SCWDS BRIEFS: Volume 21, Number 3 (October 2005)

H5N1 Influenza Virus in Wild Birds: A Fact Sheet Highly Pathogenic Avian Influenza Virus H5N1 and Wild Birds What are avian influenza viruses? What is a Highly Pathogenic Avian Influenza virus? What is “Bird Flu” and what is “HPAI H5N1”? What do we know about avian influenza viruses in wild birds? Do we have HPAI H5N1 in North America? Is there currently a public health risk associated with HPAI H5N1 in wild birds? Is there a domestic animal health risk associated with HPAI in wild birds? What is the possibility of HPAI H5N1 entering North America via migratory wild birds? What is the possibility of this virus being maintained in wild bird populations? Do we have surveillance for HPAI H5N1 in the United States? Additional information on HPAI can be found at these websites: The recognized geographic and species distribution of chronic wasting disease (CWD) has expanded since early September 2005 to include Hampshire County in the eastern panhandle of West Virginia National Fish and Wildlife Health Initiative Guiding Principles Eastern Equine Encephalitis (EEE) virus was isolated from seven white-tailed deer in southwestern Michigan during September 2005 During the past summer, more than 500 head of livestock in North Dakota and South Dakota were lost to one of the largest recorded anthrax outbreaks in U.S. history. Most of the losses were in cattle, but horses, bison, and farm-reared elk also were affected. Dr. John Fischer, Director of SCWDS, has received this year’s Special Recognition Award from the International Association of Fish and Wildlife Agencies (IAFWA). Dr. William Randolph Davidson is retiring in November 2005.

Within-winter movements: a common phenomenon in the Common Pochard Aythya ferina

Waterbirds are often observed to move between different wintering sites within the same winter—for example, in response to food availability or weather conditions. Within-winter movements may contribute to the spreading of diseases, such as avian influenza, outside the actual migration period. The Common Pochard Aythya ferina seems to be particularly sensitive to infection with the highly pathogenic avian influenza virus H5N1 and, consequently, could play an important role as vectors for the disease. We describe here the within-winter movements of Pochards in Europe in relation to topography, climate, sex and age. We analysed data provided by the Euring data bank on 201 individuals for which records from different locations from the same winter (December–February) were available. The distances and directions moved within the winter varied markedly between regions, which could be ascribed to the differing topography (coast lines, Alps). We found no significant differences in terms of distances and directions moved between the sexes and only weak indications of differences between the age classes. In Switzerland, juveniles moved in more westerly directions than adults. During relatively mild winters, winter harshness had no effect on the distances travelled, but in cold winters, a positive relationship was observed, a pattern possibly triggered by the freezing of lakes. Winter harshness did not influence the directions of the movement. About 41% (83/201) of the Pochards that were recovered at least 1 km from the ringing site had moved more than 200 km. A substantial number of birds moved between central/southern Europe and the north-western coast of mainland Europe, and between the north-western coast of mainland Europe and Great Britain, whereas no direct exchange between Great Britain and central/southern Europe was observed. Within-winter movements of Pochards seem to be a common phenomenon in all years and possibly occur as a response to the depletion of food resources. This high tendency to move could potentially contribute to the spread of bird-transmitted diseases outside the actual migration period.

Role of Urbanization, Land-Use Diversity, and Livestock Intensification in Zoonotic Emerging Infectious Diseases

Emerging infectious diseases (EIDs) continue to significantly threaten human and animal health. While there has been some progress in identifying underlying proximal driving forces and causal mechanisms of disease emergence, the role of distal factors is most poorly understood. This article focuses on analyzing the statistical association between highly pathogenic avian influenza (HPAI) H5N1 and urbanization, land-use diversity and poultry intensification. A special form of the urban transition—peri-urbanization—was hypothesized as being associated with ‘hot-spots’ of disease emergence. Novel metrics were used to characterize these distal risk factors. Our models, which combined these newly proposed risk factors with previously known natural and human risk factors, had a far higher predictive performance compared to published models for the first two epidemiological waves in Viet Nam. We found that when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor. However, urbanization spatially combines other risk factors leading to peri-urban places being the most likely ‘hot-spots’. The work highlights that peri-urban areas have highest levels of chicken density, duck and geese flock size diversity, fraction of land under rice, fraction of land under aquaculture compared to rural and urban areas. Land-use diversity, which has previously never been studied in the context of HPAI H5N1, was found to be a significant risk factor. Places where intensive and extensive forms of poultry production are collocated were found to be at greater risk.

Role of position 627 of PB2 and the multibasic cleavage site of the hemagglutinin in the virulence of H5N1 avian influenza virus in chickens and ducks

Highly pathogenic H5N1 avian influenza viruses have caused major disease outbreaks in domestic and free-living birds with transmission to humans resulting in 59% mortality amongst 564 cases. The mutation of the amino acid at position 627 of the viral polymerase basic-2 protein (PB2) from glutamic acid (E) in avian isolates to lysine (K) in human isolates is frequently found, but it is not known if this change affects the fitness and pathogenicity of the virus in birds. We show here that horizontal transmission of A/Vietnam/1203/2004 H5N1 (VN/1203) virus in chickens and ducks was not affected by the change of K to E at PB2-627. All chickens died between 21 to 48 hours post infection (pi), while 70% of the ducks survived infection. Virus replication was detected in chickens within 12 hours pi and reached peak titers in spleen, lung and brain between 18 to 24 hours for both viruses. Viral antigen in chickens was predominantly in the endothelium, while in ducks it was present in multiple cell types, including neurons, myocardium, skeletal muscle and connective tissues. Virus replicated to a high titer in chicken thrombocytes and caused upregulation of TLR3 and several cell adhesion molecules, which may explain the rapid virus dissemination and location of viral antigen in endothelium. Virus replication in ducks reached peak values between 2 and 4 days pi in spleen, lung and brain tissues and in contrast to infection in chickens, thrombocytes were not involved. In addition, infection of chickens with low pathogenic VN/1203 caused neuropathology, with E at position PB2-627 causing significantly higher infection rates than K, indicating that it enhances virulence in chickens.

Mutations in H5N1 influenza virus hemagglutinin that confer binding to human tracheal airway epithelium

The emergence in 2009 of a swine-origin H1N1 influenza virus as the first pandemic of the 21st Century is a timely reminder of the international public health impact of influenza viruses, even those associated with mild disease. The widespread distribution of highly pathogenic H5N1 influenza virus in the avian population has spawned concern that it may give rise to a human influenza pandemic. The mortality rate associated with occasional human infection by H5N1 virus approximates 60%, suggesting that an H5N1 pandemic would be devastating to global health and economy. To date, the H5N1 virus has not acquired the propensity to transmit efficiently between humans. The reasons behind this are unclear, especially given the high mutation rate associated with influenza virus replication. Here we used a panel of recombinant H5 hemagglutinin (HA) variants to demonstrate the potential for H5 HA to bind human airway epithelium, the predominant target tissue for influenza virus infection and spread. While parental H5 HA exhibited limited binding to human tracheal epithelium, introduction of selected mutations converted the binding profile to that of a current human influenza strain HA. Strikingly, these amino-acid changes required multiple simultaneous mutations in the genomes of naturally occurring H5 isolates. Moreover, H5 HAs bearing intermediate sequences failed to bind airway tissues and likely represent mutations that are an evolutionary "dead end." We conclude that, although genetic changes that adapt H5 to human airways can be demonstrated, they may not readily arise during natural virus replication. This genetic barrier limits the likelihood that current H5 viruses will originate a human pandemic.

Generation of candidate human influenza vaccine strains in cell culture: rehearsing the European response to an H7N1 pandemic threat

Background: Although H5N1 avian influenza viruses pose the most obvious imminent pandemic threat, there have been several recent zoonotic incidents involving transmission of H7 viruses to humans. Vaccines are the primary public health defense against pandemics, but reliance on embryonated chickens eggs to propagate vaccine and logistic problems posed by the use of new technology may slow our ability to respond rapidly in a pandemic situation. Objectives: We sought to generate an H7 candidate vaccine virus suitable for administration to humans whose generation and amplification avoided the use of eggs. Methods: We generated a suitable H7 vaccine virus by reverse genetics. This virus, known as RD3, comprises the internal genes of A/Puerto Rico/8/34 with surface antigens of the highly pathogenic avian strain A/Chicken/Italy/13474/99 (H7N1). The multi-basic amino acid site in the HA gene, associated with high pathogenicity in chickens, was removed. Results: The HA modification did not alter the antigenicity of the virus and the resultant single basic motif was stably retained following several passages in Vero and PER. C6 cells. RD3 was attenuated for growth in embryonated eggs, chickens, and ferrets. RD3 induced an antibody response in infected animals reactive against both the homologous virus and other H7 influenza viruses associated with recent infection by H7 viruses in humans. Conclusions: This is the first report of a candidate H7 vaccine virus for use in humans generated by reverse genetics and propagated entirely in mammalian tissue culture. The vaccine has potential use against a wide range of H7 strains.

A question of self-preservation : immunopathology in influenza virus infection

Influenza A viruses that circulate normally in the human population cause a debilitating, though generally transient, illness that is sometimes fatal, particularly in the elderly. Severe complications arising from pandemic influenza or the highly pathogenic avian H5N1 viruses are often associated with rapid, massive inflammatory cell infiltration, acute respiratory distress, reactive hemophagocytosis and multiple organ involvement. Histological and pathological indicators strongly suggest a key role for an excessive host response in mediating at least some of this pathology. Here, we review the current literature on how various effector arms of the immune system can act deleteriously to initiate or exacerbate pathological damage in this viral pneumonia. Generally, the same immunological factors mediating tissue damage during the anti-influenza immune response are also critical for efficient elimination of virus, thereby posing a significant challenge in the design of harmless yet effective therapeutic strategies for tackling influenza virus.

An examination of the predictors of blood donors' intentions to donate during two phases of an avian influenza outbreak

BACKGROUND: Data from prior health scares suggest that an avian influenza outbreak will impact on people’s intention to donate blood; however research exploring this is scarce. Using an augmented theory of planned behavior (TPB), incorporating threat perceptions alongside the rational decision-making components of the TPB, the current study sought to identify predictors of blood donors’ intentions to donate during two phases of an avian influenza outbreak. STUDY DESIGN AND METHODS: Blood donors (N = 172) completed an on-line survey assessing the standard TPB predictors as well as measures of threat perceptions from the health belief model (HBM; i.e., perceived susceptibility and severity). Path analyses examined the utility of the augmented TPB to predict donors’ intentions to donate during a low- and high-risk phase of an avian influenza outbreak. RESULTS: In both phases, the model provided a good fit to the data explaining 69% (low risk) and 72% (high risk) of the variance in intentions. Attitude, subjective norm, and perceived susceptibility significantly predicted donor intentions in both phases. Within the low-risk phase, gender was an additional significant predictor of intention, while in the high-risk phase, perceived behavioral control was significantly related to intentions. CONCLUSION: An augmented TPB model can be used to predict donors’ intentions to donate blood in a low-risk and a high-risk phase of an outbreak of avian influenza. As such, the results provide important insights into donors’ decision-making that can be used by blood agencies to maintain the blood supply in the context of an avian influenza outbreak.

Beliefs underlying blood donors’ intentions to donate during two phases of an avian influenza outbreak

Using a Theory of Planned Behavior (TPB) framework the current study explored the beliefs of current blood donors (N=172) about donating during a low and high-risk phase of a potential avian influenza outbreak. While the majority of behavioral, normative, and control beliefs identified in preliminary research differed as a function of donors’ intentions to donate during both phases of an avian influenza outbreak, regression analyses suggested that the targeting of different specific beliefs during each phase of an outbreak would yield most benefit in bolstering donors’ intentions to remain donating. The findings provide insight in how to best motivate donors in different phases of an avian influenza outbreak.

Computational analysis of proteome of H5N1 avian influenza virus to define T cell epitopes with vaccine potential

The existing vaccines against influenza are based on the generation of neutralizing antibody primarily directed against surface proteins-hernagglutinin and neuraminidase. In this work, we have computationally defined conserved T cell epitopes of proteins of influenza virus H5N1 to help in the design of a vaccine with haplotype specificity for a target population. The peptides from the proteome of H5NI irus which are predicted to bind to different HLAs, do not show similarity with peptides of human proteorne and are also identified to be generated by proteolytic cleavage. These peptides could be made use of in the design of either a DNA vaccine or a subunit vaccine against V influenza. (c) 2007 Elsevier Ltd. All rights reserved.

Secondary structural analysis of the mRNA regions encoding the hemagglutinin cleavage site basic amino acids of the avian influenza virus H5N1 subtype samples

Here we report the codon bias and the mRNA secondary structural features of the hemagglutinin (HA) cleavage site basic amino acid regions of avian influenza virus H5N1 subtypes. We have developed a dynamic extended folding strategy to predict RNA secondar

Detection of avian influenza virus subtype H5 using a biosensor based on imaging ellipsometry

A novel method is reported for the detection of avian influenza virus subtype H5 using a biosensor based on high spatial resolution imaging ellipsometry (IE). Monoclonal antibodies specific to H5 hemagglutinin protein were immobilized on silicon wafers and used to capture virus particles. Resultant changes on the surface of the wafers were visualized directly in gray-scale on an imaging ellipsometry image. This preliminary study has shown that the assay is rapid and specific for the identification of avian influenza virus subtype H5. Compared with lateral-flow immunoassays, this biosensor not only has better sensitivity, but can also simultaneously perform multiplexed tests. These results suggest that this biosensor might be a valuable diagnostic toot for avian influenza virus detection. (c) 2009 Elsevier B.V. All rights reserved.

Detection of avian influenza virus by fluorescent DNA barcode-based immunoassay with sensitivity comparable to PCR

In this paper, a coupling of fluorophore-DNA barcode and bead-based immunoassay for detecting avian influenza virus (AIV) with PCR-like sensitivity is reported. The assay is based on the use of sandwich immunoassay and fluorophore-tagged oligonucleotides as representative barcodes. The detection involves the sandwiching of the target AIV between magnetic immunoprobes and barcode-carrying immunoprobes. Because each barcode-carrying immunoprobe is functionalized with a multitude of fluorophore-DNA barcode strands, many DNA barcodes are released for each positive binding event resulting in amplification of the signal. Using an inactivated H16N3 AIV as a model, a linear response over five orders of magnitude was obtained, and the sensitivity of the detection was comparable to conventional RT-PCR. Moreover, the entire detection required less than 2 hr. The results indicate that the method has great potential as an alternative for surveillance of epidemic outbreaks caused by AIV, other viruses and microorganisms.