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United States Department of Agriculture

Agricultural Research Service

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Research Project: Genomic Strategies for Control of Herpesviruses of Poultry

Location: Endemic Poultry Viral Diseases Research Unit

2013 Annual Report


1a.Objectives (from AD-416):
1. Discover novel Marek’s disease virus (MDV) vaccine platforms that are safe, efficacious, and cost effective by determining genetic and biological determinants of gallid herpesvirus type 2 (GaHV-2) virulence and by developing a vaccine strain of GaHV-2 that is cell free and does not require liquid nitrogen for storage and shipment.

1.1. To generate GaHV-2 recombinants containing genes encoding immunomodulators to elicit Th1 immunity.

1.2. To develop and test vaccines that mediate cell free egress.

2. Discover novel infectious laryngotracheitis virus (ILTV) vaccine platforms that are safe, efficacious, and cost effective by determining genetic and biological determinants of gallid herpesvirus type 1 (GaHV-1) virulence and by identifying mechanisms of GaHV-1 protective immunity.

2.1. To identify predictors of virulence through sequencing of ILTV genomes of attenuated and virulent isolates from the United States.

2.2. To develop and test vaccines containing deletions in genes involved in ILTV virulence but also containing host gene additions to mediated Th1 immunity.


1b.Approach (from AD-416):
1. To generate GaHV-2 recombinants containing genes encoding immunomodulators to elicit Th1 immunity. Comparative genomic will be implemented to identify genes associated with virulence. These genes will be deleted and replaced with genes encoding cytokines. Candidate vaccines will be tested in animal challenging experiments.

2. To develop and test vaccines that mediate cell-free egress. Cell free GaHV-2 vaccines will be generated by tranfering the packaging sites from the genome of a cell free avian herpesviruses into the genome of vaccine strain of GaHV-2. This hybrid genome will be encapsulated into cell free virions using either a helper virus or a packaging cell line. Chimera virions will be lyophilized and used in animal challenging experiments.

3. To identify predictors of virulence through sequencing of GaHV-1 genomes of attenuated and virulent isolates from the United States. The nucleotide sequenced of both attenuated and virulent strains of GaHV-1 will be determined using second and third generation sequencing technologies. Comparative genomic will be used to determine the polymorphorisms that occur in the attenuated genomes.

4. To develop and test vaccines containing deletions in genes involved in ILTV virulence but also containing host gene additions to mediated Th1 immunity.

Comparative genomic will be implemented to identify genes associated with virulence. These genes will be deleted and replaced with genes encoding cytokines and/or RNAi constructs in order to elicit Th1 type immunity. Candidate vaccines will be tested in animal challenging experiments with virulent field isolates.


3.Progress Report:
The project 6612-32000-062-00D titled “Genomic strategies of control of herpesviruses of poultry” for 2013 involved the generation of a Meleagrid herpesvirus 1 (MeHV-1) helper virus containing a single packaging site flanked by loxP sites. Previously using a three step recombination scheme it was believed that this construct was successfully created, however the complete nucleotide sequence of this recombinant indicated that the construct contained a deletion in a critical cis-acting site needed for packaging. In 2013 this construct was successfully repaired to be packaging competent.

Another project under the Marek’s program involved the nucleotide sequence determination of strains of gallid herpesvirus 2 exhibiting thymic atrophy phenotypes and mutants lacking this phenotype. It is anticipated that these sequences will be analyzed in the 4th quarter of 2013.

The genomic infectious laryngotracheitis virus (ILTV) program for 2012 involved the generation of reagents needed to investigate the biological function of a gene (ORF C) previously identified through a comparative analysis of virulent and vaccine strains of gallid herpesvirus type 1. These reagents include the production of recombinant ORF C protein in E. coli and the use of this protein to generated monoclonal antibodies.

In the autumn of 2012, experiments were implemented to generate a recombinant containing a deletion in the ORF C gene within a virulent ILTV background. Pathogenicity studies in the spring of 2013 indicated that this recombinant was attenuated and replicated in the trachea to the same extinct as wild type virus. Clinical protection studies were likewise encouraging. These studies indicated that an ORF C deletion of ILTV can act as a potential vaccine candidate and protect against virulent ILTV challenge. The results were presented at the International Herpesvirus Workshop in Grand Rapid, MI and the American Association of Avian Pathologists in Chicago IL. In the last quarter of 2013 we have determined whether this vaccine is safe and protective when administered in ovo.

Little progress has occurred in the generation of nucleotide sequencing data from backyard field isolate of ILTV due to their poor growth characteristics in cell culture and the likelihood that the isolates are contaminated with other avian viruses. Also little progress has occurred in the generation of an ILTV recombinant containing IL-18. This was mainly due to the lack of a technician from August 2012 through June 2013.


4.Accomplishments
1. The generation of an infectious laryngotracheitis virus (ILTV) vaccine strain containing a deletion in the gene encoding ORFC. By determining the nucleotide sequence of the TCO vaccine strain of ILTV ARS researchers in collaboration with the University of Georgia have identified a mutation which is likely to be responsible for its attenuation. ILTV is the causative agent of infectious laryngotracheitis in poultry. By deleting this gene in a genome of a virulent virus, we have generated a potential vaccine strain. Safety and clinical protection studies have indicated that this candidate is safe and can protect poultry against virulent challenge. This vaccine can also be used to differentiate animals infected with field isolates from those vaccinated. The generation of a stable deletion ILTV vaccine will provide our stakeholders with the tools to control and perhaps one day eradicate ILTV from flocks.

2. The complete nucleotide sequence of two avian herpesviruses which exhibit cell-free phenotypes was completed. By determining the nucleotide sequences of columbid herpesvirus-1 falconid herpesvirus -1 ARS researchers in collaboration with industry partners have identify genes unique to avian cell- free viruses. Using comparative genomic these genes suspected to be involved in virus egress will add to our understanding of the mechanism needed to achieve cell –free virions. The generation of a cell-free Marek’s disease vaccine will have an enormous effect on how poultry producers vaccinate against Marek’s disease.


Last Modified: 11/25/2014
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