Dengue infection in mice inoculated by the intracerebral route: neuropathological effects and identification of target cells for virus replication.

Dengue is an important arboviral infection, causing a broad range symptom that varies from life-threatening mild illness to severe clinical manifestations. Recent studies reported the impairment of the central nervous system (CNS) after dengue infection, a characteristic previously considered as atypical and underreported. However, little is known about the neuropathology associated to dengue. Since animal models are important tools for helping to understand the dengue pathogenesis, including neurological damages, the aim of this work was to investigate the effects of intracerebral inoculation of a neuroadapted dengue serotype 2 virus (DENV2) in immunocompetent BALB/c mice, mimicking some aspects of the viral encephalitis. Mice presented neurological morbidity after the 7th day post infection. At the same time, histopathological analysis revealed that DENV2 led to damages in the CNS, such as hemorrhage, reactive gliosis, hyperplastic and hypertrophied microglia, astrocyte proliferation, Purkinje neurons retraction and cellular infiltration around vessels in the pia mater and in neuropil. Viral tropism and replication were detected in resident cells of the brain and cerebellum, such as neurons, astrocyte, microglia and oligodendrocytes. Results suggest that this classical mice model might be useful for analyzing the neurotropic effect of DENV with similarities to what occurs in human.

Pathologic and molecular findings associated with atypical porcine pestivirus infection in newborn piglets.

Atypical porcine pestivirus (APPV) has been associated with congenital tremor (CT) type A-II in newborn piglets. Although the number of APPV-based studies is increasing, the associated pathologic findings in infected piglets are underreported. This study describes the histopathologic features of spontaneous APPV infection in CT-affected piglets and complements a previous report by our group. Four two-day-old piglets with CT were evaluated by histopathology, immunohistochemistry (IHC), and molecular assay. The main histopathologic findings at the brain and spinal cord included neuronal necrosis, gliosis, neuronophagia, satellitosis, demyelination, Wallerian degeneration, and Purkinje cell necrosis. An IHC assay designed to detect the proliferation of glial fibrillary acidic protein (GFAP) in affected areas of the brain and spinal cord revealed that the proliferation of GFAP + cells and fibers was predominant in APPV-infected piglets relative to asymptomatic piglets of the same age group. The RT-nested-PCR assays identified APPV RNA in the cerebrum, cerebellum, and brainstem of all piglets; other viruses known to produce similar manifestations were not detected. These results suggest that the APPV-induced histopathologic findings are predominantly degenerative and necrotic and correlate with our previous findings. Consequently, it is proposed that neuronal necrosis, gliosis, neuronophagia, and satellitosis should be considered as important histologic features of APPV-induced infection in symptomatic CT piglets.

[Retrospective analysis of cases with a diagnosis of cerebrocortical necrosis and its relation with type 5 bovine herpesvirus]. / Análisis retrospectivo de casos con diagnóstico de necrosis cerebrocortical y su relación con herpesvirus bovino tipo 5.

In order to demonstrate the association of bovine herpesvirus type 5 (BHV-5) and cerebrocortical necrosis (CCN), 89 such cases were examined in cattle from three regions of Buenos Aires Province, Argentina, registered between 1970-1999. Hematoxylin-eosin staining and BHV-5 in situ hybridization were performed on paraffin-embedded neural tissues. The severity of microscopic lesions was scored according to a 0-3 scale. Morbidity, mortality and lethality rates between groups depending on age and regions were determined. The highest prevalence of CCN was detected between 1979 and 1984, particularly during the spring. Differences in morbidity and mortality rates between groups of age and regions were not detected (P > 0.05). Amaurosis (48%), ptyalism (42%), circling (40%), ataxia (36%) and bruxism (37%) were frequently observed. Lesions were predominantly found in anterior and posterior cortex (90.6%) and diencephalon (36.5%). Meningitis and perivascular cuffing (94.4%) and focal (78%) or diffuse (73%) gliosis were predominant in cerebrum. Focal necrosis was observed in 66.6% of cases. BHV-5 was isolated from 9/19 cases since 1992 and BHV-5 DNA was detected by in situ hybridization in 3/9 cases. No virus was identified in brain tissues with severe lesions. These findings indicate the association of BHV-5 in neurological disease previously reported as CCN.

Long-lasting astrocyte reaction to persistent Junin virus infection of rat cortical neurons.

Immunoperoxidase labeling was performed in histological sections from rat brain harvested during acute (10-30 days), clinically inapparent (90-270 days) and late (450-540 days) stages of Junin virus-induced neurological disease. In frontoparietal cortex, count of viral antigen (+) neurons peaked during the acute period (27.7+/-6.8), dropped within the intermediate (4.8+/-4.0 to 1.4+/-1.1) and increased (7.6+/-4.3) at the onset of the late neurological syndrome. In infected vs. control rats, the number of GFAP (+) astrocytes maximized during the acute stage (19+/-4 vs. 11+/-5), and from the end of the intermediate (27+/-5 vs. 21+/-5) up to the late (37+/-7 vs. 26+/-6) periods. In turn, surface density of GFAP (+) material in infected samples peaked at 0.196+/-0.066, while it failed to exceed 0.090+/-0.043 in controls. Both astrocyte hypertrophy relapsing into chronicity, as depicted by surface density, and astrocyte hyperplasia preceding the onset of the late neurological syndrome, support their pathogenic contribution to disease expression.

Junin virus-induced astrocytosis is impaired by iNOS inhibition.

Because Junin virus (JV) experimental encephalitis of mice and rats is characterized by mild histopathological changes that do not seem to justify per se lethality after intracerebral infection, such a murine model seems adequate to investigate the potential role of inducible nitric oxide synthase (iNOS) as a pathogenic factor. Concomitant with a predominant astrocyte reaction, increased immunoperoxidase expression of iNOS, mitochondrial superoxide dismutase (SODm) and glutathione peroxidase (GPX) was disclosed in brain of mice infected with JV strain #44. When specific inhibition of iNOS was achieved by intraperitoneal administration of amino guanidine (AG), significantly greater mortality was observed in treated animals (70% vs. 40%), together with similar infective titers ( approximately 10(7) PFU/g) but lower astrocytosis, as shown by glial fibrillary acidic (GFAP) labeling. As regards SODm and GPX immunochemical expression in neurons, no differences were found between mice with or without AG treatment. The present results suggest that the apparent protective role of nitric oxide (NO), when synthesized by iNOS, is unrelated to reduced viral replication but rather to enhanced astrocyte activation behaving as a beneficial cell response to virus-induced CNS damage.

Inflammation is a component of neurodegeneration in response to Venezuelan equine encephalitis virus infection in mice.

Infection with the mosquito-transmitted Venezuelan equine encephalitis virus (VEE) causes an acute systemic febrile illness followed by meningoencephalitis. In this communication we characterize the cytokine profile induced in the central nervous system (CNS) in response to virulent or attenuated strains of VEE using RNase Protection Assays. Virulent VEE causes an upregulation of multiple pro-inflammatory genes including inducible nitric oxide synthase (iNOS) and tumor necrosis factor alpha (TNF-alpha). To determine if iNOS and TNF-alpha contribute to the neuropathogenesis of VEE infection, iNOS and TNF receptor knockout mice were used in VEE mortality studies and exhibited extended survival times. Finally, CNS tissue sections labeled for VEE antigen, and adjacent sections double-labeled for an astrocyte marker and apoptosis, revealed that apoptosis of neurons occurs not only in areas of the brain positive for VEE-antigen, but also in areas of astrogliosis. These findings suggest that the inflammatory response, which is in part mediated by iNOS and TNF-alpha, may contribute to neurodegeneration following encephalitic virus infection.