2008 Annual Report 1a.Objectives (from AD-416) Objective 1: Develop new mass immunizing vaccines and delivery systems to protect against H5N1 high pathogenicity avian influenza (HPAI) virus. Objective 2: Develop and supporting the use of rapid, site-deployable diagnostic technologies to accurately detect avian influenza virus. Objective 3: Conduct genomic sequencing of AI viruses and determining the biological and molecular epidemiological relevance of genetic information. Objective 4: Support ongoing targeted surveillance of wild migratory birds and poultry for HPAI in areas of greatest risk throughout North and South America, Asia and Pacific Rim. 1b.Approach (from AD-416) Objective 1: Develop mass immunizing vaccines using reverse genetics or biotechnological engineering to create an attenuated avian influenza (AI) vaccine stain and recombinant vectored vaccines that can produce protective immune responses in poultry following aerosol, oral or in ovo administration. Assist partners in finding alternative sources of renewable agricultural plant materials for raw materials needed to manufacture anti-influenza drugs. Objective 2: Develop rapid, state laboratory based or site-deployable tools and other assays that will allow rapid detection and classification of AI viruses in samples from birds and environmental specimens including new RRT-PCR tests for wild bird cloacal samples, microarray test development for AI virus classification and more sensitive penside tests for detecting avian influenza viruses using optical reading technologies. Objective 3: Conduct genome sequencing of poultry and wild bird AI viruses in SEPRL archive and from on going surveillance, and characterize them biologically. The sequence data will be analyzed to determine viral evolution, strain relationships, and determinants of virulence as well as identify diagnostic sequences and potential vaccine antigens. Selected virus strains will be studied to determine genomic changes that define host adaptation and specificity and changes necessary for AI viruses to cross to new avian and mammalian hosts. Objective 4: Conduct epidemiological studies to identify the risk factors for transmission of virus between farms and biosecurity mitigation steps to reduce transmission. Conduct targeted surveillance of wild birds and poultry in high risk areas for avian influenza and assess risk of introduction to farms. 3.Progress Report This project is in National Program 103, Animal Diseases (100%), components of Pathogen Detection and Diagnostics, Animal Immunology, Microbial Genomics, Mechanism of Disease, Epidemiology of Disease, and Strategies to Control Infectious and Non-Infectious Disease. This report documents research conducted under an Interagency agreement. Additional details of research can be found in the report for the in-house associated project 6612-32000-048-00D, Application of Biological and Molecular Techniques to the Diagnosis and Control of Avian Influenza and other Emerging Poultry Pathogens. The interagency agreement on avian influenza virus has substantially met all the program objectives. The intramural research at the Agricultural Research Service (ARS) Southeast Poultry Research Laboratory, the Avian Disease Oncology Laboratory, the National Animal Disease Center, and the Beltsville Agriculture Research Center has been completed and most of the specific cooperative agreements developed as part of this project will be completed in Fiscal Year 2008. However, several other specific cooperative agreements will continue through Fiscal Year 2009 to allow the program objectives to be met. A total of 12 specific cooperative agreements were developed to help meet the objectives of the project. The progress reports for each specific cooperative agreement are presented separately according to the project. Specific accomplishments are presented below that are primarily related to the intramural research in the project. 4.Accomplishments 1. This accomplishment is in National Program 103 Animal Health (100%), Component 4 – Countermeasures to prevent and control respiratory diseases. Problem Statement 4C: Poultry Respiratory Diseases. Major sequencing project for avian influenza viruses. Avian influenza viruses are extremely diverse in sequence and they have a wide host range. In an effort to better understand and correlate the genotype with the phenotype of avian influenza, close to 1000 avian influenza virus isolates have been submitted for full coding sequencing as part of a virus characterization program. In cooperation with Seqwright corporation, 80% of the genes have been sequenced from the 1000 viruses, and this information is being reviewed and annotated at the Southeast Poultry Research Laboratory before being released to GenBank. The availability of this genetic information and other data from animal studies looking at virus growth should provide us a sound basis for understanding how viruses infect and cause disease in different host species. 2. This accomplishment is in National Program 103 Animal Health (100%), Component 1 – Bio-defense research. Problem Statement 1B: Emerging diseases. New virus discovered in penguins. Worldwide efforts for monitoring wild bird populations for avian influenza and Newcastle disease viruses have increased since the discovery of H5N1 highly pathogenic avian influenza in wild birds in the past 5 years. In collaboration with Wildlife Conservation Society and University of Georgia Southeast Cooperative Wildlife Disease Study, a previously unknown and unclassified paramyxovirus was isolated from a rock hopper penguin sampled on the Falkland Islands in 2007. This new virus is distinct from known paramyxoviruses type 1-9 based on serology and sequencing. This new virus will be further studied to determine its possible role on health of wild bird populations in Southern Hemisphere. 3. This accomplishment is in National Program 103 Animal Health (100%), Component 4 – Countermeasures to prevent and control respiratory diseases. Problem Statement 4C: Poultry Respiratory Diseases. Chickens and turkeys have limited susceptibility to wild bird avian influenza. Wild waterfowl are major reservoirs of low pathogenicity avian influenza (LPAI) viruses, but these viruses rarely are passed to poultry. It is not known if poultry are susceptible to wild bird influenza viruses. Sixteen H4, H6 and H9 LPAI viruses obtained from wild birds were inoculated into chickens and turkeys. Eleven of the 16 viruses infected chickens and eight of 16 viruses infected turkeys, although none of the birds became ill except for one virus that caused mild upper-respiratory disease in turkeys. The viruses that did infect grew to very low quantity. These data suggest that chickens and turkeys have limited susceptibility to these wild bird LPAI viruses and when infected, low amounts of virus are produced which would limit spread within and between flocks. 4. This accomplishment is in National Program 103 Animal Health (100%), Component 4 – Countermeasures to prevent and control respiratory diseases. Problem Statement 4C: Poultry Respiratory Diseases. Previous exposure to low pathogenicity avian influenza (LPAI) provides partial protection to wild waterfowl against H5N1 high pathogenicity avian influenza (HPAI). Avian influenza viruses infect many species of wild waterfowl without causing disease. It is unknown if such infections would provide protection against H5N1 HPAI viruses. An experimental study was conducted in wood ducks to determine if exposure to low pathogenicity avian influenza viruses would produce antibodies and protect the birds from H5N1 HPAI virus challenge. Birds were exposed to H1N1, H5N2, and H5N1 LPAI viruses by intranasal inoculation followed by intranasal challenge 3 weeks later by an H5N1 HPAI virus. 100% of the wood ducks survived the HPAI virus challenge when previously exposed to an H5N1 LPAI virus from North America. Protection rates with other H5N2, H5N1 and H1N1 LPAI viruses ranged from 20-80%. Previous exposure of wild waterfowl to H5 or N1 LPAI viruses may provide partial or complete protection from H5N1 HPAI virus infection. 5. This accomplishment is in National Program 103 Animal Health (100%), Component 4 – Countermeasures to prevent and control respiratory diseases. Problem Statement 4C: Poultry Respiratory Diseases. New natural sources of shikimic acid discovered. The antiviral drug oseltamivir phosphate is the most effective treatment for human infections of H5N1 high pathogenicity avian influenza (HPAI) viruses. However, the major limiting factor in manufacturing of adequate supplies of oseltamivir is the availability of the raw materials, shikimic acid. The cooperator, Stephen F. Austin University, investigated common native plants in North America as potential new high-yield sources of shikimic acid. Over 3,000 samples representing more than 820 species from the 131 families were examined. The cooperator identified 11 genera of native American plants including six genera of angiosperms and five genera of gymnosperms with high shikimic acid yield. Sweet gums had higher levels of shikimic acid in the early growing season (April to July) than other seasons, and the leaves and fruits had highest levels of shikimic acid content. Evergreen pines showed no significant seasonal change in shikimic acid content, and leaves were the preferable harvest materials at any season. Pruning significantly improved the shikimic acid production. A feasible commercial extraction method using water and ethanol extraction and purification was developed. These studies have identified new sources of shikimic acid for manufacturing the anti-influenza drug oseltamivir phosphate. 5.Significant Activities that Support Special Target Populations None.