Hepacivirus A Infection in Horses Defines Distinct Envelope Hypervariable Regions and Elucidates Potential Roles of Viral Strain and Adaptive Immune Status in Determining Envelope Diversity and Infection Outcome.

Hepacivirus A (also known as nonprimate hepacivirus and equine hepacivirus) is a hepatotropic virus that can cause both transient and persistent infections in horses. The evolution of intrahost viral populations (quasispecies) has not been studied in detail for hepacivirus A, and its roles in immune evasion and persistence are unknown. To address these knowledge gaps, we first evaluated the envelope gene (E1 and E2) diversity of two different hepacivirus A strains (WSU and CU) in longitudinal blood samples from experimentally infected adult horses, juvenile horses (foals), and foals with severe combined immunodeficiency (SCID). Persistent infection with the WSU strain was associated with significantly greater quasispecies diversity than that observed in horses who spontaneously cleared infection (P = 0.0002) or in SCID foals (P < 0.0001). In contrast, the CU strain was able to persist despite significantly lower (P < 0.0001) and relatively static envelope diversity. These findings indicate that envelope diversity is a poor predictor of hepacivirus A infection outcomes and could be dependent on strain-specific factors. Next, entropy analysis was performed on all E1/E2 genes entered into GenBank. This analysis defined three novel hypervariable regions (HVRs) in E2, at residues 391 to 402 (HVR1), 450 to 461 (HVR2), and 550 to 562 (HVR3). For the experimentally infected horses, entropy analysis focusing on the HVRs demonstrated that these regions were under increased selective pressure during persistent infection. Increased diversity in the HVRs was also temporally associated with seroconversion in some horses, suggesting that these regions may be targets of neutralizing antibody and may play a role in immune evasion.IMPORTANCE Hepacivirus C (hepatitis C virus) is estimated to infect 150 million people worldwide and is a leading cause of cirrhosis and hepatocellular carcinoma. In contrast, its closest relative, hepacivirus A, causes relatively mild disease in horses and is frequently cleared. The relationship between quasispecies evolution and infection outcome has not been explored for hepacivirus A. To address this knowledge gap, we examined envelope gene diversity in horses with resolving and persistent infections. Interestingly, two strain-specific patterns of quasispecies diversity emerged. Persistence of the WSU strain was associated with increased quasispecies diversity and the accumulation of amino acid changes within three novel hypervariable regions following seroconversion. These findings provided evidence that envelope gene mutation is influenced by adaptive immune pressure and may contribute to hepacivirus persistence. However, the CU strain persisted despite relative evolutionary stasis, suggesting that some hepacivirus strains may use alternative mechanisms to persist in the host.

Extended-spectrum antiprotozoal bumped kinase inhibitors: A review.

Many life-cycle processes in parasites are regulated by protein phosphorylation. Hence, disruption of essential protein kinase function has been explored for therapy of parasitic diseases. However, the difficulty of inhibiting parasite protein kinases to the exclusion of host orthologues poses a practical challenge. A possible path around this difficulty is the use of bumped kinase inhibitors for targeting calcium-dependent protein kinases that contain atypically small gatekeeper residues and are crucial for pathogenic apicomplexan parasites' survival and proliferation. In this article, we review efficacy against the kinase target, parasite growth in vitro, and in animal infection models, as well as the relevant pharmacokinetic and safety parameters of bumped kinase inhibitors.

Antibody escape kinetics of equine infectious anemia virus infection of horses.

Lentivirus escape from neutralizing antibodies (NAbs) is not well understood. In this work, we quantified antibody escape of a lentivirus, using antibody escape data from horses infected with equine infectious anemia virus. We calculated antibody blocking rates of wild-type virus, fitness costs of mutant virus, and growth rates of both viruses. These quantitative kinetic estimates of antibody escape are important for understanding lentiviral control by antibody neutralization and in developing NAb-eliciting vaccine strategies.

Experimental transmission of equine hepacivirus in horses as a model for hepatitis C virus.

UNLABELLED: Equine hepacivirus (EHCV; nonprimate hepacivirus) is a hepatotropic member of the Flaviviridae family that infects horses. Although EHCV is the closest known relative to hepatitis C virus (HCV), its complete replication kinetics in vivo have not been described, and direct evidence that it causes hepatitis has been lacking. In this study, we detected EHCV in 2 horses that developed post-transfusion hepatitis. Plasma and serum from these horses were used to experimentally transmit EHCV to 4 young adult Arabian horses, two 1-month-old foals (1 Arabian and 1 Arabian-pony cross), and 2 foals (1 Arabian and 1 Arabian-pony cross) with severe combined immunodeficiency (SCID). Our results demonstrated that EHCV had infection kinetics similar to HCV and that infection was associated with acute and chronic liver disease as measured by elevations of liver-specific enzymes and/or by histopathology. Although most of these animals were coinfected with equine pegivirus (EPgV), also a flavivirus, EPgV viral loads were much lower and often undetectable in both liver and blood. Three additional young adult Arabian-pony crosses and 1 SCID foal were then inoculated with plasma containing only EHCV, and evidence of mild hepatocellular damage was observed. The different levels of liver-specific enzyme elevation, hepatic inflammation, and duration of viremia observed during EHCV infection suggested that the magnitude and course of liver disease was mediated by the virus inoculum and/or by host factors, including breed, age, and adaptive immune status. CONCLUSION: This work documents the complete infection kinetics and liver pathology associated with acute and chronic EHCV infection in horses and further justifies it as a large animal model for HCV.

Equine infectious anemia in 2014: live with it or eradicate it?

In the absence of an effective vaccine, the success of the test and removal approach for the control of equine infectious anemia (EIA) cannot be overstated, at least in those areas where testing has been traditionally routine. This article addresses 4 main aspects: what has been learned about EIA virus, host control of its replication, and inapparent carriers; international status regarding the control of EIA; diagnostic and laboratory investigation; and reducing the spread of blood-borne infections by veterinarians. An attempt is made to put these issues into practical contemporary perspectives for the equine practitioner.

Equine piroplasmosis.

Equine piroplasmosis, caused by the parasites Theileria equi and Babesia caballi, is a globally important disease, affecting a large percentage of the world's horses. This article serves as a review of these divergent parasites. Discussed are the clinical presentation of disease, diagnosis, and treatment. Special attention is given to the current disease status specifically in North America.

Comparative genomic analysis and phylogenetic position of Theileria equi.

BACKGROUND: Transmission of arthropod-borne apicomplexan parasites that cause disease and result in death or persistent infection represents a major challenge to global human and animal health. First described in 1901 as Piroplasma equi, this re-emergent apicomplexan parasite was renamed Babesia equi and subsequently Theileria equi, reflecting an uncertain taxonomy. Understanding mechanisms by which apicomplexan parasites evade immune or chemotherapeutic elimination is required for development of effective vaccines or chemotherapeutics. The continued risk of transmission of T. equi from clinically silent, persistently infected equids impedes the goal of returning the U. S. to non-endemic status. Therefore comparative genomic analysis of T. equi was undertaken to: 1) identify genes contributing to immune evasion and persistence in equid hosts, 2) identify genes involved in PBMC infection biology and 3) define the phylogenetic position of T. equi relative to sequenced apicomplexan parasites. RESULTS: The known immunodominant proteins, EMA1, 2 and 3 were discovered to belong to a ten member gene family with a mean amino acid identity, in pairwise comparisons, of 39%. Importantly, the amino acid diversity of EMAs is distributed throughout the length of the proteins. Eight of the EMA genes were simultaneously transcribed. As the agents that cause bovine theileriosis infect and transform host cell PBMCs, we confirmed that T. equi infects equine PBMCs, however, there is no evidence of host cell transformation. Indeed, a number of genes identified as potential manipulators of the host cell phenotype are absent from the T. equi genome. Comparative genomic analysis of T. equi revealed the phylogenetic positioning relative to seven apicomplexan parasites using deduced amino acid sequences from 150 genes placed it as a sister taxon to Theileria spp. CONCLUSIONS: The EMA family does not fit the paradigm for classical antigenic variation, and we propose a novel model describing the role of the EMA family in persistence. T. equi has lost the putative genes for host cell transformation, or the genes were acquired by T. parva and T. annulata after divergence from T. equi. Our analysis identified 50 genes that will be useful for definitive phylogenetic classification of T. equi and closely related organisms.

Protective effects of broadly neutralizing immunoglobulin against homologous and heterologous equine infectious anemia virus infection in horses with severe combined immunodeficiency.

Using the equine infectious anemia virus (EIAV) lentivirus model system, we previously demonstrated protective effects of broadly neutralizing immune plasma in young horses (foals) with severe combined immunodeficiency (SCID). However, in vivo selection of a neutralization-resistant envelope variant occurred. Here, we determined the protective effects of purified immunoglobulin with more potent broadly neutralizing activity. Overall, protection correlated with the breadth and potency of neutralizing activity in vitro. Four of five SCID foals were completely protected against homologous challenge, while partial protection occurred following heterologous challenge. These results support the inclusion of broadly neutralizing antibodies in lentivirus control strategies.

Decreased infectivity of a neutralization-resistant equine infectious anemia virus variant can be overcome by efficient cell-to-cell spread.

Two variants of equine infectious anemia virus (EIAV) that differed in sensitivity to broadly neutralizing antibody were tested in direct competition assays. No differences were observed in the growth curves and relative fitness scores of EIAVs of principal neutralizing domain variants of groups 1 (EIAV(PND-1)) and 5 (EIAV(PND-5)), respectively; however, the neutralization-resistant EIAV(PND-5) variant was less infectious in single-round replication assays. Infectious center assays indicated similar rates of cell-to-cell spread, which was approximately 1,000-fold more efficient than cell-free infectivity. These data indicate that efficient cell-to-cell spread can overcome the decreased infectivity that may accompany immune escape and should be considered in studies assessing the relative levels of fitness among lentivirus variants, including HIV-1.

Selection of a rare neutralization-resistant variant following passive transfer of convalescent immune plasma in equine infectious anemia virus-challenged SCID horses.

Vaccines preventing HIV-1 infection will likely elicit antibodies that neutralize diverse strains. However, the capacity for lentiviruses to escape broadly neutralizing antibodies (NAbs) is not completely understood, nor is it known whether NAbs alone can control heterologous infection. Here, we determined that convalescent immune plasma from a horse persistently infected with equine infectious anemia virus (EIAV) neutralized homologous virus and several envelope variants containing heterologous principal neutralizing domains (PND). Plasma was infused into young horses (foals) affected with severe combined immunodeficiency (SCID), followed by challenge with a homologous EIAV stock. Treated SCID foals were protected against clinical disease, with complete prevention of infection occurring in one foal. In three SCID foals, a novel neutralization-resistant variant arose that was found to preexist at a low frequency in the challenge inoculum. In contrast, SCID foals infused with nonimmune plasma developed acute disease associated with high levels of the predominant challenge virus. Following transfer to an immunocompetent horse, the neutralization-resistant variant induced a single febrile episode and was subsequently controlled in the absence of type-specific NAb. Long-term control was associated with the presence of cytotoxic T lymphocytes (CTL). Our results demonstrate that immune plasma with neutralizing activity against heterologous PND variants can prevent lentivirus infection and clinical disease in the complete absence of T cells. Importantly, however, rare neutralization-resistant envelope variants can replicate in vivo under relatively broad selection pressure, highlighting the need for protective lentivirus vaccines to elicit NAb responses with increased breadth and potency and/or CTL that target conserved epitopes.

Development of a DNA microarray for detection of expressed equine classical MHC class I sequences in a defined population.

Development of an accurate and efficient molecular-based equine MHC class I typing method would facilitate the study of T lymphocyte immune responses in horses. Here, a DNA microarray was designed to detect expressed classical MHC class I genes comprising serologically defined equine leukocyte antigen (ELA)-A haplotypes represented in a closed Arabian horse breeding herd. Initially, cloning and sequencing of RT-PCR products were used to identify sequences associated with the ELA-A1, A4, and W11 haplotypes, and one undefined haplotype, in six horses. Subsequently, sequence-specific, conserved (positive control), and random nucleotide (negative control) 23- to 27-mer oligonucleotide microarray probes were designed and spotted onto an epoxy-coated masked slide using a robotic arrayer. Bulk RT-PCR products from each horse were biotinylated by nick translation, hybridized to the array, and detected using tyramide signal amplification. The microarray consistently detected eight of nine classical MHC class I transcripts and allowed ELA haplotypic associations to be made. Cloning and sequencing of RT-PCR products were then performed in a group of ELA disparate horses and ponies, in which six novel sequences were identified. This group was used to determine the specificity of the array. Overall, the microarray was more efficient than cloning and sequencing for detecting expressed classical MHC class I sequences in this defined population of horses, and was significantly more specific than serology. These results confirmed the utility of a microarray-based method for high-resolution MHC class I typing in the horse. With additional probes the array could be useful in a broader population.

Identification of Rhodococcus equi lipids recognized by host cytotoxic T lymphocytes.

Immune adult horses have CD8(+) cytotoxic T lymphocytes (CTLs) that recognize and lyse Rhodococcus equi-infected cells in an equine lymphocyte alloantigen (ELA)-A [classical major histocompatibility complex (MHC) class I]-unrestricted fashion. As protein antigens are MHC class I-restricted, the lack of restriction suggests that the bacterial antigens being recognized by the host are not proteins. The goals of this study were to test the hypothesis that these CTLs recognize unique R. equi cell-wall lipids related to mycobacterial lipids. Initial experiments showed that treatment of soluble R. equi antigen with broadly reactive proteases did not significantly diminish the ability of the antigen to stimulate R. equi-specific CTLs. R. equi-specific CTLs were also shown to lyse target cells (equine macrophages) pulsed with an R. equi lipid extract. Analysis of the R. equi lipid by TLC and MS (MALDI-TOF and ES) indicated that the extracted antigen consisted of three primary fractions: trehalose monomycolate (TMM), trehalose dimycolate (TDM) and cardiolipin (CL). ELA-A-mismatched cells pulsed with purified TMM and CL, but not the TDM fraction, were recognized and lysed by R. equi-specific CTLs. Because of their role in immune clearance and pathogenesis, transcription of the cytokines gamma interferon (IFN-gamma) and interleukin-4 (IL-4) was also measured in response to R. equi lipids by using real-time PCR; elevated IFN-gamma, but not IL-4, was associated with host clearance of the bacteria. The whole-cell R. equi lipid and all three R. equi lipid fractions resulted in marked increases in IFN-gamma transcription, but no increase in IL-4 transcription. Together, these data support the hypothesis that immune recognition of unique lipids in the bacterial cell wall is an important component of the protective immune response to R. equi. The results also identify potential lipid antigens not previously shown to be recognized by CTLs in an important, naturally occurring actinomycete bacterial pathogen.

Evaluation of an arthroscopic approach for transection of the equine collateral sesamoidean ligament.

OBJECTIVE: To evaluate: (1) an arthroscopic technique for transection of the collateral sesamoidean ligament (CSL); and (2) the healing response using magnetic resonance (MR) and microscopic examination. STUDY DESIGN: Experimental study. ANIMALS: Adult horses (n=6). METHODS: Six sound horses with normal front foot radiographic and MR examinations were used. Lameness examination was performed before surgery and monthly for 12 months. Front foot radiography was performed at 180 and 360 days after surgery. Front foot MR was performed before, and at 7, 90, 180, and 360 days after surgery. Arthroscopic CSL desmotomy was performed on 1 forelimb. Gross and microscopic examination was performed on the CSL from both forelimbs at 360 days after surgery. Lameness scores were compared over time using the nonparametric Friedman's test for paired groups. CSL measurements were compared using paired t-tests with a 2-tailed significance level of P<.05. RESULTS: Radiographs remained normal throughout study period. Surgery resulted in lameness on the operated limb for up to 2 months, after which all horses returned to soundness. CSL transection was confirmed during arthroscopy and with MR examination 7 days after surgery. Gross and microscopic evaluation confirmed ligament healing. CONCLUSIONS: CSL desmotomy resulted in short-term lameness after surgery followed by healing of the CSL confirmed by gross and microscopic analysis.

Exposure to ambient air causes degradation and decreased in vitro potency of buparvaquone and parvaquone.

Buparvaquone and parvaquone are hydroxynaphthoquinone compounds commonly used to treat livestock infected with Theileria species such as T. parva and T. annulata. In many (sub)tropical regions, chromatic changes in medicines can result from extreme environmental conditions and improper drug storage or handling, raising the possibility of drug degradation and loss of potency. We evaluated the effects of UV light, elevated temperature, and atmospheric air on the stability and potency of both buparvaquone and parvaquone by using a combination of high performance liquid chromatography (HPLC) and a T. equi based in vitro parasite growth inhibition assay (to measure potency). Aliquots (1ml; 3 replicates per treatment) of each compound were subjected to a variety of treatments that varied in duration and intensity followed by HPLC and potency assays. Exposure to ambient air for 50 days was correlated with a significant loss of potency for both buparvaquone (4535%, P< 0.05) and parvaquone (247%, P< 0.05), while elevated temperature (37°C) and UV light exposure (24 h) had no significant impact (P> 0.05). The decrease in potency of both buparvaquone and parvaquone correlated with drug degradation (r = -0.74 and -0.88, respectively) as measured by HPLC. In practice, if there is headspace present in the vial, then ambient air will invariably enter the vial and contribute to degradation of these compounds. Such degradation may contribute to increasing drug resistance, economic losses for farmers, and animal welfare concerns for animals that are treated for Theileria infections.

Exposure to ambient air causes degradation and decreased in vitro potency of buparvaquone and parvaquone.

Buparvaquone and parvaquone are hydroxynaphthoquinone compounds commonly used to treat livestock infected with Theileria species such as T. parva and T. annulata. In many (sub)tropical regions, chromatic changes in medicines can result from extreme environmental conditions and improper drug storage or handling, raising the possibility of drug degradation and loss of potency. We evaluated the effects of UV light, elevated temperature, and atmospheric air on the stability and potency of both buparvaquone and parvaquone by using a combination of high performance liquid chromatography (HPLC) and a T. equi based in vitro parasite growth inhibition assay (to measure potency). Aliquots (1 ml; 3 replicates per treatment) of each compound were subjected to a variety of treatments that varied in duration and intensity followed by HPLC and potency assays. Exposure to ambient air for 50 days was correlated with a significant loss of potency for both buparvaquone (4535%, P <  0.05) and parvaquone (247%, P <  0.05), while elevated temperature (37°C) and UV light exposure (24 h) had no significant impact (P >  0.05). The decrease in potency of both buparvaquone and parvaquone correlated with drug degradation (r = -0.74 and -0.88, respectively) as measured by HPLC. In practice, if there is headspace present in the vial, then ambient air will invariably enter the vial and contribute to degradation of these compounds. Such degradation may contribute to increasing drug resistance, economic losses for farmers, and animal welfare concerns for animals that are treated for Theileria infections.

Failure of low-dose recombinant human IL-2 to support the survival of virus-specific CTL clones infused into severe combined immunodeficient foals: lack of correlation between in vitro activity and in vivo efficacy.

Although CTL are important for control of lentiviruses, including equine infectious anemia virus (EIAV), it is not known if CTL can limit lentiviral replication in the absence of CD4 help and neutralizing antibody. Adoptive transfer of EIAV-specific CTL clones into severe combined immunodeficient (SCID) foals could resolve this issue, but it is not known whether exogenous IL-2 administration is sufficient to support the engraftment and proliferation of CTL clones infused into immunodeficient horses. To address this question we adoptively transferred EIAV Rev-specific CTL clones into four EIAV-challenged SCID foals, concurrent with low-dose aldesleukin (180,000U/m2), a modified recombinant human IL-2 (rhuIL-2) product. The dose was calculated based on the specific activity on equine PBMC in vitro, and resulted in plasma concentrations considered sufficient to saturate high affinity IL-2 receptors in humans. Despite specific activity on equine PBMC that was equivalent to recombinant equine IL-2 and another form of rhuIL-2, aldesleukin did not support the engraftment and expansion of infused CTL clones, and control of viral load and clinical disease did not occur. It was concluded that survival of Rev-specific CTL clones infused into EIAV-challenged SCID foals was not enhanced by aldesleukin at the doses used in this study, and that in vitro specific activity did not correlate with in vivo efficacy. Successful adoptive immunotherapy with CTL clones in immunodeficient horses will likely require higher doses of rhuIL-2, co-infusion of CD4+ T lymphocytes, or administration of equine IL-2.

Dynamics of lentiviral infection in vivo in the absence of adaptive immune responses.

Understanding the dynamics of acute viral infection is crucial for developing strategies to prevent and control infection. In this study, lentiviral dynamics in a host without adaptive immunity were examined in order to determine kinetic parameters of infection and quantify the effect of neutralizing antibodies in preventing infection, using mathematical modeling of data from equine infectious anemia virus (EIAV) infection of horses with severe combined immunodeficiency (SCID). Estimated parameters were used to calculate the basic reproductive number and virus doubling time and found that the rate that antibodies neutralized virus was ~18 times greater than the virus clearance rate. These results establish EIAV replication kinetics in SCID horses and the minimal efficacy of antibodies that blocked infection. Furthermore, they indicate that EIAV is at most mildly cytopathic. This study advances our understanding of EIAV infection and may have important implications for the control of other viral infections, including HIV.