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

Abstract

BackgroundEquine 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 4 years of age, describes the infection with EHV-1 Ab4, or its deletion mutant devoid of ORF2 (Ab4ΔORF2) compared to non-infected controls (each group n = 8). The horses’ clinical presentation, virus shedding, viremia, antibody and cellular immune responses were monitored over 260 days after experimental infection.ResultsInfection with Ab4ΔORF2 reduced fever and minimized nasal virus shedding after infection compared to the parent virus strain Ab4, while Ab4ΔORF2 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 Ab4ΔORF2 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 Ab4ΔORF2. 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 Ab4ΔORF2.ConclusionsORF2 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 Ab4ΔORF2 and parent Ab4 viruses are identical.Graphical abstractGraphical 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.Electronic supplementary materialThe online version of this article (10.1186/s12917-018-1563-4) contains supplementary material, which is available to authorized users.

Equine Herpesvirus Type 1 Modulates Cytokine and Chemokine Profiles of Mononuclear Cells for Efficient Dissemination to Target Organs

“…We assume that these viral proteins (ORF2 and ORF17) are involved in modulating several pathways in PBMC, including cell signaling, adhesion, and immune pathways, thereby ORF2 and ORF17 gene deletions resulted in reduced virus transfer to EC. Our findings clearly correlate with previous in vivo findings where ORF2, ORF1/ORF71, and ORF1/ORF2 deletion mutants showed respiratory infection, comparable viremia, but no noticeable endothelial cell infection in four different experimental studies in horses [48][49][50]76].…”

“…Experimental infection with ORF1 and ORF71 deletion mutants revealed a brief period of pyrexia, low virus shedding, and decreased cytokine response (IFNα, IL-10, and soluble CD14); however, they had comparable viremia to Ab4-wt [48]. In another study, ORF2 deletion mutant was attenuated; however, it had strong immunogenicity without altering viremia in Icelandic horses [49,76]. Failure to induce T-cell response was suggested as a reason for viremia in both Ab4-wt and mutant viruses infected horses [48][49][50].…”

“…In another study, ORF2 deletion mutant was attenuated; however, it had strong immunogenicity without altering viremia in Icelandic horses [49,76]. Failure to induce T-cell response was suggested as a reason for viremia in both Ab4-wt and mutant viruses infected horses [48][49][50]. As animal experiments were performed in non-pregnant horses and no distinct neurological signs were observed following infection with Ab4-wt and mutant viruses, it is unclear that cell-associated viremia with mutant viruses can result in subsequent endothelial infection or not.…”

“…Ab4-wt infection blocks IFNγ release from PBMC and deleting ORF1 gene resulted in release of more cytokines/chemokines which shows the immune-modulating potential of ORF1 in Ab4 strain. ORF2 gene deletion also showed release of more cytokines in both in vitro and in vivo [49,76]. ORF2 gene deletion resulted in expression of higher levels of IL-1β cytokine, which is considered as a host defense against virus infection.…”

“…The ORF2 gene product is considered a virulence factor for EHV-1 [ 47 ]. Previous in vivo studies have revealed the involvement of ORF1 and ORF2 in modulating cytokine and chemokine responses in infected horses and subsequently minimizing the course of the disease [ 48 , 49 , 50 ].…”