Mode of Subacute Sclerosing Panencephalitis (SSPE) Virus Infection in Tissue Culture Cells: III. Neurovirulence of Cell-Free SSPE Viruses of Niigata-1, Kitaken-1, and Biken Strains

Cell-free viruses recovered from virus-carrying cultures of the Niigata-1, Kitaken-1, and Biken strains of SSPE virus were examined for neurovirulence. The cell-free viruses were prepared by freezing and thawing or by EDTA treatment of the virus-carrying cultures and inoculated into adult mice intracerebrally. A considerable number of the inoculated mice showed clinical signs about 1 to 5 weeks after the inoculation. The first symptom was hyperreactivity, which was followed by paresis and myoclonus. All of the affected mice fell in paralysis and finally died. The virus could be recovered from the moribund mice by cocultivation of the brain cells with Vero cells. Immunofluorescence staining of the brain tissue revealed that infected cells containing viral antigens were distributed sparsely. No inflammatory feature, however, was observed in the brain as far as examined and neutralizing antibody against SSPE virus was not detected in sera from the mice inoculated with the cell-free SSPE viruses.     

Encephalomyelitis in mice experimentally infected with Akabane virus.

Lesions in the central nervous system of mice, induced by intracerebral injection of Akabane virus, were observed by the fluorescent antibody technique and histological method. Fluorescent antigens were recognized in the cytoplasm of nerve cells, but were not detected exactly in any other part. Fluoresced nerve cells were distributed almost all over the central nervous system, especially in medulla oblongata and spinal cord. The appearance of fluorescent antigens was followed by histological changes. So-called Nissl's acute severe degeneration was observed in nerve cells in the area where the fluorescent antigens were distributed. Spongy foci were seen in medulla oblongata and spinal cord. Virus was recovered from brain and spinal cord, but not from any other visceral organ or blood. Akabane virus showed an affinity to nerve cells and caused primary nonpurulent encephalomyelitis when inoculated intracerebrally to mice.     

Effect of (E)-5-(2-bromovinyl)-2'-deoxyuridine on the growth and viral capsid antigen expression of Epstein-Barr virus-associated tumor (B-95-8) cells transplanted to nude mice

When persistently Epstein-Barr virus (EBV)-infected lymphoblastoid (B-95-8) cells were transplanted subcutaneously or intracerebrally to nude mice of either BALB/c or NIH background, tumors developed, and the tumor cells spontaneously expressed viral capsid antigen (VCA). This model was used to evaluate the in vivo anti-EBV activity of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), a highly potent and selective antiherpes agent, which was recently shown to inhibit several parameters of EBV infection in vitro. When administered intraperitoneally at 200 mg/kg/day for 4 weeks, or 500 mg/kg/day for 2 weeks, starting immediately after B-95-8 cell inoculation, BVDU effectively reduced tumor growth and VCA expression of either subcutaneously or intracerebrally inoculated B-95-8 cells.     

Dobrava, but not Saaremaa, hantavirus is lethal and induces nitric oxide production in suckling mice

Hantaviruses are the causative agents of HFRS and HCPS (hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome), two severe, and often fatal human diseases. Mortality from HFRS varies between hantaviruses; Hantaan and Dobrava show the highest, Seoul intermediate, and Puumala low mortality. Saaremaa, genetically closely related to Dobrava, is also known to induce HFRS, with low or no mortality. In this study, mice were inoculated with Dobrava and Saaremaa viruses to test for infectibility, lethality, viremia, nitric oxide production and antibody responses. Out of suckling mice intracerebrally inoculated with 50, 500 and 5,000 focus-forming units of Dobrava virus, respectively, 1/8, 2/8 and 7/8 died within 18-26 days. In all but one of the lethally infected mice high levels of replicating virus were detected, and most were positive for neutralizing antibodies and showed elevated levels of nitric oxide production. All suckling mice intracerebrally inoculated with 50, 500, or 5,000 focus-forming units of Saaremaa virus survived and all seroconverted. Clearly lower viral titers were observed for the Saaremaa virus-inoculated mice, also when sacrificed at day 18 after infection, compared to those in mice that died following Dobrava virus infection. Dobrava, Saaremaa, Puumala and Hantaan virus infections of adult mice were asymptomatic, and the anti-nucleocapsid protein IgG2a/IgG1-titer ratio was higher in mice inoculated with Dobrava virus than in those inoculated with Saaremaa virus. Elevated nitric oxide production was not detected in asymptomatically infected mice, and iNOS-/- mice, like normal mice, cleared viremia. In conclusion, we show that Dobrava virus and Saaremaa virus induce distinct differences in terms of survival, viremia, nitric oxide production and antibody responses in mice.     

Sindbis virus mutations which coordinately affect glycoprotein processing, penetration, and virulence in mice

Rapid penetration of baby hamster kidney cells was used as a selective pressure for the isolation of pathogenesis mutants of the S.A.AR86 strain of Sindbis virus. Unlike most Sindbis virus strains, S.A.AR86 is virulent in adult as well as neonatal mice. Two classes of mutants were defined. One class was attenuated in adult mice inoculated intracerebrally as well as in neonatal mice inoculated either intracerebrally or subcutaneously. Sequence analysis of the glycoprotein genes of the parent virus and three such mutant strains revealed a single point mutation which resulted in an amino acid change at position 1 in the E2 glycoprotein. The change from a serine in S.A.AR86 to an asparagine in the mutants created a new site for N-linked glycosylation which appeared to be utilized. This mutation did not retard release of infectious particles; however, mutant virions contained the E2 precursor protein (PE2) rather than the E2 glycoprotein itself. The mutants also lost the ability to bind two E2-specific monoclonal antibodies, R6 and R13. A second class of mutants was attenuated in neonatal mice upon subcutaneous inoculation but remained virulent in adults and in neonates when inoculated intracerebrally. Sequence analysis of three such strains revealed the substitution of an arginine residue for a serine at position 114 in the E2 glycoprotein. Reactivity with monoclonal antibodies R6 and R13 was reduced, yet members of this mutant class were more susceptible than S.A.AR86 to neutralization by these antibodies.     

Wild mouse ecotropic murine leukemia virus infection of inbred mice: dual-tropic virus expression precedes the onset of paralysis and lymphoma.

NFS/N mice inoculated at birth with an ecotropic murine leukemia virus (Cas-Br-MuLV) obtained from wild mice developed hind limb paralysis beginning at 7 weeks of age and nonthymic lymphomas beginning at more than 20 weeks of age. Studies of 1- to 7-week-old Cas-Br-M MuLV-infected mice showed the following: (i) a marked increase in nonecotropic MuLV-related antigens on spleen cells but not thymocytes beginning at 2 weeks; (ii) the appearance of dual-tropic mink cell focus-forming (MCF) MuLV-related gp70 in spleen but not thymus or brain cells at 4 weeks; and (iii) the isolation of infectious MCF MuLV from spleen cells of 7-week-old mice. A role for MCF MuLV in Cas-Br-M MuLV-induced nonthymic lymphomas is indicated by these studies, and a role for recombinant MuLV in neurological disease is considered.     

Alterations of acetylcholine enzymes in neuroblastoma cells persistently infected with lymphocytic choriomeningitis virus.

Persistent infection of murine neuroblastoma cells with a relatively non-cytopathic virus, lymphocytic choriomeningitis virus (LCMV), significantly lowered the cells' concentrations of choline acetyl transferase (CAT) and acetylcholine esterase (ACHE), enzymes which make or degrade acetylcholine. Quantities of acetylcholine enzymes remained depressed during the observation period of more than two years. This cellular luxury function was turned off without observable alterations in the cells' vital functions – growth rates, protein and RNA synthesis. Cloning experiments showed that CAT and ACHE levels were altered in the majority of LCMV infected neuroblastoma cells in culture and not limited to a specific subpopulation. Cells persistently infected with virus also contained receptors for neurotoxin A and α bungarotoxin. Six months after becoming infected, neuroblastoma cells having significant alterations in luxury functions stopped making infectious virus. Instead these cells now produced a defective interfering virus component. Similar events to those seen in vitro with neuroblastoma cells also occurred in vivo. Mice inoculated with LCMV at birth carried high titers of LCMV in brain tissues and viral antigens in neuronal cells as adults. Some of these mice also showed significant alterations in their ability to make and degrade acetylcholine when compared to age and sex matched controls.     

Herpes simplex virus type 1-induced hydrocephalus in mice.

Adult ICR/Slc or BALB/c mice developed hydrocephalus when attenuated herpes simplex virus type 1 (HSV-1) (strain Ska) was injected intracerebrally 2 to 4 weeks earlier and then after mice were challenged with the same virus or virulent HSV-1. Initial inoculation of the Ska strain elicited acute meningitis and ependymitis with transient mild hydrocephalus. Viral antigen was seen in the meninges and subependymal areas, and the virus was titrated during the acute phase of infection. After the second virus inoculation, more prominent inflammation was evoked in the same area, and the animals developed hydrocephalus, although viral antigen and infectious virus were hardly detected. When the mice were immunosuppressed with cyclophosphamide, they ceased to develop hydrocephalus. BALB/c nude mice did not show the same pathology, even though they were treated in the same way. When irradiated mice, which had been infected with the Ska strain intracerebrally 2 weeks earlier, received syngeneic immune spleen cells, they developed hydrocephalus. The T-cell nature of the effector cells was confirmed by the elimination of the pathology after treatment of the donor cells with anti-Thy-1.2 plus complement. No hydrocephalic mice were observed after treatment of the donor cells with anti-Lyt-1.2 plus complement, which gave further evidence of the T-cell nature of the effector cells as the Lyt-1+.2+ antigen-bearing subsets. Intervals between priming and challenge virus inoculation could be more than 18 months. The presence of purified HSV-1 envelope protein was feasible for the development of the hydrocephalic animals.     

Experimental Maedi Infection in Sheep

Eight sheep were inoculated with Icelandic maedi strain M 88; 2 sheep served as control sheep and were in close contact with the inoculated ones. Four of the sheep were inoculated via the respiratory tract with 7×106 TGID50 of strain M88 and the other 4 intracerebrally with 5×105 TGID50 of the same strain. Maedi M88 strain was isolated from peripheral blood leukocytes of all inoculated sheep. There was a striking difference between the 2 groups in the appearance of demonstrable viremia after inoculation. Viremia could be demonstrated in the intrapulmonarily inoculated sheep within 2–6 months but not until 8–11 months after inoculation in the intracerebrally inoculated ones. This finding is thought most probably to reflect a weak neurotropism of the strain used. After the first demonstration of viremia, maedi virus has been recovered quite reqularly in peripheral leukocytes of all intrapulmonarily inoculated sheep, but less regularly in the intracerebrally inoculated ones. Maedi virus was isolated from 1 of the uninoculated control sheep 15 months after inoculation. The first clinical case with a clinical appearance suggesting combined involvement of maedi and visna was found among the intrapulmonarily inoculated sheep, 8% months after inoculation. Histopathological examination and virus isolation confirmed maedi. The cause of paraplegia could not be confirmed. No histopathological changes were found and no virus isolation was made from the central nervous system of this animal. One of the intracerebrally inoculated sheep died suddenly without any observed clinical signs 11 months after inoculation. Histopathological examination revealed pulmonary lesions of maedi, but no visna lesions in the central nervous system, although maedi virus was isolated from various parts of brain. None of the other experimental sheep displayed clinical signs of maedi or visna during the observation period of 18 months.     

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.     

Peritoneal barrier to the spread of Semliki forest virus in mice

The LD50 for encephalitis caused by Semliki forest virus in 6- to 8-week-old mice is 1 plaque-forming unit (PFU) in C3H/Ten strain of mice when injected intracerebrally, iv, or in the footpad; however, the LD50 by the ip route is 4 x 10(3) PFU. In the ICR strain of mice at the same age, the LD50 for the intracerebral route is 1 PFU, 10(3) PFU for the iv and footpad routes, and 4 x 10(3) PFU for the ip route. A number of in vivo and in vitro experiments were done to explain the relative resistance to Semliki forest virus injection by the ip route. The results suggest that the viruses are adsorbed to and enter adherent cells of the peritoneal cavity but do not replicate and release progeny virus. After inoculation with the virus, viral antigens could only be observed in methanol-treated cells as a halo by immunofluorescence at or just below the plasma membrane of only a small fraction (less than 0.5%) of peritoneal adherent cells. Naturally occurring interferon-alpha/beta (less than 1 unit/ml) was found to probably play a marginal role, if any, in the resistance.     

汉坦病毒感染诱导乳鼠脑神经细胞表达热休克蛋白70

生后2～3天的昆明乳鼠,每只腹腔接种0.05mL100个半数致死量的陈株汉坦病毒,于接种后不同时间点处死动物,取其脑组织常规固定,石蜡包埋制备5μm连续组织切片,用免疫组化法检测组织中的病毒抗原及热休克蛋白70的表达,用共聚焦显微镜观察二者之间的关系。结果发现,感染了病毒的组织能够稳定地检测到热休克蛋白70的表达,而病毒阴性的组织则检测不到热休克蛋白70的表达,且二者有共定位关系,其分布与组织病变一致。这与在病毒感染的VeroE6细胞及EHF病人尸检组织中得到的结果一致,说明病毒感染可诱导热休克蛋白70的表达,后者可能具有保护组织避免或减轻损伤的作用。     

Attenuation of Sindbis virus neurovirulence by using defined mutations in nontranslated regions of the genome RNA.

We examined a panel of Sindbis virus mutants containing defined mutations in the 5' nontranslated region of the genome RNA, in the 3' nontranslated region, or in both for their growth in cultured cells and virulence in newborn mice. In cultured cells, these viruses all had defects in RNA synthesis and displayed a wide range of growth rates. The growth properties of the mutants were often very different in mouse cells from those in chicken cells or in mosquito cells. We hypothesize that host factors, presumably proteins, interact with these nontranslated regions to promote viral replication and that the mammalian protein and the chicken or mosquito protein are sufficiently divergent that alterations in the viral RNA sequence can affect the interactions with these different host proteins in different ways. Some of the mutants were temperature sensitive for plaque formation, whereas one mutant was slightly cold sensitive in its growth in chicken cells. Upon inoculation into mice, viruses that grew well in cultured mouse cells retained their virulence, but mice that succumbed usually had extended survival times. One virulent mutant that grew slightly less well in cultured mouse cells than did the parental virus produced eight times as much virus in mouse brain following intracerebral inoculation, suggesting that changes in these regulatory regions may have tissue-specific as well as host-specific effects. Viruses that were severely crippled in their growth in mouse cells in culture were usually, but not always, attenuated in their virulence. In particular, temperature sensitivity was correlated with attenuation. The effect of two mutations was found to be cumulative, and double mutants that contained mutations in both the 5' and 3' nontranslated regions were more attenuated than was either single mutant. Three of four double mutants tested were severely crippled for virus production in cultured cells and were avirulent for mice, even when inoculated intracerebrally.     

Amino acid substitutions in VP2 residues contacting sialic acid in low-neurovirulence BeAn virus dramatically reduce viral binding and spread of infection

Theiler's murine encephalomyelitis viruses (TMEV) consist of two groups, the high- and low-neurovirulence groups, based on lethality in intracerebrally inoculated mice. Low-neurovirulence TMEV result in a persistent central nervous system infection in mice, leading to an inflammatory demyelinating pathology and disease. Low- but not high-neurovirulence strains use sialic acid as an attachment factor. The recent resolution of the crystal structure of the low-neurovirulence DA virus in complex with the sialic acid mimic sialyllactose demonstrated that four capsid residues make contact with sialic acid through noncovalent hydrogen bonds. To systematically test the importance of these sialic acid-binding residues in viral entry and infection, we mutated three VP2 puff B amino acids proposed to make contact with sialic acid and analyzed the consequences of each amino acid substitution on viral entry and spread. The fourth residue is in the VP3-VP1 cleavage dipeptide and could not be mutated. Our data suggest that residues Q2161 and G2174 are directly involved in BeAn virus attachment to sialic acid and that substitutions of these two residues result in the loss of or reduced viral binding and hemagglutination and in the inability to spread among BHK-21 cells. In addition, a gain of function-revertant virus was recovered with the Q2161A mutation after prolonged passage in cells.     

Attenuated, replication-competent herpes simplex virus type 1 mutant G207: safety evaluation in mice

Herpes simplex virus type 1 (HSV-1) mutants that are attenuated for neurovirulence are being used for the treatment of cancer. We have examined the safety of G207, a multimutated replication-competent HSV-1 vector, in mice. BALB/c mice inoculated intracerebrally or intracerebroventricularly with 10(7) PFU of G207 survived for over 20 weeks with no apparent symptoms of disease. In contrast, over 80% of animals inoculated intracerebrally with 1.5 x 10(3) PFU of HSV-1 wild-type strain KOS and 50% of animals inoculated intracerebroventricularly with 10(4) PFU of wild-type strain F died within 10 days. Similarly, after intrahepatic inoculation of G207 (3 x 10(7) PFU) all animals survived for over 10 weeks, whereas no animals survived for even 1 week after inoculation with 10(6) PFU of KOS. After intracerebroventricular inoculation, LacZ expression was initially observed in the cells lining the ventricles and subarachnoid space; expression decreased until almost absent within 5 days postinfection, with no apparent loss of ependymal cells. G207 DNA could be detected by PCR in the brains of mice 8 weeks after intracerebral inoculation; however, no infectious virus could be detected after 2 days. As a model for latent HSV in the brain, we used survivors of an intracerebral inoculation of HSV-1 KOS at the 50% lethal dose. Inoculation of a high dose of G207 at the same stereotactic coordinates did not result in reactivation of detectable infectious virus or symptoms of disease. We conclude that G207 is safe at or above doses that were efficacious in mouse tumor studies.     

Isolation and Molecular Characterization of a Poliovirus Type 1 Mutant That Replicates in the Spinal Cords of Mice

The Mahoney strain of poliovirus type 1 (OM) is generally unable to cause paralysis in mice. We isolated a mouse-adapted mutant, PV1/OM-SA (SA), from the spinal cord of a mouse that had been intracerebrally inoculated with OM. SA showed mouse neurovirulence only with intraspinal inoculation, and the infected mice developed a flaccid paralysis, which was indistinguishable from that observed in poliovirus-sensitive transgenic mice inoculated with OM. SA antigens were detected in neurons of the spinal cords of the infected mice. Nucleotide (nt) sequence analysis revealed 9 nt changes on the SA genome, resulting in three amino acid (a.a.) substitutions, i.e., one each in the capsid proteins VP4 and VP1 and in the noncapsid protein 2C. To identify the key mutation site(s) for the mouse neurovirulence, virus recombinants between OM and SA were constructed by using infectious cDNA clones of these two viruses and tested for their mouse neurovirulence after inoculation via an intraspinal route. The results indicated that a mutation at nt 928 (replacement of A with G), resulting in a substitution of Met for Ile at a.a. 62 within VP4, was responsible for conferring the mouse neurovirulence phenotype of the mutant SA. The mutation in VP4 may render the virus accessible to a molecule that acts as a virus receptor and is located on the surfaces of neurons of the mouse spinal cord. This molecule appears not to be expressed in the mouse brain.     

Experimental Infection in Newborn Mice and Rats by Hemorrhagic Fever with Renal Syndrome (HFRS) Virus

Newborn mice and rats were inoculated intracerebrally (ic) or intraperitoneally (ip) with Hantaan virus (76–118 strain) or HFRS-related virus (B-1 strain). The mortality and the influence on the increase of body weight in newborn mice were higher in the groups infected with the 76–118 strain than in the groups infected with the B-1 strain, while the B-1 strain was more virulent in rats than the 76–118 strain. Virus isolation from rats inoculated with either strain was attempted 7 and 11 weeks after inoculation. Virus could be isolated from various organs of rats infected with the B-1 strain, while it was recovered from only the brain and lungs of rats infected with the 76–118 strain. Viral antigen was readily detected in various organs of rats infected with the B-1 strain, but the amount and distribution of antigens were less in rats infected with the 76–118 strain. Our results suggest that the virulence of HFRS-related virus is variable, depending on the species of infected animals as well as on the. virus strains. The virus also persists in the injected animals with high titers of antibodies for at least 11 weeks.     

Characterization of M gene-deficient rabies virus with advantages of effective immunization and safety as a vaccine strain

Matrix (M) protein of rabies virus is known to play an important role in assembly and budding of the progeny virus. We generated an M gene-deficient rabies virus, RC-HLDeltaM, using a reverse genetics system of rabies virus RC-HL strain to develop a novel type of vaccine. RC-HLDeltaM infection was confined within a single cell in mouse neuroblastoma cells. This deficient virus failed to generate the progeny virus in the cells. In contrast, RC-HLDeltaM propagated in BHK cells inductively expressing M protein. Suckling and adult mice inoculated intracerebrally with the parental RC-HL strain showed lethal infection and transient body weight loss, respectively, whereas both suckling and adult mice inoculated with RC-HLDeltaM showed no symptoms. The neutralizing antibody against rabies virus was successfully induced by intramuscular immunization with 10(5) focus-forming units of RC-HLDeltaM but not UV-inactivated RC-HL. Intranasal immunization with RC-HLDeltaM resulted in almost the same antibody titer to rabies virus as that in the case of immunization with live RC-HL strain. These findings indicate that RC-HLDeltaM is a candidate for a novel rabies vaccine that is safer and more effective than are current vaccines.     

Apoptosis in the Mouse Central Nervous System in Response to Infection with Mouse-Neurovirulent Dengue Viruses

Apoptosis has been suggested as a mechanism by which dengue (DEN) virus infection may cause neuronal cell death (P. Desprès, M. Flamand, P.-E. Ceccaldi, and V. Deubel, J. Virol. 70:4090–4096, 1996). In this study, we investigated whether apoptotic cell death occurred in the central nervous system (CNS) of neonatal mice inoculated intracerebrally with DEN virus. We showed that serial passage of a wild-type human isolate of DEN virus in mouse brains selected highly neurovirulent variants which replicated more efficiently in the CNS. Infection of newborn mice with these neurovirulent variants produced fatal encephalitis within 10 days after inoculation. Virus-induced cell death and oligonucleosomal DNA fragmentation were observed in mouse brain tissue by day 9. Infected mouse brain tissue was assayed for apoptosis by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and for virus replication by immunostaining of viral antigens and in situ hybridization. Apoptotic cell death and DEN virus replication were restricted to the neurons of the cortical and hippocampal regions. Thus, DEN virus-induced apoptosis in the CNS was a direct result of virus infection. In the murine neuronal cell line Neuro 2a, neuroadapted DEN virus variants showed infection patterns similar to those of the parental strain. However, DEN virus-induced apoptosis in these cells was more pronounced after infection with the neurovirulent variants than after infection with the parental strain.     

California serogroup and Powassan virus infection of cats

One hundred and seventy five sera from cats in Ontario, Canada, were tested for hemagglutination inhibition (HI) antibodies to three arboviruses; namely, Powassan (POW) of the Flavivirus serogroup, and Snowshoe hare (SSH) and Jamestown Canyon (JC) viruses of the California (CAL) serogroup. All sera were negative for antibodies to POW virus. Twelve cats possessed CAL serogroup antibodies including 3 with antibodies to SSH alone, 6 with antibodies to JC alone, and 3 with antibodies to both SSH and JC antigens. POW virus was inoculated into seven cats, one intracerebrally and six intravenously. Neurologic signs were not detected in any of the cats. Histologic lesions of a nonsuppurative encephalitis and encephalomyelitis were observed in the intracerebrally inoculated cat and in one of the intravenously inoculated cats, respectively. POW virus was not isolated from the brain or spinal cord of either of these two cats. HI antibodies were detected in the sera of all inoculated animals. HI antibodies were not detected in the CSF of any animal.