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RNA synthesis in BHK 21 cells persistently infected with vesicular stomatitis virus and rabies virus.

Virus-induced RNA synthesis was studied in BHK 21 cells persistently infected with vesicular stomatitis virus (VSV) and rabies virus by labelling RNA synthesized in the presence of antinomycin D. During persistent infection the species of messenger RNA synthesized were similar in size and relative proportions to those seen during acute infection, but there were some minor differences. Full-sized B virion RNA was generally not detected during persistent infection, and new species (probably DI virion RNA) appeared.

Animals

Long-term persistent vesicular stomatitis virus and rabies virus infection of cells in vitro.

BHK 21 carrier cells persistently infected with VSV Indiana for over 2 years have been shedding generally very low levels of mature infectious virus or mature T particles (averaging less than one-hundredth p.f.u./cell/day) yet most cells are producing virus antigens and are resistant to homologous superinfection. However, large amounts of biologically active T particle RNP can be recovered from cytoplasmic extracts of these carrier cells even at times when they are shedding no detectable infectious virus. This recovered cytoplasmic RNP replicates (with helper B virions) to produce mature T particles, interferes strongly after DEAE dextran-facilitated uptake and, together with B virions, allows the establishment of a persistent carrier state in exposed cells. No 'provirus' DNA copies of the VSV RNA genome are detectable (less than 1/40 copy/cell or I copy per 40 cells) in carrier cells after more than 2 years of persistent infection, and all transfection attempts have failed using DNA from these VSV carriers or DNA from carrier cells persistently infected with some other negative strand RNA viruses (measles, mumps, LCM, influenza, rabies). Infectious viruses shed after more than I year from carrier cells originally infected with wild-type B virions are small plaque mutants showing a slight temperature sensitivity. Cured cell populations can be obtained from the long term VSV carrier culture by cloning in the presence or absence of antiviral antibody.

Animals

Rabies virus and the problems of rabies vaccination in man.

The structure of rabies virus and the importance of its glycoprotein in immunization are discussed. The improvement in vaccines for use in man, culminating in the production of the human diploid vaccine is described. Nevertheless problems remain, particularly with regard to post-exposure therapy. Possible disadvantages in the use of subunit vaccines are mentioned and attention is drawn to the discovery of the rabies-related viruses.

Antibody Formation

Experimental oral and nasal transmission of rabies virus in mice.

Weanling female white Swiss mice were exposed to challenge virus standard rabies virus and street virus isolates from various domestic and wild animals. Virus was given free choice as suspension or as infected mouse brain by stomach tube, by single injection of suspension into the oral cavity of unanesthetized mice, by repeated injection into the oral cavity of anesthetized mice and by single application to the external nares of anesthetized mice. Challenge virus standard virus in mouse brain suspension and a suspension of skunk salivary glands infected with street virus (titers greater than or equal to 10(6)MICLD50/0.03 ml) consistently produced high rates of infection in mice exposed intranasally, low to high rates of infection in mice exposed by forced feeding and other artificial methods of oral exposure and very low rates of infection when given free choice. Street virus isolates passaged intracerebrally in mice had titers less than or equal to 10(4.5) MICLD50/0.03 ml and rarely caused rabies in mice exposed orally or nasally by any method. The results indicate that with the isolates used, virus of high titer (greater than or equal to 10(6)MICLD50/0.03 ml) is required to consistently produce infection in mice by the nasal route and that the mucosa of the nasal cavity probably is the chief route of infection even after oral administration.

Animals

Suppression of cell-mediated immunity by street rabies virus.

Mice lethally infected with street rabies virus failed to develop cytotoxic T cells specific for rabies virus-infected target cells, whereas high levels of cell-mediated cytotoxicity (CMC) were generated after nonfatal infection with the attenuated high egg passage (HEP) or ERA rabies virus strains. Furthermore concurrent infection with street, but not with HEP, rabies virus suppresses development of a primary (but not a secondary) CMC response specific for influenza virus. No cross-reactivity is found between effector T-cell populations from mice immunized with HEP or with influenza virus. It thus appears that street rabies virus, which is not known to replicate in the cells of immune system, induces some general defect in the primary CMC lymphocyte response, though restimulation of memory T-cell populations is unimpaired and there is no defect in antibody formation. Development of fatal rabies may reflect the operation of this selective immunosuppressive mechanism.

Animals

Clinical use of human globulin immune to rabies virus.

Studies of human globulin immune to rabies virus before licensure showed that it suppressed active antibody responses when individuals received 16 doses of duck embryo vaccine but not when they received 23 doses of duck embryo vaccine. Prospective surveillance of use of human globulin immune to rabies virus since licensure in 1974 has revealed that 40% of persons who receive 14-16 doses of duck embryo vaccine have low or undetectable antibody responses 30-90 days after initiation of the series. Ten percent of individuals receiving 21-23 doses of duck embryo vaccine have inadequate antibody responses, a percentage not significantly different from that found in recipients of 14 doses of vaccine alone. Human globulin immune to rabies virus has also been used for treatment of one case of clinical human rabies; this use was based on the observation that antibodies to rabies virus in serum do not develop until after the seventh day of clinical illness, and antibodies are absent from spinal fluid as long as 19 days after onset of symptoms. Intracranial pressure and neurological function remained stable after administration of human globulin immune to rabies virus, but two days after initiation of therapy, the patient died of progressive pulmonary dysfunction and tension pneumothorax.

Adult

Structure and function of rabies virus glycoprotein.

Of the three major proteins associated with the rabies virus membrane, only the glycoprotein (G-protein) was found to be located on the external surface of the viral membrane. A minor glycoprotein (gp 50) detected by SDS-polyacrylamide gel electrophoresis (PAGE) of rabies virus appeared to be a breakdown product of the G-protein. Glycoprotein prepared by treatment of rabies virus with Triton X100 and purified by isoelectric focusing was found to be homogeneous with respect to size and isoelectric point. The apparent molecular weight of this component isolated under nondenaturing conditions is approximately 400,000 daltons. The same material analyzed by PAGE was found to be comprised solely of polypeptide chains of the G-protein (MW 80,000 daltons). Therefore, the Triton X100-released material represents homopolymers of the G-protein. The purified rabies virus glycoprotein (G) is only structural protein of the virus that induces the formation of virus-neutralizing antibodies and which confers immunity to animals. The total protective activity of the virus was recovered in the purified G protein preparation. The protective activity of G protein increased with purification : 9 ng of G protein was required to protect 50% of the mice as compared to 1.63 microgram of the virus. The number of oligosaccharide side chains on rabies virus glycoprotein was investigated. Analysis of glycopeptides obtained by protease digestion of desialated glycoprotein revealed three discrete glycopeptides. Comparison of the protease digestion products from desialated and from untreated glycoprotein indicated a heterogeneity among the glycopeptides in the sialic acid content. Two major tryptic glycopeptides were isolated from desialated rabies virus glycoprotein and were analyzed after protease digestion; one contained two oligosaccharide side chains and the other contained a single oligosaccharide side chain.

Chemical Phenomena

[Haemagglutinin from rabies viruses (author's transl)].

Rabies virus haemagglutinin has not hitherto been obtained to our knowledge, from brain material of infected animals. A slight modification of the arbovirus technique and the use of trypsin treatment can reveal a good haemagglutinin from fixed and street strain from infected brains.

Animals

[Preparatory extraction and study of the structural proteins of rabies virus].

A technology for preparation of purified concentrates of rabies virus has been developed permitting to use simultaneously dozens of liters of tissue culture virus-containing fluid for the preparation of a concentrate. When concentrated 10(4)-fold or more, the degree of virus purification relative soluble proteins was at least 8 X 10(6). The virus yield in purified preparations was approximately 500 microgram of protein per 10 1 of the original culture fluid. Purification and preparation of a surface antigen of rabies virus, protein G, was also carried out. The yield of this protein in the resulting preparations was about 170 microgram per 10 1 of the culture fluid, the degree of purity being about 90%. A re-evaluation of the set, characteristics, and localization of rabies virus structural proteins was done. According to the experimental results, virus nucleocapsids contain 3 classes of proteins: L (a minor components with molecular weight of approximately 200,000), N (the dominating component, approximately 54,000) and NS (an intermediate component, approximately 47,000). The inner layer of virus lipoprotein membrane consists of protein M molecules (approximately 21,000) and apparently of protein A molecules (approximately 43,000) which are likely to be of cellular actin. The external spikes (peplomers) of virus particles contain molecules of glycoprotein G (approximately 65,000). Thus, the experimental results permit a conclusion that previously assumed significant differences in the protein composition of rabies viruses on the one hand and the other rhabdoviruses on the other are non-existent.

Centrifugation, Density Gradient

Isolation and purification of a polymeric form of the glycoprotein of rabies virus.

Of the three major proteins associated with the rabies virus membrane, only the glycoprotein was found to be located on the external surface of the virus membrane. Glycoprotein prepared by treatment of rabies virus with Triton X-100 and purified by isoelectric focusing was found to be homogeneous with respect to size and isoelectric point. This material, which is free of phospholipids, is able to protect in vaccination experiments against a lethal challenge infection with rabies virus. The apparent mol. wt. of this component isolated under non-denaturing conditions is approx. 400000. The same material analysed by SDS polyacrylamide gel electrophoresis (PAGE) was found to consist solely of polypeptide chains of the G protein (mol. wt. 80000). A minor glycoprotein (gp 50), detected by PAGE of the Triton X-100 released material, appeared to be a breakdown product of the G-protein. Therefore the detergent released material represents homopolymers of the G-protein. Whether the antigenic determinants reside on the monomeric subunit or are a property of the polymeric form of the G-protein is discussed.

Epitopes

Detection of antigenic differences among street and fixed rabies virus strains by the counterimmunoelectrophoresis test.

Using the counterimmunoelectrophoresis test, two street rabies virus strains (Mangosta and Apipe) revealed antigens not detected in the standard challenge virus (CVS). No antigenic differences, however, were found between CVS and another fixed rabies virus strain (strain 91) by this method. The results of serum neutralization tests revealed similar antigenic relationships among the rabies virus strains studied. These findings are discussed in terms of their significance to antigenic variations in rabies virus strains.

Counterimmunoelectrophoresis

Isolation of rabies virus from bats in Bolivia.

Rabies virus was isolated from apparently normal blood-sucking (Desmodus rotundus) and insectivorous (Artibeus planirostris) bats caught in Bolivia. The virus was identified by immunofluorescence, biological and sero-neutralisation tests.

Animals

Reevaluation of the proteins in rabies virus particles.

Protein content and localization of individual proteins of rabies virus have been studied. Four major proteins (estimated molecular weights, about 65,000, 54,000, 37,000 and 21,000), one minor component (molecular weight, about 200,000), and one intermediate (as regards its molar concentration) component (molecular weight, about 43,000) were revealed in rabies virus particles. In subviral particles accumulating in virus-infected cells, the 200,000-, 54,000-, and 37,000-dalton components were revealed. Some properties of the subviral particles allow them to be considered as viral nucleocapsids and the proteins composing them as analogs of L, N, and NS proteins of other rhabdoviruses. Thus, the protein composition of the rabies virus strain studied does not differ from that of other rhabdoviruses.

Cell Line

Rabies virus protein synthesis in infected BHK-21 cells.

Rabies virus specific polypeptide synthesis was examined under hypertonic conditions, which selectively inhibit cellular protein synthesis. The rabies virus proteins (L, G, N, M1, M2) were synthesized throughout the course of infection, with little change in their relative rates of synthesis. The rates of synthesis of the G and M1 polypeptides were more sensitive to increasing osmolarity than those of the L, N, and M2 polypeptides. Extrapolation to isotonicity of the results obtained under hypertonic conditions indicated that the molar ratios of the polypeptides synthesized under normal conditions were 0.4 (L), 64 (G), 100 (N), 75 (M1) and 35 (M2). A high-molecular-weight polypeptide (190,000), designated polypeptide L, was repeatedly detected both in infected cells and in extracellular virus. The estimated number of L polypeptide molecules per virion was 33. The synthesis of a viral glycoprotein precursor, designated gp78, , preceded the appearance of the mature viral glycoprotein in infected cells labeled with [3H]glucosamine under isotonic conditions. In cells labeled under hypertonic conditions, little or no mature viral glycoprotein was detected, but a virus-specific glycoprotein with an electrophoretic mobility similar to that of gp78 was observed. This glycoprotein could be chased into mature viral glycoprotein when the hypertonic conditions were made isotonic. These results suggest that a reversible block of viral glycoprotein synthesis occurs under hypertonic conditions.

Animals

Transcriptional mapping of rabies virus in vivo.

Synthesis of the proteins of rabies virus was studied in hamster cells infected with UV-irradiated virus. The UV target size of genes L, N, M1, and M2 was measured during primary transcription. Except for N, the target size of the remaining genes was considerably larger than that of their physical sizes. The data fit the hypothesis that four genes occupy a single transcriptional unit and that transcription of rabies virus proceeds in the order N, M1, M2, and L.

Cell Line