Isolation of a temperature-sensitive dengue-2 virus under conditions suitable for vaccine development

Dengue virus, type 2, in viremic human sera and after passage in cell cultures produces mixtures of small and large plaques when assayed in LLC-MK2 cells. Clones of dengue virus type 2 obtained by plaque selection in primary green monkey kidney cell cultures were tested for temperature sensitivity in vitro and for virulence by intracerebral inoculation of suckling mice. Sublines of a small-plaque clone were found to have lower nonpermissive temperatures than the parent virus by both plaque formation and release of infectious virus into the culture media. Small-plaque sublines were significantly less virulent in suckling mice than was the parent virus. Sublines from a large-plaque clone were not temperature sensitive and closely resembled parent virus mixed-plaque morphology. When small-plaque sublines were serially passaged using undiluted inocula, reversion occurred as evidenced by the appearance of large plaques and return of mouse virulence. Small-plaque virus could be maintained through several serial passages without reversion by using low-input inocula. Desirable passage history as well as temperature-sensitive and attentuation characteristics of the S-1 small-plaque subline make it appear suitable as a vaccine candidate virus.     

Induction of protective immunity against Japanese encephalitis in mice by immunization with a plasmid encoding Japanese encephalitis virus premembrane and envelope genes

A DNA vaccine plasmid containing the Japanese encephalitis (JE) virus premembrane (prM) and envelope (E) genes (designated pcDNA3JEME) was evaluated for immunogenicity and protective efficacy in mice. Two immunizations of 4-week-old female ICR mice with pcDNA3JEME by intramuscular or intradermal injections at a dose of 10 or 100 microg per mouse elicited neutralizing (NEUT) antibodies at titers of 1:10 to 1:20 (90% plaque reduction), and all immunized mice survived a challenge with 10,000 50% lethal doses of the P3 strain of JE virus. A single immunization with 100 microg of pcDNA3JEME did not elicit detectable NEUT antibodies but induced protective immunity. Spleen cells obtained from BALB/c mice immunized once with 10 or 100 microg of pcDNA3JEME contained JE virus-specific memory cytotoxic T lymphocytes (CTLs). BALB/c mice maintained detectable levels of memory B cells and CTLs for at least 6 months after one immunization with pcDNA3JEME at a dose of 100 microg. The CTLs induced in BALB/c mice immunized twice with 100 microg of pcDNA3JEME were CD8 positive and recognized mainly the envelope protein. These results indicate that pcDNA3JEME has the ability to induce a protective immune response which includes JE virus-specific antibodies and CTLs.     

Alphavirus-induced apoptosis in mouse brains correlates with neurovirulence

Sindbis virus induces apoptotic cell death in cultured cell lines, raising the possibility that apoptosis of infected neurons and other target cells in vivo may contribute to the resulting disease and mortality. To investigate the role of apoptosis in Sindbis virus pathogenesis, infected mouse brains were assayed by the in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling technique and for DNA ladder formation. Infection with recombinant Sindbis virus strain 633 resulted in widespread apoptosis in newborn mouse brains and spinal cords, but few apoptotic cells were observed following infection of 2-week-old animals. This finding correlates with the age-dependent mortality observed in mice. The more neurovirulent virus TE, which differs from 633 by a single amino acid in the E2 glycoprotein, induced significant apoptosis in brains and spinal cords of 2-week-old animals, consistent with its ability to cause fatal disease in older animals. Double-labeling experiments demonstrated that the apoptotic cells were also infected with Sindbis virus. Thus, Sindbis virus-induced apoptosis appears to be a result of virus infection and is likely to reflect pathogenic mechanisms for other viruses.     

Incidence and risk factors for radiographic knee osteoarthritis in middle-aged women: the Chingford Study

In susceptible mouse strains, the wild-type Daniel's (wt-DA) strain of Theiler's murine encephalomyelitis virus induces a persistent central nervous system (CNS) infection with chronic demyelination. The virus is cleared from resistant mice with no resulting demyelination. We characterized the role of the DA L* protein in late demyelination and persistent infection. The DA genome has two alternative reading frames, encoding the virus polyprotein and L*, respectively. The mutant virus DAL*-1 fails to synthesize L* and does not persist in the CNS of wt-DA-susceptible SJL/J or B10.S mice. Since class I-restricted cytotoxicity has been shown to determine resistance to virus persistence and demyelination in this model, virus-specific cytotoxicity in the CNS of DA-resistant (B6 or B10) and -susceptible (SJL/J and B10.S) mice during the acute stage of DA and DAL*-1 infection was characterized. Following intracerebral inoculation with DAL*-1, virus-specific Db- and Kb-restricted CTLs were demonstrated in the CNS of resistant B10 mice, whereas only Db-restricted CTL were found in wt-DA-inoculated mice. CTLs specific to wt-DA or DAL*-1 recognized class I-presented peptides from either of the viruses. Of particular interest, Ks-restricted virus-specific cytotoxicity-restricted CTLs were identified in the CNS of susceptible SJL/J (H-2s) and B10.S (H-2s) mice inoculated with DAL*-1. In contrast, no virus-specific CTLs were identified in the CNS of SJL/J and B10.S mice inoculated with wt-DA. We propose that L* inhibits the generation of H-2K-restricted virus-specific cytotoxicity in the CNS, permitting a persistent infection in susceptible strains, with subsequent inflammatory demyelination in the CNS similar to that in human multiple sclerosis.     

The range and distribution of murine central nervous system cells infected with the gamma(1)34.5- mutant of herpes simplex virus 1

Wild-type herpes simplex virus 1 (HSV-1) multiplies, spreads, and rapidly destroys cells of the murine central nervous system (CNS). In contrast, mutants lacking both copies of the gamma(1)34.5- gene have been shown to be virtually lacking in virulence even after direct inoculation of high-titered virus into the CNS of susceptible mice (J. Chou, E. R. Kern, R. J. Whitley, and B. Roizman, Science 250:1262-1266, 1990). To investigate the host range and distribution of infected cells in the CNS of mice, 4- to 5-week-old mice were inoculated stereotaxically into the caudate/putamen with 3 x 10(5) PFU of the gamma(1)34.5- virus R3616. Four-micrometer-thick sections of mouse brains removed on day 3, 5, or 7 after infection were reacted with a polyclonal antibody directed primarily to structural proteins of the virus and with antibodies specific for neurons, astrocytes, or oligodendrocytes. This report shows the following: (i) most of the tissue damage caused by R3616 was at the site of injection, (ii) the virus spread by retrograde transport from the site of infection to neuronal cell nuclei at distant sites and to ependymal cells by cerebrospinal fluid, (iii) the virus infected neurons, astrocytes, oligodendrocytes, and ependymal cells and hence did not discriminate among CNS cells, (iv) viral replication in some neurons could be deduced from the observation of infected astrocytes and oligodendrocytes at distant sites, and (v) infected cells were being efficiently cleared from the nervous system by day 7 after infection. We conclude that the gamma(1)34.5- attenuation phenotype is reflected in a gross reduction in the ability of the virus to replicate and spread from cell to cell and is not due to a restricted host range. The block in viral replication appears to be a late event in viral replication.     

Reduction of acetaminophen toxicity by sodium sulfate in mice

Twenty-one strains of Venezuelan encephalitis (VE) virus isolated from three habitats in Trinidad, W.I. during 1960 to 1972, were subtype III (Mucambo) VE virus by plaque-reduction neutralization tests. Like prototype Mucambo virus, each strain killed 8- to 15-week-old mice inoculated intraperitoneally. If the subtype I strain of VE virus that caused a major outbreak in Trinidad during 1943 to 1944 persisted on the island into the 1960s and early 1970s, it did not become the dominant VE virus in these three enzootic foci.     

Partial nucleotide sequence of Japanese encephalitis virus ling strain genome and comparison of the encoded structural proteins and nonstructural protein NS1 among Japanese encephalitis virus strains

Approximately 4000 nucleotides of the 5'-terminal portion of Japanese encephalitis virus (JEV) Ling strain genome were cloned and sequenced. This nucleotide sequence and its encoded C-PrM-E-NS1 polyprotein sequences were also compared with the corresponding sequences of other JE virus strains. Results demonstrated that the nucleotide sequence homology varied from 97.1 to 99.3% and the amino acid homology 98.6 to 98.9%. Based on homology, the Ling strain was closer to the Beijing-1 strain than to the SA14 and JaOArS982 strains. However, only on comparison of the E sequence, which neutralization, hemagglutination-inhibition and complement fixation antigenic determinants are located, between Ling and other JEV strains demonstrated that nucleotide sequence homology varied from 97.1% to 99.3% and amino acid homology from 98.6% to 99.2%. The Ling strain JEV is more closely related to the Beijing-1 strain than to the Nakayama NIH, SA14 and JaOArS982 strains in that order. Based on this analysis, the Taiwanese JEV strain appears to be more closely related to the Chinese strain than to the Japanese strain. Also, JEV strains isolated in humans are more closely related to each other than to JEV strains of mosquito isolates.     

Disassociation between the in vitro and in vivo effects of nitric oxide on a neurotropic murine coronavirus

Intranasal inoculation of the neuroattenuated OBLV60 strain of mouse hepatitis virus results in infection of mitral neurons in the olfactory bulb, followed by spread along olfactory and limbic pathways to the brain. Immunocompetent BALB/c mice were able to clear virus by 11 days postinfection (p.i.). Gamma interferon (IFN-gamma) may play a role in clearance of OBLV60 from infected immunocompetent BALB/c mice through a nonlytic mechanism. Among the variety of immunomodulatory activities of IFN-gamma is the induction of expression of inducible nitric oxide synthase (iNOS), an enzyme responsible for the production of nitric oxide (NO). Studies were undertaken to investigate the role of IFN-gamma and NO in host defense and clearance of OBLV60 from the central nervous system (CNS). Exposure of OBLV60-infected OBL21a cells, a mouse neuronal cell line, to the NO-generating compound S-nitroso-L-acetyl penicillamine resulted in a significant decrease in viral replication, indicating that NO interfered with viral replication. Furthermore, infection of IFN-gamma knockout (GKO) mice and athymic nude mice with OBLV60 resulted in low-level expression of iNOS mRNA and protein in the brains compared to that of OBLV60-infected BALB/c mice. Nude mice were unable to clear virus and eventually died between days 11 and 14 p.i. (B. D. Pearce, M. V. Hobbs, T. S. McGraw, and M. J. Buchmeier, J. Virol. 68:5483-5495, 1994); however, GKO mice survived infection and cleared virus by day 18 p.i. These data suggest that IFN-gamma production in the olfactory bulb contributed to but may not be essential for clearance of OBLV60 from the brain. In addition, treatment of OBLV60-infected BALB/c mice with aminoguanidine, a selective inhibitor of iNOS activity, did not result in any increase in mortality, and the mice cleared the virus by 11 days p.i. These data suggest that although NO was able to block replication of virus in vitro, expression of iNOS with NO release in vivo did not appear to be the determinant factor in clearance of OBLV60 from CNS neurons.     

A poliovirus-susceptible transgenic mouse model as a possible replacement for the monkey neurovirulence test of oral poliovirus vaccine

Two poliovirus-susceptible transgenic mouse (Tg PVR) strains, Tg1 and Tg21, were compared with the monkey test for their sensitivity to neurovirulence of live oral poliovirus vaccine (OPV). Intracerebral (i.c.) and intraspinal (i.s.) routes of inoculation were investigated to determine the most suitable combination of mouse strain and route. Evaluation of the mouse tests was performed using several indicators; clinical score and failure time were selected as the most efficient. Tg1 and Tg21 mice inoculated i.s. with type 2, and Tg21 mice inoculated i.s. with type 3 OPV were determined to be the most appropriate systems, whereas they are shown not to be suitable for type 1 OPV. The sensitivity of each of the two mouse models was at least equal to that of the monkey test, suggesting that these mouse systems might be considered as a potential replacement for the monkey test of OPV. However, more data are needed to establish regulatory criteria of acceptability for vaccine lots tested in Tg PVR mice. This is the first study conducted with Tg PVR mice with all three types of poliovirus vaccine preparations.     

Encephalitis in ferrets caused by a nonproductive strain of measles virus (D.R.) isolated from patient with subacute sclerosing panencephalitis

A nonproductive, syncytiogenic strain (D.R.) of measles virus, isolated from a patient with subacute sclerosing panencephalitis (SSPE), was inoculated intracerebrally into ferrets in an attempt to induce subacute encephalitis. Inoculation of freeze-thawed syncytia before immunization was the least effective procedure, and inoculation of live syncytia after immunization with measles virus vaccine was the most effective procedure, for induction of subacute or persistent subclinical encephalitis in the animals. After the latter procedure three of five ferrets developed subacute or subclinical encephalitis, whereas ferrets inoculated with live syncytia without prior immunization consistently contracted acute fatal encephalitis in one to two weeks. The subacute encephalitis in ferrets was characterized by high titers of antibody to measles virus in serum. At the time of sacrifice 1.25, 4.5, or 8.0 months after inoculation, brains of the ferrets showed histologic lesions similar to those characteristic of SSPE, and nonproductive syncytiogenic measles virus was recovered from the brains of two of the animals. All three ferrets had greatly increased concentrations of gamma-globulin in their brains and high levels of neutralizing and hemagglutination-inhibiting antibodies to measles virus. Only one of these animals developed clinical signs 1.25 months after inoculation.     

A field study on Nakayama and Beijing strains of Japanese encephalitis vaccines

A field study to compare the immune response of children aged 1-6 years to Nakayama and Beijing strains JE vaccines was carried out in Mae Hong Son Province, northwest Thailand, where there was low incidence of JEV infection. The first and second dose of each vaccine was given 1-2 weeks apart and the third dose was 1 year after the second dose. Seroconversion rate was similarly high, about 94% in both groups of vaccinees. At 6 and 12 months after 2 doses of vaccines, the seroconversion rates dropped in both groups of vaccinees, so there were 10-20% of children (50-65% if cross protection was considered) susceptible to JEV infections during this period. After the third dose of vaccine, the seroconversion rate rose to 100% in both groups. The GMT in Bejing strain vaccinees were slightly higher than Nakayama strain JE vaccines. To reduce the number of susceptible children during 6-12 months after the second dose and for longer protection, the primary JE immunization should be 3 doses and the timing for the third dose should be at 6 months after the second dose. Either Nakayama or Beijing strain vaccine could be used in Thailand.     

Protective efficacy of a dengue 2 DNA vaccine in mice and the effect of CpG immuno-stimulatory motifs on antibody responses

A recently described DNA vaccine for dengue (DEN) type 2 was shown to elicit high levels of neutralizing antibodies in mice. The vaccine candidate consists of the PreM and 92% of the envelope genes of DEN 2 New Guinea C strain. We further evaluated this DNA vaccine candidate by examining the effect of immuno-stimulatory CpG DNA motifs on antibody response and by studying the protective efficacy of the vaccine. The results showed that CpG motifs present in pUC 19 significantly improved the antibody response to a suboptimal dose of 3.1 micrograms of the DEN DNA vaccine. In a lethal mouse intracerebral challenge model, the vaccine provided a significant level of protection. Sixty percent of the mice immunized with the DEN DNA vaccine plus pUC 19 survived the challenge compared to only 10% in the control group that received vector plus pUC. These studies illustrate that nucleic acid immunization is a viable approach to developing a DEN vaccine and that immuno-stimulatory CpG DNA motifs can be used to lower the minimum dose required to produce an antibody response.     

NMR studies of Borrelia burgdorferi OspA, a 28 kDa protein containing a single-layer beta-sheet

Viruses such as HIV, influenza, picornavirus and others are known stimulators of apoptosis. This individual cellular elimination is a preferential host defense in regenerative tissues. In contrast, if this death occurred in nonregenerating cells, such as neurons of the central nervous system, may result in disease. The target cell for rabies virus is the neuron. Here we studied the outcome of the interaction between rabies virus (CVS-11) and mouse brain cells. Replication of rabies virus in suckling mouse brain cells resulted in brain cell apoptosis, detected by DNA fragmentation and in situ apoptosis within 25 h after infection and before evidence of intracerebral immune activation. Cell death occurred simultaneously with rabies virus replication. There were clinical signs of illness in infected newborn mice within 24 h after the appearance of DNA fragmentation and before infiltration by lymphocytes. This suggested that onset of illness started independently of the immune function. This conclusion was supported by the occurrence of massive apoptosis followed by paralysis in rabies virus-infected immunosuppressed mice. Direct, viral-induced, neuronal apoptosis was the earliest death mechanism detected in these mice. We propose that pathogenesis of this fixed strain of rabies virus in mice begins with the induction of apoptosis by rabies virus replication. Cerebral damage may then be amplified by immunological mechanisms plus an additional unidentified factor. This is followed by increased permeability of the blood brain barrier.     

Thymic dendritic cells are primary targets for the oncogenic virus SL3-3

The murine retrovirus SL3-3 causes malignant transformation of thymocytes and thymic lymphoma in mice of the AKR and NFS strains when they are inoculated neonatally. The objective of the present study was to identify the primary target cells for the virus in the thymuses of these mice. Immunohistochemical studies of the thymus after neonatal inoculation of the SL3-3 virus showed that cells expressing the viral envelope glycoprotein (gp70(+) cells) were first seen at 2 weeks of age. These virus-expressing cells were found in the cortex and at the corticomedullary junction in both mouse strains. The gp70(+) cells had the morphology and immunophenotype of dendritic cells. They lacked macrophage-specific antigens. Cell separation studies showed that bright gp70(+) cells were detected in a fraction enriched for dendritic cells. At 3 weeks of age, macrophages also expressed gp70. At that time, both gp70(+) dendritic cells and macrophages were found at the corticomedullary junction and in foci in the thymic cortex. At no time during this 3-week period was the virus expressed in cortical and medullary epithelial cells or in thymic lymphoid cells. Infectious cell center assays indicated that cells expressing infectious virus were present in small numbers at 2 weeks after inoculation but increased at 5 weeks of age by several orders of magnitude, indicating virus spread to the thymic lymphoid cells. Thus, at 2 weeks after neonatal inoculation of SL3-3, thymic dendritic cells are the first cells to express the virus. At 3 weeks of age, macrophages also express the virus. In subsequent weeks, the virus spreads to the thymocytes. This pathway of virus expression in the thymus allows the inevitable provirus integration in a thymocyte that results in a clonal lymphoma.     

Zoonotic potential of infection with Fasciola spp. by consumption of freshly prepared raw liver containing immature flukes

Mice were successfully infected with metacercariae of the Japanese Fasciola sp., resulting in the recovery of a mean number of 110 live immature flukes per mouse 4-5 days after inoculation. Twenty-four mice were then inoculated orally, each with a mean number of 68 freshly recovered immature flukes. The livers of 7 of the 24 recipient mice showed migratory lesions of capsular and subcapsular granulomatous infiltration and 2 of those mice also had haemorrhagic lesions. The lesions were typical of those caused by active migration of early immature flukes. However, no flukes were found in the livers of the recipient mice at necropsy when the flukes were aged 14 weeks. In another experiment, 10 piglets were given fresh livers of mice harbouring approximately 2000 live immature flukes aged 3-7 days. Two additional piglets were inoculated with 2000 metacercariae of Fasciola. All pigs were killed when the flukes were 14 days old. Granulomatous lesions were present in all pigs, except in those that were given livers containing flukes aged 7 days. The lesions were localized, forming well-defined foci, different from the typical migratory lesions normally observed in mouse or sheep liver at the early stage of fluke migration. From the 10 pigs given livers, 65 live flukes were recovered at necropsy, 0.29% of the estimated number of immature flukes given. From the 2 pigs which received 2000 metacercariae each, a total of 198 flukes were recovered (5%). The results of the experiments suggest that humans consuming raw liver dishes prepared from fresh livers infected with immature Fasciola spp. could become infected with liver fluke.     

A candidate Ross River virus vaccine: preclinical evaluation

A killed Ross River virus vaccine is being developed in an effort to prevent the, ca 5000 cases of epidemic polyarthritis which occur in Australia each year. The symptoms of epidemic polyarthritis commonly last 30-40 weeks and 25% of patients have symptoms for a year or more. There is no cure. Although there was some strain to strain variation, particularly after a single injection, outbred and inbred strains of mice all produced significant levels of anti-Ross River virus antibody after intramuscular (i.m.) injection with 24 h BEI inactivated, sucrose gradient purified, Ross River virus vaccine. Mice immunized i.m. with two 20 micrograms doses of vaccine or live virus produced similar levels of neutralizing antibody but the reaction of IgG 2a and IgG 2b antibody from these two groups of mice to Ross River virus proteins in western blots differed. Antibody from BALB/c mice immunized with this vaccine neutralized all strains of Ross River virus tested, in vitro, albeit to different degrees.     

Failure of protection induced by a Brazilian vaccine against Brazilian wild rabies viruses

This report shows that the SMB vaccine currently used in Brazil for human immunisation provides different degrees of protection in mice, depending on the rabies virus strain used as challenge. Using the NIH and Habel potency tests to evaluate the protective activity of rabies vaccine, we observed that vaccinated mice showed a higher resistance to a challenge with a fixed rabies virus (CVS-Challenge Virus Strain). The vaccine potency using the Habel or NIH tests was respectively > 6.4 (log 10) and 1.0 (Relative Potency-RP) when the fixed rabies virus was used for challenge, and from 2.9 to 4.3 (log 10) or 0.13 to 0.8 (RP) when different wild rabies viruses were used for challenge. The presence of virus neutralising antibodies (VNA) could not explain the differences of susceptibility after vaccination, since sera of vaccinated animals had similar VNA levels against both fixed and wild strains before virus challenge (respectively, 5.6 +/- 0.24 and 5.0 +/- 0.25 IU/ml of VNA against the fixed rabies virus and the 566-M strain of wild rabies virus in sera of mice vaccinated with 0.2 units of vaccine). Only cell-mediated immunity parameters correlated with the protection induced by vaccination. The IFN gamma titers found in sera and brain tissues of animals challenged with CVS strain were higher (from 36.7 +/- 5.7 to 293.3 +/- 46.2 IU/ml) than those found in mice challenged with 566-M virus strain (from 16.7 +/- 5.8 to 36.7 +/- 5.8). The proliferation index of spleen cells obtained with CVS stimulation reached a maximal value of 15.1 +/- 0.7 while spleen cells from vaccinated mice stimulated with 566-M virus failed to proliferate. The implications of these data in human protection by vaccination are discussed.     

Apoptosis plays an important role in experimental rabies virus infection

Cultured rat prostatic adenocarcinoma (AT3) cells infected with the challenge virus standard (CVS) strain of fixed rabies virus showed characteristic morphologic features of apoptosis, evidence of oligonucleosomal DNA fragmentation, and expression of the Bax protein. CVS-infected Bcl-2-transfected AT3 cells did not demonstrate these features. Adult ICR mice inoculated intracerebrally with CVS showed morphologic changes of apoptosis, DNA fragmentation, and increased Bax expression in neurons, with changes most marked in the hippocampus and cerebral cortex. Ultrastructurally, some neurons demonstrated morphologic features more typical of necrosis. These studies provide evidence that apoptosis plays an important role in the pathogenesis of rabies virus infection.     

Effect of the strain of Toxoplasma gondii on the development of toxoplasmic encephalitis in mice treated with antibody to interferon-gamma

To examine whether differences observed in the development of toxoplasmic encephalitis between strains of Toxoplasma gondii are determined by the numbers of cysts occurring in the brain, C57BL/10 mice were infected with ten cysts of the ME49, Beverley, or C56 strain of the parasite. At 10 weeks after infection, the numbers of cysts and inflammatory changes in the brains of the mice were examined in each of the experimental groups. The ME49 strain formed significantly more cysts than did the other two strains, with no difference in the number of cysts being noted between the Beverley and C56 strains. Mice infected with the ME49 strain showed infiltration of inflammatory mononuclear cells in both the meninges and the parenchyma of their brains, whereas mice infected with the Beverley or C56 strain showed no inflammatory changes in their brains. Treatment of mice with a monoclonal antibody to interferon-gamma (IFN-gamma) once weekly for 3 weeks beginning at 10 weeks after infection augmented the inflammatory changes in the brains of mice infected with each strain. However, the intensity of the inflammatory changes differed significantly between the mice, depending on the strain of T. gondii with which they were infected. Mice infected with the ME49 strain showed the most remarkable inflammatory changes in their brains. Mice infected with the Beverley strain developed foci of acute inflammation in their brains after treatment with an antibody to IFN-gamma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mouse hydrocephalus and encephalitis following intracerebral infection with Newcastle disease vaccine viruses

Hydrocephalus and encephalitis in 14-day-old mice was induced by an intracerebral inoculation of a high dose of live Newcastle disease vaccine viruses. The distended lateral ventricles occurred as a result of aqueduct stenosis superimposed on ciliary degeneration and destruction of ependymal cells. The brain virus titres were high on Days 3, 5 and 7 but never exceeded the titre of the original dose given. Positive serum haemagglutination-inhibition titres persisted throughout the experiment.