Phenotypic change of adult pancreatic islets has been implicated in the development of certain pancreatic cancers and in islet transplant failure. The aim of this study was to characterize intracellular events that mediate changes in adult islet phenotype. Using an in vitro islet-to-duct transformation model, canine islets were induced to undergo phenotypic transformation to duct-like epithelial structures through a two-stage process. Stage one was characterized by widespread islet cell apoptosis associated with the formation of cavitary spaces within the islets. During this stage, c-Jun N-terminal regulated kinase (JNK) and caspase-3 activities were elevated, while extracellular signal-regulated kinase (ERK) and Akt activities were decreased. The second stage of the process was characterized by an inversion in the balance in activity between these signal transduction pathways and by a concomitant decrease in apoptosis. The transformed islets were no longer immunoreactive for islet cell hormones, but expressed the duct epithelial cell marker CK-AE1/AE3. In contrast to islet cells, these duct epithelial cells were highly proliferative. To clarify the role of the identified changes in signal transduction events, we performed additional studies using pharmacological inhibitors of enzyme activity and demonstrated that inhibition of JNK and caspase-3 activity prevented cystic transformation. Our results indicate that the balance in signaling activity between ERK/Akt and JNK/caspase-3 appears to be an important regulator of islet cell death and differentiation.

Several derivatives of 2,5-diaziridinyl-3-phenyl-1,4-benzoquinone have been synthesized and their cytotoxicities in six different human cancer cell lines (H460, H596, HT29, BE, K562 and A2780) have been determined. It was observed that certain phenol-ester derivatives were significantly more cytotoxic in all of the cell lines investigated. These esters were shown to be cleaved by esterases to form a stable meta-phenol and an unstable para-phenol. The meta-phenol was also highly cytotoxic. Several of these compounds were studied in detail using DNA cross-linking, clonogenic, apoptosis and flow cytometry assays. It is proposed that although the phenol-esters and the phenols can efficiently cross-link DNA, this mechanism alone is not sufficient to explain the toxicities of these compounds.

Equine herpesvirus 2 (EHV-2) was isolated from healthy animals; therefore, the association between EHV-2 infection and respiratory disease raises the question of the role of this agent in this pathology. To date, there are no reports that relate viral excretion to health, this study then analysed 153 nasal swabs from horses in different age groups (older and younger than 1 year old) and state of health (clinically healthy and with respiratory symptoms). Results showed that the percentage of horses with viral excretion was higher within the clinically healthy group, being significative (p < 0.05) in the younger than 1 year old group, whereas the percentage of animals with respiratory symptoms did not show significant differences (p > 0.05) between age groups.

The occurrence of equine arteritis virus (EAV) induced equine abortions was studied with different laboratory methods during a 3-year period. Tissue samples from 96 aborted equine foetuses or newborn foals were collected from 57 farms located in different parts of Hungary. Virus isolation, polymerase chain reaction (PCR), immunohistochemistry and serology were used for the detection of EAV infection. The overall seroprevalence of EAV infection in mares was 65%. EAV induced abortion was diagnosed in eight (8.3%) cases from six (10.5%) herds. Abortion was sporadic in all herds except for one, where epidemic abortion happened. Fetal serology in six (75%) cases, the virus isolation in one (12.5%) case whereas PCR in all of the four investigated cases were positive. The virus could be observed with immunohistochemistry in seven (87.5%) cases mostly in the spleen followed by other organs and the allantochorion. In conclusion, PCR and immunohistochemistry seem to be the most sensitive and useful tests for the diagnosis of EAV induced equine abortion.

Equine rhinitis viruses (ERVs) are the causative agents of mild to severe upper respiratory infections in horses worldwide. Immunologically, four serotypes of ERVs have been identified. Equine rhinitis A virus (ERAV) and Equine rhinitis B virus 1 (ERBV1) are the most frequent serotypes in Europe. Both viruses have a broad host range in cultured cells with ERAV being able to infect humans. Since there is neither information on the seroprevalence of ERAV and ERBV1 in Austria nor on the zoonotic potential of ERBV1, we investigated 200 horse and 137 veterinary sera for the presence of neutralizing antibodies relating to ERAV and ERBV1. One hundred and eighty (90%) and 173 (86%) horse sera neutralized ERAV and ERBV1, respectively. In contrast, only four (2.7%) and five (3.6%) human sera showed weak neutralizing activity to ERAV and ERBV1, respectively. These results indicate that ERAV and ERBV1 are widespread in the Austrian horse population; however, the risk of acquiring zoonotic infection among veterinarians appears low.

Equine herpesvirus type 1 was determined as the etiological cause of an abortion storm in Czechia in 2003 after the virus strain was isolated from aborted fetus and identified by serological means and by PCR technique. Cloning and sequencing of the glycoprotein D confirmed the identity of the isolates and showed molecular relationships to known EHV-1 strains. Comparison of glycoprotein D sequences with corresponding sequence of EHV-1 reference strains (Kentucky-A and Ab1) revealed high nucleotide homology. The Czech isolate of EHV-1 virus does not differ significantly from the Ab1 strain regarding the glycoprotein D gene and does not bear the frameshift in the 3' terminus which occurs in the Kentucky-A strain.

REASONS FOR PERFORMING STUDY: Currently, there is no recommended immunoprophylaxis against febrile respiratory diseases due to equine herpesvirus-1 (EHV-1) and -4 (EHV-4) in horses below age 5-6 months. This is because of interference by maternally-derived antibody (MDA) of vaccines. OBJECTIVE: Unweaned equine foals are an important reservoir of EHV-1 transmission; therefore, we experimentally assessed the efficacy of a live EHV-1 vaccine in foals age 1.4-3.5 months with MDA. METHODS: Following vaccination and challenge, parameters assessed were virus shedding in nasal mucus, leucocyte-associated viraemia, circulating virus neutralising antibody activity and clinical reactions. RESULTS: Controlled challenge showed that a single intranasal dose of the vaccine afforded partial but significant protection against febrile respiratory disease, virus shedding and viraemia due to EHV-1 infection, despite virus-neutralising MDA. CONCLUSIONS AND POTENTIAL RELEVANCE: The prospective vaccine would be a significant step forward in reducing the incidence of the disease caused by EHV-1 infection.

In general, vaccines containing inactivated equine herpesvirus-1 (EHV-1) fail to prevent abortion in pregnant mares following infection with a virulent strain of EHV-1. We have tested the hypothesis that resistance to EHV-1-induced abortion in pregnant mares is associated with high frequencies of EHV-1 specific, major histocompatibility complex (MHC) class I-restricted, cytotoxic T lymphocytes (CTL) in the circulation. To test this theory, three groups of pregnant mares were assembled with varying backgrounds of infection or vaccination in an attempt to mimick the immune status of the general population. Group 1 mares (n=9) were untreated controls selected at random. Group 2 mares (n=5) were vaccinated three times intramuscularly with inactivated EHV-1. Group 3 mares (n=3) had been infected with EHV-1 on four previous occasions. The frequency of CTL in blood leucocytes was measured by limiting dilution analysis at three time points; at the beginning of pregnancy (approximately 28 weeks before infection) in the Group 2 and Group 3 mares (4-7 weeks of gestation)(Group 1 was unavailable for sampling) and then 2 weeks before (30-40 weeks of gestation) and 3 weeks after experimental infection in all the mares. Serum samples were collected to monitor complement fixing (CF) antibody titres. Mares in all three groups were infected experimentally with EHV-1 strain Ab4/8 by the intranasal route after which they were monitored clinically to determine the outcome of pregnancy and samples were collected to determine the duration of nasopharyngeal shedding and cell-associated viraemia.The untreated control mares showed low pre-infection CTL. After experimental infection, they all seroconverted, aborted and demonstrated expected clinical and virological signs. Some vaccinated mares (3/5) had elevated titres of CF antibody prior to their first vaccination. All the vaccinated mares seroconverted after vaccination and exhibited higher CTL frequencies than controls before infection. Four of the five foaled normally. The multiply infected mares had low CF antibody titres prior to infection and showed neither seroconversion nor clinical or virological signs after infection. All multiply infected mares exhibited high frequencies of CTL before infection and they all foaled normally. The CTL frequencies observed differed significantly from the expected frequencies in the control and multiply infected groups at 2 weeks pre-infection (P=0.034) and between the foaling and aborting mares at 2 weeks pre-infection (P=0.005) and 3 weeks post-infection (P=0.015). The results show a positive correlation between the number of virus-specific CTL in the peripheral blood of pregnant mares and their protection against abortion induced by EHV-1 infection. Therefore, as indicated by this study, rational approaches to the development of new vaccines for EHV-1 should stimulate cytotoxic immune responses and develop virus-specific CTL as pre-requisites for protection against abortion.

Five of 10 pregnant, lactating mares, each with a foal at foot, developed neurological disease. Three of them became recumbent, developed complications and were euthanased; of the two that survived, one aborted an equine herpesvirus type 1 (EHV-1)-positive fetus 68 days after the first signs were observed in the index case and the other gave birth to a healthy foal on day 283 but remained ataxic and incontinent. The diagnosis of EHV-1 myeloencephalitis was supported by postmortem findings, PCR identification of the virus and by serological tests with an EHV-1-specific ELISA. At the time of the index case, the 10 foals all had a heavy mucopurulent nasal discharge, and PCR and the ELISA were used to detect and monitor EHV-1 infection in them. The status of EHV-1 infection in the five in-contact mares was similarly monitored. Sera from three of the affected mares, taken seven days after the index case were negative or had borderline EHV-1-specific antibody titres. In later serum samples there was an increase in the titres of EHV-1-specific antibody in two of the affected mares. In contrast, sera from the five unaffected in-contact mares were all EHV-1-antibody positive when they were first tested seven or 13 days after the index case.

The objective of the investigations was to study the occurrence of the equine herpesvirus type 1 (EHV-1) infection in aborted equine fetuses and in newborn foals and to compare the sensitivity of virus isolation, immunohistochemistry and histology in 101 cases and of fetal serology in 68 cases in the diagnosis of the infection. Out of the 93 aborted equine fetuses and 8 weak foals, 15 (14.9%)(14 fetuses and 1 foal) proved to be EHV-1 infected by immunohistochemical and 13 (12.9%) by virological investigation. Characteristic microscopic changes were seen in several organs in all cases, while intranuclear inclusion bodies could be found only in 25 (35.2%) of the 71 virus-positive tissue samples. Four (5.9%) cases proved to be positive by fetal serological investigation, but none of these cases showed any EHV-1 specific lesions and in none of these cases could the virus be detected by virus isolation or by immunohistochemistry. According to the results, fetal serology does not seem to be a useful test in virus-positive cases, while the immunohistochemical method seems to be a reliable and a slightly more sensitive method than virus isolation in the diagnosis of EHV-1 infection.

The temperature sensitive and host range mutant clone 147 of equine herpesvirus 1 (EHV-1) was assessed for its ability to protect conventional, susceptible adult horses against respiratory infection by EHV-1 and equine herpesvirus 4 (EHV-4).Intranasal (IN) vaccination with 5.2 log(10) TCID(50) did not cause adverse clinical reactions although a limited virus shedding and viraemia (leukocytes) was observed in 11 of 15 and 10 of 15 vaccinated horses respectively. All 15 vaccinated horses showed a significant seroresponse to both EHV-1 and EHV-4 for virus neutralising (VN) antibody. None of 14 control horses shed virus or became viraemic or seroconverted prior to challenge. EHV-1 challenge (dose 6.0 log(10)) 6 weeks after vaccination resulted in pyrexia in all eight control horses while eight vaccinated horses remained unaffected. Six control horses developed nasal discharge, five of which were mucopurulent nasal discharge (mean duration 3.2 days) which also occurred in four vaccinated horses for 1 day. All eight control horses shed challenge EHV-1 at a significantly higher level (group mean titre 2.6+/-0.4 log(10) TCID(50) per sample) and for much longer (mean duration 4.8+/-1.5 days) than that (group mean titre 1.4+/-0.8 log(10) TCID(50) per sample and mean duration 1.5+/-0.5 days) in six vaccinated horses. Furthermore, all eight control horses became viraemic (mean duration 2.9 days) but viraemia did not occur in eight vaccinated horses. Following EHV-1 challenge, all eight control horses showed a significant VN antibody rise to both EHV-1 and EHV-4 but this occurred in only one vaccinated horse and to EHV-4 only. In EHV-4 challenge (dose of 4.2 log(10) TCID(50)) of a separate pair of seven vaccinated and six control horses, 6 weeks after EHV-1 vaccination resulted in pyrexia (mean duration 2.3 days) and nasal discharge (mean duration 1.8 days) in three and five control horses respectively but the only reaction observed in the vaccinated group was nasal discharge for 1 day in one animal. All six control animals shed virus (mean titre 2.5+/-0.6 log(10) TCID(50) per sample and mean duration 2+/-0.6 days) compared to one vaccinated animal. Although EHV-4 viraemia is rare, 3 of 6 control horses became viraemic after EHV-4 challenge but this was not observed in vaccinated horses. After EHV-4 challenge 3 and 5 of 6 control horses seroconverted for VN antibody to EHV-1 and EHV-4 respectively; a non-responsive control horse had high level of pre-existing VN antibody to EHV-4. However, only 1 of 7 vaccinated horses showed a significant antibody rise and only to EHV-4.