To identify and sequence glycoprotein genes of Marek's disease virus (MDV) and nonessential genes of herpesvirus of turkeys (HVT) with the main aim of constructing recombinant HVT viruses which express MDV glycoprotein genes and genes of other avian pathogens.
First objectives of the study were:
To identify by partial sequencing the Marek's disease virus (MDV) homologues ofgH, gD of herpes simplex virus (HSV);
To prepare probes to identify homologues of gD, gH and non-essential genes of herpesvirus of turkeys (HVT);
To prepare HVT DNA for construction of vectors, cloning and partial sequencing of non-essential genes.
MDV and HVT homologues of gH, gD, PK, gI genes of HSV have been identified. The RR gene of HVT has been sequenced. It has been demonstrated that HVT can tolerate the insertion of a 3.9 kbp MDV DNA at the TK locus and that authentic MDV gB is synthesised by the recombinant under the control of the MDV gB promoter.
Progress has been made in identifying genes of MDV and HVT by comparison of the predicted amino acid sequence of random DNA fragments to known herpesvirus proteins. Results have shown that HVT, a vaccine widely used in chickens, has potential as a vector and that it can accommodate at least 3.9 kbp of foreign DNA. The ability of the recombinants to replicate in vivo remains to be determined and the search for insertion sites compatible with maximum replication in vivo is continuing.
Glycoprotein genes of MDV and nonessential genes of HVT will be identified by random sequencing and comparing the translated sequences to protein databases of herpes simplex and varicella zoster viruses. This approach has been used previously in our laboratory to identify several MDV homologues of alphaherpesvirus genes including glycoprotein B (gB) which has already been sequenced. MDV glycoproteins gD and gH and their homologues in HVT will be sequenced. Knowledge of the deduced amino acid sequences will allow the preparation of MDV specific antipeptide sera which will be required to monitor expression of the MDV genes by HVT recombinants.
Initially, the thymidine kinase (TK) locus of HVT will be used as an insertion site for expressing MDV glycoprotein genes particularly gB as this is an important immunogen. Virus recombinants will be generated by homologous recombination following co-transfection of infectious HVT DNA and plasmid constructs consisting of MDV genes flanked by HVT TK sequences. Recombinants will be selected and tested for safety, replication in vivo and efficacy as vaccines. Alternative sites for gene insertion in HVT will be identified and those that allow efficient replication of recombinants in vivo and optimal expression of the genes of interest will be finally selected for constructing recombinant vaccines.
Funding SchemeCSC - Cost-sharing contracts