Recipient Organization
UNIVERSITY OF DELAWARE
(N/A)
NEWARK,DE 19717
Performing Department
Animal And Food Sciences
Non Technical Summary
Infectious laryngotracheitis (ILT) is an important avian respiratory disease. Chicken embryo origin vaccines (CEO) for ILT protect well but are moderately pathogenic and cannot be used in young birds. These live attenuated vaccines were developed decades ago, before modern molecular techniques were developed. Our hypothesis fis that these CEO vaccines contain mixtures of virus populations exhibiting varying degrees of pathogenicity and differences in sequence. The first two objectives of this proposal are designed to identify and characterize these mixed populations of viruses and to evaluate how they adapt to growth in birds, eggs, or tissue culture. The third objective is designed to develop a new generation of recombinant ILT vaccines. Taken togehter these experiments may identify a new generation of ILT vaccines that are more "stable", less pathogenic, and still provide excellent protection from virus challenge.
Animal Health Component
25%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Goals / Objectives
Infectious laryngotracheitis (ILT) is an important avian respiratory disease and is caused by an avian alpha herpesvirus of the Iltovirus genus, infectious laryngotracheitis virus (ILTV). The ILTV sequence was first reported in 2006 (Thureen and Keeler, 2006) and much remains to be learned about the genetic basis of ILTV pathogenesis. This research project has three specific objectives:Objective 1: There are two forms of live attenuated ILTV vaccines. Tissue culture origin vaccines (TCO) are relatively apathogenic but offer limited protection. Chicken embryo origin vaccines (CEO) protect well but are moderately pathogenic and cannot be used in young birds. These live attenuated vaccines were developed decades ago, before modern molecular techniques were developed. They were derived from virulent field isolates and have never been purified. Our hypothesis for this objective is that CEO vaccines contain mixtures of virus populations exhibiting varying degrees of pathogenicity and differences in sequence. Individual "clones" of a commercial CEO vaccine will be recovered by limit dilution on embryonated eggs. These CEO derivatives will be sequenced and evaluated for pathogenicity. This experiment will determine if CEO vaccines contain mixed subpopulations of virus. This process may identify a new generation of CEO vaccines that are more "stable", less pathogenic, and provide protection from challenge.Objective 2: Since the early 1990s (Guy et al. 1991) there has been a concern that ILTV increases in virulence following bird-to-bird passage. However, as a DNA virus the ILTV genome is known to be relatively stable (unlike RNA viruses such as avian influenza). Furthermore, we now have the ability to identify genome changes at the nucleotide level. Using a defined plaque-purified CEO vaccine strain passage studies in birds, embryonated eggs, and tissue culture (chicken embryo liver cells) will be performed. Besides monitoring changes in pathogenicity, complete genome sequences will be determined for the initial and final virus populations. Any changes in pathogenicity may then be correlated to specific changes in nucleotide sequence, which will form the basis for further study.Objective 3: Although ILTV is an excellent potential candidate as a poultry vaccine vector, work in this area and in the identification and study of viral genes involved in the pathogenicity of ILTV has been hampered by the lack of a robust system for constructing defined viral mutants. The handful of currently available ILTV mutants have been created using in vivo recombination methods. Complementing the efforts of other research groups which are attempting to create a BAC (bacterial artificial chromosome) clone of ILTV, we propose to construct a cosmid library of ILTV which can be used to construct defined ILTV mutants. The cosmid library will be characterized and used to generate a live recombinant ILTV strain.
Project Methods
Objective 1: Generate five (5) "pock-purified" derivatives from a commercially available CEO ILTV vaccine. Determine if these derivatives differ with respect to their DNA sequence, their pathogenicity in 3-4 week old broiler chickens, and their immunogenicity.Experiment 1: A commercially available CEO vaccine will be utilized for this objective. The virus will be initially be reconstituted according to the manufacturer's instructions. This virus stock will be serially diluted and then used to infect the chorioallantoic membranes (CAM) of 10 day embryonated chicken eggs. After one week of incubation the infected CAMs will be evaluated. When CAMs containing single "pocks" are identified, these infected regions will be excised and used to create virus stocks. This process will be repeated five times in order to create five independent and unique single pock derivatives of the CEO vaccine (UDCEOD1-UDCEOD5).Experiment 2: Total DNA (chicken and viral) will be purified from UDCEOD1-UDCEOD5-infected CAMs. The methods for propagating ILTV, and for isolating viral DNA have previously been published by our laboratory (Keeler et al., 1993). Once viral DNA is isolated a library of viral DNA fragments generated by sonication will be sequenced on an Illumina Genome Analyzer. This sequenced will be assembled into a full length viral genome sequence using a bioinformatics pipeline developed in our laboratory (manuscript in preparation). Viral DNA sequences will be analyzed and compared using DNA*. Our hypothesis is that the five unique CAM-derived CEO vaccine derivatives will differ in their primary sequence.Experiment 3: The five CEO vaccine derivatives will also be evaluated for their pathogenicity and immunogenicity in 3-4-week-old broiler chickens maintained in isolation units. Birds (10) will be inoculated via the choanal cleft with 103.5 EID50 of virus. Virulence and immunogenicity will be determined as previously described (Poulsen et al., 1998). Briefly, pathogenicity will be evaluated on a 0-5 scale from day2 through day 10 after inoculation. In addition, birds will be swabbed by the oral-pharyngeal route 5 days after inoculation to determine the presence of virus. Two weeks after inoculation, the birds will be evaluated for resistance to challenge by administering 103.5 EID50 of the NVSL ILTV challenge virus via the same route. Birds will again be evaluated for clinical signs of ILT on a 0-5 scale from day2 through day 10 after challenge.Objective 2: Evaluate changes in pathogenicity and sequence following bird, chicken embryo, and tissue culture passage of ILTV. Experiment 1: Using a defined plaque-purified CEO vaccine strain, a bird-to-bird passage study will be performed. Bird-to-bird passage of ILTV will be performed in 3-4-week-old SPF chickens maintained in isolation units. Birds (10) will be inoculated intratracheally with 103.5 EID50 of virus. On day 5 after infection, tracheal swabs will be collected and used to inoculate an additional group of birds. This procedure will be repeated 20 times. The virulence of the initial virus and of virus isolated after passages 1, 10 and 20 will be determined as previously described (Poulsen et al., 1998). In addition, the complete genome sequence will be determined for these three virus passages. Experiment 2: Using the same plaque-purified CEO vaccine derivative, an egg passage study will be performed. An experiment similar in scope to Experiment 1 will be performed in embryonated eggs. In this case 10 day embryonated eggs (5) will be inoculated with 103.5 EID50 of virus. After 7 days, the infected chorioallantoic membranes will be excised, homogenized, and used to infect another three eggs. This process will be repeated 20 times. The virulence of the initial virus and of virus isolated after passages 1, 10 and 20 will be determined as previously described (Poulsen et al., 1998). In addition, the complete genome sequence will be determined for these three virus passages. We expect that will observe differences in virus sequence, and perhaps in vivo pathogenicity, as the virus adapts to growth in eggs. The observed changes may differ from those observed when the virus is grown in birds.Experiment 3: Using the same plaque-purified CEO vaccine derivative, a tissue culture passage study will be performed. An experiment similar in scope to Experiment 1 will be performed in primary avian hepatocyte tissue culture (CEL). In this experiment, primary avian hepatocyte cultures will be obtained from 15-16 day embryonated eggs. One 100mm culture dish of cells (~1 x 107) will be inoculated with 103.5 EID50 of virus. After 2-3 days of incubation, when maximum CPE is observed, the infected cells and supernatant will be removed from the plate and used to infect another cell culture. This process will be repeated 20 times. The virulence of the initial virus and of virus isolated after passages 1, 10 and 20 will be determined as previously described (Poulsen et al., 1998). In addition, the complete genome sequence will be determined for these three virus passages. We expect that will observe differences in virus sequence, and perhaps in vivo pathogenicity, as the virus adapts to growth in tissue culture. The observed changes may differ from those observed when the virus is grown in birds or eggs.Objective 3: Construct a cosmid library of ILTV which can be used to construct defined ILTV mutants.We propose to use a cosmid approach for the construction of ILTV mutants. This approach has been used successfully with Marek's Disease virus, another avian herpesvirus (Reddy et al., 2002). Since the sequence of the ILTV genome is known 5-6 cosmid clones, each containing ~40 kb inserts, will be sufficient to provide coverage of the entire genome. Using restriction endonucleases, defined regions of the ILTV genome will be produced and cloned into cosmid vector (Stratagene Corp.). Cosmids will be packaged with the use of commercially available lambda packaging extracts (Stratagene Corp.) and cloned into Escherichia coli. To produce virus, all 5-6 cosmid inserts will be released using the restriction endonuclease NotI, combined and transfected into 60-mm dishes containing either primary avian hepatocytes (CEL) or LMH cells (an avian hepatocyte cell line) using calcium phosphate or lipofectin. After 2-4 days, cells will be trypsinized and seeded onto fresh CEL monoloayers, which will be monitored for typical ILTV cytopathic effects (CPE). Reconstructed virus will be compared to the parental virus by determining its growth characteristics, complete nucleotide sequence, and by evaluating its pathogenesis in birds.We propose to construct sets of overlapping cosmid clones from two strains of ILTV. A CEO vaccine strain will be chosen as a base for vaccine and vector development while a pathogenic field isolate (632) will be used for biological characterizations (Keeler et al., 1993).