The evolutionary emergence of pandemic influenza

Pandemic influenza remains a serious public health threat and the processes involved in the evolutionary emergence of pandemic influenza strains remain incompletely understood. Here, we develop a stochastic model for the evolutionary emergence of pandemic influenza, and use it to address three main questions. (i) What is the minimum annual number of avian influenza virus infections required in humans to explain the historical rate of pandemic emergence? (ii) Are such avian influenza infections in humans more likely to give rise to pandemic strains if they are driven by repeated cross-species introductions, or by low-level transmission of avian influenza viruses between humans? (iii) What are the most effective interventions for reducing the probability that an influenza strain with pandemic potential will evolve? Our results suggest that if evolutionary emergence of past pandemics has occurred primarily through viral reassortment in humans, then thousands of avian influenza virus infections in humans must have occurred each year for the past 250 years. Analyses also show that if there is epidemiologically significant variation among avian influenza virus genotypes, then avian virus outbreaks stemming from repeated cross-species transmission events result in a greater likelihood of a pandemic strain evolving than those caused by low-level transmission between humans. Finally, public health interventions aimed at reducing the duration of avian virus infections in humans give the greatest reduction in the probability that a pandemic strain will evolve.     

The Role of Environmental Transmission in Recurrent Avian Influenza Epidemics

Avian influenza virus (AIV) persists in North American wild waterfowl, exhibiting major outbreaks every 2–4 years. Attempts to explain the patterns of periodicity and persistence using simple direct transmission models are unsuccessful. Motivated by empirical evidence, we examine the contribution of an overlooked AIV transmission mode: environmental transmission. It is known that infectious birds shed large concentrations of virions in the environment, where virions may persist for a long time. We thus propose that, in addition to direct fecal/oral transmission, birds may become infected by ingesting virions that have long persisted in the environment. We design a new host–pathogen model that combines within-season transmission dynamics, between-season migration and reproduction, and environmental variation. Analysis of the model yields three major results. First, environmental transmission provides a persistence mechanism within small communities where epidemics cannot be sustained by direct transmission only (i.e., communities smaller than the critical community size). Second, environmental transmission offers a parsimonious explanation of the 2–4 year periodicity of avian influenza epidemics. Third, very low levels of environmental transmission (i.e., few cases per year) are sufficient for avian influenza to persist in populations where it would otherwise vanish.     

Highly pathogenic avian influenza outbreak mitigated by seasonal low pathogenic strains: Insights from dynamic modeling

The spread of highly pathogenic avian influenza (HPAI) H5N1 remains a threat for both wild and domestic bird populations, while low pathogenic avian influenza (LPAI) strains have been reported to induce partial immunity to HPAI in poultry and some wild birds inoculated with both HPAI and LPAI strains. Here, based on the reported data and experiments, we develop a two-strain avian influenza model to examine the extent to which this partial immunity observed at the individual level can affect the outcome of the outbreaks among migratory birds in the wild at the population level during different seasons. We find a distinct mitigating effect of LPAI on the death toll induced by HPAI strain, and this effect is particularly important for populations previously exposed to and recovered from LPAI. We further investigate the effect of the dominant mode of transmission of an HPAI strain on the outcome of the epidemic. Four combinations of contact based direct transmission and indirect fecal-to-oral (or environmental) routes are examined. For a given infection peak of HPAI, indirect fecal-to-oral transmission of HPAI can lead to a higher death toll than that associated with direct transmission. The mitigating effect of LPAI can, in turn, be dependent on the route of infection of HPAI.     

Genesis of pandemic influenza

During the last decade the number of reported outbreaks caused by highly pathogenic avian influenza (HPAI) in domestic poultry has drastically increased. At the same time, low pathogenic avian influenza (LPAI) strains, such as H9N2 in many parts of the Middle East and Asia and H6N2 in live bird markets in California, have become endemic. Each AI outbreak brings the concomitant possibility of poultry-to-human transmission. Indeed, human illness and death have resulted from such occasional transmissions with highly pathogenic avian H7N7 and H5N1 viruses while avian H9N2 viruses have been isolated from individuals with mild influenza. The transmission of avian influenza directly from poultry to humans has brought a sense of urgency in terms of understanding the mechanisms that lead to interspecies transmission of influenza. Domestic poultry species have been previously overlooked as potential intermediate hosts in the generation of influenza viruses with the capacity to infect humans. In this review, we will discuss molecular and epidemiological aspects that have led to the recurrent emergence of avian influenza strains with pandemic potential, with a particular emphasis on the current Asian H5N1 viruses.     

Key strategies for reducing spread of avian influenza among commercial poultry holdings: lessons for transmission to humans

Recent avian flu epidemics (A/H5N1) in Southeast Asia and case reports from around the world have led to fears of a human pandemic. Control of these outbreaks in birds would probably lead to reduced transmission of the avian virus to humans. This study presents a mathematical model based on stochastic farm-to-farm transmission that incorporates flock size and spatial contacts to evaluate the impact of control strategies. Fit to data from the recent epidemic in the Netherlands, we evaluate the efficacy of control strategies and forecast avian influenza dynamics. Our results identify high-risk areas of spread by mapping of the farm level reproductive number. Results suggest that an immediate depopulation of infected flocks following an accurate and quick diagnosis would have a greater impact than simply depopulating surrounding flocks. Understanding the relative importance of different control measures is essential for response planning.     

Optimal isolation control strategies and cost-effectiveness analysis of a two-strain avian influenza model

The most important and effective measures against disease outbreaks in the absence of valid medicines or vaccine are quarantine and isolation strategies. In this paper optimal control theory is applied to a system of ordinary differential equation describing a two-strain avian influenza transmission via the Pontryagin's Maximum Principle. To this end, a pair of control variables representing the isolation strategies for individuals with avian and mutant strains were incorporated into the transmission model. The infection averted ratio (IAR) and the incremental cost-effectiveness ratio (ICER) were calculated to investigate the cost-effectiveness of all possible combinations of the control strategies. The simulation results show that the implementation of the combination strategy during the epidemic is the most cost-effective strategy for avian influenza transmission. This is followed by the control strategy involving isolation of individuals with the mutant strain. Also observed was the fact that low mutating and more virulent virus results in an increased control effort of isolating individuals with the avian strain; and high mutating with more virulent virus results in increased efforts in isolating individuals with the mutant strain.     

Highly Pathogenic Avian Influenza Virus among Wild Birds in Mongolia

Mongolia combines a near absence of domestic poultry, with an abundance of migratory waterbirds, to create an ideal location to study the epidemiology of highly pathogenic avian influenza virus (HPAIV) in a purely wild bird system. Here we present the findings of active and passive surveillance for HPAIV subtype H5N1 in Mongolia from 2005–2011, together with the results of five outbreak investigations. In total eight HPAIV outbreaks were confirmed in Mongolia during this period. Of these, one was detected during active surveillance employed by this project, three by active surveillance performed by Mongolian government agencies, and four through passive surveillance. A further three outbreaks were recorded in the neighbouring Tyva Republic of Russia on a lake that bisects the international border. No HPAIV was isolated (cultured) from 7,855 environmental fecal samples (primarily from ducks), or from 2,765 live, clinically healthy birds captured during active surveillance (primarily shelducks, geese and swans), while four HPAIVs were isolated from 141 clinically ill or dead birds located through active surveillance. Two low pathogenic avian influenza viruses (LPAIV) were cultured from ill or dead birds during active surveillance, while environmental feces and live healthy birds yielded 56 and 1 LPAIV respectively. All Mongolian outbreaks occurred in 2005 and 2006 (clade 2.2), or 2009 and 2010 (clade 2.3.2.1); all years in which spring HPAIV outbreaks were reported in Tibet and/or Qinghai provinces in China. The occurrence of outbreaks in areas deficient in domestic poultry is strong evidence that wild birds can carry HPAIV over at least moderate distances. However, failure to detect further outbreaks of clade 2.2 after June 2006, and clade 2.3.2.1 after June 2010 suggests that wild birds migrating to and from Mongolia may not be competent as indefinite reservoirs of HPAIV, or that HPAIV did not reach susceptible populations during our study.     

Natural,Persistent Oscillations in a Spatial Multi-Strain Disease System with Application to Dengue

Many infectious diseases are not maintained in a state of equilibrium but exhibit significant fluctuations in prevalence over time. For pathogens that consist of multiple antigenic types or strains, such as influenza, malaria or dengue, these fluctuations often take on the form of regular or irregular epidemic outbreaks in addition to oscillatory prevalence levels of the constituent strains. To explain the observed temporal dynamics and structuring in pathogen populations, epidemiological multi-strain models have commonly evoked strong immune interactions between strains as the predominant driver. Here, with specific reference to dengue, we show how spatially explicit, multi-strain systems can exhibit all of the described epidemiological dynamics even in the absence of immune competition. Instead, amplification of natural stochastic differences in disease transmission, can give rise to persistent oscillations comprising semi-regular epidemic outbreaks and sequential dominance of dengue''s four serotypes. Not only can this mechanism explain observed differences in serotype and disease distributions between neighbouring geographical areas, it also has important implications for inferring the nature and epidemiological consequences of immune mediated competition in multi-strain pathogen systems.     

Low pathogenic avian influenza isolates from wild birds replicate and transmit via contact in ferrets without prior adaptation

Direct transmission of avian influenza viruses to mammals has become an increasingly investigated topic during the past decade; however, isolates that have been primarily investigated are typically ones originating from human or poultry outbreaks. Currently there is minimal comparative information on the behavior of the innumerable viruses that exist in the natural wild bird host. We have previously demonstrated the capacity of numerous North American avian influenza viruses isolated from wild birds to infect and induce lesions in the respiratory tract of mice. In this study, two isolates from shorebirds that were previously examined in mice (H1N9 and H6N1 subtypes) are further examined through experimental inoculations in the ferret with analysis of viral shedding, histopathology, and antigen localization via immunohistochemistry to elucidate pathogenicity and transmission of these viruses. Using sequence analysis and glycan binding analysis, we show that these avian viruses have the typical avian influenza binding pattern, with affinity for cell glycoproteins/glycolipids having terminal sialic acid (SA) residues with α 2,3 linkage [Neu5Ac(α2,3)Gal]. Despite the lack of α2,6 linked SA binding, these AIVs productively infected both the upper and lower respiratory tract of ferrets, resulting in nasal viral shedding and pulmonary lesions with minimal morbidity. Moreover, we show that one of the viruses is able to transmit to ferrets via direct contact, despite its binding affinity for α 2,3 linked SA residues. These results demonstrate that avian influenza viruses, which are endemic in aquatic birds, can potentially infect humans and other mammals without adaptation. Finally this work highlights the need for additional study of the wild bird subset of influenza viruses in regard to surveillance, transmission, and potential for reassortment, as they have zoonotic potential.     

Avian influenza virus

Recent outbreaks of highly pathogenic avian influenza in chickens and ducks that occurred in 9 Asian countries including Japan alarmed to realize that there is no border for infections and gave a rise to great concern for human health as well as for agriculture. This H5N1 virus jumped the species barrier and caused severe disease with high mortality in humans in Viet Nam and Thailand; 15 deaths of 22 cases and 8 of 12, respectively. A second concern was the possibility that the situation could give rise to another influenza pandemic in humans since genetic reassortment may occur between avian and human influenza viruses when a person is concurrently infected with viruses from both species. This process of gene swapping inside the human body can give rise to a new subtype of the influenza virus to which humans would not have immunity. The outbreaks also emphasized the need to continue active surveillance on avian influenza throughout the year to undertake aggressive emergency control measures as soon as an infection is detected.     

Generation of Influenza Virus from Avian Cells Infected by Salmonella Carrying the Viral Genome

Domestic poultry serve as intermediates for transmission of influenza A virus from the wild aquatic bird reservoir to humans, resulting in influenza outbreaks in poultry and potential epidemics/pandemics among human beings. To combat emerging avian influenza virus, an inexpensive, heat-stable, and orally administered influenza vaccine would be useful to vaccinate large commercial poultry flocks and even migratory birds. Our hypothesized vaccine is a recombinant attenuated bacterial strain able to mediate production of attenuated influenza virus in vivo to induce protective immunity against influenza. Here we report the feasibility and technical limitations toward such an ideal vaccine based on our exploratory study. Five 8-unit plasmids carrying a chloramphenicol resistance gene or free of an antibiotic resistance marker were constructed. Influenza virus was successfully generated in avian cells transfected by each of the plasmids. The Salmonella carrier was engineered to allow stable maintenance and conditional release of the 8-unit plasmid into the avian cells for recovery of influenza virus. Influenza A virus up to 10⁷ 50% tissue culture infective doses (TCID₅₀)/ml were recovered from 11 out of 26 co-cultures of chicken embryonic fibroblasts (CEF) and Madin-Darby canine kidney (MDCK) cells upon infection by the recombinant Salmonella carrying the 8-unit plasmid. Our data prove that a bacterial carrier can mediate generation of influenza virus by delivering its DNA cargoes into permissive host cells. Although we have made progress in developing this Salmonella influenza virus vaccine delivery system, further improvements are necessary to achieve efficient virus production, especially in vivo.     

The curse of the Pharaoh revisited: evolutionary bi-stability in environmentally transmitted pathogens

It is increasingly evident that for a number of high-profile pathogens, transmission involves both direct and environmental pathways. Much of the distinguished evolutionary theory has, however, focused on each of transmission component separately. Herein, we use the framework of adaptive dynamics to study the evolutionary consequences of mixed transmission. We find that environmental transmission can select for increased virulence when direct transmission is low. Increasing the efficiency of direct transmission gives rise to an evolutionary bi-stability, with coexistence of different levels of virulence. We conclude that the overlooked contribution of environmental transmission may explain the curious appearance of high virulence in pathogens that are typically only moderately pathogenic, as observed for avian influenza viruses and cholera.     

Deterministic Factors Overwhelm Stochastic Environmental Fluctuations as Drivers of Jellyfish Outbreaks

Jellyfish outbreaks are increasingly viewed as a deterministic response to escalating levels of environmental degradation and climate extremes. However, a comprehensive understanding of the influence of deterministic drivers and stochastic environmental variations favouring population renewal processes has remained elusive. This study quantifies the deterministic and stochastic components of environmental change that lead to outbreaks of the jellyfish Pelagia noctiluca in the Mediterranen Sea. Using data of jellyfish abundance collected at 241 sites along the Catalan coast from 2007 to 2010 we: (1) tested hypotheses about the influence of time-varying and spatial predictors of jellyfish outbreaks; (2) evaluated the relative importance of stochastic vs. deterministic forcing of outbreaks through the environmental bootstrap method; and (3) quantified return times of extreme events. Outbreaks were common in May and June and less likely in other summer months, which resulted in a negative relationship between outbreaks and SST. Cross- and along-shore advection by geostrophic flow were important concentrating forces of jellyfish, but most outbreaks occurred in the proximity of two canyons in the northern part of the study area. This result supported the recent hypothesis that canyons can funnel P. noctiluca blooms towards shore during upwelling. This can be a general, yet unappreciated mechanism leading to outbreaks of holoplanktonic jellyfish species. The environmental bootstrap indicated that stochastic environmental fluctuations have negligible effects on return times of outbreaks. Our analysis emphasized the importance of deterministic processes leading to jellyfish outbreaks compared to the stochastic component of environmental variation. A better understanding of how environmental drivers affect demographic and population processes in jellyfish species will increase the ability to anticipate jellyfish outbreaks in the future.     

Avian influenza H5N1: an update on molecular pathogenesis

Avian influenza A virus constitutes a large threat to human health. Recent outbreaks of highly pathogenic avian influenza H5N1 virus in poultry and in humans have raised concerns that an influenza pandemic will occur in the near future. Transmission from avian species to humans remains sporadic, but the mortality associated with human infection is very high (about 62%). To date, there are no effective therapeutic drugs or a prophylactic vaccines available, which means that there is still a long way to go before we can eradicate or cure avian influenza. This review focuses on the molecular pathogenesis of avian influenza H5N1 virus infection. An understanding of the viral pathogenesis may facilitate the development of novel treatments or effective eradication of this fatal disease.     

Temperature drops and the onset of severe avian influenza A H5N1 virus outbreaks

Global influenza surveillance is one of the most effective strategies for containing outbreaks and preparing for a possible pandemic influenza. Since the end of 2003, highly pathogenic avian influenza viruses (HPAI) H5N1 have caused many outbreaks in poultries and wild birds from East Asia and have spread to at least 48 countries. For such a fast and wide-spreading virulent pathogen, prediction based on changes of micro- and macro-environment has rarely been evaluated. In this study, we are developing a new climatic approach by investigating the conditions that occurred before the H5N1 avian influenza outbreaks for early predicting future HPAI outbreaks and preventing pandemic disasters. The results show a temperature drop shortly before these outbreaks in birds in each of the Eurasian regions stricken in 2005 and 2006. Dust storms, like those that struck near China's Lake Qinghai around May 4, 2005, exacerbated the spread of this HPAI H5N1 virus, causing the deaths of a record number of wild birds and triggering the subsequent spread of H5N1. Weather monitoring could play an important role in the early warning of outbreaks of this potentially dangerous virus.     

Early alterations of the receptor-binding properties of H1, H2, and H3 avian influenza virus hemagglutinins after their introduction into mammals

Interspecies transmission of influenza A viruses circulating in wild aquatic birds occasionally results in influenza outbreaks in mammals, including humans. To identify early changes in the receptor binding properties of the avian virus hemagglutinin (HA) after interspecies transmission and to determine the amino acid substitutions responsible for these alterations, we studied the HAs of the initial isolates from the human pandemics of 1957 (H2N2) and 1968 (H3N2), the European swine epizootic of 1979 (H1N1), and the seal epizootic of 1992 (H3N3), all of which were caused by the introduction of avian virus HAs into these species. The viruses were assayed for their ability to bind the synthetic sialylglycopolymers 3'SL-PAA and 6'SLN-PAA, which contained, respectively, 3'-sialyllactose (the receptor determinant preferentially recognized by avian influenza viruses) and 6'-sialyl(N-acetyllactosamine) (the receptor determinant for human viruses). Avian and seal viruses bound 6'SLN-PAA very weakly, whereas the earliest available human and swine epidemic viruses bound this polymer with a higher affinity. For the H2 and H3 strains, a single mutation, 226Q-->L, increased binding to 6'SLN-PAA, while among H1 swine viruses, the 190E-->D and 225G-->E mutations in the HA appeared important for the increased affinity of the viruses for 6'SLN-PAA. Amino acid substitutions at positions 190 and 225 with respect to the avian virus consensus sequence are also present in H1 human viruses, including those that circulated in 1918, suggesting that substitutions at these positions are important for the generation of H1 human pandemic strains. These results show that the receptor-binding specificity of the HA is altered early after the transmission of an avian virus to humans and pigs and, therefore, may be a prerequisite for the highly effective replication and spread which characterize epidemic strains.     

Broadly Protective Adenovirus-Based Multivalent Vaccines against Highly Pathogenic Avian Influenza Viruses for Pandemic Preparedness

Recurrent outbreaks of H5, H7 and H9 avian influenza viruses in domestic poultry accompanied by their occasional transmission to humans have highlighted the public health threat posed by these viruses. Newer vaccine approaches for pandemic preparedness against these viruses are needed, given the limitations of vaccines currently approved for H5N1 viruses in terms of their production timelines and the ability to induce protective immune responses in the absence of adjuvants. In this study, we evaluated the feasibility of an adenovirus (AdV)-based multivalent vaccine approach for pandemic preparedness against H5, H7 and H9 avian influenza viruses in a mouse model. Replication-defective AdV vectors expressing hemagglutinin (HA) from different subtypes and nucleoprotein (NP) from one subtype induced high levels of humoral and cellular immune responses and conferred protection against virus replication following challenge with H5, H7 and H9 avian influenza virus subtypes. Inclusion of HA from the 2009 H1N1 pandemic virus in the vaccine formulation further broadened the vaccine coverage. Significantly high levels of HA stalk-specific antibodies were observed following immunization with the multivalent vaccine. Inclusion of NP into the multivalent HA vaccine formulation resulted in the induction of CD8 T cell responses. These results suggest that a multivalent vaccine strategy may provide reasonable protection in the event of a pandemic caused by H5, H7, or H9 avian influenza virus before a strain-matched vaccine can be produced.     

Risk maps for the spread of highly pathogenic avian influenza in poultry

Devastating epidemics of highly contagious animal diseases such as avian influenza, classical swine fever, and foot-and-mouth disease underline the need for improved understanding of the factors promoting the spread of these pathogens. Here the authors present a spatial analysis of the between-farm transmission of a highly pathogenic H7N7 avian influenza virus that caused a large epidemic in The Netherlands in 2003. The authors developed a method to estimate key parameters determining the spread of highly transmissible animal diseases between farms based on outbreak data. The method allows for the identification of high-risk areas for propagating spread in an epidemiologically underpinned manner. A central concept is the transmission kernel, which determines the probability of pathogen transmission from infected to uninfected farms as a function of interfarm distance. The authors show how an estimate of the transmission kernel naturally provides estimates of the critical farm density and local reproduction numbers, which allows one to evaluate the effectiveness of control strategies. For avian influenza, the analyses show that there are two poultry-dense areas in The Netherlands where epidemic spread is possible, and in which local control measures are unlikely to be able to halt an unfolding epidemic. In these regions an epidemic can only be brought to an end by the depletion of susceptible farms by infection or massive culling. The analyses provide an estimate of the spatial range over which highly pathogenic avian influenza viruses spread between farms, and emphasize that control measures aimed at controlling such outbreaks need to take into account the local density of farms.     

Epidemiology,Evolution, and Recent Outbreaks of Avian Influenza Virus in China

Novel reassortants of H7N9, H10N8, and H5N6 avian influenza viruses (AIVs) are currently circulating in China''s poultry flocks, occasionally infecting humans and other mammals. Combined with the sometimes enzootic H5N1 and H9N2 strains, this cauldron of genetically diverse AIVs pose significant risks to public health. Here, we review the epidemiology, evolution, and recent outbreaks of AIVs in China, discuss reasons behind the recent increase in the emergence of novel AIVs, and identify warning signs which may point to the emergence of a potentially virulent and highly transmissible AIV to humans. This review will be useful to authorities who consider options for the detection and control of AIV transmission in animals and humans, with the goal of preventing future epidemics and pandemics.