Equine Arteritis Virus Has Specific Tropism for Stromal Cells and CD8⁺T and CD21⁺B Lymphocytes but Not for Glandular Epithelium at the Primary Site of Persistent Infection in the Stallion Reproductive Tract

Abstract

Equine arteritis virus (EAV) has a global impact on the equine industry as the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of equids. A distinctive feature of EAV infection is that it establishes long-term persistent infection in 10 to 70% of infected stallions (carriers). In these stallions, EAV is detectable only in the reproductive tract, and viral persistence occurs despite the presence of high serum neutralizing antibody titers. Carrier stallions constitute the natural reservoir of the virus as they continuously shed EAV in their semen. Although the accessory sex glands have been implicated as the primary sites of EAV persistence, the viral host cell tropism and whether viral replication in carrier stallions occurs in the presence or absence of host inflammatory responses remain unknown. In this study, dual immunohistochemical and immunofluorescence techniques were employed to unequivocally demonstrate that the ampulla is the main EAV tissue reservoir rather than immunologically privileged tissues (i.e., testes). Furthermore, we demonstrate that EAV has specific tropism for stromal cells (fibrocytes and possibly tissue macrophages) and CD8 ϩ T and CD21 ϩ B lymphocytes but not glandular epithelium. Persistent EAV infection is associated with moderate, multifocal lymphoplasmacytic ampullitis comprising clusters of B (CD21 ϩ ) lymphocytes and significant infiltration of T (CD3 ϩ , CD4 ϩ , CD8 ϩ , and CD25 ϩ ) lymphocytes, tissue macrophages, and dendritic cells (Iba-1 ϩ and CD83 ϩ ), with a small number of tissue macrophages expressing CD163 and CD204 scavenger receptors. This study suggests that EAV employs complex immune evasion mechanisms that warrant further investigation. IMPORTANCEThe major challenge for the worldwide control of EAV is that this virus has the distinctive ability to establish persistent infection in the stallion's reproductive tract as a mechanism to ensure its maintenance in equid populations. Therefore, the precise identification of tissue and cellular tropism of EAV is critical for understanding the molecular basis of viral persistence and for development of im-

Equine Arteritis Virus Elicits a Mucosal Antibody Response in the Reproductive Tract of Persistently Infected Stallions

“…Inflammatory lesions in the accessory sex glands of control (naive) stallions (n ϭ 4) frequently ranged from being absent to being present as minimal and focal lymphoplasmacytic infiltrates, with these lesions showing a significantly lower severity and distribution and significantly lower cumulative scores than those for long-term carrier stallions (P Ͻ 0.01), while no statistically significant differences between naive and short-term carrier stallions were observed, as previously shown (40). In addition, specific immunostaining scores for diverse inflammatory cells infiltrating the ampullae were demonstrated to be significantly higher in long-term carrier stallions than short-term carrier and control stallions (P Ͻ 0.0001) (data not shown) (40). Interestingly, the bulbourethral glands were abundantly infiltrated with plasma cells, as observed by hematoxylin and eosin (H&E) staining and confirmed by specific immunostaining, as described below ( Fig.…”

“…The clinical outcome after intranasal challenge with the EAV KY84 strain in the group of stallions evaluated in this study has been described in other studies (40,41). Briefly, all experimentally infected stallions (n ϭ 8) developed moderate to severe clinical signs of EVA following experimental infection, which lasted a median of 20 days (interquartile range [IQR] ϭ 11.25 days), and no signs of disease or reproductive dysfunction were observed after clinical recovery.…”

“…For behavioral reasons, semen collection from one of the stallions (stallion L143) was not feasible, and the carrier status could not be determined until the end of the study. The absence of infective virus, viral antigen, and RNA from tissues derived from the reproductive tract after euthanasia and the minimal inflammation observed in the accessory sex glands were indicative that this animal stopped shedding earlier in the course of the study and was considered to be a short-term carrier stallion (40).…”

“…EAV infection induced an inflammatory response in the reproductive tract of experimentally infected stallions, and an extensive characterization of the inflammatory infiltration has been previously described (40). Histological lesions in the accessory sex glands of experimentally infected stallions that were actively shedding EAV at the time of euthanasia (n ϭ 2 long-term carrier stallions) included a bilateral, moderate, multifocal lymphoplasmacytic ampullitis ( Fig.…”

“…However, EAV persists in the reproductive tract of carrier stallions regardless of the high titers of neutralizing antibodies present in the serum of these animals (15,40). The pathogenesis of persistent EAV infection, including the host factors associated with the establishment and maintenance of persistent infection, is not fully comprehended and is under extensive investigation in U.…”