Pathogenesis of mouse scrapie: dynamics of agent replication in spleen, spinal cord and brain after infection by different routes.

Groups of Compton white mice were infected with different doses of the Chandler (139A) strain of agent by one of the following routes of injection; intracerebral, intraperitoneal, intravenous, subcutaneous in the “scruff” of the neck or under the footpad. At weekly intervals, 3 mice were taken from each group and infectivity assays were performed on pools of spleen, brain and spinal cord to study the dynamics of agent replication during the early stages of incubation. All peripheral routes of infection gave a similar pattern in that agent replication started early in spleen and reached a plateau concentration of agent before replication occurred in spinal cord. Replication in spinal cord occurred after 25 to 42 per cent of the incubation period had elapsed and always preceded the onset of replication in brain (41 to 55 per cent of incubation period). This sequence is consistent with the spread of agent from spleen to spinal cord and thence to brain, possibly along neural pathways. Agent replication in brain started much sooner (13 to 19 per cent of incubation period) with the i.c. route of infection but replication in spinal cord followed the pattern found with peripheral routes of infection and occurred after 24 to 34 per cent of the incubation had elapsed.     

Pathogenesis of scrapie in mice after intragastric infection

Infection via the gastrointestinal tract is likely to be a natural route of scrapie infection in sheep. This paper describes the pathogenesis of the 139A strain of scrapie introduced intragastrically (i.g.) into CW mice. There was an almost immediate uptake of infectivity and onset of replication in Peyer's patches which preceded replication in spleen. Splenectomy had no effect on incubation period suggesting that, in contrast to the intraperitoneal route, the spleen plays little or no role in the pathogenesis of 139A scrapie administered intragastrically. Replication in the CNS was first detectable in the thoracic spinal cord and later in brain. The evidence is consistent with neural spread of infection from the gastrointestinal tract, via the enteric and sympathetic nervous systems to spinal cord. Neuroinvasion may be initiated either via infection of Peyer's patches or directly by infection of nerve endings in the gut wall. The latter possibility means that pathogenesis may be completely independent of the lymphoreticular system.     

The role of the spleen in the neuroinvasion of scrapie in mice

The pathogenesis of 139A scrapie has been studied in CW mice infected intraperitoneally (i.p.), intravenously (i.v.) or subcutaneously (s.c.). In mice splenectomised before i.p. infection, the evidence points to a neuroinvasive pathway from visceral lymph nodes (and other sites of scrapie replication in the peritoneum) to the thoracic spinal cord. However, in non-splenectomised mice, the major neuroinvasive pathway is clearly from spleen to thoracic cord because i.p. incubation periods are shorter and replication in the thoracic cord starts correspondingly earlier than in splenectomised mice. Studies of splenectomy at different times after i.p. infection show that pathogenesis becomes independent of the spleen once infection has initiated scrapie replication in the spinal cord. The simplest interpretation of all the evidence favours the spread of scrapie infection along splenic nerve fibres to the thoracic spinal cord. The same neuroinvasive pathway is suggested by the findings using the s.c. and i.v. routes of infection. In addition it was found that the 100-fold greater efficiency of infection by the i.v. compared to the i.p. route was entirely dependent on the spleen, because splenectomy before i.v. infection reduced its efficiency to the same as that found in i.p. infected (non-splenectomised) mice.     

Interaction of scrapie agent and cells of the lymphoreticular system

Summary The current study focused on the role of lymphoid elements of the lymphoreticular system in scrapie pathogenesis. In the first experiment, adherent and non-adherent splenocytes from mice infected with the 139A scrapie strain were prepared. The level of infectivity on a per cell basis was significantly higher in the adherent cell population. In a second set of experiments, thymocytes, unfractionated splenocytes, T-cell enriched and T-cell depleted fractions of splenocytes were infected in vitro with ME7 scrapie strain. There was no evidence of replication of scrapie in ME7-exposed cells in any of the preparations during the first 5–14 days post-exposure. In assays done 5 days after infection, most of the infectivity was cell-associated. These data suggest that lymphoid cells are not involved in scrapie replication. The level of IgA in the serum of 139A-infected mice was markedly reduced compared to the levels in mice injected with normal mouse brain homogenate or with the ME7 scrapie strain. The reduction in IgA levels in 139A-infected mice was evident at each of the 4 time points tested. The final experiment dealt with the question of scrapie replication in the lymphoreticular organs in mouse strains with different incubation periods for 139A after intraperitoneal injection. The results in this experiment suggest that the difference in incubation periods is related to differences in time of access of infection to the central nervous system rather than to differences in the ability of agent to replicate in spleen.     

Early events in the pathogenesis of eastern equine encephalitis virus in mice

To elucidate the pathogenesis of eastern equine encephalitis (EEE) virus infections, we used histopathology, immunohistochemistry, and in situ hybridization to track the spread and early cellular targets of viral infection in mice. Young mice were inoculated with virulent EEE virus in their right rear footpad and were followed in a time-course study for 4 days. Virulent EEE virus produced a biphasic illness characterized by an early self-limiting replication phase in peripheral tissues followed by an invariably fatal central nervous system (CNS) phase. In the early extraneural phase, there was primary amplifying replication of virus within fibroblasts at the inoculation site and within osteoblasts in active growth areas of bone that resulted in a transient high-titer viremia. Pathological changes and viral infection were observed as early as 12 hours post-infection (PI) in osteoblasts, skeletal muscle myocytes, and in fibroblasts along fascial sheaths. The severity and extent of infection in peripheral tissues peaked at day 1 PI. In the neural phase of infection, virus was first detected in the brain on day 1 PI, with rapid interneuronal spread of infection leading to death by day 4 PI. EEE virus appeared to be directly cytopathic for neurons. The very rapid onset and apparently random and widely dispersed infection in the CNS, with concurrent sparing of olfactory neuroepithelium, strongly suggests that invasion of the CNS by EEE occurs by a vascular route, rather than via peripheral nerves or the olfactory neuroepithelium. Our finding that metaphyseal osteoblasts are an early site of amplifying viral replication may explain the higher-titer viremias and higher incidence of neuroinvasion and fulminant encephalitis seen in the young, and may also explain why mature animals become refractory to encephalitis after peripheral inoculation with EEE virus.     

Characteristics of a short incubation model of scrapie in the golden hamster.

Repeated passage of the "Chandler" strain of scrapie in female golden hamsters using the intracerebral route of inoculation reduces the minimum incubation period to 60 days, about half of the minimum incubation period so far found in any of the mouse models of scrapie. The infectivity titres in brain in the clinical stage of the disease are considerably higher (greater than 8-0 -log10 LD50 i.c. units/0-05 g) than those found in mouse scrapie. The biological characteristics of this model of hamster scrapie are reported, including the effects on incubation period of route of inoculation, dose of agent, sex of hamster, ambient temperature (hibernation) and splenectomy. Some general and specific applications of this experimental model of scrapie are discussed.     

Evidence for intrinsic control of scrapie pathogenesis in the murine visual system.

Using the optic nerve to route scrapie infection into the brain reduces the initial spread of the disease to well-defined neuronal relays, and simplifies the observation of cause and effect of agent transport, replication and degeneration of the nervous system. One drawback of intraocular targeting of infection is the relatively long incubation periods required to produce clinical disease. By using highly-enriched fractions of infectivity and two models of murine scrapie, we have found that this time delay is not simply due to the limited amount of infective inoculum that can be injected into the eye. This provides evidence of intrinsic control of scrapie pathogenesis within the murine visual system.     

Pathological PrP is abundant in sympathetic and sensory ganglia of hamsters fed with scrapie.

Although the ultimate target of infection is the CNS, there is evidence that the peripheral nervous system (PNS) is involved in the pathogenesis of Transmissible Spongiform Encephalopathies (TSEs). We used immunocytochemistry to identify the presence of pathological accumulations of a host protein, PrP, in the CNS and PNS (sensory and autonomic ganglia) of hamsters orally infected with 263K scrapie. All hamsters showed pathological deposition of PrP in most brain areas, along the length of the spinal cord, in nodose (NG) and dorsal root (DRG) ganglia and in the coeliac mesenteric ganglion complex (CMGC). In one case, scant deposition was observed along a few axons of the vagus nerve. This finding suggests that, after oral challenge, TSE infectious agent uses neural pathways and ganglia of the peripheral nervous system to reach target sites in the CNS.     

Brain slice culture for analysis of developmental brain disorders with special reference to congenital cytomegalovirus infection

ABSTRACT  Cytomegalovirus (CMV) is the most significant infectious cause of congenital abnormalities of the central nervous system (CNS) with variation from the fatal cytomegalic inclusion disease to functional brain disorder. The phenotype and degree of the brain disorder depends on infection time during the developing stage, virulence, route of infection and the viral susceptibility of the cells. The pathogenesis of the CMV infection to the CNS seems to be strongly related to neural migration, neural death, cellular compositions and the immune system of the brain. To understand the complex mechanism of this disorder, we used organotypic brain slice cultures. In the brain slice culture system, migration of CM V-in-fected neuronal cells was observed, which reflects infectious dynamics in vivo. Neural progenitor cells or glial immature cells in the subventricular zone and marginal area are most susceptible to murine cytomegalovirus (MCMV) infection in this system. The susceptibility declined as the number of immature glial cells decreased with age. The immature glial cells proliferated in brain slice cultures during prolonged incubation, and the susceptibility to MCMV infection also increased in association with the proliferation of these cells. The brain slice from an immunocompromised mouse (Beige-SCID mouse) unexpectedly showed lower susceptibility than that of an immunocompetent mouse during any prolonged incubation. These results suggest that the number of immature glial cells might determine the susceptibility of CMV infection to the brain, independent of the immune system. We reviewed recent findings of CMV infection to the brain from the perspective of brain slice cultures and the possibility that this system could be a useful method to investigate mechanisms of congenital anomaly of the brain.     

Pathogenesis of scrapie: agent multiplication in brain at the first and second passage of hamster scrapie in mice.

The intracerebral (i.c.) injection of mice with a particular source of hamster passaged scrapie produced disease after an incubation period of 325 +/- 6 days (mean +/- s.e.). The incubation period at the second i.e. passage in mice was reduced to 149 +/- 2 days. Studies were made of the dynamics of agent replication at 1st and 2nd passages in mice. At first passage, there was a 'zero phase' lasting about 175 days, when no infectious agent was detected in brain (or spleen), followed by a period of agent replication which lasted 150 days. At second passage, there was no significant 'zero phase' and agent replication occupied the whole of the incubation period. The occurrence of a 'zero phase' on interspecies passage of scrapie is discussed in relation to other reports of a 'zero phase' in mouse passaged scrapie.     

Effect of bacterial flora and mouse genotype (euthymic or athymic) on scrapie pathogenesis

Euthymic and athymic female BALB/c mice, reared under either germfree or defined flora conditions, were used to investigate the pathogenesis of scrapie after intracerebral or intraperitoneal inoculation. Time in days to onset of clinical signs (Stage I), to endstage (Stage II), and the time interval between Stage I and Stage II were compared among groups. In addition, scrapie agent titers in spleen were determined at 28 and 90 days after infection, as were agent titers in spleen and brain at Stage II. Three-way analysis of variance indicated that the bacterial flora, the presence or absence of a thymus, and the route of agent inoculation interact to produce significant differences in the pathogenesis of disease. The three factors in the experimental design also influenced the spleen titers of scrapie infectivity. The variation in scrapie pathogenesis among the groups of mice is likely to be mediated by differences in their reticuloendothelial systems. These differences may alter the agent's adsorption in spleen and/or route of transport from spleen to brain.     

Scrapie-Induced Changes in the Percentage of Polymorphonuclear Neutrophils in Mouse Peripheral Blood

A decrease in the percentage of polymorphonuclear neutrophils (PMN) in the peripheral blood of mice appeared 3 days after intracerebral (IC) inoculation with scrapie mouse brain homogenate. Mice inoculated IC with normal mouse brain had PMN percentages similar to those found for uninoculated mice. This difference between normal and scrapie-inoculated mice continued throughout the preclinical phase of the disease. In the clinical phase of the disease, the percentage of PMN was either higher or lower than that found in normals. The factor causing the decrease in PMN percentages was found in the filtrates from 220-, 100-, and 50-nm filters, but not in the filtrates from a 25-nm filter. Sodium periodate treatment of the scrapie brain samples eliminated their ability to cause the decrease in PMN percentages, whereas sodium iodate had no effect. In addition to two genetically different scrapie mouse brain isolates, homogenates of mouse spleen, sheep brain, and sheep spleen from scrapie-affected animals caused a decrease in percent PMN, whereas the corresponding normal tissue homogenates did not.     

Bacteremia is required for invasion of the murine central nervous system by Listeria monocytogenes

The ability of the facultative intracellular pathogenListeria monocytogenesto penetrate the central nervous system (CNS) was studied by following the kinetics of brain invasion and histological lesions during an acute intravenous (i.v.) infection in the mouse. CNS invasion occurred during the early phase of infection and produced severe meningoencephalitis characterized by multiple granulomatous foci predominantly located in the brainstem and associated with diffuse meningitis and an intense inflammatory reaction involving the choroid plexuses. Bacterial counts in the brain could reach 104.5–105.8by day 5 of infection with 1–2×106bacteria i.v., depending upon the bacterial strain used. It was found that CNS invasion was highly dependent upon the level and the duration of bacteremia, thus indicating that persistent bacteremia is essential to induce meningoencephalitis toL. monocytogenes.     

Levels of infectivity in the blood throughout the incubation period of hamsters peripherally injected with scrapie

Summary Viremia is found in intraperitoneally scrapie-injected hamsters. The absence of a viremic peak before the beginning of scrapie replication in the brain suggests either that the spread of the agent to the brain is not via the blood or that early after infection, circulating monocytes carry the agent to the brain where it remains silent until the neural cells start replicating it.     

What can we learn from the oral intake of prions by sheep?

The central nervous system is the ultimate target of prions, the agents responsible for fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs). The neuro-invasive phase and its related clinical signs take place after a long incubation period. During this asymptomatic phase, however, active transport and replication of the infectious agent take place in peripheral sites. The oral infection route has been extensively studied because of its implication in the recent epidemic of bovine spongiform encephalopathy (BSE) in cattle and of the resulting human cases of variant Creutzfeldt-Jakob disease (vCJD). Rodent models have been useful in studying some aspects of this pathogenesis. Now, new data on the initial steps of oral infection have been obtained in sheep. This species is naturally infected with scrapie by horizontal transmission and there is strong evidence implicating the oral route. Furthermore, the existence of resistant and susceptible genotypes offers the possibility of comparative studies. The data were obtained using surgical and biochemical procedures to modulate the efficiency of oral infection and show that, in sheep, the abnormal prion protein (PrP) associated with the infectious agent crosses the intact intestinal barrier at the level of the enterocytes and then passes rapidly into lymph. These steps are identical in susceptible and resistant sheep. Thereafter, replication takes place in lymphoid structures. Other results in the same study indicate that alimentary fluids almost completely degrade the PrP of the inoculum. Though not directly transposable to human diseases, in which it is not possible to study these early stages, these data allow the elaboration of a simplified concept for the pathogenesis of TSEs. They also suggest that human contamination at the level of the oral cavity might be more important than previously suspected.     

Role of spleen macrophages in the clearance of scrapie agent early in pathogenesis

The involvement of spleen macrophages in the early stages of scrapie pathogenesis was studied by applying the 'macrophage-suicide technique' to scrapie-infected mice. This method comprises critically the intravenous administration to mice of dichloromethylene disphosphonate encapsulated into liposomes. Depletion of spleen macrophages before scrapie infection induced an increased amount of scrapie inoculum in the spleen, consequently leading to accelerated scrapie agent replication in the early phase of pathogenesis, as followed by PrPres accumulation, a specific hallmark of scrapie. The same effect was observed when spleen macrophages were depleted just before the beginning of scrapie agent replication. These findings suggest that macrophages may partly control scrapie infection in peripheral tissues by sequestration of the scrapie inoculum and may thus impair early scrapie agent replication in the spleen. In addition to macrophages, most follicular dendritic cells and B lymphocytes, which are thought to support scrapie agent replication, were also transiently depleted by dichloromethylene disphosphonate administration. This suggests that a compensatory mechanism is sufficient to ensure the persistence of infection in these early stages of pathogenesis.     

Type 3 reovirus neuroinvasion after intramuscular inoculation: direct invasion of nerve terminals and age-dependent pathogenesis

Neonatal but not adult mice are vulnerable to reovirus invasion of the central nervous system after peripheral inoculation. After hindlimb injection, type 3 reovirus travels via the sciatic nerve to replicate in spinal cord motor neurons before spread to the brain and development of lethal encephalitis. Here we provide ultrastructural evidence for direct reovirus invasion of unmyelinated neonatal motor nerve terminals within 2 h and replication in spinal cord motor neurons within 14 h after hindlimb injection of 1-day-old mice. In adult mice, resistance to reovirus lethality after intracranial (IC) injection correlates with the restriction of virus growth in cortical neurons. We found that neuroinvasion also is age dependent after intramuscular injection. Virus lethality and CNS infection decreased sharply during the first postnatal week, while lethality after IC injection continued for 2 additional weeks. Mice inoculated at 7 days of age with high virus doses suffered paralysis of the injected limb, but significant brain infection was not lethal. These results suggest that reovirus invasion of the neonatal CNS is restricted by several progressive age-dependent mechanisms.     

Encephalitic syndrome and anosmia in COVID‐19: Do these clinical presentations really reflect SARS‐CoV‐2 neurotropism? A theory based on the review of 25 COVID‐19 cases

Since the discovery of coronavirus disease 2019 (COVID‐19), a disease caused by the new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), the pathology showed different faces. There is an increasing number of cases described as (meningo)encephalitis although evidence often lacks. Anosmia, another atypical form of COVID‐19, has been considered as testimony of the potential of neuroinvasiveness of SARS‐CoV‐2, though this hypothesis remains highly speculative. We did a review of the cases reported as brain injury caused by SARS‐CoV‐2. Over 98 papers found, 21 were analyzed. Only four publications provided evidence of the presence of SARS‐CoV‐2 within the central nervous system (CNS). When facing acute neurological abnormalities during an infectious episode it is often difficult to disentangle neurological symptoms induced by the brain infection and those due to the impact of host immune response on the CNS. Cytokines release can disturb neural cells functioning and can have in the most severe cases vascular and cytotoxic effects. An inappropriate immune response can lead to the production of auto‐antibodies directed toward CNS components. In the case of proven SARS‐CoV‐2 brain invasion, the main hypothesis found in the literature focus on a neural pathway, especially the direct route via the nasal cavity, although the virus is likely to reach the CNS using other routes. Our ability to come up with hypotheses about the mechanisms by which the virus might interact with the CNS may help to keep in mind that all neurological symptoms observed during COVID‐19 do not always rely on CNS viral invasion.     

Transportation of prion protein across the intestinal mucosa of scrapie-susceptible and scrapie-resistant sheep

To determine the mechanisms of intestinal transport of infection, and early pathogenesis, of sheep scrapie, isolated gut-loops were inoculated to ensure that significant concentrations of scrapie agent would come into direct contact with the relevant ileal structures (epithelial, lymphoreticular, and nervous). Gut loops were inoculated with a scrapie brain pool homogenate or normal brain or sucrose solution. After surgery, animals were necropsied at time points ranging from 15 min to 1 month and at clinical end point. Inoculum-associated prion protein (PrP) was detected by immunohistochemistry in villous lacteals and in sub-mucosal lymphatics from 15 min to 3.5 h post-challenge. It was also detected in association with dendritic-like cells in the draining lymph nodes at up to 24 h post-challenge. Replication of infection, as demonstrated by the accumulation of disease-associated forms of PrP in Peyer's patches, was detected at 30 days and sheep developed clinical signs of scrapie at 18-22 months post-challenge. These results indicate discrepancies between the routes of transportation of PrP from the inoculum and sites of de novo-generated disease-associated PrP subsequent to scrapie agent replication. When samples of homogenized inoculum were incubated with alimentary tract fluids in vitro, only trace amounts of protease-resistant PrP could be detected by western blotting, suggesting that the majority of both normal and abnormal PrP within the inoculum is readily digested by alimentary fluids.     

Vagotomy attenuates effects of L-glucose but not of D-glucose on spontaneous alternation performance

Two peripheral signaling routes have been proposed to account for the ability of peripheral substances such as glucose to modulate memory processing in the brain. One possible signaling route is by crossing the blood-brain barrier to act directly on brain. A second route involves activation of peripheral nerves with resulting changes in neural activity carried by peripheral nerves to the brain. Because the vagus nerve is a major neural pathway between the periphery and brain, peripherally acting modulators of memory modulators may act via vagal afferents to the brain to enhance memory processing. In the present experiments, systemic injections of either D-glucose or L-glucose, a metabolically inactive enantiomer, facilitated performance of rats on a four-arm alternation task, but at very different doses (D-glucose, 250 mg/kg; L-glucose, 3,000 mg/kg). The enhanced performance seen with L-glucose, but not that seen with D-glucose, was attenuated by vagotomy. These findings suggest that the mechanisms by which these enantiomers act to enhance memory are quite different, with L-glucose acting via vagal afferents but D-glucose acting by other means, including direct modulation of central nervous system (CNS) processes by D-glucose.