Stefanie Czub

Immunization with Modified Vaccinia Virus Ankara-Based Recombinant Vaccine against Severe Acute Respiratory Syndrome Is Associated with Enhanced Hepatitis in Ferrets

ABSTRACT Severe acute respiratory syndrome (SARS) caused by a newly identified coronavirus (SARS-CoV) is a serious emerging human infectious disease. In this report, we immunized ferrets (Mustela putorius furo) with recombinant modified vaccinia virus Ankara (rMVA) expressing the SARS-CoV spike (S) protein. Immunized ferrets developed a more rapid and vigorous neutralizing antibody response than control animals after challenge with SARS-CoV; however, they also exhibited strong inflammatory responses in liver tissue. Inflammation in control animals exposed to SARS-CoV was relatively mild. Thus, our data suggest that vaccination with rMVA expressing SARS-CoV S protein is associated with enhanced hepatitis.

Invasion of the Central Nervous System in a Porcine Host by Nipah Virus

ABSTRACT Nipah virus, a newly emerged zoonotic paramyxovirus, infects a number of species. Human infections were linked to direct contact with pigs, specifically with their body fluids. Clinical signs in human cases indicated primarily involvement of the central nervous system, while in pigs the respiratory system was considered the primary virus target, with only rare involvement of the central nervous system. Eleven 5-week-old piglets were infected intranasally, orally, and ocularly with 2.5 × 105 PFU of Nipah virus per animal and euthanized between 3 and 8 days postinoculation. Nipah virus caused neurological signs in two out of eleven inoculated pigs. The rest of the pigs remained clinically healthy. Virus was detected in the respiratory system (turbinates, nasopharynx, trachea, bronchus, and lung in titers up to 105.3 PFU/g) and in the lymphoreticular system (endothelial cells of blood and lymphatic vessels, submandibular and bronchiolar lymph nodes, tonsil, and spleen with titers up to 106 PFU/g). Virus presence was confirmed in the nervous system of both sick and apparently healthy animals (cranial nerves, trigeminal ganglion, brain, and cerebrospinal fluid, with titers up to 107.7 PFU/g of tissue). Nipah virus distribution was confirmed by immunohistochemistry. The study presents novel findings indicating that Nipah virus invaded the central nervous system of the porcine host via cranial nerves as well as by crossing the blood-brain barrier after initial virus replication in the upper respiratory tract.

Microglial/macrophage expression of interleukin 10 in human glioblastomas

Interleukin 10 (IL‐10) expression has been found to be correlated with the extent of malignancy in gliomas. In vitro, IL‐10 increases proliferation and migratory capacity in human glioma cell lines. In this study, we localized the site of IL‐10 synthesis in gliomas to cells of microglial origin. Biopsy specimens from 11 patients with malignant glioma were processed on native tissues and at early cell culture passages (0–4). IL‐10 mRNA was analyzed by RT‐PCR and in situ hybridization. Protein was quantitatively assessed by ELISA in cell culture supernatants, and cells expressing IL‐10 were determined by a combination of immunohistochemistry for CD68 (specific for microglia/macrophage lineage) and IL‐10 in situ hybridization. IL‐10 mRNA decreased from passage 0 to 4 in all samples and was undetectable beyond passage 5. Such downregulation of mRNA leads to a steep decrease of IL‐10 protein in culture supernatants (below detection level, 0.05 ng/ml, beyond passage 1). The combination of in situ hybridization for IL‐10 and CD68 immunostaining revealed that only cells of the microglia/macrophage lineage produced IL‐10 mRNA. Our results identify microglia/macrophage cells as the major source of IL‐10 expression in gliomas which decreases markedly during early passages of primary cultures of human gliomas due to a progressive reduction of microglia/macrophages present. Int. J. Cancer 82:12–16, 1999.

Alterations in neurotrophin and neurotrophin receptor gene expression patterns in the rat central nervous system following perinatal Borna disease virus infection.

Infection of newborn rats with Borna disease virus (BDV) leads to persistence in the absence of overt signs of inflammation. BDV persistence, however, causes cerebellar hypoplasia and hippocampal dentate gyrus neuronal cell loss, which are accompanied by diverse neurobehavioral abnormalities. Neurotrophins and their receptors play important roles in the differentiation and survival of hippocampal and cerebellar neurons. We have examined whether BDV can cause alterations in the neurotrophin network, thus promoting neuronal damage. We have used RNase protection assay to measure mRNA levels of the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), and their trkC and trkB receptors, as well as the growth factors insulin-like growth factor I (IGF-1) and basic fibroblast growth factor (bFGF), in the cerebellum and hippocampus of BDV-infected and control rats at different time points p.i. Reduced mRNA expression levels of NT-3, BDNF and NGF were found after day 14 p.i. in the hippocampus, but not in the cerebellum, of newborn infected rats. Three weeks after infection, trkC mRNA expression levels were reduced in both hippocampus and cerebellum of infected rats, whereas decreased trkB mRNA levels were only observed in the cerebellum. Reduced trkC mRNA expression was confined to the dentate gyrus of the hippocampus, as assessed by in situ hybridization. TUNEL assay revealed massive apoptotic cell death in the dentate gyrus of infected rats at days 27 and 33 p.i. Increased numbers of apoptotic cells were also detected in the cerebellar granular layer of infected rats after 8 days p.i. Moreover, a dramatic loss of cerebellar Purkinje cells was seen after day 27 p.i. Our results support the hypothesis, that BDV-induced alterations in neurotrophin systems might contribute to selective neuronal cell death.

Relationship between viral load in blood, cerebrospinal fluid, brain tissue and isolated microglia with neurological disease in macaques infected with different strains of SIV

The role of the viral burden in the brain for the pathogenesis of human immunodeficiency virus-associated neurological disorders is still unclear. To address this issue, we have quantified the viral load in plasma, cerebrospinal fluid (CSF) and brain tissue of macaques infected with simian immunodeficiency virus (SIV). We discovered that the viral strain used for infection determines the replicative capacity in microglial cells as well as the extent of neuropathological lesions and the occurrence of neurological symptoms. Moreover, the viral load in the brain parenchyma correlated with the development of overt neurological disease whereas the one in plasma did not. By comparing the viral load in three different compartments, we demonstrated that the viral burden in the CSF is influenced both by the viral replication in the periphery as well as in the brain parenchyma. According to these results, it is not the absolute amount of viral load in the CSF but rather the viral antigen contributed by the viral production within the brain which correlates with the development of neurological disease. In longitudinal studies, we observed that this autochthonous virus production, as evidenced by a ratio of the viral load in CSF to the one in plasma, takes place for a prolonged period of time before overt neurological signs are manifested. This finding suggests that this ratio could be used as a prognostic marker for immunodeficiency virus-induced neurological disease.

Impact of various simian immunodeficiency virus variants on induction and nature of neuropathology in macaques

To investigate the possible influence of virus tropism on SIV-induced neuropathogenesis, macaques were infected with molecularly cloned SIVmac239 which replicates poorly in cultured macrophages, with SIVmac251/32H which is a macrophage-tropic biologic clone, and with SIVmac251/MPBMC which is an early passage of 32H with enhanced replication competence in macrophages. We found that inflammatory as well as neuropathologic changes were identical in all clinically affected animals, irrespective of the in vitro tropism of the inocula. Moribund animals exhibited SIV encephalitis characterized by overt infection of macrophages/microglia inside the CNS parenchyma. Additionally, neuropathology of moribund animals was characterized by extraparenchymal immunopathology (meninges, perivascular space, choroid plexus stroma) and subtle white matter degeneration with glial changes, often associated with infected macrophages in situ (except in leukoencephalopathy). However, in animals inoculated with the lymphocyte-tropic and enhanced macrophage-tropic inocula, microglia but not blood-derived macrophages were the primary cells infected. Altogether, our results underline the significance of macrophage infection for the development of SIV encephalitis, and suggest that SIVmac239 either undergoes a change in cell tropism in vivo that results in the ability to replicate in macrophages, or else macrophages become more permissive to infection by this virus in the terminal stage of immunosuppressive disease.

The thymic epithelial reticulum and interdigitating cells in SIV-induced thymus atrophy and its comparison with other forms of thymus involution.

Alterations in the thymus were investigated in the early course of SIV infection of rhesus monkeys and compared with age-related and acute accidental thymus atrophy. The SIV-induced pathology was characterized by shrinkage of the thymic parenchyma and capsule, whereas in age-related thymus atrophy, the size of the capsule remained unaltered and the emerging space was filled by fatty tissue. Acute accidental thymus involution is characterized by massive cell death of the thymocytes, but there was no increase in pycnotic thymocytes either in SIV-induced or in age-related thymus atrophy. Ultrastructural analysis revealed no major differences between the juvenile control and the aged thymus. In contrast, SIV-induced thymus atrophy exhibited severe alterations of the epithelial cells of the cortex and the interdigitating dendritic cells, which were not found in the aged thymus nor in the juvenile control cortex.

Activation of microglia cells is dispensable for the induction of rat retroviral spongiform encephalopathy

In the course of retroviral CNS infections, microglia activation has been observed frequently, and it has been hypothesized that activated microglia produce and secrete neurotoxic products like proinflammatory cytokines, by this promoting brain damage. We challenged this hypothesis in a rat model for neurodegeneration. In a kinetic study, we found that microglia cells of rats neonatally inoculated with neurovirulent murine leukemia virus (MuLV) NT40 became infected in vivo to maximal levels within 9–13 days postinoculation (bdd.p.i.). Beginning from 13 d.p.i., degenerative alterations, i.e., vacuolization of neurons and neuropil were found in cerebellar and other brain-stem nuclei. Elevated numbers of activated microglia cells—as revealed by immunohistochemical staining with monoclonal antibody EDI—were first detected at 19 d.p.i. and were always locally associated with degenerated areas but not with nonaltered, yet infected, brain regions. Both neuropathological changes and activated microglia cells increased in intensity and numbers, respectively, with ongoing infection but did not spread to other than initially affected brain regions. By ribonuclease protection assays, we were unable to detect differences in the expression levels of tumor-necrosis-factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in microglia cells nor in total brains from infected versus uninfected rats. Our results suggest that the activation of microglia in the course of MuLV neurodegeneration is rather a reaction to, and not the cause of, neuronal damage. Furthermore, overt expression of the proinflammatory cytokines TNF-α, IL-1β, and IL-6 within the CNS is not required for the induction of retroviral associated neurodegeneration in rats.

Modulation of acute coronavirus-induced encephalomyelitis in γ-irradiated rats by transfer of naive lymphocyte subsets before infection

Clinical course, recovery of infectious virus from brain tissue and histopathology of the central nervous system were examined in gamma-irradiated Lewis rats reconstituted by naive lymphocytes before infection with coronavirus MHV-4 (strain JHM). Up to 9 days past infection, no differences were seen between immunologically competent and immuno-deficient animals in terms of onset and progression of neurological disease. However, in the latter animals neurological symptoms were dominated by signs of encephalitis instead of paralytic disease as usually seen in immunocompetent animals. Nevertheless, despite high titers of infectious virus in the CNS of immunodeficient animals only mild histopathological changes were noticeable. In contrast, infectious virus in the CNS of immunologically competent animals was below the detection limit of the assay. Paralytic disease and tissue destruction were T lymphocyte mediated because gamma-irradiated rats that were reconstituted by CD4+ or CD8+ T lymphocyte enriched cells in the absence of B lymphocytes revealed an earlier onset of clinical symptoms and a more rapid deterioration of their clinical state compared to fully competent animals. Whereas in CD4+ T cell reconstituted animals infectious virus was moderately reduced and tissue destruction as well as inflammatory changes in the CNS were focal, in CD8+ T cell reconstituted animals vacuolizing white matter inflammation was diffuse without reduction of infectious virus in brain tissue. From the presented data we conclude that in the acute stage of JHMV-induced encephalomyelitis of Lewis rats: (i) tissue destruction and paralytic clinical symptomatology are mainly T cell-mediated; (ii) CD4+ T lymphocytes can directly contribute to reduction of viral load in the brain and (iii) only coordinated action of both, the T and the B cell compartment enables animals to survive the infection and recover from disease.

A collaborative Canadian–United Kingdom evaluation of an immunohistochemistry protocol to diagnose bovine spongiform encephalopathy

Collaboration was established in 2001 to evaluate a commercially available immunohistochemistry assay kit for the detection of bovine spongiform encephalopathy (BSE) disease-associated prion protein in formic acid-treated formalin-fixed samples of bovine brain. The kit protocol was evaluated at the National Centre for Foreign Animal Diseases (Winnipeg, Canada) and the Veterinary Laboratories Agency (Weybridge, U.K.). The U.K. laboratory provided paraffin-embedded blocks of brainstem (medulla oblongata at the level of the obex) from 100 positive cases defined by clinical signs and histopathology, and 100 clinically suspect but BSE-negative samples defined by histopathology and immunohistochemistry with anti-PrP monoclonal antibody R145. The Canadian laboratory provided 400 blocks from surveillance cases defined as clinically suspect but negative by histopathology and immunohistochemistry with anti-PrP antibody 6H4. Consecutive sections from each block were cut and coded. Each set of 600 slides was immunolabeled and read in each laboratory. Evaluation parameters included estimates of diagnostic sensitivity and specificity and reproducibility of the results. The kit performed with 100% sensitivity, specificity, and reproducibility in spite of minor differences between the laboratories in brain sample areas, fixation and processing, and in the immunolabeling protocol. Although enzyme linked immunosorbent assays are widely used in high throughput surveillance programs, standardized protocols and reagents for manual immunohistochemistry provide a useful adjunct to surveillance efforts, particularly in laboratories testing small numbers of samples or using immunohistochemistry for confirmation and characterization of BSE cases.