Characterization of a recombinant Newcastle disease virus expressing the glycoprotein of bovine ephemeral fever virus

Abstract

Bovine ephemeral fever (BEF) is caused by the arthropod-borne bovine ephemeral fever virus (BEFV), which is a member of the family Rhabdoviridae and the genus Ephemerovirus. BEFV causes an acute febrile infection in cattle and water buffalo. In this study, a recombinant Newcastle disease virus (NDV) expressing the glycoprotein (G) of BEFV (rL-BEFV-G) was constructed, and its biological characteristics in vitro and in vivo, pathogenicity, and immune response in mice and cattle were evaluated. BEFV G enabled NDV to spread from cell to cell. rL-BEFV-G remained nonvirulent in poultry and mice compared with vector LaSota virus. rL-BEFV-G triggered a high titer of neutralizing antibodies against BEFV in mice and cattle. These results suggest that rL-BEFV-G might be a suitable candidate vaccine against BEF.

Fig. 1

Construction and identification of rL-BEFV-G. (A) Schematic representation of the rLaSota genome and BEFV G inserted between the P and M genes. (B) Western blot demonstrating the expression of BEFV G. BHK-21 cells were infected with rLaSota or rL-BEFV-G at an MOI of 1. After 24 h, cells were collected and lysed, and proteins in the cell lysate were separated by SDS-PAGE and immunoblotted with mouse anti-NDV antibodies or BEFV G polyclonal antibody. (C) Immunofluorescence analysis of BEFV G protein expression. BHK-21 cells were infected with rLaSota or rL-BEFV-G at an MOI of 0.01. After 24 h, the cells were fixed and then stained with chicken anti-NDV antibody or BEFV G polyclonal antibody, followed by incubation with FITC-conjugated goat anti-mouse antibody or TRITC-conjugated rabbit anti-chicken antibody

Fig. 2

Cell-to-cell spread of rL-BEFV-G and NDV vector in BHK-21 cells. (A) Monolayers of BHK-21 cells were infected with either rLaSoTa or rL-BEFV-G at an MOI of 0.05. After five washes 1 h postinfection, the infected cells were incubated at 37 °C. The infected cells were examined at the indicated times post-infection using IFA with chicken serum against NDV. (B) Assay for inhibition of intercellular spread of recombinant virus in BHK-21 cells. Monolayers of BHK-21 cells were infected with either rL or rL-BEFV-G at an MOI of 0.05. After five washes 1 h postinfection, the cells were incubated with culture medium containing 100-fold-diluted mouse serum against NDV, mouse serum against BEFV, or naïve mouse serum. Infected cells were examined 72 h postinfection using IFA with chicken serum against NDV

Fig. 3

Biological characteristics of rL-BEFV-G in poultry. (A) Kinetics of rL-BEFV-G replication in embryonated eggs. Ten-day-old embryonated eggs were infected with rLa or rL-BEFV-G at 10⁵ TCID₅₀. Allantoic fluid of six eggs from each group was harvested at 24, 48, 72 and 96 h postinoculation, and virus titers were determined in EID₅₀ units in 10-day-old embryonated eggs. (B) Kinetics of rL-BEFV-G replication in MDBK cells. Monolayers of MDBK cells were infected with either egg-propagated rLaSota or rL-BEFV-G at an MOI of 0.01. After five washes 1 h postinfection, the cells were incubated with 100-fold-diluted mouse serum against NDV for 30 min to neutralize the residual viruses in the supernatants. After replacement of the medium with fresh medium, the infected cells were incubated at 37 °C in the absence (B) or presence (C) of TPCK trypsin (1 μg/ml). The culture supernatants were collected at different times, and their virus was titrated in MDBK cells with 1 μg of TPCK trypsin per ml. Significant differences between rLa and rL-BEFV-G were observed using Student’s t-test. *, P <0.05; **, P < 0.01. (D) Pathogenicity assay in SPF eggs and chickens. MDT, ICPI and IVPI were determined according to the recommended OIE method

Fig. 4

Changes in body weight in mice inoculated with rL-BEFV-G. Mice were inoculated intramuscularly (A) or intracerebrally (B) with 10⁷ TCID₅₀ of rL-BEFV-G on day 0. Mice were observed and weighed daily from day 0 to 14. All mice survived for the duration of the experiment. Body weight changes for each group are shown as ratios relative to the body weight at day 0, which was set as 100 %

Fig. 5

Serum neutralization analysis in mice. Serum samples were collected at different times after vaccination, and SN antibody titers for BEFV (A) and NDV (B) were measured. Statistically significant differences were determined using Student’s t-test. *, P < 0.05