Detection of a novel Borna disease virus-encoded 10 kDa protein in infected cells and tissues.

Borna disease (BD) is a transmissible, progressive polioencephalomyelitis primarily of horses and sheep. The genomes of two cell-adapted strains of Borna disease virus (BDV), the aetiological agent of BD, have been cloned and sequenced. According to the structural characterization achieved so far, BDV contains a non-segmented negative-sense 8.9 kb single-stranded RNA genome. In this paper we report the expression, purification and intracellular tracing of a novel non-glycosylated BDV-specific protein with a molecular mass of approximately 10 kDa (BDV p10 protein). The successful isolation of the corresponding mRNA from infected cells, amplification of the genetic region by RT-PCR and its efficient expression as a glutathione S-transferase (GST) fusion protein demonstrated that antibodies specific for the BDV p10 protein are induced in infected animals. In addition, we have produced monospecific antisera against the GST-p10 fusion protein in rabbits. This monospecific antiserum recognized the BDV p10 protein in brain cells of naturally and experimentally infected animals as well as in persistently BDV-infected cells. Antibody-mediated affinity-chromatography using the anti-p10 serum could successfully be applied to purify a ca. 10 kDa antigen from infected animal cells to such an extent that glycosylation of this component could be ruled out.

Mapping of cross-reacting and serotype-specific epitopes on the VP3 structural protein of the infectious bursal disease virus (IBDV).

The binding sites of a panel of monoclonal antibodies cross-reacting with the structural protein VP3 of the two serotypes of the infectious bursal disease virus (IBDV) could be mapped to four segments of the VP3 gene. Two of these antigenic domains also carry epitopes which are specific for one serotype only. Formation of the common or type-specific epitopes is in agreement with homologous or mismatching amino acid sequences yielding hydrophilic segments on the VP3 polypeptide. These antigenic patterns obtained by immunoblotting could be verified by a competitive ELISA.

Protection of mice against an influenza virus infection by oral vaccination with viral nucleoprotein incorporated into immunostimulating complexes.

Influenza A virus nucleoprotein (NP) was integrated into immunostimulating complexes (ISCOMs) after attachment of bacterial lipopolysaccharide to the antigen. Oral immunization with these NP-ISCOMs protected mice fully against an otherwise lethal challenge infection with an unrelated influenza virus subtype without the appearance of severe clinical signs or extensive pathological lesions in the lungs. Mice immunized with analogous bovine serum albumine-incorporated ISCOMs all died. After oral immunization, high titers of NP-specific antibodies, particularly IgA, could be detected in the bronchoalveolar fluid and in the blood serum. No cytotoxic lymphocytes could be demonstrated in the spleens or the lungs of vaccinated mice, and no anti-NP antibody-dependent cytolysis of infected host cells was mediated by complement or in the form of an antibody-dependent cell cytotoxicity. However, a vigorous delayed-type hypersensitivity reaction was produced after probing vaccinated animals with purified NP. No comparable protective immunity or antibody response was induced by a strictly intragastric administration of NP-ISCOMs. It appears, therefore, that the general and local immune response in the lungs was primarily stimulated through contact of NP-ISCOMs with the mucous membrane of the oro-pharyngeal cavity and that cytotoxic effects did not play a major role for the establishment of the protective immunity. Partial protection against a lethal challenge was observed in chickens immunized with NP-ISCOMs in the drinking water.

Isolation and immunogenic properties of a monomeric form of the HA1 subunit of the influenza virus haemagglutinin from infected cells.

A monomeric, truncated form of the HA1 subunit of the haemagglutinin of fowl plague virus can be isolated from chorioallantoic membranes of infected eggs. This type of soluble HA1 seems to be generated by the elimination of the amino-terminal 19 amino acids from the native HA1, including the disulphide linkage to the HA2 subunit. The same type of truncated HA1 could be isolated from a filtrate of the allantoic fluid of infected embryonated eggs. Antibodies prepared against this monomeric soluble form of HA1 did not inhibit haemagglutination or neutralize viral infectivity, but interfered with virus release and would be expected to impair the spread of virus after infection.

[Concentration of species alien (bovine) IgG in the blood serum of foals after the intake of non-species specific colostrum preparation]. / Konzentration von speziesfremdem (bovinem) IgG im Blutserum von Fohlen nach Aufnahme einer nichtspeziesspezifischen Kolostrumzubereitung.

Sixteen vital foals with free access to maternal colostrum received a additional non-species-specific commercial colostrum additive within the first 18 hours of their life. The additive had been prepared from bovine colostrum. At birth no bovine IgG was detectable. The concentration of bovine IgG reached its maximum 18 hours post natum with XG = 74.6 mg/dl. 96 hours after birth IgG levels had dropped to XG = 20.9 mg/dl. The correlation of bovine IgG with GGT-activity was highly significant. Formation of antibodies against bovine IgG could not be demonstrated. It is not possible to increase igG in the circulation of newborn foals to satisfactory levels with IgG of bovine origin in the used commercial colostrum additive.

Analysis of virus-specific RNA species and proteins in Freon-113 preparations of the Borna disease virus.

Treatment of homogenates from Borna disease virus (BDV)-infected brain tissue or cell cultures with Freon-113 yielded infectious particles with a buoyant density of 1.16-1.22 g/ml. Positive- and negative-stranded BDV-specific RNA species as well as three virus-specific proteins, known to be present in BDV-infected cell extracts, were demonstrated in these Freon-treated fractions. When the Freon-purified virus preparations were treated with RNase A prior to RNA extraction, only negative-stranded, genomic RNA was detected in Northern blot hybridizations using sense and antisense RNA probes. These data substantiate that BDV is a negative-stranded RNA virus.

The genetic basis for the antigenicity of the VP2 protein of the infectious bursal disease virus.

The genomic region coding for the antigenic structure responsible for the induction of neutralizing antibodies was localized in the central variable region of the VP2 gene by comparing the nucleotide sequence of five escape mutants derived from the standard infectious bursal disease virus strain Cu-1. Exchange of a single amino acid at one of the prominent hydrophilic parts of this region proved to be sufficient for altering the neutralizing properties. The reactivity of neutralizing antibodies with peptides expressed in vitro encompassing both hydrophilic areas suggests that the entire variable region is engaged in the formation of this conformation-dependent antigenic site. VP2-specific, non-neutralizing monoclonal antibodies directed against the sequence-dependent epitope of the serotype I strain Cu-1 and the serotype II strain 23/82 cross-reacted with peptides located towards the carboxy terminus of VP2; no reaction occurred with peptides derived from the amino-terminal side adjacent to the variable region.

Infectious bursal disease of poultry: antigenic structure of the virus and control.

The present knowledge of genome organisation, structural basis of pathogenicity and antigenicity of infectious bursal disease virus (IBDV) are briefly reviewed. The current situation of IBDV infection in various countries is stated and recommendations for improved vaccination schemes are given.

Virus disease as a consequence of viral pathogenicity and the anti-viral immune response.

The brief description of two virus systems, influenza and infectious bursal disease, shows enigmatically how at least two requirements must be met to render a virus pathogenic: the array of the whole genome rather than the formation of a particular "pathogenicity gene" and the capacity of the host cell to provide the appropriate microenvironment for an optimal posttranslational processing of structural proteins. In the case of influenza viruses this relates particularly to the cleavability of the haemagglutinin. Efficient virus replication in cells of vital importance, however, does not necessarily result in the development of pathological conditions, as in Borna disease, where neural cells are loaded with virus, and the disease is mediated by a T cell immune response. Immunological stimuli against this virus do not induce neutralizing antibodies which could mount a protective immunity. Infection with influenza viruses is inhibited by neutralizing antibodies, but the course of the disease in an infected organism is largely influenced by virus-specific antibodies which block virus release. It is difficult, however, to evaluate the effectiveness of this type of mechanism directed against the infected cell besides antibody-dependent and cell-mediated cytolysis.

The structural polypeptide VP3 of infectious bursal disease virus carries group- and serotype-specific epitopes.

Two independent non-overlapping epitopes could be demonstrated on the structural protein VP3 of infectious bursal disease virus by non-neutralizing monoclonal antibodies produced against serotypes I and II. Both serotypes have one epitope in common, whereas the second epitope is distinct for serotype I and serotype II.

The influenza virus-infected host cell, a target for the immune response.

The exposure of some viral antigens at the surface of infected host cells was studied as an essential stimulus for the immune response during influenza virus infections. Only antibodies directed against the HA1 of the haemagglutinin were bound to the cell surface, anti-HA2 antibodies did not gain access to the membrane. Attempts to purify the cell-associated haemagglutinin (formerly called "viromicrosomes" by, R. Rott) indicated the formation of a special form of truncated haemagglutinin. This antigen and the nucleoprotein was purified from infected chorioallantoic membranes by immunoaffinity chromatography. NP-specific epitopes could not be defined on the native NP molecule which seems to be in solution in a discoordinate and partially polymeric array. Three non-overlapping epitopes were assigned to proteolytic peptides of the NP. Purified NP could induce cross-reactive cytotoxic T-cells in mice, but these animals were not protected against a challenge infection. CTL induced by exogenous stimulation or intracellular synthesis of the NP were restricted by MHC class I in both cases. NP could be incorporated into ISCOM after amphiphatic modification of the molecule. NP-ISCOM conferred some protection to experimental mice, but did not induce CTL demonstrable in vitro.

Infectious bursal disease--B cell dependent immunodeficiency syndrome in chickens.

Infectious bursal disease of chickens can run an acute lethal course, or death can result from a B cell-dependent immunodefect due to destruction of the bursa of Fabricius following infection with infectious bursal disease virus (IBDV). This member of the Birnaviridae has been characterized, the nucleotide sequence and coding capacity of the two genomic segments of dsRNA has been determined, and the functional significance of the four structural proteins has been largely elucidated. The antigenic structure of the two main structural components permits differentiation of two serotypes; the antigenic domain responsible for the induction of neutralizing antibodies resides, in a conformation-dependent fashion, on one of these proteins. Various types of defective particles are formed and various degrees of pathogenicity appear, depending on the host cell in which the virus replicates. B cells in the bursa offer optimal conditions for virus replication. Pathogenic properties of the virus are also influenced by both genomic segments and cannot be attributed to a single gene. Many aspects of practical interest for disease control are briefly discussed.

[Scrapie, still always a puzzling infection]. / Scrapie, eine immer noch rätselhafte Infektion.

Scrapie belongs to the spongiform encephalopathies in man and animals. The nature of the infectious agent, an "unconventional virus", has not been elucidated so far. The agent starts to replicate in lymphoid tissue, reaches high titers in the brain and induces the formation of amyloid in this organ. After trials to purify the agent, the infectivity proved to be associated with a protein, which has therefore been called "prion", and with rod-like structures from brain tissue (scrapie-associated fibrils, SAF). This protein is considered to be a reaction product of the infection rather than the infectious agent itself.

Heterogeneity of the antigenic site responsible for the induction of neutralizing antibodies in infectious bursal disease virus.

Using neutralizing monoclonal antibodies, three categories of escape mutants were selected from a stock of wild-type infectious bursal disease virus (IBDV). Additional mutants were found, where alterations coexisted in two or three of these epitopes. Although each group of mutants had a distinct reaction pattern with neutralizing monoclonal antibodies, all types of mutants were neutralized by convalescent chicken sera to the same extent. In spite of the lack of homogeneity in these antigenic sites located on IBDV structural polypeptide VP2, all neutralizing monoclonal antibodies reacted with epitopes in extracts prepared from the bursa of Fabricius from animals that had died during recent outbreaks of infectious bursal disease in the F.R.G. and Africa. Since binding to VP2 of the escape mutants, demonstrable by immunoprecipitation, correlated with the neutralizing capacity of these antibodies, a combined immunoprecipitation-immunoblotting technique was established as equivalent for a neutralization assay. The results of our experiments indicate that IBDV did not undergo a major antigenic variation in these two areas of Europe and Africa. The significance of protein conformation for the interaction of VP2 with neutralizing antibodies was underlined by the finding that renatured VP2 was capable of binding neutralizing antibodies; the antibodies induced in animals by immunization with this protein, however, were not neutralizing.

Characterization and immunological properties of influenza A virus nucleoprotein (NP): cell-associated NP isolated from infected cells or viral NP expressed by vaccinia recombinant virus do not confer protection.

A nucleoprotein (NP) preparation purified from the chorioallantoic membrane of chicken eggs infected with fowl plague virus (A/FPV/Rostock/34, H7N1) yielded, in addition to the commonly known 56K protein, a 42K component that could not be detected in virus particles. After testing with a series of NP-specific monoclonal antibodies it was found that some reacted with both proteins and others were bound only by the 56K protein. Among both types of NP-specific monoclonal antibodies only a limited number were bound to infected murine cells. Some antibodies bound to cells infected with a given subtype failed to react with the surface of cells infected with a different subtype. Binding was demonstrated by cellular ELISA, radioimmunoassay and immunofluorescence. The results indicate that only restricted antigenic domains of the native NP and perhaps NP fragments are exposed at the surface of infected murine cells. Additionally, the purified NP preparation was used to immunize mice in order to determine the protective capacity of cell-associated NP. In parallel, and as a relevant control, mice were immunized with a vaccinia virus recombinant containing the gene for NP prior to challenge with infectious virus. High levels of monospecific antibodies and a cytotoxic T cell activity was found in mice immunized with purified NP or infected with the vaccinia recombinant after secondary restimulation in vitro. After treatment with specific antibodies the cytotoxic cells were shown to be classical CD8+ cytotoxic T lymphocytes. Despite the elicitation of a humoral and a cellular immune response by the forms of NP employed mice were not protected from influenza virus infection.

Immunogenic properties of ISCOM prepared with influenza virus nucleoprotein.

After covalent attachment of bacterial lipopolysaccharide to the nucleoprotein of influenza A virus, this water-soluble antigen could be incorporated firmly into ISCOM. This potent "immunostimulating complex" induced the production of high antibody titers in mice and could partially protect the animals from a lethal challenge infection. After immunization with ISCOM preparations NP-specific cytotoxic T cell activity could not be demonstrated.

The glycoprotein of influenza C virus is the haemagglutinin, esterase and fusion factor.

Of the biological activities of influenza C virus, haemagglutination, receptor inactivation and fusion, only the latter has been conclusively correlated with its surface glycoprotein (gp). We have purified the gp by octylglucoside treatment of influenza C virions followed by centrifugation into a sucrose gradient. Evidence was obtained that gp also represents the receptor-destroying enzyme of influenza C virus, which has been characterized as a neuraminate 9-O-acetylesterase: (i) it inactivated the receptors for influenza C virus on chicken erythrocytes; (ii) it had acetylesterase activity as indicated by the release of acetate from bovine submandibulary mucin; (iii) monoclonal antibodies directed against gp inhibited the acetylesterase activity of influenza C virus. Although purified gp was unable to agglutinate chicken red blood cells, it blocked haemagglutination by viruses. This finding as well as the haemagglutination inhibition activity of monoclonal anti-gp antibodies indicate that gp is also responsible for the haemagglutinating activity of influenza C virus. Thus, as the influenza C glycoprotein is the only myxovirus glycoprotein with three different activities, we propose the designation HEF in order to describe its function as a haemagglutinin (H), an esterase (E) and a fusion factor (F).

Comparative studies on structural and antigenic properties of two serotypes of infectious bursal disease virus.

The electrophoretic mobilities of the two genome segments and the structural polypeptides of the chicken strain Cu-1 (serotype I) and the turkey isolate 23/82 (serotype II) of infectious bursal disease virus were compared. There is a close antigenic relationship between the smaller of the two major structural proteins (32K) of both strains. Neutralizing monoclonal antibodies are induced by the larger protein (40K in Cu-1) which differentiates between the two serotypes. The 40K structural protein also has epitopes which do not induce neutralizing antibodies and which are common to both strains. There is evidence that the antigenic region responsible for the production of neutralizing antibodies is highly conformation-dependent. Passively administered neutralizing antibodies directed against the 40K structural polypeptide of Cu-1 confer protective immunity to susceptible chickens, whereas antibodies directed against the 32K structural protein do not have any protective effect.