SummaryIntracerebral inoculation of newborn and 5-week-old mice with Zika virus resulted in an early and marked enlargement of astroglial cells with patchy destruction of the pyriform cells of Ammon's horn. Replication of the virus was demonstrated in both neuroncs and astroglial cells. New virions appeared to be formed within networks of endoplasmic reticulum. The similarity of these ultrastructural observations to those obtained from in vivo studies of other group B arboviruses is contrasted with the widely differing findings from in vitro studies.

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A simple method was devised for negativestain transmission electron microscopy of brain infected with the agent of scrapie. Brains of infected' hamsters contained large masses of tubuloframentous structures with irregular fuzzy surfaces. Brains of mice infected with Creutzfeldt-Jakob disease agent contained similar tubulofilaments in smaller numbers. The abnormal tubulofilaments resembled but were distinguished from normal microtubules. On grids soaked in sodium dodecyl sulfate the abnormal tubuloframents were found in stages of fragmentation, an outer coat appearing to strip from the surface to reveal thinner fibrillary structures resembling scrapie-associated fibrils (SAF). The unmasked fibrils were identified as SAF by immunogold labeling, while the larger tubulofilaments were not labeled. The findings indicate that in infected brain tissue SAF may occur as an internal part of a larger structure that is disrupted by detergent and are not likely to be an artifact formed during extraction procedures.Unique "virus-like" tubulovesicular particles (Fig. 1) have been consistently observed by electron microscopy (EM) in thin sections of plastic-embedded brain tissues from several types of animals and from humans infected with the agents of scrapie and Creutzfeldt-Jakob disease (CJD) (1-6). However, progress in characterizing those particles or investigating their relationship to the etiological agents of the spongiform encephalopathies has been impeded by failure to identify them except in plastic sections, where they are relatively inaccessible to studies other than morphological analyses. We studied unembedded preparations of brain tissue by negative staining (7) with the goal of obtaining such particles in a state in which they could be concentrated and purified. Not only were abnormal tubulofilaments identified in brains of rodents with spongiform encephalopathies but also an unanticipated association was observed between those structures and filaments of the amyloid-like protein variously designated as scrapie-associated fibrils (SAF) (8) and prion protein 27-30 (PrP 27-30) (9, 10). MATERIALS AND METHODSScrapie and CJD Agents. The strains of agent used have been previously described. The 263K strain of hamsteradapted scrapie agent (11, 12) was used at the 4th passage level (13). The KFu strain of mouse-adapted CJD agent (14) was used at our 3rd passage level (15).Animals and Inoculations. Weanling female LGV/LAK golden Syrian hamsters were inoculated intracerebrally with 0.03-ml aliquots of 10% suspension of scrapie-infected brain as previously described. The inoculum of scrapie agent contained at least 107 LD50 units per 0.03 ml; animals became ill as early as 65 days later, and all had died by 90 days after inoculation. Weanling female National Institutes of Health white Swiss mice were inoculated intracerebrally with 0.03 ml of a 10-3 dilution of CJD-agent infected brain suspension; the inoculum contained at least 50 LD50 units per 0.03 ml.Mice became ill 141 days later and were killed 149 days after...

Abnormal tubulofilamentous structures have been identified in electron micrographs of thin sections and negatively stained impression grids prepared from brains of animals with scrapie and other spongiform encephalopathies, and we showed that such tubules contain a core of rdamentous structures resembling scrapie-associated fibrils (SAF). We treated impression grids from brains of scrapie-infected hamsters with several substances that bind to or cleave proteins and nucleic acids to see if they had any effect on the abnormal tubuloframentous structures. Treatment with three proteolytic enzymes reduced the caliber of the tubules from about 50 nm to 30 nm; subsequent treatment of the 30-nm tubules with DNase I left many typical SAF as well as transitional forms in which twisted SAF emerged from tubules. DNase treatment of the original thicker tubules had no effect, and no SAF were seen on grids. Treatment of the 30-nm tubules with any of three other nucleases (micrococcal, mung bean, and BAL-31) also produced SAF. However, treatment with RNase A had no effect either on the original 50-nm tubules or on the 30-nm tubules produced by proteolysis. Detergent treatment of any of the preparations produced SAF. Treatment with ethidium bromide resulted in staining of the tubules that was inhibited by magnesium ions. The data suggest that the abnormal tubulofilamentous particles found in spongiform encephalopathies may consist of an outer cylinder of protein, an inner cylinder of DNA, and an innermost core of SAF.We recently described a simple method of negative-stain transmission electron microscopy using grids touched to the freshly cut surface of brain tissue. In brains infected with the agents ofthe spongiform encephalopathies, we found abnormal tubular structures (1) that generally resembled tubulovesicular particles often seen in thin sections of plastic-embedded brain specimens (2,3). We demonstrated that treatment of those tubules with detergent released inner filaments that were morphologically identical to scrapie-associated fibrils (SAF) (4) and reacted with antisera prepared against the proteaseresistant sialoglycoprotein "prion protein [27][28][29][30] (5, 6) of which SAF are composed (7,8). We report here a study of the effects of several proteases and nucleases and of ethidium bromide on the abnormal tubules. The abnormal tubules appear to consist of at least three layers: (i) an outer coat of protease-sensitive material, (ii) an intermediate layer that is digested by a DNase and several other nucleases but not by an RNase, and (iii) an inner core of SAF. MATERIALS AND METHODSScrapie Virus. The 263K strain of scrapie agent (9) was used at the fourth passage level as described (1 20,10,5, 2.5, and 0.25 units/ml diluted in buffer recommended by the supplier; and (ix) diluent alone. Grids were immersed under 0.5 ml of solution and incubated in a moist chamber. Grids were removed at intervals from 30 min to 120 min. After a 30-min incubation, grids treated with collagenase, trypsin, or proteinase K were...