Isolation and characterization of bovine herpesvirus 4 (BoHV-4) from a cow affected by post partum metritis and cloning of the genome as a bacterial artificial chromosome.
BACKGROUND
Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus with a Worldwide distribution in cattle and is often isolated from the uterus of animals with postpartum metritis or pelvic inflammatory disease. Virus strain adaptation to an organ, tissue or cell type is an important issue for the pathogenesis of disease. To explore the mechanistic role of viral strain variation for uterine disease, the present study aimed to develop a tool enabling precise genetic discrimination between strains of BoHV-4 and to easily manipulate the viral genome.


METHODS
A strain of BoHV-4 was isolated from the uterus of a persistently infected cow and designated BoHV-4-U. The authenticity of the isolate was confirmed by RFLP-PCR and sequencing using the TK and IE2 loci as genetic marker regions for the BoHV-4 genome. The isolated genome was cloned as a Bacterial Artificial Chromosome (BAC) and manipulated through recombineering technology


RESULTS
The BoHV-4-U genome was successfully cloned as a BAC, and the stability of the pBAC-BoHV-4-U clone was confirmed over twenty passages, with viral growth similar to the wild type virus. The feasibility of using BoHV-4-U for mutagenesis was demonstrated using the BAC recombineering system.


CONCLUSIONS
The analysis of genome strain variation is a key method for investigating genes associated with disease. A resource for dissection of the interactions between BoHV-4 and host endometrial cells was generated by cloning the genome of BoHV-4 as a BAC.
Donofrio
Gaetano
G
Dipartimento di Salute Animale, Facoltà di Medicina Veterinaria, Università di Parma, via del Taglio 10, 43100 Parma, Italy. gaetano.donofrio@unipr.it
Franceschi
Valentina
V
Capocefalo
Antonio
A
Cavirani
Sandro
S
Sheldon
Iain Martin
IM
D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000821:virology*; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism; D015247:Deoxyribonuclease HindIII / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004716:Endometritis / Q000821:virology*; D004926:Escherichia coli / Q000235:genetics / Q000821:virology; D005240:Feasibility Studies; D005260:Female; D016679:Genome, Viral / Q000235:genetics*; D026021:Herpesvirus 4, Bovine / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D007150:Immunohistochemistry; D016254:Mutagenesis, Insertional; D049590:Postpartum Period; D011644:Puerperal Disorders / Q000662:veterinary; D014599:Uterus / Q000821:virology
D004247:DNA; D015247:Deoxyribonuclease HindIII
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