Sequence comparison of a bacterial artificial chromosome (BAC)-based infectious clone of the CVI988 (Rispens) strain of Marek's disease virus (CVI988-699-2) to a back-passaged isolate that has reverted to virulence (CVI988-699-2 RV)
Rojas Amortegui, Juliana
University of Delaware
Marek's disease (MD) is pathology of chickens characterized by paralysis, immumosuppression, and the rapid induction of T-cell lymphomas. MD is caused by an oncogenic alphaherpesvirus, Marek's Disease Virus (MDV). Currently, MD is controlled through vaccination with non-oncogenic vaccine strains comprised of the herpesvirus of turkeys (HVT), or a combination of HVT and MDV-2 (strain SB-1) in a bivalent mixture. However, field strains of MDV have continued to evolve in virulence, necessitating the use of an attenuated MDV-1 strain, CVI988 (Rispens), initially developed in Europe. Commercial CVI988-based vaccines were established after 33 passages in cell culture, by which passage, the virulence of CVI988 had been attenuated. Presently, CVI988 is produced by four vaccine companies and each version of CVI988 confers various levels of protection. In our lab, we have been able to generate a bacterial artificial chromosome (BAC)-based infectious clone of a low passage of CVI988 (∼p25), that provides high levels of vaccine protection (33). This particular clone (CVI988-699-2) was reisolated from a visibly-protected broiler chicken (Bird tag #699) at seven weeks post-vaccination. CVI988-699-2 however, was found to retain some level of pathogenicity upon serial back passage in SPF leghorn chickens. Our initial goal was to attenuate CVI988-699-2 RV through the targeted deletion of the glycoprotein C (gC) gene with subsequent insertion of immunoregulatory genes (IFN-α/β, IL-1β). These were to be under the control of cellular type II keratin 5 (Ker-5) or viral (UL47) promoters for specific expression in the skin. After two years of attempting this mutagenesis, we shifted the focus of this work to the mutations in CVI988-699-2 RV through examination of the whole genome sequence of this virus to the original CVI988-699-2. To determine what changes had taken place in CVI988-699-2 during back passage, we inoculated chickens with a 10X dose (∼25,000 PFU) of CVI988-699-2 and reisolated BAC-containing virus from tumors caused by this virus. We have termed this virus CVI988-699-2-RV for reverted-to-virulent. The sequence of the original CVI988-699-2 virus was performed by Dr. Stephen Spatz, USDA-SEPRL, using 454 pyrosequencing. The genome sequence of CVI988-699-2-RV was recently determined using the Pacific Biosciences (Pac-Bio) polymerase-based method, which yields up to 100 independent reads per nucleotide. In addition, Dr. Stephen Spatz determined the DNA sequence of CVI988-699-2 RV via 454 pyrosequencing with an average coverage of ∼5,000 reads per nucleotide. Comparison of the sequences of the original CVI988-699-2, CVI988-699-2 RV (Pac-Bio) and CVI988-699-2 RV (454 pyrosequencing) revealed 51 and 5 genetic changes (nucleotide substitutions, insertions/deletions and sequence duplications), respectively. The basis of this project, therefore, was focused mutations that may induce reversion to virulence of CVI988-699-2. In addition, the construction of keratinocyte-specific expression cassettes may provide useful tools in the future generation of MD vaccines that could possibly block the transmission of challenge viruses from vaccinated chickens.