Progress 10/01/04 to 09/30/09
Outputs
OUTPUTS: During the last year of the study we compared the outcome of IBV infection in vaccinated and unvaccinated birds 48 hours after challenge by the MASS 41 serotype of IBV. We looked at the effects of two different vaccines, the MASS41 specific vaccine and the Ark vaccine. During this past year we completed evaluation of our microarray data, validated the data using real time PCR and developed and used Western blots to document the predicted changes in protein levels during IBV infection. Evaluation of the microarray data revealed that IBV infection resulted in significant changes in intracellular signalling pathways involving AKT, ERK, and JNK. Furthermore, vaccination against IBV altered mRNA expression in these pathways and different types of vaccination led to differences in mRNA production. These outcomes were different than anticipated. We anticipated that the major changes in tracheal tissue after IBV infection would involve upregulation of cytokines. Though we cannot rule out the importance of cytokines in IBV infection, we can say that other intracellular signalling cascades seem to be more important in determining the oucome of disease in tracheal tissue. Because of the intracellular signalling pathways that were involved, it was necissary to validate our findings with Western blots. Thus, we adapeted Western blots specific for the phosphorylated (active) forms of AKT, ERK and JNK as well as for total AKT, ERK and JNK. It was clear that 48 hours after infection the total and active AKT, and ERK levels differed between treatment groups. Furthermore, the protein levels differeed between the two vaccinated groups. Thus, even though histopathological findings comparing the two vaccinated groups were identical, our data show significant differences in how these vaccines affect animals at a molecular level. Additionally, we conducted in vitro experiments to verify our in vivo results. We saw similar changes in our in vitro experiments (tracheal explants) and documented that the changes in AKT, and ERK levels could be detected within the first several hours of infection. Moreover, we demostrated that the effects were specific to IBV as other cellular stressors (UV light and avian influenza infection) produced different results. These findings are significant because the point to a different mechanism of IBV pathogensis than previously thought. We predicted that cytokine pathways would be the most important pathways upregulated during IBV infection. This was not the case. In fact the pathways that were upregulated are important in determining cell survivial vs apoptosis. Because of this unexpected finding, we believe that we will be able to demostrate that alterning these intracellular signalling pathways with pharmacological agents will significant differences in dease outcome. In the long term we hope to be able to exploit these pathways to develop novel control stratagies for IBV. PARTICIPANTS: Dr. Shankar Mondal-post doctoral associate Dr. Micheal Piepenbrink-post doctoral associate Ms. Shira Grul-DVM student participating in summer VIP research program TARGET AUDIENCES: We consutled both poultry produces and state poultry health official during the completion and development of this project. The ensured that the research was of interest to them and that it met a need in New York. The research directly targets poultry producers as it is aimed at developing novel IBV prevention stratagies. In designing these experiments we consulted with New York State producers so that our experimental vaccination stratagies could be based on those done in the field. Additionally, the vaccines we tested were chosen due to input from the New York State Departmet of Agriculture and Markets. Specifically, they wished to know if the Ark vaccine used in this study would protect birds against IBV. PROJECT MODIFICATIONS: We did additional experiments based on the data that we collected during our microarray experiments. Because the microarray and real time PCR data indicated that AKT, ERK and JNK signalling was important to IBV infection, we had to modify our laboratory analysis to validate these findings. This included validation of Western blot techniques and the development of a novel tracheal explant system in which to study these signal pathways in vitro.
Impacts
The findings and techniques developed in thise study have multiple impacts. First, we demostrated that intracellular signaling of AKT and ERK is important in early IBV infection and confirmed this by microarray, real time PCR and Western blot. These finding have not previously be reported in IBV and could prove to be important factors in determining disease outcome. Additionally, by using these techniques to compare outcomes of vaccination, we have descovered that though each vaccine resulted in protection of the birds from IBV, the molecular mechanism of protection appears to be different. Ark vaccine showed different levels of AKT and ERK than did MASS41 vaccineation. These findings give new insight into how vaccinations work and in the long term may prove important when developing new vaccines for chickens. Finally, we have validated new techniques for use in birds. During the completion of this project we successfully used microarray analysis to identify proteins of interest during IBV infection. We then were able to validate Western blot analysis of intracellular signalling molecules AKT, ERK and JNK in domestic chickens. This had not previously been done. Moreover, we developed a novel tracheal explant culture system in which to study IBV infection in vitro and used it to confirm and add to our in vivo results. These results are very important in realizing our long term goal to develop novel vaccine methods for IBV. Because AKT and ERK signal transduction cascades appear to be important in early IBV infection, we can target those pathways for intervention. Pharmacologic agents are commercially available that can selectively block these pathways. Using our newly developed tracheal explant system we will be able to test the toxicity of these compounds in vitro. We will also be able to determine how application of these compounds affects the outcome of IBV infection. Once the action of the compounds has been determined in vitro we hope to be able to integrate then into a vaccinaation protocol to be tested in vivo. Because of the size of the project and the need to validate techniques in avian tissues, we have not produced any publication this year. However, three publications outlineing our results are currently in preparation and we hope to have them submitted for peer review within the next three months.
Publications
- No publications reported this period
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: During the last year of this project we have made signifcant progress in multiple areas. We have 1) conducted a microarray experiment comparing the subcellular response of vaccinated and vaccinated chickens to acute infection with two strains of infectious bronchitis virus (Mass and Ark); 2) validated the microarray results from the previous funding year using real time PCR and conducted pathway analysis on the data and 3) We have finished cloning the Mass 41 genome into pBeloBac 11, corrected errors in the sequence, added a CMV promoter and a GFP marker to the construct. These results have been disseminated to New York State producers, the New York State Department of Agriculture and Markets and university researchers by a presentation at the Unit of Avian Health Advisory Council Meeting in 2008. Additionally publications are in process that will disseminate these results to the wider community. PARTICIPANTS: This project provided multiple oportunities for training of individuals, collaboration with partners and in my own professional development. During the last funding period, Dr. Shankar Mondal, my post doctoral associate worked on developing the BAC and processing microarray experiments. He is currently drafting a paper on the microarray results. He has completed his post doctoral training period and has taken a job working to control avian influenza in Bangladesh. Additionally, I have worked on learning how to analyze microarray data and conduct computer aided pathway analysis. These are skills I did not previously have. The project has also allowed for multiple collaborations. Because of the MAP kinsae data generated by our experiment, I have begun a collaboration with Dr. Mark Roberson, a collegue with long term experience in working with MAP kinases. Additionally, because of the interest by New York state poultry producers in using the Ark vaccine, I was able to collaborate with the New York State Department of Agriculture and Markets and provide data that will help them make management decisions. TARGET AUDIENCES: This data was presented to the target audience of poultry producers and veterinarians at the New York State Department of Agriculture and Markets. The initial data indicating that the Ark vaccine cross protects against Mass type infectious bronchitis virus is of great interest to both groups. There is interest in whether use of the Ark vaccine in New York would be beneficial, and this initial experiment indicates that it might be. Additionally, the initial microarray data indicating that apoptosis and MAP kinase pathways contributed to the pathogenesis of infectious bronchitis, may eventually be used to develop better control techniques for the virus. Finally, once completed, the BAC will allow us to better understand how individual viral genes contribute to infectious bronchitis virus pathogenesis. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The outcomes of the microarray experiments have be very interesting. During the previous funding year we conducted the experiment and did initial analysis of the data. During this funding year we validated the results of our analysis using real time PCR and conducted pathway analysis on the outcomes. Real time PCR validated our initial results and computer aided molecular pathway analysis indicates that the major differences in unvaccinated chickens to infection with different Mass type IBV viruses involve the apoptosis pathways as well as Map Kinase pathways. This was unexpected as our hypothesis was that cytokine responses would differ between the groups. This has significance in that possible future vaccination or prevention strategies might be better aimed at preventing apoptosis and intracellular signaling cascades rather than enhancing specific arms of the immune response. In the animal experiment conducted during this funding cycle, we aimed to further characterize how vaccinated birds respond challenge with infectious bronchitis virus. We vaccinated birds with either Mass or Ark vaccine and then challenged with Mass. We are currently analyzing this data, but it does appear that Ark vaccine will cross protect against Mass IBV. This is important to New York State because the Department of Agriculture and Markets is considering weather to allow the use of Ark vaccine in New York State. I will be consulting with them on the results of this study. Furthermore, we are currently analyzing the microarray data from this experiment. Because we have cross protection between two serotypes of infectious bronchitis, the data will yield important information regarding the mechanisms of immunity against infectious bronchitis. Finally we have made great progress in constructing a BAC containing infectious bronchitis virus. During the last funding period we cloned the genome into the BAC. During this funding period we finished sequencing our construct, corrected errors introduced during the cloning process and added a CMV promotor and GFP marker upstream of our construct. Addition of the CMV promotor will allow us a wider range of cell culture options as we try to recover virus from the BAC and the GFP marker allows us to rapidly screen cell cultures for successful transfection.
Publications
- No publications reported this period
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: During the 2006-2007 funding year we made significant progress in completing the goals outlined in our proposal. The main goal for Year 1 of this proposal was to compare the gene expression profiles in the tracheas of chickens infected with a field strain and a vaccine strain of infectious bronchitis virus (IBV) and to correlate these findings with morphologic changes seen in the infected tissues. The second goal of the project is to create a Bacterial Artificial Chromosome (BAC) containing the complete genome of the Mass 41 strain of IBV. Thirty-six, four-week-old specific pathogen free white leghorn chickens were divided into three groups containing twelve birds each. Birds received either ocular inoculations with field strain Mass 41 IBV, a vaccine strain of IBV or PBS. Birds were euthanized 48 hours after infections and their tracheas were examined for gross lesions. Samples of trachea were collected for histopathological analysis and for processing for gene expression
profiling. Sections of trachea from each bird were fixed in 10% neutral buffered formalin , processed routinely for light microscopic examination. All tissues were examined by the PI and scored based on degree of inflammation, edema and epithelial changes. RNA was extracted from the fresh tracheas using the Qiagen RNeasy fibrous tissue kits and submitted to the Cornell Core Microarray Facility for analysis. Tissues were processed using the Affymetrix Chicken genome microarray and analyzed by the PI and personnel at the core facility. Changes in gene expression that were 2 or more times increased or decreased from the controls were considered significant and are being targeted for validation using real time polymerase chain reaction. In addition to the microarray experiments we were able to make significant progress in the development of a IBV BAC. A cDNA library of the Mass41 genome was created by cloning seven overlapping segments of the genome into separate Topo TA plasmid vectors
and expressing them into E. coli. A second cDNA library was created by cloning three larger overlapping segments of the IBV genome into pBeloBac11 (New England Biolabs). Using natural and inserted restriction sites in these fragments, the genome segments were assembled to form a full length cDNA clone of the IBV genome. This full length cDNA clone was inserted into pBeloBac11, downstream of a T7 promoter. After transfection into E. coli the recombinant cDNA clone was and compared to that of the wild-type virus. The sequence was analayzed for mutations which could prevent the recombinant virus from replicating. The results of our studies were reported to the Avian Health Advisory Board during their annual meeting at Cornell, University. Representative of local poultry producers, New York State Agriculture and Markets as well as extension veterinarians were present at this meeting. Additionally, the results of the BAC development were presented at the Annual Meeting of the American
Association of Poultry Pathologists in Washington DC.
PARTICIPANTS: Dr. Elizabeth Buckles is the PI on the project and supervised all aspects. She also performed all histopathology and coordinated animal experiments. Dr.Shankar Mondal is a Post Doctoral Associate-he worked on the BAC development and helped with in vivo experiments Joy Tseng is an undergraduate student who worked in the lab. She learned basic laboratory procedures and observed some of the molecular biology experiments
Impacts
The resources allocated by this Hatch grant allowed the activities to be completed. The outcomes of the experiments performed during the funding period resulted in significant changes in knowledge regarding the pathogenesis of IBV and resulted in significant progress in the development of a BAC. Specifically, the results of the histopathology indicate that both the field strain and the vaccine strain cause damage to the tracheal epithelium of the chicken. One unexpected finding was the fact that the vaccine strain causes more cellular influx and edema in the infected tissue than did the field strain. Neither strain produces severe changes after 48 hours, however the birds infected with the vaccine strain grossly had more mucous present in the trachea, and histologically there are increased numbers of heterophils when compared to the controls. The reason for this is not known. However, the damage produced by the vaccination may be integral in producing the protective
immune response. Microarray analysis reveals that a large number of genes are differentially expressed in birds infected with both strains of the virus when compared to the controls. Eleven genes were common to all three groups, 19 to the vaccine strain compared to the control, 75 to vaccine and the field and 19 to the vaccine to the controls. These changes are currently being validated by use of real time PCR. However, the initial gene expression analysis documented a greater than two fold increase in the expression of interferon induced protein, Chemokine ligand 20, and caspases 6 and 1 and interleukin 22 receptor. Additionally there was a greater than 2 fold decrease in the expression of MHC type II, programmed cell death protein 4, Angiotensin 2 and Toll-like receptor 6. Until these findings are validated their significance cannot be fully assessed, however the upregulation of caspases suggests that apoptosis plays a role in viral pathogenesis and that down regulation of some
receptors may also play a role in viral infection. The BAC cloning is in its early stages but we have made significant progress. Development of the BAC will greatly aid in future pathogenesis studies. At least one publication by an IBV researcher asserts that IBV cannot be cloned into a BAC. However, we have succeeded. We have not encountered the problems other researchers have had when producing reverse genetic systems of coronaviruses. Specifically, we have not found that any of the Mass41 gene sequences are toxic to E. coli. Our recombinant virus has 36 point mutations when compared to wild-type virus. Three of these mutations cause a genetic frame shift, and if they remain will prevent the virus from being recovered from the BAC. Current work is focusing on correcting these mutations by using a markerless method of homologous recombination.
Publications
- Mondal, S.P. and E. L. Buckles. 2007. Cloning of the pathogenic M41 strain of avian infectious bronchitis virus as bacterial artificial chromosome (poster). Proceedings of the 144th AVMA Annual Convention, July 14-18, 2007, Washington, DC.
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