Cosmid cloning and restriction endonuclease mapping of the herpesvirus of turkeys (HVT) genome

Abstract

The genomic libraries of the herpesvirus of turkeys (HVT) presented to date do not include the entire genome. To construct a complete genomic library, HVT DNA was partially digested with Sau3A and inserted into the double cos site vector pcos2EMBL. The PstI maps derived from the partial Sau3A library demonstrate that the left region of the genome compared to the right area is overrepresented. Similar to the libraries of the HVT genome established earlier, a defined portion of the middle genomic region, however, is not contained in the partial Sau3A library. Cloning of HVT DNA in pcos2EMBL, employing BamHI for the partial digestion step, enabled to find the recombinant cosmid cBL267 which carries the BamHI-C and -D fragment as DNA insertion. As shown by Southern blot hybridization, the BamHI-C fragment ranges in a size to close the gap in the partial Sau3A library and thus guarantees the completeness of the genomic library of HVT which consists of seven overlapping cosmid clones (cBL1, cBL328, cBL11, cBL267, cBL27, cBL33, and cBL34).

Methods for the Manipulation of Herpesvirus Genome and the Application to Marek’s Disease Virus Research

“…An advantage of this method is that it does not require plaque purification, because all reconstituted viruses are recombinant (contain the gene modification), allowing this technique to be widely used in herpesvirus research. Since originally introduced, this technique has been utilized to generate a large number of mutants for HSV-1, EBV, VZV, equine herpesvirus (EHV), MDV and HVT to study gene function [38,[69][70][71][72][73][74]. 2).…”