Replication-Competent Bacterial Artificial Chromosomes of Marek's Disease Virus: Novel Tools for Generation of Molecularly Defined Herpesvirus Vaccines

Abstract

Marek's disease (MD), a highly infectious disease caused by an oncogenic herpesvirus, is one of the few herpesvirus diseases against which live attenuated vaccines are used as the main strategy for control. We have constructed bacterial artificial chromosomes (BACs) of the CVI988 (Rispens) strain of the virus, the most widely used and effective vaccine against MD. Viruses derived from the BAC clones were stable after in vitro and in vivo passages and showed characteristics and growth kinetics similar to those of the parental virus. Molecular analysis of the individual BAC clones showed differences in the structure of the meq gene, indicating that the commercial vaccine contains virus populations with distinct genomic structures. We also demonstrate that, contrary to the published data, the sequence of the L-meq of the BAC clone did not show any frameshift. Virus stocks derived from one of the BAC clones (clone 10) induced 100 percent protection against infection by the virulent strain RB1B, indicating that BAC-derived viruses could be used with efficacies similar to those of the parental CVI988 vaccines. As a DNA vaccine, this BAC clone was also able to induce protection in 6 of 20 birds. Isolation of CVI988 virus from all of these six birds suggested that immunity against challenge was probably dependent on the reconstitution of the virus in vivo and that such viruses are also as immunogenic as the in vitro-grown BAC-derived or parental vaccine viruses. Although the reasons for the induction of protection only in a proportion of birds (33.3%) that received the DNA vaccine are not clear, this is most likely to be related to the suboptimal method of DNA delivery. The construction of the CVI988 BAC is a major step towards understanding the superior immunogenic features of CVI988 and provides the opportunity to exploit the power of BAC technology for generation of novel molecularly defined vaccines.Herpesviruses are important pathogens associated with a wide range of diseases in humans and animals. Unlike a lot of human herpesvirus diseases for which safe attenuated vaccines are not available, control of many important herpesvirus diseases in veterinary medicine is dependent primarily on the use of vaccines. Most of the herpesvirus vaccines used in animals are derived from live viruses attenuated by classical methods. Although these vaccines are effective in inducing protection, there is a general desire to improve the effectiveness and safety of these vaccines, taking advantage of the advances in recombinant DNA technology. Recent applications of bacterial artificial chromosome (BAC) technology in cloning large DNA virus genomes have opened new avenues for reverse and forward genetics in basic herpesvirus research (1). While the use of BAC clones of herpesviruses is proving to be a powerful tool for the study of viral gene functions or for the identification of pathogenic determinants (22), this novel technology has not been examined for the improvement of herpesvirus vaccines. Recent success with the use of a BAC...

Expression profiles of microRNAs encoded by the oncogenic Marek's disease virus reveal two distinct expression patterns in vivo during different phases of disease

“…Although whether or not REV-LTR insertion affects the in vivo expression of MDV-1-encoded miRNAs is unknown as yet, our research may provide meaningful clues for further evaluating their functions, especially for the twelve miRNAs highlighted in this study. Based on the successful establishment of strategies for mRNA target prediction and experimental identification (Kuhn et al, 2008;Alexiou et al, 2009;Bartel, 2009), and the application of bacterial artificial chromosomes in research on MD pathogenesis (Schumacher et al, 2000;Petherbridge et al, 2003Petherbridge et al, , 2004Sun et al, 2009Sun et al, , 2010, focusing on those miRNAs shown to be expressed in vivo in this study should reveal more about the biological functions of MDV-1-encoded miRNAs.…”

Oncogenicity of Virulent Marek's Disease Virus Cloned as Bacterial Artificial Chromosomes

“…The RB-1B strain of MDV isolated in the early 1980s is a highly oncogenic strain (5,6) that consistently induces a high incidence of MD with rapid-onset tumors in visceral organs. We have previously shown that RB-1B infection in 1-week-old Rhode Island red chickens caused a 100% incidence of MD with tumors in visceral organs 6 to 7 weeks after infection (4). Taking advantage of this high oncogenic potential, we have chosen the RB-1B strain for the construction of BAC clones to identify oncogenic determinants of MDV.…”

A p53-dependent promoter associated with polymorphic tandem repeats controls the expression of a viral transcript encoding clustered microRNAs

“…The entire sequence of the putative promoter of the GaHV-2 LAT transcript was amplified from Bacmide DNA of the RB-1B strain (Petherbridge et al 2003). The initial construct extended from nucleotide 142486 to nucleotide 143448 of the reference RB-1B genome (GenBank accession number EF523390), covering the region between the telomeric repeat and the LAT-miRNA cluster.…”