Bird flu leaves the nest — adapting to a new host ( How to Kill or Cure Millions )

EEV: Reposted at request from our biological site www.healthresearchreport.me

Public release date: 26-Aug-2009

– an unadapted avian strain and an avian strain adapted to infect mice by mutations that increase the efficiency of the viral polymerase

They found that whereas the avian strain only infected the lungs, the mouse-adapted strain caused suppression of the immune system, which resulted in infection in multiple organs

–  bird-specific flu strains rarely cross species, further adaption can lead to lethal infection in humans.

Hamburg, Germany – Current research suggests that viral polymerase may provide a new therapeutic target for host-adapted avian influenza. The related report by Gabriel et al, “Spread of Infection and Lymphocyte Depletion in Mice Depends on Polymerase of Influenza Virus” appears in the September 2009 issue of the American Journal of Pathology.

Highly pathogenic avian influenza, commonly known as bird flu, is a strain of the influenza virus that has adapted to infect birds. Although bird-specific flu strains rarely cross species, further adaption can lead to lethal infection in humans.

To determine which genetic changes may lead to host adaptation, Gülsah Gabriel (currently at the Heinrich-Pette-Institute for Experimental Virology and Immunology the University of Hamburg) and Hans-Dieter Klenk at the Institute of Virology at the Philipps University of Marburg examined two strains of avian influenza, an unadapted avian strain and an avian strain adapted to infect mice by mutations that increase the efficiency of the viral polymerase. They found that whereas the avian strain only infected the lungs, the mouse-adapted strain caused suppression of the immune system, which resulted in infection in multiple organs. In addition, while the avian strain caused only mild symptoms in mice, the mouse-adapted strain led to severe illness including pneumonia and infection of the brain, followed by death. The viral polymerase may therefore provide an important target in preventing systemic flu in humans.

Gabriel et al suggest that “reduction of high virus loads by targeting the viral polymerase may play an important role in the treatment of human influenza with systemic virus spread.” In future studies, Dr. Gabriel and colleagues will aim to develop drugs interfering with virus polymerase activity.

Food additive may prevent spread of deadly new avian flu

Contact: Sharon Parmet sparmet@uic.edu 312-413-2695 University of Illinois at Chicago

A common food additive can block a deadly new strain of avian influenza virus from infecting healthy cells, report researchers at the University of Illinois at Chicago College of Medicine in the online journal, PLOS ONE.

The compound, in wide use as a preservative, binds to a part of the flu virus that has never been targeted by any existing antiviral drug, raising hopes for its effectiveness against multi-drug-resistant flu viruses.

“The recent H7N9 outbreak in China this past March had a mortality rate of more than 20 percent,” says Michael Caffrey, associate professor of biochemistry and molecular genetics at UIC. That strain, which is new, is already showing resistance to the majority of existing drugs used to treat it, Caffrey said. Preventing an outbreak that could lead to mass casualties would be difficult with the current arsenal.

“The need to develop new antiviral therapeutics now is crucial,” he said.

Flu viruses enter host cells using a special protein called hemagglutinin, which acts as a “key” that opens receptors on the cell surface. If hemagglutinin is disabled, the virus is locked out and can’t infect cells.

UIC researchers, led by Caffrey, found that the FDA-approved food additive tert-butyl hydroquinone sticks to a specific region on the hemagglutinin molecule. The additive, he said, “attaches to the Achilles’ heel of the virus—a loop-shaped portion of hemagglutinin necessary for binding to cells, making cell infection impossible.”

The loop on the hemagglutinin molecule represents a new therapeutic target, since existing drugs don’t go after it, Caffrey said.

“Any drugs that focus on the hemagglutinin loop would be totally novel to flu viruses, and so resistance, if developed, would still be a long way off.”

Caffrey and his colleagues were looking at a different class of viruses when the first outbreak of the H7N9 virus was reported in China last spring.

“Tert-butyl hydroquinone was known to have virus-blocking effects for H3 viruses,” he said. “So when the H7N9 outbreak occurred, we thought we’d see if it had any effect on H7 viruses.”

Using a novel technique, the researchers fused the hemagglutinin of the H7N9 virus to a less dangerous virus in order to study it safely. They found that tert-butyl hydroquinone was able to prevent the virus from infecting human lung cells in the lab.

The researchers are now looking for ways to enhance tert-butyl hydroquinone’s ability to prevent infection. One way might be to add it to poultry feed. Keeping the virus from spreading in chickens could reduce the likelihood of it jumping to humans, Caffrey said. While the compound is used in a variety of foods as a preservative and stabilizer, questions remain regarding its safety if consumed in very high doses.

###

 

Coauthors on the PLOS ONE paper, all of UIC, are graduate student Aleksandar Antanasijevic; postdoctoral researcher Han Cheng; Duncan Wardrop, associate professor of chemistry; and Lijun Rong, associate professor microbiology and immunology.

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.

BIOTERRORISM FEARS

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)

http://www.trust.org/item/20130807165536-m5jun/?source=hpbreaking

 

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.

KARSTEN SCHNEIDER/SCIENCE PHOTO LIBRARY

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:
Nature
DOI:
doi:10.1038/nature.2013.12925
http://www.nature.com/news/scientists-create-hybrid-flu-that-can-go-airborne-1.12925

‘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.

Reference

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

http://www3.imperial.ac.uk/newsandeventspggrp/imperialcollege/newssummary/news_8-4-2013-12-47-4

 

Paradox of Vaccination: Is Vaccination Really Effective against Avian Flu Epidemics?

Abstract

Background

Although vaccination can be a useful tool for control of avian influenza epidemics, it might engender emergence of a vaccine-resistant strain. Field and experimental studies show that some avian influenza strains acquire resistance ability against vaccination. We investigated, in the context of the emergence of a vaccine-resistant strain, whether a vaccination program can prevent the spread of infectious disease. We also investigated how losses from immunization by vaccination imposed by the resistant strain affect the spread of the disease.

Methods and Findings

We designed and analyzed a deterministic compartment model illustrating transmission of vaccine-sensitive and vaccine-resistant strains during a vaccination program. We investigated how the loss of protection effectiveness impacts the program. Results show that a vaccination to prevent the spread of disease can instead spread the disease when the resistant strain is less virulent than the sensitive strain. If the loss is high, the program does not prevent the spread of the resistant strain despite a large prevalence rate of the program. The epidemic’s final size can be larger than that before the vaccination program. We propose how to use poor vaccines, which have a large loss, to maximize program effects and describe various program risks, which can be estimated using available epidemiological data.

Conclusions

We presented clear and simple concepts to elucidate vaccination program guidelines to avoid negative program effects. Using our theory, monitoring the virulence of the resistant strain and investigating the loss caused by the resistant strain better development of vaccination strategies is possible.

Citation: Iwami S, Suzuki T, Takeuchi Y (2009) Paradox of Vaccination: Is Vaccination Really Effective against Avian Flu Epidemics? PLoS ONE 4(3):          e4915.            doi:10.1371/journal.pone.0004915

Editor: Carl Kingsford, University of Maryland, United States of America

Received: November 12, 2008; Accepted: November 26, 2008; Published: March 18, 2009

Copyright: © 2009 Iwami et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

* E-mail: takeuchi@sys.eng.shizuoka.ac.jp

Introduction

Highly pathogenic H5N1 influenza A viruses have spread relentlessly across the globe since 2003. They are associated with widespread death of poultry, substantial economic loss to farmers, and reported infections of more than 300 people with a mortality rate of 60% [1]. Influenza prevention and containment strategies can be considered under the broad categories of antiviral, vaccine, and non-pharmaceutical measures [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13]. A major public health concern is the next influenza pandemic; yet it remains unclear how to control such a crisis.

Vaccination of domestic poultry against the H5N1 subtype of avian influenza has been used in several countries such as Pakistan, Hong Kong, Indonesia, China, and Vietnam [14], [15], [16]. Using vaccination to reduce the transmission rate might provide an alternative to mass culling, by reducing both the susceptibility of healthy birds and the infectiousness of infected birds [14], [17], [18]. However, incomplete protection at the bird level can cause the silent spread of the virus within and among birds [11]. Furthermore, vaccines might provide immunological pressure on the circulating strains, which might engender the emergence of drifted or shifted variants with enhanced potential for pathogenicity in humans [1]. Therefore, although vaccination programs have been recommended recently, some field evidence indicates that vaccination alone will not achieve eradication. Moreover, if not used appropriately, vaccination might result in the infection becoming endemic [11], [17].

An important issue related to influenza epidemics is the potential for the emergence of vaccine-resistant influenza viruses. The vaccine-resistant strain, in general, causes a loss of the protection effectiveness of vaccination [19], [20], [21], [22] (there is experimental evidence of the loss of the protection effectiveness for antiviral-resistant strains [23]). Consequently, a vaccination program that engenders the emergence of the resistant strain might promote the spread of the resistant strain and undermine the control of the infectious disease, even if the vaccination protects against the transmission of a vaccine-sensitive strain [20], [21], [22].

For example, in China, despite a compulsory program for the vaccination of all poultry commencing in September 2005, the H5N1 influenza virus has caused outbreaks in poultry in 12 provinces from October 2005 to August 2006 [14], [15], [22]. Genetic analysis revealed that an H5N1 influenza variant (Fujian-like, FJ like), which is a previously uncharacterized H5N1 virus sublineage, had emerged and subsequently became the prevalent variant in each of the provinces, replacing those previously established multiple sublineages in different regions of southern China. Some data suggest that the poultry vaccine currently used in China might only generate very low neutralizing antibodies to FJ-like viruses (seroconversion rates remain low and vaccinated birds are poorly immunized against FJ-like viruses) in comparison to other previously cocirculating H5N1 sublineages [20], [22]. That evidence implies the possibility that the emergence and replacement of FJ-like virus was preceded by and facilitated by the vaccination program, although the mechanism remains unknown epidemiologically and virologically (some researchers consider that the emergence and replacement of FJ-like virus are questionable [24], [25]).

Furthermore, the H5N2 vaccines have been used in Mexico since 1995 [17], [19], [21]. Phylogenetic analysis suggests the presence of (previously uncharacterized) multiple sublineages of Mexican lineage isolates which emerged after the introduction of the vaccine. Vaccine protection studies further confirmed in vitro serologic results indicating that commercial vaccine was not able to prevent virus shedding when chickens were challenged with the multiple sublineage isolates [19], [21]. Therefore, the vaccine protective efficacy would be impaired and the use of this specific vaccine would eventually become obsolete. That fact also implies that the vaccine promotes the selection of mutation in the circulating virus.

The emergence of a vaccine-resistant strain presents the risk of generating a new pandemic virus that is dangerous for humans through an avian-human link because of the spread of vaccine-resistant strain. The dynamics of competition between vaccine-sensitive and vaccine-resistant strains is, in general, complex [8], [9]. Actually, outcomes of the dynamics might be influenced by several factors, including a loss of protection effectiveness, a competitive advantage of vaccine-resistant strain, and a prevalence rate of vaccination. Understanding the dynamics of a spread of vaccine-resistant is therefore crucial for implementation of effective mitigation strategies.

Several theoretical studies have investigated the impact of an emergence of a resistant strain of antiviral drug such as M2 inhibitors and NA inhibitors during an influenza pandemic among humans [2], [3], [8], [9], [10], [12], [26]. However, to our knowledge, no study has used a mathematical model to investigate the application of vaccination program among poultry in the context of an emergence of a vaccine-resistant strain. It remains unclear whether a vaccination program can prevent the spread of infectious disease when the vaccine-resistant strain emerges and how a loss of immunization by vaccination within birds infected with the vaccine-resistant strain affects the spread of infectious disease among birds. Nobody can give a simple and clear explanation to capture the problems described above in a theoretical framework (using numerical simulations, many qualitative and quantitative but sometimes very complex studies have investigated effects of antiviral drugs [3], [8], [9], [10], [12], [26]). Furthermore, we remain skeptical that a vaccination program can reduce the number of total infectious individuals even if the vaccination protects against transmission of a vaccine-sensitive strain. We developed a simple mathematical model to evaluate the effectiveness, as a strategy to control influenza epidemic, of a vaccination program among poultry which can engender the emergence of a vaccine-resistant strain.

Methods

Herein, we describe a homogeneous population model of infectious disease and its control using a vaccination program in the presence of a vaccine-resistant strain (Fig. 1).

thumbnail

Figure 1. Model structure for the emergence of vaccine-resistant strain during a vaccination program: Susceptible birds (X) become infected with vaccine-sensitive (Y) and vaccine-resistant (Z) strains at rates in direct relation to the number of respective infectious birds.

We assume that vaccinated birds (V) can be protected completely from the vaccine-sensitive strain, but are partially protected from vaccine-resistant strains with a loss of protection effectiveness of the vaccination (σ). See the Mathematical model section for corresponding equations.

doi:10.1371/journal.pone.0004915.g001

All birds in the effective population are divided into several compartments, respectively including susceptible birds (X), vaccinated birds (V), birds infected with vaccine-sensitive strain (Y), and birds infected with vaccine-resistant strain (Z). We assume that susceptible birds are born or restocked at a rate of c per day and that all birds are naturally dead or removed from the effective population at a rate of b per day.

In the absence of vaccination, transmission occurs at a rate that is directly related to the number of infectious birds, with respective transmission rate constants ω and φ from infected birds with the vaccine-sensitive strain and with the vaccine-resistant strain. The infectiousness of vaccine-sensitive and vaccine-resistant strain are assumed to be exponentially distributed, respectively, with mean durations of 1/(b+my) and 1/(b+mz) days. Actually, my and mz respectively signify virulence of vaccine-sensitive and vaccine-resistant strains.

At the beginning of the vaccination program, X moves directly to V by the vaccination. However, after some period after the initial vaccination, the direct movement might vanish because almost all birds are vaccinated. Therefore, we can assume that vaccination is only administered to the newly hatched birds. The newly hatched birds are vaccinated at the rate 0≤p≤1 (more appropriately, p is proportional). Actually, p represents the prevalence rate of the vaccination program.

To simplify the theoretical treatment, as described in [11], we assume that the vaccinated birds can be protected completely from the vaccine-sensitive strain (note that the assumption is not necessary for our results: see Supplementary Information: Text S1, Fig. S10, S11). Actually, in laboratory experience, many avian influenza vaccines confer a very high level of protection against clinical signs and mortality (90–100% protected birds) [21]. However, many factors determine whether a vaccinated bird becomes infected, including age, species, challenge dose, health, antibody titre, infections of immunosuppressive diseases, and cross-reactivity of other avian influenza serotypes [11], [27], [28], [29]. On the other hand, we assume that the vaccinated birds are partially protected from the vaccine-resistant strain at the rate (proportion) 0≤1−σ≤1 because of cross-reactivity of immune systems [19], [20], [22], [23], [29] (e.g., σ = 0 represents complete cross immunity against vaccine-resistant strains). Actually, σ represents a loss of protection effectiveness of the vaccination caused by a vaccine-resistant strain.

Mathematical model

We extended the standard susceptible–infective model [30] including the effect of a vaccination program that can engender the emergence of a vaccine-resistant strain. Our mathematical model is given by the following equations: (1) Model (1) is a simplified one that is used in [31]. We considered a mechanism for the emergence and replacement of the FJ-like virus over a large geographical region in China using a more complex patch-structured model in the heterogeneous area [31]. Here we investigate the impact of the vaccination program in a homogeneous area and specifically examine the role of epidemiological parameters such as the prevalence rate of the vaccination program (p) and the loss of protection effectiveness of the vaccination (σ) in the spread of the disease.

Estimation of epidemiological parameters

Baseline values of model parameters and their respective ranges used for simulations are presented in Table 1 and 2. These parameters are based on avian influenza epidemics among poultry in The Netherlands in 2003 [32], [33], [34].

thumbnail

Table 1. Description of physical characteristics, transmission, infectious, and vaccination parameters of the model with their baseline values and ranges used for simulations.

doi:10.1371/journal.pone.0004915.t001

thumbnail

Table 2. Basic reproductive numbers and invasion reproductive numbers before the vaccination program.

doi:10.1371/journal.pone.0004915.t002

The initial population size was c/b = 984 birds at the 2003 epidemic [34]. Usually, the mean lifespan of poultry is about 2 years. However, we assume that the mean duration of a bird being in effective population is about 1/b = 100 days because of migration and marketing. Therefore, the birth or restocking rate of birds is c = 9.84 birds per day. Estimated infectious period and transmission parameters are 1/(b+my) = 13.8 days and ω = 4.78×10−4 day−1 individual−1, respectively, [34]. These physical characteristics, in addition to infectious and transmission parameters, are used in our model as parameters of the vaccine-sensitive strain.

The epidemiological and biological feature of antiviral drug-resistance is well reported in [23]. The transmissibility and virulence of drug-resistant strains are usually lower than those of the wild strain because of its mutation cost [8], [10], [23], [35]. Actually, antiviral drugs are also used for prophylaxis drug intervention as vaccination [8], [10], [12]. Herein, we use some reduced value of transmissibility (φ/ω = 0.58) and the increased value of infectious period of the vaccine-sensitive strain ((b+my)/(b+mz) = 1.32) for parameters of vaccine-resistant strain (sensitivity analyses are given in Supplementary Information: Text S1, Fig. S6, S7, S8, S9).

Reproductive numbers

A measure of transmissibility and of the stringency of control policies necessary to stop an epidemic is the basic reproductive number, which is the number of secondary cases produced by each primary case [30]. We obtain basic reproductive quantities of vaccine-sensitive strain and vaccine-resistant strain before vaccination program (superscript n means no vaccination). In fact, during the vaccination program, the basic reproductive numbers depend on the rate of prevalence of the vaccination program. We derived these basic reproductive numbers depending on the prevalence rate in Supplementary Information: Text S1. With the estimated parameters in Table 1 the basic reproductive number of vaccine-sensitive and vaccine-resistant strain are and , respectively (note that corresponds to an estimated value in [34]).

Furthermore, to clarify the concept of competition among strains simply, we introduce the invasion reproductive number for the vaccine-resistant strain before the vaccination program , which signifies an expected number of new infectious cases with the vaccine-resistant strain after a spread of a vaccine-sensitive strain among birds. The invasion reproductive number is considered as a competitive condition (relative fitness), which represents some advantage measure of the vaccine-resistant strain against the vaccine-sensitive strain. The estimated invasion reproductive number of the vaccine-resistant strain is . During the vaccination program, the invasion reproductive number also depends on the prevalence rate of the vaccination program (see Supplementary Information: Text S1).

Results

We consider a scenario in which a vaccine-resistant strain can emerge (i.e., be eventually selected) during a vaccination program designed to be effective against the spread of a vaccine-sensitive strain. This implies that : otherwise the vaccine-resistant strain can not emerge at all (see Supplementary Information: Text S1, Fig. S1, S2, S3). Acquisition of resistance ability usually engenders a strain which, in the absence of a pharmaceutical intervention, is less fit than the sensitive strain [8], [9], [12], [35]. Therefore, . We generally assume the following conditions for reproductive numbers before the vaccination program (our baseline parameter values are satisfied with these assumptions):

The assumption precludes the possibility that a pre-existing vaccine-resistant strain beats the vaccine-sensitive strain before the vaccination program because .

Evaluation of the effect of a vaccination program

Although vaccination is an important tool to control epidemics, the use of vaccination might engender a spread of a vaccine-resistant strain. To demonstrate the interplay between these opposing effects, we simulated our model to determine the final size of an epidemic (total infected individuals Y+Z at equilibrium level) over vaccination prevalence (0≤p≤1) in Fig. 2 (we use our baseline parameter values except for mz). We assume that the loss of the protection effectiveness is 35% (σ = 0.35: this value can be chosen arbitrarily with little effect on the meaning of the results). The estimated infectious period of the vaccine-sensitive strain is 13.8 days [34] (see Table 1). Therefore, the virulence of vaccine-sensitive strain is my = 0.062 day−1. Results show that the patterns of the final size can be divided into two cases, which depend strongly on the virulence of the vaccine-resistant strain. If the virulence of the vaccine-resistant strain is lower than that of vaccine-sensitive strain (e.g., we choose mz = 0.045), then increasing the prevalence rate of vaccination from 13.5% to 30.3% can increase the final size (green line at top figure in Fig. 2). On the other hand, if the virulence is higher (mz = 0.065), increasing the prevalence always decreases the final size (bottom figure in Fig. 2). These two patterns are qualitatively preserved for different virulence of the vaccine-resistant strain.

thumbnail

Figure 2. Final size of epidemics related with the prevalence rate of the vaccination: The top figure represents that the vaccination is not always effective in the case of lower virulence of vaccine-resistant strain.

The bottom figure represents that the vaccination is always effective in the case of higher virulence of the vaccine-resistant strain. We assume that σ = 0.35, mz = 0.045 (top) and mz = 0.065 (bottom). These values of σ and mz are not so influential on the result. The blue, green, and red lines respectively signify situations in which only the vaccine-sensitive strain exists, both the vaccine-sensitive and the vaccine-resistant strains exist, and only the vaccine-resistant strain exists.

doi:10.1371/journal.pone.0004915.g002

In [8], [9], although they consider the emergence of an antiviral drug-resistant virus, a similar tendency (increasing the treatment level increases the final size of the epidemic) was obtained through complex models that are difficult to treat mathematically. The mathematical model presented herein demonstrates that the patterns of final size over vaccination prevalence only depend on the virulence of the vaccine-resistant strain as follows (see Supplementary Information: Text S1). Increasing the prevalence rate increases the final size when only both strains co-exist if the virulence of vaccine-resistant strain is lower than that of vaccine-sensitive strain (my>mz). That is to say, the vaccination is effective when either a vaccine-sensitive or a vaccine-resistant strain exists. On the other hand, if the virulence of vaccine-resistant strain is higher than that of vaccine-sensitive strain (my<mz), the final size always decreases as the prevalence rate increases. The other parameters can not change these patterns. In fact, many studies have ignored the impact of the virulence of the vaccine-resistant strain. In [7], we also found that the virulence of mutant strain determines a choice of the optimal prevention policy for avian influenza epidemic. Therefore, we suggest that, to monitor and investigate the virulence evolution between the vaccine-sensitive and vaccine-resistant strain is important to develop avian flu epidemic plans. In fact, if the vaccine-resistant strain has higher virulence than the vaccine-sensitive strain, the vaccination program is always effective, even though the program engenders the emergence of a vaccine-resistant strain. On the other hand, if the vaccine-resistant strain has lower virulence, we must carefully manage vaccination to prevent the spread of a vaccine-resistant strain.

Impact of loss of protection effectiveness of vaccination

To ensure an effective vaccination program, the vaccine must protect vaccinated animals against clinical signs of the disease and prevent mortality [21]. However, the vaccine-resistant strain causes a loss of the protection effectiveness of the vaccination [19], [20], [21], [22], [37]. We investigate an impact of the loss of the protection on change of final size of the epidemic over the vaccination prevalence. Assume, hereafter, that the virulence of vaccine-resistant strain is lower than that of vaccine-sensitive strain (my>mz): otherwise, the vaccination is always effective (our baseline parameter values are satisfied with my>mz). Actually, a resistant strain seems to have reduced virulence in general [8], [10], [23], [35].

We conduct a simulation using our model to elucidate the change of the final size with the loss of the protection effectiveness 5%, 15%, and 80% over vaccination prevalence in Fig. 3. Results showed that the patterns of the change are divisible into three cases. In theory, we can estimate the threshold values of the loss of the protection which determines the patterns (see Supplementary Information: Text S1, Fig. S4):

thumbnail

Figure 3. Impact of the loss of the protection effectiveness of the vaccination on the change of the final size of the epidemic: The losses of the protection in the top, middle, and bottom figure are σ = 0.05, 0.15, and 0.8, respectively.

The top (0≤σσ*) and middle () figures portray the possibility of eradication of the infectious disease through the vaccination program. However, in the bottom figure (), the vaccination engenders a failure to prevent the spread of the disease. The patterns of the change are divisible into these three cases, depending on the loss of the protection. The blue, green, and red lines respectively correspond to the situation in which only the vaccine-sensitive strain exists, both the vaccine-sensitive and the vaccine-resistant strains exist, and only the vaccine-resistant strain exists.

doi:10.1371/journal.pone.0004915.g003

In fact, σ* = 0.056 and in our simulation from Table 1. When the loss of the protection is between 0% and σ* = 5.6% (5%: the top figure in Fig. 3), the vaccination can control the epidemic with the prevalence rate of 84.7% without the emergence of a resistant strain (a vaccine-resistant strain never emerges in the population). Therefore, increasing the prevalence rate of vaccination always decreases the final size of the epidemic. For the loss of the protection is between σ* = 5.6% and (15%: the middle figure in Fig. 3), the vaccination eventually prevents the spread of the disease with 94.1% of vaccination prevalence in spite of the emergence of the resistant strain. Increasing the prevalence rate from 31.5% to 44.1% increases the final size. Therefore, the vaccination is not always effective. However, when the loss of the protection is between and 100% (80%: the bottom figure in Fig. 3), the vaccination no longer controls the disease (even if the prevalence rate is 100%) and the vaccine-resistant strain spreads widely through the population instead of the vaccine-sensitive strain. In this case, the vaccination only slightly provides beneficial effects for preventing the spread of the disease. Therefore, the loss of the protection effectiveness of vaccination plays an important role in preventing the spread of the disease.

Vaccination can facilitate spread of disease

Sometimes a considerable spread of the resistant strain partially compromises the benefits of a vaccination program [19], [20], [22], [37]. For example, even if we can completely execute the vaccination program (p = 1), the final size of the epidemic can become larger than that before the vaccination program (p = 0) by the emergence of vaccine-resistant strain (bottom figure in Fig. 3). This implies that the vaccination, which is expected to prevent the spread of the disease, can instead help the spread of the disease. If the loss of the protection effectiveness of vaccination is high (σ*σ≤1), the vaccination might increase the final size over vaccination prevalence compared with that before the vaccination program (vaccination always decreases the final size if 0≤σσ* (top figure in Fig. 3)). Here we can also calculate such a risk of help, which depends on the loss of the protection (see Supplementary Information: Text S1). Let

Actually, σc = 0.236 in our simulation is from Table 1. When the loss of the protection is between 23.6% and 100%, we found that the vaccination program is attended by the risk that the final size becomes larger than that before the vaccination program (see Supplementary Information: Text S1).

Difficulty of prediction of a prevalent strain

Vaccination is well known to engender “silent carriers or excretors” if the vaccine can not completely protect the vaccinated animals against clinical signs of the disease [16], [21]. The existence of silent carriers or excretors is dangerous because they become a virus reservoir and shed the virus into their environment, causing potential outbreaks among their own and other species. Furthermore, even if a vaccination is effective in a bird (individual level), an incomplete vaccination program for all birds (population level) can engender the “silent spread” of an infectious disease [1], [11]. Additionally, we found that it is difficult for us to predict a prevalent strain even if we can completely estimate the basic reproductive number of vaccine-sensitive and vaccine-resistant strains during the vaccination program (although estimations, usually, are almost impossible). Even when the basic reproductive number of the vaccine-resistant strain is less than that of the vaccine-sensitive strain (), the vaccine-resistant strain can beat the vaccine-sensitive strain and spread widely through the population (see Supplementary Information: Text S1, Fig. S5). Therefore, a non-ideal vaccination program might make a prediction of prevalent strain difficult.

Optimal prevalence rate of vaccination program

In the absence of a vaccine-resistant strain, a goal of vaccination program is to reduce the basic reproductive number of vaccine-sensitive strain to be less than 1. We assume that . Therefore, the vaccination can eradicate the vaccine-sensitive strain if at least 84.7% of the birds in poultry are vaccinated effectively based on the fraction of [30]. However, in the presence of the resistant strain, the simple theory is inapplicable to an optimal prevalence rate of vaccination program. Here we define the optimal prevalence rate of a vaccination program which minimizes both the final size of the epidemic and the prevalence rate (see Supplementary Information: Text S1).

We calculate the optimal prevalence rate, which depends on the loss of the protection effectiveness of the vaccination in Fig. 4 (sensitivity analyses are given in Supplementary Information: Text S1, Fig. S6). At the point where the loss of the protection effectiveness is greater than some threshold value σo, the optimal prevalence rate changes catastrophically from high prevalence rate to a low prevalence rate. Here

thumbnail

Figure 4. Optimal prevalence rate of vaccination program: Increasing of the loss of the protection effectiveness engenders a catastrophic change in the optimal prevalence rate.

The optimal rate increases as the loss increases if the loss of the protection effectiveness is small (0≤σσo). This implies that a small loss of the protection effectiveness can be compensated by a high optimal prevalence rate of the vaccination program. On the other hand, if the loss is large (σoσ≤1), the optimal rate decreases as the loss of the protection effectiveness increases. This eventuality implies that a large loss of the protection effectiveness is no longer compensated by the high optimal prevalence rate of the vaccination program. Therefore, a low prevalence rate, which does not engender the emergence of a vaccine-resistant strain becomes optimal because the poor vaccine engenders the increase of final size of the epidemic because of the spread of the resistant strain.

doi:10.1371/journal.pone.0004915.g004

Actually, σo = 0.461 in our simulation from Table 1. The optimal prevalence rate is 84.6% when the loss of the protection effectiveness is between 0% and 5.6%. In addition, if the loss rate is between 5.6% and 20.1%, then the optimal prevalence rate increases from 84.6% to 100%. Furthermore, if the loss rate is between 20.1% and 46.1%, then the optimal prevalence rate must always be 100%. Consequently, as long as the loss of the protection effectiveness is small (0%–46.1%), the loss can be compensated by a high optimal prevalence rate of the vaccination program. However, if the loss rate is greater than 46.1%, the loss is no longer compensated by the high prevalence rate of the vaccination program. The optimal prevalence rate changes catastrophically from 100% to 10.2%. Afterward, as the loss rate increases from 46.1% to 100%, the optimal prevalence rate decreases from 10.2% to 4.72% (the low prevalence rate becomes optimal). This is true because the poor vaccine (with a large loss of the protection) engenders the emergence of the vaccine-resistant strain for the high prevalence rate; in addition, the spread of the resistant strain increases the final size of the epidemic. Therefore, the loss of the protection effectiveness strongly impacts also on the optimal prevalence rate.

Variation of final size of epidemic according to the vaccination program

In countries where poultry are mainly backyard scavengers, optimum vaccination coverage might be difficult to achieve [21]. The final size of the epidemic might be increased and the program might fail if the optimal prevalence rate of the vaccination program can not be achieved. However, if we can achieve optimum vaccination coverage, the final size is greatly reduced. The final size of the epidemics can be variable depending on the prevalence rate. Here we calculate the optimal (smallest) and worst (largest) final size of the epidemic over the vaccination prevalence (see Supplementary Information: Text S1) in Fig. 5 (black and yellow bars respectively represent the optimal and worst final size). The variation of the final size is between black and yellow bars shown in Fig. 5 (sensitivity analyses are given in Supplementary Information: Text S1, Fig. S7).

thumbnail

Figure 5. Variation of the final size of the epidemic over the vaccination prevalence: The black bar represents the optimal (smallest) final size of the epidemic.

The yellow bar represents the worst (largest) final size of the epidemic over the vaccination prevalence. The variation of the final size depending on the prevalence rate is between black and yellow bars. If the loss of protection effectiveness is small, then the variation is very large. On the other hand, if the loss becomes large, then the variation decreases. Therefore, the final size of the epidemic is strongly affected by the vaccination coverage and the loss of protection effectiveness: a bad vaccination program (far from the optimal prevalence rate) increases the final size and prevents eradication of the disease.

doi:10.1371/journal.pone.0004915.g005

If the loss of protection effectiveness is small, then the variation is very large. The vaccination program can eradicate the disease or reduce the final size of the epidemic to a very small size if we can execute the vaccination program near the optimal prevalence rate. The variation is sensitive for the prevalence rate. Therefore, we must carefully manage the vaccination program to control the disease when the loss is small. However, as the loss of protection effectiveness increases, the variation decreases. In particular, when the loss is medium, the reduction of the variation is remarkable. In addition, the reduction of the variation remains almost unchanged when the loss is large. This implies that the variation becomes insensitive if the loss is high. In this case, even if we can execute the vaccination program near the optimal prevalence rate, the effect of the program is not large. Therefore, although the final size is strongly affected by the vaccination coverage and a non-optimal vaccination program (far from the optimal prevalence rate) increases the final size, in general, good vaccine treatment with small loss of protection effectiveness has a great possibility for disease control. Demonstrably, poor vaccine application has little or no benefit.

Effects of non-pharmaceutical intervention

Avian influenza vaccination need not be used alone to eradicate the disease: additional non-pharmaceutical intervention is beneficial. Additional interventions must include culling infected animals, strict quarantine, movement controls and increased biosecurity, extensive surveillance [11], [16], [21], [34], [37]. We investigate the effects of some additional non-pharmaceutical intervention measures on the vaccination program. The effects are considered by changing model parameters (1).

In the European Union (EU), regulations for the control of avian influenza strains are imposed by EU council directive 92/40/EEC [34]. Virus output is reduced by the killing and removal of infected poultry flocks (culling). During the H7N7 epidemic in The Netherlands in 2003, this and other approaches were executed. To investigate the effectiveness of the control measures, A. Stegeman et al. quantified the transmission characteristics of the H7N7 strain before and after detection of the first outbreak of avian influenza in The Netherlands in 2003 [34]. In Table 1, we present the chosen epidemiological parameters, which are estimated on the H7N7 epidemic before notification of the circulation of the avian influenza (these parameters are not affected by the additional control measures). Here we choose other epidemiological parameters for vaccine-sensitive strain which are estimated by the H7N7 epidemic after the notification in [34] (these parameters are affected by the additional control measures) to evaluate an effect of the non-pharmaceutical intervention on the vaccination program. The estimate of the transmission parameter ω decreases considerably from 4.78×10−4 day−1 individual−1 to 1.70×10−4 day−1 individual−1 by the control measures. Furthermore, the estimate of the infectious period 1/(b+my) is also reduced from 13.8 days to 7.3 days. Therefore, control measures can reduce the basic reproductive number from 6.53 to 1.22 [34]. In addition, we assume, for example, that the relative transmissibility of vaccine-resistant strains is φ/ω = 0.7 and that the relative infectious period of vaccine-resistant strain is (b+my)/(b+mz) = 1.32 (these values are not strongly influential on our results).

We calculated the threshold values of the loss of protection effectiveness of the vaccination and present them in Table 3 when the vaccination program accompanies non-pharmaceutical intervention. Results show that the non-pharmaceutical intervention markedly reduces the risk of the emergence of the vaccine-resistant strain because σ* changes from 5.6% to 37.2%. In addition, the possibility that the vaccination program eventually eradicates the spread of the disease increases because changes from 20.1% to 88.6%. Furthermore, because σc changes from 23.6% to 100%, the vaccination program always decreases the final size of the epidemic compared with that before the vaccination program, even if the size increases when both strains co-exist. When the vaccination program accompanies non-pharmaceutical intervention, even if the loss of protection effectiveness is increased considerably by the vaccine-resistant strain, the loss can almost be compensated by the high optimal prevalence rate of the vaccination program: σo changes from 46.1% to 96.8%.

thumbnail

Table 3. Threshold values of the loss of protection effectiveness of the vaccination.

doi:10.1371/journal.pone.0004915.t003

Figure 6 portrays the optimal prevalence rate of a vaccination program (top figure) and the optimal final size of the epidemic (bottom figure) with (pink curve and bar) or without (black curve and bar) the non-pharmaceutical intervention. The non-pharmaceutical intervention makes it easy to achieve an optimal prevalence rate and to prevent the spread of the disease. Moreover, catastrophic change does not occur until the loss of protection effectiveness becomes very high (top figure in Fig. 6). Furthermore, the optimal final size is also dramatically reduced by the additional intervention (bottom figure in Fig. 6). Even if vaccination without the additional intervention can not prevent the spread of the disease, the vaccination with the intervention can eradicate the disease (for example σ = 60%). Therefore, non-pharmaceutical intervention improves weak points of vaccination programs such as the difficult control of optimal vaccination coverage, the small applicability of the program with respect to the loss of protection effectiveness caused by the vaccine-resistant strain, and so on.

thumbnail

Figure 6. Effects of non-pharmaceutical intervention: The top figure shows the optimal prevalence rate of the vaccination program with (pink curve) or without (black curve) non-pharmaceutical intervention.

The non-pharmaceutical intervention readily achieves the optimal prevalence rate and hinders the catastrophic change. The bottom figure shows the optimal final size of the epidemic with (pink bar) or without (black bar) the non-pharmaceutical intervention. The intervention also dramatically reduces the final size of the epidemic.

doi:10.1371/journal.pone.0004915.g006

Time-course of the spread of the disease

Finally, we investigate the time-course of spread of the disease according to vaccination and non-pharmaceutical interventions for 500 days in the presence of a vaccine-resistant strain. The results are presented in Fig. 7. We consider that the vaccination program and non-pharmaceutical interventions are executed after the vaccine-sensitive strain spreads and becomes endemic (around 200 days). Furthermore, the vaccine-resistant strain is assumed to occur in a few individuals after the start of the vaccination program (around 260 days). We assume that the prevalence rate of the vaccination program is p = 50%, the loss of protection effectiveness is σ = 80%; the other parameters are the same as those used in the descriptions above. These values of p and σ are not influential on our results (sensitivity analyses are shown in Supplementary Information: Text S1, Fig. S8, S9).

thumbnail

Figure 7. Time-course of the spread of the disease with vaccination and non-pharmaceutical interventions: We calculate epidemic curves with a vaccination program for 500 days.

The vaccination program and non-pharmaceutical intervention are started after the vaccine-sensitive strain becomes endemic (around 200 days). We assume that the vaccine-resistant strain occurs after the start of vaccination (around 260 days). The top, middle, and bottom figures respectively depict time courses of infection without the vaccination program, with only the vaccination program, and with both the vaccination program and the non-pharmaceutical intervention. The blue and red curves respectively represent the number of infected individuals with vaccine-sensitive and vaccine-resistant strains. We assume that the prevalence rate of vaccination program is p = 0.5, the loss of protection effectiveness is σ = 0.8.

doi:10.1371/journal.pone.0004915.g007

The top figure in Fig. 7 depicts the epidemic curve without the vaccination program. It is apparent that the vaccine-sensitive strain (the blue curve) becomes endemic at around 200 days after a pandemic phase of the disease if we execute no intervention policy. The middle figure portrays the time-course of spread of the disease, assuming the vaccination program alone. A vaccine-resistant strain (the red curve) emerges and spreads widely through the population by replacing the vaccine-sensitive strain. It becomes endemic at around 450 days. This result shows the possibility that the emergence and replacement of the resistant strain can be facilitated by the vaccination program, as in some vaccination programs [19], [21], [22]. We can observe that it takes about several months for the resistant strain to beat the sensitive strain (see the middle figure in Fig. 7). Actually, the replacement time of the resistant strain was reported as several months in the China and Mexico epidemics [19], [21], [22]. The final size of the simulated epidemic is larger than that before (without) the vaccination program because the loss of protection effectiveness σ = 80% is greater than (see Fig. 3). In this case, the vaccination program negatively affects the control of infectious disease. The bottom figure presents the time-course of the spread of the disease with both the vaccination program and non-pharmaceutical interventions. The vaccine-sensitive strain is dramatically reduced and the vaccine-resistant strain hardly spreads in the population; therefore, both strains are eventually controlled at a low level by the interventions. Thus, non-pharmaceutical interventions can help the vaccination program and control the resistant strain to spread in the population.

Discussion

A serious problem of vaccination strategy is the emergence of vaccine-resistant strains [19], [20], [21], [22]. Even if a resistant strain emerges, a vaccination program must be managed to control the spread of the disease. In the absence of the resistant strain, our mathematical model certainly shows that a large prevalence of the vaccination program might markedly reduce an epidemic curve and the final size of the epidemic. Therefore, we can control infectious diseases as in previous models [30]. However, in the presence of the emergence of a vaccine-resistant strain, the vaccination program can not simply control the spread of the disease. The control of the infectious disease through vaccination becomes more difficult.

The paradoxical result obtained here is that if the virulence of vaccine-resistant strain is less than that of vaccine-sensitive strain, the final size of the epidemic might increase as the prevalence rate of the vaccination program increases (see Fig. 2). A vaccination that is expected to prevent the spread of the disease can instead foster the spread of the disease. Although qualitatively similar results were obtained through more complex models [8], [9], which can be treated analytically only to a slight degree, one of our important results is the clear and simple concept illustrating the value and pitfalls of vaccination programs; the concept can help farmers and administrators to avoid negative effects from paradoxical phenomena.

We investigated how the loss of protection effectiveness impacts a vaccination program’s results in the lower virulence case. If the loss of protection effectiveness is between 0 and , the vaccination program can eventually eradicate the disease, even if a vaccine-resistant strain emerges (see Fig. 3). In particular, if the loss is between 0 and σ*, the program prevents even the emergence of the resistant strain. However, when the loss is greater than , the program no longer prevents the wide spread of the resistant strain in spite of the large prevalence rate of the program. Furthermore, if the loss is between σc and 1, the program presents the risk that the final size will become larger than that without the vaccination program. Therefore, in the context of the emergence of the resistant strain, we must carefully execute the program to exercise a positive effect of the vaccine effectively. Additionally, we investigated the optimal prevalence rate of the vaccination program, its final size, and the worst-case final size (see Fig. 4, 5 and Supplementary Information: Text S1). The catastrophic change of the optimal prevalence rate and the variation of the final size depending on the loss of protection effectiveness were confirmed.

From our theoretical analysis, we propose that monitoring the virulence of the resistant strain and investigating the loss resulting from a resistant strain can have important consequences for developing a vaccination strategy. In particular, all thresholds derived herein are only constructed using basic reproductive numbers and transmissibilities that prevail before the vaccination program, which can be estimated using epidemiological data (it is usually almost impossible to estimate basic and invasion reproductive numbers during vaccination programs). Therefore, using our theory, we were able to calculate various risks in the vaccination program using the available data (Table 3) and propose how we might use a poor vaccine, which has a large loss of protection effectiveness, against the resistant strain to maximize the effects of the program (Fig. 4, 5, and 6). For the results reported here, we assumed that the vaccinated birds can perfectly protect the infection from the vaccine-sensitive strain. Although that assumption is not unreasonable [21], in Supplementary Information: Text S1, Fig. S10, S11, we present an investigation of the effect of the loss of protection effectiveness against the vaccine-sensitive strain. Qualitatively similar results were obtained using numerical simulations.

Vaccination is now being used extensively to aid the prevention of emergence or to control the spread of avian influenza [14]. However, if the vaccinations are not used appropriately, prevention and control will be negatively affected by the vaccination program [1], [11], [19], [21], [22]. Actually, when the vaccine-resistant strain emerges, our model predicts various risks in the program. Therefore, to eradicate the infectious disease effectively by vaccination, early detection of the resistant strain, monitoring of its virulence and loss of protection effectiveness of vaccination caused by the resistant strain, and attendance of non-pharmaceutical interventions, in addition to collaboration among farmers, industry, public health authorities, and the government are all required.

Supporting Information

Figure S1.

(0.42 MB EPS)

Figure S2.

(0.42 MB EPS)

Figure S3.

(0.44 MB EPS)

Figure S4.

(0.47 MB EPS)

Figure S5.

(0.43 MB EPS)

Figure S6.

(1.01 MB EPS)

Figure S7.

(1.02 MB EPS)

Figure S8.

(1.83 MB EPS)

Figure S9.

(1.72 MB EPS)

Figure S10.

(0.96 MB EPS)

Figure S11.

(1.35 MB EPS)

Text S1.

(0.09 MB PDF)

Author Contributions

Analyzed the data: SI TS YT. Contributed reagents/materials/analysis tools: SI TS YT. Wrote the paper: SI.

References

  1. Gambotto A, Barratt-Boyes SM, de Jong MD, Neumann G, Kawaoka Y (2008) Human infection with highly pathogenic H5N1 influenza virus. Lancet  371: 1464–1475.                        Find this article online                    
  2. Alexander ME, Bowman CS, Feng Z, Gardam M, Moghadas SM, et al.  (2007) Emergence of drug resistance: implications for antiviral control of pandemic influenza. Proc R Soc Lond B  274: 1675–1684.                        Find this article online                    
  3. Colizza V, Barrat A, Barthelemy M, Valleron A-J, Vespignani A (2007) Modeling the worldwide spread of pandemic influenza: baseline case and containment interventions. PLoS Med  4(1): e13.                        Find this article online                    
  4. Ferguson NM, Cummings DAT, Cauchemez S, Fraser C, Riley S, Meeyai A, Iamsirithaworn S, Burke DS (2005) Strategies for containing an emerging influenza pandemic in Southeast Asia. Nature  437: 209–214.                        Find this article online                    
  5. Ferguson NM, Cummings DAT, Fraser C, Cajka JC, Cooley PC, Burke DS (2006) Strategies for mitigating an influenza pandemic. Nature  442: 448–452.                        Find this article online                    
  6. Iwami S, Takeuchi Y, Liu X (2007) Avian-human influenza epidemic model. Math Biosci  207: 1–25.                        Find this article online                    
  7. Iwami S, Takeuchi Y, Korobeinikov A, Liu X (2008) Prevention of avian influenza epidemic: What policy should we choose?. J Theor Biol  252: 732–741.                        Find this article online                    
  8. Lipsitch M, Cohen T, Murray M, Levin BR (2007) Antiviral resistance and the control of pandemic influenza. PLoS Med  4(1): e15.                        Find this article online                    
  9. Moghadas SM, Bowman CS, Rost G, Wu J (2008) Population-wide emergence of antiviral resistance during pandemic influenza. PLoS ONE  3(3): e1839.                        Find this article online                    
  10. Regoes RR, Bonhoeffer S (2006) Emergence of drug-resistant influenza virus: population dynamical considerations. Nature  312: 389–391.                        Find this article online                    
  11. Savill NJ, St. Rose SG, Keeling MJ, Woolhouse ME (2006) Silent spread of H5N1 in vaccinated poultry. Nature  442: 757.                        Find this article online                    
  12. Stilianakis NI, Perelson AS, Hayden FG (1998) Emergence of drug resistance during an influenza epidemic: insights from a mathematical model. J Infec Dis  177: 863–873.                        Find this article online                    
  13. Tiensin T, Nielen M, Vernooij H, Songserm T, Kalpravidh W, et al.  (2007) Transmission of the highly pathogenic avian influenza virus H5N1 within flocks during the 2004 epidemic in Thailand. J Infec Dis  196: 1679–1684.                        Find this article online                    
  14. Capua I (2007) Vaccination for notifiable avian influenza in poultry. Rev Sci Tech Off Int Epiz  26: 217–227.                        Find this article online                    
  15. Marangon S, Cecchinato M, Capua I (2008) Use of vaccination in avian influenza control and eradication. Zoo Pub Health  55: 65–72.                        Find this article online                    
  16. Tiensin T, Chaitaweesub P, Songserm T, Chaisingh A, Hoonsuwan W, et al.  (2005) Highly pathogenic avian influenza H5N1, Thailand, 2004. Emerg Infect Dis  11: 1664–1672.                        Find this article online                    
  17. Capua I, Marangon S (2004) Vaccination for avian influenza in Asia. Vaccine  22: 4137–4138.                        Find this article online                    
  18. Capua I, Marangon S (2006) Control of avian influenza in poultry. Emerg Infect Dis  12: 1319–1324.                        Find this article online                    
  19. Lee CW, Senne DA, Suarez DL (2004) Effect of vaccine use in the evolution of Mexican lineage H5N2 avian influenza virus. J Virol  78: 8372–8381.                        Find this article online                    
  20. Pasquato A, Seidah NG (2008) The H5N1 influenza variant Fujian-like hemagglutinin selected following vaccination exhibits a compromised furin cleavage: neurological Consequences of highly pathogenic Fujian H5N1 strains. J Mol Neurosci  35: 339–343.                        Find this article online                    
  21. Peyre M, Fusheng G, Desvaux S, Roger F (2008) Avian influenza vaccines: a practical review in relation to their application in the field with a specifically examine the Asian experience. Epidemiol Infect  14: 1–21.                        Find this article online                    
  22. Smith GJD, Fan XH, Wang J, Li KS, Qin K, et al.  (2006) Emergence and predominance of an H5N1 influenza variant in China. Proc Nat Acad Sci U S A  103: 16936–16941.                        Find this article online                    
  23. Hayden FG (2001) Perspectives on antiviral use during pandemic influenza. Phil Trans R Soc Lond B  356: 1877–1884.                        Find this article online                    
  24. Guan Y, Smith GJD, Peiris JSM, Webster RG (2007) Comments on the Fujianlike strain of avian influenza H5N1 – reply. Poultry Sci  86: 437–438.                        Find this article online                    
  25. Leung FC (2007) Comments on the Fujian-Like strain of avian influenza H5N1. Poultry Sci  86: 435–436.                        Find this article online                    
  26. McCaw JM, Wood JG, McCaw CT, McVernon J (2008) Impact of emerging antiviral drug resistance on influenza containment and spread: influence of subclinical infection and strategic use of a stockpile containing one or two drugs. PLoS ONE  3(6): e2362.                        Find this article online                    
  27. Seo SH, Webster RG (2001) Cross-reactive, cell-mediated immunity and protection of chickens from lethal H5N1 influenza virus infection in Hong Kong poultry markets. J Virol  75: 2516–2525.                        Find this article online                    
  28. Swayne DE, Beck JR, Garcia M, Stone HD (1999) Influence of virus strain and antigen mass on efficacy of H5 avian influenza inactivated vaccines. Avian Pathol  28: 245–255.                        Find this article online                    
  29. van den Berga T, Lambrechta B, Marche S, Steenselsa M, Borma SV, et al.  (2007) Influenza vaccines and vaccination strategies in birds. Com Immunol Microbiol Infec Dis  31: 121–165.                        Find this article online                    
  30. Anderson RM, May RM (1991) Infectious disease of humans: dynamics and control. Oxford University Press.
  31. Iwami S, Takeuchi Y, Liu X, Nakaoka SA geographical spread of vaccineresistance in avian influenza epidemics, In Revision                        Find this article online                    
  32. Elbers AR, Fabri TH, de Vries TS, de Wit JJ, Pijpers A, Koch G (2004) The highly pathogenic avian influenza A (H7N7) virus epidemic in The Netherlands in 2003 – lessons learned from the first five outbreaks. Avian Dis  48: 691–705.                        Find this article online                    
  33. Elbers AR, Koch G, Bouma A (2005) Performance of clinical signs in poultry for detection of outbreaks during the avian influenza A (H7N7) epidemic in The Netherlands in 2003. Avian Pathol  34: 181–187.                        Find this article online                    
  34. Stegeman A, Bouma A, Elbers ARW, de Jong MCM, Nodelijk G, et al.  (2004) Avian influenza A virus (H7N7) epidemic in The Netherlands in 2003: course of the epidemic and effectiveness of control measures. J Infect Dis  190: 2088–2095.                        Find this article online                    
  35. Handel A, Regoes RR, Antia R (2006) The role of compensatory mutations in the emergence of drug resistance. PLoS Com Biol  2(10): e137.                        Find this article online                    
  36. Capua I, Alexander DJ (2004) Human Health Implications of Avian Influenza Viruses and Paramyxoviruses. Eur J Clin Microbiol Infect Dis  23: 1–6.                        Find this article online                    
  37. Webster RG, Kawaoka Y, Bean WJ, Beard CW, Brugh M (1985) Chemotherapy and vaccination: a possible strategy for the control of highly virulent influenza virus. J Virol  55: 173–176.                        Find this article online

Pigs in southern China infected with avian flu: Recent Infections of H1N1 & H3N2

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

Researchers report for the first time the seroprevalence of three strains of avian influenza viruses in pigs in southern China, but not the H5N1 avian influenza virus.  Their research, published online ahead of print in the Journal of Clinical Microbiology, has implications for efforts to protect the public health from pandemics.

Influenza A virus is responsible both for pandemics that have killed millions worldwide, and for the much less severe annual outbreaks of influenza. Because pigs can be infected with both human and avian influenza viruses, they are thought to serve as “mixing vessels” for genetic reassortment that could lead to pandemics, and pigs have been infected experimentally by all avian H1-H13 subtypes. But natural transmission of avian influenza to pigs has been documented only rarely.

In the study, from 2010-2012, Guihong Zhang and colleagues of the College of Veterinary Medicine, South China Agricultural University, Guangzhou, People’s Republic of China, tested 1080 21-25 week old pigs for H3, H4, H5, and H6 subtypes of avian influenza virus, and H1 and H3 subtypes of swine influenza virus. Thirty-five percent of the serum samples were positive for H1N1, and 19.7 percent were positive for H3N2 swine flu virus, and 0.93 percent, 1.6 percent, and 1.8 percent were positive, respectively, for the H3, H4, and H6 subtypes of avian influenza A virus. However, no serum samples collected in 2001 were positive for any of these viruses, indicating that transmission into swine was recent.

Given the recent transmission of avian influenzas into swine, “We recommend strongly that the pork industry worldwide should monitor the prevalence of influenza in pigs, considering their important role in transmitting this virus to humans,” says Zhang.

Previously, novel reassortant H2N3 influenza viruses were isolated from US pigs, which “were infectious and highly transmissible in swine and ferrets without prior adaptation,” according to a 2009 paper in the Journal of Molecular and Genetic Medicine by Wenjun Ma et al. Those viruses resembled, but were not identical to the H2N2 human pandemic virus of 1957.

###

A copy of the manuscript can be found online at http://bit.ly/asmtip1212d.  Formal publication is scheduled for the February 2013 issue of the Journal of Clinical Microbiology.

(S. Su, W. Qi, J. Chen, W. Zhu, Z. Huang, J. Xie, and G. Zhang, 2012. Seroepidemiological evidence of avian influenza A virus transmission in pigs in southern China. J. Clin. Microbiol. Online ahead of print 21 November 2012.)

The Journal of Clinical Microbiology 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.

H1N1 Pandemic Virus Does Not Mutate Into ‘Superbug’ in UMd. Lab Study

2009 study posted for filing

 

COLLEGE PARK, Md. – A laboratory study by University of Maryland researchers suggests that some of the worst fears about a virulent H1N1 pandemic flu season may not be realized this year, but does demonstrate the heightened communicability of the virus.

 

Using ferrets exposed to three different viruses, the Maryland researchers found no evidence that the H1N1 pandemic variety, responsible for the so-called swine flu, combines in a lab setting with other flu strains to form a more virulent ‘superbug.’ Rather, the pandemic virus prevailed and out-competed the other strains, reproducing in the ferrets, on average, twice as much.

 

The researchers believe their study is the first to examine how the pandemic virus interacts with other flu viruses. The findings are newly published in an online scientific journal designed to fast-track science research and quickly share results with other investigators, PLOS Currents.

 

“The H1N1 pandemic virus has a clear biological advantage over the two main seasonal flu strains and all the makings of a virus fully adapted to humans,” says virologist Daniel Perez, the lead researcher and program director of the University of Maryland-based Prevention and Control of Avian Influenza Coordinated Agricultural Project.

 

“I’m not surprised to find that the pandemic virus is more infectious, simply because it’s new, so hosts haven’t had a chance to build immunity yet. Meanwhile, the older strains encounter resistance from hosts’ immunity to them,” Perez adds.

 

Some of the animals who were infected with both the new virus and one of the more familiar seasonal viruses (H3N2) developed not only respiratory symptoms, but intestinal illness as well. Perez and his team call for additional research to see whether this kind of co-infection and multiple symptoms may account for some of the deaths attributed to the new virus.

 

Among other research findings, the pandemic virus successfully established infections deeper in the ferret’s respiratory system, including the lungs. The H1 and H3 seasonal viruses remained in the nasal passages.

 

“Our findings underscore the need for vaccinating against the pandemic flu virus this season,” Perez concludes. “The findings of this study are preliminary, but the far greater communicability of the pandemic virus serves as a clearly blinking warning light.”

 

Perez and his team used samples of the H1N1 pandemic variety from last April’s initial outbreak of the so-called swine flu.

 

The research is funded by the National Institute of Allergy and Infectious Diseases, part of the National Institutes of Health.

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.

http://www.courthousenews.com/2012/10/23/51581.htm

Bird flu virus remains infectious up to 600 days in municipal landfills H5N1

2009 study posted for filing

 

Environmental Science & Technology

 

Amid concerns about a pandemic of swine flu, researchers from Nebraska report for the first time that poultry carcasses infected with another threat — the “bird flu” virus — can remain infectious in municipal landfills for almost 2 years. Their report is scheduled for the June 15 issue of ACS’ semi-monthly journal Environmental Science & Technology.

 

Shannon L. Bartelt-Hunt and colleagues note that avian influenza, specifically the H5N1 strain, is an ongoing public health concern. Hundreds of millions of chickens and ducks infected with the virus have died or been culled from flocks worldwide in efforts to control the disease. More than 4 million poultry died or were culled in a 2002 outbreak in Virginia, and the carcasses were disposed of in municipal landfills. Until now, few studies have directly assessed the safety of landfill disposal.

 

“The objectives of this study were to assess the survival of avian influenza in landfill leachate and the influence of environmental factors,” says the report. The data showed that the virus survived in landfill leachate — liquid that drains or “leaches” from a landfill — for at least 30 days and up to 600 days. The two factors that most reduced influenza survival times were elevated temperature and acidic or alkaline pH. “Data obtained from this study indicate that landfilling is an appropriate method for disposal of carcasses infected with avian influenza,” says the study, noting that landfills are designed to hold material for much longer periods of time.

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

2009 study posted for filing

Contact: Lucy Goodchild
lucy.goodchild@imperial.ac.uk
44-207-594-6702
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.

Research on enhanced transmissibility in H5N1 influenza: Should the moratorium end?

Public Release: 9-Oct-2012

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

How can scientists safely conduct avian flu research if the results could potentially threaten, as well as save, millions of lives? In a series of commentaries appearing on Tuesday, October 9 in mBio®, the online open-access journal of the American Society for Microbiology, prominent microbiologists and physicians argue the cases both for and against lifting a voluntary moratorium on experiments to enhance the ability of the H5N1 virus to move from mammal to mammal, so-called “gain-of-function” research, and discuss the level of biosecurity that would be appropriate for moving that research forward.

In January 2012, in response to the controversy caused by the unprecedented recommendation iof an advisory board to the government to redact methods sections  of two research studies showing how genetic changes could make H5N1 become transmissible between mammals, a group of influenza researchers agreed to a voluntary pause on any research involving highly pathogenic avian influenza H5N1 viruses leading to the generation of viruses that are more transmissible in mammals. Despite both articles eventually being published in full in May and June 2012, the research moratorium remains in place.

“The scientific community and the greater society that it serves are currently engaged in a vigorous debate on whether and how to carry out experiments that could provide essential information for preparedness against a pandemic of avian influenza. To foster discussion and to provide a venue to record the arguments for or against this moratorium, mBio® has commissioned a series of views from experts in the field,” write Arturo Casadevall of the Albert Einstein School of Medicine, editor-in-chief of mBio®; and Thomas Shenk of Princeton University, Chair of the ASM Publications Board, in an introductory editorial.

Enhancing and analyzing the transmissibility of the H5N1 virus could, on the one hand, provide insights that could help prevent or treat a future outbreak of H5N1 , or, on the other hand, it may provide a roadmap for a “bad actor” to deliberately bring about an influenza pandemic or lead to an inadvertent release of a virus with enhanced transmissibility.

Authors of the commentaries are prominent scientists, including:

  • Ron Fouchier of Erasmus MC Rotterdam in The Netherlands, Adolfo García-Sastre of the Mount Sinai School of Medicine, and Yoshihiro Kawaoka of the University of Wisconsin-Madison, lead authors of the two papers that began the controversy, argue that in the eight months since the moratorium was agreed upon, the international research community has had sufficient time to review biosafety and biosecurity measures and that H5N1 transmission studies ought to proceed. 
  • Anthony Fauci, Director of the National Institute of Allergy and Infectious Diseases contributes his voice as a representative of an organization that is a key funder of influenza research. Although Fauci acknowledges that the benefits of gain-of-function research outweigh the risks, he argues that scientists have yet to fully meet their responsibility for engaging the public in weighing these matters and making the case for proceeding. He outlines how the U.S. government plans to augment policy guidelines related to “dual use research of concern” like the experiments on enhanced influenza transmission. 
  • Marc Lipsitch and Barry R. Bloom of the Harvard School of Public Health explain why they view H5N1 with enhanced transmissibility as a “potential pandemic pathogen,”representing an even greater threat to global health than Ebola and other biosafety level 4 (BSL-4) pathogens. They argue that research on enhanced H5N1 and other potential pandemic pathogens requires a new, more stringent set of guidelines for safety, thorough public discussion of the risks and benefits involved, and global guidelines for laboratory procedures, among other measures to minimize the risk of laboratory-released infections or epidemics. 
  • Ian Lipkin of Columbia University argues that once research on enhanced strains of H5N1 continues it may be advisable to conduct the work only in BSL-3 Ag laboratories that meet additional, enhanced guidelines for handling agents with pandemic potential. Lipkin proposes that any course should be charted in consultation with and oversight from the global scientific and regulatory community. 
  • Stanley Falkow of Stanford University provides perspective on the H5N1 research moratorium based on his own experiences with a similar situation in the 1970s, when research in recombinant DNA techniques was halted while a committee of scientists and non-scientists could establish a set of guidelines for conducting the work safely. Falkow argues that research on H5N1 viruses with enhanced transmissibility should move forward once scientists work with the public to establish standardized guidelines using common sense and scientific creativity.

“This is a historic time in science,” says Casadevall. mBio® has solicited the views of experts in the field, he says, in order to provide a venue for recording the arguments for and against continuing H5N1 gain-of-function research. “Society is asking for a pause of research that is perhaps the best defense against pandemics because of concern about both biosafety and biosecurity.” With the research moratorium continuing well past the 60-days originally planned, it is time these conflicting views were aired in a public forum, he says.

###

mBio® is an open access online journal published by the American Society for Microbiology to make microbiology research broadly accessible. The focus of the journal is on rapid publication of cutting-edge research spanning the entire spectrum of microbiology and related fields. It can be found online at http://mBio.asm.org.

The American Society for Microbiology is the largest single life science society, composed of over 39,000 scientists and health professionals. ASM’s 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

Researchers Map Molecular Details That Encourage H1N1 Transmission To Humans

The 2009 H1N1 pandemic influenza virus appears to have required certain mutations in order to be transmitted to humans, according to a paper in the September Journal of Virology. The research could prove extremely valuable for efforts to predict human outbreaks.

The 2009 influenza pandemic was caused by a swine influenza virus that mutated in a way that made it transmissible among humans. The researchers, led by Hualan Chen of the Harbin Veterinary Research Institute, Harbin, China, have determined the probable details of the mutations that led to human transmission.

In this study, Chen, who is director of the National Avian Influenza Reference Laboratory at the Institute, and her collaborators have shown that two specific mutations in each of two proteins appear to be critical to transmission to, and among humans. One of those mutations, of a single amino acid in the virus’ hemagglutinin protein, gives the virus the ability to bind to human receptors, and enables transmission in mammals via droplets of respiratory fluids.

That amino acid, in the 226th slot in the protein, is glutamine. The researchers showed its importance by causing a mutation from glutamine, the amino acid in that position seen in viruses from infected humans, to argenine, as seen in swine. Working in cell cultures, the researchers showed that the switch dampened the virus’ ability to bind the human receptor, while boosting its ability to bind to the avian receptor. They showed further that the change rendered the virus non-transmissible via respiratory droplets in guinea pig models, and unable to replicate in the lungs of ferrets—results that suggest, but do not prove that the same may happen in humans.

Also in guinea pigs, changing an amino acid in the virus’ PB2 protein abolished transmission in guinea pigs via respiratory droplets, while that change, plus another single amino acid change in the hemagglutinin protein, killed such transmission in ferrets.

It gets still more convoluted. The same amino acid in the PB2 protein that enables virus transmission via respiratory droplets, which is located at position 271 in that protein, can also encourage the afore-mentioned mutation in hemagglutinin position 226 to glutamine, which enables the virus to cleave to the human receptor.

The value of all this information, says Chen, is that it provides a means for predicting outbreaks of human-transmissible H1N1.

(Y. Zhang, Q. Zhang, Y. Gao, X. He, H. Kong, Y. Jiang, Y. Guan, X. Xia, Y. Shu, Y. Kawaoka, Z. Bu, and H. Chen, 2012. Key molecular factors in hemagglutinin and PB2 contribute to efficient transmission of the 2009 H1N1 pandemic influenza virus. J. Virol. 86:9666-9674.)

Download a copy of the article at http://bit.ly/asmtip0912b

The pandemic potential of H9N2 avian influenza viruses

Re-Post for Filing 2008

Contact: Beth Cavanaugh
bcavana@umd.edu
Public Library of Science

Since their introduction into land-based birds in 1988, H9N2 avian influenza A viruses have caused multiple human infections and become endemic in domestic poultry in Eurasia. This particular influenza subtype has been evolving and acquiring characteristics that raise concerns that it may become more transmissible among humans. Mechanisms that allow infection and subsequent human-to-human transmission of avian influenza viruses are not well understood.

In a new study published August 13 in the journal PLoS ONE, Daniel Perez (of the University of Maryland) and colleagues used ferrets to characterize the mechanism of replication and transmission of recent avian H9N2 viruses. The researchers show that some currently circulating avian H9N2 viruses can transmit to naïve ferrets placed in direct contact with infected ferrets. However, aerosol transmission was not observed, a key factor in potentially pandemic strains.

More importantly, Perez and colleagues show that a single amino acid residue (Leu226) at the receptor-binding site (RBS) of the hemagglutinin (HA) surface protein plays a major role in the ability of these viruses to transmit. They also found that an avian-human H9N2 reassortant virus increases virulence, pathology and replication in ferrets. These results suggest that the establishment and prevalence of H9N2 viruses in poultry could pose a significant threat for humans.

 

###

Contact:

Beth Cavanaugh
University of Maryland Press Office
Email: bcavana@umd.edu

Daniel Perez
University of Maryland
Email: dperez1@umd.edu

Citation: Wan H, Sorrell EM, Song H, Hossain MJ, Ramirez-Nieto G, et al. (2008) Replication and Transmission of H9N2 Influenza Viruses in Ferrets: Evaluation of Pandemic Potential. PLoS ONE 3(8): e2923. doi:10.1371/journal.pone.0002923

PLEASE ADD THE LINK TO THE PUBLISHED ARTICLE IN ONLINE VERSIONS OF YOUR REPORT (URL live from Aug 13): http://dx.plos.org/10.1371/journal.pone.0002923

PRESS-ONLY PREVIEW: http://www.plos.org/press/pone-03-08-perez.pdf

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

Abstract

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 2.3.2.1, 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 2.3.2.1 (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 2.3.2.1 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

References and Notes

1.↵

S. Herfst

et al

., Science336, 1534 (2012).

Abstract/FREE Full Text

2.↵

M. Imai

et al

., Nature, published online 2 May 2012; 10.1038/nature10831.

CrossRef

3.↵

A. Vines

et al

., The role of influenza A virus hemagglutinin residues 226 and 228 in receptor specificity and host range restriction. J. Virol.72, 7626 (1998).

Abstract/FREE Full Text

4.↵

S. Chutinimitkul

et al

., In vitro assessment of attachment pattern and replication efficiency of H5N1 influenza A viruses with altered receptor specificity. J. Virol.84, 6825 (2010).

Abstract/FREE Full Text

5.↵

M. Hatta

et al

., Growth of H5N1 Influenza A Viruses in the Upper Respiratory Tracts of Mice. PLoS Pathog.3, e133 (2007).

CrossRef

6.↵Materials and methods are available as supplementary materials on Science Online.

7.↵

S. E. Luria,

M. Delbrück

, Mutations of bacteria from virus sensitivity to virus resistance. Genetics28, 491 (1943).

CrossRefMedlineWeb of Science

8.

C. J. Mode,

T. Raj,

C. K. Sleeman

, Simulating the emergence and survival of mutations using a self regulating multitype branching processes. J. Probab. Stat.2011, 1 (2011).

CrossRef

9.

J. M. Coffin

, HIV population dynamics in vivo: Implications for genetic variation, pathogenesis, and therapy. Science267, 483 (1995).

Abstract/FREE Full Text

10.↵

A. S. Perelson,

L. Rong,

F. G. Hayden

, J. Infect. Dis., published online 23 March 2012; 10.1093/infdis/jis265.

11.↵

Y. Sidorenko,

U. Reichl

, Structured model of influenza virus replication in MDCK cells. Biotechnol. Bioeng.88, 1 (2004).

CrossRefMedlineWeb of Science

12.↵

L. Möhler,

D. Flockerzi,

H. Sann,

U. Reichl

, Mathematical model of influenza A virus production in large-scale microcarrier culture. Biotechnol. Bioeng.90, 46 (2005).

CrossRefMedlineWeb of Science

13.↵

E. R. Weibel

, Morphometry of the human lung: The state of the art after two decades. Bull. Eur. Physiopathol. Respir.15, 999 (1979).

MedlineWeb of Science

14.↵

Writing Committee WHO

, Update on Avian Influenza A (H5N1) virus infection in humans. N. Engl. J. Med.358, 261 (2008).

CrossRefMedlineWeb of Science

15.↵

P. R. Murcia

et al

., Intra- and interhost evolutionary dynamics of equine influenza virus. J. Virol.84, 6943 (2010).

Abstract/FREE Full Text

16.↵

T. Kuiken

et al

., Host species barriers to influenza virus infections. Science312, 394 (2006).

Abstract/FREE Full Text

17.↵

S. Bonhoeffer,

M. A. Nowak

, Pre-existence and emergence of drug resistance in HIV-1 infection. Proc. Biol. Sci.264, 631 (1997).

Abstract/FREE Full Text

18.↵

S. Wain-Hobson

, The fastest genome evolution ever described: HIV variation in situ. Curr. Opin. Genet. Dev.3, 878 (1993).

CrossRefMedline

19.↵

W. G. Hill,

A. Robertson

, The effect of linkage on limits to artificial selection. Genet. Res.8, 269 (1966).

MedlineWeb of Science

20.↵

A. Antón

et al

., Selection and viral load kinetics of an oseltamivir-resistant pandemic influenza A (H1N1) virus in an immunocompromised patient during treatment with neuraminidase inhibitors. Diagn. Microbiol. Infect. Dis.68, 214 (2010).

CrossRefMedline

21.↵

M. D. de Jong

et al

., Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia. Nat. Med.12, 1203 (2006).

CrossRefMedlineWeb of Science

22.↵

C. A. Nidom

et al

., Influenza A (H5N1) viruses from pigs, Indonesia. Emerg. Infect. Dis.16, 1515 (2010).

Medline

23.

J. Keawcharoen

et al

., Avian influenza H5N1 in tigers and leopards. Emerg. Infect. Dis.10, 2189 (2004).

MedlineWeb of Science

24.

X. Qi

et al

., Molecular characterization of highly pathogenic H5N1 avian influenza A viruses isolated from raccoon dogs in China. PLoS ONE4, e4682 (2009).

CrossRefMedline

25.↵

L. Reperant

et al

., Rev. Sci. Tech.1, 137 (2009).

26.↵

N. M. Ferguson,

C. Fraser,

C. A. Donnelly,

A. C. Ghani,

R. M. Anderson

, Public health risk from the avian H5N1 influenza epidemic. Science304, 968 (2004).

Abstract/FREE Full Text

27.↵

T. Y. Aditama

et al

., Avian influenza H5N1 transmission in households, Indonesia. PLoS ONE7, e29971 (2012).

CrossRefMedline

28.↵

Y. Yang,

M. E. Halloran,

J. D. Sugimoto,

I. M. Longini Jr.

, Detecting human-to-human transmission of avian influenza A (H5N1). Emerg. Infect. Dis.13, 1348 (2007).

MedlineWeb of Science

29.↵

M. P. Atkinson,

L. M. Wein

, Quantifying the routes of transmission for pandemic influenza. Bull. Math. Biol.70, 820 (2008).

CrossRefMedlineWeb of Science

30.↵

P. Fabian

et al

., Influenza virus in human exhaled breath: An observational study. PLoS ONE3, e2691 (2008).

CrossRefMedline

31.↵

R. Tellier

, Aerosol transmission of influenza A virus: A review of new studies. J. R. Soc. Interface6 (suppl. 6), S783 (2008).

CrossRef

32.↵

T. Ord,

R. Hillerbrand,

A. Sandberg

, Probing the improbable: Methodological challenges for risks with low probabilities and high stakes. J. Risk Res.13, 191 (2010).

CrossRefWeb of Science

33.↵

J. A. Lednicky

et al

., Ferrets develop fatal influenza after inhaling small particle aerosols of highly pathogenic avian influenza virus A/Vietnam/1203/2004 (H5N1). Virol. J.7, 231 (2010).

CrossRefMedline

34.↵

D. J. Spiegelhalter,

H. Riesch

, Philos. Trans. R. Soc. London Ser. A269, 4730 (2011).

35.↵

J. Bahl

et al

., Continued evolution of highly pathogenic avian influenza A (H5N1): Updated nomenclature. Influenza Other Respir. Viruses6, 1 (2012).

CrossRefMedlineWeb of Science

36.↵

S. Guindon

et al

., New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0. Syst. Biol.59, 307 (2010).

Abstract/FREE Full Text

37.↵

D. Posada,

K. A. Crandall

, MODELTEST: Testing the model of DNA substitution. Bioinformatics14, 817 (1998).

Abstract/FREE Full Text

38.↵

D. J. Zwickl, thesis, University of Texas (2006).

39.↵

J. Steel,

A. C. Lowen,

S. Mubareka,

P. Palese

, Transmission of influenza virus in a mammalian host is increased by PB2 amino acids 627K or 627E/701N. PLoS Pathog.5, e1000252 (2009).

CrossRefMedline

40.

Z. Li

et al

., Molecular basis of replication of duck H5N1 influenza viruses in a mammalian mouse model. J. Virol.79, 12058 (2005).

Abstract/FREE Full Text

41.

E. J. Schrauwen

et al

., The multibasic cleavage site in H5N1 virus is critical for systemic spread along the olfactory and hematogenous routes in ferrets. J. Virol.86, 3975 (2012).

Abstract/FREE Full Text

42.

T. Kuiken,

J. K. Taubenberger

, Pathology of human influenza revisited. Vaccine26, (Suppl 4), D59 (2008).

CrossRefMedlineWeb of Science

43.

Y. Hatta

et al

., Viral replication rate regulates clinical outcome and CD8 T cell responses during highly pathogenic H5N1 influenza virus infection in mice. PLoS Pathog.6, e1001139 (2010).

CrossRefMedline

44.

E. M. Sorrell

et al

., Predicting ‘airborne’ influenza viruses: (trans-) mission impossible? Curr. Opin. Virol.1, 635 (2011).

Medline

45.

L. A. Loeb

, Mutator phenotype may be required for multistage carcinogenesis. Cancer Res.51, 3075 (1991).

FREE Full Text

46.

K. Shinya

et al

., Avian flu: influenza virus receptors in the human airway. Nature440, 435 (2006).

CrossRefMedline

47.

D. van Riel

et al

., H5N1 virus attachment to lower respiratory tract. Science312, 399 (2006).

Abstract/FREE Full Text

48.

A. Mehle,

J. A. Doudna

, Adaptive strategies of the influenza virus polymerase for replication in humans. Proc. Natl. Acad. Sci. U.S.A.106, 21312 (2009).

Abstract/FREE Full Text

49.

S. J. Stray,

G. M. Air

, Apoptosis by influenza viruses correlates with efficiency of viral mRNA synthesis. Virus Res.77, 3 (2001).

CrossRefMedlineWeb of Science

50.

P. Baccam,

C. Beauchemin,

C. A. Macken,

F. G. Hayden,

A. S. Perelson

, Kinetics of influenza A virus infection in humans. J. Virol.80, 7590 (2006).

Abstract/FREE Full Text

51.

J. K. Taubenberger

et al

., Characterization of the 1918 influenza virus polymerase genes. Nature437, 889 (2005).

CrossRefMedline

52.

Y. Gao

et al

., Identification of amino acids in HA and PB2 critical for the transmission of H5N1 avian influenza viruses in a mammalian host. PLoS Pathog.5, e1000709 (2009).

CrossRefMedline

53.

L. M. Chen

et al

., In vitro evolution of H5N1 avian influenza virus toward human-type receptor specificity. Virology422, 105 (2012).

CrossRefMedlineWeb of Science

54.

P. Auewarakul

et al

., An avian influenza H5N1 virus that binds to a human-type receptor. J. Virol.81, 9950 (2007).

Abstract/FREE Full Text

55.

Y. Watanabe

et al

., Acquisition of human-type receptor binding specificity by new H5N1 influenza virus sublineages during their emergence in birds in Egypt. PLoS Pathog.7, e1002068 (2011).

CrossRefMedline

56.

Z. Y. Yang

et al

., Immunization by avian H5 influenza hemagglutinin mutants with altered receptor binding specificity. Science317, 825 (2007).

Abstract/FREE Full Text

57.

J. Stevens

et al

., Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus. Science312, 404 (2006).

Abstract/FREE Full Text

58.

J. Stevens

et al

., Recent avian H5N1 viruses exhibit increased propensity for acquiring human receptor specificity. J. Mol. Biol.381, 1382 (2008).

CrossRefMedlineWeb of Science

59.

S. Yamada

et al

., Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to human-type receptors. Nature444, 378 (2006).

CrossRefMedline

60.

Y. Iwasa,

F. Michor,

M. A. Nowak

, Stochastic tunnels in evolutionary dynamics. Genetics166, 1571 (2004).

Abstract/FREE Full Text

61.

D. B. Weissman,

M. M. Desai,

D. S. Fisher,

M. W. Feldman

, The rate at which asexual populations cross fitness valleys. Theor. Popul. Biol.75, 286 (2009).

CrossRefMedlineWeb of Science

62.

D. B. Weissman,

M. W. Feldman,

D. S. Fisher

, The rate of fitness-valley crossing in sexual populations. Genetics186, 1389 (2010).

CrossRefMedlineWeb of Science

63.

R. Durrett,

D. Schmidt

, Waiting for two mutations: With applications to regulatory sequence evolution and the limits of Darwinian evolution. Genetics180, 1501 (2008).

Abstract/FREE Full Text

64.

R. Durrett,

D. Schmidt,

J. Schweinsberg

, A waiting time problem arising from the study of multi-stage carcinogenesis. Ann. Appl. Probab.19, 676 (2009).

CrossRefWeb of Science

65.

M. Lynch

, Scaling expectations for the time to establishment of complex adaptations. Proc. Natl. Acad. Sci. U.S.A.107, 16577 (2009).

CrossRef

66.↵

N. L. Komarova,

A. Sengupta,

M. A. Nowak

, Mutation-selection networks of cancer initiation: tumor suppressor genes and chromosomal instability. J. Theor. Biol.223, 433 (2003).

CrossRefMedlineWeb of Science

67.

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.