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Purpose: To compare the efficacy of the vertical lid split (VLS) to the standard lateral canthotomy and cantholysis (LC/C) for orbital compartment syndrome (OCS) in the cadaveric model.Methods: Simulated OCS was achieved in seven fresh frozen cadaveric orbits. Orbital pressure (OP) was monitored in one control orbit and six interventional orbits. Initial OP was recorded before three right orbits underwent lateral canthotomy with superior and inferior cantholysis, and three left orbits underwent vertical lid split of the upper and lower eyelids. In all 7 orbits, OP was recorded for a total of 16 min. The main outcome measure was the amount of OP reduction at timed intervals.Results: Beginning OP in the control orbit was 109 mmHg, and average initial OP of the LC/C and VLS orbits were 90 and 103 mmHg, respectively. The control orbit maintained high OP without intervention. One minute after LC/C, OP decreased an average of 58.7 mmHg (65.2%; range 48-65 mmHg). One minute following VLS, OP decreased an average of 63 mmHg (61.0%; range 39-102 mmHg). At 16 min, OP reduction in the LC/C orbits averaged 65.3 mmHg (72.6%; range 56-71 mmHg), and OP reduction in the VLS orbits averaged 78 mmHg (75.5%; range 54-121 mmHg). Both interventions produced a comparable reduction in OP.Conclusions: Vertical lid split was found to be as effective as LC/C in reducing OP. The technical simplicity of the VLS lends itself well to utilization by physicians who are unfamiliar with eyelid surgery.

Equine rotavirus group A (ERVA) is one of the most common causes of foal diarrhea. Starting in February 2021, there was an increase in the frequency of severe watery to hemorrhagic diarrhea cases in neonatal foals in Central Kentucky. Diagnostic investigation of fecal samples failed to detect evidence of diarrhea-causing pathogens including ERVA. Based on Illumina-based metagenomic sequencing, we identified a novel equine rotavirus group B (ERVB) in fecal specimens from the affected foals in the absence of any other known enteric pathogens. Interestingly, the protein sequence of all 11 segments had greater than 96% identity with group B rotaviruses previously found in ruminants. Furthermore, phylogenetic analysis demonstrated clustering of the ERVB with group B rotaviruses of caprine and bovine strains from the USA. Subsequent analysis of 33 foal diarrheic samples by RT-qPCR identified 23 rotavirus B-positive cases (69.69%). These observations suggest that the ERVB originated from ruminants and was associated with outbreaks of neonatal foal diarrhea in the 2021 foaling season in Kentucky. Emergence of the ruminant-like group B rotavirus in foals clearly warrants further investigation due to the significant impact of the disease in neonatal foals and its economic impact on the equine industry.

Optic nerve sheath meningiomas (ONSM) and intra-conal orbital lymphomas are common entities on the differential of a retrobulbar optic nerve involving space-occupying lesion. In this study, we compare the pre-surgical diagnosis, based on clinical presentation and neuroimaging, to the surgical pathology results of intra-conal orbital lymphomas and ONSM. This is an IRB approved retrospective chart review of orbital lymphomas and optic nerve sheath meningiomas biopsied by a single surgeon over a 4-year period at a single institution. Pre-surgical diagnosis and surgical pathology were compared. Fifteen cases of orbital lymphoma were identified. Fourteen were excluded based on extra-conal location. The single histologically confirmed intra-conal orbital lymphoma had a pre-surgical diagnosis of ONSM. Four cases of optic nerve sheath meningioma were identified. Three of the 4 cases of histologically confirmed ONSM had a pre-surgical diagnosis of ONSM. One of the 4 had a pre-surgical diagnosis of lymphoma. Diagnosis based on surgical pathology differed from the pre-surgical diagnosis in 2 out of 5 cases showing that clinical diagnosis does not always correlate with histologic diagnosis. Although both diseases are typically managed with radiation therapy, the treatment dosage and systemic disease implications are very different. These findings emphasis the importance of biopsy in the diagnosis of orbital lesions surrounding the optic nerve.

BackgroundCrawford tube placement is commonly used to achieve patency of nasolacrimal ducts for epiphora secondary to nasolacrimal duct obstruction. The nasal passages of pediatric patients are narrower than adults, and the result is a relatively higher risk of intranasal complications (e.g., synechiae, bleeding) with Crawford tube placement. There is evidence that general anesthesia may negatively affect the neurocognitive function and behavioral development of children, which prompts efforts to decrease operation times for potential health benefits and also potentially to reduce health care costs. Analysis ofresearch reports supports the use of nasal endoscopy to reduce intranasal complications with Crawford tube placement; however, no publications currently address the effect of nasal endoscopy concurrent with Crawford tube placement on operative times on pediatric patients or the resulting effects on health care costs.ObjectiveTo determine the difference in procedure time and cost between Crawford tubes placed traditionally and those placed with endoscopic assistance in pediatric patients.MethodsA chart review was performed from January 1, 2011 to December 31, 2016 for cases using CPT codes 68815 or 31231. Within this group of patients, the patient in whom nasal endoscopy was performed were placed in the “endoscopic” group and the patients without endoscopy were placed in the “traditional” group. Procedure times were noted, and the t-test was performed to examine for any statistically significant difference in operative times. Estimates of anesthesia cost savings were made. We identified 24 patients in the traditional group and 7 patients in the endoscopic group.ResultsThe average operative time for the traditional group was 27.3 minutes compared with 14.0 minutes for the endoscopic group (p = 0.02). The cost comparison data revealed no significant difference with the traditional group averaging $9369 per procedure and the endoscopic group averaging $8891 (p = 0.51).ConclusionAn endoscopically assisted Crawford tube placement resulted in patients who had less time under general anesthesia compared with the traditional technique at no difference in cost.

Equine arteritis virus (EAV) has the unique ability to establish long-term persistent infection in the reproductive tract of stallions and be sexually transmitted. Previous studies showed that long-term persistent infection is associated with a specific allele of the CXCL16 gene (CXCL16S) and that persistence is maintained despite the presence of local inflammatory and humoral and mucosal antibody responses. Here, we performed transcriptomic analysis of the ampullae, the primary site of EAV persistence in long-term EAV carrier stallions, to understand the molecular signatures of viral persistence. We demonstrated that the local CD8+ T lymphocyte response is predominantly orchestrated by the transcription factors eomesodermin (EOMES) and nuclear factor of activated T-cells cytoplasmic 2 (NFATC2), which is likely modulated by the upregulation of inhibitory receptors. Most importantly, EAV persistence is associated with an enhanced expression of CXCL16 and CXCR6 by infiltrating lymphocytes, providing evidence of the implication of this chemokine axis in the pathogenesis of persistent EAV infection in the stallion reproductive tract. Furthermore, we have established a link between the CXCL16 genotype and the gene expression profile in the ampullae of the stallion reproductive tract. Specifically, CXCL16 acts as a “hub” gene likely driving a specific transcriptional network. The findings herein are novel and strongly suggest that RNA viruses such as EAV could exploit the CXCL16/CXCR6 axis in order to modulate local inflammatory and immune responses in the male reproductive tract by inducing a dysfunctional CD8+ T lymphocyte response and unique lymphocyte homing in the reproductive tract., Author summary A distinctive feature of equine arteritis virus (EAV) is its ability to establish long-term persistent infection in the stallion reproductive tract in the presence of strong immune and inflammatory responses. The data presented herein provides insight into the molecular signature of the inflammatory response during persistent infection in the male reproductive tract, and shows that long-term persistence is associated with the predominance of specific CD8+ T lymphocyte transcription factors that drive the inflammatory process in the reproductive tract, along with the upregulation of inhibitory receptors and CXCL16/CXCR6, a chemokine axis strongly implicated in EAV persistence. Furthermore, the host’s CXCL16 genotype drives the changes in transcriptional factors that favors EAV persistent infection. These findings have a broad translational importance in the immunopathogenesis of EAV and other persistent viral infections in the male reproductive tract of animals and humans, as well as in the prevention and treatment of such infections.

Purpose The safety profile of the transcutaneous medial canthal incision for access to the medial orbit is assessed with a focus on the risk of post-operative iatrogenic epiphora. Methods A retrospective chart review of patients undergoing medial orbitotomy via the transcutaneous medial canthal incision was performed. Patients with a minimum of 3 months of follow-up were included and post-operative complications were assessed and characterized. Results One-hundred-fifty patients were included in the study. A total of 4 complications were identified, including one each of the following: nasolacrimal duct obstruction, hypertrophic scar, suture granuloma and soft tissue infection. Only the nasolacrimal duct obstruction required surgical intervention. Discussion Access to the medial orbit has been achieved through a variety of approaches, each with their own benefits and risk profile. The transcaruncular approach has increased in usage as a means to avoid a visible cutaneous scar and decrease the risk of iatrogenic epiphora, however, there are specific patients who may have relative contraindications to this approach. The current study demonstrates the low risk profile of the transcutaneous medial canthal incision, specifically the minimal risk of iatrogenic damage to the nasolacrimal outflow system. This approach is another useful tool which orbit surgeons should be familiar with to offer as an option to patients requiring medial orbitotomy.

Purpose To examine the incidence of intracapsular hemorrhage in orbital fracture repair with non-fixated nylon sheet implants. Methods A retrospective chart review of 227 patients presenting from January 2013 to December 2016 for orbital fracture repair with nylon sheet implants. Results Of the 331 orbital fractures repaired over 4 years, a total of 227 met inclusion criteria. The average implant thickness was 0.38 mm and no implants were fixated. Four total implants (1.8%) were removed due to complications; one each secondary to exploration for ongoing postoperative diplopia, immediate post-operative orbital hemorrhage, a cystic mass anterior to the implant, and pain. There were no cases of intracapsular hemorrhage nor infection for any of the 227 patients over 4 years. Conclusions To the authors knowledge, this represents the largest case series to date to assess the rate of intracapsular hemorrhage in non-fixated nylon sheet orbital implants. In the 227 cases reviewed over a 4-year period, there were no cases of intracapsular hemorrhage. This suggests a much lower complication rate than previously reported. Precis A case series of 227 patients who underwent orbital fracture repair with non-fixated nylon sheet implants.

Summary Equine arteritis virus is the causative agent of equine viral arteritis, a respiratory and reproductive disease that affects the members of the family Equidae. The virus was first isolated from the lung of an aborted fetus after an extensive outbreak of respiratory disease and abortion on a Standardbred breeding farm near Bucyrus, Ohio, in 1953. Since then, periodic outbreaks of equine viral arteritis have been reported in a number of countries around the world. This disease may result in significant economic loss to the equine industry due to the occurrence of abortion in pregnant mares, neonatal mortality, and establishment of the carrier state in stallions. This article provides an extensive review on equine arteritis virus, epidemiology, disease, pathogenesis, and prevention and control measures.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory and reproductive disease of horses. Most importantly, EAV induces abortion in pregnant mares and can establish persistent infection in up to 10-70% of the infected stallions, which will continue to shed the virus in their semen. The objective of this study was to develop and evaluate a reverse transcription insulated isothermal polymerase chain reaction (RT-iiPCR) for the detection of EAV in semen and tissue samples. The newly developed assay had a limit of detection of 10 RNA copies and a 10-fold higher sensitivity than a previously described real-time RT-PCR (RT-qPCR). Evaluation of 125 semen samples revealed a sensitivity and specificity of 98.46% and 100.00%, respectively for the RT-qPCR assay, and 100.00% and 98.33%, respectively for the RT-iiPCR assay. Both assays had the same accuracy (99.2%, k=0.98) compared to virus isolation. Corresponding values derived from testing various tissue samples (n=122) collected from aborted fetuses, foals, and EAV carrier stallions are as follows: relative sensitivity, specificity, and accuracy of 88.14%, 96.83%, and 92.62% (k=0.85), respectively for the RT-qPCR assay, and 98.31%, 92.06%, and 95.08% (k=0.90), respectively for the RT-iiPCR assay. These results indicate that RT-iiPCR is a sensitive, specific, and a robust test enabling detection of EAV in semen and tissue samples with very considerable accuracy. Even though the RT-qPCR assay showed a sensitivity and specificity equal to virus isolation for semen samples, its diagnostic performance was somewhat limited for tissue samples. Thus, this new RT-iiPCR could be considered as an alternative tool in the implementation of EAV control and prevention strategies.

Studies in our laboratory have shown that horses can be divided into two distinct groups based on whether they possess a subpopulation of CD3 + T lymphocytes susceptible to in vitro infection by equine arteritis virus (EAV) or not. It has been also demonstrated that stallions with the CD3 + T lymphocyte susceptibility phenotype to in vitro EAV infection are at higher risk of becoming persistently infected carriers compared to those that lack this phenotype. Furthermore, experimental EAV infection of horses with either the in vitro CD3 + T lymphocyte susceptibility or resistance phenotype showed a significant difference between the two groups in terms of proinflammatory and immunomodulatory cytokine mRNA expression. A genome-wide association study (GWAS) demonstrated that these phenotypes are associated with the CXCL16 gene located in equine chromosome 11 (ECA11). We demonstrated that the membrane bound form of the protein encoded by EqCXCL16 gene function as a primary receptor molecule for EAV. Moreover, the secretory form of the CXCL16 protein is the ligand for the chemokine receptor CXCR6 that is predicted to be expressed on CD4 + and CD8 + T cells, NKT cells, and NK cells. Thus, equine CXCL16 and CXCR6 may be two major cellular proteins associated with the EAV carrier state in stallions. Finally, analysis of multiple tissues from EAV carrier stallions unequivocally confirmed the ampullae of the vas deferens as the primary site of EAV persistence with dual immunohistochemistry studies showing that EAV is localized in fibrocytes and mononuclear cells (T and B lymphocytes) but not in the glandular epithelium.

SummaryReasons for performing study An analysis of the factors leading to equine disease events was used to support the development of international recommendations for mitigating the risk of disease dissemination through sport horse movements (high health, high performance – ‘HHP' horses). Objectives A review was undertaken to identify the factors resulting in equine disease events following international movement of horses to draw lessons in support of the development of international recommendations for the safe movements of a specific subpopulation of horses: the HHP sport horses. Study design Systematic review carried out in accordance with the PRISMA statement. Methods The review covered disease events that occurred from 1995 to 2014, identified from the databases of the World Organisation for Animal Health (OIE) and international surveillance reports. Results Overall, 54 disease events were identified, of which 7 were contained in post arrival quarantine and the others resulted in the introduction of pathogens into importing countries. For 81% of the introductions, the OIE recommendations applicable to the diseases involved had not been complied with. Subclinical infections are a challenge for international trade: 88% of the regulated movements that resulted in introductions involved infected horses that showed no clinical signs at the time of import. Biosecurity and management practices in resident equine populations were identified as important mitigating factors in preventing disease spread to the local horse population. Conclusions The global increase in international horse movements, if not appropriately regulated and supervised by competent veterinary authorities and respective equine industry partners, could potentially lead to increased global spread of infectious equine diseases. Appropriate mitigation measures and compliance with OIE import recommendations for specific diseases can significantly reduce this risk. The recommendations proposed under the HHP approach take into account the mitigation measures identified by this review as important factors in preventing pathogen introduction and spread.

The 'high-health, high-performance' (HHP) horse concept has been developed by the World Organisation for Animal Health (OIE) together with the F6ddration Equestre Internationale and the International Federation of Horseracing Authorities. This concept is outlined in the OIE Terrestrial Animal Health Code (Chapter 4.16). It aims to address impediments to the international movement of competition horses through a harmonised, practically feasible, globally applicable framework based on simplified certification requirements for the temporary importation of HHP horses and for their return to their country of usual residence. Based on the principle of compartmentalisation, the high health status of these horses would be established by the application, at all times, of stringent health management practices and biosecurity measures to create and maintain a functional separation between horses within the defined compartment and all other equids. These provisions are intended to mitigate the risk of disease spread for most OIE-listed diseases. For six OIE-listed diseases (African horse sickness, equine influenza, equine infectious anaemia, equine piroplasmosis, glanders and Venezuelan equine encephalomyelitis), the OIE recommends disease-specific mitigation measures, which have been included in a model HHP Veterinary Certificate, to provide additional guarantees to mitigate the risk of disease spread. This article presents the HH P disease risk mitigation strategy. It demonstrates how continuous observance of the HHP biosecurity measures and health management practices provides a scientific rationale for limiting the list of diseases for which HHP horses should be screened with respect to their temporary importation for competition purposes.

To assess the practice patterns of current members of the American Society of Ophthalmic Plastic and Reconstructive Surgery (ASOPRS) in the treatment of medically refractive endophthalmitis (MRE). A 17-question survey included the procedure of choice for MRE, the preferred type of orbital implant, the timing of implant placement, and the incidence of postoperative complications. 107 ASOPRS members participated in the study. In the setting of MRE, 72% preferred evisceration versus 28% who preferred enucleation. Fifty-nine percent of responders preferred enucleation if the MRE extended to orbital tissues versus 27% who would eviscerate. Among those that would place an orbital implant at the initial surgery, 65% would do so during an enucleation and 58% would do so during an evisceration. If an orbital implant was placed at the initial surgery, 52% of responders preferred a silicone implant, while 17% preferred hydroxyapatite implant. A minority of responders (6%) reported "yes" to ever having infectious complications after primary enucleation, compared to 10% after primary evisceration. Overall, 12% of responders noted more frequent complications (other than infectious) when an enucleation was performed compared to 5% for eviscerations. Current opinions on the surgical management of MRE show a trend toward evisceration, with 65% of ASOPRS respondents placing an orbital implant at the time of an enucleation and 58% at the time of an evisceration. Enucleation was preferred when involvement of orbital tissues was apparent. Both evisceration and enucleation remain a viable treatment option for MRE. The most appropriate initial approach is best determined by careful patient selection and informed decision-making by the patient.

Equine arteritis virus (EAV) can establish long-term persistent infection in the reproductive tract of stallions and is shed in the semen. Previous studies showed that long-term persistence is associated with a specific allele of the CXCL16 gene ( CXCL16S ) and that persistent infection is maintained despite the presence of a local inflammatory and humoral and mucosal antibody responses. In this study, we demonstrated that equine seminal exosomes (SEs) are enriched in a small subset of microRNAs (miRNAs). Most importantly, we demonstrated that long-term EAV persistence is associated with the downregulation of an SE-associated miRNA (eca-mir-128) and with an enhanced expression of CXCL16 in the reproductive tract, a putative target of eca-mir-128. The findings presented here suggest that SE eca-mir-128 is implicated in the regulation of the CXCL16/CXCR6 axis in the reproductive tract of persistently infected stallions, a chemokine axis strongly implicated in EAV persistence. This is a novel finding and warrants further investigation to identify its specific mechanism in modulating the CXCL16/CXCR6 axis in the reproductive tract of the EAV long-term carrier stallion. IMPORTANCE Equine arteritis virus (EAV) has the ability to establish long-term persistent infection in the stallion reproductive tract and to be shed in semen, which jeopardizes its worldwide control. Currently, the molecular mechanisms of viral persistence are being unraveled, and these are essential for the development of effective therapeutics to eliminate persistent infection. Recently, it has been determined that long-term persistence is associated with a specific allele of the CXCL16 gene ( CXCL16S ) and is maintained despite induction of local inflammatory, humoral, and mucosal antibody responses. This study demonstrated that long-term persistence is associated with the downregulation of seminal exosome miRNA eca-mir-128 and enhanced expression of its putative target, CXCL16, in the reproductive tract. For the first time, this study suggests complex interactions between eca-mir-128 and cellular elements at the site of EAV persistence and implicates this miRNA in the regulation of the CXCL16/CXCR6 axis in the reproductive tract during long-term persistence.

In an effort to improve a competitive blocking enzyme-linked immunosorbent assay (cELISA) for antibody detection to Equine arteritis virus (EAV), antigen purified by anion-exchange membrane chromatography capsule (AEC) was evaluated. Virus purification by the AEC method was rapid and easily scalable. A comparison was made between virus purified by the AEC method with that obtained by differential centrifugation based on the following: 1) the relative purity and quality of EAV glycoprotein 5 (GP5) containing the epitope defined by monoclonal antibody 17B7, and 2) the relative sensitivity of a commercial antibody cELISA with the only change being the 2 purified antigens. On evaluation by Western blot using GP5-specific monoclonal antibody 17B7, the AEC-purified EAV contained 86% GP5 monomer whereas the differentially centrifuged EAV contained

This study was undertaken to investigate the bacterial and fungal microflora on the external genitalia of a population of healthy male donkeys in the state of Michigan, USA. The aim was to identify and determine the frequency of occurrence of these microorganisms using seven different isolation media and standard microbiological procedures. The sites (urethral fossa [fossa glandis], dorsal diverticulum of the urethral sinus, distal urethra, and penile surface) in the distal reproductive tract were cultured and each isolated microorganism identified. Ten different genera of gram-positive bacteria, eight different genera of gram-negative bacteria, and two genera of fungi were isolated from the external genitalia of the 43 donkeys in this study. All 43 donkeys yielded gram-positive bacteria (2–8 species) from all four sites sampled. Arcanobacterium spp., Corynebacterium spp., and Bacillus spp. were the most frequently isolated gram-positive bacteria. Gram-negative bacteria were cultured from 16 (37.2%) of the 43 donkeys, with Acinetobacter lwoffii (16.3%), Oligella urethralis (11.6%), and Taylorella asinigenitalis (9.3%), the most frequently isolated. Fungi were cultured from only 5 (11.6%) of the 43 donkeys, with Rhizopus spp. isolated from 3 (7.0%) and Cladosporium spp. from 2 (4.7%) individuals. The testes and epididymides collected from 40 donkeys at time of castration were culture negative. Few differences were found in the bacterial flora between prepubertal and mature intact and castrated donkeys. Of notable interest was the scarcity of known equine pathogens across the population tested and isolation of T. asinigenitalis from normal donkeys, especially prepubertal individuals and previously castrated males.

Equine arteritis virus (EAV) has the ability to establish persistent infection in the reproductive tract of the stallion (carrier) and is continuously shed in its semen. We have recently demonstrated that EAV persists within stromal cells and a subset of lymphocytes in the stallion accessory sex glands in the presence of a significant local inflammatory response. In the present study, we demonstrated that EAV elicits a mucosal antibody response in the reproductive tract during persistent infection with homing of plasma cells into accessory sex glands. The EAV-specific immunoglobulin isotypes in seminal plasma included IgA, IgG1, IgG3/5, and IgG4/7. Interestingly, seminal plasma IgG1 and IgG4/7 possessed virus-neutralizing activity, while seminal plasma IgA and IgG3/5 did not. However, virus-neutralizing IgG1 and IgG4/7 in seminal plasma were not effective in preventing viral infectivity. In addition, the serological response was primarily mediated by virus-specific IgM and IgG1, while virus-specific serum IgA, IgG3/5, IgG4/7, and IgG6 isotype responses were not detected. This is the first report characterizing the immunoglobulin isotypes in equine serum and seminal plasma in response to EAV infection. The findings presented herein suggest that while a broader immunoglobulin isotype diversity is elicited in seminal plasma, EAV has the ability to persist in the reproductive tract, in spite of local mucosal antibody and inflammatory responses. This study provides further evidence that EAV employs complex immune evasion mechanisms during persistence in the reproductive tract that warrant further investigation.

Orbital surgeons are quite familiar with the diagnosis and management of paranasal sinus mucoceles due their frequent involvement of the orbit. These benign masses form and expand following sinus outflow obstruction secondary to various causes including trauma, inflammation and malignancy, amongst others. The authors present two cases of isolated orbital mucoceles without associated sinus outflow obstruction. There were notably no connections between the paranasal sinuses and the lesions. The mucoceles were diagnosed following orbitotomies and excisional biopsies with confirmatory histopathology. These rare orbital lesions should be considered when evaluating cystic orbital lesions, and the orbital surgeon should be familiar with the proposed pathophysiology and treatment recommendations.

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 + T and CD21 + B lymphocytes but not glandular epithelium. Persistent EAV infection is associated with moderate, multifocal lymphoplasmacytic ampullitis comprising clusters of B (CD21 + ) lymphocytes and significant infiltration of T (CD3 + , CD4 + , CD8 + , and CD25 + ) lymphocytes, tissue macrophages, and dendritic cells (Iba-1 + and CD83 + ), 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. IMPORTANCE The 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 improved prophylactic or treatment strategies. This study significantly enhances our understanding of the EAV carrier state in stallions by unequivocally identifying the ampullae as the primary sites of viral persistence, combined with the fact that persistence involves continuous viral replication in fibrocytes (possibly including tissue macrophages) and T and B lymphocytes in the presence of detectable inflammatory responses, suggesting the involvement of complex viral mechanisms of immune evasion. Therefore, EAV persistence provides a powerful new natural animal model to study RNA virus persistence in the male reproductive tract.

Equine arteritis virus (EAV) is the causal agent of equine viral arteritis (EVA), a respiratory and reproductive disease of equids. Some strains of EAV can cause fever, leukopenia, and dependent edema of the limbs, scrotum, and preputium in the acutely infected stallion. We hypothesized that fever and scrotal edema observed during the acute phase of the infection, but not the presence of EAV, have an adverse effect on semen quality. A group of seven stallions were intranasally inoculated with the Kentucky 84 (KY84) strain of EAV. Stallions were monitored for clinical signs of EVA until 42 days postinoculation (dpi). Semen was collected every other day for the first 15 days and 2 times a week up to 79 dpi. Additional samples were collected at 147, 149, and 151 dpi. Semen from each stallion was evaluated on the basis of motion characteristics, total number of spermatozoa, membrane integrity, and morphology. Virus infectivity titers were determined in RK-13 cells. Significant decreases in sperm quality were observed between 9 and 76 dpi. LOWESS (locally weighted scatterplot smoothing) curves for each horse were fit and integrated to quantify spermatozoa exposure to fever, virus, and edema over a period of 67 days before each ejaculation. Linear mixed models were then fit to isolate the effects of each factor on semen quality. Scrotal edema and fever were found to exert independent effects on all the semen quality parameters (P ≤ 0.002), whereas virus seems to exert little to no direct effect, as virus titers remained high long after semen quality returned to baseline.

The objective of the present study was to validate a previously described competitive enzyme-linked immunosorbent assay (cELISA) to detect antibody to Equine arteritis virus (EAV) based on GP5-specific nonneutralizing monoclonal antibody (mAb) 17B79 using the World Organization for Animal Health (OIE)–recommended protocol, which includes the following 5 in-house analyses. 1) The assay was calibrated with the OIE-designated reference serum panel for EAV; 2) repeatability was evaluated within and between assay runs; 3) analytical specificity was evaluated using sera specific to related viruses; 4) analytical sensitivity was evaluated with sera from horses vaccinated with an EAV modified live virus (MLV) vaccine; and 5) the duration of cELISA antibody detection following EAV vaccination was determined. The positive cELISA cutoff of ≥35% inhibition (%I) was confirmed by receiver operating characteristic plot analysis. Analytical sensitivity of the cELISA was comparable to the serum neutralization (SN) assay in that it detected EAV-specific antibody as early as 8 days postvaccination. The duration of EAV-specific antibody detected by cELISA was over 5 years after the last vaccination. This cELISA could detect EAV-specific antibody in serum samples collected from horses infected with various EAV strains. In the field trial performed by American Association of Veterinary Laboratory Diagnosticians–accredited state laboratories and OIE laboratory, the diagnostic specificity of the cELISA was 99.5% and the diagnostic sensitivity was 98.2%. The data using various serum panels also had consistently significant positive correlation between SN titers and cELISA %I results. The results further confirm that the EAV antibody cELISA is a reliable, simple alternative to the SN assay for detecting EAV-specific antibodies in equine sera.

Purpose To assess current members of the American Society of Ophthalmic Plastic and Reconstructive Surgery (ASOPRS) regarding preference in surgical techniques for orbital decompression in Graves' disease. Methods A 10-question web-based, anonymous survey was distributed to oculoplastic surgeons utilizing the ASOPRS listserv. The questions addressed the number of years of experience performing orbital decompression surgery, preferred surgical techniques, and whether orbital decompression was performed in collaboration with an ENT surgeon. Results Ninety ASOPRS members participated in the study. Most that completed the survey have performed orbital decompression surgery for >15 years. The majority of responders preferred a combined approach of floor and medial wall decompression or balanced lateral and medial wall decompression; only a minority selected a technique limited to 1 wall. Those surgeons who perform fat decompression were more likely to operate in collaboration with ENT. Most surgeons rarely remove the orbital strut, citing risk of worsening diplopia or orbital dystopia except in cases of optic nerve compression or severe proptosis. The most common reason given for performing orbital decompression was exposure keratopathy. The majority of surgeons perform the surgery without ENT involvement, and number of years of experience did not correlate significantly with collaboration with ENT. Conclusions The majority of surveyed ASOPRS surgeons prefer a combined wall approach over single wall approach to initial orbital decompression. Despite the technological advances made in the field of modern endoscopic surgery, no single approach has been adopted by the ASOPRS community as the gold standard.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of horses and other equid species. Following natural infection, 10–70% of the infected stallions can become persistently infected and continue to shed EAV in their semen for periods ranging from several months to life. Recently, we reported that some stallions possess a subpopulation(s) of CD3+ T lymphocytes that are susceptible to in vitro EAV infection and that this phenotypic trait is associated with long-term carrier status following exposure to the virus. In contrast, stallions not possessing the CD3+ T lymphocyte susceptible phenotype are at less risk of becoming long-term virus carriers. A genome wide association study (GWAS) using the Illumina Equine SNP50 chip revealed that the ability of EAV to infect CD3+ T lymphocytes and establish long-term carrier status in stallions correlated with a region within equine chromosome 11. Here we identified the gene and mutations responsible for these phenotypes. Specifically, the work implicated three allelic variants of the equine orthologue of CXCL16 (EqCXCL16) that differ by four non-synonymous nucleotide substitutions (XM_00154756; c.715 A → T, c.801 G → C, c.804 T → A/G, c.810 G → A) within exon 1. This resulted in four amino acid changes with EqCXCL16S (XP_001504806.1) having Phe, His, Ile and Lys as compared to EqCXL16R having Tyr, Asp, Phe, and Glu at 40, 49, 50, and 52, respectively. Two alleles (EqCXCL16Sa, EqCXCL16Sb) encoded identical protein products that correlated strongly with long-term EAV persistence in stallions (P, Author Summary A variable proportion of EAV infected stallions (10–70%) may become persistently infected and continuously shed the virus exclusively in their semen after recovery from acute infection. Previous studies in our laboratory have shown that stallions with the CD3+ T lymphocyte susceptibility phenotype to in vitro EAV infection are at higher risk of becoming persistently infected carriers compared to those that lack this phenotype. Here genetic and experimental studies were used to demonstrate that CXCL16 in the horse codes for two proteins, one associated with resistance and the other associated with susceptibility of CD3+ T lymphocytes to EAV infection. The two proteins are the result of four nucleotide substitutions in exon 1 of the equine CXCL16 gene. These alleles determine the outcome of in vitro infection of CD3+ T lymphocytes with EAV and are strongly associated with the establishment and maintenance of long-term carrier state in stallions. In vitro studies demonstrated that one form of CXCL16 protein (CXCL16S) is one of the cellular receptors for EAV and has higher scavenger activity and adhesion ability as compared to the form of the protein associated with resistance (CXCL16R).

and negative, respectively. Regarding the buffy coat samples, iiRTPCR detected EIAV nucleic acid in 31/56 AGID positive samples, while 108/109 AGID negative samples tested negative by iiRTPCR. Total agreement was 84.24%. When whole blood samples were evaluated, a total of 16 iiRT-PCR positive and 15 iiRT-PCR negative out of 31 AGID positive samples were obtained. Additionally, EIAV nucleic acid was not detected in 25 AGID negative samples .Total agreement was 73.21% for this sample type. When evaluating the accuracy of EIAV iiRT-PCR and qPCR, 28/165 and 130/165 buffy coat samples tested positive and negative by both assays, respectively. Five iiRT-PCR positive samples were negative by qPCR and two qPCR positive samples were negative by iiRTPCR. Thus, EIAV iiRT-PCR showed more than 95% agreement with qPCR results. It has been demonstrated that the absence of clinical signs is correlated with very low, frequently undetectable viremia. Therefore, EIAV iiRT-PCR appears to be a promising tool to identify infected horses including those experiencing low infectivity titers in blood. Furthermore, as initial EIAV replication rates are frequently high, recently infected equids pose a considerable transmission risk long before seroconversion. In our study, a horse was determined seronegative by AGID, but was identified as EIAV infected by iiRT-PCR. Thus, EIAV iiRT-PCR could be considered as an alternative diagnostic tool in the implementation of control strategies during an EIA outbreak in the low prevalence area of our country.

Previous studies in our laboratory have identified equine CXCL16 (EqCXCL16) to be a candidate molecule and possible cell entry receptor for equine arteritis virus (EAV). In horses, the CXCL16 gene is located on equine chromosome 11 (ECA11) and encodes a glycosylated, type I transmembrane protein with 247 amino acids. Stable transfection of HEK-293T cells with plasmid DNA carrying EqCXCL16 (HEK-EqCXCL16 cells) increased the proportion of the cell population permissive to EAV infection from in vitro . The binding of biotinylated virulent EAV strain Bucyrus at 4°C was significantly higher in HEK-EqCXCL16 cells than nontransfected HEK-293T cells. Finally, the results demonstrated that EAV preferentially infects subpopulations of horse CD14 + monocytes expressing EqCXCL16 and that infection of these cells is significantly reduced by pretreatment with Gp anti-EqCXCL16 pAb. The collective data from this study provide confirmatory evidence that the transmembrane form of EqCXCL16 likely plays a major role in EAV host cell entry processes, possibly acting as a primary receptor molecule for this virus. IMPORTANCE Outbreaks of EVA can be a source of significant economic loss for the equine industry from high rates of abortion in pregnant mares, death in young foals, establishment of the carrier state in stallions, and trade restrictions imposed by various countries. Similar to other arteriviruses, EAV primarily targets cells of the monocyte/macrophage lineage, which, when infected, are believed to play a critical role in EVA pathogenesis. To this point, however, the host-specified molecules involved in EAV binding and entry into monocytes/macrophages have not been identified. Identification of the cellular receptors for EAV may provide insights to design antivirals and better prophylactic reagents. In this study, we have demonstrated that EqCXCL16 acts as an EAV entry receptor in EAV-susceptible cells, equine monocytes. These findings represent a significant advance in our understanding of the fundamental mechanisms associated with the entry of EAV into susceptible cells.

Equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) are members of family Arteriviridae; they are highly species specific and differ significantly in cellular tropism in cultured cells. In this study we examined the role of the two major envelope proteins (GP5 and M) of EAV and PRRSV in determining their cellular tropism. We generated three viable EAV/PRRSV chimeric viruses by swapping the N-terminal ectodomains of these two proteins from PRRSV IA1107 strain into an infectious cDNA clone of EAV (rMLVB4/5 GP5ecto, rMLVB4/5/6 Mecto and rMLVB4/5/6 GP5&Mecto). The three chimeric viruses could only infect EAV susceptible cell lines but not PRRSV susceptible cells in culture. Therefore, these data unequivocally demonstrate that the ectodomains of GP5 and M are not the major determinants of cellular tropism, further supporting the recent findings that the minor envelope proteins are the critical proteins in mediating cellular tropism (Tian et al., 2012).

A single-nucleotide polymorphism (A 2254 or G 2254 ) in open reading frame 30 (ORF30) has been linked to the neuropathogenic phenotype of equine herpesvirus-1 (EHV-1). Identification of this polymorphism led to the development of a real-time PCR (rPCR) assay using allelic discrimination (E 2 ) to distinguish between potentially neuropathogenic and nonneuropathogenic EHV-1 strains (G. P. Allen, J. Vet. Diagn. Invest. 19:69–72, 2007). Although this rPCR assay can detect and genotype EHV-1 strains, subsequent studies demonstrated that it lacks the sensitivity for the routine detection of viral nucleic acid in clinical specimens. Therefore, a new allelic discrimination EHV-1 rPCR assay (E 1 ) was developed by redesigning primers and probes specific to ORF30. The E 1 and E 2 rPCR assays were evaluated using 76 archived EHV isolates and 433 clinical specimens from cases of suspected EHV-1 infection. Nucleotide sequence analysis of ORF30 was used to confirm the presence of EHV-1 and characterize the genotype (A 2254 or G 2254 ) in all archived isolates plus 168 of the clinical samples. The E 1 assay was 10 times more sensitive than E 2 , with a lower detection limit of 10 infectious virus particles. Furthermore, all A 2254 and G 2254 genotypes along with samples from three cases of dual infection (A 2254 +G 2254 ) were correctly identified by E 1 , whereas E 2 produced 20 false dual positive results with only one actual mixed A 2254 +G 2254 genotype confirmed. Based on these findings, E 1 offers greater sensitivity and accuracy for the detection and A/G 2254 genotyping of EHV-1, making this improved rPCR assay a valuable diagnostic tool for investigating outbreaks of EHV-1 infection.

Previously, we have shown that horses could be divided into susceptible and resistant groups based on an in vitro assay using dual-color flow cytometric analysis of CD3 + T cells infected with equine arteritis virus (EAV). Here, we demonstrate that the differences in in vitro susceptibility of equine CD3 + T lymphocytes to EAV infection have a genetic basis. To investigate the possible hereditary basis for this trait, we conducted a genome-wide association study (GWAS) to compare susceptible and resistant phenotypes. Testing of 267 DNA samples from four horse breeds that had a susceptible or a resistant CD3 + T lymphocyte phenotype using both Illumina Equine SNP50 BeadChip and Sequenom's MassARRAY system identified a common, genetically dominant haplotype associated with the susceptible phenotype in a region of equine chromosome 11 (ECA11), positions 49572804 to 49643932. The presence of a common haplotype indicates that the trait occurred in a common ancestor of all four breeds, suggesting that it may be segregated among other modern horse breeds. Biological pathway analysis revealed several cellular genes within this region of ECA11 encoding proteins associated with virus attachment and entry, cytoskeletal organization, and NF-κB pathways that may be associated with the trait responsible for the in vitro susceptibility/resistance of CD3 + T lymphocytes to EAV infection. The data presented in this study demonstrated a strong association of genetic markers with the trait, representing de facto proof that the trait is under genetic control. To our knowledge, this is the first GWAS of an equine infectious disease and the first GWAS of equine viral arteritis.

The objective was to evaluate the potential risks associated with embryo transfer from mares bred with equine arteritis virus (EAV) infective semen. Twenty-six mares were embryo donors, whereas 18 unvaccinated and EAV antibody seronegative mares were embryo recipients. Of the 26 donor mares, 15 were unvaccinated and seronegative for antibodies to EAV and 11 were vaccinated for the first time with a commercially available modified live virus vaccine against EVA before breeding and subsequent embryo transfer. All donor mares were bred with EAV-infective semen from a stallion persistently infected with the virus. Twenty-four embryos were recovered 7 d post-ovulation; all were subjected in sequential order to five washes in embryo flush medium, two trypsin treatments, and five additional washes in embryo flush medium (prior to transfer). Twelve and seven embryos (Grades 1 or 2) were transferred from the non-vaccinated and vaccinated donors, respectively, and pregnancy was established in 3 of 12 and 2 of 7. Perhaps trypsin reduced embryo viability and pregnancy rate. The uterine flush fluid of 11 mares (9 of 15 and 2 of 11 from non-vaccinated and vaccinated donor groups, respectively) was positive for EAV by VI (confirmed by real-time RT-PCR); the wash fluid from the embryos of nine of these mares was negative following 10 washes and two trypsin treatments. However, the embryo wash fluid from two mares was still positive for EAV after all 10 washes and the two trypsin treatments, and one embryo was positive for EAV. Two of 18 recipient mares had seroconverted to EAV 28 d after embryo transfer. Virus was not detected in any fetal tissues or fluids harvested after pregnancies were terminated (60 d). In conclusion, we inferred that the washing protocol of 10 washes and two trypsin treatments did not eliminate EAV from all embryos; due to limitations in experimental design, this requires confirmation. Furthermore, there may be a risk of EAV transmission associated with in vivo embryo transfer from a donor mare inseminated with EAV infective semen.

This study was undertaken to re-evaluate safety aspects of the commercial modified live virus vaccine against equine viral arteritis (Arvac) in stallions. Ten seronegative stallions were administered a single dose of vaccine, whereas three others were used as controls. Presence of vaccine virus in blood, semen, and nasopharyngeal and conjunctival secretions was determined by virus isolation and reverse transcription-polymerase chain reaction assay. No adverse effects were observed after vaccination. Vaccine virus was detected in buffy-coat cultures of all stallions for an average of 4.3 days, and in nasopharyngeal swabs from 40% of the stallions for an average of 3.2 days. No virus was detected in conjunctival swabs. Small quantities of virus were detected in the semen of one stallion on days 4 and 6 postvaccination. Vaccinated stallions showed a marked increase in neutralizing antibody titers within 5 to 8 days after vaccination. The stallions in the control group remained uninfected. Finding very low levels of virus in the semen of one stallion provides justification for withholding semen from a first-time vaccinated stallion for a minimum of 14 days to avoid the potential risk of virus transmission or having vaccine virus detected in semen frozen from a recently vaccinated stallion. There was no evidence of persistence of vaccine virus in the reproductive tract of the vaccinated stallions. Virus isolation proved more sensitive than reverse transcription-polymerase chain reaction for detection of virus in buffy-coat and nasopharyngeal specimens. Peak antibody responses were reached at a little more than a week after vaccination and these remained undiminished for the remainder of the study.

The objective of the current study was to determine the capability of 3 recently described one-step TaqMan real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assays targeting the nucleoprotein (NP), matrix (M), and hemagglutinin (HA) genes of H3N8 Equine influenza virus (EIV NP, EIV M, and EIV HA3 assays, respectively) to detect Canine influenza virus (CIV). The assays were initially evaluated with nucleic acid extracted from tissue culture fluid (TCF) containing the A/canine/FL/43/04 strain of Influenza A virus associated with the 2004 canine influenza outbreak in Florida. The EIV NP, EIV M, and EIV HA3 assays could detect CIV nucleic acid at threshold cycle (Ct) values of 16.31, 23.71, and 15.28, respectively. Three assays using TCF or allantoic fluid (AF) samples containing CIV ( n = 13) and archived canine nasal swab samples ( n = 20) originally submitted for laboratory diagnosis of CIV were further evaluated. All TCF and AF samples, together with 10 nasal swab samples that previously tested positive for virus by attempted isolation in embryonated hens' eggs or Madin–Darby canine kidney cells, were positive in all 3 real-time RT-PCR assays. None of the 3 assays detected the H1N1 Swine influenza virus strain in current circulation. These findings demonstrate that previously described real-time RT-PCR assays targeting NP, M, and H3 HA gene segments of H3N8 EIV are also valuable for the diagnosis of CIV infection in dogs. The assays could expedite the detection and identification of CIV.

Extensive cell culture passage of the virulent Bucyrus (VB) strain of equine arteritis virus (EAV) to produce the modified live virus (MLV) vaccine strain has altered its tropism for equine CD3 + T lymphocytes and CD14 + monocytes. The VB strain primarily infects CD14 + monocytes and a small subpopulation of CD3 + T lymphocytes (predominantly CD4 + T lymphocytes), as determined by dual-color flow cytometry. In contrast, the MLV vaccine strain has a significantly reduced ability to infect CD14 + monocytes and has lost its capability to infect CD3 + T lymphocytes. Using a panel of five recombinant chimeric viruses, we demonstrated that interactions among the GP2, GP3, GP4, GP5, and M envelope proteins play a major role in determining the CD14 + monocyte tropism while the tropism for CD3 + T lymphocytes is determined by the GP2, GP4, GP5, and M envelope proteins but not the GP3 protein. The data clearly suggest that there are intricate interactions among these envelope proteins that affect the binding of EAV to different cell receptors on CD3 + T lymphocytes and CD14 + monocytes. This study shows, for the first time, that CD3 + T lymphocytes may play an important role in the pathogenesis of equine viral arteritis when horses are infected with the virulent strains of EAV.

In 2006-2007, equine viral arteritis (EVA) was confirmed for the first time in Quarter Horses in multiple states in the USA. The entire genome of an equine arteritis virus (EAV) isolate from the index premises in New Mexico was 12 731 nt in length and possessed a previously unrecorded unique 15 nt insertion in the nsp2-coding region in ORF1a and a 12 nt insertion in ORF3. Sequence analysis of additional isolates made during this disease occurrence revealed that all isolates from New Mexico, Utah, Kansas, Oklahoma and Idaho had 98.6-100.0 % (nsp2) and 97.8-100 % (ORF3) nucleotide identity and contained the unique insertions in nsp2 and ORF3, indicating that the EVA outbreaks in these states probably originated from the same strain of EAV. Sequence and phylogenetic analysis of several EAV isolates made following an EVA outbreak on another Quarter Horse farm in New Mexico in 2005 provided evidence that this outbreak may well have been the source of virus for the 2006-2007 occurrence of the disease. A virus isolate from an aborted fetus in Utah was shown to have a distinct neutralization phenotype compared with other isolates associated with the 2006-2007 EVA occurrence. Full-length genomic sequence analysis of 18 sequential isolates of EAV made from eight carrier stallions established that the virus evolved genetically during persistent infection, and the rate of genetic change varied between individual animals and the period of virus shedding.

A significant consequence of equine arteritis virus (EAV) infection of horses is persistence of the virus in a variable percentage of infected stallions. We recently established an in vitro model of EAV persistence in cell culture for the purpose of furthering our understanding of EAV biology in general and viral persistence in the stallion in particular. In this study we investigated whether persistently infected HeLa cells could be cured of EAV infection by treatment with an antisense peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) designed to target the 5'-terminal region of the EAV genome. We found that persistently infected HeLa cells passaged three times in the presence of 5-10 microM EAV-specific PPMO produced no detectable virus. The PPMO-cured HeLa cells were free of infectious virus, viral antigen and EAV RNA as measured by plaque assay, indirect immunofluorescence assay and RT-PCR, respectively. Furthermore, when re-challenged with EAV at several passages after discontinuation of PPMO treatments, PPMO-cured HeLa cells were found to be refractory to re-infection and to the re-establishment of viral persistence. While these findings demonstrate that PPMO can be used to eliminate persistent EAV infection in cell culture, the efficacy of PPMO against EAV in vivo remains to be addressed.

Summary Reasons for performing study:A serological study conducted in 1995 revealed that 7 stallions at the Lipizzaner Centre, Gauteng, South Africa, were seropositive for antibody to equine arteritis virus (EAV). A Lipizzaner stallion imported into South Africa from Yugoslavia in 1981 had previously (1988) been confirmed to be an EAV carrier. Despite being placed under life-long breeding quarantine, EAV had been transmitted between stallions at the Lipizzaner Centre. Objectives: To investigate the phylogenetic relationships between the strain of EAV shed in the semen of the original carrier stallion and strains recovered from the semen of 5 other stallions; and to investigate the means whereby lateral transmission of EAV occurred among 7 in-contact, nonbreeding stallions at the Centre. Methods: EAV was isolated from semen collected from the seropositive stallions using RK-13 cells. Viral RNA was reverse transcribed and amplified by polymerase chain reaction using ORF5-specific primers, subjected to sequence and phylogenetic analysis. Results: Phylogenetic analysis of strains of EAV recovered from the semen of 6 persistently infected stal lions confirmed that all viruses were closely related and probably derived from a common ancestor, i.e. the stallion imported from Yugoslavia. Lateral transmission subsequently occurred among 7 in-contact, nonbreeding stallions at the Centre. It is speculated that these stallions may have been exposed to virus from be dding or fo mites contaminated with semen. Conclusions: These data confirm that lateral transmission of EAV can occur from shedding stallions to susceptible, in-contact horses, including other stallions, which may become persistently infected with the virus. Potential relevance: The findings are consistent with lateral spread of a single, unique strain of EAV among a group; and suggest that transmission of EAV may be initiated by infection of one or more stallions with virus on bedding or other fomites contaminated with EAV-infected semen.

The objective of this study was to develop and evaluate new TaqMan real-time reverse transcription-PCR (rRT-PCR) assays by the use of the minor groove binding probe to detect a wide range of equine influenza virus (EIV) strains comprising both subtypes of the virus (H3N8 and H7N7). A total of eight rRT-PCR assays were developed, targeting the nucleoprotein (NP), matrix (M), and hemagglutinin (HA) genes of the two EIV subtypes. None of the eight assays cross-reacted with any of the other known equine respiratory viruses. Three rRT-PCR assays (EqFlu NP, M, and HA3) which can detect strains of the H3N8 subtype were evaluated using nasal swabs received for routine diagnosis and swabs collected from experimentally inoculated horses. All three rRT-PCR assays have greater specificity and sensitivity than virus isolation by egg inoculation (93%, 89%, and 87% sensitivity for EqFlu NP, EqFlu M, and EqFlu HA3 assays, respectively). These assays had analytical sensitivities of ≥10 EIV RNA molecules. Comparison of the sensitivities of rRT-PCR assays targeting the NP and M genes of both subtypes with egg inoculation and the Directigen Flu A test clearly shows that molecular assays provide the highest sensitivity. The EqFlu HA7 assay targeting the H7 HA gene is highly specific for the H7N7 subtype of EIV. It should enable highly reliable surveillance for the H7N7 subtype, which is thought to be extinct or possibly still circulating at a very low level in nature. The assays that we developed provide a fast and reliable means of EIV diagnosis and subtype identification of EIV subtypes.

Two previously developed TaqMan fluorogenic probe-based 1-tube real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assays (T1 and T2) were compared and validated for the detection of Equine arteritis virus (EAV) nucleic acid in equine semen and tissue culture fluid (TCF). The specificity and sensitivity of these 2 molecular-based assays were compared to traditional virus isolation (VI) in cell culture. The T1 real-time RT-PCR had a higher sensitivity (93.4%) than the T2 real-time RT-PCR (42.6%) for detection of EAV RNA in semen. However, the T1 real-time RT-PCR was less sensitive (93.4%) than the World Organization for Animal Health (OIE)-prescribed VI test (gold standard). The sensitivity of both PCR assays was high (100.0% [T1] and 95.2% [T2]) for detecting EAV RNA in TCF. In light of the discrepancy in sensitivity between either real-time RT-PCR assay and VI, semen that is negative for EAV nucleic acid by real-time RT-PCR that is from an EAV-seropositive stallion should be confirmed free of virus by VI. Similarly, the presence of EAV in TCF samples that are VI-positive but real-time RT-PCR-negative should be confirmed in a 1-way neutralization test using anti-EAV equine serum or by fluorescent antibody test using monoclonal antibodies to EAV. If the viral isolate is not identified as EAV, such samples should be tested for other equine viral pathogens. The results of this study underscore the importance of comparative evaluation and validation of real-time RT-PCR assays prior to their recommended use in a diagnostic setting for the detection and identification of specific infectious agents.

The development and validation of a microsphere immunoassay (MIA) to detect equine antibodies to the major structural proteins of equine arteritis virus (EAV) are described. The assay development process was based on the cloning and expression of genes for full-length individual major structural proteins (GP5 amino acids 1 to 255 [GP5 1-255 ], M 1-162 , and N 1-110 ), as well as partial sequences of these structural proteins (GP5 1-116 , GP5 75-112 , GP5 55-98 , M 88-162 , and N 1-69 ) that constituted putative antigenic regions. Purified recombinant viral proteins expressed in Escherichia coli were covalently bound to fluorescent polystyrene microspheres and analyzed with the Luminex xMap 100 instrument. Of the eight recombinant proteins, the highest concordance with the virus neutralization test (VNT) results was obtained with the partial GP5 55-98 protein. The MIA was validated by testing a total of 2,500 equine serum samples previously characterized by the VNT. With the use of an optimal median fluorescence intensity cutoff value of 992, the sensitivity and specificity of the assay were 92.6% and 92.9%, respectively. The GP5 55-98 MIA and VNT outcomes correlated significantly ( r = 0.84; P < 0.0001). Although the GP5 55-98 MIA is less sensitive than the standard VNT, it has the potential to provide a rapid, convenient, and more economical test for screening equine sera for the presence of antibodies to EAV, with the VNT then being used as a confirmatory assay.

International audience; Genetic variation and phylogenetic relationships among 22 French isolates of equine arteritis virus (EAV) obtained over four breeding seasons (2001-2004) were determined by sequencing open reading frames (ORFs) 2a-7. The ORFs 2a-7 of 22 isolates differed from the prototype virulent Bucyrus strain of EAV by between 14 (99.5% identity) and 328 (88.7% identity) nucleotides, and differed from each other by between 0 (100% identity) and 346 (88.1% identity) nucleotides, confirming genetic diversity among EAV strains circulating in France. Phylogenetic analysis based on the partial ORF5 sequences (nucleotides 11296-11813) of 22 French isolates and 216 additional EAV strains available in GenBank clustered the global isolates of EAV into two distinct groups: North American and European. The latter could be further divided into two large subgroups: European subgroup 1 (EU-1) and European subgroup 2 (EU-2). Phylogenetic analysis based on 100 EAV ORF3 sequences yielded similar results. Of the 22 French EAV isolates, the 11 isolates obtained before January 28, 2003 clustered with either the EU-1 (9 isolates) or EU-2 (2 isolates) subgroup. In contrast, by the criteria used in this study, the 11 isolates obtained after January 30, 2003 belong to the North American group, strongly suggesting that these strains were recently introduced into France.

Strains of Equine arteritis virus (EAV) differ in the severity of the disease that they induce in horses. Infectious cDNA clones are potentially useful for identification of genetic determinants of EAV virulence; to date, two clones have been derived from a cell culture-adapted variant of the original (Bucyrus) isolate of EAV, and it has previously been shown that recombinant virus derived from one of these (rEAV030) is attenuated in horses. A complete cDNA copy of the genome of the virulent Bucyrus strain of EAV has now been assembled into a plasmid vector. In contrast to rEAV030, recombinant progeny virus derived from this clone caused severe disease in horses, characterized by pyrexia, oedema, leukopenia, high-titre viraemia and substantial nasal shedding of virus. The availability of infectious cDNA clones that produce recombinant viruses of different virulence to horses will facilitate characterization of the virulence determinants of EAV through reverse genetics.

Equid herpesvirus 1 (EHV-1) is one of the most economically important equine viral pathogens. Its clinical manifestations in horses vary from acute upper respiratory tract disease, abortion, or neonatal death, to neurological disease termed equine herpesviral myeloencephalopathy, which may lead to paralysis and a fatal outcome. Successful identification of EHV-1 infection in horses depends on a variety of factors such as suitable case selection with emphasis on timing of sample collection, selection of appropriate sample(s) based on the clinical manifestations, application of relevant diagnostic technique(s) and/or test(s), and careful evaluation and interpretation of laboratory results. Several traditional serologic and virus isolation assays have been described; however, these assays have inherent limitations that prevent rapid and reliable detection of EHV-1. The advent of molecular biologic techniques has revolutionized the diagnosis of infectious diseases in humans and animal species. Specifically, polymerase chain reaction (PCR)–based assays have allowed detection of nucleic acid in clinical specimens precisely and rapidly as compared to the traditional methods that detect the agent or antigen, or agent-specific antibodies in serum. The new molecular methods, especially real-time PCR, can be a very useful means of EHV-1 detection and identification. Veterinarians involved in equine practice must be aware of the advantages and disadvantages of various real-time PCR assays, interpretation of viral genetic marker(s), and latency in order to provide the best standard of care for their equine patients.

A high-passage rabbit kidney RK-13 cell line (HP-RK-13[KY], originally derived from the ATCC CCL-37 cell line) used in certain laboratories worldwide is contaminated with noncytopathic bovine viral diarrhea virus (ncpBVDV). On complete genome sequence analysis, the virus strain was found to belong to BVDV group 1b.

Strains of equine arteritis virus (EAV) differ in their virulence phenotypes, causing anywhere from subclinical infections to severe disease in horses. Here, we describe the in silico design and de novo synthesis of a full-length infectious cDNA clone of the horse-adapted virulent Bucyrus strain (VBS) of EAV encoding mCherry along with in vitro characterization of the progeny virions (EAV sVBSmCherry) in terms of host-cell tropism, replicative capacity and stability of the mCherry coding sequences following sequential passage in cell culture. The relative stability of the mCherry sequence during sequential cell culture passage coupled with a comparable host-cell range phenotype (equine endothelial cells, CD3(+) T cells and CD14(+) monocytes) to parental EAV VBS suggest that EAV-sVBSmCherry-derived virus could become a valuable research tool for identification of host-cell tropism determinants and for characterization of the viral proteins involved in virus attachment and entry into different subpopulations of peripheral blood mononuclear cells. Furthermore, this study demonstrates that advances in nucleic acid synthesis technology permit synthesis of complex viral genomes with overlapping genes like those of arteriviruses, thereby circumventing the need for complicated molecular cloning techniques. In summary, de novo nucleic acid synthesis technology facilitates innovative viral vector design without the tedium and risks posed by more-conventional laboratory techniques.

Equine arteritis virus (EAV) causes a persistent infection of the reproductive tract of carrier stallions. The authors determined the complete genome sequences of viruses (CW96 and CW01) that were present 5 years apart in the semen of a carrier stallion (CW). The CW96 and CW01 viruses respectively had only 85·6 % and 85·7 % nucleotide identity to the published sequence of EAV (EAV030). The CW96 and CW01 viruses had two 1 nt insertions and a single 1 nt deletion in the leader sequence, and a 3 nt coding insertion in ORF1a; thus their genomes included 12 708 nt as compared to the 12 704 nt in EAV030. Variation between viruses present in the semen of stallion CW and EAV030 was especially marked in the replicase gene (ORF1a and 1b), and the greatest variation occurred in the portion of ORF1a encoding the nsp2 protein. The ORFs 3 and 5, which respectively encode the GP3 and GP5 envelope proteins, showed greatest variation amongst ORFs encoding structural EAV proteins. Comparative sequence analyses of CW96 and CW01 indicated that ORFs 1a, 1b and 7 were highly conserved during persistent infection, whereas there was substantial variation in ORFs 3 and 5. Although the variation that occurs in ORF5 results in the emergence of novel phenotypic viral variants as determined by neutralization assay, all variants were neutralized by high-titre polyclonal equine antisera, suggesting that immune evasion is unlikely to be responsible for the establishment of persistent EAV infection of carrier stallions. Northern blot analyses of RNA extracted from cell culture propagated viruses isolated from 10 different persistently infected stallions failed to demonstrate any large genomic deletions, suggesting that defective interfering particles are also unlikely to be important in either the maintenance or clearance of persistent EAV infection of the reproductive tract of carrier stallions.

An 80-year-old woman with a painful, poorly seeing right eye underwent retrobulbar chlorpromazine injection for pain control. After the injection, the patient's symptoms improved; however, a neurotrophic ulcer developed within 2 weeks after the procedure. It is postulated that chlorpromazine may lead to sensory denervation to the cornea with the subsequent development of neurotrophic keratopathy, as observed in this case. Awareness of this potential adverse effect is important for proper patient safety, education, and postinjection management.