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 and is caused by an avian alpha herpesvirus infectious laryngotracheitis virus (ILTV). In its acute form, ILT is characterized by signs of respiratory distress in birds, accompanied by gasping and expectoration of bloody exudate. In addition, the mucous membranes of the trachea become swollen and hemorrhagic. The epizootic form of the disease spreads rapidly and can affect up to 90-100% of an infected flock. Mortality generally averages between 10 and 20%.By performing the first detailed and comprehensive examination of the chicken's transcriptional response to infection with a virulent strain of ILTV and correlating the clinical response of infected birds to their transcriptional response, it is hoped that better methods for controlling and limiting ILTV infection may be developed. This research will help to answer the question of what transcriptional changes are occurring in the chicken in response to ILTV.Under commercial conditions, a clinical diagnosis of infectious laryngotracheitis (ILT) can be complicated by co-infections with other viral agents (either pathogens or vaccines) and/or the development of secondary bacterial infections.Combined with suboptimal environmental or management conditions, a complex multi-factored respiratory disease complex (RDC) often develops. It is difficult to reproduce the complex etiology of RDC under defined laboratory conditions due to the inability to reproduce the complex microbial and environmental environment found in a commercial broiler facility. One reason why RDC is difficult to reproduce is that the microbial ecology of the respiratory tract of healthy and diseased birds in a commercial setting is poorly characterized. Therefore, the purpose of this study is to identify, compare and contrast the respiratory microbiome (including the bacterial, bacteriophage, eukaryotic virus, and fungal components) of a healthy broiler flocks from flocks diagnosed with ILT or RDC.Ultimately, control of ILT will depend on the development of safer and more effective vaccines. It is likely that these vaccines will be live recombinant strains of ILTV that have the efficacy and immunogenicity of current CEO vaccines but the pathogenicity of current TCO vaccines.Unfortunately, the development of ILTV as a vaccine vector and the ability of researchers to target the disruption of specific genes in order to study the biology of the virus has been hampered by the lack of a robust system for constructing defined viral mutants. We propose to construct sets of overlapping cosmid clones from two strains of ILTV to aid in the construction of ILTV mutants for vaccine development and studies on the biology of the virus.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
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 and much remains to be learned about the genetic basis of ILTV pathogenesis. This research project has three specific objectives:Objective 1: A detailed examination of the chicken's transcriptional response to ILTV has not been undertaken to date . The goal of the first objective is to infect a group of broiler chickens with a virulent strain of ILTV and to observe how the birds respond clinically (respiratory symptoms, histopathology), serologically (seroconversion) and transcriptionally (RNASeq and microRNASeq) in tissues affected by the virus (trachea, lung, and spleen).Objective 2: Respiratory diseases in commercial poultry are a clinical manifestation of a broader dysbiosis of the respiratory microbial community. Although the bacterial component of the healthy broiler chicken has been characterized, there are limited tools available to identify the eukaryotic virus, bacteriophage, and fungal composition of the broiler respiratory tract. BiomeSeq is a computational tool utilizes utilizes a sequence similarity-dependent approach and a comprehensive workflow on nucleotide data generated through high throughput sequencing platforms to determine the composition of the eukaryotic virus, bacterial, bacteriophage, and fungal microbiomes. The goal of this objective is to generate a comprehensive view of the respiratory microbiome from birds diagnosed with infectious laryngotracheitis and/or respiratory disease complex.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:Experiment 1: The first part of this project chickens will be infected with a virulent strain of ILTV. The chickens' clinical response to the virus will be determined and tissue samples will be collected (spleen, trachea, lung). The two-week experiment will use 37 three-week old straight run broiler chickens. Eight birds will constitute an uninfected control group, while the remaining 29 birds were inoculated with 0.03 ml of LT-Blen (CEO vaccine) and 103.5 EID50 of NVSL challenge virus via the choanal cleft.Birds will be scored daily on a one to five scale for severity of clinical symptoms. At day -1 and day 14, blood will be collected to determine if the birds mounted an antibody response to the virus. Tracheal swabs will also be collected. These will be used to confirm virus presence or clearance (by qPCR or virus isolation in eggs) on days 0, 5, and 14. Trachea and eyelids will be collected on day 5 in formalin jars examined by histochemistry for ILTV pathognomic intranuclear inclusion bodies. Trachea, lung, and spleen samples will be collected on the days 1, 2, 3, 5, 7, 10, and 14 for RNA extraction and transcriptome analysis.Experiment 2: The second phase of the objective is to extract mRNA and small RNA from the tissue samples. mRNA and small RNA will be extracted from tissues using the mirVana miRNA Isolation Kit. Alternatively, total RNA will be extracted using RNeasy kits (Qiagen) according to the manufacturers' instructions. Once the mRNA and small RNA is extracted, they will be sent to Novogene Corp. who will construct and sequence the RNASeq and microRNASeq libraries. The sequencing data received from Novogene will be analyzed through the programs TopHat and Cuffslinks. TopHat will align RNA-seq reads to the reference genome of a chicken and Cuffslinks will assemble the mapped reads into possible transcripts and then generate a final transcriptome profile. From this, it can be observed through pathway analysis what genes are expressed and repressed over the course of the pathogenicity experiment the transcriptional response of the chickens.Objective 2Experiment 1: Tracheal swabs will be collected from respiratory clinical isolates submitted to the UDPHS Lasher Laboratory at the University of Delaware. The clinical diagnosis of all flocks will be ILT or RDC or a ILT complicated by RDC. Tracheal swabs will be collected. Individual swabs will be colelcted in 3 ml of buffer PV1 (Qiagen) and immediately frozen at -80C.After thawing on ice, samples will be gently homogenized, split evenly into two tubes and then centrifuged (7000 X g, 20min; 4°C) to form pellets. Total RNA will be isolated from one pellet using the Qiagen Viral Nucleic Acid extraction kit following the manufacturer's protocol. DNA was isolated from the duplicate pellet using the Qiagen Blood and Tissue Kit following the manufacturer's protocol. Library construction and sequencing using the Illumina HiSeq platform and producing 1 X 100 single-end reads will be performed at the University of Delaware Sequencing Core Facility.Raw DNA-seq and RNA-seq reads will be processed using BiomeSeq (BMC Genomics, submitted). Individual sequence files are first analyzed for per-base sequence quality, per-sequence quality, sequence length distribution, duplicate sequences, and overrepresented Kmers. Reads with a quality phred score below 30, reads under 100 base pairs and adapter sequences were removed. The remaining reads were then aligned to the reference host genome (Gallus gallus Annotation Release 104) using Bowtie2 alignment algorithm. Only unmapped reads were extracted and analyzed further. This step removes host genome contamination from the data, increasing analytical efficiency. Determining the amount of host genome sequence in the library is also required when quantifying the results. The remaining reads are then aligned to microbial databases including a bacteriophage, a fungal and an avian-specific viral genome database.Objective 3Experiment 1. 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.