Research Structure Aim

"Prevention and Control of Avian Influenza in the U.S." Research Structure

SPECIFIC AIMS

Specific Aim 1. TOP

Objective 1 : TOP To determine the molecular basis for adaptation of influenza A viruses from wild aquatic birds in land-based poultry, particularly chickens, turkeys and quail. (Jack Gelb, Jr., Conrad Pope, Brian S. Ladman, University of Delaware, Newark, DE; Erica Spackman, Southeast Regional Poultry Laboratory ARS, USDA, Athens, GA; and Richard Slemons, The Ohio State University, Columbus, OH. Original Title: Viral Adaptation to Host Species-Effect of AIV Passage within a Host on Viral Populations and Sequence Changes)

The critical first event in the development of HPAI is efficient replication of a LPAI. We will study the molecular basis of the events that allow the transmission of AI from wild waterfowl and the ensuing adaptation to and perpetuation in land-based poultry. We will investigate the ability of a wild waterfowl H5 virus to adapt to commercial poultry, specifically the broiler chicken and the meat type turkey. These species best represent the highest numbers of commercial poultry in the US and other countries. Our AICAP year 1 research showed that three H7N2 LP isolates from the Delmarva peninsula, were clearly more virulent for broilers than for SPF or commercial type leghorns (Gelb, J., Jr., et al 2005 unpublished). Undoubtedly, there are genetic differences between meat and egg type poultry that are responsible for susceptibilities to avian influenza viruses. As an internal control for our studies, we propose to use the same species of waterfowl (mallard) from which the original virus (low path H5N1) was recovered in order to minimize adaptive genetic changes.

1. Perform AI passage in four poultry host species and assess viral pathogenicity associated with adaptation. Goal: To adapt a LPAI A/duck/H5N1/ Maryland/2006 (H5N1) to four poultry host species resulting in 4 divergent viruses. Little is known about the relative frequency and the range of initial efficiency with which aquatic bird influenza can replicate in chickens. Recent studies indicate that quail are more susceptible than chickens to most AI viruses from feral waterfowl(61, 62). A H5 AI was selected as it represents typical isolates recently found in natural migratory bird populations in the US. Commercial type meat chickens and turkeys were selected as they represent the major populations used in the poultry industry in the US and other countries and in which LPAI H5 and H7 have shown to become HPAI. Mallards will be used as controls; as they are the same species as the original source of the LPAI H5N1 virus. Quail will be used due to their apparent susceptibility to viruses from the wild bird reservoir(45, 61, 62).

2. Determine the ability of serially passaged, species adapted AI to cross species barrier. Assess viral sequence changes associated with AI passage and adaptation in different poultry host species. Chickens, turkeys, quail, and ducks will be challenged with the 10th passage of the chicken (similarly with the turkey, quail and duck) adapted LP H5N1 virus. Histopathology and immunochemistry of different tissues will be analyzed as well as serum to determine the degree of seroconversion. Full AI genome sequencing will be performed on original H5N1 LP strain and the four viruses following 10 passages in each of the respective poultry species. Sequence changes will be associated with pathogenicity findings established previously. Specific probes will be designed to examine and quantify the changes in AI viral populations of the original and passaged AIs that are associated with host species adaptation.

Anticipated results and significance. We expect to determine whether adaptation of a duck H5N1 virus in four different bird species leads to the emergence of virus populations with distinct molecular changes. We also expect to determine whether adaptation to a single species results in expansion of the host range of the H5N1 virus and/or perhaps in a strain with enhanced virulence. Our studies will reveal whether serial passage in different bird species leads to a random or a defined set of molecular changes that either expands or narrows the host range of the virus.

Objective 2: TOP To determine the molecular parameters for interspecies transmission and virulence of swine influenza viruses in turkeys and vice-versa and the potential susceptibility of swine to other avian influenza strains.

Transmission of avian influenza viruses to pigs and the ensuing roles that pigs may play in pandemic influenza virus creation (serving as hosts for adaptation of avian viruses to replication in mammals and for avian and human virus reassortment) have received extensive research attention (56, 71, 72, 101, 102, 114). However, virus transmission between birds and pigs is a bi-directional process(3, 5, 44, 50, 107). Pig-to-bird transmission poses economically important disease concerns for domestic turkeys and also raises the possibility for widespread distribution of influenza viruses across the human population via the migration of free-ranging waterfowl(34, 40, 56, 57). In the US, the turkey industry includes production of over 270 million birds and an annual gross income of over $2.8 billion(4). Six LPAI outbreaks in turkeys in the US between 1978-2002 led to $179 million in losses(4). Beyond such direct monetary costs, a potential mechanism for enhancing the infectivity of HPAI viruses for human beings is via initial replication in pigs. Interestingly, turkeys could act also as amplifiers of influenza viruses that can transmit to humans as suggested by Hinshaw et al(28). Nonetheless, little research has been done to understand the viral factors that control virus movement 'backwards' from pigs to birds. We seek to address this gap in our knowledge of influenza virus host range determinants.

1. To identify the determinants of interspecies transmission of H3N2 TR viruses between swine and turkeys at the molecular level. (Yehia M. Saif and Chang Won Lee, Ohio Agricultural Research and Development Center (OARDC), The Ohio State University (OSU), Wooster, OH. Consultant: Daniel R. Perez, University of Maryland, College Park, MD. Original Title: Molecular Determinants of Interspecies Transmission of H3N2 Triple Reassortant Influenza A Viruses).

In 2003 and 2004 a new lineage of swine influenza viruses (H3N2 triple reassortants (TR)) were isolated for the first time from turkeys in the US(14, 91). Turkey hens infected with these viruses showed no clinical signs, but experienced complete cessation of egg production. In AICAP 1, we compared antigenically and genetically different H3N2 TR viruses of swine and turkey origin. Additionally, we studied their interspecies transmission between swine and turkeys, as well as their intraspecies transmission amongst different avian species. Based on our findings we hypothesize that molecular changes in one or more genes of the TR viruses determine the ability of these viruses to transmit between swine and turkeys. We also hypothesize that these TR viruses had retained the ability to bind to human receptors (SA 2,6gal) which could be a potential risk if these viruses evolve and cause serious diseases in human. Finally, we believe that molecular changes at the RBD of the HA gene determine the potential of this virus to infect some of the avian species (turkeys and chickens) but not others (ducks).

Experimental Plan: Characterization of gene(s) and amino acid(s) that contribute to the interspecies transmission of H3N2 TR viruses between swine and turkeys. Four H3N2 TR viruses of turkey and swine origin and one human virus will be rescued by reverse genetics(52, 53). We will perform 'directed' reassortment between these viruses to determine the genes that are responsible for efficient replication efficiency, pathogenesis and inter- and intraspecies transmission in pigs and turkeys. We will perform site directed mutagenesis at one or more amino acid residue(s) based on sequence comparison to determine their contribution to transmission. We anticipate that some genes/mutations will lead to the 'gain or loss of function' for their ability to transmit between the two species.

We will study the distribution of influenza A viruses receptors in different age turkeys (from 1 day to 40 weeks). We will perform lectin-based staining studies, to determine the presence of SA2,3gal and SA2,6gal receptors in different tissues(97). We will also study the distribution of various epithelial cell types (ciliated versus non-ciliated) on the tracheal surface as described(96). Receptor binding specificity of H3N2 TR viruses will be assayed using the solid-phase enzyme linked assay as previously described. Mutations will be generated at amino acid residues at the RBD, mainly on residues 190, 226 and 228 to determine the role in receptor specificity.

Anticipated results and significance: We anticipate that various reassortants will behave differently in their transmissibility between swine and turkeys. Hence, we can identify single gene(s) and amino acid residues that play a major role in this transmission. Findings from these experiments are important in terms of epidemiology of H3N2 TR viruses. These viruses are now endemic in the swine population in the US and they are continually isolated from poultry. Such continual evolution while maintaining receptor-binding specificity to human receptors could be of high potential risk for infecting humans.

2. Role of PB1-F2 in Virulence, Transmission and Host Adaptation of Influenza A Viruses of Swine and Turkeys. (Elankumaran Subbiah, Virginia Tech University, Blacksburg, VA; Daniel R. Perez, University of Maryland, College Park, MD.)

Of particular interest to this proposal is the 11th viral protein, PB1-F2, which was discovered recently from a camouflaged alternative reading frame within segment 2(25). PB1-F2 protein localizes to mitochondria and results in cell death by apoptosis(8, 13). The size of PB1-F2 polypeptides ranges from 79 to 101 amino acids (aa); most isolates encode versions of either 87 or 90 aa(112). The frequency of the 79-aa PB1-F2 is 5%(112). A functional PB1-F2 is expressed by 92% of all segment-2 sequences, i.e., a polypeptide >78 aa. The proportion of intact PB1-F2 varies according to host (humans 90%, swine 76%, other mammals 100%, birds 95%)(112). All pandemic strains of influenza possess an intact PB1-F2 and the high mortality rates associated with them has been attributed in part to PB1-F2 (3). Knocking out the PB1-F2 open reading frame attenuated the ability of A/Puerto Rico8/34 virus to induce apoptosis in immune cells(18) and reduced the virulence of the virus in a mouse model(110). PB1-F2 protein may play a role in priming the host for secondary bacterial infections(58). The severity of recent H3N2 infections in swine and turkeys is unprecedented, including abortions in pregnant sows. Secondary bacterial infections commonly increase the severity of H3N2 infections. The hypothesis to be tested is that genomic signatures in genes other than the surface proteins can modulate the host immune response imparting enhanced pathogenicity and ability to transfer and adapt between hosts. To test this hypothesis, we will examine the role of PB1-F2 protein in virulence, cross-species transmission and host range adaptation employing novel triple reassortant domestic H3N2 viruses.

2a. Define the role of PB1-F2 in the virulence and transmission of H3N2 viruses of swine and turkeys. We have used reverse genetics to create the A/turkey/Ohio/313053/04 (H3N2) and A/Swine/Minnesota/593/99 (H3N2) wild type viruses and isogenic mutants and chimeric viruses of swine and turkey H3N2 viruses will be prepared to study the role of PB1-F2 in pathogenesis. We propose to mutate full-length PB1-F2 gene in A/turkey/Ohio/313053/04 (H3N2) and A/Swine/Minnesota/593/99 (H3N2) to a truncated version. Additionally, we will replace the PB1-F2 genes of these viruses to that of A/Hong Kong/156/97 (H5N1) avian influenza virus and A/Brevig Mission/1/1918 (H1N1) human influenza virus to make chimeric viruses and study their pathogenesis and transmission in swine, quail, and turkeys. PB1-F2 expression from chimeric and knock-out mutants will be tested using specific anti-PB1-F2 polyclonal antibody.

To compare the pathogenesis and transmission of PB1-F2 knock-out mutants and chimeric viruses, we will perform infection studies in commercially available outbred SPF pigs, turkeys, and Japanese quail. Virus will be titrated from nasal swabs (pigs), tracheal and cloacal swabs (quail and turkeys). At the end of the experiment, virus content in respiratory and extra-respiratory tissues will be determined. Viruses recovered from contact animals/birds from various groups will be fully sequenced to assess the genetic stability after cross-species transmission. We will also choose one host-adapted variant isolated from transmission studies and propagate it by five sequential intranasal passages in the new host to evaluate host stability. Virus content in the lungs at each passage level will be titrated and the genetic stability of the virus at each passage level will be examined by whole genome sequencing.

Anticipated Results and Significance. Our studies will provide the minimum requirements for the PB1-F2 protein to play a role in virulence, host range restriction and adaptation of influenza in biological and ecological significant host species. The overall results will provide avenues for novel intervention strategies against avian and mammalian influenza viruses.

Pitfalls and limitations. It is possible that PB-1F2 knock-out mutants may not result in a decreased virulence. However, we expect to see differences in virus clearance and increased rates of transmission. Further, we expect an increased virulence pathotype in chimeric viruses. The incorporation of H5N1 and the 1918 H1N1 PB1-F2 into H3N2 genes may result in the enhanced virulence of H3N2 viruses for other species. To safeguard against the escape of these viruses into the environment, these experiments will be conducted in the ABSL-3 Ag facilities at the Virginia-Maryland Regional College of Veterinary Medicine, College Park.

Objective 3. TOP To determine the molecular basis for the differences in virulence and disease outcome of different domestic land-based and aquatic birds infected with HPAI. (Alicia Solorzano, Daniel R. Perez, University of Maryland, College Park, MD. Original title: Innate immune responses of different poultry species towards highly pathogenic avian influenza)

HPAI are characterized by high morbidity and mortality in gallinaceous species(2). In contrast, disease signs in wild birds infected with HPAI are rare and a spill over into this population makes prevention and eradication of these viruses particularly difficult. Interestingly, the newly emerged Asian H5N1 viruses have shown an unusual ability to kill waterfowl, which in itself poses major biological and ecological challenges. The molecular mechanisms responsible for the difference in mortality in wild birds between the previously circulating viruses and the newer strains is poorly understood. It is tempting to speculate that differences in the innate immune response in different avian species are in part responsible for the outcome of the disease. A lethal outcome may be explained by either a weak innate immune response not capable of counteracting the virus replication or, as it has been proposed in the case of H5N1 infection in humans, due to a "cytokine storm", in which an exacerbated response of secreted pro-inflammatory cytokines leads to severe immunopathology(20).

We propose to analyze the innate immune responses of different poultry species infected with HPAI. We will use two strains with clearly distinct phenotypes in different avian species. The influenza A/Vietnam/1203/04 (H5N1) strain has been shown to be able to kill chickens, geese and ducks(33), whereas the A/Hong Kong/483/97 (H5N1) virus has been shown to be lethal for chickens, partially lethal for geese and avirulent for ducks(76). We will establish correlates between the profile of the immune response and the outcome of illness in different avian species. Understanding the modulation of the immune response will provide insights and lead to potential intervention strategies to control the replication and spread of HPAI in avian species.

3a. Cloning and sequence of the innate immune response genes from different avian species.

  • Primary cell lines, induction of the immune response in vitro and cloning of innate immune response genes

We will produce primary cell lines from eight poultry species: chicken, turkey, quail, goose, duck, pheasant, pigeon and ostrich. Avian primary fibroblast will be obtained from embryonated eggs and cultured following the protocols established in our laboratory. We will stimulate the innate immune response by treating alternatively with different stimulants as described(43, 81, 99). We will clone thirteen genes related to the innate immune response by homology, designing specific or degenerate primers or characterization through a cDNA library.

3b. Characterization of the innate immune response in vitro and in vivo from different avian species infected with HPAI H5N1 viruses of different pathotypes. The first response against viral infection is the type I IFN. We will measure the innate immune response by two methods, Bioassay and qRT-PCR as described(78). Quantitative and time course of gene expression will be followed by qRT-PCR with primers specific for IFN and other genes related to the innate immune response. The differences in gene regulation are essential to recognize the potential role of each gene in the outcome of disease (morbidity and mortality). We will standardize in vitro conditions compatible with all species to establish correlates of susceptibility and virulence in different hosts infected with the same or different HPAI H5N1 viruses.

We will perform H5N1 infections in vivo in chickens and ducks in order to establish the molecular mechanism involved in disease progression. We will collect different organs at different time points PI and the RNA will be used for qRT-PCR to measure mRNA levels of the genes of interest. The comparison between the in vitro and the in vivo data will allow us to establish correlates between lethality in vivo and modulation of innate immune responses in vitro. Our studies will have a significant impact for the future prediction of virulence and/or lethality of avian influenza strains in different animal species.

Summary. TOP These studies exploit the natural environments provided by different animal species to understand the molecular changes related to adaptation of avian influenza viruses in land-based birds. They also provide a venue to better understand the cross species transmission of influenza viruses between mammals (pigs) and birds. The power of reverse genetics will be used to dissect the molecular events that take place for avian influenza viruses to become endemic in land-based poultry. Thus, as we have previously demonstrated with quail, we will generate the necessary scientific evidence from which to establish agricultural and production management practices aimed at preventing the emergence of influenza A viruses in poultry. The use of state-of-the-art molecular biology, reverse genetics, and classical virology techniques will reveal molecular markers associated with interspecies transmission of influenza A viruses in land-based birds and pigs. To our knowledge, the studies on PB1-F2 and innate immune responses in biologically and ecologically relevant animal species are the first aimed at understanding the role of virus-host interactions for pathogenesis and transmission of influenza viruses. Our studies will determine the importance of intermediate hosts in the emergence of influenza strains with novel phenotypes and provide the necessary basic information to implement intervention strategies against avian influenza.

Specific Aim 2. TOP

Objective 4. TOP Understanding the Natural History of Avian Influenza Viruses in Wild Birds and the Temporal-Spatial-Host-Genomic Relationships of Avian Influenza Viruses Infecting Wild Birds and Poultry (Richard Slemons and Nathan Watermeier, The Ohio State University, Columbus, OH; Blanca Lupiani, Texas A&M University, College Station, TX. Cost-Sharing Collaborators: Joseph Giambrone, Teresa Dormitorio, Auburn University; Carol Cardona, UC-Davis; Cindy Driscoll, Larry Hindman, Maryland Department of Natural Resources; Jack Gelb, University of Delaware; Rose Foster, David Graber, Missouri Avian Influenza Task Force; George Happ, Jon Rundstadler, University of Alaska-Faibanks; David Halvorson, Ian Rubinoff, University of Minnesota; Jeffrey Taubenberger, National Institutes of Health; David Spiro; J. Craig Venter Institute)

The ultimate goal of AICAP is to help prevent future avian influenza (AI) outbreaks in poultry and wild birds in the US. This contribution is to be reached by reducing the probability of avian influenza virus introductions into these populations and detecting emerging viral threats. This research will focus on gaining knowledge about the natural history and the viral properties of AI viruses maintained in wild birds and by identifying temporal-spatial-host-viral genomic signature relationships associated with interspecies transmission of AI. This new knowledge will be distributed through multi-level and multi-format education formats. These education efforts will target audiences and individuals including, but not limited to: wildlife experts, zoo managers and health care providers, pet and exotic bird owners, poultry producers, poultry health experts, government officials and public health officials and researchers. This program will not prevent all future outbreaks, but, at a minimum, if it prevents even one outbreak (which we will never know) it will be cost-effective by reducing the expenditures resulting from control programs requiring the use of vaccination, depopulation, indemnity, controlled marketing and losses due to marketing disruptions. We believe increasing prevention efforts can aid in combating LP and HP AI threats in the U.S and abroad.

Significant progress was made during the initial 2 1/2 years of the AICAP 1. The most significant accomplishments were the establishment of the first continent-wide research network with aimed at defining the natural history of AIs in wild birds and providing education and outreach based results. As a result this research network reaches from Alaska to Delaware to Alabama to California and resulted in the sampling of 5,900 and 6,609 wild birds from which there were 143 and 254 actual AI isolates recovered and analyzed during 2005 and 2006, respectively. Each research group recovered isolates which included the first low pathogenic, North American lineage H5N1 AIV isolates recovered in the US during 2005 and 2006. Findings reported in a paper submitted to Science (Dugan et al.) are one example how bioinformatics analysis of our wild bird-origin AI isolates are contributing new knowledge about the natural history of AIs. With limited funding we were able to develop a master database structure; data entry is up to date through 2006 for 5 states and all network participants have been invited to a database management workshop at The Ohio State University to learn how to use the system by the end of July, 2007. In June 2007 AICAP 1 began sending our network data to the US Department of Interior, USGS, National Wildlife Health Center for inclusion in the National HPAI Early Detection Data System (HEDDS) News, which is posted on the internet weekly. Most importantly, we have graduate, veterinary and undergraduate students at multiple universities gaining expertise currently needed by FAO, USAID, World Bank and other organizations.

AICAP 2 will continue to build upon the foundation laid during AICAP 1. Master Database: during the renewal period there will be an increased emphasis on improving, expanding and utilizing the Master GIS Database through direct contacts between researchers and database manager/GIS specialist and at yearly workshops where bioinfomatics assistance will be provided by cost-sharing collaborators. Providing science-based information is central to the ultimate successes of the continent-wide network for AI research in wild birds. This effort master database supports all contributing collaborators in the network. The comprehensive geographic information data management system is designed to handle the framework of a North America wide data collection model for wild bird field surveillance work for AI. The system will consist of a standardized data entry system including a web-based Intranet data entry system. A geospatial relational database management system has been built using MySQL. The data entry system supports ODBC clients for authentication and connection to MySQL. Data for use in querying, reporting, mapping and analysis will be assisted with applications using MA Access and ESRI ArcIMS/SDE for web-based GIS mapping. The system is being designed to promote data integrity, security and privacy of the data being collected. Field and Laboratory: The established field and laboratory research efforts will continue to be upgraded as needed at annual workshops and via e-mail communication.

For AICAP 2 we will:

  • Expand the continent wide, type A influenza virus research network.
  • Continue to characterize the antigenic and genomic properties of isolated AIs using molecular and bioinformatics tools.
  • Use antigenic and genetic data to track the movement of wild bird-origin AIs and their genes in wild bird populations across the four migratory bird flyways, and to poultry and vicecersa.
  • Maintain and expand the inclusiveness of the master GIS Decision Support System.
  • Explore the potential for development of models to predict the spread of wild bird-origin AIs and novel AI introductions in the US.
  • Provide input and selected isolates for experiments examining interspecies transmission, reverse genetics and vaccine development.
Assuring adequate progress will be achieved on reaching these deliverables will require mutually beneficial, two-way sharing, collaborative, multi-disciplined research projects among researchers in many universities and collaborators in government agencies.

Objective 5. TOP To continue delivering and developing educational programs on in house depopulation, viral inactivation, and composting methods for use in the case of catastrophic mortality or depopulation. (Eric R. Benson, Robert L. Alphin, and George W. Malone, University of Delaware, Newark, DE. Collaborator: Nathaniel Tablante, University of Maryland, College Park, MD. Original Title: Integrated Development and Distribution of Catastrophic Poultry Emergency Procedures)

The project team has concentrated on the development of procedures for response to avian influenza outbreaks and on training. During AICAP 1, the project team had two focuses, development of disinfection treatments and a national catastrophic disease response-training program. The disinfection research led to additional external funding and two refereed publications. The training program far exceeded the initial projections and lead to additional domestic and limited international training opportunities. The project team has made over 50 research and educational presentations based on the information developed during the AICAP program. In AICAP 2, the project team is continuing a scientific research and increasingly international training effort.

  • Lombardi M.E., B.S. Ladman, R.L. Alphin and E.R. Benson. 2007. Inactivation of Avian Influenza Virus Using Common Soaps, Detergents, Chemicals, and Disinfectants. Avian Diseases. In review
  • Benson E.R., G.W. Malone, R.L. Alphin, K. Johnson, and E. Staicu. 2007. Application of In-house Composting on Viral Inactivity of Newcastle Disease Virus. Poultry Science. In review.
  • Lombardi, M.E. 2007. Inactivation of avian influenza virus using common soaps, detergents, chemicals, and disinfectants. Undergraduate Thesis. University of Delaware.

During AICAP 1, we evaluated decontamination of poultry houses and equipment, which was performed in a four-phase process. Phase 1 consisted of a laboratory trial evaluating the ability of selected commercially disinfectants against both AI and NDV. Phase 2, completed in Year 2, involved evaluating the efficacy of commercially available disinfectants in the field on NDV. Phase 3 includes valuation of disinfection of mechanical equipment and is currently being conducted. Phase 4 evaluated the inactivation of virus in a windrow during cold weather in Years 2 (2006) and 3 (2007). Experimental details of this study are provided in the original application included in the appendix.

As mentioned in the preliminary data section, a half-day comprehensive classroom-training program was developed in the Spring of 2005. During the past 1 1/2 years this training program was continuously modified and updated as new information became available. Content of the training material includes: current status of avian influenza worldwide, human health guidelines for responders to an avian influenza outbreak; requirements, options and procedures for mass depopulation; disposal options, procedures and cost with comprehensive details for in-house windrow composting. The vast majority of material used in this training was based on lessons-learned from those who have dealt with this issue including the presenter's personal experience. Demand for this train-the-trainer program far exceeded the initial projections. A total of 42 sessions were held in 26 different states, 5 in Canada (supported by the poultry industry) and 2 in Brazil (supported by the Brazilian government).

During AICAP 2, the project team will conduct an integrated scientific and international extension project. The project will specifically address the continued development, improvement, and dissemination of catastrophic poultry emergency procedures. The project specifically will

  • Continue evaluating the use of foam applied compost additives, compost covers, alternative bulking agents and carcass to carbon mixture;
  • Continue developing international catastrophic poultry mortality training materials.

The use of foam applied compost additives, compost covers, alternative bulking agents and carcass to carbon mixture will be evaluated in four systematic studies. Study 1 will evaluate the interaction between compost additive and foam. Study 2 will evaluate the performance of available bulking agents including litter, sawdust, woodchips, mulch and/or straw. Study 3 will evaluate the alternative compost pile mixes of carcass to carbon ratios from 1:1 to 1.2. Study 4 will evaluate the use of compost covers including fleece, filter fabric and/or polyethylene sheet in the context of outdoor compost piles. An estimate of carcass degradation will be performed at two and four weeks. Compost moisture and nutrient composition will be determined at four weeks. Compost temperatures will be monitored at 30 cm and 90 cm depths at two locations per treatment. Sample packages containing NDV inoculated breast meat will be placed approximately 1 ft (30 cm) inside the pile and covered with litter material. The samples will be frozen immediately after sampling and evaluated using standard re-isolation procedures.

We will develop and maintain a comprehensive web based emergency response educational module. The emergency response module will include the Delaware model, biosecurity, depopulation (culling), and in-house composting. Additional modules including bioterrorism may be added as appropriate. The depopulation training will be comprehensive and include depopulation and disposal options. A logic model based approach utilizing inputs, outputs and outcomes - impacts will be used to maximize the effectiveness of the materials developed. The training program will be intended for industry level personnel including veterinarians, flock supervisors, and similar.

In Year 1, the modules will be prepared in a distance learning compatible presentation with audio and made available through a password-protected portion of the University of Delaware Avian Biosciences Center. In Year 2, an additional module will be developed for Central and South America. In Year 3, an additional module will be developed for a third region based on emerging worldwide needs. The region will be selected in Year 2 through discussions with the AICAP Executive Committee and UD Avian Biosciences Center. The Avian Bioscience Center will be linked to the AICAP Web portal (www.aicap.umd.edu) and access will be provided as well from the latter site. In addition, during Years 1 through 3, modules will be added into the eXtensions system.

Summary. TOP The success of any major program aimed at controlling any given infectious disease is measured by the prevalence of the agent at the beginning and at the end of the program. This will be true for all except the surveillance conducted in wild free-ranging waterfowl in which no interventions will be attempted nor will any changes be expected. Working closely with the live bird marketing system and producers that supply those markets as well as the producers of wild captive birds, we determined the risks factors that promote the perpetuation of AI in land-based poultry. During AICAP 1, we implemented education programs aimed at a defined target audience including gamebird producers and similar through our AI risk analysis and prevention program. Likewise, our program on virus inactivation, mass depopulation and composting had major success among the large commercial poultry sector. We will continue analyzing the dynamics and prevalence of AI viruses in the wild bird reservoir and its movements to domestic poultry spcies Our phylogenetic studies as well as our contribution to the overall pool of AI information gathered by other groups will provide evidence on the overall movement and spread of viruses in different states and across the country. By the end of the 3-year period of this proposal, the education programs (traditional- and electronic-based) that we will be developed and implemented will definitely raise public awareness among market owners, suppliers, wholesalers, dealers of commercial poultry and the general public of the importance of AI for poultry and public health. Without scientific evidence it is difficult to change cultural, management, traditional, and market practices. Our studies will change the way the general public perceives AI and will help in the implementation of bioregulatory strategies that will limit the emergence of novel and more virulent influenza A virus strains.

Specific Aim 3. TOP To develop critical diagnostic tests and vaccines for avian influenza control. This objective seeks to exploit the expertise and knowledge from the previous specific aims to address two key high priority needs: 1) Development of highly sensitive and rapid diagnostic tests for avian influenza during surveillance and outbreaks capable of discriminating between non-vaccinated and vaccinated birds as well as infected birds. 2) Development of alternative strategies for vaccination against avian influenza exploiting the latest molecular approaches available including reverse genetics and recombinant expression vectors.

Objective 6. TOP Development of alternative diagnostic tools for AI. Rapid detection of avian influenza (AI) virus and antibodies in individual birds and flocks is essential for early detection and regulatory surveillance(89, 92). AI surveillance in commercial poultry is carried out by detecting the presence of antibodies to NP and M1, two group specific antigens, using agar gel immunodiffusion (AGID) tests and by detection of virus by traditional virus isolation method or real-time RT-PCR (RRT-PCR)(55, 105). In order to determine the viral subtype responsible for the infection, all positive serum samples and viruses must be further subtyped using the hemagglutination inhibition (HI) and neuraminidase inhibition (NI) tests(55, 105). In total, these tests take over 4 days to complete, slowing down an effective control response. In addition, AI subtyping of chicken or turkey sera requires laboratories to be equipped with appropriate biosafety facilities to propagate the viruses required to conduct HI and NI assays. Similarly, subtyping of the virus detected by RRT-PCR requires virus isolation in eggs followed by HI, NI or RRT-PCR tests with specific probe and primer sets(79). An urgent need exists for improved diagnostic methods that allow more rapid detection and efficient subtyping of AI virus infections. The underlying objective of the proposals presented below is to develop improved diagnostic tests that allow simultaneous detection and subtyping of AI virus infections (both virus and antibodies) as well as the development of species-independent serologic tests. These tests can be used in conjunction with vaccines to differentiate infected from vaccinated animals (DIVA). The PIs of the enclosed proposals have agreed to exchange reference and field samples for validation of the proposed tests. The goal of the AICAP diagnostics group is to accelerate the progress of each individual project.

1a. Development of Luminex and ELISA based immunoassay for the subtyping of sera from avian influenza virus infected chickens and turkeys. (Blanca Lupiani, Texas A&M University, College Station, TX)

Our long-term goal is to develop immunoassays for rapid detection and subtyping of AI virus specific antibodies in chickens and turkeys. We will develop Luminex and ELISA based assays for the detection of H5 (North American strain), H6, H7 and H9 antibodies in chickens and turkeys. We propose the following specific objectives: 1) To express avian influenza virus recombinant hemagglutinin (H5 (North America strain), H6, H7, and H9 using an alphavirus (VEE) replicon (VEErep) 2) To develop Luminex and ELISA based assays for detection of chicken antibodies to different HA proteins. The Luminex test proposed has the advantage of multiplexing capabilities(37, 63, 94) and therefore allows for the subtyping of all HAs in a single test. On the other hand, the ELISA test has the advantage of not requiring significant investments in expensive equipment and can be carried out in any laboratory.

During AICAP 1, we developed a fluorescence microsphere based immunoassay (based on the Luminex xMAP technology) for the detection of chicken antibodies to NP, M1 and NS1 influenza virus proteins. Conditions were optimized for the coating of the fluorescent beads with baculovirus expressed and purified NP, M1 and NS1 proteins, and their reactivity with AI positive and negative reference sera (NVSL) was evaluated. The signal obtained for NP protein was significantly higher than that observed for M1 and NS1 suggesting that NP is the antigen of choice for the detection of AI type specific antibodies. Initial work with a VEErep expressed H5 protein, in an ELISA based test, indicated no cross reactivity with chicken sera specific for other avian pathogens and only some level of cross reactivity with chicken sera specific for subtype H2 strongly supporting the feasibility of the proposed project.

1b. Validation of avian influenza serological tests for differentiating infected from vaccinated animals (DIVA). (Maricarmen Garcia, University of Georgia, Athens, GA)

The detection of antibodies directed against the NA or the non-structural protein NS1 antibodies has been utilized as a tool for this DIVA strategy(7, 11, 41, 83). In our laboratory we have developed indirect NA (N1 and N2 subtypes) and NS1 ELISAs for the use in a DIVA coordinated strategy. With funds from AICAP 1 we evaluated an N1, N2 and NS1 ELISA using baculovirus purified antigens. The detection of NA and NS1 antibodies by ELISA was specific and reproducible in sera from infected birds. For AICAP 2, we will validate control strategies for AI using a coordinated vaccination and monitoring approach. To support this approach monoclonal antibodies (mAbs) will be generated against two neuraminidase subtypes (N1 and N2). These mAbs will be used to increase the sensitivity of already established assays in a competitive approach(22, 23, 73, 113). In addition, it will be evaluated if the DIVA vaccination strategy to detect N1 and N2 antibodies can be applied for chickens as it has been successfully utilized to differentiate vaccinated/infected turkeys in the field(12).

1c. Development of a species-independent ELISA for detection of influenza antibodies directed against H6/H7/H9 subtypes. (Egbert Mundt, University of Georgia, Athens, GA)

To gain a better understanding of the epidemiology of AI virus based on surveillance studies in wild and domestic animals other than birds and for studies of the presence of AI virus antibodies in domestic poultry, species-independent ELISA systems(21, 80) would be most valuable and can be performed in semi-automatic approaches. The proposed assays (H7, H6, H9) are applicable in a broad range of laboratories since the necessary equipment is generally present. The ELISA systems to be developed will use a competitor antibody of one species that competes with the binding of antibodies in the test sample (cELISA) in a species-independent way. To this end HA will be obtained by generating recombinant baculoviruses. The subsequently purified antigens will be used for the establishment of monoclonal antibodies. Finally both components will be used for the establishment of the cELISA systems. The assays for the different HAs (H5 from AICAP 1, H6, H7, H9) can be combined on one plate so that serum samples can be tested in parallel for the presence of antibodies against four different subtypes. In initial experiments, we will develop cELISA with currently available mAbs and baculovirus expressed H5 protein.

2. Detection of nucleic acids. Real-Time RT-PCR (RRT-PCR) provides a rapid and feasible alternative to virus isolation in embryonating chicken eggs and subtyping by HI test(79). Typically, the RRT-PCR test screens for the presence of the matrix gene and, if positive, additional RRT-PCR tests are carried out to determine if the virus present is of H5 and H7 subtypes. The high cost of the assay and the limitation in multiplexing is major a disadvantage. To overcome this bottleneck the newly emerging microsphere-based suspension array technology (xMAP technology), which provides high specificity and sensitivity, will be applied. This system permits the multiplexing of up to 100 different assays within a single sample.

2a. Development of a multiplex microsphere-based assay for the detection and differentiation of different hemagglutinin subtypes of influenza virus. (Chang Won Lee, Ohio State University, Wooster, OH)

We propose to develop a rapid and precise multiplex assay for the identification and subtyping of different HA subtypes of avian influenza virus by combining two proven technologies, branched DNA (bDNA) signal amplification technology (a sandwich nucleic acid hybridization assay) and xMAP technology(10, 38). Using this technology the RNA will be detected directly and amplification of the signal will be performed later using the bDNA signal amplification. This approach can be used for swab samples, allantoic fluid, or cell culture supernatant to accurately detect and measure viral RNA. In a preliminary study (3-plex assay), we were able to detect different HA subtypes and differentiate H5 and H7 HA subtypes at the same time. The M specific probe detected isolates of 13 different HA subtypes tested (n=65), the Asian H5N1-lineage specific H5 probe detected all 9 H5N1 viruses and the H7 probe detected North American H7 viruses that are currently circulating in the US. Purified RNA samples, nasal swabs and allantoic fluid (BPL inactivated allantoic fluid for highly pathogenic H5N1 viruses) samples showed similar sensitivity and non-specific reaction was not observed. The detection limit was approximately 103 EID50. Considering the flexibility, ability to multiplex and automated high throughput capabilities at an affordable price, we expect this system will be useful for screening tests for reference laboratories, such as NVSL and NAHLN laboratories. The significant reduction in time necessary to identify and differentiate influenza virus will be a major asset in surveillance efforts.

2b. Multiplex detection of avian influenza HA and NA types using a microsphere assay. (Mark Jackwood, University of Georgia, Athens, GA)

We will focus on a pan influenza test, against the matrix protein as well as specific H5, H7, N1, and N2 subtypes. The multiplexed microsphere assay will be paired with RT-PCR amplification of the whole RNA transcriptome in a clinical sample using the TransPlextm amplification kit (Rubicon Genomics, Ann Arbor, MI). All AI specific amplicons will be detected allowing immediate typing of one or more isolates in a single sample using a single test. The multiplex assay will be compared in sensitivity and specificity to RRT-PCR. This methodology can also be expanded to include any respiratory RNA virus. We have developed microsphere assays for detection of antibodies against infectious bronchitis virus (IBV), Newcastle disease virus (NDV), reovirus, Mycoplasma gallisepticum (MG), Salmonella enteritidis, and Salmonella typhimurium. A multiplex assay with NDV and MG as well as an assay with NDV, IBV, and reovirus was developed and tested(9).

Collaborations: All assays described focus on the simultaneous detection of several subtypes of AI virus. For all five approaches the presence of a set of samples (either serum or nucleic acid) for the validation (specificity, accuracy, precision, detection limit, linearity, robustness) of the assays is necessary. Such sets will be either established in individual laboratories for development of the tests or will be provided by different resources for a first validation of the appropriate assay. This approach will aid in the validation of each of the proposed assays. Several of the PIs in the AICAP are performing animal experiments for investigation on AI not funded by the AICAP [NIAID Center of Excellence for Influenza Research and Surveillance (Mundt); contracts with USDA-ARS (Lee, Jackwood); DHS (Lupiani), other sources (Cardona)], in their laboratories. Furthermore in other projects as part of the AICAP animal experiments will be performed. During the course of these experiments samples will be generated and provided to the investigators of the AICAP diagnostics group to compare sensitivity and specificity between the established assays. Sample will be also available from members of the diagnostic group obtained from SPF chickens and turkeys, which have been infected with infectious agents other than AI (e.g. IBV, ILTV, APMV, IBDV) for validation of the specificity of the assays. Based on results with defined samples, tests will be validated with field samples. To this end with the support of diagnostic labs (PDRC at University of Georgia, Texas Veterinary Medical Diagnostic Laboratory (TVMDL), California Animal Health and Food Safety Laboratory (CAHFS), Indiana Animal Disease Diagnostic Laboratory (ADDL), ADDL at Ohio Department of Agriculture) such a set of serum samples negative for AI virus will be compiled and distributed within the group. After validation, the tests will be evaluated as described in the technology transition plan (see below). Based on an agreement with the OIE reference laboratory for AI and NDV in Germany (Federal Research Institute for Animal Health, Insel Riems, Germany) serological tests will be investigated for their ruggedness. A very important mechanism to support the development of tests is the exchange of reagents (e.g. proteins, monoclonal antibodies) generated during the development of each of the proposed assays. Based on collaborations within the AICP diagnostic group, those tools will be provided and tested for the applicability in other approaches. The exchange of these reagents will be carried out using MTAs between the involved Institutions.

Technology transition plan: Invention disclosures will be filed to protect intellectual property. A dossier of finalized GLP-like protocols and manufacturing concepts will be packaged for delivery to major commercial manufacturers and vendors of biologics. The concept for the benefits of the diagnostic tests outlined in this proposal will be discussed with USDA/APHIS leadership representing end users, the USDA/APHIS/Center for Veterinary Biologics at Ames (IA) representing the regulatory authority for animal health, and presented at the annual meeting of the United States Animal Health Association where producers, marketing, academic and federal officials convene to recommend implementation of new technology for diagnosis of AI virus infection in commercial poultry. We will encourage interested commercial manufacturers and vendors to license diagnostic tests outlined in this proposal and assume responsibility for product development and federal approval for distribution to state and federal officials for detection and diagnosis of avian influenza virus in commercial poultry.

Anticipated results: The implementation of these objectives will require active collaboration between the molecular biology and immunology groups on one hand, and the infectious diseases and epizootiology groups working in the LBM and commercial poultry facilities. As soon as the tests are validated for influenza virus experimental diagnostic use, we will either engage our technology transfer offices in getting private sector involvement to develop these tests into licensed commercial products, or directly transfer the technologies to the appropriate diagnostic laboratories. To maximize the efficient utilization of resources, a decision will be made each year of the grant to select the most promising technologies for further development with enhanced resources.

Objective 7. Vaccines. TOP
Rationale: There is very little doubt that AI transmission is effectively prevented in sero-positive flocks, reviewed in(27). Inactivated oil-emulsion AI vaccines are very effective in protecting poultry against disease and reducing the spread of HPAI. Major scientific breakthroughs in the field of reverse genetics of negative strand viruses and recombinant DNA technology may provide the ideal candidates for the future implementation of vaccination alternatives to help control AI disease and spread of HPAI and LPAI. More importantly, some of these new technologies may provide the long-sought mucosal immunity against AI. We will continue expanding on two alternative strategies for vaccination against HPAI (and LPAI) using 1) a replication deficient recombinant adenovirus vector(74), 2) a recombinant live lentogenic Newcastle Disease Virus vector (NDV)(32). A third alternative aimed at developing live attenuated influenza viruses have become independently funded through independent grants: C-W Lee, AICAP 1 and USDA cooperative agreement; and D.R. Perez and A. Garcia-Sastre, AICAP 1, USDA and NIH grants. Two pilot projects have been selected to increase the alternative vaccine spectrum/strategies that can have a major impact for the poultry and swine industries.

1. Protective properties of adenovirus vectored vaccines against HPAI and LPAI viruses. (Haroldo Toro, Auburn University, Ellen Collisson, Western University Health Sciences; David Suarez, Southeast poultry Research Labs)

We reported previously that protective immunity against AI can be elicited in chickens in a single-dose regimen by in ovo vaccination with a replication-competent adenovirus (RCA)-free human adenovirus serotype 5 (Ad5)-derived vector encoding an avian H5 hemagglutinin (HA) (AdTW68.H5)(93). RCA-free Ad5 vectored AI vaccines have numerous advantages over other vaccines including cost effective production in cell cultures, capacity for cost effective mass vaccination using automated in ovo injectors, compliance with a DIVA strategy, replication incompetence, and no possibility of reassortment of the vaccine and field strains of AI(24, 93). For AICAP 2 we plan to accomplish the following specific objectives: (1) Develop and evaluate RCA-free Ad vaccines vectoring codon-optimized synthetic AI HA genes. An RCA-free Ad vector expressing a codon optimized (to the chicken cell tRNA) synthetic H5 HA gene was developed (AdTW68.H5ck) which induced higher antibody titers in vaccinated chickens than those obtained after vaccination with the cognate HA gene. (2) Evaluate immunity of chickens vaccinated in ovo with Ad-vectored AI vaccines. Broiler chickens are routinely vaccinated in ovo against Marek's disease virus (MDV). Thus, vaccination with Ad vectored AI vaccines would likely have to be applied simultaneously with MDV vaccines. We will evaluate both humoral and cellular immune responses of chickens vaccinated with a combination of a commercial MDV vaccine and the AdTW68.H5 experimental vaccine. (3) Evaluate immunity of chickens vaccinated dually with adenovirus vectors expressing H5 and H7 HA antigens. Chickens sequentially vaccinated with AdTW68.H5 and AdChNY94.H7 developed antibody responses against both HA proteins. We will evaluate humoral and cellular immune responses of chickens vaccinated in ovo with both vaccine constructs simultaneously. (4) Evaluate protective immunity conferred by spray vaccination with AdTW68.H5ck. Our recent results show that systemic and mucosal antibodies are effectively induced by ocular application of the RCA-free AdTW68.H5 vaccine. Vaccination by coarse spray is likely the most efficient route for mass vaccine delivery to chicken populations already placed in the field. We will evaluate immune responses and protection against HPAI challenge in chickens vaccinated via spray with AdTW68.H5ck. Antibody and cellular T responses of spray-vaccinated chickens will be compared with the response of ocularly-vaccinated chickens.

2. Enhancing Avian Influenza Vaccine Efficacy Using Toll-like Receptor Ligands for Rapid Induction of immune Response (Pilot Project: Sanjay Reddy, Texas A&M University, College Station, TX)

The overarching goal of the proposed research is to enhance immunogenicity of inactivated avian influenza (AI) vaccine for rapid induction of protective immunity in poultry. We propose to test the hypothesis that inoculation of chickens with a single dose of an inactivated whole AI virus vaccine formulated with synthetic ligands for Toll-like Receptor (TLR) 3 and 7 will stimulate rapid induction of robust AI-specific neutralizing antibodies and thereby improve vaccine efficacy(48, 66, 67). This hypothesis is based on the observation that infection of chickens with live low pathogenic AI virus (LPAI) stimulates rapid induction of antibodies detectable four days post infection. This rapid response is due, in part, to the ability of the AI viral RNA to stimulate innate immune responses through TLRs, which results in secretion of pro-inflammatory cytokines and chemokines that influence adaptive immune responses(66, 111). Specifically, the AI viral replicative intermediate double stranded (ds) RNA and single stranded (ss) RNA that accumulates within infected cells triggers TLR3 and TLR7/8, respectively. In this study, we will evaluate the ability of poly I:C (a synthetic dsRNA analog) and R848 (a synthetic ssRNA analog), to enhance the immunogenicity of an inactivated AI vaccine for eliciting rapid immunity and reduction of virus shedding upon challenge.

3. Recombinant Lactococcus-based Vaccine for the Prevention of Avian Influenza Virus infection (Pilot Project: Bruce Geller, Manoj Pastey, Oregon State University, Corvallis, OR)

This is a proof-of-concept plan to develop an oral vaccine for commercial poultry that will help control HPAI. This oral vaccine is a live, recombinant, nonpathogenic bacterium, Lactococcus lactis, which is an FDA-approved GRAS organism used to make fermented dairy products, but has recently been developed for use as an oral vaccine delivery system(46, 49, 64, 70, 104). Three conserved peptides, one each from influenza virus A surface antigens, M2, HA, and NA will be expressed on the surface of L. lactis. The DNA coding region for each antigen will be cloned in tandem into a gene for a membrane protein in L. lactis. Expression of each antigen on the surface of L. lactis will be measured by ELISA. Chickens will be orally vaccinated with live L. lactis that express the influenza virus A antigens. Antigen-specific humoral immune responses will be measured in the serum, saliva, and feces. Vaccinated animals will be challenged with an infectious dose of influenza virus type A. Infection will be monitored by viral titer in tissues, histological pathology, recovery time, and survival.

4. Development of a novel Newcastle disease virus vectored vaccine for Avian Influenza A subtype H9 virus. (Siba K Samal, Baibaswata Nayak, and Daniel R Perez, University of Maryland, College Park)

H9N2 influenza viruses have become established across Eurasia in which they are responsible for outbreaks of disease characterized by drops in egg production, diarrhea, anorexia, reduced water consumption and weight loss. Mortality due to secondary infections has been observed during H9N2 outbreaks(15, 26, 42, 51, 54, 60, 69, 108). In addition, H9N2 viruses may have been involved in favoring the geographic spread of other important subtypes, including the Asian H5N1 viruses and have also been associated with transmission and respiratory disease in humans. We have previously used the recombinant (rNDV) strain LaSota(31) to express the HA genes of H5 and H7 AI viruses and we are currently completing challenge studies in chickens using in ovo and oral routes of vaccine administration. For AICAP 2 we will construct rNDV vaccines containing the HA gene of a prototypic H9 subtype alone or in combination with a H5 subtype. After recovery and characterization, the recombinants will be used to evaluate the immunogenicity and protective efficacy of rNDV-H9 and rNDV-H9H5 in chickens. The differences in virus and antibody titers pre- and post-challenge, composite clinical scores and weight loss will be evaluated by using standard statistical data analysis software ANOVA(6, 47).

Summary . TOP Influenza A viruses continue to pose a major threat to the poultry industry and public health. In fact, the Office International des Epizooties (OIE) has recently (May 2003) revised its international standard on avian influenza concerned with the risk that a spread of avian influenza may present to the international community not only from economic but also from public health points of view. Uncontrolled avian influenza in the US could paralyze the industry and compromise our position as the leading exporter of poultry in the world. As part of the preparedness for AI outbreaks, it will be important to maintain an active network that effectively monitors influenza activity in poultry across the country, using the most versatile and fastest methods of detection available. Likewise, access to reliable vaccine strategies that can be quickly implemented to control the spread of AI during outbreaks is another important national priority. Our research on better and faster diagnostic methods will result in the replacement of virus isolation in eggs and AGID, both tedious and heavily time-consuming, as the standard methods for AI diagnosis. The technologies that have been and will be developed during this program are already contributing to improve the speed in the diagnosis of AI. We intend to continue with this line of investigation in order to provide a combination of easy-to-use as well as sophisticated molecular tools for the detection of AI in poultry. We will demonstrate the efficacy of our newly developed vaccines in experimental settings. Our vaccines studies will contribute to a better understanding of the possible application of vaccination to control the emergence and spread of potentially LPAI and HPAI viruses.




> Next
LITERATURE SEARCHED