et al.  Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses.  Science 293(5536):1840-42 (2001).

Press releases, fact sheets and other NIAID-related materials are available on the NIAID Web site at http://www.niaid.nih.gov.

Superflu is being brewed in the lab (Article H5N1 2004 Requested Repost)

Contact: Claire Bowles claire.bowles@rbi.co.uk 44-207-331-2751 New Scientist

Superflu is being brewed in the lab

AFTER the worldwide alarm triggered by last year’s SARS outbreak, it might seem reckless to set about creating a potentially far more devastating virus in the lab. But that is what is being attempted by some researchers, who argue that the dangers of doing nothing are even greater.

We already know that the H5N1 bird flu virus ravaging poultry farms in Asia can be lethal on the rare occasions when it infects people. Now a team is tinkering with its genes to see if it can turn into a strain capable of spreading from human to human. If they manage this, they will have created a virus that could kill tens of millions if it got out of the lab.

Many researchers say experiments like this are needed to answer crucial questions. Why can a few animal flu viruses infect humans? What makes the viruses deadly? And what changes, if any, would enable them to spread from person to person and cause pandemics that might prove far worse than that of 1918? Once we know this, they argue, we will be better prepared for whatever nature throws at us.

Others disagree. It is not clear how much we can learn from such work, they argue. And they point out that it is already possible to create a vaccine by other means (see page 36). The work is simply too dangerous, they say. “I’m getting bombarded from both sides,” says Ronald Atlas, head of the Center for Deterrence of Biowarfare and Bioterrorism at the University of Louisville in Kentucky. “Some say that this sort of research is dangerous because of the risk of the virus escaping or being using in bioterrorism, and others that it’s good science.”

Some researchers refuse to discuss their plans. But Jacqueline Katz at the US Centers for Disease Control (CDC)in Atlanta, Georgia, told New Scientist her team is already tweaking the genes of the H5N1 bird flu virus that killed several people in Hong Kong in 1997, and those of the human flu virus H3N2. She is testing the ability of the new viruses to spread by air and cause disease in ferrets, whose susceptibility to flu appears to be remarkably similar to ours. Albert Osterhaus of Erasmus University in Rotterdam in the Netherlands plans to test altered viruses on rodents and macaque monkeys. Other groups are also considering similar experiments, he says.  If such work were to show that H5N1 could cause a human pandemic, everything that is happening in Asia would be even more alarming, Osterhaus argues. If, on the other hand, it failed to transform H5N1 into a highly contagious human virus, we could relax. “It becomes a veterinary health problem, not a public health problem. That would be an enormous relief.”

But Wendy Barclay of the University of Reading in the UK, who “thought long and hard” about trying to create a pandemic flu virus before abandoning the idea, disagrees. “If you get a negative, how can you be sure that you have tested every option?” she says. Health authorities would still have to take the precaution of creating H5N1 vaccines.

Barclay concedes, however, that creating a virus that spreads in people might tell us how real the threat is. For instance, do you need one mutation for H5N1 to adapt to humans, or dozens?

Osterhaus is more optimistic. “Within the next decade, the whole thing will be solved,” he says. “We will know the rules.” In other words, once experts understand what the genetic sequence of any flu virus means, they could predict which animals it can infect, how severe it will be, and how easily it will spread.

Yet any new viruses could only be tested in human cell cultures or in animals, not on people. None of these methods exactly reflects how flu behaves in humans. This has led some flu experts to argue that attempts to create a pandemic virus should be put on hold until there is agreement on the best way of testing it.  And there is an even more fundamental objection to such experiments: the processes used to create the viruses may be too artificial. Researchers who want to see if H5N1 can be pandemic can take two approaches. One is to tinker with the genome of the bird flu virus to mimic mutations that might occur naturally. This can be done precisely using a technique called reverse genetics (see page 38) The other approach is to mix bird flu genes with those of human flu viruses, either using reverse genetics or through random reassortment in cells infected with both types.

Although reassortment sounds more natural, there’s a problem. “Reassortments can be made very easily in the lab using cells or animals,” says flu expert Graeme Laver, formerly at the Australian National University in Canberra. “But one of the big mysteries is that [human] viruses that appear by reassortment are extremely rare in nature. There is something else involved that we don’t understand.”

Then there is the question of safety. The worst-case scenario is that researchers might end up engineering extremely dangerous viruses that would never have evolved naturally. In 2001, for instance, Australian researchers created a mousepox virus far more virulent than any wild strains. This scenario is unlikely, but not impossible, says virologist Earl Brown of the University of Ottawa, Canada. “You could create something that is right out of whack, but I’d be surprised.”

For those reasons, several prominent flu researchers told New Scientist that the H5N1 experiments must be done at the highest level of containment: Biosafety Level 4, or BSL-4  (see right). But the CDC work is being done at BSL-3Ag, an intermediate level between BSL-3 and BSL-4. Workers wear half-suits with masks or hoods to prevent infection, for instance, rather than full-body suits as in BSL-4.  “US Department of Agriculture guidelines specify that work with highly pathogenic avian strains be done in BSL-3+ (also known as BSL-3Ag) laboratories,” a CDC spokeswomen says. One of the reasons is that the H5N1 virus is regarded as a non-contagious, treatable disease in humans. But this is not necessarily true of all of the genetically engineered strains that might be created. And drug supplies would quickly run out if an escaped virus triggered a major epidemic (see page 39).

A recent report by the US National Academy of Sciences recommends a series of checks be put in place to control such research. It says a panel of leading scientists and security experts should be set up to regulate it. “Some public representation is another option,” says Atlas, who helped draw up the report. At the moment, however, such experiments can be carried out without any special consultation.   Methods like reverse genetics might also be used to create new variants of other diseases. “You can make some pretty unusual things- new viruses that would never have existed in nature,” says Barclay. “It’s not just an issue for flu.”


Rachel Nowak, Melbourne Additional reporting by Michael Le Page

New Scientist issue: 28 February 2004


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Detailed How To: The Potential for Respiratory Droplet–Transmissible A/H5N1 Influenza Virus to Evolve in a Mammalian Host

* This is information has been made public, I am leaving the figures out...

Science 22 June 2012:
Vol. 336 no. 6088 pp. 1541-1547
DOI: 10.1126/science.1222526


Avian A/H5N1 influenza viruses pose a pandemic threat. As few as five amino acid substitutions, or four with reassortment, might be sufficient for mammal-to-mammal transmission through respiratory droplets. From surveillance data, we found that two of these substitutions are common in A/H5N1 viruses, and thus, some viruses might require only three additional substitutions to become transmissible via respiratory droplets between mammals. We used a mathematical model of within-host virus evolution to study factors that could increase and decrease the probability of the remaining substitutions evolving after the virus has infected a mammalian host. These factors, combined with the presence of some of these substitutions in circulating strains, make a virus evolving in nature a potentially serious threat. These results highlight critical areas in which more data are needed for assessing, and potentially averting, this threat.

Recent studies have shown that the A/Indonesia/5/2005 avian A/H5N1 influenza virus may require as few as five amino acid substitutions (1), and the A/Vietnam/1203/2004 A/H5N1 influenza virus requires four substitutions and reassortment (2), to become transmissible between ferrets via respiratory droplets. Here, we assess the likelihood that these substitutions could arise in nature. We first analyzed A/H5N1 sequence surveillance data to identify whether any of these substitutions are already circulating. We then explored the probability of the virus evolving the remaining substitutions after a spillover event of an avian virus into a single mammalian host and in a short chain of transmission between mammalian hosts.

The minimal set of substitutions identified by (1) (the Herfst et al. set) contains two receptor-binding amino acid substitutions, Q222L and G224S (H5 numbering used throughout) in the hemagglutinin (HA), known to change the virus from the more avian-like alpha-2-3–linked sialic acid specificity to the more humanlike alpha-2-6–linked sialic acid (3, 4). The remaining three substitutions in the set are T156A in HA, which disrupts the N-linked glycosylation sequon spanning positions 154 to 156; H103Y in the HA trimer-interface; and E627K in the PB2, which is a common mammalian polymerase adaptation (5). (Numbers refer to amino acid positions in the mature H5 proteins; for example, Q222L indicates that glutamine at position 222 was replaced by leucine. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.)

The four amino acid substitutions in HA identified by (2) (the Imai et al. set) also contain two receptor-binding amino acid substitutions, N220K and Q222L, one of which is in common with the Herfst et al. set and which together are known to change the sialic acid linkage preference to the more human-like alpha-2-6 linkage (2). The remaining two substitutions are N154D, which disrupts the same N-linked glycosylation sequon as the T156A substitution in the Herfst et al. set, and T315I in the stalk region.

Of the three receptor-binding substitutions in the two sets, only N220K in the Imai et al. set has been detected by means of surveillance in consensus sequencing of the HA of A/H5N1 viruses, and only in 2 of 3392 sequences [both avian viruses, one from 2007 in Vietnam, one from Egypt in 2010 (Fig. 1, B and F, black arrows)]. The T315I stalk substitution and H103Y trimer interface substitution have each been detected once in two viruses from China in 2002 (Fig. 1, A and B, orange arrows). T315I has been detected in two pre-1997 H5N1 viruses, four H5N2 viruses, two H5N3 viruses, and two H5N9 viruses. H103Y has been detected in five H5N2 viruses and one H5N3 virus. The remaining substitutions, N154D and T156A in the HA glycosylation sequon and E627K in PB2, however, are common and occur in 942 of 3392, 1803 of 3392, and 432 of 1612 sequences, respectively. A summary of the substitutions detected in surveillance is shown in fig. S1 and table S1. For viruses in which both HA and PB2 have been sequenced, 338 of 1533 have lost the 154-to-156 glycosylation sequon and have E627K in PB2. These viruses have been collected in at least 28 countries in Europe, the Middle East, Africa, and Asia.

Fig. 1

(A to L) Phylogenetic trees of the A/H5N1 HA1 nucleotide sequences. The sequences are split into three trees: 2022 avian H5 sequences from East and Southeast (E and SE) Asia (top row); 1097 avian H5 sequences from Europe, the Middle East, and Africa (middle row); and 385 human H5 sequences (bottom row). Each sequence is color coded by the minimum number of nucleotide mutations required to obtain the four amino acid substitutions in HA in the Herfst et al. set (column 1), to obtain the four amino acid substitutions in the Imai et al. set (column 2), to disrupt the N-linked glycosylation sequon spanning positions 154 to 156 in HA (column 3), and to obtain E627K in the PB2 segment of the corresponding virus in these HA trees (column 4). In columns 1 and 2, blue indicates five nucleotide changes, green indicates four, and orange indicates three. In columns 3 and 4, yellow viruses require one mutation, and pink require zero mutations. Gray indicates PB2 not sequenced. Clades as defined by (35) are marked to the right of the branches; the red portion of the vertical clade-identification lines indicates strains sampled in 2010 or 2011. The viruses indicated by black arrows are two nucleotides from the Imai et al. set. The virus indicated in (A) by the orange arrow has the H103Y substitution, and the virus indicated in (B) by the orange arrow has the T315I substitution. The blue circle indicates A/Indonesia/5/2005, and the red circle indicates A/Vietnam/1203/2004, the starting viruses used by (1) and (2), respectively. The initial trees were constructed with PhyML version 3.0 (36), with A/Chicken/Scotland/1959 as the root, using GTR+I+Γ4 [determined by ModelTest (37)] as the evolutionary model. GARLI version 0.96 (38) was run on the best tree from PhyML for 1 million generations to optimize tree topology and branch lengths. “Zoom-able” versions of these trees are shown in fig. S1 to show detail

The HA glycosylation sequon substitutions, N154D and T156A, have drifted in and out of the avian virus population over time, suggesting that they may be under little selective pressure in birds. The other substitutions—which are rare in birds, particularly those that change the sialic acid linkage preference—are likely to be negatively selected in birds.

Phylogenetic trees of the A/H5N1 HA are shown in Fig. 1, color-coded by the number of nucleotide mutations required to obtain the five Herfst et al. set (column 1) and four Imai et al. set (column 2) of substitutions in HA. Obtaining these mutations does not necessarily mean the virus will be transmissible through respiratory droplets between ferrets because the genetic background of each strain is different from the strain used by Herfst et al. (Fig. 1A, blue circle) and the strain used by Imai et al. (Fig. 1J, red circle). Other than for clade, the variation in color in Fig. 1, columns 1 and 2, is due to the presence (mostly in East and Southeast Asia) or absence (mostly outside of East and Southeast Asia) of the glycosylation sequon at positions 154 to 156.

The sequenced viruses that are closest to the Herfst et al. set are in clade (Fig. 1A and fig. S1A). These HAs have acquired a silent nucleotide mutation that makes the amino acid substitution G224S require only one nucleotide mutation instead of the two mutations for other strains. It is the requirement of these two nucleotide mutations that makes viruses usually farther from the Herfst et al. set than the Imai et al. set. The viruses in clade have been sampled in Nepal, Mongolia, Japan, and Korea from 2009 to 2011. Seventeen out of 94 of these viruses have been sequenced in PB2 (Fig. 1D), and none have the E627K substitution. Thus, the closest known viruses to the Herfst et al. set by consensus sequencing are four nucleotide substitutions away.

The majority of H5 viruses in clade 2.2 (and its subclades) are three nucleotide mutations from the Imai et al. set in HA (Fig. 1, F and J). These viruses have been sampled in Europe from 2005 to 2007, in the Middle East (including Egypt) from 2005 to 2011, and in Africa from 2005 to 2007. Viruses sampled in 2010 and 2011 are indicated by the red portion of the vertical line delimiting the clade (Fig. 1 and by the time series in fig. S1, F and J). If it is the loss of glycosylation that is important, rather than any other effect of N154D, then as shown in Fig. 1, column 3, almost all the non-Asian viruses have lost the glycosylation sequon, and thus all these viruses would potentially be functionally three nucleotides from the Imai et al. set in HA.

The viruses indicated by the black arrows in Fig. 1, B and F (one from Vietnam in 2007 and one from Egypt in 2010), have the N220K receptor-binding substitution and have lost the glycosylation sequon at positions 154 to 156. Thus, these two viruses are two nucleotide substitutions from the Imai et al. set in HA, and are the viruses closest to having the full Imai et al. set in HA detected to date by means of consensus sequencing.

Surveillance has detected humans with A/H5N1 viruses four nucleotide mutations from the full Herfst et al. set and three from the Imai et al. set in HA. Viruses isolated from human A/H5N1 infections (Fig. 1, bottom row) are generally the same number of mutations in HA away from the Herfst et al. and Imai et al. sets, by means of consensus sequencing, as their most closely related avian viruses. The within-host evolution modeling below indicates that any host adaptation substitutions would only reach a small proportion of the total virus population in the first spillover host and, although potentially critical in the host-adaptation process, would not be detected with consensus sequencing. Thus, the absence of evidence of host-adaption through consensus sequencing is not evidence for the absence of potentially critical adaptation to the mammalian host. See (6) for details of human strains and their most genetically similar avian A/H5N1 viruses.

To explore the probability of accumulating the remaining nucleotide mutations after the avian virus has been transmitted to a human (or other mammalian host), we constructed a mathematical model (7–10) of the within-host evolutionary dynamics of the virus. In the model, errors made by the virus polymerase are the source of mutation (10−5 mutations per site, per genome replication), the initial virus population expands exponentially [each infected cell produces 104 virions (11, 12), and 1010 cells can be infected (13)] until it reaches 1014 virions, after which the virus population size stays roughly constant, and selection is modeled by use of differences in expected numbers of progeny (fig. S2 and table S2) (6). The results of the model are largely insensitive to the number of cells that can be infected, maximum virus population size, and whether the virus population remains roughly constant or declines (figs. S3 to S5). Typical infections were simulated out to 5 days corresponding to the approximate time of peak viral load, and long-duration infections to 14 days (14).

It is not possible to calculate the level of risk precisely because of uncertainties in some aspects of the biology. We used the model to compare the relative effects of factors that could increase or decrease the probability of accumulating mutations and to identify areas for further investigation that are critical for more accurate risk assessment. We compare and contrast the effects of factors that can increase the probability of accumulating mutations and thus evolving a respiratory droplet–transmissible A/H5N1 influenza virus in a mammalian host, and factors that could decrease the probability of evolving a such a virus. The factors we considered that can increase the probability are random mutation, positive selection, long infection, alternate functionally equivalent substitutions, and transmission of partially adapted viruses as a proportion of the within-host diversity both in the avian-mammal and the mammal-mammal transmission events (10, 14–18). The factors we considered that can decrease the probability are an effective immune response, deleterious substitutions, and order-dependence in the acquisition of substitutions. We considered these factors for starting viruses differing in the number of mutations that separates them from a respiratory droplet–transmissible A/H5N1 virus—viruses that require five, four, three, two, or one mutations at specific positions in the virus HA, reflecting that zero, one, two, three, or four of the mutations are already present in the avian population and thus are present at the start of the infection in mammals. We treat each amino acid substitution as if it can be acquired by a single-nucleotide mutation, as is the case for the circulating viruses closest to acquiring the Herfst et al. or Imai et al. sets [see (6) for the general case].

First, we considered random mutation. Even without any positive selection pressure, the random process of mutations introduced by the virus polymerase in the expanding population of viruses will on average produce viruses that contain the required single, double, or triple mutations and even some quadruple mutants. These mutants will arise after a few days of an infection in a host in which the virus replicates efficiently (Fig. 2A) and would be delayed if replication is impaired (fig. S5). However, the existence of a virus within-host does not mean that it will transmit because it might exist only as a small proportion of the total virus population and thus have little chance of being excreted (Fig. 2B). The minimum proportion of mutant virus required to make transmission likely is not known, but increased proportion translates into increased probability of transmission; thus, we focused on proportion of mutant virus in the total virus population. These proportions (equivalent to the probability of a single virion to be a mutant), both here and below, cannot yet be precisely determined—they are sensitive to some biological parameters that are not yet known accurately and some that are specific to a particular virus or mutant. For such parameters, we tested a range of the current best estimates and focused on the relative, rather than the absolute, effects (6).

Fig. 2

Fig. 2

Expected absolute numbers and proportions of respiratory droplet–transmissible A/H5N1 virions within a host initially infected by strains that require five (blue), four (green), three (orange), two (red), or one (purple) mutation (or mutations) to become respiratory droplet–transmissible, calculated from the deterministic model. (A) The absolute number of respiratory droplet–transmissible A/H5N1 viruses in a host. The intersections with the gray line indicate the point when at least one virus in each host is expected to have the required mutations. The change in slope is due to the transition in the virus population from exponential expansion to constant size. (B) Expected proportion of respiratory droplet–transmissible A/H5N1 viruses in the total virus population over time in the random mutation case (when all mutations are fitness-neutral).

Second, we considered positive selection. Some of the substitutions identified by (1) and (2) have been shown to increase within-host virus fitness—specifically, the loss of glycosylation at positions 154 and 156 and E627K in PB2. However, given the absence of specific information on the within-host selective advantage or disadvantage conferred by each substitution, or combination of substitutions, we considered two cases of positive selection: one in which each individual substitution confers an additive advantage (hill-climb) and one in which only viruses that have acquired all substitutions have an advantage (all-or-nothing). We considered a total advantage of 1.1-, 2-, or 10-fold in each genome replication step for the full set of respiratory droplet transmission–enabling substitutions (table S2 and fig. S6) (A twofold advantage at each genome replication step translates into an approximately 100-fold increase in mutant virus titer after 36 hours.) In the all-or-nothing scenario, a strong increase in proportion occurs for viruses that have acquired all mutations because of its substantial fitness advantage over the rest of the population. The rate at which all-or-nothing selection increases the proportion of respiratory droplet–transmissible A/H5N1 viruses, as compared with the neutral case, is mostly independent of the number of mutations required (Fig. 3A). In contrast, for hill-climb selection the rate of increase above the neutral case decreases when fewer mutations are required (Fig. 3A). This difference between the all-or-nothing and hill-climb is because the fitness differential from the starting virus to the respiratory droplet–transmissible A/H5N1 virus decreases as the number of needed mutations decreases (if some of the mutations are already present in the avian host) (table S2) (6). We consider this hill-climb case to be the most likely situation during the host-adaptation we modeled (in the absence of deleterious substitutions). However, we have also compared the two selection scenarios when the starting fitnesses of all-or-nothing and hill-climb are the same independent of the starting number of necessary mutations, and discuss the subtle tradeoff between the fitness advantage of, and clonal-interference among, intermediate mutants (figs. S7 and S8) (6, 19).

Fig. 3

Factors that increase or decrease the proportion of respiratory droplet–transmissible A/H5N1 virus based on starting viruses that require five (blue), four (green), three (orange), two (red), or one (purple) mutation (or mutations) to become respiratory droplet–transmissible. (A) The effect of hill-climb and all-or-nothing positive selection compared with random mutation alone. (B) The effect of avian–mammal transmission of partially adapted virus as a result of intra-host diversity (100 viruses start the infection, one of which has a mutation) and the effect of alternate substitutions with 10 functionally equivalent sites for a virus requiring five mutations (blue), nine sites for a virus requiring four mutations (green), eight sites for a virus requiring three mutations (orange), seven sites for a virus requiring two mutations (red), and six sites for a virus requiring one mutation (purple), both with hill-climb selection, compared with hill-climb selection alone. (C) The effect of two of the required substitutions being individually deleterious (for these two specific substitutions, either substitution alone reduces the replicative fitness of the virus to zero) and the effect of complete order dependence of acquiring substitutions, both with hill-climb selection as compared with hill-climb selection alone.

Third, we considered long infection. Because both random mutation and positive selection increase the expected proportion of mutated virions with every viral generation, the longer a host is infected, the greater the proportion of a particular mutant (Fig. 4) (15). Human A/H5N1 infections lasting 14 days or longer have been reported especially in children, the elderly, and the immunocompromised (14) and have been associated with the evolution of oseltamivir resistance (20). It might be that only immunocompromised individuals can typically transmit the virus late in a long infection. The increasing proportion of mutant virus is only dependent on continued virus production and is independent of whether the virus load stays constant or declines (fig. S4) (21). The variance in the proportion of mutant virus (Fig. 4, pale regions) increases with each additional mutation required because of the increased number of combinatorial options and the greater selective advantage of mutant viruses as compared with wild-type viruses in the hill-climb scenario. The pale regions only reflect the within-model variance in results, as indicated by the different runs of the stochastic model, and not uncertainty as a result of other factors; sensitivity of the outcomes for model parameters such as the error rate and the number of virions produced by each infected cell are explored in (6) (fig. S5).

Fig. 4

Proportion of respiratory droplet–transmissible A/H5N1 virus in a long infection with virus replication for 14 days in the presence of hill-climb selection. Bold lines show results from a probability-based deterministic model of virus mutation, the pale region (composed of lines) shows 10,000 stochastic model simulations for each starting virus. Starting viruses require either five (blue), four (green), or three (orange) mutations to become respiratory droplet–transmissible. For the stochastic simulations, the lines start when the first virion that has the required mutations appears.

Fourth, we considered functionally equivalent substitutions. The sets of substitutions required for a respiratory droplet–transmissible A/H5N1 virus identified by (1) and (2) are unlikely to be the only combinations of substitutions capable of producing a respiratory droplet–transmissible A/H5N1 virus. If particular biological traits could be achieved by other substitutions, this would increase the expected proportions of respiratory droplet–transmissible A/H5N1 viruses. This is likely to be the case, given that there are multiple substitutions that can cause changes in receptor-binding specificity and two sites where substitutions will result in loss of glycosylation: positions 154 and 156 (table S3). If five substitutions could be from any 10 specific positions in the virus genome (or if two already existed in nature, three from any eight), then there would be 252 (or 56) combinations, and this would raise the proportion of respiratory droplet–transmissible A/H5N1 virus within a host by ~102.5 (or ~101.5) above the case of positive selection alone after 5 days (Fig. 3B, figs. S9 and S10, and table S4).

Fifth, we considered the avian-to-mammal transmission of partially adapted mutants. We specifically considered the case in which one of the required mutations exists as a small proportion of the avian within-host viral population, or in the viral populations from the >20 mammalian hosts in which A/H5N1 infections have been observed (22–25), so that they would not be detected by the usual consensus sequencing techniques. If the mutant is one of the 100 virions that seed an infection (16, 17), then with positive selection the probability of acquiring the remaining mutations increases by 103 after 5 days of infection above the case of positive selection alone (Fig. 3B). If the proportion of mutants in the seeding population is 10−4 however, the increase in proportion of respiratory droplet transmissible A/H5N1 virions in the mammalian host is small (fig. S11).

Sixth, we considered mammal-to-mammal transmission of partially adapted viruses. Transmission of viruses between mammals that have some but not all of the substitutions necessary for respiratory droplet transmission potentially increases the risk of evolving a respiratory droplet–transmissible A/H5N1 virus, but this increase is modulated by the difficulty of transmitting partially adapted strains and the loss of diversity at transmission. Two primary factors strongly modulate the effect of transmission on the accumulation of mutations. First, transmission could decrease the accumulation of mutations by the loss of low-proportion mutants because only a limited portion of the virus population will be transmitted. Second, transmission could increase the accumulation of respiratory droplet transmission–enabling substitutions by concentrating a transmissible virus during excretion from or seeding into a host—for example, if the adapted virus has increased tropism for the mammalian upper respiratory tract and therefore concentrated in the nose and throat. Thus, the effect of transmission can range from negligible, if mutants are culled by the loss of diversity at transmission, to substantial, if selection favors mutants at transmission (table S5). Given that A/H5N1 virus infections have been observed in >20 mammalian species, there is a potentially large pool of nonhuman hosts in which short chains of transmission could play a role in the emergence of respiratory droplet–transmissible A/H5N1 viruses.

In contrast to these factors that could increase the rate of accumulating substitutions, we next discuss factors that could decrease this rate.

First, we considered an effective immune response. An immune response that substantially shortened an infection would decrease the probability of the accumulation of mutations; however, there are many reported cases of infections up to and beyond 5 days (14, 21). Variation in the number of virions produced by each infected cell does not affect the deterministic calculations of the proportion of mutants. However, if this number is substantially lower for the stochastic simulations—for example, 25 (6) as compared with 10,000 (used for most of the figures)—the slower growth and lower total number of viruses could substantially delay the appearance of mutants within a host. As the number of required mutations increases, stochastic effects caused by the slower growth decrease the proportion of these mutants (fig. S5) (6).

Second, we considered deleterious intermediate substitutions. The receptor binding and trimer-interface or stalk substitutions required by (1) and (2) are, as we have seen, either rare or absent in influenza viruses isolated to date. The receptor-binding substitutions, although deleterious in birds, would be expected to be advantageous in humans. However, the details of this host-adaptation are not yet elucidated, and so we also consider the possibility that there are deleterious intermediate substitutions and explore a variety of scenarios (figs. S12 and S13). When two of the required substitutions are individually deleterious (for these two specific substitutions, either substitution alone reduces the replicative fitness of the virus to zero), this slows the rate of accumulation of mutations for the three-mutation case by less than the amount that hill-climb positive selection increases the rate above the neutral case (Fig. 3C). When three substitutions are required (all single and double substitutions reduce the replicative fitness of a mutant virus to zero), this can lower the accumulation rate ~102 below the neutral case (fig. S12). Deleterious (or advantageous) substitutions other than the respiratory droplet–transmissible A/H5N1 substitutions can, to a first approximation, be ignored in calculating proportions because such substitutions would on average affect all viruses equally and thus would not specifically affect the accumulation of respiratory droplet–transmissible A/H5N1 mutations (6).

Third, we considered order dependence in the acquisition of substitutions. It is not currently known whether the acquisition of some or all of the respiratory droplet transmission–enabling substitutions is dependent on the order in which viruses accumulate those substitutions. For example, the gain of 2-6-receptor binding might be required before loss of 2-3-receptor binding. If there were any order dependence, it would slow down the rate of accumulation of mutations. However, even in the most extreme scenario in which there is a single specific order in which the substitutions must be acquired, and any other order results in a virion with a replicative fitness of zero, if fewer than four mutations are required, the effect on the rate of accumulation of mutations is less than that of the deleterious scenario described above (Fig. 3C and figs. S14 and S15).

In addition to the substitutions in HA, the Imai et al. virus was a reassortment with an A/H1pdm09 virus. The probability of a reassortment event is difficult to determine given current knowledge. In one study (26), it has been estimated to be more likely than the likelihood of acquiring a single mutation as calculated here.

Highly pathogenic avian A/H5N1 viruses have been infecting humans for over a decade, with ~600 reported cases to date (and possibly many more that have not been reported), but there have yet to be known cases of efficient human-to-human transmission (27, 28). One hypothesis for the lack of sustained transmission is that it is not possible for A/H5N1 viruses to become respiratory droplet–transmissible in mammals; (1) and (2) have shown that this may not be the case in ferrets. Another hypothesis is that the number of mutations necessary for respiratory droplet–transmissibility might be so great that such a virus would be unlikely to evolve. We show here that in biologically plausible scenarios, respiratory droplet–transmissible A/H5N1 viruses can evolve during a mammalian infection. Given that respiratory droplet transmission between mammals is possible and that respiratory droplet–transmissible A/H5N1 mutants are likely to evolve in infected individuals, the primary impediment to transmission could be whether the respiratory droplet–transmissible A/H5N1 viruses comprise a sufficient proportion of the within-host viral population to actually transmit.

The minimum proportion of virus required for transmission is not known, but increased proportion likely translates into increased probability of transmission. There cannot be respiratory droplet transmission if there are no viruses in the air. Given a peak excretion rate of ~107 viruses per day (29, 30), a proportion of which are likely to become aerosolized (31), mutants at proportions near or above 10−7 might thus be among the particles excreted. Each of the factors analyzed above has a potentially substantial effect on the rate of accumulating mutations (Fig. 3), and the effects of each can be additive. With plausible combinations of these factors, a virus that requires three mutations reaches proportions at which a few respiratory droplet–transmissible A/H5N1 viruses are likely to be among the particles excreted. For a virus that requires five mutations, it may only reach such proportions with more extreme combinations of factors or if an event occurs that is not encompassed by the model (32). However, it is known that influenza viruses are capable of respiratory droplet transmission in animal models at low infectious doses (33), and that transmission routes other than in respiratory droplets could be important; thus despite the three key current unknowns about transmission (6), even low numbers of excreted respiratory droplet–transmissible A/H5N1 virus may be relevant for emergence. In addition, the probability of emergence increases when more mammals are infected when this also corresponds with a rise in potential transmission events. The output of the model is a guide to understand the approximate effects of different factors and should not be interpreted as actual proportions of virus and probabilities of transmission, given the uncertainty inherent in parameter estimates and model structure, and the inherent unpredictability of rare events (34).

These results highlight four areas of investigation that are critical to more accurately assess and monitor the risk of a respiratory droplet–transmissible A/H5N1 virus emerging and to increase our understanding of virus emergence in general. Some of this work is already ongoing, planned, or suggested. The work of Herfst et al. (1) and Imai et al. (2) and the analyses here help to prioritize particular areas.

First, additional surveillance in higher-risk regions where viruses require fewer nucleotide mutations to acquire respiratory droplet transmission–enabling substitutions (Fig. 1 and fig. S1) (and in regions connected by travel, trade, and migratory flyways) is key for monitoring the emergence of a respiratory droplet–transmissible A/H5N1 virus. Surveillance of nonhuman mammalian hosts, especially any that harbor long infections or live in large groups, is important for the early identification of mammalian adaptation. Additionally, studies are needed on the accumulation of mutations within-host and in short chains of transmission in mammals (22–25), even when endemic circulation has not been observed.

Second, deep sequencing of avian and other nonhuman virus samples is necessary to accurately estimate the prevalence of the respiratory droplet transmission–enabling amino acid substitutions in nature. Deep sequencing of human samples, particularly at multiple time points from individuals with long infections, would be useful for evaluating within-host evolution, for estimating selective advantage of substitutions, and for testing the underlying dynamics and assumptions of the model (15). Respiratory droplet–transmissible A/H5N1 mutations present in a proportion higher than the polymerase error rate—exceeding approximately 10−5, but far below the threshold for detection with consensus sequencing and thus not detectable with current surveillance practices—would increase the risk of a respiratory droplet–transmissible A/H5N1 evolving. Thus, sequencing deeper than that currently routinely achieved for RNA viruses (ideally detecting mutations at 0.1% frequency and lower for detailed studies) is necessary to more accurately assess the risk posed by intra-host variability (15).

Third, experiments are needed to determine which substitutions, besides the already identified receptor-binding substitutions by (1) and (2), are capable of producing respiratory droplet–transmissible A/H5N1 viruses, including the important case of functionally equivalent substitutions or alternative sets of substitutions that would require fewer nucleotide mutations than those of the Herfst et al. or Imai et al. sets. This work will be important for calculating risk and for monitoring in surveillance.

Fourth, further studies are needed to elucidate the changes in within-host fitness and between-host transmissibility associated with each respiratory droplet transmission–enabling substitution and combination of substitutions. These studies are necessary for determining the dynamics of within-host selection [including data on, and modeling of, the effects of glycan heterogeneity between the upper and lower respiratory tract (6)] and the potential for transmission of partially adapted viruses. It is important to determine the strength of selection at transmission because it can increase the proportion of respiratory droplet transmission–enabling substitutions. Further work is needed to refine the estimate for virus excretion and the minimum human infectious dose (29).

Numerous avian A/H5N1 viruses have been sampled in the past 2 years that are four nucleotide mutations from acquiring the Herfst et al. set of HA and PB2 substitutions and three nucleotide mutations from acquiring the Imai et al. set in HA (the Imai et al. set also requires a reassortment event). Precise estimates of the probability of evolving the remaining mutations for the virus to become a respiratory droplet–transmissible A/H5N1 virus cannot be accurately calculated at this time because of gaps in knowledge of the factors described above. However, the analyses here, using current best estimates, indicate that the remaining mutations could evolve within a single mammalian host, making the possibility of a respiratory droplet–transmissible A/H5N1 virus evolving in nature a

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Acknowledgments: C.A.R. was supported by a University Research Fellowship from the Royal Society. The authors acknowledge an Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) VICI grant, European Union (EU) FP7 programs EMPERIE (223498) and ANTIGONE (278976), Human Frontier Science Program (HFSP) program grant P0050/2008, Wellcome 087982AIA, the Bill and Melinda Gates Foundation (OPPGH5383), and NIH Director’s Pioneer Award DP1-OD000490-01. R.A.M.F was supported by National Institute of Allergy and Infectious Diseases (NIAID)–NIH contract HHSN266200700010C. A.E.X.B. was supported by a long-term fellowship from the HFSP. E.M., G.N., and Y.K. are supported by the Bill and Melinda Gates Foundation (OPPGH5383) and NIAID-NIH grant R01 AI 069274; in addition, Y.K. was supported by a Grant-in-Aid for Specially Promoted Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by ERATO. Y.K. and G.N. have a financial interest as founders of FluGen and hold a patent on influenza virus reverse genetics. Y.K and G.N. have a paid consulting relationship with Theraclone; Y.K. also has a paid consulting relationship with Crucell. Y.K. has received speaker’s honoraria from Chugai Pharmaceuticals, Novartis, Daiichi-Sankyo Pharmaceutical, Toyama Chemical, Wyeth, GlaxoSmithKline, and Astellas and grant support from Chugai Pharmaceuticals, Daiichi Sankyo Pharmaceutical, Toyama Chemical, Otsuka Pharmaceutical Company A.D.M.E.O. (on behalf of Viroclinics Biosciences BV) has advisory affiliations with GlaxoSmithKline, Novartis, and Roche. A.D.M.E.O. is Chief Scientific Officer of Viroclinics Biosciences BV. We thank S. Cornell, E. Ghedin, R. Johnstone, L. Reperant, and D. M. Smith for helpful discussions and the reviewers for their detailed and thoughtful comments.