Neuropathogenic and non-neuropathogenic EHV-1 strains induce the accumulation of hyperphosphorylated Tau in primary murine neurons.

Equid herpesvirus 1 (EHV-1) causes respiratory disease, abortion and neurological disorders in horses. Similarly, to other alphaherpesviruses, EHV-1 is neurotropic and establishes latency in the neurons of its natural host. Despite the fact that many studies have been devoted to the pathogenesis of various clinical forms of EHV-1 infection, mechanisms of the neuronal damage are still not fully understood. The aim of this study was to define the phosphorylation status of tau protein in neuronal cell culture infected with EHV-1. Phosphorylation of tau was tested at tau-ser199/ser202, tau-ser404, tau-ser262, tau-thr181, tau-thr217 and tau-thr205 sites. We described, for the first time, that EHV-1 infection leads to the accumulation of hyperphosphorylated tau in primary murine neurons. We showed that non-neuropathogenic and neuropathogenic EHV-1 strains specifically induce hyperphosphorylation of tau-ser199/ser202, tau-ser404 and tau-thr205 during long-term infection and after a controlled activation of productive infection. Keywords: tau protein; hyperphosphorylation; equid herpesvirus 1 (EHV-1); neuronal cell culture.

Equine herpesvirus type 1 affects mitochondrial network morphology and reactive oxygen species generation in equine dermal cell line.

In the present study, the influence of the infection with equine herpesvirus type 1 (non-neuro-pathogenic and neuropathogenic strains of EHV-1) on the morphology and distribution of mitochondrial network in equine dermal cell line was investigated. Our results indicate that EHV-1-infection caused changes in the mitochondrial morphology manifested mostly by fission and reactive oxygen species generation.

Application of NucleoCounter for the comprehensive assessment of murine cultured neurons during infection with Equine Herpesvirus type 1 (EHV-1).

The NucleoCounter NC-3000, a portable high-speed cell counting device based on the principle of fluorescence microscopy, provides the alternative method for standard flow cytometry. The main objective of the study was to apply an efficient technique for the assessment of the primary murine neurons culture infected with either neuropathogenic or non-neuropathogenic strains of Equine Herpesvirus type 1 (EHV-1). Using the NucleoCounter NC-3000 we have observed a decrease in mitochondrial potential and reduction in cells viability but we have not observed changes in the cell cycle of cultured neurons infected with all EHV-1 strains.

Function of myosin during entry and egress of equid herpesvirus type 1 in primary murine neurons.

Equid herpesvirus type 1 (EHV-1) is a major pathogen of horses with a worldwide distribution, which can cause various clinical signs ranging from mild respiratory disease to neurological disorders. To initiate an effective infection, EHV-1 evolved a broad spectrum of mechanisms exploiting the host cell, including its actin filaments. An actin-myosin-driven transport has been described to precede cellular entry of different viruses. Therefore, in the present study we investigated the role of actin motor protein - myosin, during replication of two EHV-1 strains: Jan-E (wild-type EHV-1 strain isolated from aborted equine fetus) and Rac-H (attenuated strain highly adapted in cell cultures in vitro) in primary murine neurons. In order to investigate this, we used two inhibitors: blebbistatin (BLB; non-muscle myosin II inhibitor) and 2,3-butanedione monoxime (BDM; inhibitor of myosin ATPase). Our results demonstrated that limitation of Jan-E EHV-1 replication occurred in cells treated with myosin inhibitor, which confirmed the important role of actin motor proteins during the entry and egress of EHV-1 virions. Application of blebbistatin did not affect Rac-H EHV-1 replication, while BDM caused reduction of replication in murine neurons. Based on these results it can be assumed that EHV-1 virion movement was myosin-dependent.

Primary murine neurons as in vitro model for studying neuroinfections caused by human adenoviruses.

Adenoviral infections of the central nervous system are rare, but they are characteristic for their high mortality rate. People with impaired immunity and children are particularly vulnerable. A few reports of neuroinfections caused by adenoviruses are found in literature. In this study the tropism of the human adenoviruses type 4, 5, 7 to primary cultures of murine neurons and the influence of infection on the neuron morphology and actin cytoskeleton was examined. The A549 non-small-cell lung cancer cell line was used as a reference line. Viral effects upon the cell culture were evaluated by direct immunofluorescence. The levels of adenovirus replication in the above-mentioned cell cultures were determined by real-time PCR. In the current study we demonstrated for the first time that human adenovirus (HAdV) type 4, 5 and 7 exhibits tropism for neurons cultured in vitro followed by the extensive replication of all serotypes in the primary culture of murine neurons. Immunofluorescent labelling and confocal microscopy revealed the changes in cell morphology, destruction of actin cytoskeleton and cell lysis as the final stage of infection. According to the obtained results we can assume that productive cycle of HAdV in the studied cell cultures occurred. We also observed accumulation of nuclear actin within nuclei of infected cells which may indicate that it plays role in adenovirus infection and replication in neurons and A549 cells.

Application of scanning cytometry and confocal-microscopy-based image analysis for investigation the role of cytoskeletal elements during equine herpesvirus type 1 (EHV-1) infection of primary murine neurons.

Equine herpesvirus type 1 (EHV-1), a member of Alphaherpesvirinae, has a broad host range in vitro, allowing for study of the mechanisms of productive viral infection, including intracellular transport in various cell cultures. In the current study, quantitative methods (scanning cytometry and real-time PCR) and confocal-microscopy-based image analysis were used to investigate the contribution of microtubules and neurofilaments in the transport of virus in primary murine neurons separately infected with two EHV-1 strains. Confocal-microscopy analysis revealed that viral antigen co-localized with the ß-tubulin fibres within the neurites of infected cells. Alterations in ß-tubulin and neurofilaments were evaluated by confocal microscopy and scanning cytometry. Real-time PCR analysis demonstrated that inhibitor-induced (nocodazole, EHNA) disruption of microtubules and dynein significantly reduced EHV-1 replication in neurons. Our results suggest that microtubules together with the motor protein - dynein, are involved in EHV-1 replication process in neurons. Moreover, the data presented here and our earlier results support the hypothesis that microtubules and actin filaments play an important role in the EHV-1 transport in primary murine neurons, and that both cytoskeletal structures complement each-other.

Replication kinetics of neuropathogenic and non-neuropathogenic equine herpesvirus type 1 (EHV-1) strains in primary murine neurons and ED cell line.

Equine herpesvirus type 1 (EHV-1) causes respiratory infections, abortion and neurological disorders in horses. Molecular epidemiology studies have demonstrated that a single-point mutation in DNA polymerase gene, resulting in an amino acid variation (N752/D752), is significantly associated with the neuropathogenic potential of EHV-1 strains. The aim of the study was to elucidate if there are any differences between neuropathogenic (EHV-1 26) and non-neuropathogenic (Jan-E and Rac-H) EHV-1 strains in their ability to infect neuronal cells. For the tested EHV-1 strains, cytopathic effect (CPE) was manifested by changed morphology of cells, destruction of actin cytoskeleton and nuclei degeneration, which led to focal degeneration. Moreover, EHV-1 26 strain caused fusion of the infected cells to form syncytia in culture. Real-time PCR analysis demonstrated that both neuropathogenic and non-neuropathogenic EHV-1 strains replicated in neurons and ED cells (equine dermal cell line) at a similar level. We can assume that a point mutation in the EHV-1 polymerase does not affect viral replication in this cell type.

Equid herpesvirus type 1 (EHV-1) disrupts actin cytoskeleton during productive infection in equine leukocytes.

Equid herpesvirus type 1 (EHV-1) is a prevalent causative agent of equine diseases worldwide. After primary replication in the respiratory epithelium the virus disseminates systemically through a peripheral blood mononuclear cell (PBMC)-associated viraemia. EHV-1 is the only alphaherpes- virus known so far which is capable of establishing latent infection not only in neurons but also in immune system cells (mainly in lymphocytes and macrophages). Since leukocytes are not the target cells for viral replication but are used to transport EHV-1 to the internal organs, the questionremains how the virus avoids the immune response and whether it could potentially be associated with virus-induced cytoskeletal rearrangements. Therefore, the aim of this study was to investigate the progress of EHV-1 replication in leukocytes stimulated by phytohemagglutinin and the impact of EHV-1 infection on the actin cytoskeleton. Using the real-time PCR method we evaluated the quantity of viral DNA from samples collected at indicated time points post infection. In order to examine possible changes in actin cytoskeleton organization due to EHV-1 infection, we performed immunofluorescent staining using TRITC-phalloidin conjugate. The results showed that EHV-1 rep- licates in leukocytes at a restricted level but with the accompaniment of chromatin degradation. Simultaneously, infection with EHV-1 caused disruption of the actin cytoskeleton; this was particularly apparent in further stages of infection. Disruption of the actin cytoskeleton may lead to the limited release of the virus from the cells, but may be also beneficial for the virus, since at the same time it potentially impairs the immune function of leukocytes.

Autophagy in cultured murine neurons infected with equid herpesvirus 1.

In this study we investigated the relationship of equid herpesvirus 1 (EHV-1) infection to autophagy in primary culture of murine neurons. Infection with both Jan-E and Rac-H strains of EHV-1 resulted in the formation of autophagosomes in the cytoplasm during early stages of infection, while in late stages of infection autophagosomes were mainly concentrated around the nucleus what suggests the induction of nuclear envelope-derived autophagy (NEDA). No significant effect of an authophagy inhibitor-chloroquine on final virus titers demonstrated that autophagy is not essential for EHV-1 replication.

Equine herpesvirus type 1 (EHV-1)-induced rearrangements of actin filaments in productively infected primary murine neurons.

Equine herpesvirus type 1 (EHV-1) causes respiratory disease, abortion and neurological disorders in horses. In the present study, we investigated reorganization of the cytoskeleton in neurons infected with two EHV-1 strains: Jan-E (wild-type strain) and Rac-H (attenuated strain). The studies were performed on primary murine neurons, which are an excellent model for studying neurotropism and neurovirulence of EHV-1. We have demonstrated for the first time that EHV-1 infection causes rearrangements in the actin network of neurons that are dependent on the virus strain and its adaptation to cell culture in vitro. Immunofluorescent labeling and confocal microscopy revealed the formation of long, thin projections in neurons infected with the Jan-E strain, which was probably associated with enhanced intracellular spread of the virus. The EHV-1 Rac-H strain caused disruption of the microfilaments system and general depolymerization of actin, but treatment of neurons with cytochalasin D or latrunculin A resulted in limitation of viral replication. It can therefore be assumed that actin filaments are required only at the early stages of infection. Our results allow us to suggest that the actin cytoskeleton participates in EHV-1 infection of primary murine neurons but is not essential, and that other components of the cytoskeleton and/or cellular mechanisms may be also involved during EHV-1 infection.

Primary cultures of murine neurons for studying herpes simplex virus 1 infection and its inhibition by antivirals.

Herpes simplex virus 1 (HSV-1) establishes life-long latency in peripheral neurons, where productive replication is suppressed. To study the specific relationship between the virus and peripheral neurons that would not be affected by other cells usually present in in vivo systems, we present an in vitro model system based on primary cultures of murine neurons. This model system can be used for characterization of various virus strains and testing of cytotoxicity and inhibitory activity of acyclovir (ACV), cidofovir (CDV) and other antivirals.

Influence of importin alpha/beta and exportin 1 on equine herpesvirus type 1 (EHV-1) replication in primary murine neurons.

Viruses replicating in the nucleus need to cross the nuclear membrane barrier during infection, therefore disruption of specific nuclear transport pathways is crucial for their replication cycle. In the present study we have investigated the influence of nucleo-cytoplasmic transport inhibitors -- ivermectin and leptomycin B, on EHV-1 replication in primary murine neurons. Obtained results suggest that the examined proteins -- exportin 1 and importin alpha/beta may participate, but are not required, during EHV-1 infection. Based on these results, it can be assumed that EHV-1 is able to use other receptors for nucleo-cytoplasmic transport.

HybProbes-based real-time PCR assay for rapid detection of equine herpesvirus type 2 DNA.

Equid herpesvirus type 2 (EHV-2) together with equid herpesvirus type 5 are members of Gammaherpesvirinae subfamily, genus Rhadinovirus. EHV-2 is one of major agents causing diseases of horses common worldwide. A possible role of EHV-2 in reactivating latent equid herpesvirus type-1 has been suggested, because reactivation of latent EHV-1 was always accompanied by EHV-2 replication. Variety techniques, including cell culture, PCR and its modifications, have been used to diagnose EHV-2 infections. The aim of this study was to develop, optimize and determine specificity of real-time PCR (qPCR) for EHV-2 DNA detection using HybProbes chemistry and to evaluate clinical samples with this method. The analytical sensitivity of assay was tested using serial dilutions of viral DNA in range between 70 and 7x10(5) copies/ml. The limit of detection (LOD) was calculated using probit analysis and was determined as 56 copies/ml. In further studies 20 different clinical samples were tested for the presence of EHV-2. Described in-house qPCR method detected viral DNA in 5 of 20 specimens used. The results of this work show that developed HybProbes-based real-time PCR assay is very reliable and valuable for detection and quantification of equid herpesvirus type 2 DNA in different clinical samples. The high level of sensitivity, accuracy and rapidity provided by the LightCycler 2.0 instrument are favorable for the use of this system in the detection of EHV-2 DNA in veterinary

The xCELLigence system for real-time and label-free analysis of neuronal and dermal cell response to equine herpesvirus type 1 infection.

Real-time cell electronic sensing (RT-CES) based on impedance measurements is an emerging technology for analyzing the status of cells in vitro. It allows label-free, real time monitoring of the biological status of cells. The present study was designed to assess dynamic data on the cell processes during equine herpesvirus type 1 (EHV-1) infection of ED (equine dermal) cells and primary murine neuronal cell culture. We have demonstrated that the xCELLigence system with dynamic monitoring can be used as a rapid diagnostic tool both to analyze cellular behavior and to investigate the effect of viral infection.

Apoptotic and necrotic changes in cultured murine neurons infected with equid herpesvirus 1.

Equid herpesvirus 1 (EHV-1), like other members of the Alphaherpesvirinae, is a neurotropic virus, that causes latent infections in the nervous system of the natural host. All alphaherpesviruses have developed sophisticated strategies to interfere with the host cell apoptotic mechanisms, but the ability of EHV-1 to induce apoptosis in neurons has not been determined yet. In this study, apoptotic and necrotic changes in cultured murine neurons were methods identifying key stages of apoptosis. These methods have demonstrated characteristic apoptosis features, like DNA fragmentation, chromatin condensation, membrane blebbing and cell shrinkage in the infected cells. It seems likely that apoptosis was the predominant way of cell death in EHV-1-infected murine neurons. However, we showed also that during acute EHV-1 infection the majority of infected neurons remained unchanged and survived for more than eight weeks in culture, suggesting some protective mechanisms induced by the virus. Furthermore, it was shown that infection of neurons with EHV-1 has no significant influence on the level of the caspase 3, 7, and 8. We speculate that the control of apoptosis may be the key mechanism regulating the balance between productive and latent infection at the site of virus persistence.

Equine herpesvirus type 1 (EHV-1) replication in primary murine neurons culture.

Equine herpesvirus-1 (EHV-1) infections cause significant economic losses for equine industries worldwide as a result of abortion, respiratory illness, and neurologic disease in all breeds of horses. The occurrence of abortions caused by EHV-1 has repeatedly been confirmed in Poland, but neurological manifestations of the infection have not been described yet. Also it is unknown how the infection of neurons with non-neuropathogenic strains is regulated. To further understand the virus-neuron interaction we studied two strains of EHV-1 in murine primary neuron cell cultures. Both strains were isolated from aborted fetuses: Rac-H, a reference strain isolated by Woyciechowska in 1959 (Woyciechowska 1960) and Jan-E isolated by Banbura et al. (Banbura et al. 2000). Upon infection of primary murine neuronal cell cultures with Jan-E or Rac-H strains, a cytopathic effect was observed, manifested by a changed morphology and disintegration of the cell monolayer. Positive results of immunofluorescence, nPCR and real-time PCR tests indicated high virus concentration in neurons, meaning that both EHV-1 strains were likely to replicate in mouse neurons in vitro without the need for adaptation. Moreover, we demonstrated that some neurons may survive (limited) virus replication during primary infection, and these neurons (eight weeks p.i.) harbour EHV-1 and were still able to transmit infection to other cells.

Equine herpesvirus type 1 quantification in different types of samples by a real-time PCR.

Equine herpesvirus type 1 (EHV-1) is one of the major viral agents causing diseases in horses common worldwide. A variety of techniques, including PCR, have been used to diagnose EHV-1 infections. In this paper, an attempt of real-time PCR has been described, which uses specific fluorochrome-labeled TaqMan probes for detection of viral DNA. This method does not require post-amplification manipulations, thereby reducing the risk of cross-contamination. The assay was sensitive enough to detect EHV-1 sequences in different clinical samples, as well in mice neuronal cell cultures. The technique was also very specific--here was no cross reaction with other human and equine herpesviruses. Compared to previously used nested PCR technique, the test was more sensitive and should be useful for the common diagnosis based on its specificity and rapidity.