Develop new prevention and control strategies for poultry respiratory diseases. Station sub-objectives: <ol>
<li> Establishment of continously growing avian cell line <li> Recombiant vaccine development for avian viral pathogen <BR> 2.1 Reverse genetic systems for RNA viruses <BR>2.2 Virulency defective vaccines for DNA virus
NON-TECHNICAL SUMMARY: Avian viral disease, such as avian influenza, Newcastle disease, infectious bronchitis, pneumovirus, Marek's disease, or laryngotracheitis cause economic losses in poultry industries by decrease productivities. Effective detection and prevention of avian viruses are of the utmost importance to maintain the US leadership in world poultry product markets. Development of more rapidly produced and more effective vaccine is required to minimize the loss in poultry production. This project for optimal vaccine production will address a stable and safer baseline for protection of avian infectious viral pathogens. By doing so, the improvement in sustainability of US poultry may be realized by the much safer control of long-term threat of virus spread (as either an epidemic or even pandemic).
APPROACH: Various regulatory factors will be modulated by genetic engineering technologies in primary chicken cells to establish continuously growing cell lines. Negatively regulating factors in both cell cycle and cell lifespan will be down-regulated using gene knock-down technology, while positively regulating factors will be overexpressed using various eukaryotic expression vectors containing strong promoter. For reverse genetic system, RNA genome of specific avian virus will be converted to cDNA by reverse transcriptase, and double stranded genome will be assembled into plasmid vector containing specialized promoter. Further recombinant virus propagation will be perform in continuously growing avian cell substrates. Mutagenesis will be performed on specific regions of the viral genes to be able to determine if these changes are detrimental to the infectivity/virulency of RNA virus. To construct the virulency defective-, but infectious DNA viruses, various viral genes including surface glycoproteins will be removed using homologous recombination between purified viral DNA genome and various reporter plasmids, that contain selectable markers. In the entire processes, the immortal chicken cell lines will be used as cellular substrates for virus propagation and diagnostic uses to detect viruses.
PROGRESS: 2007/01 TO 2007/12
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; email@example.com) and Douglas N. Foster (UMN; firstname.lastname@example.org) are participated in this project. TARGET AUDIENCES: US poultry industry; Animal/poultry vaccine industry PROJECT MODIFICATIONS: None
IMPACT: 2007/01 TO 2007/12
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.