Cell type-specific resistance of trigeminal ganglion neurons towards apoptotic stimuli

Trigeminal ganglion (TG) neurons are important target cells for many alphaherpesviruses and constitute a major site of virus latency and reactivation. Earlier we showed that porcine TG neurons are remarkably more resistant towards (apoptotic) cell death resulting from infection by the swine alphaherpesvirus pseudorabies virus (PRV) compared to a broad range of other primary porcine cell types and that this resistance does not depend on the strongly anti-apoptotic US3 viral protein kinase (Geenen, K., Favoreel, H.W., Nauwynck, H.J., 2005a. Higher resistance of porcine trigeminal ganglion neurons towards pseudorabies virus-induced cell death compared with other porcine cell types in vitro. J. Gen. Virol. 86, 1251-1260). Although other viral anti-apoptotic proteins may be involved in survival of TG neurons during PRV infection, an additional factor may be that TG neurons possess a cell type-dependent capacity to withstand apoptosis compared to other cell types. To investigate this, we treated uninfected porcine TG cultures, swine kidney cells, and porcine superior cervical ganglion (SCG) neurons with several apoptosis-inducing reagents (staurosporine, camptothecin and genistein). None of these reagents were able to trigger substantial apoptotic cell death in TG neurons, whereas non-neuronal TG cells, swine kidney cells, and SCG neurons showed a clear dose-dependent increase in apoptosis using either of these reagents. In conclusion, sensory TG neurons may contain a cell type-specific capacity to withstand different apoptotic assaults, including infection with an alphaherpesvirus.     

Pseudorabies virus induces a rapid up-regulation of nitric oxide synthases in the nervous system of swine

Nitric oxide (NO) is a free radical gas with important roles in the host's immune response against viral infections. In this study, we examined the kinetics and distribution of nitric oxide synthase (NOS) expression during the early steps of infection of the porcine nervous system by the alphaherpesvirus pseudorabies virus (PRV). To this end, we examined changes in the expression of the three major NOS isoforms, neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS), by immunohistochemistry in the trigeminal ganglia and brain of pigs inoculated intranasally with a virulent PRV strain. The results obtained show that infection of the porcine nervous system by PRV induced a rapid and progressive increment in NOS expression that coincided in timing, location, and magnitude with those of virus propagation in the nervous tissue. A major finding of this study was that PRV caused not only nNOS and iNOS induction in a variety of cell types, but also eNOS up-regulation in endothelial cells and neurons; therefore, all possible sources of NO are activated and probably contribute to the overproduction of NO during infection with the neurotropic alphaherpesvirus PRV in its natural host.     

A homologous in vitro model to study interactions between alphaherpesviruses and trigeminal ganglion neurons

A key aspect in the life cycle of alphaherpesviruses is their neurotropic behaviour. Sensory neurons of the trigeminal ganglion (TG) are important target cells for many alphaherpesviruses (including herpes simplex virus 1, pseudorabies virus (PRV), bovine herpesvirus 1) and constitute major sites for latent infections. The aim of this study was to develop an in vitro model that simulates the in vivo infection pattern of TG neurons by alphaherpesviruses. To this end, we developed a homologous in vitro two-chamber model using PRV and porcine TG neurons. TG of 4- to 6-week-old piglets were dissociated and cultured in the inner chamber of the in vitro model, which is separated from the outer chamber by a medium- and virus-impermeable silicon barrier. Outgrowth of axons from neuronal cell bodies in the inner chamber through the silicon barrier into the outer chamber could be observed after 2-3 weeks of cultivation. Subsequent addition of PRV to the outer chamber resulted in exclusive infection of the TG neurons by transport of virus through the axons, subsequently giving rise to productively infected TG neurons that transmitted virus to contacting neurons and non-neuronal cells in the inner chamber. Thus, we established a homologous in vitro model that mimics the natural route of alphaherpesvirus infection of TG neurons that can be used to study interactions between these viruses and this pathogenetically very important cell type.     

Replication and immunosuppressive effects of Pseudorabies virus on swine peripheral blood mononuclear cells.

The infectivity and potential immunosuppressive effects of Pseudorabies virus (PRV) was evaluated in swine peripheral blood mononuclear cells (PBMC). Virus progeny titers and viral DNA synthesis at various intervals post-inoculation revealed the replication of PRV in both peripheral blood monocytes and lymphocytes; however, replication in lymphocytes was restricted compared with monocytes. PRV infection resulted in the damage and death of monocytes. Although PRV did not appear to affect the viability of the lymphocytes, PRV infection suppressed lymphocyte functions such as proliferation and interleukin-2 (IL-2) synthesis in response to Concanavalin A. This immunosuppression was dependent upon the multiplicity of infection (MOI) of infectious PRV. UV-inactivated PRV was not immunosuppressive. There was no effect of PRV on natural killer (NK) cell activity. The reduction of lymphocyte proliferation by PRV was not reversible by the addition of supernatant containing porcine IL-2 and non-infected monocytes to the infected cultures. The results from these in vitro studies demonstrate that PRV can infect and cause immunosuppressive effects on swine PBMC. These effects may explain the potential role of PRV in predisposing infected pigs to secondary infection and support the hypothesis that PRV can spread systemically by infected PBMC in blood and lymph.     

Variation of Aujeszky's disease viruses in wild swine in USA

In the United States of America, Aujeszky's disease (pseudorabies) has been eradicated from all domestic swine. Some re-emergence of infection occurred as vaccine use diminished. Sporadic outbreaks have also occurred because of the reservoir of infection in feral swine that have spread across the southern two-thirds of the country and Hawaii. In order to be able to understand the origins of re-emerging virus, sequence analysis of variable genes in pseudorabies virus (PRV) has been used to differentiate strains. Most PRV from feral swine can be distinguished from virus circulating in domestic pigs during the national epizootic. However, several feral swine isolates of PRV from south central states are closely related or identical in sequence to strains from domestic pigs. Extensive study by PCR for the presence of virus in the oral cavity of feral pigs disclosed that the viral DNA is distributed widely in tonsils salivary glands, taste buds and even mucosa in the vicinity of tusks. Clearly the virus in feral swine has multiple mechanisms of transmission to insure persistent infection and the threat of re-emergence in domestic swine continues.     

Directional spread of an alpha-herpesvirus in the nervous system

Pseudorabies virus (PRV), an alpha-herpesvirus, is capable of spreading between synaptically connected neurons in diverse hosts. In this report, two lines of experimentation are summarized that provide insight into the mechanism of virus spread in neurons. First, techniques were developed to measure the transport dynamics of capsids in infected neurons. Individual viral capsids labeled with green fluorescent protein (GFP) were visualized and tracked as they moved in axons away from infected neuronal cell bodies in culture during egress. Second, the effects of three viral membrane proteins (gE, gI and Us9) on the localization of envelope, tegument, and capsid proteins in infected, cultured sympathetic neurons were determined. These three proteins are necessary for spread of infection from pre-synaptic neurons to post-synaptic neurons in vivo (anterograde spread). Us9 mutants apparently are defective in anterograde spread in neural circuits because essential viral membrane proteins such as gB are not transported to axon terminals to facilitate spread to the connected neuron. By contrast, gE and gI mutants manifest their phenotype because these proteins most likely function at the axon terminal of the infected neuron to promote spread. These two sets of experiments are consistent with a model for herpesvirus spread in neurons first suggested by Cunningham and colleagues where capsids and envelope proteins, but not whole virions, are transported separately into the axon.     

Pseudorabies virus infection in mink: a host-specific pathogenesis

Pseudorabies virus (PRV) is an alphaherpesvirus that causes a neurological disease in many wild and domestic animals. The neuropathology elicited by PRV is quite consistent regardless of the host with the only exception of mink, in which it is characterized by a vasculopathy rather than by an encephalitis. In this study, we aimed to investigate the underlying pathogenic mechanism(s) of PRV infection in mink by using immunohistochemistry and laser capture microdissection (LCM) on material from naturally and experimentally infected animals. The inflammatory reaction induced by PRV was minimal or absent not only in the nervous system, where we identified a low number of macrophages and a few T lymphocytes, but also in the primary replication site, the oropharyngeal mucosa; however, the number of PRV-infected cells detected by immunohistochemistry was extremely high both in the peripheral mucosa and in the nervous tissue. On the other hand, the vascular pathology included parenchymal hemorrhages of various degrees and, in specific cortical areas of the brain, fibrinoid degeneration of the capillary walls. Detection of viral antigens by immunohistochemistry revealed infection of endothelial cells of capillaries situated both in the oropharyngeal mucosa and in the brain stem; the presence of PRV DNA in vessels was further demonstrated by PCR performed on LCM samples of brain capillaries. These results can be interpreted as supporting the idea that the different pathology of the disease in mink may be the consequence of an increased endotheliotropism of PRV in this species. Infection of the vessel wall may then lead to vascular pathology and impairment in endothelial cell function, resulting in a weak immune response to infection.     

Immunofluorescence studies on the pathogenesis of hemagglutinating encephalomyelitis virus infection in pigs after oronasal inoculation

Hemagglutinating encephalomyelitis virus (HEV; also designated vomiting and wasting disease virus) was inoculated oronasally in 14 colostrum-deprived pigs at the day of birth. Anorexia and vomition were seen after 4 days. Pigs were killed at different times after inoculation, and the results of the examination by immunofluorescent antibody technique revealed that the epithelial cells of nasal mucosa, tonsils, lungs, and small intestine served as sites of primary viral replication. After the local replication near the sites of entry, the virus spread via peripheral nervous system to the CNS. During the incubation period, viral antigens were detected in the trigeminal ganglion,the inferior vagal ganglion, the superior cervical ganglion, the intestinal nervous plexuses, the solar ganglion, and the dorsal root ganglia of the lower thoracic region. In the brain stem, the infection started in the trigeminal and vagal sensory nuclei and spread to other nuclei and to the rostral part of the brain stem. In later stages of the infection, viral spread into the cerebrum, cerebellum, and spinal cord was sometimes also observed. Viral replication in nervous plexuses of the stomach was not present during the incubation period, but was detected in all except 1 of the pigs that were ill when killed. The question whether the vomition is induced centrally by viral replication in the brain stem or is due to viral replication in peripheral nervous tissues remains unanswered.     

In vivo and in vitro reactivation of latent pseudorabies virus in pigs born to vaccinated sows

Latency of pseudorabies virus (PRV) was established in 8 of 9 pigs born to 2 vaccinated sows. Pigs had high, low, or no maternal antibody titers at the time of the initial inoculation. At postinoculation months 3 to 4, latent PRV could be reactivated in vivo by the administration of large doses of corticosteroids. In most pigs, the stress-simulating treatment resulted in recrudescence of virus shedding after lag periods of 4 to 11 days. In 3 pigs, virus shedding was without clinical signs of disease, whereas clinical signs that developed in 4 pigs appeared to be due to the corticosteroid treatment, rather than to the reactivation of PRV. Pigs with a log10 neutralizing antibody titer of less than or equal to 2.55 at the onset of corticosteroid treatment had a booster response. Reactivated PRV spread to sentinel pigs housed with the inoculated pigs. Reactivation of PRV was also demonstrated in vitro. Explant cultures of trigeminal ganglia from pigs killed between postinoculation months 4 to 5 produced infectious virus. Restriction endonuclease analysis indicated that the reactivated PRV was indistinguishable from virus isolated shortly after the primary infection. Seemingly, pigs with maternal antibodies can become latently infected and therefore may be regarded as potential sources of dissemination of PRV.     

猪伪狂犬病病毒潜伏感染检测方法研究进展

猪伪狂犬病是由伪狂犬病病毒引起的 ,是以仔猪的发病死亡和怀孕母猪繁殖障碍为特征的猪病 ,是危害养猪业的重要疫病之一。该病在世界上分布广泛 ,并有不断蔓延扩大的趋势。任何年龄的猪在耐过伪狂犬病病毒急性感染后均能形成潜伏感染 ,并可在体内终生潜伏 ,且不表现临床症状 ,在一定条件下 ,潜伏状态的病毒能被激活 ,引起复发性感染并向外散毒。这种伪狂犬病病毒潜伏 -激活循环机制决定了伪狂犬病病毒在猪群中的永远存在。猪一旦感染必须视为伪狂犬病病毒散播的潜在来源。因而对潜伏感染猪的剔除对于根除伪狂犬病病毒感染具有重要意义。国内外对潜伏感染的检测方法主要有鉴别血清学诊断、潜伏病毒的激活与检测、组织块培养技术、核酸探针技术以及PCR技术。本文主要综述了这几种方法的研究情况 ,并比较了其优缺点     

Distribution of neutral amino acid transporter ASCT 1 in the non-neuronal tissues of mice

Distribution of ASCT1, a neutral amino acid transporter, in non-neuronal peripheral tissues of adult and developing mice was examined by immunohistochemistry and immunoelectron microscopy. Immunoreactivity for ASCT1 in the digestive system was localized in basal cells of stratified squamous epithelia from oral parietes to nonglandular region of the stomach, chief cells of the glandular stomach, acinar cells of the salivary gland and exocrine pancreas, and Paneth's cells of the small intestine, in all of which the basolateral membrane was selectively immuno-labeled. In the liver of adult mice, ASCT1 immunoreactivity was detected on the plasma membrane of hepatocytes surrounding central veins, and a temporal expansion of immunoreactive hepatocytes was observed in the embryonic and CCl4-treated adult livers. ASCT1 was also localized on the plasma membranes of proximal uriniferous tubule epithelial cells in the kidney of adult mice, and those of supporting cells in the medulla of adrenal gland. These results suggest that ASCT1 is expressed in various non-neuronal peripheral tissues in mice, and it contributes to the amino acid transport throughout non-neuronal tissues.     

Study of the potential involvement of pseudorabies virus in swine respiratory disease.

In order to investigate the potential involvement of pseudorabies virus (PRV) in swine respiratory disease, nine week old pigs were intranasally inoculated with the PRV strain 4892. Two doses of infection were used: 10(4.5) median tissue culture infectious doses (TCID50)/pig and 10(3.5) TCID50/pig, with ten pigs per group. In the group of pigs inoculated with 10(4.5) TCID50, seven out of ten pigs died within six days after inoculation. The mortality rate in the group of pigs inoculated with the lower dose was only two out of ten and, there were several pigs in this group that showed signs of respiratory distress besides some mild nervous signs. Pseudorabies virus was isolated from various tissues collected postmortem, including alveolar macrophages. Virus localization in tissues was also detected by in situ hybridization. The histopathological examination of the respiratory tract tissues revealed a pathological process that was progressing from mild pneumonia to severe suppurative bronchopneumonia. The isolation of virus from alveolar macrophages provides support to the hypothesis that replication of PRV during the course of infection produces an impairment of the defense mechanisms in the respiratory tract.     

Factors associated with spread of pseudorabies virus among breeding swine in quarantined herds.

Knowledge of the factors that place susceptible gilts at highest risk of pseudorabies virus (PRV) infection in a quarantined herd is crucial to reduce spread of PRV within the herd. Cohorts of PRV seronegative gilts were monitored in 17 herds that were endemically infected with PRV to determine the location of breeding females at the time of infection with PRV and identify herd characteristics and management and housing factors that may influence spread of PRV in the breeding section of swine herds endemically infected with PRV. Blood samples were collected every 1 to 2 months for an average of 13.6 months. In addition, blood was collected from a representative sample of finishing pigs (greater than or equal to 20 weeks old) 3 times per year to determine their serologic PRV status. Incidence rates and relative risks of PRV infection were estimated for 4 areas of the breeding section: gestation barn, gilt pool, farrowing room, and breeding area. Overall, 28, 11, 8, and 2 females became infected with PRV in each of these areas, respectively. The greater number of females infected in the gestation barns, compared with the number of females infected in other locations, is probably a consequence of being at risk for a longer period rather than of a higher incidence rate. Herd size, common housing for gilts in the gilt pool and sows, and serologic pattern of PRV infection in finishing pigs were associated with the detection of spread of PRV in the breeding section of the 17 herds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histopathology and immunohistochemistry of bats infected by Australian bat lyssavirus

OBJECTIVE: To describe the lesions and distribution of viral antigens in bats infected by Australian bat lyssavirus. DESIGN: A retrospective histopathological and immunohistochemical study of bats naturally infected with the virus. PROCEDURE: Tissues from 37 infected bats were examined. Nineteen flying foxes (fruit bats) and two insectivorous bats were examined in detail. Brains of another 16 flying foxes were poorly fixed and were examined less fully. RESULT: Lesions varied considerably between individuals and, where present, were mostly those of nonsuppurative meningoencephalomyelitis and ganglioneuritis similar to lesions seen in rabies and rabies-like diseases. The number of cells with intracytoplasmic inclusion bodies (Negri bodies) was variable; none were seen in some bats. Intracytoplasmic vacuolation of neurons was a common finding. Lesions occurred throughout the central nervous system but were most frequent and severe in the hippocampus, thalamus and midbrain, and medulla oblongata and pons. Indirect immunoperoxidase tests for lyssavirus antigen reactions varied in intensity and distribution, but also occurred mostly in the hippocampus, thalamus and midbrain, and medulla oblongata and pons. In peripheral tissues, reactions were seen in autonomic ganglia, in nerve plexuses of the gastrointestinal tract, in nervous tissues within muscles and immediately adjacent to individual muscle fibres, in an adrenal medulla, and in epithelial tissues in one of eight salivary glands examined. CONCLUSION: The main lesion in Australian bat lyssavirus infection is nonsuppurative inflammation similar to that seen in rabies and other rabies-like diseases, except that the number of Negri bodies is more variable. Reactions to immunoperoxidase tests for lyssavirus vary in intensity and distribution and may occur in both central and peripheral nervous systems. These reactions do not always occur in the salivary glands, even if brain infection is present.     

Propagation of scrapie in peripheral nerves after footpad infection in normal and neurotoxin exposed hamsters

As is known from various animal models, the spread of agents causing transmissible spongiform encephalopathies (TSE) after peripheral infection affects peripheral nerves before reaching the central nervous system (CNS) and leading to a fatal end of the disease. The lack of therapeutic approaches for TSE is partially due to the limited amount of information available on the involvement of host biological compartments and processes in the propagation of the infectious agent. The in vivo model presented here can provide information on the spread of the scrapie agent via the peripheral nerves of hamsters under normal and altered axonal conditions. Syrian hamsters were unilaterally footpad (f.p.) infected with scrapie. The results of the spatiotemporal ultrasensitive immunoblot-detection of scrapie-associated prion protein (PrP(Sc)) in serial nerve segments of both distal sciatic nerves could be interpreted as a centripetal and subsequent centrifugal neural spread of PrP(Sc) for this route of infection. In order to determine whether this propagation is dependent on main components in the axonal cytoskeleton (e.g. neurofilaments, also relevant for the component ;a' of slow axonal transport mechanisms), hamsters were treated -in an additional experiment- with the neurotoxin beta,beta-iminodiproprionitrile (IDPN) around the beginning of the scrapie infection. A comparison of the Western blot signals of PrP(Sc) in the ipsilateral and in the subsequently affected contralateral sciatic nerve segments with the results revealed from IDPN-untreated animals at preclinical and clinical stages of the TSE disease, indicated similar amounts of PrP(Sc). Furthermore, the mean survival time was unchanged in both groups. This in vivo model, therefore, suggests that the propagation of PrP(Sc) along peripheral nerves is not dependent on an intact neurofilament component of the axonal cytoskeleton. Additionally, the model indicates that the spread of PrP(Sc) is not mediated by the slow component ;a' of the axonal transport mechanism.     

Viral antigen distribution in the central nervous system of cattle persistently infected with bovine viral diarrhea virus

Distribution of viral antigens in the central nervous system of 25 cattle with a persistent bovine viral diarrhea virus (BVDV) infection was studied. Using a polyclonal antiserum produced in pigs and the direct immunofluorescence and immunoperoxidase technique, BVDV antigen was located exclusively in neurons. Predilection sites for viral persistence were cerebral cortex and hippocampus; in other areas of brain and spinal cord, viral antigens were in single neurons or small groups of neurons. There was no morphological evidence of cellular alteration due to viral persistence. Perivascular lymphocytic infiltrations were in affected nervous tissue. It is concluded that the central nervous system is an important location for persistence of BVDV.     

Epidemiology, diagnosis, and treatment of blastomycosis in dogs and cats

Blastomycosis is one of the most common systemic fungal diseases in dogs in North America, but it is rarely diagnosed in cats. The typical route of infection is inhalation of aerosolized conidia of Blastomyces dermatitidis. From the respiratory tract, the developing yeast form may disseminate throughout the body and affect multiple organ systems, most commonly the lymphatic, skeletal and central nervous systems, eyes and skin. Disseminated disease often is associated with nonspecific signs of illness including lethargy, inappetence and fever, as well as signs referable to specific organ systems like chronic cough and dyspnea, peripheral lymphadenopathy, endophthalmitis, and central nervous signs. Diagnosis is typically made by detection of Blastomyces dermatitidis yeast in affected tissues by fine-needle aspiration cytology or histopathology. The treatment of choice is itraconazole. Prognosis is fair in dogs without central nervous disease and guarded in cats.     

Equine herpes myeloencephalopathy

The neurologic form of EHV-1 infection appears to be the result of central nervous system infarction caused by vasculitis, which is initiated in endothelial cells of small blood vessels. The etiologic agent is equine herpesvirus-1, subtype 1. There is some evidence to suggest that the neurologic form of the disease actually results from reactivation of a previous infection. Whether the vasculitis that causes the central nervous system injury is the direct result of the infection or an immune response to the infection has not been determined. The clinical signs are rapid in onset, nonprogressive, and many horses may improve. The diagnosis must often remain tentative, particularly in horses that recover, because there is no single reliable confirmatory test. The prognosis is generally good, although recovery may be slow and incomplete. Supportive therapy is essential, and administration of corticosteroids may be useful. There is no specific therapy for the virus or for the vasculitis. Currently no vaccine can be claimed to protect against the central nervous system form of the disease. Vaccination is recommended, however, to reduce the incidence of respiratory disease, abortion, and neonatal death on the farm. Repeated vaccination is necessary to maintain presumably protective antibody concentrations. Vaccination every 3 to 4 months may decrease the incidence of EHV-1 infection on the farm and therefore may indirectly prevent the occurrence of the neurologic form of the disease.     

Pseudorabies virus infection in wild boars in Japan

In Japan, most pig populations are now free from pseudorabies virus (PRV) due to the recent success of an extensive eradication program. However, PRV infection persists in Japanese wild boars (Sus scrofa leucomystax), representing another potential reservoir for the virus in Japan. In this study, the seroprevalence of PRV in wild boars captured in three different prefectures was ascertained. A virus neutralization (VN) test showed that 6 of 173 serum samples (3%) were positive for VN antibody; glycoprotein E-ELISA revealed infection with the wild-type, but not the available vaccine strain, PRV. These results indicate that PRV has continued to spread among wild boars in Japan.