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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

2012 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 Marek’s disease virus vaccine program for 2012 involved the generation of a Meleagrid herpesvirus 1 (MeHV-1) helper virus containing a single packaging site flanked by loxP sites. This involved a three step recombination scheme. In the first step a donor plasmid containing an antibiotic resistant gene (Bla) flanked by neighboring pac sequences was constructed and used to delete one of the pac sites in the MeHV-1 genome. In the second step a donor plasmid containing a different antibiotic resistant gene (Kan) flanked by neighboring pac sequences was constructed and used to delete the other pac site. Lastly a third donor plasmid was constructed that contained a pac next to an antibiotic resistant gene (Zeo) flanked by loxP sites. This Zeo donor plasmid was used to rescue a single pac site into the double pac deletion MeHV-1genome thus restoring its packaging phenotype.

Another project under the Marek’s program involved the generation of a Marek’s disease virus amplicon containing an origin of replication, a packaging site and a marker gene (green fluorescent protein). In as series of transfection/infection experiments we have shown that it is possible to insert concatemers of the amplicon within the nucleocapsids of the Marek’s disease virions and passage these onto nascent cells. The genomic ILTV program for 2012 involved comparative analysis of virulent and vaccine strains of gallid herpesvirus type 1. In the autumn of 2011, the nucleotide sequences of six vaccine strains [derivative of chicken embryo origin (CEO) and tissue culture origin (TCO)] was determined using hybrid next generation sequencing technology. Throughout 2011 comparative sequence analysis between the vaccine strains and virulent strains indicated surprising conservation at the amino acid lengths of the majority of open reading frames. However, numerous single nucleotide polymorphisms were identified and it is suspected that virulent isolates were the result of reversion of the vaccines to generate virulent progeny. Furthermore we have identified a gene within the TCO genome that contains a premature stop codon which results in a truncation protein for the ORF-C gene. The results were presented at the 9th International Marek’s Disease Symposium in Berlin, Germany. A research article detailing this research has been submitted to the journal Virology. In the first quarter of 2012 we have determined the nucleotide sequences of two strains of ILTV that were isolated from “backyard” flocks. These flocks have never been exposed to ILTV vaccine strains so it is hoped that they will represent true virulent isolates and not vaccinal revertants.


4.Accomplishments
1. Infectious laryngotracheitis vaccine strain tissue culture origin (TCO) contains a truncated ORF-C protein. By determining the nucleotide sequence of the TCO vaccine strain of infectious laryngotracheitis virus (ILTV) ARS researchers in collaboration with the University of Georgia have identify a mutation which may be responsible for its attenuation. ILTV is the causative agent of infectious laryngotracheitis in poultry. By identifying mutation in vaccine strain it will be possible to generate newer vaccine that contain markers, thus allowing for the development of tests that will be able to differentiate animals infected with field isolates from those vaccinated with modified live vaccines. The generation of stable marker ILTV vaccines will provide needed help in controlling ILTV by our stakeholders.

2. The nucleotide sequence determination of two virulent strains of infectious laryngotracheitis was completed in 2011. By determining the nucleotide sequence of virulent infectious laryngotracheitis virus (ILTV) isolates from flock never exposed to vaccine strains ARS researchers in collaboration with the University of Georgia have identified genetic changes associated with virulence. The sequences of these virulent strains has been instrumental in the identification of genes associated with virulence and will provide the blueprints for the generation of new vaccine containing deletion in these genes. The identification of genetic polymorphisms will impact vaccine development for the control of infectious laryngotracheitis. These sequences will be released into the public domain in the 4 quarter of 2011.


Last Modified: 4/19/2014
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