2009 Annual Report 1a.Objectives (from AD-416) 1. Characterize mucosal immunity induced by natural infection and vaccination with both high and low pathogenicity AIV to identify innate and adaptive immune indicators of protection. 2. Characterize the cellular and humoral immune responses to mucosal vaccination, and develop improved methods for identifying cells, cytokines and antibody from mucosal sites. 3. Assess immune variability of the host, both in different poultry species and lines, and non-poultry avian species, by examining the frequency of genetic polymorphism in host genes related to innate and adaptive immunity (toll-like receptors, cytokines, chemokines). 4. Using reverse genetics approaches for avian influenza and Newcastle Disease Virus, examine the role of individual viral genes in host gene response. 1b.Approach (from AD-416) New vaccine approaches for controlling avian influenza virus (AIV) in poultry will be developed following application of antigen via different routes of exposure to the mucosal system. Characterization of the mucosal immune response following vaccination and challenge will be used to extend the understating of the role of local immunity against AIV. Novel mucosal vaccines will be developed and tested with and without adjuvants to enhance immunological response to vaccination. The protective role of serum and mucosal antibodies will be ascertained by passive administration of antibodies to naive birds followed by challenge. The role of cell mediated immunity against avian influenza will be determined following adaptive transfer of isolated lymphocyte fractions from birds previously exposed to live avian influenza and provided to naïve birds prior to challenge. The pathogenomic basis of protection will be delineated by genomic characterization through sequence analysis of immune regulatory factors, including cytokines and toll-like receptors, from immunologically competent and naïve birds. Reverse genetics will be used for vaccine development and pathogenesis studies with AIV by replacement or inactivation of genes involved in virulence and evasion of the host immune response. A comparison of pathogenesis from recombinant viruses will allow characterization of proteins and motifs involved with establishment of viral infection on mucosal surfaces which may be targets for vaccine development. 3.Progress Report The Mucosal Immunology project has been active both nationally and internationally to meet the objectives and milestones of the research project. Major accomplishments have been achieved in all four objectives. Accomplishments included:. 1)the development of mucosal vaccines to protect poultry against avian influenza viruses;. 2)the construction of novel recombinant vaccine technologies for control of avian influenza in poultry;. 3)the use of reverse genetics technology to study pathogenesis of avian influenza viruses in poultry and waterfowl; and. 4)the study of genomic markers for increased innate resistance to avian influenza viruses. Collaborative research continues with national and international partners to continue to study the immune response of poultry to avian influenza. University partners include, but not limited to, the University of Georgia, the University of Delaware, the University of Arkansas, and the Ohio State University. Collaborative work with industry has included projects with CEVA Biomune, and Goldsboro Milling Company. Internationally, collaboration with the U.S. Department of Agriculture, Office of International Research Programs and the All Russian Research Institute for Animal Health has continued to support the development of mucosal vaccines and immunology against avian influenza. 4.Accomplishments 1. LACK OF BROAD H7 VACCINE PROTECTION AMONG DIVERSE H7 AVIAN INFLUENZA FIELD VIRUSES. Avian influenza (AI) viruses of H7 subtype cause mild to severe disease in poultry and is a regulated disease. To better understand protection, we vaccinated chickens with recombinant fowlpox vaccine with one of three different H7 gene inserts (Australian lineage, Vic/85; Eurasian lineage, Italy/99; North American lineage VA/02) and then challenged with Italy/99 high pathogenicity avian influenza virus. Good protection to virulent challenge was only observed in chickens vaccinated with the Italy/99 vaccine and not the other two vaccines. This suggests a single H7 AI vaccine will not protect against the diverse types of H7 field viruses present worldwide and vaccine may need to be from the same H7 lineage as the field/challenge viruses to provide protection. 2. CROSS REACTIVE ANTIBODY AND CYTOTOXIC T LYMPHOCYTES FROM AVIAN INFLUENZA INFECTED CHICKENS PROTECTS AGAINST CHALLENGE FROM A VARIETY OF AVIAN INFLUENZA ISOLATES. Immunity against avian influenza (AI) is largely based on the induction of neutralizing antibodies produced against the viral hemagglutinin protein, although host lymphocytes can also detect and destroy infected cells helping to clear virus from the host. In these studies, chickens were infected with a recent H9N2 AI isolate, and antibody and lymphocyte cross reactivity against homologous (H9N2) and heterologous (H6N2 and H5N9) AI viruses were determined. Results indicate antibodies produced against H9N2 AI displayed better cross reactivity to the H6N2 isolate than the H5N9 isolate. Additionally, lymphocytes from H9N2-infected chickens displayed cross reactivity with all of the isolates tested here. Taken together, these studies provide insight into the cross reactive nature of avian lymphocytes against AI viruses which can be utilized for vaccine development. 3. THE ROLE OF AVIAN INFLUENZA VIRUS NON-STRUCTURAL PROTEIN 1 (NS1) IN THE PATHOGENICITY OF HIGHLY PATHOGENIC AVIAN INFLUENZA (HPAI) VIRUS H5N1 IN DUCKS. The avian influenza (AI) virus NS1 protein is known to suppress the immune response in influenza virus-infected hosts, allowing more virus replication which contributes to increased pathogenicity and virus spread. In order to determine if the NS1 protein of the AI virus contributes to the increased virulence observed with H5N1 HPAI viruses in ducks, avian influenza viruses were constructed in the laboratory with NS1 genes from different parent viruses. The different NS1 genes had the same effect on pathogenicity and on the expression of immune-related duck genes upon infection. This suggests that other viral genes, apart from the NS1 protein, are most likely contributing to the increased pathogenicity of H5N1 HPAI viruses in ducks. Understanding the origin of the differences in pathogenicity between viruses helps to identify new targets for control of AI. 4. CYTOKINE RESPONSE FROM CHICKENS DIFFERING IN MYXOVIRUS RESISTANCE (MX) GENE DIFFERENCES AGAINST AVIAN INFLUENZA VIRUS. Myxovirus-resistance (Mx) proteins are a group of related proteins produced by host cells in response to viral infections and some of the Mx proteins in mammals have been shown to limit replication of influenza and other viruses. Chickens have the MX gene, but the protein is believed to be non-functional as an antiviral gene in some birds. The idea of using selective breeding of poultry to contain the functional Mx gene is an attractive approach to improve genetic resistance of chickens to avian influenza (AI) viruses. Following infection with highly pathogenic AI viruses, differences in mean death time were observed between different Mx-specific chickens as well as differential cytokine expression patterns between chickens differing in the Mx gene. The Mx gene appears to play a role in the innate immunity of chickens to avian influenza, but the Mx gene provides only partial protection to chickens to highly pathogenic avian influenza virus. 5. DETERMINED THE CONTRIBUTION OF AI VIRAL GENES IN THE PATHOGENICITY OF AVIAN INFLUENZA VIRUS IN DUCKS. The pathogenicity of H5N1 avian influenza (AI) viruses in ducks has increased over time, with recent viruses being much more pathogenic. Avian influenza viruses are highly variable in genetic sequence, and it is difficult to correlate specific genetic changes with pathogenicity. We constructed influenza viruses with specific sets of genes using a method called reverse genetics, and compared the contribution of individual viral genes in the increased pathogenicity observed with H5N1 AI viruses in ducks. Two genes, the hemagglutinin and the neuraminidase, considerably affected pathogenicity, reflected in increased disease and mortality in the ducks. The identification of viral genes associated with pathogenicity of avian influenza virus is basic to our understanding of the disease and implementing control measures. 6. EARLY PROTECTION IN DUCKS AGAINST H5N1 HIGHLY PATHOGENIC AVIAN INFLUENZA (HPAI) CONFERRED BY VACCINATION. Domestic ducks that are grazed in open fields, free range ducks, have been implicated in the spread and persistence of H5N1 HPAI viruses. Vaccination is being used as a tool to control avian influenza (AI) in domestic ducks; however protection from vaccination needs to be obtained early because ducks are released to the field at one month of age. By comparing different schedules of vaccination, it was determined that vaccinating at 7 days and boosting at 21 days of age protected ducks against disease and mortality after a virulent H5N1 HPAI challenge at a month of age. This study provides practical recommendations for vaccinating free range ducks with commercial vaccines that will help in the control of AI. 7. SEROLOGIC CROSS REACTIVTIY OF TURKEYS VACCINATED FOR H1N1 AVIAN INFLUENZA AGAINST THE 2009 SWINE-ORIGIN H1N1 INFLUENZA VIRUS. Beginning in April 2009, a novel H1N1 influenza virus has caused acute respiratory disease in humans, first in Mexico and then spreading around the world. The presence of avian and swine influenza virus genes in the 2009 novel H1N1 virus raises the potential for infection in poultry following exposure to infected humans or swine. In the U.S., turkeys and swine may be raised together in close proximity and are typically vaccinated against field isolates of avian influenza (AI), including the H1N1 subtype. Serum from turkeys vaccinated against H1N1 AI viruses displayed good reactivity against the vaccine virus, however, limited cross reactivity to the 2009 influenza virus was observed. This indicates H1N1-vaccinated turkeys are likely not protected against swine-origin H1N1 using the currently available vaccines. 8. DIFFERENCES IN RESPONSE TO VACCINATION AGAINST H5N1 HIGHLY PATHOGENIC AVIAN INFLUENZA (HPAI) BETWEEN PEKIN AND MUSCOVY DUCKS DEMONSTRATE IMMUNE RESPONSE DIFFERENCES. Vaccination is being used to control H5N1 HPAI in poultry including domestic ducks, but avian influenza (AI) vaccination in ducks in the field has shown mixed results in different duck species. Clear differences in response to H5N1 AI vaccination were found between two different domestic duck species; Muscovy (Cairina moschata) and Pekin ducks (Anas platyrhynchos). Differences in the severity of clinical signs, antibody titers, and viral shedding after vaccination were observed between these two duck species, with Muscovy ducks having less protection after vaccination and longer duration of virus shedding. Differences in innate immune-related host responses to AI infection were observed in vaccinated and non-vaccinated ducks, which may explain the differences observed in response to vaccination. This information helps our understanding of the immune response in ducks to consider improved vaccine strategies for control of HPAI in this and other avian species. 9. CHARACTERIZATION OF TRIPLE REASSORTANT H3N2 AVIAN INFLUENZA VIRUSES FROM TURKEYS IN THE UNITED STATES. Triple reassortant (TR) H3N2 avian influenza viruses have become endemic in the U.S. turkey population. These viral infections do not usually result in mortality but can cause severe clinical disease in turkeys leading to reduced egg production and poor body weight gain, accounting for huge economic losses. Turkey origin TR H3N2 viruses isolated from different geographical locations and years shared high genetic and antigenic similarity, and replicated and transmitted efficiently in turkeys, but they exhibited poor replication and transmissibility in chickens and ducks. In 26-week-old layer turkeys, one of the TR H3N2 strains tested caused complete cessation of egg production within 13 days post infection. The endemicity of the TR H3N2 viruses in turkeys and associated economic importance underscore the need for their enhanced monitoring, surveillance and control. Review Publications Swayne, D.E., Kapczynski, D.R. 2008. Strategies and challenges for eliciting immunity against avian influenza virus in birds. Immunological Reviews. 225:314-331. Swayne, D.E. 2009. Avian influenza vaccines and therapies for poultry. Comparative Immunology Microbiology and Infectious Diseases. 32:351-363. Kapczynski, D.R., Gonder, E., Liljebjelke, K.A., Lippert, R., Petkov, D., Tilley, B. 2009. Vaccine induced protection from egg production losses in commercial turkey breeder hens following experimental challenge with a triple reassortant H3N2 avian influenza virus. Avian Diseases. 53:7-15. Sarmento, L., Afonso, C.L., Estevez, C., Wasilenko, J.L., Pantin Jackwood, M.J. 2008. Differential host gene expression in cells infected with highly pathogenic H5N1 avian influenza viruses. Veterinary Immunology and Immunopathology. 125:291-302. Lipatov, A.S., Kwon, Y., Pantin Jackwood, M.J., Swayne, D.E. 2009. Pathogenesis of H5N1 influenza virus infections in mice and ferret models differs according to respiratory tract or digestive tract system exposure. Journal of Infectious Diseases. 199:717-725. Wasilenko, J.L., Sarmento, L., Pantin Jackwood, M.J. 2009. A single substitution in amino acid 184 of the NP protein alters the replication and pathogenicity of H5N1 avian influenza viruses in chickens. Archives of Virology. 154:969-979. Pillai, S.S., Pantin Jackwood, M.J., Jadhao, S.J., Suarez, D.L., Wang, L., Yassine, Y., Saif, Y., Lee, C. 2009. Pathobiology of triple reassortant H3N2 influenza viruses in breeder turkeys and its potential implication for vaccine studies in turkeys. Vaccine. 27:819-824. Pantin Jackwood, M.J., Swayne, D.E. 2009. Pathogenesis and pathobiology of avian influenza virus infection in birds. OIE Scientific and Technical Review. 28(1):113-136.