Inhibition of serum neutralization (SN) test, to assay the gE2-14 antigenic properties expressed and secreted by BoHV-4CMV-IgKE2-14ΔTK infected cells. (a) Diagram showing the principle of the assay, where BVDV neutralizing antibodies pre-incubated with medium containing gE2-14 are blocked, allowing the virus to infect and destroy the cell monolayer. (b) The quantitative assay performed in a 96-multiwell plate where 4 sera (1, 2, 3, and 4) containing neutralizing antibody against BVDV were tested at different dilutions in the presence of gE2-14 (+Sera +E2) and in the absence of gE2-14 (+Sera -E2). Control virus was established in the absence of sera and presence of gE2-14 and in the absence of sera and gE2-14. Crystal violet staining allows macroscopic evaluation of the integrity or the destruction of the cell monolayer. (c) Bar graph showing the quantification of serum neutralization made on the basis of three different experiments (** = P < 0.001). Results are expressed as the log2 of the highest dilution of the serum that inhibited the development of virus-induced cytopathic effect in cell culture.