M.W. Bańbura

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.

Opposite effects of two different strains of equine herpesvirus 1 infection on cytoskeleton composition in equine dermal ED and African green monkey kidney Vero cell lines: application of scanning cytometry and confocal-microscopy-based image analysis in a quantitative study

Viruses can reorganize the cytoskeleton and restructure the host cell transport machinery. During infection viruses use different cellular cues and signals to enlist the cytoskeleton for their mission. However, each virus specifically affects the cytoskeleton structure. Thus, the aim of our study was to investigate the cytoskeletal changes in homologous equine dermal (ED) and heterologous Vero cell lines infected with either equine herpesvirus 1 (EHV-1) strain Rac-H or Jan-E. We found that Rac-H strain disrupted actin fibers and reduced F-actin level in ED cells, whereas the virus did not influence Vero cell cytoskeleton. Conversely, the Jan-E strain induced polymerization of both F-actin and MT in Vero cells, but not in ED cells. Confocal-microscopy analysis revealed that α-tubulin colocalized with viral antigen in ED cells infected with either Rac-H or Jan-E viruses. Alterations in F-actin and α-tubulin were evaluated by confocal microscopy, Microimage analysis and scanning cytometry. This unique combination allowed precise interpretation of confocal-based images showing the cellular events induced by EHV-1. We conclude that examination of viral-induced pathogenic effects in species specific cell lines is more symptomatic than in heterologous cell lines.

Influence of long-term equine herpesvirus type 1 (EHV-1) infection on primary murine neurons—the possible effects of the multiple passages of EHV-1 on its neurovirulence

Equine herpesvirus 1 (EHV-1), like other members of the Alphaherpesvirinae subfamily, is a neurotropic virus causing latent infections in the nervous system of the natural host. In the present study, we have investigated EHV-1 replication (wild-type Jan-E strain and Rac-H laboratory strain) during long-term infection and during the passages of the virus in cultured neurons. The studies were performed on primary murine neurons, which are an excellent in vitro model for studying neurotropism and neurovirulence of EHV-1. Using real-time cell growth analysis, we have demonstrated for the first time that primary murine neurons are able to survive long-term EHV-1 infection. Positive results of real-time PCR test indicated a high level of virus DNA in cultured neurons, and during long-term infection, these neurons were still able to transmit the virus to the other cells. We also compared the neurovirulence of Rac-H and Jan-E EHV-1 strains after multiple passages of these strains in neuron cell culture. The results showed that multiple passages of EHV-1 in neurons lead to the inhibition of viral replication as early as in the third passage. Interestingly, the inhibition of the EHV-1 replication occurred exclusively in neurons, because the equine dermal (ED) cells co-cultivated with neuroculture medium from the third passage showed the presence of large amount of viral DNA. In conclusion, our results showed that certain balance between EHV-1 and neurons has been established during in vitro infection allowing neurons to survive long-term infection.

Mechanisms of endocytosis utilized by viruses during infection.

Viruses, despite being relatively simple in structure and composition, have evolved a broad spectrum of mechanisms to exploit the host cell. To initiate effective infection, viruses or viral genomes have to enter cells. Recently studies have shown that apart from the direct fusion at the plasma membrane, endocytosis is more often the preferred means of entry into the host cell. Endocytosis is a complex phenomenon, that includes multiple pathways of membrane trafficking, such as clathrin-mediated endocytosis, caveolin-mediated endocytosis, macropinocytosis and phagocytosis. Endosomes offer a convenient and often rapid transit system across the plasma membrane and cytoplasm via the cellular microtubular network. They also provide protection to the virus from detection by the hosts innate immune defences. What is important, viruses are able to utilize not just one, but multiple uptake routes. Identification of these processes and factors will not only allow a better insight into pathogenic mechanism, but may identify novel targets for future therapeutic development. This review provides insight on recent developments in the rapidly evolving field of viral entry.

Acyclovir and trichostatin A modulate EHV-1 replication in murine neurons in vitro

Equine herpesvirus type 1 (EHV-1) is one of the most important viral pathogens of horses worldwide (2). It may cause respiratory disease, sporadic or epizootic abortions, or, recently more often, neurological disease known as equine herpesvirus myeloencephalopathy (EHM), which may be life-threatening and results in significant economic losses to the equine industry (1, 13, 15, 18, 19). It has been suggested that a single-nucleotide polymorphism in the EHV-1 DNA polymerase gene, which leads to amino acid variation (N752/D752), may be associated with outbreaks of EHM (3, 11, 13). D752 strains of EHV-1, which are statistically more often isolated from cases of EHM, were called neuropathogenic strains. However, it is worth mentioning that all EHV-1 strains show neurotropism and are capable of establishing latency in peripheral neurons. Moreover, EHV-1 may also establish latency in leukocytes (10). The main role of latency is to maintain the viral genome for a long time inside host cells, at the same time avoiding the immune response. On the other hand, the virus may reactivate and start productive replication at any time, especially during stress, which leads to the dissemination of progeny virions (17). The current approach to the control of EHV-1 infections is based on biosecurity measures and vaccination, but it is not sufficient. Immunity after infection or vaccination is usually incomplete and short-lived, and once latency has been established, the virus cannot be eliminated from host cells. Although some progress has been made in understanding the adaptive immunity to EHV-1, innate immunity remains poorly characterized, despite the fact that it is critically important for inducAcyclovir and trichostatin A modulate EHV-1 replication in murine neurons in vitro1)

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.

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 β-tubulin fibres within the neurites of infected cells. Alterations in β-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.

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.