A Recombinant Turkey Herpesvirus Expressing the F Protein of Newcastle Disease Virus Genotype XII Generated by NHEJ-CRISPR/Cas9 and Cre-LoxP Systems Confers Protection against Genotype XII Challenge in Chickens.

In this study, we developed a new recombinant virus rHVT-F using a Turkey herpesvirus (HVT) vector, expressing the fusion (F) protein of the genotype XII Newcastle disease virus (NDV) circulating in Peru. We evaluated the viral shedding and efficacy against the NDV genotype XII challenge in specific pathogen-free (SPF) chickens. The F protein expression cassette was inserted in the unique long (UL) UL45-UL46 intergenic locus of the HVT genome by utilizing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 gene-editing technology via a non-homologous end joining (NHEJ) repair pathway. The rHVT-F virus, which expressed the F protein stably in vitro and in vivo, showed similar growth kinetics to the wild-type HVT (wtHVT) virus. The F protein expression of the rHVT-F virus was detected by an indirect immunofluorescence assay (IFA), Western blotting, and a flow cytometry assay. The presence of an NDV-specific IgY antibody was detected in serum samples by an enzyme-linked immunosorbent assay (ELISA) in SPF chickens vaccinated with the rHVT-F virus. In the challenge experiment, the rHVT-F vaccine fully protects a high, and significantly reduced, virus shedding in oral at 5 days post-challenge (dpc). In conclusion, this new rHVT-F vaccine candidate is capable of fully protecting SPF chickens against the genotype XII challenge.

Influence of the addition of antibiotics on survival of herpesvirus of turkeys.

To determine the influence of the antibiotics ceftiofur sodium from two different laboratories (A and B) and gentamycin sulfate on a Marek's disease commercial vaccine herpesvirus of turkey (HVT), samples were assayed by titration in chicken embryo fibroblasts (CEF). Viruses were tested in vitro to establish the average number of plaque-forming units before and after different periods of incubation with the addition of the antibiotic. These tests showed no effect of gentamycin or ceftiofur A or B on HVT titers when treatments were for 1 hr or less. However, ceftiofur B decreased the titer at 2 hr. The in vivo effects of the antibiotics were determined by vaccinating 15 one-day-old chickens with HVT plus gentamycin or ceftiofur A or B. Birds were considered viremic at 1 wk postvaccination when one or more plaques were detected in CEF 5 days after inoculation of peripheral blood lymphocytes. Viremia levels were similar between 1 and 16 wk after vaccination with HVT with ceftiofur A or B. The pH values (7.5) were the same in vaccines with and without antibiotics.