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In a previous study, an apathogenic strain of bovine herpesvirus 4 (BoHV-4) cloned as a bacterial artificial chromosome and expressing a chimeric peptide (gE2/gD) as a secreted form was described. Recombinant virus-inoculated animals produced antibodies against bovine viral diarrhea virus (BVDV) gE2 and BoHV-1 gD. However, neutralizing antibodies were produced only against BVDV, not against BoHV-1. In the present work a recombinant BoHV-4 expressing a membrane-linked form of gE2/gD chimeric peptide was constructed, and inoculated rabbits produced serum-neutralizing antibodies against both BVDV and BoHV-1. Protein cell sorting and targeting are a very important issue when immunodominant antigens are engineered for recombinant virus vaccine development.

The biological characteristics of BoHV-4 make it a good candidate as a gene delivery vector for vaccination purposes. These characteristics include little or no pathogenicity, unlikely oncogenicity, the ability to accommodate large amounts of foreign genetic material, the ability to infect several cell types from different animal species, such as sheep, goats, swine, cats, dogs, rabbits, mink, horses, turkeys, ferrets, monkeys, hamsters, rats, mice, and chickens. In this report, the feasibility to use BoHV-4 based vector in chicken was investigated. Although BoHV-4 was able to replicate, leading to a cytopathic effect in a chicken cell line and infect the chorion allantoic membrane of embryonated eggs, however it was not pathogenic even when a large dose of virus was injected into the chicken. An immune response could be produced against heterologous antigen delivered by a recombinant BoHV-4. These data suggest the feasibility of using BoHV-4 based vector for vaccination purposes in chickens.

Gene transfer into hepatocytes is highly desirable for the long-term goal of replacing deficient proteins and correcting metabolic disorders. Bovine herpesvirus 4 (BoHV-4) based vector capability to transduce rat liver cells in vitro and in vivo was assessed. For the in vitro study, a buffalo rat liver cell line was successfully transduced by BoHV-4 and although did not show toxicity, the immediate early two viral gene was transcribed and cells harboring the intact viral genome could be pharmacologically selected, but no viral replication took place. For the in vivo study, adult male rats were inoculated intraportally and intraparenchimally with a BoHV-4 expressing enhanced green fluorescent protein and liver sections were analyzed through fluorescent microscopy. Although the liver parenchyma could not be transduced, the endothelial layer of the liver vasculature showed a robust transgene expression without toxicity. Successful BoHV-4 based vector transduction of primary cultures of rat hepatocytes suggests that extrinsic factors, and not hepatocytes per se, are the cause of such lack of transducibility. The present study serves as a starting point for study of the use of BoHV-4 based vectors to target gene delivery to vascular endothelial cells.