Deletion of the ORF2 gene of the neuropathogenic equine herpesvirus type 1 strain Ab4 reduces virulence while maintaining strong immunogenicity

Abstract

Background: Equine herpesvirus type 1 (EHV-1) induces respiratory infection, abortion, and neurologic disease with significant impact. Virulence factors contributing to infection and immune evasion are of particular interest. A potential virulence factor of the neuropathogenic EHV-1 strain Ab4 is ORF2. This study on 24 Icelandic horses, 2 to 4years of age, describes the infection with EHV-1 Ab4, or its deletion mutant devoid of ORF2 (Ab4[increment]ORF2) compared to non-infected controls (each group n=8). The horses' clinical presentation, virus shedding, viremia, antibody and cellular immune responses were monitored over 260days after experimental infection. Results: Infection with Ab4[increment]ORF2 reduced fever and minimized nasal virus shedding after infection compared to the parent virus strain Ab4, while Ab4[increment]ORF2 established viremia similar to Ab4. Concurrently with virus shedding, intranasal cytokine and interferon α (IFN-α) production increased in the Ab4 group, while horses infected with Ab4[increment]ORF2 expressed less IFN-α. The antibody response to EHV-1 was evaluated by a bead-based multiplex assay and was similar in both infected groups, Ab4 and Ab4[increment]ORF2. EHV-1 specific immunoglobulin (Ig) G1 was induced 8 days after infection (d8 pi) with a peak on d10-12 pi. EHV-1 specific IgG4/7 increased starting on d10 pi, and remained elevated in serum until the end of the study. The intranasal antibody response to EHV-1 was dominated by the same IgG isotypes and remained elevated in both infected groups until d130 pi. In contrast to the distinct antibody response, no induction of EHV-1 specific T-cells was detectable by flow cytometry after ex vivo re-stimulation of peripheral blood mononuclear cells (PBMC) with EHV-1 in any group. The cellular immune response was characterized by increased secretion of IFN-γ and interleukin10 in response to ex vivo re-stimulation of PBMC with EHV-1. This response was present during the time of viremia (d5-10 pi) and was similar in both infected groups, Ab4 and Ab4[increment]ORF2. Conclusions: ORF2 is a virulence factor of EHV-1 Ab4 with impact on pyrexia and virus shedding from the nasal mucosa. In contrast, ORF2 does not influence viremia. The immunogenicity of the Ab4[increment]ORF2 and parent Ab4 viruses are identical. Graphical abstract: Graphical abstract - Deletion of ORF2 reduces virulence of EHV-1 Ab4. Graphical summary of the main findings of this study: ORF2 is a virulence factor of EHV-1 Ab4 with impact on pyrexia and virus shedding from the nasal mucosa.[Figure not available: see fulltext.]