Monensin-resistant mouse Balb/3T3 cell mutant with aberrant penetration of vesicular stomatitis virus.

A mutant (MO-5) resistant to monensin (an ionophoric antibiotic) derived from the mouse Balb/3T3 cell line, was a poor host for vesicular stomatitis virus (VSV) or semliki forest virus (SFV) multiplication. The yield of VSV particles in MO-5 is one 100-fold reduced as is VSV-dependent RNA synthesis. In contrast to a pH-remedial mutant, the abortive production of infectious VSV particles in MO-5 cells was not restored by low pH treatment. The pH values in the endosome and the lysosome of MO-5 cells were 5.2 and 5.4, respectively, values that were comparable to the pH value in Balb/3T3 cells. Assays with [3H]uridine-labeled VSV indicated similar binding of VSV in MO-5: percoll gradient centrifugation analysis of [35S]methionine-labeled VSV-infected Balb/3T3 showed accumulation of VSV in the lysosome fraction 20 min after VSV infection, whereas VSV can be found mainly in endosome/Golgi fraction of MO-5 cells after 40 to 60 min on the percoll gradients. Degradation of [35S]methionine-labeled VSV was observed at a significant rate in Balb/3T3 cells, but not in MO-5 cells. The monensin-resistant somatic cell may thus provide a genetic route to study the mechanism of endocytosis or transport of enveloped viruses.

Auditory brain stem responses in relation to the clinical symptoms of schizophrenia.

Auditory brain stem responses (ABSR) were examined systematically both in normal and schizophrenic subjects. All ABSR wave forms were evoked by click stimuli, which were delivered binaurally through headphones both to normal subjects and to a nondeteriorated group of schizophrenics. However, in the group of schizophrenics with marked deterioration of personality, not all wave forms were elicited, and the amplitudes were low. The characteristics of the ABSR wave forms correlated well with the clinical symptoms of the schizophrenic illness. The cause of these ABSR wave form changes in schizophrenics is discussed.

Hepatitis B virus harboring nucleotide deletions in the core promoter region and genotype B correlate with low viral replication activity in anti-HBe positive carriers.

BACKGROUND: Emergence of anti-HBe following seroconversion of HBe antigen indicates reduced hepatitis B virus (HBV) replication in the liver and low infectivity in the natural course of infection. However, some patients show continued replication or reactivation even in the presence of anti-HBe. OBJECTIVE: To clarify the cause of HBV replication, we investigated genotype differences and mutations in the core promoter and precore region in relation to virus titer. STUDY DESIGN: Using quantification of HBV DNA, nucleotide sequencing of the core promoter and precore region, and genotyping with the S gene by restriction fragment length polymorphism (RFLP), we analyzed sera of 26 anti-HBe positive carriers (28 serum samples). RESULTS: Various mutations were detected including C to T point mutation at nt 1653, A to T and G to A contiguous point mutations at nt 1762 and 1764 in the core promoter region, and G to A point mutation at nt 1896 in the precore region, but no common mutations were detected that were directly related to the virus titer from earlier reported mutations. In contrast, the mean titer of genotype B virus was 1.5 x 10(5) copies per ml and that of mutant HBV of genotype C having 8 base pairs (8-bp) deletion (nt 1768-1775) in the core promoter region was 7.9 x 10(4) copies per ml (mean titer). These titers showed commonly lower than that of genotype C virus without 8-bp deletion (median titer 5.0 x 10(6) copies per ml). Transition of genotype from C to B after viral reactivation and reduction of proportion of 8-bp deletion mutant at reactivation period was observed in a patient who demonstrated exacerbation of liver dysfunction due to immunosuppressive therapy and increased viral replication. CONCLUSIONS: These results confirm those of our earlier study describing low replication ability of 8-bp deletion mutant HBV in vitro, and also indicate that the presence of genotype B correlates with reduced titer of HBV.

Genotyping of hepatitis C virus in the Dominican Republic.

Genotyping of hepatitis C virus (HCV) of liver disease patients in the Dominican Republic was performed. Eighty-four samples positive for HCV antibody, which were confirmed by ELISA, particle agglutination, and recombinant immunoblot assay III tests, were subjected to HCV genotyping by polymerase chain reaction using type-specific primers located in the nonstructural protein 5 region. Of the 84 samples tested, 50 (59%) were found to have genotype 1a/I and this genotype was the most frequent type detected in the present study. The numbers of isolates of genotypes 1b/II, 2a/III, 2b/IV, and 3a/V were three (3.6%) six (7.1%), two (2.4%), and two 2.4%), respectively. The number of samples having mixed genotype populations was 16 (19%). The possible causes of the high prevalence of genotype 1a/I in the Dominican Republic compared with other countries and of the high detection ratio of samples having mixed genotypes are discussed.

Circadian variation in amikacin clearance and its effects on efficacy and toxicity in mice with and without immunosuppression.

A once-daily dosage regimen has been recently recommended in the use of aminoglycoside antibiotics since they induce a postantibiotic effect. In choosing this regimen, one must determine the most appropriate time of day for administration of the drug. We investigated the effects of the timing of amikacin (AMK) administration on the kinetics, the efficacy against intraperitoneal infection with Pseudomonas aeruginosa, and the toxicity of AMK in mice with and without immunosuppression. We found circadian variations in the kinetics, efficacy, and toxicity of the drug in mice. Male and female ICR mice, which were housed under a light-dark (12:12 h) cycle with free food and water intake, were injected subcutaneously with AMK sulfate 50 mg/kg body wt. There was a circadian variation in AMK clearance for both sexes with the maximum value in the dark phase and the minimum in the light phase after a single administration. When AMK 500 mg/kg/day was repeatedly administered once daily for 30 days, higher toxicity was demonstrated in mice injected with the drug at the time of day with lower AMK clearance, although no difference was demonstrated in the toxicity between the two time points with different AMK clearance when AMK 1,500 mg/kg was administered in a single dose. The ED50 of AMK to cure the infected mice in the midlight phase (13:00 h) with lower clearance was significantly lower than that in the middark phase (01:00 h) with higher clearance. In contrast, the ED50 in the early light phase (09:00 h) was significantly lower than that in the early dark phase (21:00 h), although AMK clearance was no different between these two different time points. In mice premedicated with cyclophosphamide to suppress immune functions, the difference in the ED50 of AMK was still demonstrated between 13:00 and 01:00 h, but not between 09:00 and 21:00 h. The present study shows not only that there were circadian variations in both AMK clearance and toxicity after repeated administration, but also that there was a circadian variation in the efficacy of AMK in mice infected with P. aeruginosa. These results suggest that the timing of drug administration should be considered in pharmacotherapy with AMK and that the most appropriate time of administration in mice and nocturnal animals may be in the midlight (resting) phase. They also suggest that the ED50 of AMK against P. aeruginosa infection may be influenced not only by the circadian variation in pharmacokinetics but also by the variations in immune systems suppressed by cyclophosphamide.

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.

Comparison of rabies virus G proteins produced by cDNA-transfected animal cells that display either inducible or constitutive expression of the gene.

By using a retrovirus expression vector, pZIP-NeoSV(X)1, we introduced a cloned cDNA of the rabies virus G gene into BHK-21 cells and the NA cell clone originated from the murine neuroblastoma C1300 line. Using the neomycin resistance gene of the vector, we isolated several G418-resistant transformants of BHK-21 and NA cells (referred to as G-BHK and G-NA cells, respectively). G-BHK cells constitutively produced G proteins, whereas G-NA cells produced the proteins only when treated with sodium butyrate. G proteins synthesized in these transformants were transported normally to the surface of the cell, but they displayed different electrophoretic mobilities, which were shown to originate from differences in the number and structure of the carbohydrate moieties of the protein; G-BHK cells produced highly glycosylated and sialylated G proteins, whereas less glycosylated and much less sialylated G proteins were produced by G-NA cells as observed in virus-infected NA and BHK-21 cells, indicating that the glycosylation and sialylation of the G protein depend on the cellular conditions under which the protein was produced. In the absence of sodium butyrate the G protein was not detectable in G-NA cells either by immunoblot assay or fluorescent antibody staining, but the cells were fairly sensitive to syngeneic rabies virus-specific cytotoxic T lymphocytes, although the sensitivity was much increased by treatment with sodium butyrate.

Demonstration of hemolytic and fusion activities of influenza C virus.

Influenza C virus showed a marked hemolytic activity when incubated with murine erythrocytes at 37 degrees C in acidic medium. The virus-specific hemolysis was most efficient at pH 5.0. Extensive cell fusion also occurred when the erythrocytes were treated with the virus at acidic pH. When propagated in MDCK cells, the virus had an extremely low infectivity and did not display hemolytic activity in any pH range. When the inactive virus was subjected to mild trypsin treatment, hemolytic activity was drastically manifested, accompanying a drastic increase in infectivity. The glycoprotein in the inactive virus was cleaved into smaller components by trypsin treatments. These results indicated that the envelope of influenza C virus can fuse with the cellular membrane under acidic conditions and that the activation of influenza C virus by cleavage was due to the appearance of this envelope fusion activity.

Protective effect of measles virus inoculation on subacute sclerosing panencephalitis virus-infected mice.

The Biken strain of subacute sclerosing panencephalitis (SSPE) virus caused a fatal neurologic disease in adult mice after intracerebral inoculation. However, the mice were completely protected from the disease when a high dose of measles virus was given intracerebrally after the SSPE virus infection. The measles virus inoculation induced interferon production and immune responses. An experiment with athymic nude mice showed that interferon and anti-measles antibody were able to prolong the incubation period of the disease but not to protect the SSPE virus-infected nude mice from death. For complete protection, T lymphocytes appeared to be essential. The present study suggested that the protective effect of measles virus inoculation is basically due to the induction of immune responses and that SSPE virus infection in mice is susceptible to immune reactions.

pH-dependent hemolysis and cell fusion of rhabdoviruses.

Human erythrocytes pretreated with fungal semialkali protease or trypsin became susceptible to hemagglutination by vesicular stomatitis virus (VSV) and rabies virus. Both viruses exhibited extensive hemolytic and fusion activities against erythrocytes pretreated with these enzymes. The hemolysis and fusion were pH dependent and the activities were most apparent at pH 5.0 and decreased with increase in pH. However, VSV still exhibited slight hemolytic activity at neutral pH. Hemolysis was also dependent on the dose of virus and was inhibited by treatment of the viruses with antiviral antibody. Results of sodium dodecyl sulfate polyacrylamide gel electrophoresis of erythrocyte membranes suggested that most of the carbohydrates were removed from the membrane proteins by the treatment with proteolytic enzymes.

[Immunosuppressive factor(s) present in crude preparations of human chorionic gonadotropin].

The present study was undertaken to purify and characterize the immunosuppressive factor(s) present in commercially available crude human chorionic gonadotropin (crude hCG). Amberlite CG-50 column chromatography of crude hCG(2,680 IU/mg) produced hCG(7,130 IU/mg) and non hCG(130 IU/mg) fractions. The hCG fraction was further fractionated by Sephadex G-100 column chromatography and a highly purified fraction with a potency of 18,600 IU/mg was obtained. The non hCG fraction was separated into two fractions, F-1 and F-2, on Sephadex G-75. Each fraction was examined for its inhibitory effect on the incorporation of 3H-thymidine by normal human peripheral blood mononuclear cells stimulated with phytohemagglutinin (PHA test) or mixed lymphocyte reaction (MLR test). The original crude hCG showed considerable inhibition in both PHA and MLR tests, but the purified hCG showed no inhibition. F-1 fraction, having a molecular weight (M.W.) ranging from 75,000 to 100,000 daltons, was the most potent in the inhibition of all the other fractions. The inhibitory activity in the F-1 fraction was dose-dependent and relatively stable when exposed to heat and trypsin treatment, but completely inactivated by neuraminidase treatment. These results strongly suggest that the macromolecular substance(s), with a M.W. of 75,000 to 100,000, which is present in crude hCG but different from genuine hCG, has potent immunosuppressive effects and that sialic acid residues in the substance(s) are related to the manifestation of these effects.

Enhanced antibody responses in mice by combined administration of interferon with rabies vaccine.

Exogenous interferon administered to mice at the time of, and 6 hours after the first dose of 3 daily vaccinations accelerated and enhanced the IgM and IgG antibody responses to rabies virus. The effect of interferon was not evident when the interferon was administered later in the vaccination schedules and was abrogated by prior administration of anti-interferon antibody to the mice. The number of IgM antibody secreting cells in the spleen was significantly greater in mice treated with interferon than in controls.

Further studies on an improved haemagglutination inhibition test with higher sensitivity for rabies virus antibody.

The efficiency of the removal of non-specific inhibitors of rabies virus haemagglutinin by treatment with colloidal silicic acid, which was proposed in an earlier study, was examined in a number of test samples. The non-specific inhibitors were removed in 289 out of 296 normal human sera (97.6%) by this treatment to a level that was undetectable at the 1:4 starting dilution in the haemagglutination inhibition test. Antigenic differences among three strains of rabies virus were detected in the haemagglutination inhibition test and the antibody titres to the homologous antigens were apparently higher than those to heterologous antigens.

Murine T cell clones directed to rabies virus: isolation and some of their properties.

Seventeen Thy-1+ cell clones were induced in A/J mice immunized with the HEP-Flury strain of rabies virus after repeated stimulations with antigens in vitro. Ten clones with cell surface phenotypes Thy-1+, Lyt-1-,2+ were cytotoxic T lymphocytes (CTL) which lysed the virus-infected target cells under H-2 restriction. Target cells expressed the G and M2 structural proteins of rabies virus on their surface; however, target lysis by CTL clones was not blocked by anti-rabies antibody or by monoclonal antibodies to these proteins. All of the CTL clones efficiently and equally lysed target cells infected with three different strains of rabies virus and were cross-reactive for target cells infected with one (Duvenhage virus) of three different rabies serogroup viruses. Another five clones having phenotype Thy-1+, Lyt-1+,2- did not show any cytotoxic activity. The proliferation response of these clones to antigen stimulation was virus-specific and H-2-restricted. These clones were able to grow in culture medium without any or with the addition of low concentrations of T cell growth factor, in contrast to CTL clones, and were considered to be helper T lymphocytes (HTL). Both CTL and HTL clones produced gamma-interferon in response to antigen stimulation. The remaining two clones were Thy-1+, Lyt-1-,2-, asialo-GM1+, and were not cytotoxic to target cells even in the presence of anti-rabies antibody but were cytotoxic to YAC-1 cells. Further studies with these clones should allow us to investigate more closely the role of T cells in the pathogenesis of rabies.

Methods of increasing the sensitivity of the haemagglutination inhibition test for rabies virus antibody.

A new method for the removal of non-specific inhibitors of rabies virus haemagglutinin has been developed. Treatment with colloidal silicic acid (Aerosil) or with acetone plus Aerosil reduced the non-specific inhibitors in human, mouse, and dog sera to a level that was undetectable at the 1:4 starting dilution in the haemagglutination inhibition test.Bromelain-treated goose erythrocytes were much more susceptible to haemagglutination by rabies virus than were untreated erythrocytes, and the sensitivity of the haemagglutination inhibition test was considerably increased by using bromelain-treated erythrocytes. Low levels of antibodies in sera from immunized human subjects were detected with higher sensitivity by combining Aerosil treatment of the sera with the use of bromelain-treated goose erythrocytes in the haemagglutination inhibition test.

Conserved nucleotide sequence of rabies virus cDNA encoding the nucleoprotein.

The cDNAs of rabies virus (the CVS strain) encoding the structural proteins (G, N, NS, and M) were cloned. Of these clones, the nucleotide sequence of the cDNA encoding the nucleoprotein was determined to compare with those of other strains of rabies virus. The comparison confirmed that the nucleotide sequences and deduced amino acid sequences are highly conserved among strains including an avirulent strain.

Effect of exogenous interferon on rubella virus production in carrier cultures of cells defective in interferon production.

An established cell line (Vero) defective in interferon production was used to evaluate the role of interferon in chronic rubella virus infections of cell cultures. Inoculation of Vero cells with a low multiplicity of virus resulted in the development of carrier cultures which had the characteristics of a regulated infection. Although added interferon did not alter rubella virus production in carrier cultures of cells capable of producing interferon, such added interferon caused a dramatic reduction of virus production in the carrier cultures of Vero cells. There was a reduction of the fraction of cells producing virus in Vero carrier cultures, but not in carrier cultures of other cells upon incubation in the continual presence of rubella virus antibodies. In addition, the fraction of infected cells fluctuated in carrier cultures in Vero cells. The data indicate that interferon is not necessary for maintaining a chronic rubella virus infection in vitro and suggest an instability of the virus genome in Vero cells.