Latency & Immune evasion strategies

The ability to establish life-long latency in their respective host is one of the trademark properties of herpesviruses (41). Alphaherpesviruses utilize long-term latency as an epidemiological strategy avoiding an active host immune system to ensure their spread and survival within the host population (35, 36, 42, 43). During latency, the virus is expressed at a low, nearly undetectable rate, yet is able to re-infect the host and in turn be spread to other horses while the host is not eliciting symptoms, obvious virus shedding or cell-associated viremia (2, 35, 42). Latently infected horses are termed silent shedders and pose a threat to susceptible horses by transmitting the virus through nasal secretions or aborted foetuses (2, 35,

36, 42). EHV-1 infection can periodically be re-activated from the latent state either spontaneously or after induction by external stimuli e.g. relocation and transport, training and competitions, illnesses, operations and treatment (2, 35, 36, 41, 44, 45). In particular, treatment with corticosteroids has shown to re-activate latent virus (2, 35, 36, 41, 44, 45).

Generally, a productive EHV-1 infection activates viral gene expression (35). During latency viral gene expression is restricted and synthesis of viral factors is not possible, hence infectious viral particles are absent (35). The primary site harbouring circulating latent EHV-1 are considered to be lymphocytes as established in numerous studies (35, 46, 47). Eighty percent of CD5+/CD8+ have been verified to enable latency; CD8-/CD4- have also been found to enable establishment of latency (35, 38). Other studies have proven latent EHV-1 to be harboured within the sensory nerve cell bodies in the trigeminal ganglia (35, 45, 48, 49). During latency, the viral genome is concealed while latency-associated transcripts, antisense to IE viral genes or regulatory early viral genes are detectable (2, 35, 38, 41, 49). The exact molecular and physiological mechanisms are still poorly understood and transformation to a latent stage is considered a deliberate biological behaviour (35). Suggestion however include, that lymphocytes advance toward active transcription resulting in DNA restoration and active virus replication (45) or IE gene promoter being trans-activated in presence of another equine Herpesvirus (38). Understanding molecular and physiological factors reactivating the virus are vital to comprehend, not only to grasp reactivation and reproduction thereafter, but also for epidemiological reasons (43).

Immune evasion strategies

The conventional immune response to epithelial cells becoming infected by a respiratory virus begins with the induction of synthesis of interferon (IFN) and interleukin-6 (2). These induce the up-regulation of major histocompatibility complex (MHC) class 1 molecules, which will bind viral peptides and present them on a virus-infected cell’s surface for recognition by

lymphocytes (2). Meanwhile, antiviral resistance in uninfected cells is stimulated by IFN and interleukin-6 (2). Pro-inflammatory cytokines are released by macrophages and neutrophils, thereby restraining infection, elevating body temperature and recruiting phagocytic and natural killer (NK) cells (2). Macrophages will also synthesise IFNα and interleukin-12, which recruit further NK cells, which will exert their cytotoxic activity on infected cells (12). NK cells synthesise IFNγ, thereby urging the development of the adaptive immune system elicited in nasal associate lymphoid tissue and mucosal associated lymphoid tissue (2). The synthesis of serum or mucosal antibodies, is induced by viral antigen in lymphoid tissue and are exerting their neutralising activity and enhance humoral antibody dependent cell cytotoxicity (ADCC) (2). Virus-specific cytotoxic T- Lymphocytes (CTL) are stimulated as well and lyse virus-infected cells (2). All of these immune effector mechanisms are orchestrated by chemical messengers termed cytokines (50, 51).

Throughout evolution, herpesviruses, including EHV-1, co-evolved with their host and developed immune evasion strategies to circumvent each of the effector mechanisms as well as the cytokine orchestration (2, 50): during the early phase of infection, customarily, immediate protection is provided by the innate immune response. EHV-1 developed strategies to avoid this innate or non-specific immune response by tempering with its recognition mechanisms:

glycoprotein C of EHV-1 binds to a protein within the immune system which usually activates the complement pathway and would, under normal circumstances, generate a membrane-attack-complex. By binding to this protein, this mechanism is blocked.

EHV-1 circumvents the specific immune response by avoiding the virus neutralizing antibodies by becoming intracellular and undetectable rapidly within a few hours after infection (43, 50). Moreover, EHV-1 glycoproteins form a complex mimicking an antibody and thereby blocking antibody–mediated functions such as complement activation or ADCC (43, 50). EHV-1 infected peripheral blood mononuclear cells (PBMCs) cannot express the virus on their

surface, also leaving these cells undetectable to the immune system and insensitive to ADCC and CTL (50). A further evasion strategy of EHV-1 is the interference with NK cell-mediated lysis (50): if cells do not present viral proteins or MHC-1 post infection and are rendered insensitive to ADCC and CTL recognition and destruction, they may remain vulnerable to lysis by NK cells (50). Yet, herpesviruses have developed evasion strategies to avoid detection and activation by NK cells as well (50): EHV-1 is able to lower the concentration of MHC-1 presented on the cell surface, thereby avoiding recognition by NK cells (43, 50, 51).

Downregulation of MHC-1 has been found to be strain dependent (43): while EHV-1 viral Ab4 induced significant downregulation, viral strain RacL11 only led to mild downregulation (43).

Additionally, it is not only virus strain dependent but also locus or allele dependent (43).

EHV-1 also evades or interferes with the cytokine network by either mimicking cytokines or blocking their activity: usually, cytokines act as chemical messengers between cells during an innate or adaptive immune answer (43, 50). Upon secretion, they bind to cytokine receptors immune cells and induce their functions (43, 50). Viruses might mimic these cytokines’ activities by emitting cytokine binding proteins themselves or introducing cytokines with inhibitory action on immune cells (50). EHV-1 employed an immune suppressive activity in ponies which was believed to be connected with a circulating cytokine, transforming growth factor (TGF), exerting multiple inhibitory effects on immune cells (50). Once again, it is the viral surface proteins interfering with cytokine mediated immune response (50). Viral surface protein gB has the ability to bind to certain chemokines and thereby obstructs interaction with receptors, proper signal transduction and migration (43, 50). Ultimately, it will inhibit chemokine-mediated inflammatory reactions (50).