2008 Annual Report 1a.Objectives (from AD-416) 1. Develop cassette concept for avian influenza hemagglutinin gene insert for recombinant fowl poxvirus vaccines. 2. Update the H5 AI insert in current licensed recombinant fowl poxvirus product. 3. Developing vaccination protocols for commercial poultry to maximize inactivated AI and recombinant fowl pox vaccine efficacies. 1b.Approach (from AD-416) Avian influenza (AI) hemagglutinin genes for insertion in recombinant fowl poxvirus (rFP) vaccines will be accomplished based on collection, sequencing and analyzing current H5 AI field viruses. New rFP vaccines will be constructed and tested for efficacy in both high and low pathogenicity AI chicken and duck challenge models with measurement of protection being prevention of illness and death, increasing resistance to infection, reduction in number of infected birds and a decrease in the amount of challenge virus shed from respiratory and alimentary tracts. The rFP and inactivated AI vaccines will be tested in commercial poultry with development of priming and boost vaccination protocols to optimize immunity and protection. 3.Progress Report Progress related to Objective 2 of the in-house project: 2. Develop vaccines that effectively stop outbreaks, allow differentiation from natural infection and can be administered in a cost effective manner. Vaccines against avian influenza (AI) are valuable in the prevention and control of the disease and can be used in eradication strategies. During FY2008, a study was conducted in chickens using a licensed recombinant fowl poxvirus vaccine with an AI H5 gene insert (rFP-AI-H5) at 1 day of age and a booster vaccination with inactivated A/chicken/Legok/03 H5N1 oil emulsion vaccine at 3 weeks of age and then challenged by 3 different Indonesian H5N1 high pathogenicity avian influenza (HPAI) viruses. The vaccines protected chicken from all 3 challenge viruses with survival of 80-100% of the birds. The vaccinated birds had detection of challenge virus in orophayrnx of one of thirty vaccinated birds at 2 days post-challenge while all 30 sham birds had virus excretion from the oropharynx. These vaccines and vaccination protocol can be useful in an AI control program against antigenic variant Asian-origin H5N1 HPAI viruses. Avian influenza vaccines protect chickens from morbidity and mortality and reduce, but do not completely prevent replication of wild AI viruses in the respiratory and intestinal tracts of vaccinated chickens. For accurate surveillance programs, infected birds must be identified within the vaccinated population. In this study, chickens were immunized with a commercial recombinant fowlpox virus vaccine containing an H5 hemagglutinin gene from A/turkey/Ireland/83 (H5N8) AI virus (rFP-H5). Chickens immunized with the rFP-H5 vaccine did not develop agar gel immunodiffusion (AGID) antibodies because the vaccine lacks AI nucleoprotein and matrix genes (NP/M), but H5 hemagglutination inhibition (HI) antibodies were present indicating the birds were vaccinated but not infected with the live AI virus. The most consistent and highest HI titers were observed when using A/turkey/Ireland/83 (H5N8) high pathogenicity (HP) AI virus strain as the beta-propiolactone (BPL)-inactivated HI test antigen which matched the hemagglutinin gene insert in the rFP-H5 vaccine. In addition, higher HI titers were observed if ether or a combination of ether/BPL-inactivated virus was used in place of the BPL-inactivated virus. The rFP-H5 vaccinated chickens survived HPAI challenge and antibodies were detected by both AGID and HI tests. In conclusion, we demonstrated that the rFP-H5 vaccine allowed easy serological differentiation of infected from non-infected birds in vaccinated populations of chickens when using standard AGID and HI tests.