Report of the equine herpesvirus-1 Havermeyer Workshop, San Gimignano, Tuscany, June 2004

Amongst the infectious diseases that threaten equine health, herpesviral infections remain a world wide cause of serious morbidity and mortality. Equine herpesvirus-1 infection is the most important pathogen, causing an array of disorders including epidemic respiratory disease abortion, neonatal foal death, myeloencephalopathy and chorioretinopathy. Despite intense scientific investigation, extensive use of vaccination, and established codes of practice for control of disease outbreaks, infection and disease remain common. While equine herpesvirus-1 infection remains a daunting challenge for immunoprophylaxis, many critical advances in equine immunology have resulted in studies of this virus, particularly related to MHC-restricted cytotoxicity in the horse. A workshop was convened in San Gimignano, Tuscany, Italy in June 2004, to bring together clinical and basic researchers in the field of equine herpesvirus-1 study to discuss the latest advances and future prospects for improving our under-standing of these diseases, and equine immunity to herpesviral infection. This report highlights the new information that was the focus of this workshop, and is intended to summarize this material and identify the critical questions in the field. (c) 2006 Elsevier B.V. All rights reserved.

Equine herpesvirus-2 E10 gene product, but not its cellular homologue, activates NF-kappaB transcription factor and c-Jun N-terminal kinase.

We have previously reported on the death effector domain containing E8 gene product from equine herpesvirus-2, designated FLICE inhibitory protein (v-FLIP), and on its cellular homologue, c-FLIP, which inhibit the activation of caspase-8 by death receptors. Here we report on the structure and function of the E10 gene product of equine herpesvirus-2, designated v-CARMEN, and on its cellular homologue, c-CARMEN, which contain a caspase-recruiting domain (CARD) motif. c-CARMEN is highly homologous to the viral protein in its N-terminal CARD motif but differs in its C-terminal extension. v-CARMEN and c-CARMEN interact directly in a CARD-dependent manner yet reveal different binding specificities toward members of the tumor necrosis factor receptor-associated factor (TRAF) family. v-CARMEN binds to TRAF6 and weakly to TRAF3 and, upon overexpression, potently induces the c-Jun N-terminal kinase (JNK), p38, and nuclear factor (NF)-kappaB transcriptional pathways. c-CARMEN or truncated versions thereof do not appear to induce JNK and NF-kappaB activation by themselves, nor do they affect the JNK and NF-kappaB activating potential of v-CARMEN. Thus, in contrast to the cellular homologue, v-CARMEN may have additional properties in its unique C terminus that allow for an autonomous activator effect on NF-kappaB and JNK. Through activation of NF-kappaB, v-CARMEN may regulate the expression of the cellular and viral genes important for viral replication.

Equine herpesvirus protein E10 induces membrane recruitment and phosphorylation of its cellular homologue, bcl-10.

v-E10, a caspase recruitment domain (CARD)-containing gene product of equine herpesvirus 2, is the viral homologue of the bcl-10 protein whose gene was found to be translocated in mucosa-associated lymphoid tissue (MALT) lymphomas. v-E10 efficiently activates the c-jun NH(2)-terminal kinase (JNK), p38 stress kinase, and the nuclear factor (NF)-kappaB transcriptional pathway and interacts with its cellular homologue, bcl-10, via a CARD-mediated interaction. Here we demonstrate that v-E10 contains a COOH-terminal geranylgeranylation consensus site which is responsible for its plasma membrane localization. Expression of v-E10 induces hyperphosphorylation and redistribution of bcl-10 from the cytoplasm to the plasma membrane, a process which is dependent on the intactness of the v-E10 CARD motif. Both membrane localization and a functional CARD motif are important for v-E10-mediated NF-kappaB induction, but not for JNK activation, which instead requires a functional v-E10 binding site for tumor necrosis factor receptor-associated factor (TRAF)6. Moreover, v-E10-induced NF-kappaB activation is inhibited by a dominant negative version of the bcl-10 binding protein TRAF1, suggesting that v-E10-induced membrane recruitment of cellular bcl-10 induces constitutive TRAF-mediated NF-kappaB activation.

Diversity of genomic electropherotypes of naturally occurring equine herpesvirus 1 isolates in Argentina

The genomes of 10 equine herpesvirus 1 (EHV-1) strains isolated in Argentina from 1979 to 1991, and a Japanese HH1 reference strain were compared by restriction endonuclease analysis. Two restriction enzymes, BamHI and BglII, were used and analysis of the electropherotypes did not show significant differences among isolates obtained from horses with different clinical signs. This suggests that the EHV-1 isolates studied, which circulated in Argentina for more than 10 years, belong to a single genotype.

First detection of the equine herpesvirus 1 neuropathogenic variant in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

First defection of the equine herpesvirus 1 neuropathogenic variant in Brazil

This report describes the first detection of an equine herpesvirus 1 (EHV-1) neuropathogenic variant (G 2254/D 752) in Brazil from a case of fatal equine herpesvirus myeloencephalopathy (EHM) in a mare. The results of nucleotide sequencing of the EHV-1 ORF30 gene showed that two other Brazilian EHV-1 isolates from EHM cases are representatives of the non-neuropathogenic variant (A 2254/N 752), suggesting that other unidentified factors are probably also involved in the neuropathogenicity of EHV-1 in horses. These findings will contribute to the epidemiological knowledge of EHV-1 infection in Brazil.

Equine respiratory viruses in foals in New Zealand

AIMS: To identify the respiratory viruses that are present among foals in New Zealand and to establish the age at which foals first become infected with these viruses. METHODS: Foals were recruited to the study in October/ November 1995 at the age of 1 month (Group A) or in March/ April 1996 at the age of 4-6 months (Groups B and C). Nasal swabs and blood samples were collected at monthly intervals. Nasal swabs and peripheral blood leucocytes (PBL) harvested from heparinised blood samples were used for virus isolation; serum harvested from whole-blood samples was used for serological testing for the presence of antibodies against equine herpesvirus (EHV)-1 or -4, equine rhinitis-A virus (ERAV), equine rhinitis-B virus (ERBV), equine adenovirus 1 (EAdV-1), equine arteritis virus (EAV), reovirus 3 and parainfluenza virus type 3 (PIV3). Twelve foals were sampled until December 1996; the remaining 19 foals were lost from the study at various times prior to this date. RESULTS: The only viruses isolated were EHV 2 and EHV 5. EHV 2 was isolated from 155/157 PBL samples collected during the period of study and from 40/172 nasal swabs collected from 18 foals. All isolations from nasal swabs, except one, were made over a period of 2-4 months from January to April (Group A), March to April (Group B) or May, to July (Group C). EHV 5 was isolated from either PBL, nasal swabs, or both, from 15 foals on 32 occasions. All foals were positive for antibodies to EHV 1 or EHV 4, as tested by serum neutralisation (SN), on at least one sampling occasion and all but one were positive for EHV 1 antibodies measured by enzyme-linked immunosorbent assay (ELISA) on at least one sampling occasion. Recent EHV 1 infection was evident at least once during the period of study in 18/23 (78%) foals for which at least two samples were collected. SN antibodies to ERBV were evident in 19/23 (83%) foals on at least one sampling occasion and 15/23 foals showed evidence of seroconversion to ERBV Antibodies to ERAV were only detected in serum samples collected from foals in Group A and probably represented maternally-derived antibodies. Haemagglutination inhibition (HI) antibody titres greater than or equal to 1:10 to EAdV-1 were evident in 21/23 (91%) foals on at least one sampling occasion and 16/23 foals showed serological evidence of recent EAdV-1 infection. None of the 67 serum samples tested were positive for antibodies to EAV, reovirus 3 or PIV3. There was no clear association between infection with any of the viruses isolated or tested for and the presence of overt clinical signs of respiratory disease. CONCLUSIONS: There was serological and/or virological evidence that EHV-1, EHV-2, EHV-5, EAdV-1 and ERBV infections were present among foals in New Zealand. EHV-2 infection was first detected in foals as young as 3 months of age. The isolation of EHV-2 from nasal swabs preceded serological evidence of infection with other respiratory viruses, suggesting that EHV-2 may predispose foals to other viral infections.

Viruses associated with outbreaks of equine respiratory disease in New Zealand

AIM: To identify viruses associated with respiratory disease in young horses in New Zealand. METHODS: Nasal swabs and blood samples were collected from 45 foals or horses from five separate outbreaks of respiratory disease that occurred in New Zealand in 1996, and from 37 yearlings at the time of the annual yearling sales in January that same year. Virus isolation from nasal swabs and peripheral blood leukocytes (PBL) was undertaken and serum samples were tested for antibodies against equine herpesviruses (EHV-1, EHV-2, EHV-4 and EHV-5), equine rhinitis-A virus (ERAV), equine rhinitis-B virus (ERBV), equine adenovirus 1 (EAdV-1), equine arteritis virus (EAV), reovirus 3 and parainfluenza virus type 3 (PIV3). RESULTS: Viruses were isolated from 24/94 (26%) nasal swab samples and from 77/80 (96%) PBL samples collected from both healthy horses and horses showing clinical signs of respiratory disease. All isolates were identified as EHV-2, EHV-4, EHV-5 or untyped EHV Of the horses and foals tested, 59/82 (72%) were positive for EHV-1 and/or EHV-4 serum neutralising (SN) antibody on at least one sampling occasion, 52/82 (63%) for EHV-1-specific antibody tested by enzyme-linked immunosorbent assay (ELISA), 10/80 (13%) for ERAV SN antibody, 60/80 (75%) for ERBV SN antibody, and 42/80 (53%) for haemagglutination inhibition (HI) antibody to EAdV-1. None of the 64 serum samples tested were positive for antibodies to EAV, reovirus 3 or PIV3. Evidence of infection with all viruses tested was detected in both healthy horses and in horses showing clinical signs of respiratory disease. Recent EHV 2 infection was associated with the development of signs of respiratory disease among yearlings [relative risk (RR) = 2.67, 95% CI = 1.59-4.47, p = 0.0171]. CONCLUSIONS: Of the equine respiratory viruses detected in horses in New Zealand during this study, EHV 2 was most likely to be associated with respiratory disease. However, factors other than viral infection are probably important in the development of clinical signs of disease.

Hevosen herpesvirus 1 (EHV-1) : neljän syndrooman virus : tapausselostus ja kirjallisuuskatsaus

Summary: Equine herpesvirus 1 (EHV-1) - the virus with four syndromes : a case report and a review of literature

Serum Iron Parameters and Acute Experimental EHV-1 Infection in Horses

Background Research in humans has demonstrated that high serum iron (sFe) concentration can predispose to infection, and many infections subsequently result in alterations of host sFe. A decrease in sFe concentration is an early and sensitive indicator of systemic inflammation caused by tissue necrosis, bacterial infections, or endotoxemia in horses. Serum iron parameters in acute equine herpesvirus type 1 (EHV-1) infection have not been evaluated previously. Objectives To document the sFe response to EHV-1 infection and to determine whether or not significant differences in sFe concentration exist between EHV-1 infected horses that develop neurologic disease and those that do not. Animals A total of 14 horses experimentally infected with EHV-1. Methods Data were collected as an ancillary data set during a blinded experimental EHV-1 infection. Horses were infected with the rAb4 strain of EHV-1. Temperature, neurologic score, packed cell volume (PCV), and sFe parameters (sFe concentration, % saturation, and total iron-binding capacity) were recorded daily for 2weeks. Data were evaluated using Wilcoxon signed rank tests and Wilcoxon rank sum tests with Bonferroni corrections. Conclusions and Clinical Relevance Serum iron concentration decreases significantly in a biphasic pattern after EHV-1 infection. There was no significant difference in sFe concentration in horses that developed neurologic disease and those that did not in these experimentally infected animals. Serum iron parameters may be useful in monitoring the clinical course of viral infections such as EHV-1.

Human Herpesvirus-6 And Cytomegalovirus Dna In Liver Donor Biopsies And Their Correlation With Hla Matches And Acute Cellular Rejection.

Herpesvirus reactivation is common after liver transplantation. Analyze the presence of cytomegalovirus (HCMV) and human herpesvirus-6 (HHV-6) DNA in liver donor biopsies, seeking to better understand issues involving human donor leukocyte antigens (HLA)-A, B and DR, as well as correlations with acute cellular rejection. Fifty-nine liver transplantation patients were investigated for the presence of HCMV and HHV-6 DNA in liver donor biopsies, using the Nested-PCR technique. The clinical donor information and HLA matches were obtained from the São Paulo State Transplant System. The recipients' records regarding acute cellular rejection were studied. Seven (11.8%) biopsies were positive for HCMV DNA and 29 (49%) were positive for HHV-6 DNA. In 14 donors with HLA-DR 15 nine had HHV-6 DNA positive liver biopsy with a tendency for significant association (p=0.09), 22 recipients developed acute cellular rejection and 9/22 were positive for HLA-DR 15 (p=0.03; χ(2)=4.51), which was statistically significant in univariate analysis and showed a tendency after multivariate analysis (p=0.08). HHV-6 DNA was prevalent in liver donors studied as well as HLA-DR 15. These findings suggest that patients with HLA-DR 15 in liver donor biopsies develop more rejection after liver transplantation.

Human Herpesvirus Infections Of The Central Nervous System: Laboratory Diagnosis Based On Dna Detection By Nested Pcr In Plasma And Cerebrospinal Fluid Samples.

Infections of the central nervous systems (CNS) present a diagnostic problem for which an accurate laboratory diagnosis is essential. Invasive practices, such as cerebral biopsy, have been replaced by obtaining a polymerase chain reaction (PCR) diagnosis using cerebral spinal fluid (CSF) as a reference method. Tests on DNA extracted from plasma are noninvasive, thus avoiding all of the collateral effects and patient risks associated with CSF collection. This study aimed to determine whether plasma can replace CSF in nested PCR analysis for the detection of CNS human herpesvirus (HHV) diseases by analysing the proportion of patients whose CSF nested PCR results were positive for CNS HHV who also had the same organism identified by plasma nested PCR. In this study, CSF DNA was used as the gold standard, and nested PCR was performed on both types of samples. Fifty-two patients with symptoms of nervous system infection were submitted to CSF and blood collection. For the eight HHV, one positive DNA result-in plasma and/or CSF nested PCR-was considered an active HHV infection, whereas the occurrence of two or more HHVs in the same sample was considered a coinfection. HHV infections were positively detected in 27/52 (51.9%) of the CSF and in 32/52 (61.5%) of the plasma, difference not significant, thus nested PCR can be performed on plasma instead of CSF. In conclusion, this findings suggest that plasma as a useful material for the diagnosis of cases where there is any difficulty to perform a CSF puncture.

Damage assessment of the equine sperm membranes by fluorimetric technique

To validate a practical technique of simultaneous evaluation of the plasma, acrosomal and mitochondrial membranes in equine spermatozoa three fluorescent probes (PI, FITC-PSA and MITO) were associated. Four ejaculates from three stallions (n=12) were diluted in TALP medium and split into 2 aliquots, 1 aliquot was flash frozen in liquid nitrogen to induce damage in cellular membranes. Three treatments were prepared with the following fixed ratios of fresh semen: flash frozen semen: 100:0 (T100), 50:50 (T50), and 0:100 (T0). A 150-µL aliquot of diluted semen of each treatment was added of 2 µL of PI, 2 µL of MITO and 80 µL of FITC-PSA; incubated at 38.5ºC/8 min, and sperm cells were evaluated by epifluorescent microscopy. Based in regression analysis, this could be an efficient and practical technique to assess damage in equine spermatozoa, as it was able to determine the sperm percentage more representative of the potential to fertilize the oocyte.

Non-Detection of Human Herpesvirus 8 (HHV-8) DNA in HHV-8-Seropositive Blood Donors from Three Brazilian Regions

Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), is the etiologic agent of all forms of Kaposi's sarcoma, primary effusion lymphoma and the plasmablastic cell variant of multicentric Castleman disease. In endemic areas of sub-Saharan Africa, blood transfusions have been associated with a substantial risk of HHV-8 transmission. By contrast, several studies among healthy blood donors from North America have failed to detect HHV-8 DNA in samples of seropositive individuals. In this study, using a real-time PCR assay, we investigated the presence of HHV-8 DNA in whole-blood samples of 803 HHV-8 blood donors from three Brazilian states (Sao Paulo, Amazon, Bahia) who tested positive for HHV-8 antibodies, in a previous multicenter study. HHV-8 DNA was not detected in any sample. Our findings do not support the introduction of routine HHV-8 screening among healthy blood donors in Brazil. (WC = 140).