New method for making human-based gelatin for gelatin-type desserts, marshmallows, candy and innumerable other products

Public release date: 13-Jul-2011 (HRR-Requested Repost)


Scientists are reporting development of a new approach for producing large quantities of human-derived gelatin that could become a substitute for some of the 300,000 tons of animal-based gelatin produced annually for gelatin-type desserts, marshmallows, candy and innumerable other products. Their study appears in ACS’s Journal of Agriculture and Food Chemistry.

Jinchun Chen and colleagues explain that animal-based gelatin, which is made most often from the bones and skin of cows and pigs, may carry a risk of infectious diseases such as “Mad Cow” disease and could provoke immune system responses in some people. Animal- based gelatin has other draw-backs, with variability from batch to batch, for instance, creating difficulties for manufacturers. Scientists thus have sought alternatives, including development of a human-recombinant gelatin for potential use in drug capsules and other medical applications. Continue reading “New method for making human-based gelatin for gelatin-type desserts, marshmallows, candy and innumerable other products”

Change in Human Social Behavior in Response to a Common Vaccine ( Flu Vaccine )


– In the 2 days immediately after influenza immunization, study participants socially encountered almost twice as many other humans as they did in the 2 days before immunization



PURPOSE: The purpose of this study was to test the hypothesis that exposure to a directly transmitted human pathogendflu virusdincreases human  social behavior  presymptomatically. This  hypothesis  is grounded in empirical evidence that animals infected with pathogens rarely behave like uninfected animals, and in evolutionary theory as applied to infectious disease. Such behavioral changes have the potential to increase parasite transmission and/or host solicitation of care. Continue reading “Change in Human Social Behavior in Response to a Common Vaccine ( Flu Vaccine )”

Vivax malaria may be evolving around natural defense ( 2.5 billion people worldwide are at risk )

Contact: Kevin Mayhood 216-368-4442 Case Western Reserve University

3 gene mutations appear to be invasion mechanisms

             IMAGE:   Plasmodium vivax has traditionally infected red blood cells of hosts in the Duffy positive blood group but Duffy negative people have been resistant.

Click here for more information.     

CLEVELAND—Researchers at Case Western Reserve University and Cleveland Clinic Lerner Research Institute have discovered recent genetic mutations in a parasite that causes over 100 million cases of malaria annually—changes that may render tens of millions of Africans who had been considered resistant, susceptible to infection.

Peter A. Zimmerman, professor of international health, biology and genetics at the Case Western Reserve School of Medicine, and David Serre, a scientific staff member of the Genomic Medicine Institute at Lerner and assistant professor of genomics at Case Western Reserve, report their findings at the American Society of Tropical Medicine and Hygiene annual meeting today (11/15).

They and fellow researchers describe the changes in the Plasmodium vivax genome in papers scheduled to be published in the journal PLoS Neglected Tropical Disease on Nov. 21 and Dec. 5.

To learn the functions of the mutations, and whether the parasite is evolving around a natural defense, Zimmerman and Serre have received a $3.5 million grant from the National Institute of Allergy and Infectious Disease at the National Institutes of Health. They will begin their field study in early 2014.

“We’ve found a duplication of a gene known to enable the parasite to infect red blood cells and two possible additional components to a more complex red cell invasion mechanism,” Zimmerman said.

Researchers have long thought that P. vivax infects a person one way: a protein on the parasite, called the Duffy binding protein, latches onto a Duffy receptor on the surface of the person’s red blood cell and works itself through the membrane. People who lack the receptor are called Duffy negative and are resistant to infection.

But, during the last decade, reports of cases of Duffy negative patients with P. vivax infections have been on the rise in several parts of the world.

P. vivax has been called benign malaria because it is less lethal than malaria caused by Plasmodium falciparum. But unlike its cousin, P. vivax can hide from treatment in a host’s liver and repeatedly emerge to cause relapses of debilitating headaches, nausea and fever. This chronic malaria often triggers a cycle of poverty for sufferers left unable to work for long periods. By weakening the immune system, the disease contributes to death.

The Malaria Atlas Project estimates 2.5 billion people worldwide are at risk for P. vivax malaria.

P. vivax does not grow well in the laboratory, so to try to understand how the parasite lives and operates, the researchers gathered samples from malaria patients and focused on its genome.

They found a duplication of the Duffy binding protein in half of 189 P. vivax infection samples taken in Madagascar.  Other researchers’ prior efforts to sequence the P. vivax genome missed the duplication but all indications are it’s a recent change, Serre said.

“The way we date duplications is to compare differences between the two parts: the more different they are, the older they are,” he explained. “They accumulate mutations. The two parts of this duplication have, among 8,000 base pairs, only one difference.”

Often a second copy of a gene enables an organism to outmaneuver a defense, Serre continued. “Instead of making a supergene, a duplication is simpler for nature.”

The researchers suspect the mutation is spreading from Madagascar through travelers. They found the duplication in less than 10 percent of samples from Cambodia and Sudan.

The new components found on the P. vivax genome are two proteins that closely resemble binding proteins used by related malaria parasites to enter immature and mature red blood cells. Both were present in samples from Cambodia, Brazil, Mauritania and North Korea.

The new proteins were absent in a 2008 sequencing of P. vivax, which is used as a reference genome, suggesting the developments are recent.

“Binding proteins and receptors are locks and keys,” Zimmerman said. “If the parasite has one key and there’s one lock, you may be able to block that. But if it has more keys and there are more locks, there are multiple ways in.”

The researchers say the duplication may be a cause of the growing infections among Duffy negative people, but it’s too early to tell.

Zimmerman, Serre and colleagues aim to find the answer with the newly-funded research project. They’ll begin by studying blood samples taken from 1,500 patients at each of two locations in Madagascar.

They and colleagues have great concern that a loss of resistance to P. vivax infection will now enable the parasite to travel the 250 miles across the Mozambique Channel to Africa. There, falciparum malaria is wrecking havoc on a population that has for the most part lived P. vivax-free. In some regions of the continent, 100 percent of the population is Duffy negative.

The researchers will conduct similar studies on P. vivax carrying the new proteins, in samples taken from Asia, Africa and South America.

In addition to studying patients, they plan to study the mutated parasites in the lab. Parasites that live a day or two could have enough time to invade new blood cells, but not many. Brian Grimberg, assistant professor of international health at the Case Western Reserve School of Medicine, is developing a scanning process that will enable the team to look through millions of red blood cells in a few minutes and spot newly infected cells. They will test the parasites in Duffy negative and Duffy positive red cells.

Zimmerman and Serre believe the work could help lead to a vaccine—that’s the overall goal. The mechanisms P. vivax uses to attach and enter a cell could be targets.


How zinc starves lethal bacteria to stop infection

Contact: Dr Christopher McDevitt 61-449-823-946 University of Adelaide

Australian researchers have found that zinc can ‘starve’ one of the world’s most deadly bacteria by preventing its uptake of an essential metal.

The finding, by infectious disease researchers at the University of Adelaide and The University of Queensland, opens the way for further work to design antibacterial agents in the fight against Streptococcus pneumoniae.

Streptococcus pneumoniae is responsible for more than one million deaths a year, killing children, the elderly and other vulnerable people by causing pneumonia, meningitis, and other serious infectious diseases.

Published today in the journal Nature Chemical Biology, the researchers describe how zinc “jams shut” a protein transporter in the bacteria so that it cannot take up manganese, an essential metal that Streptococcus pneumoniae needs to be able to invade and cause disease in humans.

“It’s long been known that zinc plays an important role in the body’s ability to protect against bacterial infection, but this is the first time anyone has been able to show how zinc actually blocks an essential pathway causing the bacteria to starve,” says project leader Dr Christopher McDevitt, Research Fellow in the University of Adelaide’s Research Centre for Infectious Diseases.

“This work spans fields from chemistry and biochemistry to microbiology and immunology to see, at an atomic level of detail, how this transport protein is responsible for keeping the bacteria alive by scavenging one essential metal (manganese), but at the same time also makes the bacteria vulnerable to being killed by another metal (zinc),” says Professor Bostjan Kobe, Professor of Structural Biology at The University of Queensland.

The study reveals that the bacterial transporter (PsaBCA) uses a ‘spring-hammer’ mechanism to bind the metals. The difference in size between the two metals, manganese and zinc, causes the transporter to bind them in different ways. The smaller size of zinc means that when it binds to the transporter, the mechanism closes too tightly around the zinc, causing an essential spring in the protein to unwind too far, jamming it shut and blocking the transporter from being able to take up manganese.

“Without manganese, these bacteria can easily be cleared by the immune system,” says Dr McDevitt. “For the first time, we understand how these types of transporters function. With this new information we can start to design the next generation of antibacterial agents to target and block these essential transporters.”


The research has been funded by the Australian Research Council and the National Health and Medical Research Council.

Media Contact:

Dr Christopher McDevitt Research Fellow Research Centre for Infectious Diseases School of Molecular and Biomedical Science The University of Adelaide Mobile: +61 449 823 946

Professor Bostjan Kobe Professor of Structural Biology Australian Infectious Diseases Research Centre School of Chemistry & Molecular Biosciences The University of Queensland Phone : +61 7 3365 2132

Robyn Mills Media Officer The University of Adelaide Phone: +61 8 8313 6341 Mobile: +61 410 689 084 robyn.mills@

Video – Health Research Reports 9 SEP 2013

Arginine performs as well as established drugs for Diabetes
* American Scientific journal Enocrinology Sep 2013
Nutritional Supplements reduce hospital stays by 21%
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Sirtuin in the brain delays the process of aging
* Cell Metabolism Sep 2013
H1N2 influenza vaccine disables the bodies defense against H1N1 Swine flu
* Science Translational Medicine Aug 2013

Plant-Based Compound May Inhibit HIV Infection, Research Shows


Posted: July 19, 2013 at 5:00 am, Last Updated: July 23, 2013 at 6:49 am

By Michele McDonald

Yuntao Wu. Creative Services photo

Yuntao Wu. Creative Services photo

A compound found in soybeans may become an effective HIV treatment without the drug resistance issues faced by current therapies, according to new research by George Mason University researchers.

It’s in the early stages, but genistein, derived from soybeans and other plants, shows promise in inhibiting the HIV infection, says Yuntao Wu, a professor with the George Mason-based National Center for Biodefense and Infectious Diseases and the Department of Molecular and Microbiology.

Still, that doesn’t mean people should begin eating large amounts of soy products. “Although genistein is rich in several plants such as soybeans, it is still uncertain whether the amount of genistein we consume from eating soy is sufficient to inhibit HIV,” Wu says.

Genistein is a “tyrosine kinase inhibitor” that works by blocking the communication from a cell’s surface sensors to its interior. Found on a cell’s surface, these sensors tell the cell about its environment and also communicate with other cells. HIV uses some of these surface sensors to trick the cell to send signals inside. These signals change cell structure so that the virus can get inside and spread infection.

But genistein blocks the signal and stops HIV from finding a way inside the cell. It takes a different approach than the standard antiretroviral drug used to inhibit HIV.

“Instead of directly acting on the virus, genistein interferes with the cellular processes that are necessary for the virus to infect cells,” Wu says. “Thus, it makes the virus more difficult to become resistant to the drug. Our study is currently it its early stage. If clinically proven effective, genistein may be used as a complement treatment for HIV infection.”

Wu and researcher Jia Guo in the lab. Creative Services photo

Wu and researcher Jia Guo in the lab. Creative Services photo

Wu sees possibilities in this plant-based approach, which may address drug toxicity issues as well. Because genistein is plant-derived, it may be able to sidestep drug toxicity, a common byproduct of the daily and lifelong pharmaceutical regimen faced by patients with HIV to keep the disease at bay, Wu says. Typically, patients take a combination of multiple drugs to inhibit the virus. The frequency can lead to drug toxicity. Plus, HIV mutates and becomes drug-resistant.

Wu and his team are working at finding out how much genistein is needed to inhibit HIV. It’s possible that plants may not have high enough levels, so drugs would need to be developed, Wu says.

Wu’s research is feeling the financial squeeze these days due to sequestration and budget cuts within the National Institutes of Health, he says. His lab has turned to novel ways to fund the HIV research, including the genistein project. A bicycle ride dubbed NYC DC AIDS Research Ride raised money for Wu’s lab a few years ago and has stepped up its efforts with a new fundraiser.

Other George Mason researchers on the genistein project include Jia Guo, Taban Rasheed, Alyson Yoder, Dongyang Yu, Huizhi Liang, Fei Yi and Todd Hawley.Xuehua Xu and Tian Jin from the National Institute of Allergy and Infectious Diseases in Rockville, Md., and Binhua Ling from Tulane University Health Sciences Center are also working on the research.

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



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.


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:


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.


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).


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.


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].


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



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


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).


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.


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.


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):


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.


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


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.


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).


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.


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%.


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


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.


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.


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).


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.


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.


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.

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Figure S2.

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Figure S3.

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Figure S4.

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Figure S5.

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Figure S6.

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Figure S7.

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Figure S8.

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Figure S9.

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Figure S10.

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Figure S11.

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Text S1.

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Author Contributions

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


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Antibiotics have long-term impacts on gut flora/ Up to 2 years

2010 study posted for filing

Contact: Laura Udakis 44-118-988-1843 Society for General Microbiology

Short courses of antibiotics can leave normal gut bacteria harbouring antibiotic resistance genes for up to two years after treatment, say scientists writing in the latest issue of Microbiology, published on 3 November.

The researchers believe that this reservoir increases the chances of resistance genes being surrendered to pathogenic bacteria, aiding their survival and suggesting that the long-term effects of antibiotic therapy are more significant than previously thought.

Antibiotics that are prescribed to treat pathogenic bacteria also have an impact on the normal microbial flora of the human gut. Antibiotics can alter the composition of microbial populations (potentially leading to other illnesses) and allow micro-organisms that are naturally resistant to the antibiotic to flourish.

The impact of antibiotics on the normal gut flora has previously been thought to be short-term, with any disturbances being restored several weeks after treatment. However, the review into the long-term impacts of antibiotic therapy reveals that this is not always the case. Studies have shown that high levels of resistance genes can be detected in gut microbes after just 7 days of antibiotic treatment and that these genes remain present for up to two years even if the individual has taken no further antibiotics.

The consequences of this could be potentially life-threatening explained Dr Cecilia Jernberg from the Swedish Institute for Infectious Disease Control who conducted the review. “The long-term presence of resistance genes in human gut bacteria dramatically increases the probability of them being transferred to and exploited by harmful bacteria that pass through the gut. This could reduce the success of future antibiotic treatments and potentially lead to new strains of antibiotic-resistant bacteria.”

The review highlights the necessity of using antibiotics prudently. “Antibiotic resistance is not a new problem and there is a growing battle with multi-drug resistant strains of pathogenic bacteria. The development of new antibiotics is slow and so we must use the effective drugs we have left with care,” said Dr Jernberg. “This new information about the long-term impacts of antibiotics is of great importance to allow rational antibiotic administration guidelines to be put in place,” she said.

Viruses cooperate or conquer to cause maximum destruction: They Change Behaviour to overcome our attempt to control them

Contact: Louise Vennells 44-013-927-22062 University of Exeter

Scientists have discovered new evidence about the evolution of viruses, in work that will change our understanding about the control of infectious diseases such as winter flu

Scientists have discovered new evidence about the evolution of viruses, in work that will change our understanding about the control of infectious diseases such as winter flu.

Researchers at the University of Exeter’s conducted experiments to manipulate a virus to see if it could evolve the ability to switch its behaviour according to how many other viruses infect a host.

Previous research has focussed on trying to force harmful microbes to become less threatening to human health as they evolve. But the new research, which was carried out in collaboration with the University of Oxford, proves viruses can readily develop the ability to adjust their behaviour to maximise their spread, in response to whether they are infecting as a single entity or in combination with other viruses.

Helen Leggett, a postgraduate researcher at the University of Exeter, was the lead scientist on the work, which is published online on December 13th in the journal Current Biology. She said: “Scientists are constantly searching for ways to limit the damage viruses can cause, to help reduce the impact of illnesses like winter flu and to respond to the next pandemic. Our work proves that regardless of how we try to manipulate viruses, they will always switch their behaviour to serve their own purposes and kill as many cells as possible. This study involved a relatively simple virus. If it can evolve so quickly, it’s reasonable to assume that a lot of other viruses and parasites can, too.”

The study was funded by the European Research Council, the Leverhulme Trust and the Natural Environment Research Council, while Helen Leggett is supported by the Biotechnology and Biological Sciences Research Council. The work also shed light on why organisms cooperate with each other. The virus would only cooperate with viruses which were related to it. When it infected alone it would clone itself within the cell, and would cooperate with those new viruses. In this context, cooperation meant killing the host relatively slowly so that the virus could replicate more.  But when it interacted with other viruses which were not related, it killed the cell faster, allowing it to out-replicate and dominate the other viruses.

Homicide spreads like infectious disease

Contact(s): Andy Henion Media Communications office: (517) 355-3294 cell: (517) 281-6949, April Zeoli Criminal Justice office: (517) 353-9554

Homicide moves through a city in a process similar to infectious disease, according to a new study that may give police a new tool in tracking and ultimately preventing murders.

Using Newark, N.J., as a pilot case, a team of Michigan State University researchers led by April Zeoli successfully applied public health tracking methods to the city’s 2,366 homicides between 1982 and 2008. They found the killings were not randomly located but instead followed a pattern, evolving from the city’s center and moving southward and westward over time.

Like a flu bug that spreads to susceptible groups such as children and the elderly, homicide clusters in Newark – often fueled by gangs and guns – spread to areas consisting largely of poor and minority residents. Over time, the concentration of homicides effectively disappeared from one area and settled in another.

“By using the principles of infectious disease control, we may be able to predict the spread of homicide and reduce the incidence of this crime,” said Zeoli, public health researcher in MSU’s School of Criminal Justice.

The study is one of the first to use analytic software from the field of medical geography to track long-term homicide trends. Zeoli said the method can be done in real time which would allow police to identify emerging hotspots.

The researchers also identified areas of Newark that had no homicide clusters during the 26-year time frame of the study, despite being surrounded by deadly violence.

“If we could discover why some of those communities are resistant,” Zeoli said, “we could work on increasing the resistance of our communities that are more susceptible to homicide.”

Joining Zeoli on the study were criminal justice researchers Jesenia Pizarro and Christopher Melde and medical geographer Sue Grady.

The study is published in Justice Quarterly, a research journal.

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

Public Affairs News Service

Tuesday, Nov. 27, 2012

Writer: James  E.  Hataway, 706/542-5222, Contact: Biao He, 706/542-2855,

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

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

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

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

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

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

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

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

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

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

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

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

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

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

New DNA vaccine technology poised to deliver safe and cost-effective disease protection


Contact: Richard Harth
Arizona State University

New and increasingly sophisticated vaccines are taking aim at a broad range of disease-causing pathogens, targeting them with greater effectiveness at lower cost and with improved measures to ensure safety.

To advance this quest, a research team led by Roy Curtiss, director of the Center for Infectious Diseases and Vaccinology, and Wei Kong, a research assistant professor, at Arizona State University’s Biodesign Institute have taken a dramatic step forward, revealing the design of a universal platform for delivering highly potent DNA vaccines, by employing a cleverly re-engineered bacterium to speed delivery to host cells in the vaccine recipient.

“The technology that we’re describing in this paper can be used to develop a vaccine against any virus, any parasite, any fungus, whereas this was never possible before the development of recombinant attenuated bacterial strains like those produced in our lab,” Curtiss says.

The experimental vaccine described in the new research demonstrated complete protection from influenza in mice, but Wei Kong, the leading author of the new study stresses that the innovative technique could be applied to the rapid manufacture of effective vaccines against virtually any infectious invader at dramatically reduced cost and without risk to either those vaccinated or the wider public.

“By delivering the DNA vaccine using a recombinant attenuated bacterium, we can get 10,000-100,000 doses per liter of culture,” Kong says, an improvement of 3-4 orders of magnitude over use of the naked plasmid DNA, which must be painstakingly isolated from bacteria before injection.

The group’s research results appear in the online Early Edition (EE) of the Proceedings of the National Academy of Sciences, the week of November 5, 2012.


Designing a vaccine that is both safe and effective presents a kind of Catch-22 for researchers. Live pathogenic strains typically generate a robust immune response, mimicking natural infection, but many challenges exist in terms of ensuring such strains do not cause illness or escape into the environment, where they have the potential to remain viable. Killed pathogen strains or vaccines produced from pathogen subunits sacrifice some of their immunogenic effectiveness for enhanced safety, and may require subsequent booster doses to ensure continued effectiveness.

The Curtiss team has worked to combine safety and effectiveness in orally administered vaccines that can be produced at a fraction of the cost of traditional methods. To do this, they have pioneered techniques using Salmonella—the notorious agent associated with food-borne illness—as a cargo vessel to deliver a suite of disease antigens to the recipient. The result has been the development and ongoing refinement of so-called RASVs (for recombinant attenuated Salmonella vaccines), capable of provoking an intense, system-wide immune response and conferring effective immunity.

One of the key innovations developed earlier by Wei Kong and other members of the Curtiss group, is a specialized Salmonella strain that can be timed to self-destruct in the body once it has carried out its immunization duties. To create this strain, the researchers modified the bacterium in such a way that it can only survive on a non-naturally occurring form of sugar. Once the Salmonella cells exhaust their store of specialized sugar, supplied to them as part of the vaccine, they are unable to maintain the integrity of their cell walls and they essentially implode. “This crucial safety feature ensures that Salmonella are unable to persist as living organisms to survive if excreted into the environment,” says Kong.

This self-destruct feature can be fine-tuned so that the bacteria fully colonize host cells, provoking a strong response from both humoral and cell-mediated arms of the immune system. Inside host tissues, recombinant Salmonella are able to synthesize protective antigens, releasing their contents when they become unstable and lyse into the intracellular fluid or cytosol.

The group demonstrated the effectiveness of this delayed-lysis bacteria in vaccine experiments with a variety of pathogens, including influenza and mycobacteria (causative agent of tuberculosis) and an RASV vaccine developed in the Curtiss lab against infant pneumonia is currently in FDA Phase I clinical trials. This earlier work focused on producing protective protein antigens in a bacterium, which would subsequently release a bolus of these antigens when the bacterial cell lysed within host cells and tissues.

In the latest research, the group sought to turn a delayed-lysis Salmonella strain into a universal DNA vaccine delivery vehicle. DNA vaccines stimulate cellular and humoral immune responses to protein antigens through the direct introduction of genetic material, prompting host cells to manufacture specific gene products. This is a crucial advance as it allows for the production of antigens that undergo host cell modification through the addition carbohydrates—a process known as glycosylation. Such modified antigens, which occur in a broad range of pathogenic viruses, fungi and parasites require synthesis by host cells, rather than by the attenuated bacteria.

“Here, we were able to deliver a vaccine whose DNA sequence induces the immunized individual to make the protective glycoprotein the way you would during a viral infection,” Curtiss says. Previous efforts to achieve this advance for delivery of DNA vacines by bacteria date to 1995, but only now has such work come to fruition.

A number of key modifications to the delayed-lysis RASV were required for this feat, and the Kong and Curtiss team has worked intensively over the past 5 years to achieve them. A hyperinvasive form of Salmonella was constructed through recombinant DNA methods in order to maximize the vaccine vector’s ability to invade host cells and become internalized.

Following host cell uptake, Salmonella are encased in a membrane-bound endosome known as the Salmonella Containing Vacuole. The RASV was further modified to permit escape from the endosome so that the mature bacterium could spew its immunogenic contents into the host cell’s cytosol.

Finally, further revisions to the Salmonella strain were applied to diminish the pathogen’s ability to cause host cell death, which would prevent the DNA vaccine from migrating to the host cell nucleus to induce the synthesis of protective antigens necessary for the immune response.

The authors note that their orally-administered RASV is markedly superior to earlier efforts which introduced DNA vaccines by means of intramuscular injection or gene gun. These methods fail to deliver the vaccine to both mucosal tissues and certain internal lymphoid tissues, vital to a sustained, protective immunity. “We can protect mice to doses of influenza that would be lethal were they not effectively immunized,” Curtiss says, adding that “RASV safety has been established in mice just two hours old as well as in pregnant and immunodeficient mice”.

Influenza spreads around the world in seasonal epidemics, resulting in about three to five million yearly cases of severe illness and about 250,000 to 500,000 yearly deaths, rising to millions in some pandemic years. Current manufacture of influenza vaccines requires use of chick embryos or cell culture methods. Global capacity is limited, making sufficient vaccine to immunize everyone impossible. Adding to concerns about managing future naturally occurring influenza epidemics is the potential for bioterrorists to produce weaponized influenza strains created using plasmid-based reverse genetics systems. “Increasing the speed of producing a matching vaccine is key in the context of response to an influenza epidemic,” Kong says.

The ability to rapidly engineer and scale up effective vaccines for influenza and other potentially lethal pathogens will require innovative approaches to vaccine design, manufacture and application. The universal DNA vaccine platform outlined in the new study represents an important advance.

“The vast majority of viruses including influenza, measles, mumps and HIV all have glycosylated proteins. You could never deliver protective immunity using a bacterium to produce those protein antigens,” Curtiss says. “But now we have the opportunity to produce vaccines against such pathogens,” Kong says. Further, the technique permits large quantities of DNA vaccine to be produced rapidly at low cost, freeze-dried and stockpiled to be used when needed.


Dr Roy Curtiss is also a professor in the College of Liberal Arts and Sciences, School of Life Sciences

Written by: Richard Harth
Science Writer: The Biodesign Institute

Roy Curtiss, (480) 727-0445
Wei Kong, (480) 727-9591

Aspirin Misuse May Have Made 1918 Flu Pandemic Worse

2009 study posted for filing


The devastation of the 1918-1919 influenza pandemic is well known, but a new article suggests a surprising factor in the high death toll: the misuse of aspirin. Appearing in the November 1 issue of Clinical Infectious Diseases and available online now, the article sounds a cautionary note as present day concerns about the novel H1N1 virus run high.


High aspirin dosing levels used to treat patients during the 1918-1919 pandemic are now known to cause, in some cases, toxicity and a dangerous build up of fluid in the lungs, which may have contributed to the incidence and severity of symptoms, bacterial infections, and mortality. Additionally, autopsy reports from 1918 are consistent with what we know today about the dangers of aspirin toxicity, as well as the expected viral causes of death.


The motivation behind the improper use of aspirin is a cautionary tale, said author Karen Starko, MD. In 1918, physicians did not fully understand either the dosing or pharmacology of aspirin, yet they were willing to recommend it. Its use was promoted by the drug industry, endorsed by doctors wanting to “do something,” and accepted by families and institutions desperate for hope.


“Understanding these natural forces is important when considering choices in the future,” Dr. Starko said. “Interventions cut both ways. Medicines can save and improve our lives. Yet we must be ever mindful of the importance of dose, of balancing benefits and risks, and of the limitations of our studies.”

‘Dung of the devil’ plant roots point to new swine flu drugs: Showed greater potency against influenza A (H1N1) than a prescription antiviral drugs

2009 study posted for filing

Contact: Michael Woods 202-872-6293 American Chemical Society

Scientists in China have discovered that roots of a plant used a century ago during the great Spanish influenza pandemic contains substances with powerful effects in laboratory experiments in killing the H1N1 swine flu virus that now threatens the world. The plant has a pleasant onion-like taste when cooked, but when raw it has sap so foul-smelling that some call it the “Dung of the Devil” plant. Their report is scheduled for the Sept. 25 issue of ACS’ Journal of Natural Products, a monthly publication.

In the study, Fang-Rong Chang and Yang-Chang Wu and colleagues note that the plant, Ferula assa-foetida, grows mainly in Iran, Afghanistan and mainland China. People used it as a possible remedy during the1918 Spanish flu pandemic that killed between 20 to 100 million people. Until now, however, nobody had determined whether the plant does produce natural antiviral compounds.

Chang and Wu identified a group of chemical compounds in extracts of the plant that showed greater potency against influenza A (H1N1) than a prescription antiviral drug available for the flu. “Overall, the present study has determined that sesquiterpene coumarins from F. assa-foetida may serve as promising lead components for new drug development against influenza A (H1N1) viral infection,” the authors write.



ARTICLE #1 FOR IMMEDIATE RELEASE “Influenza A (H1N1) Antiviral and Cytotoxic Agents from Ferula assa-foetida”


CONTACT: Fang-Rong Chang, Ph.D. Yang-Chang Wu, Ph.D. Kaohsiung Medical University. Kaohsiung 807, Taiwan, Republic of China Phone: 886-7-312-1101, ext. 2197 Fax: 886-7-311-4773 E-mail: or

No Antibodies, No Problem



Researchers Identify How Mosquito Immune System Attacks Specific Infections


Researchers at the Johns Hopkins Bloomberg School of Public Health have determined a new mechanism by which the mosquitoes’ immune system can respond with specificity to infections with various pathogens, including the parasite that causes malaria in humans, using one single gene. Unlike humans and other animals, insects do not make antibodies to target specific infections. According to the Johns Hopkins researchers, mosquitoes use a mechanism known as alternative splicing to arrange different combinations of binding domains, encoded by the same AgDscam gene, into protein repertoires that are specific for different invading pathogens. The researchers’ findings were published October 18 in the journal Cell Host & Microbe and could lead to new ways to prevent the spread of a variety of mosquito born illnesses.


Mosquitoes and other insects use their primitive innate immune systems to successfully fight infections with a broad spectrum of viruses, bacteria, fungi and parasites, despite the lack of antibodies that are part of the more sophisticated human immune system. The effectiveness of the human immune system is to a large degree based on the ability to produce an enormous variety of antibodies containing different immunoglobulin domains that can specifically tag and label a pathogen for destruction. This great variety of pathogen-binding antibodies is achieved by combining different immunoglobulin gene segments and further mutate them through mechanisms called somatic recombination and hypermutation. While mosquitoes also have genes encoding immunoglobulin domains, they lack these specific mechanisms to achieve pathogen recognition diversity.


The Johns Hopkins researchers discovered a different way by which mosquitoes can combine immunoglobulin domains of a single gene called AgDscam (Anopheles gambiae Down Syndrome Cell Adhesion Molecule) to produce a variety of pathogen-binding proteins. The AgDscam gene is subjected to a mechanism called alternative splicing that combines different immunoglobulin domains into mature AgDscam proteins, depending on which pathogen has infected the mosquito. The researchers showed that this alternative splicing is guided by the immune signal transducing pathways (analogous to electrical circuits) that they previously demonstrated to activate defenses against different malaria parasites and other pathogens. While alternative splicing of the AgDscam gene does not nearly achieve the degree of pathogen recognition diversity of human antibodies, it does nevertheless vastly increase the variety of pathogen binding molecules.


“Using antibodies to fight infection is like fishing with a harpoon—it’s very targeted. The mosquito’s innate immune system is more like fishing with a net—it catches a bit of everything,” explained George Dimopoulos, PhD, senior investigator of the study and professor with the Johns Hopkins Malaria Research Institute. “However, we discovered that immune pathway-guided alternative splicing of the AgDscam gene renders the mosquito’s immune net, so to speak, more specific than previously suspected. The mosquito’s immune system can come up with approximately 32,000 AgDscam protein combinations to target infections with greater specificity.”


Dimopoulos and his group are developing a malaria control strategy based on mosquitoes that have been genetically modified to possess an enhanced immune defense against the malaria parasite Plasmodium. One obstacle to this approach is the great variety of Plasmodium strains that may interact somewhat differently with the mosquito’s immune system.


“Some of these strains may not be detected by the engineered immune system proteins that mediate their killing. Our new discovery may provide the means to create genetically modified mosquitoes that can target a broader variety of parasite strains, like casting a net rather than shooting with a harpoon,” said Dimopoulos.


Malaria kills more than 800,000 people worldwide each year. Many are children.


“Anopheles NF-kB –Regulated Splicing Factors Direct Pathogen-Specific Repertoires of the Hypervariable Pattern Recognition Receptor AgDscam” was written by Yuemei Dong, Chris M. Cirimotich, Andrew Pike, Ramesh Chandra and George Dimopoulos.


The research was supported by grants from the National Institutes of Health/National Institute of Allergy and Infectious Disease, the Calvin A. and Helen H. Lang Fellowship, and the Johns Hopkins Malaria Research Institute.


Media contact: Tim Parsons, director of Public Affairs, at 410-955-7619 or





Survivors of 1918 flu pandemic protected with a lifetime immunity to virus

Contact: Mount Sinai Newsroom
The Mount Sinai Hospital / Mount Sinai School of Medicine

New research has discovered that infection and natural exposure to the 1918 influenza virus made survivors immune to the disease for the remaining of their lives. Antibodies produced by cells isolated from these survivors served as an effective therapy to protect mice from the highly lethal 1918 infection. The study entitled “Neutralizing antibodies derived from the B cells of 1918 influenza pandemic survivors,” was released for advanced online publication by the journal Nature. Researchers at Mount Sinai School of Medicine’s Department of Microbiology contributed to the research findings. An estimated 50 million people were killed by the 1918 flu pandemic worldwide.

“Ninety years after survivors encountered the 1918 pandemic influenza virus, we collected antibody-producing B cells from them, and successfully isolated B cells that produce antibodies that block the viral infection,” said contributing author Dr. Christopher Basler, PhD, Associate Professor of Microbiology at Mount Sinai School of Medicine. “The antibodies produced by these cells demonstrated remarkable power to block 1918 flu virus infection in mice, proving that, even nine decades after infection with this virus, survivors retain protection from it.”

“The fact that you can isolate these anti-1918 memory B cells so long after infection will hopefully provide the impetus to further study the mechanisms behind long lived immunity,” said Dr. Osvaldo Martinez, post-doctoral fellow at Mount Sinai School of Medicine.

For this study, 32 individuals who were born before 1918 and lived through the influenza pandemic were recruited by Dr. Eric Altschuler at the University of Medicine and Dentistry of New Jersey to donate blood which was tested by Dr. Basler’s lab for the presence of antibodies that recognize the 1918 virus. Dr. James Crowe and colleagues at Vanderbilt University produced antibodies from these individuals’ blood cells and provided these to Dr. Basler’s lab where the potent neutralizing activity against 1918 virus was demonstrated. Antibodies were also provided to Dr. Terrence Tumpey at the CDC to test in mice the strength of the antibodies derived from the 1918 survivors.

“Our findings show that survivors of the pandemic have highly effective, virus neutralizing antibodies to this powerful virus, and humans can sustain circulating B memory cells to viruses for up to 9 decades after exposure,” said Dr. Tshidi Tsibane, post-doctoral fellow, Department of Microbiology, Mount Sinai School of Medicine. “These findings could serve as potential therapy for another 1918-like virus.”


Vanderbilt University, Mount Sinai School of Medicine, University of Medicine and Dentistry of New Jersey, Centers for Disease Control and Prevention and The Scripps Research Institute collaborated on this research study.

About The Mount Sinai Medical Center The Mount Sinai Medical Center encompasses The Mount Sinai Hospital and Mount Sinai School of Medicine. The Mount Sinai Hospital is one of the nation’s oldest, largest and most-respected voluntary hospitals. Founded in 1852, Mount Sinai today is a 1,171-bed tertiary-care teaching facility that is internationally acclaimed for excellence in clinical care. Last year, nearly 50,000 people were treated at Mount Sinai as inpatients, and there were nearly 450,000 outpatient visits to the Medical Center.

Mount Sinai School of Medicine is internationally recognized as a leader in groundbreaking clinical and basic-science research, as well as having an innovative approach to medical education. With a faculty of more than 3,400 in 38 clinical and basic science departments and centers, Mount Sinai ranks among the top 20 medical schools in receipt of National Institute of Health (NIH) grants

Mutation causes defective Natural Killer cells


Natural Killer (NK) cells defend the body against infectious diseases and cancer by recognizing and killing stressed or infected cells and patients with NK deficiencies are susceptible to severe viral infections. In this issue of the Journal of Clinical Investigation, researchers at Baylor College of Medicine report on a patient with an NK cell deficiency caused by a mutation in CD16, which codes for a protein on the surface of NK cells that recognizes antibodies. To determine the exact role of CD16 in NK cell cytotoxicity, Jordan Orange and colleagues studied the effect of mutant CD16 in a human NK cell line. The mutant CD16 was unable to interact with another NK cell protein, CD2, which is required for cytotoxic activity in NK cells. Patients carrying this mutation were highly susceptible to viral infection. This study identifies a potential cellular mechanism that underlies human congenital immunodeficiency.


Human immunodeficiency-causing mutation defines CD16 in spontaneous NK cell cytotoxicity


Jordan Orange

Baylor College of Medicine, Houston, TX, USA

Phone: 832-824-1319; E-mail:

View this article at:

Pain drug can kill resistant tuberculosis: Researchers claim may never be tested in TB clinical trials.

Public release date: 10-Sep-2012
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Contact: Lauren Woods
New York- Presbyterian Hospital/Weill Cornell Medical Center/Weill Cornell Medical College

Researchers find low cost drug wipes out drug resistant TB, but worry it may not reach patients in need

NEW YORK (September 10, 2012) — An off-patent anti-inflammatory drug that costs around two cents for a daily dose in developing countries has been found by researchers at Weill Cornell Medical College to kill both replicating and non-replicating drug resistant tuberculosis in the laboratory — a feat few currently approved TB drugs can do, and resistance to those is spreading.

Their findings, published online by the journal PNAS, point to a potential new therapy for the more than 500,000 people worldwide whose TB has become resistant to standard drug treatments. But the researchers worry that the effective drug, oxyphenbutazone, may never be tested in TB clinical trials.

Weill Cornell’s Dr. Carl Nathan and his research team found what they call the “completely surprising” ability of oxyphenbutazone to kill drug resistant TB after testing thousands of approved drugs against the bacteria. This repurposing of agents already on the market can lead to quicker testing for new uses.

“This agent might help save lives if there was a way to test it in TB patients,” says Dr. Nathan. Oxyphenbutazone went on the market as a patented drug for arthritis-like pain in the early 1950s, and lost its patent and market dominance by the 1970s.

“It is difficult today to launch clinical studies on a medication that is so outdated in the United States, that it is mainly used here in veterinary medicine to ease pain,” says the study’s senior author, Dr. Nathan, chairman of the Department of Microbiology and Immunology, the R.A. Rees Pritchett Professor of Microbiology, and the director of The Abby and Howard Milstein Program in the Chemical Biology of Infectious Disease at Weill Cornell. “No drug firm will pay for clinical trials if they don’t expect to make a profit on the agent. And that would be the case for an off-patent drug that people can buy over the counter for pain in most of the world.”

He adds that oxyphenbutazone, best known under the trademark name of Tandearil, does have some established toxicities, “and is not a drug you should take for aches and pains if a safer alternative is available.” But the drug’s major toxicities appear to be less frequent than the major side-effects of the drug regimens that are currently used to treat TB, he says.

Treating the TB that Hides

Mycobacterium tuberculosis is unusual among disease-causing bacteria in that it naturally infects just humans. One-third of the world’s population is infected with TB, but the bacteria typically remain dormant in a person with a healthy immune system.

Nonetheless, TB becomes active in enough people that it is the leading cause of death in humans from a bacterial infection. It is difficult to treat, and the bacteria can become resistant to therapy. TB treatment in a drug-sensitive patient takes six months, using a combination of agents. If the TB is sensitive to these first-line agents and the therapy is completed with full-strength, non-counterfeit drugs, up to 95 percent of patients can be cured.

However, if a patient’s TB becomes resistant to these drugs, second-line agents are administered every day for two years or more. “These second-line drugs are often toxic and expensive, and are not readily available in developing countries, where most of the infections occur,” Dr. Nathan says. Mortality in drug resistant TB patients can be as high as 80 percent.

A major issue in treating TB is that the bacteria can “hide out” in the body in a non-replicating form, even when a TB patient is undergoing treatment.

To find agents that could attack non-replicating TB, Dr. Nathan’s research team first identified four conditions that keep bacteria in that state within the human body: low oxygen, mild acidity, a fat instead of sugar to eat and a small amount of the natural defense molecule nitric oxide.

The research team replicated those conditions in the test tube and then methodically tested the effectiveness of thousands of agents against the bacteria. After testing 5,600 drugs, researchers found oxyphenbutazone.

Researchers then delved into the mechanism by which oxyphenbutazone kills TB and found that the conditions that allow the bacterium to remain dormant modify the drug to the point that it starts reacting against both non-replicating and replicating TB. “When this happens, TB can’t defend itself and dies,” Dr. Nathan says.

But the researchers were unable to test oxyphenbutazone in mice, because the animals metabolize the drug to an inactive form far faster than humans.

“This makes testing the drug for TB use in humans problematic since the FDA requires preclinical animal testing studies for safety and efficacy,” Dr. Nathan says. “Yet there is a long track record of oxyphenbutazone’s relatively safe use in hundreds of thousands of people over decades.”

Dr. Nathan and his team are continuing their research, testing hundreds of thousands of compounds for their action against TB. His team has already found another approved drug, nitazoxanide, to be effective against the bacteria, publishing his findings in 2009.

Nitazoxanide, a drug with an excellent safety record, is still on patent for use against some infections caused by other microbes. Discussions have been held about testing it in TB, Dr. Nathan says, but have stalled because of the same problem as oxyphenbutazone. The drug is metabolized so quickly in mice that it cannot be tested against experimental TB in that species.

For both oxyphenbutazone and nitazoxanide, Dr. Nathan argues that the requirement for testing in animals with experimental TB should be waived, because these agents work against TB in the test tube, have already been used with relative safety in people and might address an urgent need for treatment of a contagious disease with high mortality and few other treatment options.


This research was supported by the Tuberculosis Drug Accelerator Program of the Bill and Melinda Gates Foundation and the Abby and Howard P. Milstein Program in Chemical Biology of Infectious Disease.

Co-authors of the study include: Dr. Ben Gold, Dr. Maneesh Pingle, Julia Roberts, Dr. Mark Rundell, Dr. Thulasi Warrier, Dr. Aditya Venugopal, Dr. Crystal Darby, Xiuju Jiang, Dr. J. David Warren, Amy Cunningham-Bussel, Poonam Rath, Tamutenda Chidawanyika, Dr. Selin Somersan and Dr. W. Clay Bracken from Weill Cornell; Dr. Steven J. Brickner of S. J. Brickner Consulting; Dr. Ouathek Ouerfelli and Dr. Nilesh Shah from Memorial Sloan–Kettering Cancer Center; Dr. Eric L. Nuermberger from Johns Hopkins Hospital; and Dr. Joseph Fernandez, Ronald Realubit, Dr. J. Fraser Glickman, and Dr. Haiteng Deng from The Rockefeller University.

Weill Cornell Medical College

Weill Cornell Medical College, Cornell University’s medical school located in New York City, is committed to excellence in research, teaching, patient care and the advancement of the art and science of medicine, locally, nationally and globally. Physicians and scientists of Weill Cornell Medical College are engaged in cutting-edge research from bench to bedside, aimed at unlocking mysteries of the human body in health and sickness and toward developing new treatments and prevention strategies. In its commitment to global health and education, Weill Cornell has a strong presence in places such as Qatar, Tanzania, Haiti, Brazil, Austria and Turkey. Through the historic Weill Cornell Medical College in Qatar, the Medical College is the first in the U.S. to offer its M.D. degree overseas. Weill Cornell is the birthplace of many medical advances — including the development of the Pap test for cervical cancer, the synthesis of penicillin, the first successful embryo-biopsy pregnancy and birth in the U.S., the first clinical trial of gene therapy for Parkinson’s disease, and most recently, the world’s first successful use of deep brain stimulation to treat a minimally conscious brain-injured patient. Weill Cornell Medical College is affiliated with NewYork-Presbyterian Hospital, where its faculty provides comprehensive patient care at NewYork-Presbyterian Hospital/Weill Cornell Medical Center. The Medical College is also affiliated with the Methodist Hospital in Houston. For more information, visit

Reconstructed 1918 influenza virus has yielded key insights, scientists say

Contact: Nalini Padmanabhan
NIH/National Institute of Allergy and Infectious Diseases



The genetic sequencing and reconstruction of the 1918 influenza virus that killed 50 million people worldwide have advanced scientists’ understanding of influenza biology and yielded important information on how to prevent and control future pandemics, according to a new commentary by scientists at the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health, and several other institutions.

By sequencing the 1918 virus, researchers were able to confirm that the viruses that caused influenza pandemics in 1957, 1968, and 2009 were all descended in part from the 1918 virus. Studies showed that the 2009 pandemic virus had structural similarities with the 1918 virus and explained why younger people, who had never been exposed to the 1918 virus or its early descendants, were most vulnerable to infection by the 2009 influenza virus. As a result, public health officials were able to target limited vaccine supplies to predominantly younger people, who needed vaccine protection most, rather than the elderly, who were at lower risk of infection in 2009, but are traditionally the most important target group for vaccination. Further, determining the physical structure of parts of the 1918 virus, particularly the portions that are consistent across influenza viruses, has informed the ongoing development of candidate “universal” influenza vaccines that may be given infrequently yet protect broadly against multiple influenza viruses. In addition, by comparing the 1918 virus to related influenza viruses found in animals, scientists have learned some of the changes necessary for influenza viruses to adapt from an animal to a human host. This has led to more targeted surveillance of certain influenza viruses in animals that may be more likely to move to humans.

More generally, the authors say that reconstruction of the 1918 influenza virus has furthered scientific understanding of how novel influenza viruses emerge and evolve. Additionally, study of the 1918 influenza virus has helped clarify the critical effects of the human immune system’s response to viral infection and the importance of bacterial co-infections that often follow the influenza infection. In sum, the authors write, learning more about the 1918 pandemic influenza virus has led to important insights that could help prevent or mitigate seasonal and pandemic influenza.




JK Taubenberger et al. Reconstruction of the 1918 influenza virus: Unexpected rewards from the past. mBio DOI: 10.1128/mBio.00201-12 (2012).



Study co-authors Jeffery K. Taubenberger, M.D., Ph.D., section chief in NIAID’s Laboratory of Infectious Diseases; and David M. Morens, M.D., senior advisor to the NIAID Director, are available to discuss this study.



To schedule interviews, please contact Nalini Padmanabhan, (301) 402-1663,

For more information about NIAID’s research involving the 1918 influenza virus, see NIAID’s web page on the History of the 1918 Pandemic (

NIAID conducts and supports research—at NIH, throughout the United States, and worldwide—to study the causes of infectious and immune-mediated diseases, and to develop better means of preventing, diagnosing and treating these illnesses. News releases, fact sheets and other NIAID-related materials are available on the NIAID Web site at

About the National Institutes of Health (NIH): NIH, the nation’s medical research agency, includes 27 Institutes and Centers and is a component of the U.S. Department of Health and Human Services. NIH is the primary federal agency conducting and supporting basic, clinical, and translational medical research, and is investigating the causes, treatments, and cures for both common and rare diseases. For more information about NIH and its programs, visit

NIH…Turning Discovery Into Health

Pheromone from the summer cypress Killed Mosquitoes (West Nile virus) in trials Everytime

*Reposted at Request, data known since 1999….  The government will not justify the expense in regards to human life and the environment…Engineering Evil

Contact: Claire Bowles 44-171-331-2751 New Scientist

A burning bush could smite New York’s mosquitoes

An ornamental bush prized by gardeners in Europe and the US contains a surprise weapon against the mosquitoes spreading West Nile virus, the brain disease that has broken out in New York (p 13). The bush might also provide a cheap way for the world’s poorest countries to fight filariasis, a disfiguring parasitic disease that affects 15 million people in Africa alone.

Oil from the summer cypress, better known as the burning bush because it blushes a deep red in autumn, contains a substance which, when converted into a pheromone, can be used to lure mosquitoes to their deaths.

New Yorkers could have used this trick to combat the mosquitoes spreading the West Nile virus. The virus attacks the central nervous system and 3 of the 12 people with the illness have already died. Another 102 suspectedcases are being investigated. New York health officials are fighting the outbreak by killing mosquitoes with conventional sprays of the powerful insecticides malathion and pyrethroid.

Burning bush oil might have provided a more benign solution. Researchers from Britain and Nigeria have turned oil from the plant’s seeds into a pheromone. This biological messenger, which is odourless to humans, could lure female mosquitoes away from the places where people live.

A team from the Rothamsted site of the Institute of Arable Crops Research in Harpenden, Hertfordshire, struck lucky when team leader John Pickett noticed that the burning bush makes a fatty acid strikingly similar to the pheromone that attracts the mosquitoes (Journal of Agricultural Food Chemistry, vol 47, p 3411).

The pheromone is produced naturally when rafts of eggs are laid in stagnant water by Culex quinquefasciatus, the mosquito species which spreads filariasis and is suspected of spreading West Nile virus. “It’s a cue for female mosquitoes, telling them that there’s a safe place where they can lay their eggs,” says Mike Birkett, a member of the Rothamsted team.

The pheromone from the burning bush could have been used in New York to lure mosquitoes to their death in drums of water laced with pesticide, says Birkett. Pickett is confident that the pheromone attracts females of all Culex species.

The team at Rothamsted has tested synthetic versions of the same pheromone, called (5R,6S)-6-acetoxy-5-hexadecanolide. “It’s been tested in field trials in Africa, Japan and the US, and it works every time,” says Birkett. The problem is that the synthetic version is too expensive to manufacture. So instead, the team screened plants for substances which could be turned into the pheromone much more cheaply.

It doesn’t need to be purified from the oil, making it cheap and easy to handle. When Jenny Mordue of the University of Aberdeen tested the oil-based pheromone in the laboratory, she found that it attracted female mosquitoes as powerfully as the natural substance.

###Author: Andy Coghlan

Issue 2nd October 99


Study illuminates how the plague bacteria causes disease

Contact: Heidi Hardman 617-397-2879 Cell Press

The bacteria responsible for the plague and some forms of food poisoning “paralyze” the immune system of their hosts in an unexpected way, according to a new study in the September 8, 2006 issue of the journal Cell, published by Cell Press.

The researchers found that these bacteria, which belong to the genus Yersinia, harbor a protein that mimics an apparently unrelated mammalian enzyme. That copycat protein blocks host cells’ capacity to change shape and move, abilities important for cells of the immune system to track down and “eat” foreign invaders, the researchers explained.

The discovery marks the second way in which this protein, called YpkA, compromises the immune system. Earlier studies suggested that another portion of YpkA–which may have been derived from a mammalian enzyme and later co-opted by Yersinia–has activity that also influences cell shape by a separate, though incompletely understood mechanism.

The findings offer important new insight into the factors that lend Yersinia their ability to spawn disease, the researchers said. The results might also contribute to new strategies for fighting the bug.

“Yersinia injects several virulence factors into its host,” said C. Erec Stebbins of Rockefeller University. “If we can discover which ones are critical, we might identify the pathogen’s Achilles heel–an attractive target for antibacterial or anti-virulence compounds.”

“We were quite excited to see such a critical and unexpected factor in the virulence of Yersinia–a bacteria historically responsible for some of the worst diseases,” he added. Although improvements in sanitation have eliminated acute problems from diseases caused by Yersinia, concerns remain about the possibility that an untreatable strain might arise or that the bacteria might come into use as a biological weapon, he said.

Nearly 200 million people are estimated to have died in the plague epidemics that devastated the ancient world, the researchers said. The successful weaponization of plague in the former Soviet Union bioweapons program also made the pathogen a primary biodefense concern. Additional medical concerns have arisen from the evolution of multidrug-resistant strains of the plague bacterium found in patients from several locations.

The plague bacterium Yersinia pestis is closely related to Y. enterocolitica and Y. pseudotuberculosis, which are food-borne agents that cause inflammation of the stomach and intestines. All Yersinia bacteria have a virulence plasmid, which is necessary to cause disease. Plasmids are extra DNA molecules frequently found in bacteria containing genes that can be passed from one bacterial strain to another and that may confer an evolutionary advantage, such as antibiotic resistance.

In the case of Yersinia, the plasmid harbors numerous genes, including a large number that contribute to the ability of diverse pathogens to deliver virulence factors into host cells. One of these genes is YpkA, a protein with multiple domains, including one closely related to an enzyme, a type of kinase, not typically found in bacteria. Earlier studies found that mutations that eliminate this activity reduce but do not eliminate YpkA’s ability to disrupt cell shape by modifying their cytoskeletal support system.

In the current study, the researchers solved the high-resolution crystal structure of a second YpkA domain, the “Rho-GTPase binding domain” along with the host protein, “Rac1,” with which it interacts.

“The Yersinia structure was doing things to Rac1 that the host proteins normally do,” Stebbins said, suggesting that the domain acted as a mimic.

Further examination confirmed the domain to be a mimic of mammalian “guanidine nucleotide dissociation inhibitor” (GDI) proteins with a critical role in the bacteria’s ability to disrupt cell structure. The domain paralyzes cells by acting as an “off-switch” for host proteins involved in modifying cell shape, Stebbins said.

Mutations that prevented the bacterial proteins’ interaction with the host protein significantly impaired YpkA’s ability to disrupt the cytoskeleton. Moreover, a mutant strain of Y. pseudotuberculosis that lacked the GDI activity caused significantly fewer problems for infected mice compared to normal bacteria.

“Earlier studies that focused only on the protein’s kinase activity had missed half the picture,” Stebbins said. “The GDI domain seems to have an even bigger effect on host cells in culture, and a significant impact on virulence.”

The results also add to broader themes in the evolution of bacterial diseases, the researchers added.

“It is becoming increasingly clear that a common strategy used by bacterial pathogens to manipulate host cell biology is the mimicry of their own biochemical processes,” Stebbins said.


The researchers include Gerd Prehna and C. Erec Stebbins of Rockefeller University in New York, NY; Maya I. Ivanov and James B. Bliska and of Stony Brook University in Stony Brook, NY.

This work was funded in part by research funds to C.E.S. from the Rockefeller University and PHS grants 1U19AI056510 (to C.E.S) and RO1AI433890 (to J.B.B) from the National Institute of Allergy and Infectious Diseases.

Prehna et al.: “Virulence in Yersinia Is Dependent on a Bacterial Mimic of Host Rho-Family Nucleotide Dissociation Inhibitors.” Publishing in Cell 126, 869–880, September 8, 2006. DOI 10.1016/j.cell.2006.06.056

Repost at Request 2006

Smart bio-weapons are now possible

* Repost for Filing

David Hears The Guardian,  Tuesday 20 May 2003 10.41 EDT

Viruses and bacteria could be genetically engineered to evade the human immune system, to create a more effective biological weapon, a leading researcher into bio-weapons said yesterday.

In the past 30 years biotechnology has been revolutionised by molecular biology and genetic engineering. These techniques, used to control infectious diseases, can also be used to create more effective biological weapons.

Speaking at the conference on the future of weaponry, Professor Kathryn Nixdorff, of the University of Darmstadt, said that dangerous micro-organisms had already been produced inadvertently during attempts to modify vaccines and viruses.

Russian researchers had created a strain of anthrax bacilli capable of evading immune mechanisms: hamsters injected with the engineered strain were not protected by the usual anthrax vaccine.

Australian researchers trying to develop a vaccine to prevent pregnancy in mice stumbled upon a new and more virulent form of mousepox virus which inhibited the production of a class of lymphocytes needed to combat the infection.

Although humans were not susceptible to infection by mousepox virus there was concern that the human pox virus could be similarly manipulated to make it more deadly.

There were several ways in which modifying micro-organisms had potential military use. Bugs could be given a resistance to antibiotics, they could be made more resistant to the environment and thus longer lasting, and they could be made more lethal.

But she dismissed the suggestion that information gained from the sequencing of the human genome could be used to create a biological weapon specific to a particular racial or ethnic group.

“At present this seems unlikely for several reasons,” she said. “It has been pointed out in several reports that races do not exist from a genetic perspective; there is generally more genetic variation within groups than between groups.

“Indeed, it has been suggested that a re-examination of the race concept is due.”

There was concern that the genome sequence information could be misused. A research team was reported to have built the polio virus from sequence information publicly available, but this was a relatively simple virus and the feat could not be readily repeated with more complex ones

Q Fever microbe’s genome is deciphered

Study sheds light on potential bioterror agent, Coxiella burnetii

Rockville, MD — Scientists at The Institute for Genomic Research (TIGR) and their collaborators have deciphered and analyzed the complete genome sequence of Coxiella burnetii, a potential bioterror agent that causes Q Fever.

C. burnetii, which was first isolated as the cause of Q Fever in Australia in 1937, is typically found in farm animals but also infects humans, including an epidemic that sickened many soldiers in Europe during World War II.  Typically, Q Fever does not kill people, but causes fever and other flu-like symptoms.

Because C. burnetii is an obligate intracellular pathogen – unable to replicate unless it is inside the cells of another living organism — it is difficult to study in laboratories. That makes the complete DNA sequence an extremely valuable resource for researchers.

“The genome sequence offers a treasure trove of information that will allow scientists to develop a much higher-resolution picture of Coxiella’s biology and its ability to cause disease,” says John F. Heidelberg, the TIGR scientist who supervised the project.

The paper will be published online this week by the Proceedings of the National Academy of Sciences (PNAS), and will appear in the journal’s April 29 issue. The sequencing project was supported by the Defense Advanced Research Projects Agency (DARPA) and the National Institute of Allergy and Infectious Diseases (NIAID).

The TIGR study found that the C. burnetii genome appears to be in the early stages of “reduction” – a process during which degraded or non-functional genes are slowly eliminated as the organism becomes more dependent on its host for nutrition. Even so, scientists say, the Q Fever microbe does not appear to be quite as dependent on its human or animal host as other intracellular pathogens that cause leprosy, chlamydia, typhus, and Legionnaire’s Disease.

“This may mean that Coxiella became an intracellular pathogen more recently than other, somewhat similar pathogens,” says Rekha Seshadri, the TIGR staff scientist who is the lead author of the PNAS paper. “Comparing Coxiella with the genomes of other intracellular pathogens reveals fundamental differences in the organization and evolution of their chromosomes, as well as in their strategies for surviving inside host cells.”

The analysis found numerous genes that appear to be involved in the pathogen’s virulence and interactions with its host.  In addition, Seshadri says, researchers found that the C. burnetii genome – which includes multiple “mobile elements” of DNA – seems to be less stable than that of other obligate intracellular pathogens such as Rickettsia and Chlamydia. The evidence of mobile elements and gene degradation suggests greater genome flux.

The study was led by TIGR researchers in collaboration with other scientists, including James E. Samuel of the Department of Medical Microbiology and Immunology at Texas A&M University System Health Science Center, in College Station, TX; Herbert A. Thompson of the Centers for Disease Control and Prevention (CDC) in Atlanta, GA; and Robert A. Heinzen of the NIAID’s Rocky Mountain Laboratories in Hamilton, MT.

“The availability of the genome sequence for Coxiella burnetii will provide a quantum leap to the investigators engaged in understanding the biology of this pathogen,” said Samuel of Texas A&M. “The most immediate application of the genome sequence will be testing of the predicted essential genes for Coxiella’s survival.”

Samuel said the genome sequence – coupled with NIAID’s heightened emphasis on vaccine development for Q Fever and other Category B potential bioterror agents – is likely to accelerate the development of better vaccines and diagnostic tests using genomic and proteomic approaches.

The CDC’s Thompson, co-editor of the book Q Fever: The Biology of Coxiella burnetii, says, “Q Fever research on all fronts will benefit from this new knowledge.” Over the last several years, he says, “consideration for both the organism Coxiella and its disease manifestations have been neglected” – mainly because of the difficulties involved with studying and manipulating the organism in the lab. “Knowledge of the genome should significantly aid our inquiries and guide us as we attempt to remedy these shortcomings.”

Heinzen, of NIAID’s Rocky Mountain Laboratory, says that having Coxiella’s complete genome sequence “is a major advance that will allow scientists to more easily conduct functional studies of genes potentially involved in virulence.”  He said the pathogen interests scientists because it has “an amazing ability to survive in the environment and to resist degradation by its macrophage host cell.”

TIGR, which in 1995 published the first complete genome sequence of a free-living organism, is a world leader in microbial genomics. The institute has completed the full sequences of about 50 organisms or microbial strains – including the potential bioterror agents Bacillus anthracis and Brucella suis – and is now working on numerous other sequencing projects.

C. burnetii is listed as a potential bioweapon/bioterror agent because it is highly infective, relatively stable, and because Q Fever’s flu-like disease symptoms make early diagnosis difficult. During the 1950s and 1960s, the U.S. military conducted research into the potential use of C. burnetii as a biowarfare agent. But that stockpile was destroyed after the U.S. government halted its biowarfare program in 1969. The former Soviet Union also conducted Q Fever bioweapons research during

* Reposted for Filing

New salmonella-based ‘clean vaccines’ aid the fight against infectious disease:To accomplish this, a recombinant strain of Salmonella was constructed using genes from another pathogen, Francisella tularensis

* They are using genes from tularensis ” inhaling as few as 10 bacteria could be potentially deadly ” I feel uncomfortable with the Gates foundation funding support utilizing a Bioweapon strain of  Rabbit Fever?


New salmonella-based ‘clean vaccines’ aid the fight against infectious disease

A powerful new class of therapeutics, known as recombinant attenuated Salmonella vaccines (RASV), holds great potential in the fight against fatal diseases including hepatitis B, tuberculosis, cholera, typhoid fever, AIDS and pneumonia.

Now, Qingke Kong and his colleagues at the Biodesign Institute at Arizona State University, have developed a technique to make such vaccines safer and more effective. The group, under the direction of Dr. Roy Curtiss, chief scientist at Biodesign’s Center for Infectious Diseases and Vaccinology, demonstrated that a modified strain of Salmonella showed a five-fold reduction in virulence in mice, while preserving strong immunogenic properties.

Their findings appear in the cover story of the current issue of the Journal of Immunology.

Streptococcus pneumoniae, an aerobic bacterium, is the causative agent of diseases including community-acquired pneumonia, otitis media, meningitis, and bacteremia. It remains a leading killer—childhood pneumonia alone causing some 3 million fatalities annually, mostly in poorer countries.

Existing vaccines are inadequate for protecting vulnerable populations for several reasons. Heat stabilization and needle injection are required, which are often impractical for mass inoculation efforts in the developing world. Repeated doses are also needed to induce full immunity. Finally, the prohibitively high costs of existing vaccines often deprive those who need them most.  The problem is exacerbated by the recent emergence of antibiotic-resistant strains of pneumococcus causing the disease, highlighting the urgency of developing safe, effective, and lower-cost antipneumococcal vaccines.

One of the most promising strategies for new vaccine development is to use a given pathogen as a cargo ship to deliver key antigens from the pathogen researchers wish to vaccinate against. Salmonella, the bacterium responsible for food poisoning, has proven particularly attractive for this purpose, as Curtiss explains: “Orally-administered RASVs stimulate all three branches of the immune system stimulating mucosal, humoral, and cellular immunity that will be protective, in this case, against a majority of pneumococcal strains causing disease.”

Recombinant Salmonella is a highly versatile vector—capable of delivering disease-causing antigens originating from viruses, bacteria and parasites.  An attenuated Salmonella vaccine against pneumonia, developed in the Curtiss lab, is currently in FDA phase 1 clinical trials.

In the current research, the team describe a method aimed at retaining the immunogenicity of an anti-pneumonia RASV while reducing or eliminating unwanted side effects sometimes associated with such vaccines, including  fever and intestinal distress. “Many of the symptoms associated with reactogenic Salmonella vaccines are consistent with known reactions to lipid A, the endotoxin component of the Salmonella lipopolysaccharide (LPS),” the the major surface membrane component , Kong explained.   “In this paper, we describe a method for detoxifying the lipid A component of LPS in living cells without compromising the ability of the vaccine to stimulate a desirable immune response.”

To achieve detoxification, Salmonella was induced to produce dephosphoylated lipid A, rendering the vaccine safer, while leaving its ability to generate a profound, system-wide immune response, intact.

To accomplish this, a recombinant strain of Salmonella was constructed using genes from another pathogen, Francisella tularensis, a bacterium associated with tularemia or rabbit fever. Salmonella expressing lipid A 1-phosphatase from tularensis (lpxE) showed less virulence in mice, yet acted to inoculate the mice against subsequent infection by wild-type Salmonella.

In further experiments, the group showed that Salmonella strains could also be constructed to additionally synthesize pneumococcal surface protein A (PspA)—a key antigen responsible for generating antibodies to pneumonia. Again, the candidate RASV displayed nearly complete dephosphorylation of lipid A, thereby reducing toxicity.

Following inoculation with the new Salmonella strain, mice produced a strong antibody response to PspA and showed greatly improved immunity to wild-type Streptococcus pneumoniae, compared with those inoculated with Salmonella lacking  the PspA antigen. Tissue culture studies showing reduction of inflammatory cytokines following application of modified lipid A further buttressed the results.

Francisella LpxE was shown to effectively strip the 1-phosphate group from Salmonella‘s lipid A, without loss of the bacterium’s capacity for colonization. The research holds promise for constructing modified live attenuated Salmonella vaccine strains for humans, with dephosphoylated lipid A providing additional safety benefits.



The research was supported by grants from the Bill and Melinda Gates Foundation and the National Institute of Health

* Reposted on Request

Tamiflu survives sewage treatment ( oseltamivir )

Contact: Jerker Fick 46-480-446-225 Public Library of Science

Swedish researchers have discovered that oseltamivir (Tamiflu); an antiviral drug used to prevent and mitigate influenza infections is not removed or degraded during normal sewage treatment. Consequently, in countries where Tamiflu is used at a high frequency, there is a risk that its concentration in natural waters can reach levels where influenza viruses in nature will develop resistance to it. Widespread resistance of viruses in nature to Tamiflu increases the risk that influenza viruses infecting humans will become resistant to one of the few medicines currently available for treating influenza.

”Antiviral medicines such as Tamiflu must be used with care and only when the medical situation justifies it,” advises Björn Olsen, Professor of Infectious Diseases with the Uppsala University and the University of Kalmar. “Otherwise there is a risk that they will be ineffective when most needed, such as during the next influenza pandemic.”

The Swedish research group demonstrated that oseltamivir, the active substance in Tamiflu, passes virtually unchanged through sewage treatment.

“That this substance is so difficult to break down means that it goes right through sewage treatment and out into surrounding waters,” says Jerker Fick, Doctor in Chemistry at Umeå University and the leader of this study.

The Swedish research group also revealed that the level of oseltamivir discharged through sewage outlets in certain countries may be so high that influenza viruses in nature can potentially develop resistance to the drug.

“Use of Tamiflu is low in most countries, but there are some exceptions such as Japan, where a third of all influenza patients are treated with Tamiflu,” explains Jerker Fick.

Influenza viruses are common among waterfowl, especially dabbling ducks such as mallards. These ducks often forage for food in water near sewage outlets. Here they can potentially encounter oseltamivir in concentrations high enough to develop resistance in the viruses they carry.

“The biggest threat is that resistance will become common among low pathogenic influenza viruses carried by wild ducks.” adds Björn Olsen.  These viruses could then recombinate with viruses that make humans sick to create new viruses that are resistant to the antiviral drugs currently available.

The Swedish researchers advise that this problem must be taken seriously so that humanity’s future health will not be endangered by too frequent and unnecessary prescription of the drug today.



This press release refers to an upcoming article in PLoS ONE. The release has been provided by the article authors and/or their institutions.  Any opinions expressed in this are the personal views of the contributors, and do not necessarily represent the views or policies of PLoS. PLoS expressly disclaims any and all warranties and liability in connection with the information found in the release and article and your use of such information.

Their study was published in the high-ranked journal PLoS ONE. The entire article is available free online, and can be read at the following address:

Citation: Fick J, Lindberg RH, Tysklind M, Haemig PD, Waldenstro¨m J, et al (2007) Antiviral Oseltamivir Is not Removed or Degraded in Normal Sewage Water Treatment: Implications for Development of Resistance by Influenza A Virus. PLoS ONE 2(10): e986. doi:10.1371/journal.pone.0000986


* Requested Repost

Protein enhances lethality of influenza virus – PB1-F2

Contact: Nancy Wampler 617-386-2121 Cell Press

Clues from the past may influence preparations for the future

Often called the most devastating epidemic in the recorded history of the world, the 1918 influenza virus pandemic was responsible for more than 40 million deaths across the globe. The incredible lethality of the 1918 flu strain is not well understood, despite having been under intense scrutiny for many years. Now, a new study published by Cell Press in the October issue of the journal Cell Host & Microbe unravels some of the mystery surrounding the devastating 1918 pandemic and provides key information that will help prepare for future pandemics.

It is relatively rare for an influenza virus to be virulent enough to cause death in healthy humans. Many deaths associated with influenza are caused by the combined influence of viral disease and the following secondary bacterial infection. Although the 1918 pandemic strain was one of the few influenza viruses capable of killing healthy victims on its own, the majority of fatal cases from the “Spanish Flu” can be attributed to secondary bacterial pathogens rather than primary viral disease. This important interaction between influenza viruses and bacteria is not well understood.

Dr. Jonathan A. McCullers from the Department of Infectious Diseases at St. Jude Children’s Research Hospital in Memphis, Tennessee and colleagues examined this interaction by studying a newly discovered influenza A virus (IAV) protein, called PB1-F2. The gene encoding PB1-F2 is present in nearly all IAVs, including highly pathogenic avian IAVs that have infected humans and the IAV associated with the 1918 pandemic. “PB1-F2 was recently shown to enhance viral pathogenicity in a mouse infection model, raising questions about its effects on the secondary bacterial infections associated with high levels of influenza morbidity and mortality,” explains Dr. McCullers.

The researchers found that expression of PB1-F2 increased the incidence of and exacerbated secondary bacterial pneumonia in a mouse model. Intranasal delivery of a synthetic peptide derived from a portion of PB1-F2 had the same effects. Further, an influenza virus engineered to express a version of PB1-F2 identical to that in the 1918 pandemic strain was more virulent in mice and led to more severe bacterial pneumonia, explaining in part both the unparalleled virulence of the 1918 strain and the high incidence of fatal pneumonia during the pandemic.

The finding that PB1-F2 promotes lung pathology in primary viral infection and secondary bacterial infection also provides critical information for the future. “Given the importance of IAV as a leading cause of virus-induced morbidity and mortality year in and year out, and its potential to kill tens of millions in the inevitable pandemic that may have its genesis in the viruses currently circulating in southeast Asia, it is imperative to understand the role of PB1-F2 in IAV pathogenicity in humans and animals,” says Dr. McCullers. “These findings also reinforce the recent suggestion of the American Society for Microbiology that nations should stockpile antibiotics for the next pandemic, since many of the deaths during this event are likely to be caused by bacterial super-infections.”



The researchers include Julie L. McAuley of Department of Infectious Diseases,  St. Jude Children’s Research Hospital in Memphis; Felicita Hornung of Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases in Bethesda; Kelli L. Boyd of Animal Resources Center, St. Jude Children’s Research Hospital in Memphis; Amber M. Smith of Department of Mathematics, University of Utah in Salt Lake City; Raelene McKeon of Department of Infectious Diseases,  St. Jude Children’s Research Hospital in Memphis; Jack Bennink and Jonathan W. Yewdell of Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases in Bethesda; and Jonathan A. McCullers of Department of Infectious Diseases,  St. Jude Children’s Research Hospital in Memphis.

This work was supported by the NIH (grants AI-66349 and AI-54802), the NIAID intramural research program, and the American Lebanese Syrian Associated Charities (ALSAC).

McAuley et al.: “Expression of the 1918 Influenza A Virus PB1-F2 Enhances the Pathogenesis of Viral and Secondary Bacterial Pneumonia.” Publishing in Cell Host & Microbe 2, 240–249, October 2007. DOI 10.1016/j.chom.2007.09.001

* Reposted For Filing

Turmeric Spices Up Virus Study – it shows promise in fighting devastating viruses

Posted: August 15, 2012 at 10:47 am, Last Updated: August 15, 2012 at 1:33 pm

By Michele McDonald

Aarthi Narayanan

Aarthi Narayanan. Photo by Evan Cantwell

The popular spice turmeric packs more than just flavor — , Mason researchers recently discovered.

Curcumin, found in turmeric, stopped the potentially deadly Rift Valley Fever virus from multiplying in infected cells, says Aarthi Narayanan, lead investigator on a new study and a research assistant professor in Mason’s National Center for Biodefense and Infectious Diseases.

Mosquito-borne Rift Valley Fever virus (RVF) is an acute, fever-causing virus that affects domestic animals such as cattle, sheep and goats, as well as humans. Results of the study were publishedthis month in the Journal of Biological Chemistry.

“Growing up in India, I was given turmeric all the time,” says Narayanan, who has spent the past 18 months working on the project. “Every time my son has a throat infection, I give (turmeric) to him.”

There’s more work to do before curcumin-based pharmaceuticals become commonplace, Narayanan emphasizes. She plans to test 10 different versions of curcumin to determine which one works the best. She also intends to apply the research to other viruses, including HIV.

Narayanan has long wanted to explore the infection-fighting properties of turmeric, in particular its key component, curcumin. “It is often not taken seriously because it’s a spice,” she says.

But science is transforming the spice from folk medicine to one that could help a patient’s body fight off a virus because it can prevent the virus from taking over healthy cells. These “broad-spectrum inhibitors” work by defeating a wide array of viruses.

Turmeric is often used as a spice in curry dishes. Photo by Sanjay Acharya from Wikipedia Commons

“Curcumin is, by its very nature, broad spectrum,” Narayanan says. “However, in the published article, we provide evidence that curcumin may interfere with how the virus manipulates the human cell to stop the cell from responding to the infection.”

Kylene Kehn-Hall, a co-investigator on the study, adds, “We are very excited about this work, as curcumin not only dramatically inhibits RVFV replication in cell culture but also demonstrates efficacy against RVFV in a mouse model.”

Narayanan and her colleagues study the connection between a virus and how it impacts the host — human or animal. Symptoms clue in the researcher about the body’s inner workings. Rift Valley Fever and Venezuelan Equine Encephalitis kick off with flu-like symptoms.

Symptoms can make it challenging for someone to recover. The body usually starts with an exaggerated inflammatory response because it doesn’t know where to start to rid itself of the virus, she says.

“Many times, the body goes above and beyond what is necessary,” Narayanan says. “And that’s not good because it’s going to influence a bunch of cells around the infection, which haven’t seen the bug. That’s one way by which disease spreads through your body. And so it is very important to control the host because a lot of times the way the host responds contributes to the disease.”

Controlling the symptoms means more than simply making the patients feels better. “You’re giving the antiviral a chance to work. Now an antiviral can go in and stop the bug. You’re no longer trying to keep the host alive and battling the bug at the same time.”

Narayanan works with a graduate student in Mason’s National Center for Biodefense and Infectious Diseases. Photo by Evan Cantwell

Once Narayanan knows how the body responds to a virus, it’s time to go after the bug itself.

She’s applying this know-how to a family of viruses called Bunyaviruses, which feature Rift Valley fever, and such alphaviruses as Venezuelan equine encephalitis and retroviruses, which notably include HIV.

She delves into uncovering why and how each virus affects the patient. “Why are some cell types more susceptible to one type of infection than another?”

HIV goes after the immune system. Bunyaviruses will infect a wide range of cells but do maximum damage to the liver. “What is it about the liver that makes it a sitting duck compared to something like the brain?” Narayanan asks.

Ultimately, curcumin could be part of drug therapies that help defeat these viruses, Narayanan says.

“I know this works. I know it works because I have seen it happen in real life,” Narayanan says. “I eat it every day. I make it a point of adding it to vegetables I cook. Every single day.”

Other Mason researchers involved in the study are Charles Bailey, Ravi Das, Irene Guendel, Lindsay Hall, Fatah Kashanchi, Svetlana Senina and Rachel Van Duyne. Several researchers from other institutions also collaborated.

Write to Michele McDonald  at

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

Contact: Jeff Minerd 301-402-1663 NIH/National Institute of Allergy and Infectious Diseases

A small genetic change makes flu virus deadly

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

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

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

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

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

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

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

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


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

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


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

Press releases, fact sheets and other NIAID-related materials are available on the NIAID Web site at

Commentary on The Video: Fundamentalist Vaccine Penatgon Using Vaccines to Alter Human Behavior VMAT2 Gene – Followed by, Change in Human Social Behavior in Response to a Common Vaccine Abstract , & Article On VMAT2

There is currently no way to confirm the data in this Video……. Chances are without any confirming data, it will be deemed a fake… This video, and the Vaccine Data has been circulating close to two years now. If it is a hoax, I will have to say it is pretty elaborate. Keep in mind they are working on a whole slew of vaccines for addictions. Effecting everythng from Endorphins, Dopamine, and serotonin. So behavior modification in a syringe is already a reality.

I first lead with the video and then an actual abstract on the subject.

Link To FunVax Interview

Ann Epidemiol. 2010 Oct;20(10):729-33.

Change in human social behavior in response to a common vaccine.


Graduate Program in Biomedical Anthropology, Department of Anthropology, Binghamton University (SUNY), Vestal, NY, USA.



The purpose of this study was to test the hypothesis that exposure to a directly transmitted human pathogen-flu virus-increases human social behavior presymptomatically. This hypothesis is grounded in empirical evidence that animals infected with pathogens rarely behave like uninfected animals, and in evolutionary theory as applied to infectious disease. Such behavioral changes have the potential to increase parasite transmission and/or host solicitation of care.


We carried out a prospective, longitudinal study that followed participants across a known point-source exposure to a form of influenza virus (immunizations), and compared social behavior before and after exposure using each participant as his/her own control.


Human social behavior does, indeed, change with exposure. Compared to the 48 hours pre-exposure, participants interacted with significantly more people, and in significantly larger groups, during the 48 hours immediately post-exposure.


These results show that there is an immediate active behavioral response to infection before the expected onset of symptoms or sickness behavior. Although the adaptive significance of this finding awaits further investigation, we anticipate it will advance ecological and evolutionary understanding of human-pathogen interactions, and will have implications for infectious disease epidemiology and prevention.

Copyright © 2010 Elsevier Inc. All rights reserved.

[PubMed – indexed for MEDLINE]
Article on The VMAT2 Gene

Is the Capacity for Spirituality Determined by Brain Chemistry?Geneticist’s Book ‘The God Gene’ Is Disputed by Scientists, Embraced by Some Religious LeadersBy Bill Broadway Washington Post Staff Writer Saturday, November 13, 2004; Page B09

Dean H. Hamer has received much criticism for his new book, “The God Gene: How Faith is Hardwired Into Our Genes.”

Evangelicals reject the idea that faith might be reduced to chemical reactions in the brain. Humanists refuse to accept that religion is inherent in people’s makeup. And some scientists have criticized Hamer’s methodology and what they believe is a futile effort to find empirical proof of religious experience.

But Hamer, a behavioral geneticist at the National Institutes of Health and the National Cancer Institute, stands by research he says shows that spirituality — the feeling of transcendence — is part of our nature. And he believes that a universal penchant for spiritual fulfillment explains the growing popularity of nontraditional religion in this country and the presence of hundreds of religions throughout the world.

“We think that all human beings have an innate capacity for spirituality and that that desire to reach out beyond oneself, which is at the heart of spirituality, is part of the human makeup,” Hamer, 53, said in an interview at his Northwest Washington townhouse. “The research suggests some people have a bit more of that capacity than others, but it’s present to some degree in everybody.”

“The God Gene,” published in September and featured in Time magazine’s Oct. 25 cover story, is a sequel to “Living With Our Genes,” a 1998 book in which Hamer examined the genetic basis of such behavioral traits as anxiety, thrill-seeking and homosexuality. Hamer said his previous research, most notably his work on anxiety, encouraged him to look into the genetic propensity for religious belief.

What he found was that the brain chemicals associated with anxiety and other emotions, including joy and sadness, appeared to be in play in the deep meditative states of Zen practitioners and the prayerful repose of Roman Catholic nuns — not to mention the mystical trances brought on by users of peyote and other mind-altering drugs.

At least one gene, which goes by the name VMAT2, controls the flow to the brain of chemicals that play a key role in emotions and consciousness. This is the “God gene” of the book’s title, and Hamer acknowledges that it’s a misnomer. There probably are dozens or hundreds more genes, yet to be identified, involved in the universal propensity for transcendence, he said.

Furthermore, the scientific linkage of a gene with chemicals that affect happiness or sadness does not answer the question “Is there a God?” but rather “Why do we believe in God?”

“Our genes can predispose us to believe. But they don’t tell us what to believe in,” said Hamer, whose current research involves HIV/AIDS.

Critics in the scientific community argue that Hamer’s conclusions are simplistic and speculative, relying too much on anecdotal evidence and too little on testing of the VMAT2 gene to determine other possible connections to behavior. They also wonder whether his findings can be replicated, a necessity in scientific research.

“The field of behavioral genetics is littered with failed links between particular genes and personality traits,” said Carl Zimmer, a science author who reviewed the book in last month’s Scientific American.

Some religious leaders welcome the idea of a genetic basis for spirituality and say it validates long-held teachings.

“I wondered for a long time why [the concept of] a genetic implant hasn’t been put in print or been part of a conversation in the broad theological community,” said Bishop John B. Chane of the Episcopal Diocese of Washington. Chane associates Hamer’s findings with the Apostle Paul’s statement, “There are a variety of gifts but the same spirit.”

Chane also welcomes the notion of genetic universality as a new, deeper way of promoting understanding among people of different faiths — particularly Judaism, Christianity and Islam, all of which trace their beginnings to the same father, Abraham.

Others, such as Bishop Adam J. Richardson Jr. of the Washington area district of the African Methodist Episcopal Church, said that it’s hard to quantify matters of the spirit and that attributing behavior to one’s genetic makeup “can be a frightful thing.” By analogy, saying that people are predisposed to be spiritual also means that criminals are genetically wired to be criminals and have no hope of rehabilitation.

“Why not just put them in prison and throw away the key?” he asked.

Richardson said there’s also the danger of people losing hope, of believing their genetic makeup limits their development and personal growth. “In my own system, we do have choice. We always have choice,” the bishop said.

Hamer said his own religious development began in a Congregationalist church, which he abandoned when he became a scientist. But he discovered new spiritual meaning when he began researching this book — through, in part, Zen meditative practices he learned at a Zen center near Kyoto, Japan.

He likens spirituality to the capacity for language: Humans are genetically predisposed to have it, but the language people speak and the religion they practice are learned rather than inherited characteristics.

People are designed to communicate through language, but they speak English, French or Chinese because of the part of the world they grew up in. Similarly, genetic makeup urges people to believe in a Creator or find spiritual fulfillment, but culture, history and environment determine whether one is a Christian, Hindu, Jew, Buddhist or Muslim.

Although people can change or abandon that religious affiliation, they cannot rid themselves of the genetic propensity to be spiritual. But people can build on and develop that innate spirituality through meditation, prayer and creative arts such as music and painting. These practices can be done inside or outside organized religion, he said.

Hamer said he has received numerous comments from people who say that the dichotomy of spirituality and religion makes sense. “I always knew this, that I was inclined to be spiritual, even though I’ve always had a problem with religion,” they tell him.

“I see more and more people doing things like yoga,” Hamer said. “They do it initially because they want to get more flexible and look good and feel great. Then they find that once they spend some time sitting on a mat, doing nothing but concentrating on their body and clearing their mind of everything else, they say, ‘That feels kind of good.’ ”

Such feelings can lead to an intuitive sense of God’s presence, Hamer said. “We do not know God; we feel Him.”

Organized religion can become so codified, so caught up with learned rituals, that the focus on spirituality gets lost, Hamer said. The resurgence of Pentecostalism and other emotion-based religions is one sign of the staying power of inherited spirituality, he said.

Megachurches, too, are part of this phenomenon and have widespread appeal because of the emotional aspects of worship, he said. “They have lots of music, video screens, the whole multimedia thing going on,” he said. “They’re tapping into that [innate spirituality]. It’s fun and allows people to get into that spiritual frame of mind.”

Hamer said more research has to be done to determine whether there is a genetic basis for other religion-related phenomena, including the existence of archetypes, the similarity of creation stories in various religions and the common characteristics of fundamentalism in Christianity, Judaism and Islam.

Also left hanging is why women score much higher than men on transcendence tests.

“I’m not completely sure about that,” Hamer said. “I just know that it’s true. Women are more attuned to their emotional connections, and that’s at the heart of spirituality.”

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

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

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


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

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

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

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

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

Fig. 1

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

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

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

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

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

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

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

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

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

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

Fig. 2

Fig. 2

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

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

Fig. 3

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

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

Fig. 4

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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