In-Silico prediction and characterization of Elephant endotheliotropic herpesvirus proteins for development of immunodiagnostics

Elephant Endotheliotropic Herpesvirus (EEHV) is a major cause of fatal hemorrhagic disease in juvenile elephants, highlighting the urgent need for reliable serological diagnostic tools for early detection and epidemiological surveillance. Effective EEHV immunodiagnostics require a conserved, stable, and immune reactive antigen capable of consistent antibody detection across viral strains. As EEHV is a non-cultivable virus encoding approximately 115 structural and non-structural proteins, careful selection of suitable diagnostic targets is essential. In the present study, an in silico approach was employed to evaluate the diagnostic potential of selected EEHV proteins. The study moves beyond descriptive in-silico characterization by integrating antigenicity, physicochemical stability, glycosylation profiling, and rational truncation to identify recombinant antigens with direct applicability in EEHV immunodiagnostic platforms. The full-length DNA polymerase (1047 aa), glycoprotein B (gB; 847 aa), and glycoprotein L (gL; 304 aa) were analyzed for sequence conservation, physicochemical properties, antigenicity, and glycosylation patterns. Multiple sequence alignment revealed a high degree of conservation among EEHV1A strains, with approximately 99% sequence identity across all three genes, underscoring their evolutionary stability and functional importance. Glycosylation prediction identified minimal post-translational modification potential within the N-terminal regions, enabling rational truncation. Based on these analyses, truncated fragments of DNA polymerase (95 aa), gB (387 aa), and gL (164 aa) were selected for recombinant antigen development. The selected fragments exhibited favorable physicochemical properties, including stability, hydrophilicity, positive net charge, and high antigenicity scores (0.5209, 0.5271, and 0.2572, respectively). These findings support the suitability of truncated EEHV proteins, particularly DNA polymerase, as recombinant antigens for competitive ELISA development and provide a rational framework for EEHV serological assay design.