2008 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 This project falls within the 2005-2010 Animal Health National Program (NP103) Action Plan, Component 1 - Bio-defense research (Foreign and Emerging Animal Diseases) and Component 4 - Countermeasures to prevent and control respiratory diseases. More specifically the research addresses Agency Performance Measure 3.2.1 (Provide scientific information to protect animals from pests, infectious diseases, and other disease-causing entities that affect animal and human health) and 3.2.3 (Develop and transfer tools to the agricultural community, commercial partners, and Federal agencies to control or eradicate domestic and exotic diseases that affect animal and human health). The research generated during FY2008 addressed each of the four objectives above. In objective one, Scientists performed multiple experiments aimed at comparing the route of delivery of inactivated vaccines and examined protective efficacy following challenge. Overall most of the mucosal vaccines tested induced an immune response, however only a few were able to confer any protection against highly pathogenic avian influenza (AI) challenge. We also examined the influence of adjuvants in the formulation of mucosal AI vaccines and demonstrated that a number of them were able to enhance the immune response. Both of the milestones associated with this objective were fully met. Milestones for objective two were fully met, as numerous bird studies were completed in chickens and ducks that examined the cytokine responses in lung, spleen, bursa or Peyers patch with real-time reverse transcriptase-polymerase chain reaction (RRT-PCR). Overall these results are extending our knowledge of how different bird species recognize and respond to AI. In objective three, we have completed some sequencing of the Mx and TLR7 genes from bird species. Our findings to date indicate a single polymorphism in the Mx protein can extend the mean death time in commercial chickens. Although the birds are still susceptible to high pathogenicity avian influenza (HPAI), the observations that a single change in one protein can have such a dramatic effect in the birds is promising. We plan to continue the work in this area to examine polymorphisms in other avian immune response genes. In objective four, we performed comparisons with three different microarray platforms with tissues taken from chickens infected with HPAI H5N1. The results suggest an unregulated proinflammatory response, coupled with a delayed interferon response may play a role in the inability of the birds to overcome infection. Future studies are planned in subsequent years. 4.Accomplishments 1. MICROARRAY ANALYSIS OF EARLY RESPONSE GENES OF CHICKENS FOLLOWING HIGHLY PATHOGENIC AVIAN INFLUENZA (HPAI) INFECTION. Within the last few years, outbreaks of HPAI have originated in Asia and spread through several Middle Eastern, African and European countries, resulting in one of the most serious animal disease incident in recent history. To better understand the mechanisms of pathogenesis of these highly pathogenic avian influenza viruses (HPAIV) in poultry we have infected chickens with the Egret/Hong Kong/757.2/02 isolate of H5N1 HPAIV. Evidence of a pronounced inflammatory response that resembled mammalian cytokine responses was observed in infected birds. In contrast, this inflammatory response was absent in vaccinated birds. An involvement of multiple inducers and responders of the interferon pathway was evident. Semiquantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis was used to confirm expression at the ribonucleic acid (RNA) levels, and immunohistochemistry confirmed expression of critical genes at the protein level. This accomplishment is in the National Program 103, Animal Health (100%). Component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Disease, and Component 4: Countermeasures to Prevent and Control Respiratory Diseases, Problem Statement 4C: Poultry Respiratory Diseases. 2. THE ROLE OF THE NS1 PROTEIN IN THE PATHOGENESIS OF AVIAN INFLUENZA VIRUSES IN DUCKS. The innate immune response to viruses has a crucial role in the outcome of infection and manifestation of disease. The non-structured 1 (NS1) protein of influenza viruses has the ability to antagonize interferon and other immune related genes in mammals and in chickens. However, the role of NS1 protein in ducks has not been determined. To better understand the differences in pathogenicity of high pathogenicity avian influenza (HPAI) viruses in ducks and the role of the NS1 protein, we investigated the ability of wild-type and recombinant viruses to replicate in duck cells and regulate gene expression. Wild-type viruses showed differences in replication kinetics and host gene expression; however, no effect on replication or host gene expression was observed due to recombination of the NS1 genes. These findings expand our understanding of the pathogenesis of avian influenza viruses (AIVs) in ducks. This accomplishment is in the National Program 103, Animal Health (100%). Component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Disease, and Component 4: Countermeasures to Prevent and Control Respiratory Diseases, Problem Statement 4C: Poultry Respiratory Diseases. 3. IDENTIFICATION OF A MINIMAL PROTECTIVE VACCINE DOSE FOLLOWING MUCOSAL VACCINATION WITH INACTIVATED H5 VACCINE. Most available commercial vaccines for avian influenza (AI) virus for application in poultry are inactivated vaccines because of the concern about reversion to virulence with the use of attenuated AI vaccines. Because avian influenza virus (AIV) initiates infection of the host on mucosal surfaces, ideally a vaccine that induces a strong mucosal infection would provide the best protection. We tested differing concentrations of inactivated AI virus in vaccines applied to mucosal surfaces. Most all birds receiving low concentrations were not protected; however, the highest does tested increased mean death time following challenge with highly pathogenic AI. These results serve as basis for developing candidate mucosal vaccines to induce protective immunity against AI. This accomplishment is in the National Program 103, Animal Health (100%). Component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Disease, and Component 4: Countermeasures to Prevent and Control Respiratory Diseases, Problem Statement 4C: Poultry Respiratory Diseases. 4. REDUCED VACCINE DOSE AGAINST AVIAN INFLUENZA NOT A GOOD IDEA. Vaccination is an emergency tool that can be used to confront outbreaks of H5N1 high pathogenicity avian influenza (HPAI), but the number of vaccine doses is limited. To determine if the available vaccine doses could be stretched by using reduced vaccine dose, but maintain adequate efficacy, a vaccination-challenge study was conducted in chickens. At full, half, one-fourth and 1/10 of the avian influenza (AI) vaccine doses, all AI vaccinated chickens were protected from disease and death, but using less vaccine than the full dose had some negative effects including reduced serological titers, more chickens excreting challenge virus, and higher quantities of challenge virus growth and excretion from intestines and respiratory system. Most importantly, the mean protective dose (PD50) was 1/50 dose and using the international regulatory standard of 50 PD50 for Newcastle disease vaccine as a guide, the full dose of the AI vaccine would be the minimum dose acceptable for use in the field. Use of the full vaccine dose is especially important because protection in commercial chickens in the field is typically less than seen in experimental studies in specific pathogen free chickens in the laboratory. This accomplishment is in the National Program 103, Animal Health (100%). Component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Disease, and Component 4: Countermeasures to Prevent and Control Respiratory Diseases, Problem Statement 4C: Poultry Respiratory Diseases. 5. POLYMORPHISM IN CHICKEN MX GENE INFLUENCES SUSCEPTIBILITY TO AVIAN INFLUENZA VIRUS INFECTION. The Mx protein is produced by host cells in response to interferon, and has been shown to confer protection against influenza in mammalian studies, but the role of the Mx protein in protection from influenza in poultry is currently not known. We evaluated whether the resistant- or susceptible- form of Mx protein influenced pathogenesis of high pathogenicity avian influenza (HPAI) in chickens of a commercial broiler line. Following challenge with HPAI strain A/Ck/Queretaro/95 almost all the chicks of both genotypes died; however, in the resistant Mx group median survival was 4 days compared with 2 days in the Mx susceptible group. Analysis of cytokine transcripts in spleens revealed that pro-inflammatory and interferon levels were significantly higher in the resistant chicks compared with the susceptible group. Overall, these studies provide the first evidence that Mx has a role in protection against avian influenza infection in chickens. This accomplishment is in the National Program 103, Animal Health (100%). Component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Disease, and Component 4: Countermeasures to Prevent and Control Respiratory Diseases, Problem Statement 4C: Poultry Respiratory Diseases. 6. COMPARISON OF DIFFERENT ADJUVANTS ON HUMORAL IMMUNE RESPONSE TO MUCOSAL AVIAN INFLUENZA VACCINES. Adjuvants are compounds added to vaccines to increase the host immune response to the antigenic portion of the vaccine. Because avian influenza viruses initiate infection on mucosal surfaces, immune stimulation at the site of infection should provide enhanced protection from disease. Chickens were mucosally vaccinated via intranasal route with inactivated avian influenza virus formulated with various adjuvants. Results demonstrated increased humoral immune responses in birds receiving the antigen with specific adjuvants; in contrast, other adjuvants tested failed to enhance immunity. The studies provide direct evidence for including adjuvants to enhance the mucosal immune response of chickens to avian influenza vaccines. This accomplishment is in the National Program 103, Animal Health (100%). Component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Disease, and Component 4: Countermeasures to Prevent and Control Respiratory Diseases, Problem Statement 4C: Poultry Respiratory Diseases. 7. COMPARISON OF ANTIBODY LEVELS FOLLOWING IN OVO VACCINATION WITH INACTIVATED AVIAN INFLUENZA FORMULATED WITH VARIOUS ADJUVANTS. In ovo vaccination is an industry practice to cost-effectively apply vaccines to chicks within the egg prior to hatch to induce an early immune response. Most in ovo vaccines utilize a live virus in the vaccine formulation due to the inherent lack of immunity of inactivated virus vaccines. Because live viruses are prohibitive for use in avian influenza (AI) vaccines, we formulated inactivated AI vaccines for application in ovo by formulating with adjuvants to enhance the immune response to the inactivated antigen. Following in ovo vaccination, birds were hatched and monitored over time for antibodies produced against avian influenza. The results demonstrated that certain adjuvants were able to enhance the humoral immune response against avian influenza. The adjuvants identified in this study will be used to formulate candidate avian influenza vaccines that can be mass applied to commercial poultry to protect against disease. This accomplishment is in the National Program 103, Animal Health (100%). Component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Disease, and Component 4: Countermeasures to Prevent and Control Respiratory Diseases, Problem Statement 4C: Poultry Respiratory Diseases. 8. MICROARRAY ANALYSIS OF GUT-ASSOCIATED LYMPHOID TISSUE FOLLOWING VACCINATION AND CHALLENGE OF CHICKENS WITH HIGHLY PATHOGENIC AVIAN INFLUENZA. Avian influenza (AI) viruses infect mucosal surfaces of chickens, including those found in the gut. Chickens contain a number of immunologically-distinct gut-associated lymphoid tissues, termed Peyers patches, which serve as antigen processing stations in the gut. In these studies, microarray analysis of Peyers patches were determined to establish the immune response profile of gut-associated tissue from naïve and vaccinated chickens following challenge with highly pathogenic AI. There were 149 genes with greater than 2-fold change in the naïve groups of birds compared to only 2 genes in the vaccinated-challenged group. Overall expression pattern showed that the AI infection group showed higher changes in gene expression (including both enhanced and suppressed genes). Interestingly, there were 48 genes which were more than 2 fold down-regulated in the vaccinated-challenged group indicating a controlled immune response following challenge in vaccinated birds. This accomplishment is in the National Program 103, Animal Health (100%). Component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Disease, and Component 4: Countermeasures to Prevent and Control Respiratory Diseases, Problem Statement 4C: Poultry Respiratory Diseases. 9. H5 INACTIVATED VACCINE PROTECTED TURKEYS FROM H5N1 HPAI VIRUS. H5 inactivated vaccines in USDA Emergency Vaccine Bank protected chickens from H5N1 high pathogenicity (HP) avian influenza (AI) viruses, but protection in turkeys in unknown. Turkeys were vaccinated with an inactivated H5 AI vaccine from the USDA Emergency Vaccine Bank and were challenged intranasally with a high dose of a 2004 Thailand H5N1 high pathogenicity avian influenza (HPAI) virus. The vaccine protected turkeys from clinical signs and death. In addition, the vaccine reduced the growth of the challenge virus in vaccinated turkeys in intestines and respiratory tract. These data indicate the H5 killed AI vaccine in the USDA Emergency Vaccine Bank will protect turkeys from H5N1 HPAI virus. This accomplishment is in the National Program 103, Animal Health (100%). Component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Disease, and Component 4: Countermeasures to Prevent and Control Respiratory Diseases, Problem Statement 4C: Poultry Respiratory Diseases. 10. DIFFERENT METHODS OF INACTIVATION OF AVIAN INFLUENZA VIRUS IN SAMPLES FOR SUBSEQUENT USE IN RESEARCH ASSAYS OUTSIDE OF BIOSAFETY FACILITIES WERE EVALUATED. Biosafety facilities are required when handling samples infected with highly pathogenic avian influenza viruses. For research purposes, samples frequently need further processing and working in biosafety facilities can limit investigations in which the use of additional equipment is required. In order to transfer virus infected samples from biosafety leveled laboratories, avian influenza viruses (AIVs) need to be efficiently inactivated. We tested chemical and physical procedures normally used preceding research applications, for their ability to inactivate these viruses. We found that chemical inactivation with paraformaldehyde at different concentrations, as well as heat treatment of cell lysates and cells, efficiently inactivated the viruses as shown by embryo mortality analysis and hemagglutination assays; however treatment alone with detergent containing lyses buffer did not. The use of paraformaldehyde and heat as high pathogenicity avian influenza (HPAI) virus inactivation methods proved to be suitable for preparation of samples for subsequent use in biological assays outside biosafety facilities. This accomplishment is in the National Program 103, Animal Health (100%). Component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Disease, and Component 4: Countermeasures to Prevent and Control Respiratory Diseases, Problem Statement 4C: Poultry Respiratory Diseases. 11. THE USE OF WHOLE GENOME CHICKEN MICROARRAY HELPED TO DETECT DIFFERENTIALLY EXPRESSED GENES ASSOCIATED WITH H5N1 AVIAN INFLUENZA VIRUS INFECTIONS IN DUCKS. The Asian H5N1 highly pathogenic avian influenza (HPAI) viruses have changed from producing mild respiratory infections in ducks, to some strains producing severe disease and mortality. We examined the differences in host response to infection with H5N1 HPAI viruses with different pathogenicity in ducks by determining differential gene expression in tissues of infected ducks using a chicken genome microarray and detected a large number of genes showing up or down-regulation. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to confirm the regulated expression of several of the differentially expressed genes. The results obtained suggested that different mechanisms are potentially induced by avian influenza (AI) viruses to modulate the host response to infection. The differentially expressed genes identified in this study are candidates for further hypothesis-driven investigation of the mechanisms involved in resistance to AI viruses in ducks. This accomplishment is in the National Program 103, Animal Health (100%). Component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Disease, and Component 4: Countermeasures to Prevent and Control Respiratory Diseases, Problem Statement 4C: Poultry Respiratory Diseases. 12. DIFFERENCES IN THE INNATE IMMUNE RESPONSE DETERMINED IN DUCKS INFECTED WITH DIFFERENT H5N1 AVIAN INFLUENZA VIRUSES. The innate immune response is responsible for detecting invading viruses during the initial stages of infection, which is crucial in determining disease resistance or susceptibility. Comparison of the expression of genes related to the innate immune response in young and older ducks was done in tissues from ducks infected with avian influenza viruses of different pathogenicity. The results indicate differential cytokine expression following infection. Both the age of the ducks and the virus strain affected the outcome of infection and the innate immune response. This study underlines the importance of the innate immune response in ducks to infection with avian influenza and how this virus may regulate this response in different ways. This accomplishment is in the National Program 103, Animal Health (100%). Component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Disease, and Component 4: Countermeasures to Prevent and Control Respiratory Diseases, Problem Statement 4C: Poultry Respiratory Diseases. 13. COMPARISON OF CYTOKINE RESPONSES BETWEEN CHICKENS AND DUCKS FOLLOWING HPAI INFECTION IN SPLEEN. Chickens and ducks show a markedly different response following H5N1 avian influenza infection. Chickens are extremely susceptible to all highly pathogenic H5N1 viruses, while ducks display differential susceptibility depending on the isolate. These studies were designed to identify the differences in the immune response in the spleen (a major immune-related response organ) by measuring cytokine expression immediately following infection. The results demonstrated that chickens displayed hyper-proinflammatory cytokine responses with delayed interferon upregulation. In contrast, resistant ducks induced increased interferon and cytokines which were at a level necessary to resolve the infection. These studies emphasize the differences of innate immune responses in ducks compared to chickens, and provide a molecular basis for disease resistance. This accomplishment is in the National Program 103, Animal Health (100%). Component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Disease, and Component 4: Countermeasures to Prevent and Control Respiratory Diseases, Problem Statement 4C: Poultry Respiratory Diseases. 14. IDENTIFYING INFECTED CHICKENS WITHIN A VACCINATED POPULATION. Avian influenza (AI) vaccines protect chickens from morbidity and mortality and reduce, but do not completely prevent replication of AI viruses in the field. For accurate surveillance programs, infected birds must be identified within the vaccinated populations and eliminated. In this study, chickens were immunized with a commercial biotechnologically advanced fowlpox virus vaccine containing an H5 hemagglutinin gene (rFP-H5) from an AI virus. Chickens immunized with the rFP-H5 vaccine did not develop agar gel immunodiffusion (AGID) antibodies because the vaccine lacked AI nucleoprotein and matrix genes (NP/M), but H5 hemagglutination inhibition (HI) antibodies were present indicating the birds were vaccinated and not infected with the live AI virus. The vaccinated chickens survived high pathogenicity AI virus challenge and antibodies were detected by both AGID and HI tests. This indicates 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 and could be used as an aid in AI eradication efforts. This accomplishment is in the National Program 103, Animal Health (100%). Component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Disease, and Component 4: Countermeasures to Prevent and Control Respiratory Diseases, Problem Statement 4C: Poultry Respiratory Diseases. 15. PROTECTIVE AVIAN INFLUENZA IN OVO VACCINATION WITH NON REPLICATING HUMAN ADENOVIRUS VECTOR. Vaccination for avian influenza is costly to perform because current commercial vaccines require the birds to be individually vaccinated, and alternative mass vaccination strategies are needed. A novel vaccine strategy of using a replication incompetent human adenovirus vaccine vector expressing the hemagglutinin gene of avian influenza was developed and shown to provide protection to virulent challenge to chicken after in ovo vaccination. The in ovo vaccination route is routinely performed in the United States as a hands-free method of mass vaccination in chickens and provides an alternative to commercial vaccines. This accomplishment is in the National Program 103, Animal Health (100%). Component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Disease, and Component 4: Countermeasures to Prevent and Control Respiratory Diseases, Problem Statement 4C: Poultry Respiratory Diseases. 16. CHARACTERIZATION OF INFLUENZA VIRUS VARIANTS WITH DIFFERENT SIZES OF THE NON-STRUCTURAL (NS) GENES AND THEIR POTENTIAL AS A LIVE INFLUENZA VACCINE IN POULTRY. Vaccination for avian influenza is costly to perform because current commercial vaccines require the birds to be individually vaccinated, and alternative mass vaccination strategies are needed. Attenuated avian influenza viruses have not been used as live vaccines because of the concern about reversion to virulence. However, influenza viruses were developed and evaluated with collaborators at The Ohio State University viruses that had different sized deletions in the NS1 protein that attenuates the virus by disrupting the viruses ability to resist the action of interferon. The use of deletion mutants appeared to provide stable mutants that are unlikely to revert to viruelence because of the large segment of the gene that was removed. This study provides a potential approach to attenuate avian influenza viruses so that they can be used as live vaccines that could likely be administered as a mass vaccination. This accomplishment is in the National Program 103, Animal Health (100%). Component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Disease, and Component 4: Countermeasures to Prevent and Control Respiratory Diseases, Problem Statement 4C: Poultry Respiratory Diseases. 17. IMMEDIATE EARLY RESPONSES OF AVIAN TRACHEAL EPITHELIAL CELLS TO INFECTION WITH HIGHLY PATHOGENIC AVIAN INFLUENZA VIRUS. Avian influenza virus (AIV) targets the epithelial cells of the upper respiratory tract. In order to develop new AIV control strategies it is necessary to understand the underlying mechanism of viral infection at mucosal respiratory sites. Infection of chickens with highly pathogenic avian influenza virus (HPAIV) CK/HK/220/97 and Egret/HK/757.2/02 causes 100% mortality and in ducks these isolates cause a range of effects from minor respiratory disease to 100% mortality. While in vivo systems are best for studying disease response, primary cell culture systems provide a valuable intermediate between animal studies and the use of cell lines to study under highly controlled conditions the mechanisms of infection and the immediate host responses. Chicken and duck tracheal epithelial cells systems (TEC) were used to study early host responses to AIV infection on TEC. Chicken and ducks TEC supported viral replication and re-infection but the capacity of the two viruses to replicate was not equal. A 42 thousand oligonucleotide chicken microarray system was used to characterize differences in gene expression between chicken tracheal epithelial cells infected with these two highly pathogenic AIV and identified the expression of virus-specific host responses as early as six hours post infection. These differences may help explain the differences in pathogenicity between both viruses. This accomplishment is in the National Program 103, Animal Health (100%). Component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Disease, and Component 4: Countermeasures to Prevent and Control Respiratory Diseases, Problem Statement 4C: Poultry Respiratory Diseases. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of Non-Peer Reviewed Presentations and Proceedings 1 Review Publications Toro, H., Tang, D.C., Suarez, D.L., Sylte, M.J., Pfeiffer, J., Van Kampen, K.R. 2007. Protective avian influenza in ovo vaccination with non-replicating human adenovirus vector. Vaccine. 25:2886-2891. Swayne, D.E., Avellaneda, G.E., Mickle, T.R., Pritchard, N., Cruz, J., Bublot, M. 2007. Improvements to the hemagglutination inhibition test for serological assessment of recombinant Fowlpox-H5-avian-influenza vaccination in chickens and its use along with an agar gel immunodiffusion test for differentiating infected from noninfected vaccinated animals. Avian Diseases. 51:697-704. Pantin Jackwood, M.J. 2008. Immunohistochemical staining of avian influenza viruses in tissues. In: Spackman, E., editor. Avian Influenza Virus. Methods in Molecular Biology. Humana Press, Totowa, NJ. p. 77-83. Kapczynski, D.R. 2008. Evaluating the cell mediated immune response of avian species to avian influenza viruses. In: Spackman, E., editor. Avian Influenza Virus. Totowa, NJ: Humana press, Inc. p. 108-121. Kapczynski, D.R., Kogut, M.H. 2008. Measurement of avian cytokines with real time RT-PCR following infection with avian influenza. In: Spackman, E., editor. Avian Influenza Virus. Totowa, NJ: Humana Press, Inc. p. 122-129. Steel, J., Burmakina, V., Thomas, C., Spackman, E., Garcia-Sastre, A., Swayne, D.E., Palese, P. 2008. A combination in-ovo vaccine for avian influenza virus and Newcastle disease virus. Vaccine. 26:522-531. Swayne, D.E., Kapczynski, D.R. 2008. Vaccines, vaccination and immunology for avian influenza viruses in poultry. In: Swayne, D.E. editor. Avian Influenza. Ames, Iowa: Blackwell Publishing. p. 407-451. Toro, H., Tang, D.C., Suarez, D.L., Shi, Z. 2008. Protection of chickens against avian influenza with non-replicating adenovirus-vectored vaccine. Vaccine. 26:2640-2646. Goetz, S., Spackman, E., Hayhow, C., Swayne, D.E. 2008. Assessment of reduced vaccine dose on efficacy of an inactivated avian influenza vaccine against an H5N1 high pathogenicity avian influenza virus. Journal of Applied Poultry Research. 17:145-150. Joseph, T., Mcauliffe, J., Lu, B., Vogel, L., Swayne, D.E., Jin, H., Kemble, G., Subbarao, K. 2008. A live attenuated cold adapted influenza A H7N3 virus vaccine provides protection against homologous and heterologous H7 viruses in mice and ferrets. Virology. 378(1):123-132.