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.     

Studies of the lymphoreticular system in the pathogenesis of scrapie: the role of spleen and thymus.

It has been established that a high proportion of extraneural replication of scrapie agent occurs in tissues associated with the lymphoreticular system, such as spleen. The question arises as to which components of the system are responsible for scrapie replication and whether such components can be identified by means of physiological manipulations. A series of experiments in mice showed that splenectomy deprived the host of a significant fraction of its capacity to permit ME7 scrapie agent to replicate, resulting in increases (10 to 30 per cent) of incubation period following intraperitoneal infection. This lost capacity remained for at least 60 days of age after neonatal splenectomy, although a smaller fraction of the total replicative capacity was vested in the spleen of the newborn than in that of mice aged 5 or more days; prolongation of incubation period in splenectomized newborn mice was over 9 per cent irrespective of the interval to injection.Experiments involving thymectomy were performed to discover whether the thymus-dependent portion of the lymphoid system was responsible for some of the spleen's ability to permit scrapie agent to replicate. The results failed to show any involvement of the thymus and its dependent lymphoid system in the replication and pathogenesis of scrapie in mice.     

Replication of herpes simplex virus type 1 and 2 in the medulla of the adrenal gland after vaginal infection of mice

Summary After vaginal infections of mice with neuroinvasive strains of herpes simplex virus type 1 and 2 (HSV-1, HSV-2) virus replicates in the epithelium of the vagina, in the paravaginal ganglia, in the spinal cord and finally in the brain and in the adrenal glands. However, viral antigens could be demonstrated only in the medulla of the adrenal glands but not in the cortex, as assessed by immunohistochemistry (IHC). HSV could not be isolated from liver, spleen, uterus, and ovaries. This contrasts to the intraperitoneal (i.p) route of infection with replication in different visceral organs including the adrenal gland's cortex.     

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.     

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.     

Neuro-invasion of Chandipura virus mediates pathogenesis in experimentally infected mice

Neuro-tropism is a major feature in many viral infections. Chandipura virus produces neurological symptoms in naturally infected young children and experimentally infected suckling mice. This study was undertaken to find out the neuro-invasive behaviour of Chandipura virus in suckling mice. The suckling mice were infected with the virus via footpad injection. Different tissues were collected at 24-h intervals up to 96-h post infection and processed for virus quantification and histological study. Further confirming the virus predilection to nerves tissues, the adult mice were inoculated with the virus via different routes. The suckling mice experimental results revealed a progressive replication of virus in spinal cord and brain. The progressive-virus replication was not observed in the other tissues like kidney, spleen, liver etc. Histo-pathological lesions noticed in the spinal cord and brain tissues suggested the extensive damages in these tissues. In adult mice experiment, the virus replication observed only in the brain of the mice infected via intra-cerebral route. From this study, we conclude that nervous tissues are predilection sites for Chandipura virus replication in suckling and adult mice. In suckling mice, virus might transmit through nervous tissues for dissemination. In contrast, the adult mice the nervous terminal might not pick up the virus through footpad infection. The pathogenesis in mice might be due to the virus replication mediated damage in the central nervous system.     

Early and late pathogenesis of natural scrapie infection in sheep

The pathogenesis of scrapie infection was studied in sheep carrying the PrP(VRQ)/PrP(VRQ) genotype, which is associated with a high susceptibility for natural scrapie. The sheep were killed at sequential time points during a scrapie infection covering both the early and late stages of scrapie pathogenesis. Various lymphoid and neural tissues were collected and immunohistochemically examined for the presence of the scrapie-associated prion protein PrP(Sc), a marker for scrapie infectivity The first stage of scrapie infection consisted of invasion of the palatine tonsil and Peyer's patches of the caudal jejunum and ileum, the so-called gut-associated lymphoid tissues (GALT). At the same time, PrP(Sc) was detected in the medial retropharyngeal lymph nodes draining the palatine tonsil and the mesenteric lymph nodes draining the jejunal and ileal Peyer's patches. From these initial sites of scrapie replication, the scrapie agent disseminated to other non-GALT-related lymphoid tissues. Neuroinvasion started in the enteric nervous system followed by retrograde spread of the scrapie agent via efferent parasympathetic and sympathetic nerve fibres innervating the gut, to the dorsal motor nucleus of the vagus in the medulla oblongata and the intermediolateral column of the thoracic spinal cord segments T8-T10, respectively.     

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.     

Pathogenesis of scrapie is faster when infection is intraspinal instead of intracerebral

Previous studies of mice infected peripherally with 139A scrapie showed that scrapie agent initially replicates outside the CNS and that invasion of the CNS occurs several weeks later by neural spread of infection along visceral autonomic fibres to the mid-thoracic cord, and thence to brain. Direct intracerebral infection of brain bypasses the need for extraneural replication and gives shorter incubation periods than peripheral routes. However, it was also found that the duration of the scrapie replication phase in brain, before clinical disease develops, is actually shorter with peripheral routes than with the intracerebral route. We have now investigated this surprising observation using the intraspinal route to reproduce just the neural phase of scrapie pathogenesis seen after peripheral infection. In studies of three strains of scrapie (263K, 139A and ME7) in either hamsters or mice, we have fulfilled the prediction that incubation periods should be shorter after intraspinal infection than after intracerebral infection. Detailed studies of 139A scrapie showed that the shorter incubation period by the intraspinal route could be accounted for by the shorter duration of the scrapie replication phase in brain before clinical disease developed. As a consequence, the severity of the vacuolar lesions in brain at the clinical stage of all three scrapie models was less after intraspinal infection than after intracerebral infection but the severities of vacuolation after intraspinal and intraperitoneal infection were remarkably similar. We speculate that (a) the site of injection (or of invasion) of the central nervous system determines which neural pathways become accessible for the spread of scrapie infection, and that (b) the duration of the neural phase of scrapie pathogenesis is related to the complexity of the pathways between the site of invasion and the clinical target areas in which, it is suggested, scrapie must replicate for disease to develop.     

Theileria sergenti infection in the Bo-RBC-SCID mouse model

We have previously developed the Bo-RBC-SCID mouse model forTheileria sergenti infection. In the present study, this model was further examined to delineate the mode of parasite infection. The Bo-RBC-SCID mice were prepared by periodically transfusing uninfected bovine erythrocytes (Bo-RBCs) into splenectomized SCID mice via the intraperitoneal (i.p.) route. The mice, separated into three groups, were inoculated i.p., intravenously (i.v.), or subcutaneously (s.c.) withT. sergenti-infected Bo-RBCs. Examination of samples of peripheral blood demonstrated that the parasite infected mice inoculated via any one of the three routes. The mice inoculated i.v., however, developed parasitemia earlier than those inoculated i.p. or s.c. When Bo-RBC-SCID mice prepared without splenectomy were infected withT. sergenti, a high-level parasitemia appeared only once. After that, not only the level of parasitemia but also the number of Bo-RBCs in the peripheral blood rapidly decreased despite the continuation of Bo-RBC transfusions. The results suggest thatT. sergenti proliferates primarily in the circulating blood in Bo-RBC-SCID mice and that in response to the parasites growth, the spleen may play an important role in the removal of both parasitized and unparasitized Bo-RBCs from the blood circulation.     

Adaptation of the agent of scrapie to hamsters

The agent of scrapie (Compton strain) can be transmitted from mice to hamsters; the incubation period of the disease is 5–6 months. Passage of the agent of scrapie through suspensions of brain tissue was repeated 10 times. The scrapie agent was found in the spinal cord and spleen but it could not be found in the liver, kidneys, adrenals, and lungs of the infected animals in the last stage of the disease.Division of General Epidemiology, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR O. V. Baroyan.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 2, pp. 199–201, February, 1976.     

A defective Th1 response of the spleen in the initial phase may explain why splenectomy helps prevent a Listeria infection

Listeria monocytogenes (Listeria) are known to grow and proliferate in the liver while a splenectomy induces host resistance against a Listeria infection despite the fact that a splenectomy inhibits the Th1 response. Therefore, the mechanism by which a splenectomy helps to prevent the growth of Listeria still remains to be elucidated. After an i.v. challenge of Listeria (1 x 10(6) CFU) in C57BL/6 mice, Listeria rapidly increased in the spleen but not in the liver until 48 h. However, after this initial phase, Listeria remarkably grew in the liver. In contrast, when the mice received a splenectomy beforehand, no remarkable growth of Listeria in the liver was observed after Listeria challenge despite the fact that serum IFN-gamma and IL-12 levels at 24 h after Listeria challenge were significantly lower than those in the sham mice. However, the liver leucocytes from mice by 6 h after infection produced a substantial amount of IFN-gamma while spleen MNC did not, whereas spleen leucocytes at 24 h after Listeria challenge did. Consistently, the IFN-gamma and IL-12 levels in the tissue homogenates of the spleen were significantly lower than in those of the liver until 6 h after infection. This defective spleen Th1 response in the early phase of Listeria infection was corrected by an IL-18 i.p. injection just after the Listeria challenge. Our findings suggest that Listeria exploit the defective Th1 environment of the spleen in the initial phase and afterwards overcome the host defense mechanism of the liver.     

Pattern of infection and selective loss of Ia positive cells in suckling and adult mice inoculated with lactic dehydrogenase virus

Summary Lactic dehydrogenase virus (LDV) infected cells were localised in cryostat sections of infected adult and suckling mice by fluorescent antibody staining. In almost every organ except brain and spinal cord, LDV infected cells were present in interstitial connective tissue, including dermis and submucosa of gastrointestinal and urinogenital tracts. They were also present in liver sinusoids and red pulp of spleen. The tissue distribution, shape, and fluorescence staining pattern of infected cells were similar in adult and suckling mice. The reactivity with monoclonal antibody to mouse macrophages (F4/80) and to Ia antigens indicated that infected cells were Ia antigen positive macrophages, and this was confirmed in double labelling experiments. Peak numbers of LDV infected cells were seen in every organ 12–18 hours post infection (p.i.), disappearing rapidly thereafter until at 48 hours p.i. they could no longer be detected. At the same time Ia positive cells fell to undetectable levels, although macrophages were still present in reduced numbers. At 7 days p.i. the total number of macrophages in sections had returned to normal, but the number of Ia positive cells remained at low levels.Macrophages recovered from peritoneal cavity and spleen of intraperitoneally infected mice were also studied. Ia positive cells had virtually disappeared from peritoneal cavity at 24–48 hours, and from spleen at 24–72 hours. Three weeks after infection the proportion of Ia positive cells was still low when compared with that of normal mice suggesting selective loss of these cells.With 4 Figures     

Selective vulnerability of neural cells and age-related susceptibility to OC43 virus in mice

Suckling CD 1 mice infected intracerebrally or extraneurally with OC43 virus developed a lethal neurotropic infection with high titres of virus in the brain. Examination of infected brain by routine H & E staining revealed no necrosis even in extensively infected tissue. Resistance to infection developed with increasing age, and by 20 days of age mice were completely insusceptible to i.c. inoculation. Virus replication was also demonstrable by FA staining, in spinal cord, dorsal root ganglia and retina. All other tissues were insusceptible and in particular, macrophages from both susceptible and resistant mice were found to be resistant to infection both in vivo and in vitro. Immunosuppression rendered 15 day old mice more susceptible to infection but adult mice remained insusceptible. The transfer of immune or non immune spleen cells from resistant mice did not confer resistance to newborn mice. Treatment of resistant mice with anti interferon globulin (AIG) did not render them more susceptible. These results indicate that the immune response is partially responsible for the development of resistance to OC43 infection but that it is only partially protective and other factors must also be required. The basis for the unique susceptibility of neural tissues in suckling mice is being investigated.     

Opposite Effects of BCG on Spleen and Lymph Node Cells: Lymphocyte Proliferation and Immunoglobulin Synthesis

C57BL/6 mice were immunized intravenously (i.v.), intraperitoneally (i.p.), or subcutaneously with one dose of Bacillus Calmette-Guérin (BCG). At various time intervals after injection, the lymphocyte response, as measured by thymidine incorporation into DNA, and the number of immunoglobulin-secreting cells were determined in vitro before and after mitogenic stimulation with phytohemagglutinin, concanavalin A, or lipopolysaccharide. In unstimulated cultures, the spontaneous thymidine incorporation and immunoglobulin synthesis of spleen cells were increased to some extent in mice infected i.p. or i.v. with BCG, as compared with noninfected mice. In contrast, after mitogenic stimulation, a marked depression of the proliferative response of spleen cells to both T- and B-cell mitogens and a marked inhibition of LPS-induced immunoglobulin secretion were observed in mice infected i.v. and to a lesser extent in those infected i.p. The depression of lymphoblastogenesis in spleens was fully established 15 days after infection and persisted for a long period of time. When unfractionated or plastic-adherent spleen cells from BCG-infected mice were cultured with normal spleen cells, a strong depression of their reactivity to phytohemagglutinin, concanavalin A, and lipopolysaccharide was observed. After the removal of cells adherent to plastic, the response was partially restored in the nonadherent population from mice infected i.p., but not in that from mice infected i.v. After mitogenic stimulation, lymph node cells of mice inoculated subcutaneously showed a response to mitogen higher than that of normal cells. These results thus demonstrate that, depending on the route of administration, BCG exerts very different effects.     

Persistent infection of mice by lactate dehydrogenase-elevating virus: transient virus replication in macrophages of the spleen

In order to assess whether the spleen is the major site of replication of lactate dehydrogenase-elevating virus (LDV) in mice during the acute phase of infection, LDV replication in the spleen was measured by electron microscopy and fluorescent antibody staining of tissue sections and northern hybridization of total spleen RNA with an LDV-specific cDNA probe, and the effect of splenectomy on LDV replication was determined. LDV RNA and antigens and infected cells, presumably macrophages, were present in the spleen in high concentrations 18-25 h post infection, but then rapidly disappeared to undetectable levels during the next 1-2 days. Thus, LDV clearly replicates in the spleen during the initial phase of infection, but LDV replication in the spleen is transient due to the cytocidal nature of LDV replication and destruction of all permissive macrophages in the spleen. Furthermore, spleen macrophages do not seem to represent the major source of LDV released into the circulation, since LDV viremia as well as anti-LDV antibody production were the same in splenectomized and control animals for at least 28 days postinfection.     

Injection of the sciatic nerve with TMEV: a new model for peripheral nerve demyelination

Demyelination of the human peripheral nervous system (PNS) can be caused by diverse mechanisms including viral infection. Despite association of several viruses with the development of peripheral demyelination, animal models of the condition have been limited to disease that is either autoimmune or genetic in origin. We describe here a model of PNS demyelination based on direct injection of sciatic nerves of mice with the cardiovirus, Theiler's murine encephalomyelitis virus (TMEV). Sciatic nerves of FVB mice develop inflammatory cell infiltration following TMEV injection. Schwann cells and macrophages are infected with TMEV. Viral replication is observed initially in the sciatic nerves and subsequently the spinal cord. Sciatic nerves are demyelinated by day 5 post-inoculation (p.i.). Injecting sciatic nerves of scid mice resulted in increased levels of virus recovered from the sciatic nerve and spinal cord relative to FVB mice. Demyelination also occurred in scid mice and by 12 days p.i., hindlimbs were paralyzed. This new model of virus-induced peripheral demyelination may be used to dissect processes involved in protection of the PNS from viral insult and to study the early phases of lesion development.     

Serum immunoglobulin concentrations before and after splenectomy in patients with homozygous beta-thalassaemia.

Serum IgM, IgA, and IgG concentrations were measured in 19 patients suffering from blood-dependent homozygous beta-thalassaemia. The study was prospective and the same patients served as presplenectomy controls. IgG levels rose rapidly and continuously after splenectomy IgA levels also rose but not so sharply. IgM fell to low levelr pattern was found in the cross-sectional part of the study comprising 39 splenectomised and 51 non-splenectomised patients who were tested once.     

Neurotropism of swine haemagglutinating encephalomyelitis virus (coronavirus) in mice depending upon host age and route of infection

Mice aged 1, 4 or 8 weeks were inoculated with haemagglutinating encephalomyelitis virus (HEV), strain 67N, by the intracerebral (i.c.), intranasal (i.n.), intraperitoneal (i.p.), subcutaneous (s.c.), intravenous (i.v.) or oral route, with different doses. In 1-week-old mice, mortality and mean time to death were mostly the same regardless of the inoculation route, except for the oral route, which appeared to be the least effective. The virus killed 4-week-old mice readily by all routes of inoculation except the oral, and 8-week-old mice by i.c., i.n. or s.c. inoculation. In descending order of efficacy, the routes of HEV infection were: i.c., i.n., s.c., i.p., i.v. and oral. To follow the spread of HEV from peripheral nerves to the central nervous system (CNS), the virus was inoculated subcutaneously into the right hind leg of 4-week-old mice. The virus was first detected in the spinal cord on day 2, and in the brain on day 3. The brain titres became higher than those of the spinal cord, reaching a maximum of 10(7)PFU/0.2 g when the animals were showing CNS signs. Viral antigen was first detected immunohistochemically in the lumbar spinal cord and the dorsal root ganglion ipsilateral to the inoculated leg; it was detected later in the pyramidal cells of the hippocampus and cerebral cortex, and in the Purkinje cells of the cerebellum but not in the ependymal cells, choroid plexus cells or other glial cells. The infected neurons showed no cytopathological changes.     

Disruption of the gene encoding the gammaHV68 v-GPCR leads to decreased efficiency of reactivation from latency

Murine gammaherpesvirus 68 (gammaHV68; MHV68) infection of mice has been a useful model for characterizing the role of conserved herpesvirus genes in pathogenesis. One of the well conserved genes among gamma2-herpesvirus, gene 74, encodes a viral G-protein coupled receptor (v-GPCR). To examine the role of the gammaHV68 v-GPCR in pathogenesis we have generated a mutant virus in which 440 base pairs of the gene 74 open reading frame have been deleted (gammaHV68v-GPCRDelta440). This deletion did not affect the growth of the virus in single or multiple rounds of replication in vitro, nor acute replication in vivo as assessed by plaque assay of spleens and lungs on days 4, 7 and 9 post-infection (p.i.). The ability of the v-GPCR mutant virus to establish latency and to reactivate from latency was quantitated on days 16 and 42 p.i. While there was no detectable difference in the ability of the mutant virus to either establish latency or reactivate from latency on day 16 p.i., as compared to wild-type gammaHV68 and marker rescue virus, there was a significant decrease in the efficiency of virus reactivation by day 42 p.i. Notably, mice infected with the mutant virus lacking the v-GPCR contained a higher frequency of viral genome positive cells in the peritoneum by day 42 p.i. than mice infected with either wild type or marker rescue virus. However, analysis of virus reactivation demonstrated that approximately the same frequency of cells reactivated virus from mice infected with either the gammaHV68 v-GPCR mutant, wild-type virus, or marker rescue virus. From these experiments we conclude that the gammaHV68 v-GPCR is dispensable for acute virus replication in vivo, but does play a role in reactivation from latency.