Viral genes and cellular markers associated with neurological complications during herpesvirus infections.

Despite the importance of neurological disorders associated with herpesviruses, the mechanism by which these viruses influence the central nervous system (CNS) has not been definitively established. Owing to the limitations of studying neuropathogenicity of human herpesviruses in their natural host, many aspects of their pathogenicity and immune response are studied in animal models. Here, we present an important model system that enables studying neuropathogenicity of herpesviruses in the natural host. Equine herpesvirus type 1 (EHV-1) is an alphaherpesvirus that causes a devastating neurological disease (EHV-1 myeloencephalopathy; EHM) in horses. Like other alphaherpesviruses, our understanding of virus neuropathogenicity in the natural host beyond the essential role of viraemia is limited. In particular, information on the role of different viral proteins for virus transfer to the spinal cord endothelium in vivo is lacking. In this study, the contribution of two viral proteins, DNA polymerase (ORF30) and glycoprotein D (gD), to the pathogenicity of EHM was addressed. Furthermore, different cellular immune markers, including alpha-interferon (IFN-α), gamma-interferon (IFN-γ), interleukin-10 (IL-10) and interleukin-1 beta (IL-1ß), were identified to play a role during the course of the disease.

Experimental infection with equine herpesvirus type 1 (EHV-1) induces chorioretinal lesions.

Equine herpesvirus myeloencephalitis (EHM) remains one of the most devastating manifestations of equine herpesvirus type 1 (EHV-1) infection but our understanding of its pathogenesis remains rudimentary, partly because of a lack of adequate experimental models. EHV-1 infection of the ocular vasculature may offer an alternative model as EHV-1-induced chorioretinopathy appears to occur in a significant number of horses, and the pathogenesis of EHM and ocular EHV-1 may be similar. To investigate the potential of ocular EHV-1 as a model for EHM, and to determine the frequency of ocular EHV-1, our goal was to study: (1) Dissemination of virus following acute infection, (2) Development and frequency of ocular lesions following infection, and (3) Utility of a GFP-expressing virus for localization of the virus in vivo. Viral antigen could be detected following acute infection in ocular tissues and the central nervous system (experiment 1). Furthermore, EHV-1 infection resulted in multifocal choroidal lesions in 90% (experiment 2) and 50% (experiment 3) of experimentally infected horses, however ocular lesions did not appear in vivo until between 3 weeks and 3 months post-infection. Taken together, the timing of the appearance of lesions and their ophthalmoscopic features suggest that their pathogenesis may involve ischemic injury to the chorioretina following viremic delivery of virus to the eye, mirroring the vascular events that result in EHM. In summary, we show that the frequency of ocular EHV-1 is 50-90% following experimental infection making this model attractive for testing future vaccines or therapeutics in an immunologically relevant age group.

Strain impact on equine herpesvirus type 1 (EHV-1) abortion models: viral loads in fetal and placental tissues and foals.

Equine herpesvirus-1 (EHV-1) continues to cause both sporadic and epidemic abortions despite extensive vaccination. Lack of progress in the development of protective vaccines may be hindered by the lack of equine abortion models that employ contemporary EHV-1 strains. The objective of our experiments was to compare a contemporary EHV-1 strain with a previously described challenge strain, and to quantify EHV-1 loads in various maternal and fetal tissues. Infection experiments were performed in two groups of 7 pregnant pony mares at 270-290 days of gestation with a contemporary EHV-1 strain (University of Findlay 2003 isolate - OH03) or an EHV-1 strain isolated over 30 years ago, and previously described in abortion models (Ab4). All mares in both groups exhibited nasal viral shedding and viremia. Infection with OH03 resulted in 1/7 abortion and infection with Ab4 resulted in 5/7 abortions. In the OH03 challenge, placentas of foals delivered at term showed little detectable virus, while the aborted fetus expressed high levels of virus infection in the spleen and liver, lower levels in the lung and thymus, and lowest levels in the chorioallantois. After Ab4 challenge, high viral loads were detected in fetal and placental tissues in abortions. In the two normal deliveries, the chorioallantois contained virus levels comparable with the chorioallantois of aborted foals and both foals shed EHV-1 starting on day 4 of life, but were clinically healthy. Our results demonstrate the continued importance of strain selection for abortion models, and this study is the first report of viral load quantification using contemporary methods. Extremely high EHV-1 loads in decidua from abortions illustrate the infection risk posed to other horses.

Evaluation of infectivity of a canine lineage H3N8 influenza A virus in ponies and in primary equine respiratory epithelial cells.

OBJECTIVE: To evaluate whether an equine-derived canine H3N8 influenza A virus was capable of infecting and transmitting disease to ponies. ANIMALS: 20 influenza virus-seronegative 12- to 24-month-old ponies. PROCEDURES: 5 ponies were inoculated via aerosol exposure with 10(7) TCID(50) of A/Canine/Wyoming/86033/07 virus (Ca/WY)/pony. A second group of 5 ponies (positive control group) was inoculated via aerosol exposure with a contemporary A/Eq/Colorado/10/07 virus (Eq/CO), and 4 sham-inoculated ponies served as a negative control group. To evaluate the potential for virus transmission, ponies (3/inoculation group) were introduced 2 days after aerosol exposure and housed with Ca/WY- and Eq/CO-inoculated ponies to serve as sentinel animals. Clinical signs, nasal virus shedding, and serologic responses to inoculation were monitored in all ponies for up to 21 days after viral inoculation. Growth and infection characteristics of viruses were examined by use of Madin-Darby canine kidney cells and primary equine and canine respiratory epithelial cells. RESULTS: Ponies inoculated with Ca/WY had mild changes in clinical appearance, compared with results for Eq/CO-inoculated ponies. Additionally, Ca/WY inoculation induced significantly lower numbers for copies of the matrix gene in nasal secretions and lower systemic antibody responses in ponies than did Eq/CO inoculation. The Ca/WY isolate was not transmitted to sentinel ponies. CONCLUSIONS AND CLINICAL RELEVANCE: Inoculation of ponies with the canine H3N8 isolate resulted in mild clinical disease, minimal nasal virus shedding, and weak systemic antibody responses, compared with responses after inoculation with the equine H3N8 influenza isolate. These results suggested that Ca/WY has not maintained infectivity for ponies.

Evaluation of immune responses following infection of ponies with an EHV-1 ORF1/2 deletion mutant.

Equine herpesvirus-1 (EHV-1) infection remains a significant problem despite the widespread use of vaccines. The inability to generate a protective immune response to EHV-1 vaccination or infection is thought to be due to immunomodulatory properties of the virus, and the ORF1 and ORF2 gene products have been hypothesized as potential candidates with immunoregulatory properties. A pony infection study was performed to define immune responses to EHV-1, and to determine if an EHV-1 ORF1/2 deletion mutant (ΔORF1/2) would have different disease and immunoregulatory effects compared to wild type EHV-1 (WT). Infection with either virus led to cytokine responses that coincided with the course of clinical disease, particularly the biphasic pyrexia, which correlates with respiratory disease and viremia, respectively. Similarly, both viruses caused suppression of proliferative T-cell responses on day 7 post infection (pi). The ΔORF1/ORF2 virus caused significantly shorter primary pyrexia and significantly reduced nasal shedding, and an attenuated decrease in PBMC IL-8 as well as increased Tbet responses compared to WT-infected ponies. In conclusion, our findings are (i) that infection of ponies with EHV-1 leads to modulation of immune responses, which are correlated with disease pathogenesis, and (ii) that the ORF1/2 genes are of importance for disease outcome and modulation of cytokine responses.

Onset and duration of immunity to equine influenza virus resulting from canarypox-vectored (ALVAC) vaccination.

Equine influenza virus remains an important problem in horses despite extensive use of vaccination. Efficacy of equine influenza vaccination depends on the onset and duration of protective immunity, and appropriate strain specificity of the immune response. This study was designed to test the protective immunity resulting from vaccination with the North American commercial ALVAC equine influenza vaccine (RECOMBITEK Influenza, Merial, USA)(1) against challenge with American lineage influenza viruses. In experiment 1, 12 ponies were vaccinated twice, at a 35 day interval, using the ALVAC-influenza vaccine expressing the HA genes of influenza A/eq/Newmarket/2/93 and A/eq/Kentucky/94 (H3N8), and 11 ponies served as unvaccinated controls. Six months after the second vaccination, all ponies were challenged with A/eq/Kentucky/91. In experiment 2, 10 ponies received one dose of the ALVAC-influenza vaccine, 10 ponies served as unvaccinated controls, and all ponies were challenge infected with A/equine/Ohio/03, 14 days after vaccination. Parameters studied included serological responses, and clinical disease and nasal viral shedding following challenge infection. In experiment 1, following the two-dose regimen, vaccinated ponies generated high titered anti-influenza virus IgGa and IgGb antibody responses to vaccination and demonstrated statistically significant clinical and virological protection to challenge infection compared to controls. Infection with A/eq/Kentucky/91 produced unusually severe signs in ponies in the control group, requiring therapy with NSAID's and antibiotics, and leading to the euthanasia of one pony. In experiment 2 following the one-dose regimen, vaccinates generated IgGa responses pre-challenge, and anamnestic IgGa and IgGb responses after challenge. Vaccinates demonstrated statistically significant clinical and virological protection to challenge infection compared to controls. The results of this study clearly demonstrate the early onset, and 6-month duration of protective immunity resulting from ALVAC-influenza vaccination against challenge with American lineage equine influenza viruses.

Comparison of four methods to quantify Equid herpesvirus 1 load by real-time polymerase chain reaction in nasal secretions of experimentally and naturally infected horses.

The objective of the current study was to compare the performance of 4 methods to quantify Equid herpesvirus 1 (EHV-1) by real-time polymerase chain reaction (PCR) in nasal secretions from experimentally and naturally infected horses. Nasal secretions were collected on the challenge day and daily thereafter for 13 days from 4 experimentally infected horses. Additional nasal swabs were collected from 30 horses with clinical signs consistent with natural EHV-1 infection. Absolute quantitation of EHV-1 target molecules was performed using standard curves for EHV-1 and equine glyceraldehyde-3-phosphate dehydrogenase, and DNA yield, and was expressed as EHV-1 glycoprotein B (gB) gene copies per million nucleated nasal cells, EHV-1 gB gene copies per entire swab, EHV-1 gB gene copies per 1 microl of purified DNA, and EHV-1 gB gene copies per 1 ng of template DNA. The study results showed that all 4 calculation methods yielded comparable results between experimentally and naturally infected horses, and that the different methods were significantly correlated with each other. Reporting of quantitative results for EHV-1 viral load in nasal swabs collected from infected horses constitutes an important advance in both the research and diagnostic fields, allowing one to determine the infectious risk of affected horses, disease stage, or response to antiviral therapy. However, protocols that normalize the PCR results against a preselected volume of DNA or nasal secretions are likely to be more prone to variations than protocols that calculate the load for the entire swab, incorporate a housekeeping gene, or use a constant amount of extracted DNA.

Comparison of temperature readings from a percutaneous thermal sensing microchip with temperature readings from a digital rectal thermometer in equids.

OBJECTIVE: To compare temperature readings from an implantable percutaneous thermal sensing microchip with temperature readings from a digital rectal thermometer, to identify factors that affect microchip readings, and to estimate the sensitivity and specificity of the microchip for fever detection. DESIGN: Prospective study. ANIMALS: 52 Welsh pony foals that were 6 to 10 months old and 30 Quarter Horses that were 2 years old. PROCEDURES: Data were collected in summer, winter, and fall in groups 1 (n = 23 ponies), 2 (29 ponies), and 3 (30 Quarter Horses), respectively. Temperature readings from a digital rectal thermometer and a percutaneous thermal sensing microchip as well as ambient temperature were recorded daily for 17, 23, and 19 days in groups 1 through 3, respectively. Effects of ambient temperature and rectal temperature on thermal sensor readings were estimated. Sensitivity and specificity of the thermal sensor for detection of fever (rectal temperature, >or= 38.9 degrees C [102 degrees F]) were estimated separately for data collection at ambient temperatures 15.6 degrees C. RESULTS: Mean ambient temperatures were 29.0 degrees C (84.2 degrees F), -2.7 degrees C (27.1 degrees F), and 10.4 degrees C (50.8 degrees F) for groups 1 through 3, respectively. Thermal sensor readings varied with ambient temperature and rectal temperature. Rectal temperatures ranged from 36.2 degrees to 41.7 degrees C (97.2 degrees to 107 degrees F), whereas thermal sensor temperature readings ranged from 23.9 degrees (75 degrees F) to 42.2 degrees C (75 degrees to 108 degrees F). Sensitivity for fever detection was 87.4%, 53.3%, and 58.3% in groups 1 to 3, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: The thermal sensor appeared to have potential use for initial screening of body temperature in equids at ambient temperatures > 15.6 degrees C.

Detection and quantification of equine herpesvirus-1 viremia and nasal shedding by real-time polymerase chain reaction.

Equine herpesvirus-1 (EHV-1) infection is common in young horses throughout the world, resulting in respiratory disease, epidemic abortion, sporadic myelitis, or latent infections. To improve on conventional diagnostic tests for EHV-1, a real-time polymerase chain reaction (PCR) technique was developed, using primers and probes specific for the EHV-1 gB gene. Amplification efficiencies of 100% +/- 5% were obtained for DNA isolated from a plasmid, infected peripheral blood mononuclear cells (PBMCs), and nasal secretions from infected ponies. The dynamic range of the assay was 8 log10 dilutions, and the lower limit of detection was 6 DNA copies. Fifteen ponies, seronegative for EHV-1, were experimentally infected with EHV-1, and nasal samples were used to quantify shedding of virus by both virus isolation and real-time PCR analysis. Virus isolation identified nasal shedding of EHV-1 in 12/15 ponies on a total of 25 days; real-time PCR detected viral shedding in 15/15 ponies on 75 days. Viremia was quantified using PBMC DNA, subsequent to challenge infection in 3 additional ponies. Viremia was identified in 1/3 ponies on a single day by virus isolation; real-time PCR detected viremia in 3/3 ponies on 17 days. When real-time PCR was used to analyze PBMC DNA from 11 latently infected ponies (documented by nested PCR), EHV-1 was not detected. We conclude that real-time PCR is a sensitive and quantitative test for EHV-1 nasal shedding and viremia and provides a valuable tool for EHV-1 surveillance, diagnosis of clinical disease, and investigation of vaccine efficacy.

Differential regulation of neutrophil chemotaxis to IL-8 and fMLP by GM-CSF: lack of direct effect of oestradiol.

Neutrophils are a normal constituent of the female reproductive tract and their numbers increase in the late secretory phase of the menstrual cycle prior to menses. Several cytokines are produced in female reproductive tract tissue. In particular granulocyte-macrophage colony-stimulating factor (GM-CSF), a potent activator of neutrophils, is secreted in high concentrations by female reproductive tract epithelia. We previously observed that GM-CSF synergizes strongly with interleukin-8 (IL-8) in enhancing chemotaxis of neutrophils. Thus we investigated whether pretreatment of neutrophils with GM-CSF would prime subsequent chemotaxis to IL-8 in the absence of GM-CSF. Surprisingly, a 3-hr pulse of GM-CSF severely diminished chemotaxis to IL-8, whereas N-formyl-methyl-leucyl-phenylalanine (fMLP)-mediated chemotaxis was retained. Conversely, when cells were incubated without GM-CSF they retained IL-8-mediated migration but lost fMLP chemotaxis. These changes in chemotaxis did not correlate with expression of CXCR1, CXCR2 or formyl peptide receptor. However, IL-8-mediated phosphorylation of p44/42 mitogen-activated protein kinase was greatly reduced in neutrophils that no longer migrated to IL-8, and was diminished in cells that no longer migrated to fMLP. Oestradiol, which is reported by some to exert an anti-inflammatory effect on neutrophils, did not change the effects of GM-CSF. These data suggest that neutrophil function may be altered by cytokines such as GM-CSF through modulation of signalling and independently of surface receptor expression.

Synergy between IL-8 and GM-CSF in reproductive tract epithelial cell secretions promotes enhanced neutrophil chemotaxis.

Neutrophils occur in tissues of the female reproductive tract (FRT) under non-infected conditions. These cells generally enter tissues under the influence of chemoattractants called chemokines. Primary epithelial cells (EC) from FRT were a potent source of chemokines, IL-8 being the chief neutrophil chemoattractant secreted. Blocking with neutralizing anti-IL-8 showed that IL-8 did not account for all of the chemoattraction observed. A mixture of 25 ng/mL rIL-8 and 1 ng/mL rGM-CSF mediated 2.7-fold more chemotaxis than that expected if the two agents were additive. We then found that GM-CSF was produced by EC in amounts that synergised strongly with IL-8 to enhance chemotaxis. Treatment of uterine EC conditioned medium with saturating doses of anti-IL-8 plus anti-GM-CSF antibodies produced an 84% inhibition of chemotaxis. These findings demonstrate that the majority of neutrophil chemoattractant activity produced by FRT EC results from the synergistic effects of IL-8 and GM-CSF.