Early and late pathogenic events of newborn mice encephalitis experimentally induced by itacaiunas and curionópolis bracorhabdoviruses infection

Abstract

In previous reports we proposed a new genus for Rhabdoviridae and described neurotropic preference and gross neuropathology in newborn albino Swiss mice after Curionopolis and Itacaiunas infections. In the present report a time-course study of experimental encephalitis induced by Itacaiunas and Curionopolis virus was conducted both in vivo and in vitro to investigate cellular targets and the sequence of neuroinvasion. We also investigate, after intranasal inoculation, clinical signs, histopathology and apoptosis in correlation with viral immunolabeling at different time points. Curionopolis and Itacaiunas viral antigens were first detected in the parenchyma of olfactory pathways at 2 and 3 days post-inoculation (dpi) and the first clinical signs were observed at 4 and 8 dpi, respectively. After Curionopolis infection, the mortality rate was 100% between 5 and 6 dpi, and 35% between 8 and 15 dpi after Itacaiunas infection. We identified CNS mice cell types both in vivo and in vitro and the temporal sequence of neuroanatomical olfactory areas infected by Itacaiunas and Curionopolis virus. Distinct virulences were reflected in the neuropathological changes including TUNEL immunolabeling and cytopathic effects, more intense and precocious after intracerebral or in vitro inoculations of Curionopolis than after Itacaiunas virus. In vitro studies revealed neuronal but not astrocyte or microglial cytopathic effects at 2 dpi, with monolayer destruction occurring at 5 and 7 dpi with Curionopolis and Itacaiunas virus, respectively. Ultrastructural changes included virus budding associated with interstitial and perivascular edema, endothelial hypertrophy, a reduced and/or collapsed small vessel luminal area, thickening of the capillary basement membrane, and presence of phagocytosed apoptotic bodies. Glial cells with viral budding similar to oligodendrocytes were infected with Itacaiunas virus but not with Curionopolis virus. Thus, Curionopolis and Itacaiunas viruses share many pathological and clinical features present in other rhabdoviruses but distinct virulence and glial targets in newborn albino Swiss mice brain.

Figure 1. Phase-contrast (A, C, E) and fluorescence photomicrographs (B, D, F) of primary neuronal cultures to illustrate normal noninfected cell morphology (A, B) and cytopathic effects 3 days after inoculation of Curionopolis virus (C, D) and 4 days after inoculation of Itacaiunas virus (E, F).

Arrows point to neurite fragmentation and circles indicate refringent points probably corresponding to apoptotic nuclei (A, C). Arrows and arrowheads indicate infected soma and dendrites, respectively (D, F). Neurons were stained by indirect immunofluorescence for anti-neurofilament antibodies. Secondary antibodies were conjugated with fluorescein isothiocyanate.

Figure 2. Combined interferential contrast and fluorescent photomicrographs of positive and negative controls (A, B) and Curionopolis- (C, D) or Itacaiunas (E,F)-infected neurons after TUNEL immunolabeling.

Apoptotic nuclei (arrows) are observed in control cultures exposed to UV light (A) and after Curionopolis (D) and Itacaiunas (F) virus inoculation at 4 and 5 days post-inoculation, respectively. Negative control cultures (B) and infected cultures at 1 day post-inoculation (C, E) present few stained nuclei.

Figure 3. Transmission electron photomicrographs of ultrathin sections obtained from primary neuronal cultures.

Control cultures of normal cells (A) and after Itacaiunas (B) and Curionopolis (C, D) infection at 4 and 5 days post-inoculation, respectively. Apoptotic cells (C) and virus budding (rectangle and arrows) (C, D) after Curionopolis infection. Itacaiunas virus particles (arrows) in culture at 5 days post-inoculation (B). N: nucleus; AN: apoptotic nucleus.

Figure 4. Photomicrographs of hematoxylin-eosin-stained sections from control (A) and infected brain sections at 96 h post-inoculation with Itacaiunas virus (B–D) and 60 h post-inoculation with Curionopolis virus (E–H).

Multiple foci of congestion with sparse distribution of leukocytes characterized by the margination phenomenon (leukocytes becoming flat and the plasma membrane sticking to the capillary endothelium) (B); hypertrophic endotheliocytes (ellipse) and perivascular edema (stars) (C). Apoptotic like-cells with nuclear pyknosis and cytoplasmic condensation were detected among tissue vacuolation (arrows) (D). Meningeal congestion and edema with lymphomononuclear cells (arrows) (E). Groups of pyknotic cells presenting cytoplasmic condensation (circles), mixed with normal cells and vacuolated parenchyma (stars) (F). Recent hemorrhagic points (presence of red blood cells) presenting parenchymatous edema (lozenge), mainly in the areas of worst cell damage (84 h post-inoculation) (G). Nuclear fragmentation or karyorrhexis (arrows) and edema (lozenge) (96 h post-inoculation) (H).

Figure 5. Transmission electron photomicrographs of ultrathin sections obtained from control (A) and mouse brain infected intracerebrally with Curionopolis for 36 (B,C), 60 (D) and 96 h (E), and with Itacaiunas for 24 (F), 60 (G), 72 (H), 96 (I) and 108 h (J).

Normal tissue with intact neuronal soma and appendages (A); viral particles (arrow), interstitial edema (stars) and cellular rarefaction (lozenge) are seen 36 h post-inoculation (p.i.) (B, C); necrotic cells were observed at 60 h p.i. (D); intense perivascular edema (stars), hyperplastic endotheliocytes and reduced vessel luminal area (E); well-preserved brain parenchyma and vessels at 24 h p.i. (F); viral particles, endotheliocyte hyperplasia, and mild interstitial edema (stars) at 60 h p.i. (G); membrane viral budding in rich polyribosomes oligodendrocyte-like cell at 72 h p.i. (H); brain parenchyma at 96 h p.i. presenting a large number of viral particles (I); apoptotic features were more marked at 108 h p.i. (J). AC = apoptotic cell, M = mitochondria, OL = oligodendrocyte, EC = endothelial cells, VL = vascular lumen, N = cell nucleus, NC = necrotic cells.

Figure 6. Bright-field (A, G) and interferential contrast (B–F, H) photomicrographs of infected mouse brain sections illustrating viral antigen-immunolabeled cells 2 (A–D) and 4 (E–G) days after inoculation with Curionopolis virus and TUNEL immunolabeling at 6 days (G, H).

Low (square) (A), medium (B) and high (C) power photomicrographs of labeled olfactory bulb neurons. High power images illustrating isolated neurons of the olfactory bulb with immunolabeled soma (arrow) and other neuronal appendages (arrowheads) (C); immunolabeled meningeal cells are also indicated (arrow) (D); cortical (E) and thalamic (F) neurons immunostained with viral antigens distributed in the cell appendages. TUNEL-positive neurons in infected brain sections (TUNEL POD procedure) 6 days after inoculation with Curionopolis virus into the ventral olfactory bulb (G, H). The arrows indicate immunostained neuronal nuclei.

Figure 7. Bright-field (A, D, F) and interferential contrast (B, C, E, G) photomicrographs of Itacaiunas virus-infected mouse brain at 4 (A–C), 6 (D, E) and 8 (F, G) days post-inoculation.

Low (A) and medium (B) power photomicrographs of the olfactory neuronal group (smaller rectangle). Details of immunolabeled neurons of the frontal cortex (C) (arrows and arrowheads). Low (D) (rectangle) and medium (E) power photomicrographs of a group (arrows) of hippocampal neurons showing low viral antigen condensation (arrowhead). TUNEL-positive midbrain neurons of infected brain sections (TUNEL POD procedure) 12 days after inoculation with Itacaiunas virus (F, G). The arrows indicate immunostained neuronal nuclei.