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OBJECTIVE To evaluate the effects of treatment of horses with standard platelet inhibitors on ex vivo inhibition of platelet activation by equine herpesvirus type I (EHV-I). ANIMALS II healthy adult horses. PROCEDURES In a double-blinded, placebo-controlled crossover study, horses were treated orally for 5 days with theophylline (5 mg/kg, q 12 h), pentoxifylline (10 mg/kg, q 12 h), clopidogrel bisulfate (4 mg/kg, q 24 h), acetylsalicylic acid (20 mg/kg, q 24 h), or placebo. Horses received all treatments, each separated by a 3-week washout period. Platelet-rich plasma was prepared from citrated blood samples obtained before each treatment session and 4 hours after each final drug dose. Platelets were exposed to 2 EHV-I strains (at I plaque forming units/cell) or positive (thrombin-convulxin) and negative control substances for 10 minutes, then platelet activation was assessed by determining the percentages of P-selectin-positive platelets and platelet-derived microparticles (PDMPs; small events positive for annexin V) with flow cytometry. Platelet aggregation in response to 10μM ADP was also assessed. RESULTS No significant differences in median percentages of P-selectin-positive platelets and PDMPs in EHV-I-exposed platelets were identified between measurement points (before and after treatment) for all drugs, nor were differences identified among drugs at each measurement point. Only clopidogrel significantly inhibited platelet aggregation in response to ADP in platelet-rich plasma samples obtained after that treatment session. CONCLUSIONS AND CLINICAL RELEVANCE Treatment of horses with standard platelet inhibitors had no effect on EHV-I-induced platelet α-granule exteriorization or microvesiculation and release of PDMPs ex vivo, suggesting these drugs will not prevent platelet activation induced directly by EHV-I in vivo.

Equid herpesvirus type 1 (EHV-1) causes outbreaks of abortion and neurological disease in horses. One of the main causes of these clinical syndromes is thrombosis in placental and spinal cord vessels, however the mechanism for thrombus formation is unknown. Platelets form part of the thrombus and amplify and propagate thrombin generation. Here, we tested the hypothesis that EHV-1 activates platelets. We found that two EHV-1 strains, RacL11 and Ab4 at 0.5 or higher plaque forming unit/cell, activate platelets within 10 minutes, causing α-granule secretion (surface P-selectin expression) and platelet microvesiculation (increased small events double positive for CD41 and Annexin V). Microvesiculation was more pronounced with the RacL11 strain. Virus-induced P-selectin expression required plasma and 1.0 mM exogenous calcium. P-selectin expression was abolished and microvesiculation was significantly reduced in factor VII- or X-deficient human plasma. Both P-selectin expression and microvesiculation were re-established in factor VII-deficient human plasma with added purified human factor VIIa (1 nM). A glycoprotein C-deficient mutant of the Ab4 strain activated platelets as effectively as non-mutated Ab4. P-selectin expression was abolished and microvesiculation was significantly reduced by preincubation of virus with a goat polyclonal anti-rabbit tissue factor antibody. Infectious virus could be retrieved from washed EHV-1-exposed platelets, suggesting a direct platelet-virus interaction. Our results indicate that EHV-1 activates equine platelets and that α-granule secretion is a consequence of virus-associated tissue factor triggering factor X activation and thrombin generation. Microvesiculation was only partly tissue factor and thrombin-dependent, suggesting the virus causes microvesiculation through other mechanisms, potentially through direct binding. These findings suggest that EHV-1-induced platelet activation could contribute to the thrombosis that occurs in clinically infected horses and provides a new mechanism by which viruses activate hemostasis.

The alphaherpesvirus, equine herpesvirus type 1 (EHV-1), is a highly prevalent cause of equine infectious abortion and encephalomyelopathy. These syndromes have been attributed to ischemic necrosis from thrombosis in placental and neural vessels, although the mechanisms underlying thrombosis are unknown. After inhalation, EHV-1 establishes a peripheral blood mononuclear cell-associated viremia, with monocytes being a target of infection. Monocytes are also the main source of tissue factor (TF) in diseased states. Since TF is the primary activator of coagulation, increased monocyte TF expression could be involved in EHV-1-associated thrombosis. We hypothesized that EHV-1 infection would induce TF-dependent procoagulant activity in equine monocytes. Monocyte-enriched fractions of blood were infected with abortigenic (RacL11, NY03) and neuropathogenic (Ab4) EHV-1 strains. All strains induced procoagulant activity, to variable degrees, within 1 to 4 h, with maximal activity at 24 h, after infection. Virus-induced procoagulant activity was similar to that seen with lipopolysaccharide, a known stimulant of TF-mediated procoagulant responses. Virus-induced procoagulant activity was factor VIIa-dependent and temporally associated with TF gene transcription, implicating TF as the main driver of the activity. Procoagulant activity was mildly decreased (30-40%) when virus was inactivated by ultraviolet light or when infected cells were treated with aphidicolin, a virus DNA polymerase inhibitor, suggesting early events of virus infection (attachment, entry or intracellular trafficking) are the primary stimulus of procoagulant activity. Our results indicate that EHV-1 rapidly stimulates procoagulant activity in equine monocytes in vitro. The EHV-1-induced procoagulant activity in monocytes may contribute to clinical thrombosis in horses with EHV-1 infection.

To better understand the pathogenesis of human herpesvirus 6 (HHV-6), it is important to elucidate the functional aspects of immediate-early (IE) genes at the initial phase of the infection. To study the functional role of the HHV-6B IE gene encoding U95, we generated a U95-Myc fusion protein and screened a pretransformed bone marrow cDNA library for U95-interacting proteins, using yeast-two hybrid analysis. The most frequently appearing U95-interacting protein identified was GRIM-19, which belongs to the family of genes associated with retinoid-interferon mortality and serves as an essential component of the oxidative phosphorylation system. This interaction was verified by both coimmunoprecipitation and confocal microscopic coimmunolocalization. Short-term HHV-6B infection of MT-4 T-lymphocytic cells induced syncytial formation, resulted in decreased mitochondrial membrane potential, and led to progressively pronounced ultrastructural changes, such as mitochondrial swelling, myelin-like figures, and a loss of cristae. Compared to controls, RNA interference against U95 effectively reduced the U95 mRNA copy number and abrogated the loss of mitochondrial membrane potential. Our results indicate that the high affinity between U95 early viral protein and GRIM-19 may be closely linked to the detrimental effect of HHV-6B infection on mitochondria. These findings may explain the alternative cell death mechanism of expiration, as opposed to apoptosis, observed in certain productively HHV-6B-infected cells. The interaction between U95 and GRIM-19 is thus functionally and metabolically significant in HHV-6B-infected cells and may be a means through which HHV-6B modulates cell death signals by interferon and retinoic acid.

The coatomer complex is involved in intracellular protein transport and comprises an assembly of seven polypeptide subunits designated alpha, beta, beta', gamma, delta, epsilon, and zeta COP. Rooted phylogenetic trees constructed from the full-length cDNA and amino acid sequences of 49 COP entities in different eukaryotes from yeast to man generally revealed striking conservation of each subunit through evolution. Both nucleotide and protein trees displayed close relationships between alpha and beta' subunits, between beta and gamma subunits, and between delta and zeta subunits, implying evolution from common ancestors as well as functional similarity. Interestingly, although 6 out of 7 epsilon-COP genes appeared to be grouped and related to the beta-COP genes, 4 out of 7 epsilon