Quantitative determination of the dissemination of Flury rabies virus in the central nervous system of the guinea-pig after intramuscular inoculation in the hind leg

Summary A quantitative determination was made of the virus content of the different portions of the central nervous system of guinea-pigs, inoculated with the Flury strain of rabies virus in the muscles of the hind leg. The titration was carried out by intracranial inoculation into mice with material obtained from guinea-pigs killed at various intervals during the incubation period and during the clinical disease and also from guinea-pigs which had succumbed to the infection. The results demonstrate that the virus first multiplies in the lumbosacral cord and afterwards spreads in the direction of the brain. The virus concentration however, remains at any time higher in the lumbosacral cord, being the first to be invaded; this is found during the incubution period, as well as during the paralytic stage and after death.     

Immune response to a recombinant capsid protein of striped jack nervous necrosis virus (SJNNV) in turbot Scophthalmus maximus and Atlantic halibut Hippoglossus hippoglossus, and evaluation of a vaccine against SJNNV.

Immunisation by intraperitoneal injection of an oil-emulgated recombinant partial capsid protein (rT2) from striped jack nervous necrosis virus (SJNNV) was performed on adult turbot Scophthalmus maximus and Atlantic halibut Hippoglossus hippoglossus. A specific humoral immune response was recorded in both species, and the levels of rT2-specific antibodies increased markedly in all groups during the 20 wk experiment. A challenge model for SJNNV was established by intramuscular injection of juvenile turbot. The turbot developed viral encephalopathy and retinopathy (VER), also known as viral nervous necrosis (VNN), with cumulative mortality in the range of 25 to 66%, after intramuscular inoculation with SJNNV propagated in the striped snake head cell line (SSN-1). Although neither clinical signs nor mortality were registered, SJNNV was neuroinvasive after bath exposure. The infection after both modes of challenge was verified by means of immunohistochemistry and RT-PCR, and SJNNV was reisolated in cell culture. The results indicate that SJNNV may have entered the central nervous system (CNS) by axonal transport through motor nerves after intramuscular inoculation. A vaccine efficacy test was performed on juvenile turbot, employing oil emulsified rT2 as a test vaccine and intramuscular inoculation of SJNNV. Significant protection was observed when the challenge was performed 10 wk post-vaccination.     

Susceptibility of chickens to avian encephalomyelitis virus. IV. Behavior of the virus in laying hens.

Some laying hens 6 months of age were inoculated subcutaneously or orally with a chick embryo--adapted strain of avian encephalomyelitis virus and examined for propagation of the virus in the body. When inoculated subcutaneously, the virus appeared in liver, spleen, ovarian follicle, and muscle at the site of inoculation 1 day, in kidney and lumbar part of the spinal cord 3 days, in the pancreas 5 days, in heart, duodenum, and cervical part of the spinal cord 7 days, and in the brain 11 days after inoculation. After its appearance, it increased gradually in amount in liver, spleen, pancreas, muscle at the site of inoculation, and cervical and lumbar parts of the spinal cord, but remained at a low level in any other organ. When examined 14 days after inoculation and later, it was distributed mainly in the central nervous system. It was detected from 12 of 16 organs examined. The highest virus level in each organ was 10(2.6)/0.1 g in pancreas and lumbar part of the spinal cord, which were followed by muscle at the site of inoculation (10(2.0)/0.1 g), spleen (10(1.8)/0.1 g), cervical part of the spinal cord, heart, and liver in the order listed. When inoculated orally, the virus was found sporadically in spleen, pancreas, kidney, cecum, ovarian follicle, and lumbar part of the spinal cord. The virus level was low in these organs, of which pancreas, kidney, and lumbar part of the spinal cord showed the highest virus level, or 10(1.3)/0.1 g.     

Susceptibility of chickens to avian encephalomyelitis virus. III. Behavior of the virus in growing chicks.

A chick embryo-adapted strain of avian encephalomyelitis virus was inoculated subcutaneously and orally into 40-day-old (middle-aged) and 110-day-old (advanced-aged) chicks to examine the behavior of the virus in the chick body. In the middle-aged chicks, the virus appeared in the muscle at the site of inoculation, liver, spleen, pancreas, lumbar and cervical portions of the spinal cord, and brain 1 approximately 9 days after subcutaneous inoculation, and remained mostly in the central nervous system up to 17 days after the inoculation. The virus was found in large amounts in the muscle at the site of inoculation (10(3.1)), lumbar portion (10(2.5)) and cervical portion (10(2.1)) of the spinal cord, brain (10(1.9)), and in minute amounts in the other organs examined. It appeared in 11 of 21 organs examined. In the middle-aged chicks inoculated by the oral route, the virus was detected transiently in small amounts from esophagus, pancreas, and rectum 4 approximately 14 days after inoculation. In the advanced-aged chicks inoculated by the subcutaneous route, the virus was detected in titer of 10(2.1) approximately 10(3.0) from the muscle at the site of inoculation 2 approximately 7 days after inoculation. The virus was also found sporadically in several organs up to 17 days after inoculation. In the advanced-aged chicks inoculated by the oral route, no virus appeared in any organ, but these chicks turned to be weakly positive for neutralizing antibody in the 4th or later week after inoculation.     

Pathogenesis of mouse hepatitis virus infection. The role of nasal epithelial cells as a primary target of low-virulence virus, MHV-S.

The pathogenesis of mouse hepatitis virus (MHV-S) infection in suckling and weanling mice was comparatively studied after intranasal inoculation. In sucklings, infectious virus as well as specific antigen was first detected in the nasal mucosa at 12 hr, then in the nerve cells of the olfactory bulbs. At this stage viral particles were demonstrated both in the supporting cells and olfactory cells of the nasal mucosa. In the posterior part of the brain and spinal cord, virus was detected on days 3 to 4 postinoculation when viral growth was clearly demonstrable in the liver, spleen and intestines. In weanlings too, infection was first established in the nasal mucosa, shedding infectious virus in the nasal washing until day 6 postinoculation, and later infection spread to the brain and spinal cord. In weanling mice, however, neither infectious virus nor viral antigen was detected in the liver or other visceral organs, while serum neutralizing antibody became detectable on day 5 postinoculation, increasing in titer thereafter. Histopathologically degenerative and necrotic changes were observed in the nasal mucosa and central nervous system of both age groups of animals coincidentally with the presence of viral specific antigen, while inflammatory response was much less prominent in sucklings. In the liver, spleen and intestines, however, some lesions were observed only in sucklings.     

Extensive Attenuation of Rabies Virus by Simultaneously Modifying the Dynein Light Chain Binding Site in the P Protein and Replacing Arg333 in the G Protein

Rabies virus (RV) is a highly neurotropic virus that migrates from the portal of entry to the central nervous system (CNS). The cytoplasmic dynein light chain (LC8), which is involved in a variety of intracellular motile events, was shown to interact with RV phosphoprotein (P). In order to determine the functional significance of this interaction, P residues 143 to 149 or 139 to 149 encompassing a conserved LC8-interacting motif (K/RXTQT) were deleted from recombinant viruses SAD-L16 and SAD-D29. These viruses are identical except for a replacement of the arginine at position 333 (R333) of the RV glycoprotein by an aspartic acid in SAD-D29. SAD-L16 virus is fully pathogenic for mice, whereas SAD-D29 is nonpathogenic for adult mice but retained pathogenicity for suckling mice. The deletions introduced into the LC8 binding site abolished the P-LC8 interaction and blocked LC8 incorporation into virions. All the mutants propagated in cell culture as efficiently as the parent strains. The pathogenicity of the mutants was then compared with that of the parent viruses by inoculating suckling mice. SAD-L16 derivatives were as pathogenic as their parent virus after intramuscular inoculation, indicating that LC8 is dispensable for the spread of a pathogenic RV from a peripheral site to the CNS. In contrast, SAD-D29-derived deletion mutants were attenuated by as much as 30-fold after intramuscular inoculation but remained as pathogenic as the parent virus when inoculated directly into the brain. This remarkable attenuation after intramuscular but not after intracranial inoculation suggested that abolishing the P-LC8 interaction reduces the efficiency of peripheral spread of the more attenuated SAD-D29 strain. These results demonstrate that elimination of the LC8 ligand and simultaneous substitution of R333 considerably attenuate RV pathogenicity and may be helpful in designing and developing highly safe live-RV-based vaccines.     

Susceptibility of chickens to avian encephalomyelitis virus. V. Behavior of a field strain in laying hens.

Some 12-month-old laying hens were inoculated orally or subcutaneously with 10(4.7) EID50 of a field strain of avian encephalomyelitis virus. They were examined for propagation of the virus in the body at regular intervals of time. When two hens were sacrificed daily in the group of oral inoculation, the virus was found in liver, pancreas, and esophagus in both hens 1 day, in brain, lumbar part of the spinal cord, heart, spleen, pharynx, larynx, glandular stomach, muscle, and blood in one of the two hens 1 day, and in various parts of the body 3 approximately 9 days after inoculation. After that, the virus was detected almost continually from the central nervous system and abdominal parenchymatous organs in nearly all the hens examined up to the end of the observation period, or 21 days after inoculation. Virus detection from the digestive tract and ovarian follicle, however, decreased in frequency and virus titer was reduced remarkably with the lapse of time after inoculation. When the largest amount of virus was determined in each organ, it was the largest, or 10(6.5) EID50/0.1 g, in the liver and about 10(5.0) EID50/0.1 g in spleen, pancreas, kidney, and ovarian follicle. There was little difference in virus propagation and its course between the group of subcutaneous inoculation and that of oral administration.     

Prophylactic and therapeutic treatment with acyclovir of genital herpes in the guinea pig

The antiviral drug, acyclovir, was tested on experimentally infected guinea pigs with either of two herpes simplex virus type 1 (HSV-1) isolates following intravaginal inoculation. The drug was continuously infused subcutaneously utilizing an osmotic pump. Infusion was begun either prior to virus inoculation (prophylactic) or after virus inoculation at the time of first appearance of lesions (therapeutic). Prophylactic treatment markedly reduced the severity of the genital lesions, the appearance of acute neurologic sequellae, and the virus excretion in the genital tract of guinea pigs infected with either of the two strains tested. Therapeutic acyclovir treatment, however, did not decrease the incidence of acute neurologic sequellae with one of the two HSV-1 strains tested, nor did it reduce the severity of the genital lesions of either strain. These neurologic sequellae may be due to insufficient levels of ACV in the central nervous system as the concentration of ACV in the dorsal root ganglia was found to exceed that of the plasma, but only trace amounts of acyclovir were present in the brain and spinal cord. Continuous perfusion of ACV gave far higher tissue levels than intermittent injections. These findings suggest that prophylactic ACV is far more effective than therapeutic treatment for genital herpes in the guinea pig model.     

Involvement of the Rabies Virus Phosphoprotein Gene in Neuroinvasiveness

Rabies virus (RABV), which is transmitted via a bite wound caused by a rabid animal, infects peripheral nerves and then spreads to the central nervous system (CNS) before causing severe neurological symptoms and death in the infected individual. Despite the importance of this ability of the virus to spread from a peripheral site to the CNS (neuroinvasiveness) in the pathogenesis of rabies, little is known about the mechanism underlying the neuroinvasiveness of RABV. In this study, to obtain insights into the mechanism, we conducted comparative analysis of two fixed RABV strains, Nishigahara and the derivative strain Ni-CE, which cause lethal and asymptomatic infections, respectively, in mice after intramuscular inoculation. Examination of a series of chimeric viruses harboring the respective genes from Nishigahara in the genetic background of Ni-CE revealed that the Nishigahara phosphoprotein (P) gene plays a major role in the neuroinvasiveness by mediating infection of peripheral nerves. The results obtained from both in vivo and in vitro experiments strongly suggested that the Nishigahara P gene, but not the Ni-CE P gene, is important for stable viral replication in muscle cells. Further investigation based on the previous finding that RABV phosphoprotein counteracts the host interferon (IFN) system demonstrated that the Nishigahara P gene, but not the Ni-CE P gene, functions to suppress expression of the beta interferon (IFN-β) gene (Ifn-β) and IFN-stimulated genes in muscle cells. In conclusion, we provide the first data strongly suggesting that RABV phosphoprotein assists viral replication in muscle cells by counteracting the host IFN system and, consequently, enhances infection of peripheral nerves.     

Transneuronal labeling of neurons in the adult rat central nervous system following inoculation of pseudorabies virus into the colon

Transneuronal tracing with pseudorabies virus (PRV) was used to identify sites in the central nervous system involved in the neural control of colon function. PRV-immunoreactive (IR) cells were primarily localized to the caudal lumbosacral (L6-S1) and caudal thoracic-rostral lumbar (T13-L1) spinal segments with the distribution varying according to survival time (72-96 h). In the lumbosacral spinal cord at all time points examined, significantly (PА.005) greater numbers of PRV-IR cells were present in the region of the sacral parasympathetic nucleus (SPN) of the S1 spinal segment compared to that of the L6 segment. These studies also revealed morphologically distinct cell types with a differential distribution (probably interneurons and preganglionic parasympathetic neurons) in the region of the SPN in the L6-S1 spinal segments following colon inoculation. PRV-labeled neurons were located at various levels of the neuraxis and at many sites had a distribution similar to that following injection of virus to other urogenital organs. However, some unique sites in the dorsal motor nucleus of the vagus, nucleus of the solitary tract, nucleus ambiguus and area postrema were also identified. To determine if labeling in these caudal medullary sites was mediated by spinal or vagal pathways, the colon was inoculated with PRV in animals with a complete spinal cord (T8) transection (5-7 days prior). Following spinal transection, PRV-infected cells were detected in the same caudal medullary regions; however, labeling in other regions (e.g., Barrington's nucleus) was eliminated or significantly reduced. These studies have yielded several novel observations concerning the central neural control of colonic function: (1) the preganglionic efferent and primary afferent innervation of the colon arises primarily from the S1 spinal segment; (2) the distribution of PRV-infected neurons in the central nervous system following colon inoculation was similar to that following PRV inoculation of other urogenital organs; (3) Barrington's nucleus, which has been identified previously as the pontine micturition center, may have a role in colonic function; and (4) PRV infection in Barrington's nucleus following colon inoculation is mediated by bulbospinal pathways whereas labeling in caudal medullary regions is mediated, at least in part, by vagal pathways.     

Attenuation of neurovirulence, biodistribution, and shedding of a poliovirus:rhinovirus chimera after intrathalamic inoculation in Macaca fascicularis

A dependence of poliovirus on an unorthodox translation initiation mode can be targeted selectively to drive viral protein synthesis and cytotoxicity in malignant cells. Transformed cells are naturally susceptible to poliovirus, due to widespread ectopic upregulation of the poliovirus receptor, Necl-5, in ectodermal/neuroectodermal cancers. Viral tumor cell killing and the host immunologic response it engenders produce potent, lasting antineoplastic effects in animal tumor models. Clinical application of this principle depends on unequivocal demonstration of safety in primate models for paralytic poliomyelitis. We conducted extensive dose-range-finding, toxicity, biodistribution, shedding, and neutralizing antibody studies of the prototype oncolytic poliovirus recombinant, PVS-RIPO, after intrathalamic inoculation in Macaca fascicularis. These studies suggest that intracerebral PVS-RIPO inoculation does not lead to viral propagation in the central nervous system (CNS), does not cause histopathological CNS lesions or neurological symptoms that can be attributed to the virus, is not associated with extraneural virus dissemination or replication and does not induce shedding of virus with stool. Intrathalamic PVS-RIPO inoculation induced neutralizing antibody responses against poliovirus serotype 1 in all animals studied.     

Axonal and transsynaptic (transneuronal) spread of Herpesvirus simiae (B virus) in experimentally infected mice.

In order to study the pathogenesis of B virus infection of the nervous system, newborn and young mice were inoculated by four different routes: 1. Intramuscular (i.m.) in the forelimb; 2. I.m. in the hindlimb; 3. Subcutaneous (s.c.) in the abdominal wall; 4. Intraperitoneal (i.p.). Spread of virus was followed by immunohistochemical demonstration of viral antigen in tissue sections of the peripheral and central nervous system. Three distinct patterns emerged: 1. After i.m. limb inoculations, virus progressed along the ipsilateral dorsal column, the bilateral spinothalamic and bilateral spinoreticular systems and along central autonomic pathways. 2. After s.c. inoculation, the dorsal column was spared, otherwise the spread was similar to that following i.m. inoculations. 3. After i.p. inoculation, virus spread in the spinal cord bilaterally, mainly along spinothalamic and central autonomic pathways. The peripheral motoneurons were conspicuously spared, even in the i.m. inoculation mode. In the brain stem, B virus antigen appeared bilaterally, at multiple sites. In the cerebrum, virus infected cells appeared first in the thalamus, hypothalamus and the motor cortex. The mode of spread from spinal levels was mainly orthograde along the ascending systems (dorsal columns, spinothalamic, spinoreticular tracts), but also retrograde along descending systems (pyramidal tract, central autonomic pathways). Oligosynaptic systems transmitted virus more quickly than the polysynaptic ones. In the involvement of various neuronal systems in virus spread, a certain selectivity, sparing the peripheral motoneuron and the cerebellar systems, could be assessed.     

Persistence of mouse hepatitis virus A59 RNA in a slow virus demyelinating infection in mice as detected by in situ hybridization.

Mouse hepatitis virus strain A59 produces chronic central nervous system demyelination in rodents. As late as 6 months after intracerebral inoculation of mice 4 to 6 weeks old, when infectious virus cannot be recovered and viral antigens cannot be detected in the central nervous systems and livers of these animals, primary demyelination is still evident. Using cloned virus-specific DNAs and the highly sensitive and specific technique of in situ hybridization, we have detected low levels of mouse hepatitis virus A59 RNA in the central nervous systems and livers of mice 10 months after inoculation. We suggest that viral persistence may play a role in mouse hepatitis virus A59-induced chronic demyelination.     

Invasion of the central nervous system in a porcine host by nipah virus

Nipah virus, a newly emerged zoonotic paramyxovirus, infects a number of species. Human infections were linked to direct contact with pigs, specifically with their body fluids. Clinical signs in human cases indicated primarily involvement of the central nervous system, while in pigs the respiratory system was considered the primary virus target, with only rare involvement of the central nervous system. Eleven 5-week-old piglets were infected intranasally, orally, and ocularly with 2.5 x 10(5) PFU of Nipah virus per animal and euthanized between 3 and 8 days postinoculation. Nipah virus caused neurological signs in two out of eleven inoculated pigs. The rest of the pigs remained clinically healthy. Virus was detected in the respiratory system (turbinates, nasopharynx, trachea, bronchus, and lung in titers up to 10(5.3) PFU/g) and in the lymphoreticular system (endothelial cells of blood and lymphatic vessels, submandibular and bronchiolar lymph nodes, tonsil, and spleen with titers up to 10(6) PFU/g). Virus presence was confirmed in the nervous system of both sick and apparently healthy animals (cranial nerves, trigeminal ganglion, brain, and cerebrospinal fluid, with titers up to 10(7.7) PFU/g of tissue). Nipah virus distribution was confirmed by immunohistochemistry. The study presents novel findings indicating that Nipah virus invaded the central nervous system of the porcine host via cranial nerves as well as by crossing the blood-brain barrier after initial virus replication in the upper respiratory tract.     

Inhibition of murine retrovirus-induced neurodegeneration in the spinal cord by explant culture.

The neurovirulent chimeric mouse ecotropic retrovirus FrCasE causes a rapid neurodegenerative disease of the central nervous system (CNS) characterized by the appearance of spongiform lesions in motor areas 10 days after neonatal inoculation. To study the details of the pathogenic process, we examined the ability of an ex vivo spinal cord model to recapitulate disease. Organotypic spinal cord slice cultures were established from IRW mice 7 days after neonatal inoculation. This corresponds to a time when virus expression in the CNS is first detectable but spongiform changes have yet to evolve. Infectivity associated with these cultures peaked at 7 days in vitro and persisted at this level for 6 weeks. FrCasE infection of the spinal cord slices was primarily found associated with microglial cells. Infection of neurons, astrocytes, oligodendroglia, and endothelial cells was not observed; however, significant astrogliosis was found. Despite the presence of extensive microglial infection in close association with spinal motor neurons in organotypic cultures, no virus-specific spongiform degenerative changes were observed. These results suggest that removal of motor neurons from the developing CNS, despite maintaining the local cytoarchitectural relationships, prevents the virus from eliciting its pathological effects. Possible reasons for the interruption of lesion development are discussed.     

Early adrenal infection by herpes simplex virus type-1 (Miyama + GC strain): special reference to inoculation dose and spread from the adrenal to the central nervous system

Male C3H/HeN mice, aged 5 weeks, were inoculated intraperitoneally (i.p.) with different doses (1 x 10(3), 1 x 10(5), 5 x 10(5), 1 x 10(6) pfu) of the herpes simplex virus type-1 (HSV-1) (Miyama + GC strain). The LD50 of this virus was 10(2) pfu (i.p.) per mouse. All the mice in each group died 12 days after inoculation. Adrenal necrosis was found to be dose-dependent, the threshold dose being 5 x 10(5) pfu. In addition, encephalitis and inflammatory cell infiltration in abdominal ganglia appeared in 3-4 days after inoculation. By the plaque method, HSV-1 was detected first in the adrenal glands, then in neurons in the spinal cord and the brain. These findings suggest that in mice inoculated with doses of virus sufficient to infect the adrenal gland, HSV-1 spreads to the central nervous system through peripheral nerves after replication in the adrenal.     

Limited in vivo susceptibility of the South American monkey -Cebus apella- to type 1 poliovirus: physical and histopathological observations after intraspinal inoculation

Type 1 polioviruses (an attenuated strain, Sabin 1 LSc 2ab and a virulent strain, Mahoney) were inoculated intraspinally into the South American Cebus monkey Cebus apella. Neither physical symptoms nor histological changes in the central nervous system were observed after inoculation of attenuated Sabin strain. But the virulent Mahoney strain caused flaccid paralysis in two of three monkeys. In these two paralyzed monkeys, definite specific histological changes were observed with spreading of the lesions to places far from the inoculation site, i.e., the cervical cord and brain. These results suggest that Cebus apella has limited susceptibility to type 1 poliovirus.     

Viral Properties of Scrapie

ALTHOUGH scrapie agent has many unusual features^1–4, some aspects of its behaviour are analogous to viral properties⁵. The manner in which the agent invades the nervous system after peripheral inoculation immediately recalls such typically neurotropic viruses as poliomyelitis or rabies. After inoculation into the fore-limb changes occur first in the cervical cord; following injection into the leg they appear first in the lumbar cord⁶. We now report another way in which scrapie behaves as a classical virus.