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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Endemic Poultry Viral Diseases Research » Research » Publications at this Location » Publication #368361

Research Project: Genetic and Biological Determinants of Avian Herpesviruses Pathogenicity, Transmission, and Evolution to Inform the Development of Effective Control Strategies

Location: Endemic Poultry Viral Diseases Research

Title: Reconstitution and mutagenesis of avian infectious laryngotracheitis virus from cosmid and yeast centromeric plasmid clones

item Spatz, Stephen
item GARCIA, MARICARMEN - University Of Georgia
item FUCHS, WALTER - Friedrich-Loeffler-institut
item LONCOMAN, CARLOS - Friedrich-Loeffler-institut
item VOLKENING, JEREMY - Base2bio
item Ross, Teresa
item RIBLET, SYLVA - University Of Georgia
item Kim, Taejoong
item LIKENS, NATHAN - USDA National Plant Disease Recovery System
item METTENLEITER, TOMAS - Friedrich-Loeffler-institut

Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/10/2023
Publication Date: 4/6/2023
Citation: Spatz, S.J., Garcia, M., Fuchs, W., Loncoman, C., Volkening, J., Ross, T.A., Riblet, S., Kim, T.N., Likens, N., Mettenleiter, T. 2023. Reconstitution and mutagenesis of avian infectious laryngotracheitis virus from cosmid and yeast centromeric plasmid clones. Journal of Virology. 97(4). Article e01406-22.

Interpretive Summary: Infectious laryngotracheitis is an economically important pathogen of chicken in which more efficacious vaccines are needed. Current live attenuated vaccines and vectored vaccines lack safety and efficacy, respectively. Since infectious bacterial artificial chromosome -based clones of infectious laryngotracheitis virus (ILTV) containing the complete genome with intact origins of replication are not feasible, we present the reconstitution of infectious ILTV from a collection of both yeast and E. coli clones and the identification of a nonessential insertion site within a redundant packaging site ( called ipac2). These constructs and the methodology for manipulation of these clones in yeast and E. coli will facilitate the development of improved live virus vaccines by deleting genes encoding virulence factor and ILTV based viral vectors for the expression of immunogens of other avian pathogens.

Technical Abstract: The genomes of numerous herpesviruses have been cloned as infectious bacterial artificial chromosomes. However, attempts to clone the complete genome of infectious laryngotracheitis virus (ILTV), formally known as Gallid alphaherpesvirus-1, have been met with limited success. In this study, we report the development of a cosmid/ yeast centromeric plasmid (YCp) genetic system to reconstitute ILTV. Overlapping cosmid clones were generated that encompassed 90% of the 151-Kb ILTV genome. Viable virus was produced by cotransfecting leghorn male hepatoma (LMH) cells with these cosmids and a YCp recombinant containing the missing genomic sequences - spanning the TRS/UL junction. An expression cassette for green fluorescent protein (GFP) was inserted within the redundant inverted packaging site (ipac2), and the cosmid/YCp-based system was used to generate recombinant replication-competent ILTV. Viable virus was also reconstituted with a YCp clone containing a BamHI linker within the deleted ipac2 site, further demonstrating the nonessential nature of this site. Recombinants deleted in the ipac2 site formed plaques undistinguished from those viruses containing intact ipac2. The 3 reconstituted viruses replicated in chicken kidney cells with growth kinetics and titers similar to the USDA ILTV reference strain. Specific pathogen-free chickens inoculated with the reconstituted ILTV recombinants succumbed to levels of clinical disease similar to that observed in birds inoculated with wildtype viruses, demonstrating the reconstituted viruses were virulent.