Complementary lethal invasion of the central nervous system by nonneuroinvasive herpes simplex virus types 1 and 2.

It is well-known that viral thymidine kinase (TK) expression is important for the maximum demonstration of virulence of herpes simplex virus (HSV). In this study, we have investigated interactions of a TK- mutant of virulent HSV type 2 (HSV-2) (syn+) and a nonneuroinvasive HSV-1 (syn) in mice. When the mice were inoculated with each virus alone in their rear footpads, no mice were killed even after infection with high doses of viruses (greater than 10(6) PFU per mouse), whereas 100% of the mice died when inoculated with 10(5) PFU of a 1:1 mixture of HSV-2 TK- mutant and nonneuroinvasive HSV-1. The 1:1 mixture exhibited even more virulence than the parental HSV-2; the mean survival time of coinfected mice was significantly shorter than that of mice inoculated with 10(5) PFU of the virulent HSV-2. We have also examined the genotypes and phenotypes of viruses isolated from the central nervous system of coinfected mice. Of 50 isolates, 7 were judged to be recombinants from their restriction endonuclease cleavage patterns, but all were nonneuroinvasive. In addition, all syn+ viruses (23 clones) tested were found to have TK- phenotypes, indicating that the majority of viruses present in the central nervous system were TK- viruses, since about 90% of viruses detected in spinal cords and brains exhibited syn+ phenotypes. These results strongly suggest that the lethal invasion of the central nervous system by HSV-2 TK- and nonneuroinvasive HSV-1 was the consequence of in vivo complementation between the two viruses.     

Age-dependent resistance to lethal alphavirus encephalitis in mice: analysis of gene expression in the central nervous system and identification of a novel interferon-inducible protective gene,mouse ISG12

Several different mammalian neurotropic viruses produce an age-dependent encephalitis characterized by more severe disease in younger hosts. To elucidate potential factors that contribute to age-dependent resistance to lethal viral encephalitis, we compared central nervous system (CNS) gene expression in neonatal and weanling mice that were either mock infected or infected intracerebrally with a recombinant strain, dsTE12Q, of the prototype alphavirus Sindbis virus. In 1-day-old mice, infection with dsTE12Q resulted in rapidly fatal disease associated with high CNS viral titers and extensive CNS apoptosis, whereas in 4-week-old mice, dsTE12Q infection resulted in asymptomatic infection with lower CNS virus titers and undetectable CNS apoptosis. GeneChip expression comparisons of mock-infected neonatal and weanling mouse brains revealed developmental regulation of the mRNA expression of numerous genes, including some apoptosis regulatory genes, such as the proapoptotic molecules caspase-3 and TRAF4, which are downregulated during development, and the neuroprotective chemokine, fractalkine, which is upregulated during postnatal development. In parallel with increased neurovirulence and increased viral replication, Sindbis virus infection in 1-day-old mice resulted in both a greater number of host inflammatory genes with altered expression and greater changes in levels of host inflammatory gene expression than infection in 4-week-old mice. Only one inflammatory response gene, an expressed sequence tag similar to human ISG12, increased by a greater magnitude in infected 4-week-old mouse brains than in infected 1-day-old mouse brains. Furthermore, we found that enforced neuronal ISG12 expression results in a significant delay in Sindbis virus-induced death in neonatal mice. Together, our data identify genes that are developmentally regulated in the CNS and genes that are differentially regulated in the brains of different aged mice in response to Sindbis virus infection.     

Murine infection by vesicular stomatitis virus: initial characterization of the H-2d system.

BALB/c mice and congenic H-2Ld-deficient BALB/c-H-2dm2 (dm2) mice were experimentally infected intranasally with isolates of vesicular stomatitis virus (VSV). The survival of infected hosts, viral replication in lungs and brains, and histopathologic in the two mouse strains were compared. In both strains of mice, mortality occurred during the period 7 to 10 days postinfection. However, dm2 mice were relatively resistant to lethal infections. Viral replication occurred at low levels in the lungs of both strains and did not evoke significant pathologic changes. In contrast, viral replication in the brains was much greater; in the BALB/c strain, this was accompanied by more frequent and more severe pathologic changes. In general, mice surviving at day 10 had effectively cleared virus from central nervous system but not respiratory sites. Evidence is presented that viral replication occurs first in the nasal cavity and is transmitted both to the lungs and to the olfactory bulb where focal cytopathology occurs. Virus enters the ventricles, causing encephalitis; necrosis occurs around the ventricles and in the lumbosacral region of the spinal cord. Necrotic lesions were accompanied by mononuclear infiltration. Mice immunized with virus of the same serotype or with a vaccinia virus hybrid encoding the VSV glycoprotein were protected from lethal infection; in contrast, mice immunized with heterotypic virus were susceptible to challenge.     

Prolonged replication in the mouse central nervous system of reoviruses isolated from persistently infected cell cultures.

We examined pathogenic characteristics of plaque-purified reoviruses isolated from persistently infected L-cell cultures (PI viruses) after intracranial inoculation into newborn mice. The PI viruses were isolated from independent cultures initiated with high-passage stocks of the wild-type (wt) strain, type 3 Dearing. The virulence of most PI viruses was equivalent to that of the wt strain. However, replication of PI viruses in the central nervous system of infected mice was prolonged to 25 (but not 50) days postinoculation. Thirty-eight percent (n = 186) of mice inoculated with the PI viruses had residual virus detectable in brain tissue 25 days after inoculation, in contrast to only 16% (n = 57) of mice inoculated with wt virus (P = 0.009). Mean residual brain titers were more than 20-fold higher in mice inoculated with PI viruses compared with wt virus (4.3 x 10(4) versus 2.1 x 10(3); P = 0.006). Tropism of PI virus within the brain resembled that of wt virus, and the distribution of PI virus antigen in the brain did not change over time. The extent of necrosis in the brains of mice harboring PI virus 25 days after inoculation was minimal, despite continued presence of high titers of infectious virus. The latter observation resembles the absence of cytopathicity seen in L-cell cultures persistently infected with reovirus. These observations suggest that the interaction of PI viruses with cells can be altered in vivo as well as in cell culture, but virus is eventually cleared from the infected animal.     

Characterization of a variant virus selected in rat brains after infection by coronavirus mouse hepatitis virus JHM.

The intracerebral inoculation of Lewis rats with the murine coronavirus MHV-JHM leads in the majority of animals to acute encephalitis and death within 14 days. Viral RNAs isolated from the brains of animals 5 to 7 days after infection were compared by Northern blot analysis with the RNAs produced during the lytic infection of Sac(-) or DBT cells with wild-type MHV-JHM (wt virus). Reproducibly, the subgenomic mRNAs 2 and 3 but no other viral RNAs were significantly larger in the brain-derived material. All viruses isolated from infected brain material displayed and maintained this altered mRNA profile when cultivated in Sac(-) or DBT cells. A virus isolated from the infected brain material, MHV-JHM clone 2 (cl-2 virus), has been further characterized. This isolate grew in tissue culture and induced cytopathic effects comparable to those induced by wt virus. However, the mRNAs 2 and 3 produced in cl-2 virus-infected cells had molecular weights ca. 150,000 larger than those produced in cells infected with wt virus. There was no detectable difference in genome-sized RNA (mRNA 1) or subgenomic mRNAs 4, 5, 6, and 7 as determined by electrophoresis in agarose gels. T1-resistant oligonucleotide analysis of genomic RNA revealed one additional and one missing oligonucleotide in the fingerprint of cl-2 virus compared with wt virus. The oligonucleotide fingerprints of intracellular mRNA 3 were identical for both viruses. Pulse-labeling with [35S]methionine in the presence of tunicamycin showed that the primary translation product of mRNA 3, the E2 apoprotein, was ca. 15,000 larger in molecular weight in cl-2 virus-infected cells. These data show that viruses with larger mRNAs 2 and 3 (the latter encoding an altered E2 glycoprotein) are selected for multiplication in rat brains. Mechanisms for the generation of such variants and the possible nature of their selective advantage are considered.     

Spontaneous and induced leukemias of myeloid origin in recombinant inbred BXH mice

BXH-2 recombinant inbred (RI) mice produce high titers of B-ecotropic murine leukemia virus beginning early in life and have a high incidence of non-T-cell leukemias that occur before 1 year of age. The leukemias that develop are in some cases associated with hind limb paralysis. In addition, a dualtropic mink cell focus-forming virus has been isolated from leukemic cells of BXH-2 mice. Immunological and cytochemical characterization of the BXH-2 leukemias showed that they are of the myeloid lineage. To assess the oncogenicity of the BXH-2 viruses, newborn mice of several BXH RI strains were inoculated at birth with biologically cloned B-ecotropic or mink cell focus-forming murine leukemia virus. These studies demonstrated that the B-ecotropic virus can induce myeloid leukemias in other BXH RI strains, whereas the dualtropic mink cell focus-forming isolates were nononcogenic in the strains tested. DNA-DNA reassociation analysis indicated that the organotropism of the B-ecotropic murine leukemia virus is confined to lymphoid tissues. Southern analysis of tumor DNAs showed that there was amplification of ecotropic virus-specific sequences in BXH-2 myeloid tumors and in all leukemias induced in other BXH RI strains by inoculation of the BXH-2 B-ecotropic virus. Although B-ecotropic virus is expressed in central nervous tissues of paralyzed BXH-2 mice, we were unable to induce the disorder in several BXH RI strains inoculated intracranially at birth with either the B-ecotropic or dualtropic virus. These results suggest that the paralysis that occurs in BXH-2 mice is due to the infiltration of leukemic cells into the central nervous system.     

Role of herpes simplex virus 1 Us3 in viral neuroinvasiveness

Us3 is a serine–threonine protein kinase that is encoded by herpes simplex virus 1 (HSV‐1). In experimental animal models of HSV infection, peripheral and intracranial inoculations can be used to study viral pathogenicity in peripheral sites (e.g., eyes and vagina) and central nervous systems (CNSs), respectively. In addition, peripheral inoculation can be used to investigate this virus' ability to invade the CNS (neuroinvasiveness) from peripheral sites. HSV‐1 Us3 has previously been shown to be critical for viral pathogenicity in both peripheral sites and CNSs of mice. However, the role of HSV‐1 Us3 in viral neuroinvasiveness has not yet been elucidated. In the present study, the yields of a Us3 null mutant virus and its repaired virus in the eyes, trigeminal ganglia, and brains of mice following ocular inoculation were examined. It was found that, although the repaired virus appeared in the brains of mice 3 days after infection, peak replication occurring 7 days after infection, no viral replication of the Us3 null mutant virus was detectable. These findings indicate that HSV‐1 Us3 plays a crucial role in the ability of the virus to invade the brain from the eyes. Thus, HSV‐1 Us3 is a significant neuroinvasiveness factor in vivo.     

Biological basis of rabies virus neurovirulence in mice: comparative pathogenesis study using the immunoperoxidase technique.

The CVS strain of fixed rabies virus causes acute, fatal encephalomyelitis in young adult ICR mice. Variant RV194-2, which was selected from CVS virus in cell culture with a neutralizing antiglycoprotein monoclonal antibody, has a single amino acid change in the glycoprotein. The infections caused by CVS virus and RV194-2 virus were compared in mice for 14 days postinoculation of 5 x 10(7) PFU into the right masseter muscle. All CVS virus-infected mice died (mean time to death, 7.9 days), compared with a mortality rate of 8.5% for RV194-2 virus-infected mice. RV194-2 virus spread to the ipsilateral trigeminal ganglion during the first 2 days postinoculation, and both viruses spread to the ipsilateral motor nucleus of the trigeminal nerve in the pons. Both viruses spread centrifugally and caused infection of bilateral trigeminal ganglia on day 3. The viruses spread throughout the central nervous system (CNS) at similar rates, but CVS virus infected many more neurons than did RV194-2 virus. Rabies virus antigen was observed in only occasional CNS neurons after day 6 of RV194-2 virus infection. By this time, CVS virus had caused severe widespread infection. In this model, virulence depends on improved efficiency of viral spread between CNS neurons rather than the rate of spread or topographical distribution of the infection.     

Expression of ecotropic murine leukemia virus in the brains of C58/M, DBA2/J, and in utero-infected CE/J mice.

In C58 and AKR mice, endogenous N-tropic, ecotropic murine leukemia virus (MuLV) proviruses become activated in rare cells during embryogenesis. Resultant replication-competent progeny viruses then actively infect a large number of cells throughout the fetus, including cells in the developing central nervous system. By in situ hybridization analyses, we have assessed the presence of ecotropic MuLV RNA in the brains of C58 mice as a function of age. Only a few ecotropic MuLV-positive cells were observed in weanling mice, but the number of positive cells in the brain increased progressively with increasing age of the mice. Throughout the lives of the mice, the ecotropic MuLV RNA-positive cells were primarily located in well-defined white-matter tracts of the brain (commissura anterior, corpus callosum, fimbria hippocampi, optical tract, and striatum) and of the spinal cord. Cells of the subventricular zone also expressed ecotropic MuLV RNA, and in older mice a small number of positive cells were present in the grey matter. Infection of endogenous ecotropic MuLV provirus-less CE/J mice in utero with ecotropic MuLV clone AKR-623 resulted in the extensive infection of brain cells. The regional distribution of ecotropic MuLV RNA-containing cells was the same as observed in the brains of C58 mice, in which cells became infected by endogenously activated virus, but the number of positive cells was higher.     

Hypothermia-inducing peptide promotes recovery of vesicular stomatitis virus from persistent animal infections.

A single injection of the hypothermia-inducing neuropeptide bombesin resulted in an excellent recovery system for reisolating viruses from Swiss albino mice infected with vesicular stomatitis virus even up to 90 days after infection. The virus was recovered from a cell homogenate prepared from whole brain tissue 24 h after intracerebral injection of bombesin; brain cells were cocultivated with BHK-21 cell monolayers and then plaqued on BHK-21 cells at 31 degrees C. All of the recovered viruses were identified as vesicular stomatitis virus by antibody neutralization and peptide analyses of some of the structural proteins. However, some of the recovered viruses were altered with regard to tryptic peptide maps, temperature sensitivity, and central nervous system disease induced compared with the viruses used to initiate the infection. Most of the recovered viruses induced a similar disease when reinoculated intracerebrally into mice, characterized by hind-leg paralysis 4 to 6 days after infection. Two of the recovered viruses were lethal, however, resulting in a relatively rapid generalized wasting disease and death in 3 to 4 days.     

Pathogenesis of a lethal mixed infection in mice with two nonneuroinvasive herpes simplex virus strains.

We previously reported that simultaneous inoculation of mice on abraded rear footpads with two nonneuroinvasive viruses (herpes simplex virus type 1 ANG and KOS) resulted in the deaths of 62% of the animals (R. T. Javier, F. Sedarati, and J. G. Stevens, Science 234:746-748, 1986). In the current study, to better understand the events responsible for the pathogenesis of this virus mixture, we investigated replicative capacity and spread of the virus mixture within specific tissues. We found that, compared with neuroinvasiveness of ANG or KOS alone, neuroinvasiveness of the virus mixture related to significantly increased amounts of the virus within spinal cords and brains of the mice. This finding indicates that ANG and KOS have defects in their capacities to spread and replicate within spinal cords. We also examined whether the increased neuroinvasiveness of the virus mixture related to complementation between viruses in tissues of the nervous system, generation and selection of neuroinvasive recombinants, or both. It was found that, although neuroinvasive recombinant viruses could be detected in the spinal cords of the infected animals, most of the viruses (both recombinants and nonrecombinants) isolated from all tissues tested were nonneuroinvasive (i.e., no mice died as a result of footpad infection with high doses of such plaque-purified isolates). As a result of these findings, we propose that the virulence of the virus mixture is a consequence of the complementation as well as the generation and selection of neuroinvasive recombinants in spinal cords of these mice.     

Infection of primary human microglia and monocyte-derived macrophages with human immunodeficiency virus type 1 isolates: evidence of differential tropism.

To ascertain whether viruses present at the time of primary viremia can infect the central nervous system and to determine if microglial tropism is distinct from tropism for monocyte-derived macrophages (MDM), 27 human immunodeficiency virus type 1 (HIV-1) isolates obtained from acutely infected individuals, as well as laboratory strains, were assayed for their ability to replicate in primary adult microglial cultures and in MDM. Most of the isolates replicated equally well in both microglia and MDM, but several isolates replicated preferentially in one of the two cell types, differing by as much as 40-fold in p24gag production. This indicated that while MDM and microglial tropism overlap, a subset of isolates is particularly tropic for one of the two cell types. One isolate was further adapted to microglia by 15 sequential passages, raising the peak p24 concentration produced by 1,000-fold. In addition, the passaged virus induced marked cytopathologic changes (vacuolization and syncytium formation) in infected microglial cultures. Sequence comparison of the V3 loop of unpassaged and multiply passaged virus revealed amino acid changes shown to be associated with isolates from patients with HIV dementia. Our data support the hypothesis that HIV-1 infection can be established in the central nervous system by viruses present early in HIV infection, that some of these viruses are particularly tropic for microglia, and that adaptation in this cell type can result in the selection of a pool of predominantly microglia-tropic (neurotropic) viruses.     

Highly lytic and persistent lentiviruses naturally present in sheep with progressive pneumonia are genetically distinct.

Ovine and caprine lentiviruses share the capacity to induce slowly progressive and inflammatory diseases of the central nervous system (leukoencephalitis or visna), lungs (progressive pneumonia or maedi), and joints (arthritis) in their natural hosts. Studies on their replication indicated that ovine lentiviruses and caprine arthritis-encephalitis virus (CAEV) recently isolated in the United States establish persistent infection in ovine and caprine fibroblasts, whereas older prototype ovine lentiviruses such as Icelandic visna virus or American progressive pneumonia virus irreversibly lyse fibroblast cultures. Since all of the recent isolates were found to be persistent, Narayan et al. (J. Gen. Virol. 59:345-356, 1982) concluded that the highly lytic viruses were only tissue-culture-adapted strains. In the present report, we isolated new ovine lentiviruses from French sheep with naturally occurring progressive pneumonia which are either highly lytic (five isolates), as are the Icelandic strains of visna virus, or persistent (one isolate), as are CAEV or American persistent ovine lentiviruses. Protein and nucleic acid content analyses of these new highly lytic (type I) and persistent (type II) isolates indicated that type I and type II ovine lentiviruses were genetically distinct, type I and type II viruses being closely related to the Icelandic strains of visna virus and to CAEV, respectively. We conclude that (i) highly lytic ovine lentiviruses, such as the Icelandic prototype strains of visna virus and persistent lentiviruses more related to CAEV, are naturally present in the ovine species, and (ii) irreversible cell lysis induced by highly lytic viruses does not result from a tissue culture adaptation of field isolates that were originally persistent but is instead the consequence of a genetic content distinct from that of persistent viruses.     

Eastern equine encephalitis in the United States, 1971: Past and prologue

During 1971, surveillance for equine encephalitis in the United States was increased due to an epizootic of Venezuelan equine encephalomyelitis. Of 1,982 specimens from 1,551 equines, 76 isolates of eastern equine encephalitis (EEE) virus were recovered from 67 individuals. The virus was isolated from 50/176 brains, 8/74 spleens, 14/1,127 sera, and 4/147 whole bloods from infected equines in 12 of the 31 states bounded by or east of the Mississippi River and in Texas and Iowa; no specimens were received from 9 of these 31 states. Thus, EEE virus was isolated from equines in 12 of 22 of these states. Determinations of antibody to EEE and western equine encephalitis (WEE) viruses indicated a relatively high prevalence of infection with EEE virus in the eastern USA and similarly high prevalence of antibody to WEE virus in the western USA. These data indicate that equine infections with EEE virus in the eastern USA are considerably more common than previous surveillance data have suggested. Increased surveillance and submission of specimens to diagnostic laboratories for diagnosis of EEE virus infections in equines are suggested so that a greater proportion of the thousands of unspecified equine encephalitis cases occurring in the United States each year can be laboratory confirmed.     

Molecular analysis of neurovirulent strains of Sindbis virus that evolve during persistent infection of scid mice.

To understand the role of tissue-specific adaptation and antibody-induced selectional pressures in the evolution of neurovirulent viruses, we analyzed three strains of Sindbis virus isolated from the brains of persistently infected scid mice and four strains of Sindbis virus isolated from the brains of scid mice with viral reactivation following immune serum treatment. For each viral isolate, we tested neurovirulence in weanling BALB/c mice and sequenced regions of the E2 and E1 envelope glycoprotein genes that are known to contain important determinants of Sindbis virus neurovirulence. One strain isolated from a persistently infected scid mouse and two strains isolated from scid mice with viral reactivation were neurovirulent, resulting in mortality in 80 to 100% of weanling BALB/c mice. All three neurovirulent strains contained an A-->U change at nucleotide 8795, which predicts a Gln-->His substitution at E2 amino acid position 55. No nucleotide changes were detected in the other sequenced regions of the E2 and E1 envelope glycoprotein genes or in the avirulent isolates. Our findings indicate that tissue-specific adaptations, rather than antibody-induced selectional pressures, are a critical determinant of the evolution of neurovirulent strains of Sindbis virus and provide evidence that E2 His-55 is an important neuroadaptive mutation that confers neurovirulence properties on Sindbis virus.     

Characterization of scrapie agent isolated from sheep in Japan

A pathogenic agent isolated in mice from the brain of a sheep affected by scrapie-like disease was characterized. The incubation period of the disease in the primary transmission from the sheep to mice was longer than in the secondary and the tertiary transmission in the same strain of mice. Progressive dilution of the inoculum caused prolongation of the incubation period. The infectivity of the agent in a 10% brain homogenate persisted, but decreased about 10(3) to 10(4) times after heating at 100 C for 30 min. Histological changes in the diseased mouse brains consisted of vacuolation of the nerve cells and spongiform degeneration in the gray matter of the central nervous system. Fine rod-shaped granulae with a length of 3 to 5 nm were observed within the swollen neuropil, axon, and perivascular astrocytic process. No serum antibodies against available mouse viruses, parainfluenza type 1 virus, lymphocytic choriomeningitis virus, and mouse reovirus type 3, were detected in any mice used in the experiments. These findings demonstrate that the disease of the sheep was the first case of scrapie in Japan.     

Effects of viral infection on contraction of the diaphragm in mice

Isometric contractile properties of isolated phrenic nerve-diaphragm muscle preparations were used to study the effects of picornavirus infections on diaphragm muscle function. Properties of muscles from virus-inoculated and control mice were similar during brief contractions. However, when subjected to a series of fatiguing contractions by indirect or direct stimulation, muscles of mice inoculated with a paralytic variant of encephalomyocarditis (EMC) virus showed a greater rate of fatigue and a reduced capacity to recover from fatigue than did muscles from uninoculated control mice or muscles from mice inoculated with a nonparalytic coxsackievirus B3 (CVB3). Mice paralyzed by EMC virus infection had high titers of virus in the brain and similar titers of virus in diaphragm muscle as found in diaphragm muscles of CVB3-inoculated mice. The results indicate that EMC virus infection of mice leads to increased fatigability of the diaphragm muscle and that there are both neural and muscular components of this enhanced fatigue.     

Infection with Cytotoxic T-Lymphocyte Escape Mutants Results in Increased Mortality and Growth Retardation in Mice Infected with a Neurotropic Coronavirus

C57BL/6 mice infected with mouse hepatitis virus strain JHM (MHV-JHM) develop a chronic demyelinating encephalomyelitis several weeks after inoculation. Previously, we showed that mutations in the immunodominant CD8 T-cell epitope (S-510-518) could be detected in nearly all samples of RNA and virus isolated from these mice. These mutations abrogated recognition by T cells harvested from the central nervous systems of infected mice in direct ex vivo cytotoxicity assays. These results suggested that cytotoxic T-lymphocyte (CTL) escape mutants contributed to virus amplification and the development of clinical disease in mice infected with wild-type virus. In the present study, the importance of these mutations was further evaluated by infecting naive mice with MHV-JHM variants isolated from infected mice and in which epitope S-510-518 was mutated. Compared to mice infected with wild-type virus, variant virus-infected animals showed higher mortality and morbidity manifested by decreased weight gain and neurological signs. Although a delay in the kinetics of virus clearance has been demonstrated in previous studies of CTL escape mutants, this is the first illustration of significant changes in clinical disease resulting from infection with viruses able to evade the CD8 T-cell immune response.     

Slow development of measles virus (Edmonston strain) infection in the brain of nude mice

The Edmonston strain of measles virus caused neurologic disease in athymic nude mice by intracerebral inoculation. The incubation periods of the disease, however, were extremely long, ranging from 59 to 140 days when the mice were inoculated with 10(4) plaque forming units (PFU) of the virus. The Edmonston strain was highly infectious in the nude mouse brain since virus infection was established even with 1 PFU of the virus. Virus titers in the brains of infected mice increased with the time of incubation. These results indicate that the extremely long incubation period of the disease is ascribed to very slow development of virus infection in the mouse brain. On the other hand, the incubation periods of the Biken strain of SSPE virus were very short (generally within 2 weeks) even with inoculations of 1 PFU of the virus. However, the extent of the dissemination of infection in brains was not significantly different between the two viruses as examined by immunofluorescent staining.