Progress 10/01/06 to 09/30/09
Outputs
OUTPUTS: During the entire period of this project, numerous chicken primary cells were isolated from various embryo tissues, in addition to embryo fibroblastic (CEF) cells. These different primary cells along with the immortal CEF cell lines were compared to determine the best permissive host for the in vitro propagation of ILTV. Primary cells derived from kidney, liver, lung, and muscle tissue, plus fibroblasts, and the DF-1 CEF cell line were permissive to ILTV infection. However, virus titers were detected only when ILTV was propagated in kidney, liver, and lung primary cells. ILTV titers produced from both liver (50000 pfu/ml) and kidney (40000 pfu/ml) cells were higher than ILTV titers propagated from embryonic lung cells (2000 pfu/ml). To further investigate host-ILTV interaction, microarray analysis was performed to find differentially expressed host genes by ILTV infection in cultured chicken cells. This work was in collaboration with Huaijun Zhou at TAMU, and Jun Jin Song at UARK. Agilent 4X44 K chicken oligo custom microarray, which was developed by Dr. Zhou, was used. As results, 789 genes showed highly differential expressions in the time course of ILTV infection in lung cells. The differential expression due to ILTV infection include genes responsible for immune responses, cell cycle regulation, and cellular metabolism. A bioinformatics tool (Ingenuity Pathway Analysis) was used to analyze gene functionalities in the scope of biological functions and pathways on the group of 789 genes that exhibited highly variable expression. A bioinformatics tool (Ingenuity Pathway Analysis) was used to analyze gene functionalities in the scope of biological functions and pathways on the differentially expressed genes in ILTV infection, resulting in that genes were classified into a number of functional groups including cancer, genetic disorder, cellular growth and proliferation, and cell death. This project was terminated and a new project (NC1180) was initiated in the similar objectives. PARTICIPANTS: Byung-Whi Kong (PI), Poultry Science, University of Arkansas. Jeong Yoon Lee (Graduate Student, PhD degree), Poultry Science, Cell and Molecular Biology Graduate Program, University of Arkansas. Rakesh Rachamadugu (Graduate Student, PhD degree), Poultry Science, Cell and Molecular Biology Graduate Program, University of Arkansas. Ann Wooming (Program Technician), Poultry Science, University of Arkansas. Collaborators: Walter Bottje, Poultry Science, University of Arkansas. Young Min Kwon, Poultry Science, University of Arkansas. Gisela Erf, Poultry Science, University of Arkansas. Jun Jin Song, Mathmatics, University of Arkansas. Huaijun Zhou, Poultry Science, Texas A&M University. Douglas Foster, Animal Science, University of Minnesota. TARGET AUDIENCES: US poultry industry; Animal/poultry vaccine industry PROJECT MODIFICATIONS: Project was termianted. A new project (NC1180) was initiated with similar objectives.
Impacts
A virus-free immortalized chicken cell substrate, which can efficiently propagate poultry viruses inclucing ILTV, would be suitable for the use in the manufacture of animal vaccine products. The overall impact of a successful outcome will benefit the poultry industrybe by developing a better control strategy against poultry viral diseases. And, the development of functional genomics techniques, including microarray analysis, enables to be realized that genome wide screening methods to identify differentially expressed genes can be powerful tools to understand host regulatory pathways in host-virus interactions. The results of microarray analysis provided the comprehensive knowledge from global gene expression, functionalities, to biological networks among differentially expressed genes in the host response to ILTV infections. Knowledge in host-virus interactions will be utilized to develop a better control strategy for infectious viral disease in poultry industry.
Publications
- Kong BW, Foster LK and Foster DN. Construction of infectious recombinant avian metapneumovirus vectors containing tractable biomarkers. Gobbles. 2008.
- Lee JY, Song JJ, Zhou H, and Kong BW. Transcriptional profiling of host gene expression by the infection of laryngotracheitis virus in chicken embryo lung cells. 2009. Annual Meeting of American Society for Virology, Vancouver, Canada.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Microarray analysis was performed to find differentially expressed host genes by ILTV infection in cultured chicken cells. This work is in collaboration with Huaijun Zhou at TAMU, and Jun Jin Song at UARK. ILTV was infected cultured primary chicken lung cells and cells were harvested at 1, 3, 5, and 7 days post infection. Fluorescently labeled probes generated from RNA samples were hybridized with Agilent 4X44 K chicken oligo custom microarray, which was developed by Dr. Zhou. Preliminary data before the statistical analysis showed that 11493 genes out of 44K array showed considerable signal intensities sorted by SNR (signal to noise ratio) >3. Of 11493 genes, 791 genes showed highly differential expressions in the time course of ILTV infection in lung cells, while 6865 genes and 4437 genes showed moderate variations or no variations, respectively, during the ILTV infection. Differentiallt expressed genes during ILTV infection include genes responsible for immune responses (cytokines, chemokines, MHC), cell cycle regulation (cyclin B2, CDK1, CKI3), metallo proteinase (MMPs), and cell metabolisms. Differential expressions shown at microarray analysis for 20 genes of 791 genes were validated by realtime reverse transcription (RT)-PCR, resulting in that patterns of differential expression shown in microarray analysis were matched closely to differential patterns determined by realtime RT-PCR. And, ILT encoding miRNAs were successfully isolated from ILTV infected cultured cells. Small RNAs derived from ILTV infected cells were used to prepare cDNA for 454 FLX sequencing. This resulted in 38,851 sequence reads containing 9,197 independent readings. 36 individual sequence reads were counted more than 100 times, resulting in 19880 total reads. More than 33,000 reads represented chicken cellular miRNAs, which belong to 475 chicken miRNAs previously deposited in MiRBase. We also recovered 164 ILTV derived miRNAs. Twelve ILTV encoding miRNA sequences were obtained and those were derived from eight ILTV miRNA precursor hairpins. Interestingly, the genomic locations of ILTV encoding miRNAs were biased in the two specific regions of non-coding repeated sequences. PARTICIPANTS: Byung-Whi Kong (PI), Poultry Science, University of Arkansas. Jeong Yoon Lee (Graduate Student, PhD degree), Poultry Science, Cell and Molecular Biology Graduate Program, University of Arkansas. Rakesh Rachamadugu (Graduate Student, PhD degree), Poultry Science, Cell and Molecular Biology Graduate Program, University of Arkansas. Ann Wooming (Program Technician), Poultry Science, University of Arkansas. Collaborators: Walter Bottje, Poultry Science, University of Arkansas. Young Min Kwon, Poultry Science, University of Arkansas. Gisela Erf, Poultry Science, University of Arkansas. Jun Jin Song, Mathmatics, University of Arkansas. Huaijun Zhou, Poultry Science, Texas A&M University. Douglas Foster, Animal Science, University of Minnesota. Hsiao-Ching Liu, Animal Science, North Carolina State University. TARGET AUDIENCES: US poultry industry Animal/poultry vaccine industry PROJECT MODIFICATIONS: None
Impacts
The development of functional genomic techniques, including microarray analysis, enables to be realized that genome wide screening methods to identify differentially expressed genes can be powerful tools to understand host regulatory pathways in host-virus interactions. Furthermore, the host regulatory elements can be utilized to control viral disease. Likewise, charaterization of the functional roles of virus encoding microRNAs will be applied to modulate virual pathogenesis by regulating gene expressions for both virus- and host genes.
Publications
- Kong BW, Carlson DF, Fahrenkrug SC, and Foster DN. 2008. Application of the Sleeping Beauty transposon system to avian cells. Animal Genetics. 39(2):180-6
- Kong BW and Lee JY. 2008.Recombinant vaccine for infectious laryngotracheitis virus (ILTV). Plant and Animal Genome XVI. San Diego, CA
- Lee JY, Wooming A and Kong BW. 2008. Comparison of primary chicken embryo cells for propagation of infectious laryngotracheitis virus (ILTV). Annual Meeting of Poultry Science Association. Niagara Falls, Ontario, Canada
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: To perform research for Objective 1, which is establishment of continuously growing avian cell lines, first, various primary chicken embryo tissue cells were isolated from either 12 day- or 18 day-old specific pathogen free avian supply (SPAFAS) embryos, which were purchased from Charles River Laboratories, North Franklin, CT, for specifically testing propagation of avian infectious laryngotracheitis virus (ILTV). Infectious fowl ILTV (USDA-APHIS-NVSL; lot#: LT83-2) was used as virus stock, which was provided by B. Hargis (University of Arkansas). ILTV propagaion in cultured chicken cells was determined by virus titers were measured by plaque assay. To find suitable homologous cellular substrates, numerous chicken primary cells were isolated from lung, trachea, liver, heart, muscle, intestine, kidney, and bursal tissue, in addition to embryonic fibroblastic (CEF) cells. These different primary cells along with the immortal DF-1 CEF cell line were compared to determine the
best permissive host for the in vitro propagation of ILTV. Primary cells derived from kidney, liver, lung, and muscle tissue, plus fibroblasts, and the DF-1 CEF cell line generated positive CPE. However, virus titers using the plaque assay with the LMH chicken liver tumor cell line (which is widely used for ILTV titering), were detected only when ILTV was propagated in kidney, liver, and lung cells. ILTV titers produced from both liver (50000 pfu/ml) and kidney (40000 pfu/ml) cells were higher than ILTV titers propagated from embryonic lung cells (2000 pfu/ml), while other cells derived from muscle, trachea, bursa, heart, and intestine did not propagate ILTV efficiently. In primary CEF and immortal DF-1 CEF cell lines, ILTV titers were not detectable through plaque assays on LMH cells. Currently, studies on the development of immortal chicken cell lines are being conducted in candidate primary cells derived from kidney, liver, and lung using various molecular constructs that can be
modulate chicken cell cycle regulation.
PARTICIPANTS: Byung-Whi Kong (UARK) serves as PI. In UARK, Ann Wooming and Jeong Yoon Lee are participated as program technician and graduate student, respectively. As collaborators, Billy M. Hargis (UARK; bhargis@uark.edu) and Douglas N. Foster (UMN; foste001@umn.edu) are participated in this project.
TARGET AUDIENCES: US poultry industry; Animal/poultry vaccine industry
PROJECT MODIFICATIONS: None
Impacts
Investigations continue into modulations of cellular factors in the candidate primary cells, such as kidney, liver, and lung, using molecular techniques that can generate continuously growing cell line. A virus-free immortalized chicken cell substrate, which can efficiently propagate poultry viruses inclucing ILTV, would be suitable for the use in the manufacture of animal vaccine products. A certifiable and well characterized continuously growing (immortal) cell line is expected to become primary importance to industry to reduce or eliminate the need for egg or primary animal tissues and subsequent establishment of cell cultures. The implementation of a continuous cell line derived from primary animal tissues should enable manufacturers of biological vaccines to assure better control over production processes, increase product safety and consistency, and ultimately reduce costs. The overall impact of a successful outcome will be improved control of poultry viral
diseases that will benefit the poultry industry. Impact of the research will be derived from new vaccine development based on stable in vitro cell culture system using immortal cell lines.
Publications
- Kong BW, Foster LK, and Foster DN. 2007. Establishment of an immortal turkey turbinate cell line suitable for avian metapneumovirus propagation. Virus Res. 127(1):106-15.
- Kong BW, Foster LK, and Foster DN. 2007. Species specific deletion of the viral attachment glycoprotein of avian metapneumovirus. Virus Res. In press.
- Kong BW, Foster LK, and Foster DN. 2007. A method for the rapid isolation of virus from cultured cells. Biotechniques. In press.
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