Measles Virus Nucleocapsid Research Articles

Canine distemper virus transcripts detected in the bone cells of dogs with metaphyseal osteopathy

Using the technique of in situ hybridisation, we have recently extended our observations that canine distemper virus (CDV) is present in the bone cells of patients with Paget's disease, and have shown that CDV is also detectable in the bone cells of dogs that are naturally infected with the virus. Since hybridisation was localised to bone cells within the metaphyses of the affected dogs, we investigated the possibility that CDV might be involved in the canine metaphyseal bone disorder, meitaphyseal osteopathy. Bone samples from three cases of metaphyseal osteopathy were examined for the presence of the CDV nucleocapsid (CDV-N) gene and the measles virus nucleocapsid (MV-N) gene, using 35S-labelled sense and antisense riboprobes. As with our previous findings in Paget's disease of bone, only the antisense probe was found to hybridise to the osteoblasts and osteoclasts within the affected metaphyses. No hybridisation was seen with the CDV-N sense and MV-N probes in any of the samples tested. Bone samples were also taken from one of the cases to check for the presence of the CDV-N gene using the polymerase chain reaction (PCR). Our findings with in situ hybridisation were confirmed by PCR and subsequent Southern blotting and probing with a 32P-labelled cDNA probe. The detection of CDV RNA within the bone cells of dogs with metaphyseal osteopathy suggests that this virus may be a cause of the disease and provides further, indirect evidence that CDV might be responsible for the bony abnormalities seen in Paget's disease of bone.

Detection of canine distemper virus in bone cells in the metaphyses of distemper-infected dogs.

In the light of recent evidence implicating canine distemper virus (CDV) as a possible etiologic agent in Paget's disease of bone, we thought that it would be of interest to examine distemper-infected bone in the natural host. Samples from the long bones, spleen, and bladder of four distemper-infected and three uninfected dogs were examined for the presence of CDV nucleocapsid and phosphoprotein genes and the measles virus (MV) nucleocapsid gene using the technique of in situ hybridization with radioactively labeled riboprobes. Two of the four distemper-infected dogs showed strongly positive hybridization with both of the CDV probes. The signal was present in marrow cells, in osteoblasts, in osteocytes, and particularly in osteoclasts. No hybridization was seen over the cartilage cells of the growth plate, and there was a clear line of demarcation at the point of invasion of osteoclasts and vascularization. The spleen and bladder samples from infected dogs also showed positive hybridization. There was no hybridization with the MV probe in any of the distemper-infected tissue. Samples from the uninfected dogs showed no evidence of hybridization with either the CDV or MV probes. These results show that CDV can infect bone cells of the natural host and provide further support for the theory that CDV may play a role in human Paget's disease of bone.

Study of transcription in measles virus-infected Vero cells using cDNA probes prepared from poly(A)RNA from uninfected and infected cells.

From af primary plasmid cDNA library prepared from measles virus-infected Vero cell poly(A)RNA, 435 clones selected at random were used to examine the sensitivity and specificity of cDNA probes derived from total poly(A)RNA from uninfected and infected Vero cells. The correlation between the abundance level of a particular species in the cDNA probe and the hybridization signal strength generated by the corresponding cDNA clone on a filter was reliably determined only when at least three independently prepared filters were examined. Variation in the amount of target plasmid was the most important cause of spurious signals. Variation in cDNA insert length did not disturb the signal strength within certain limits. cDNA species with abundance levels down to 0.08-0.01% were able to produce a hybridization signal above background. Unspecific cross-hybridization was shown to define the sensitivity limit of mixed cDNA probes. Despite the many false signals present at different stages, cDNA probes provided valuable information: the cDNA probes were used to monitor relative RNA expression levels and to clone five different measles virus transcripts and 2 host cell transcripts more abundantly expressed in infected cells. The abundance levels of the measles virus nucleocapsid, phosphoprotein, matrix, fusion protein and haemagglutinin genes were 1.5%, 1.5%, 1%, 0.75% and 0.5%, respectively, of the total cDNA library.

Detection of measles virus genomic sequences in sspe brain tissue by the polymerase chain reaction

The polymerase chain reaction (PCR) was modified to detect RNA genomic sequences by generating cDNA copies of these sequences as a preliminary step. Oligonucleotide primer pairs complementary to sequences in each of the five major structural protein genes of the measles virus (nucleocapsid protein, phosphoprotein, matrix protein, fusion protein, and hemagglutinin protein) were synthesized. PCR products were tentatively identified by visualization of bands of the appropriate size by ethidium bromide staining after gel electrophoresis, and identity was confirmed by subsequent restriction enzyme cleavage of the products at predetermined sites to yield fragments of predicted size. This method successfully amplified 400-500 base regions from each of these five genes in RNA extracts of wild measles virus cultured in Vero cells and in RNA extracted from most of the SSPE brain tissues tested, but not in RNA from any control brain tissues. Measles virus genome was detected in SSPE brain tissues stored frozen for as long as 27 years and formalin-fixed paraffin-embedded subacute sclerosing panencephalitis (SSPE) brain tissues as old as 9 years. This method provides a simple, rapid and highly sensitive means of detecting and identifying sequences of RNA genomes by PCR. The success of this method in detecting measles virus in SSPE brain tissue suggests that PCR is appropriate to investigate the possible presence of RNA viruses in other neurological disorders of unknown etiology.

Ultrastructural and Immunohistochemical Evidence of Measles Virus in Active Otosclerosis

Because of the similarity between otosclerosis and Paget's disease of bone, and the mounting evidence of a viral cause in Paget's disease, we have investigated a possible viral cause for otosclerosis. Transmission electron microscopy of stapes footplate fragments with active otosclerosis has revealed structures morphologically identical with measles virus nucleocapsid in osteoblasts and preosteoblasts. Immunofluorescence and immunoperoxidase studies have confirmed the presence of measles nucleocapsid antigen in active lesions. Application of sera from patients with subacute sclerosing panencephalitis, a defective measles virus infection of the central nervous system, resulted in positive immunoreaction in areas of active otosclerosis.

Restriction of measles virus gene expression in acute and subacute encephalitis of Lewis rats

Measles virus (MV) replication in brain tissue of Lewis rats with acute (AE) and subacute (SAME) encephalitis was characterized by biochemical techniques. Messenger RNAs specific for measles virus nucleocapsid (N), phospho (P)-, matrix (M), fusion (F), and haemagglutinin (H) protein were detected in all brain extracts examined. The quantity of the individual MV mRNA species was quite different in comparison to lytically infected Vero cells. A steep gradient of MV transcripts was found in brain tissue which is most likely due to strongly attenuated transcription of mRNAs along the viral genome, representing particularly low transcription of the glycoprotein genes. In addition, in vitro translation assays only revealed synthesis of N and P protein in consistent fashion. The mRNAs for the glycoproteins did not direct the synthesis of detectable viral proteins whereas the M mRNA revealed some activity in animals with AE. The data indicate a strong restriction of the MV envelope gene expression in infected brain tissue, which is independent of the incubation time and type of the central nervous system (CNS) disease. This phenomenon which is similar to the findings observed in measles inclusion body encephalitis and subacute sclerosing panencephalitis suggest that host factors may initially be responsible for the initiation of transcriptional and translational alterations.

T-cell recognition of measles virus haemagglutinin studied in a mouse model.

BALB/c mice were pretreated with cyclophosphamide and immunized 2 days later with inactivated, purified measles virus (MV) mixed with dimethyl dioctadecyl ammonium bromide (DDA). Seven days later, lymph nodes (LN) were removed and lymphocytes cultured in the presence of purified MV antigens. MV haemagglutinin (H) was found to be a major antigen responsible for proliferation of the lymphocytes. Incorporation of purified H into liposomes significantly enhanced the proliferative response compared with purified H alone. Response to MV nucleocapsid protein was only moderate, and insertion of this protein into liposomes did not improve the response. As an attempt to analyse T-cell epitopes of MV H, three synthetic peptides previously found to elicit a strong antibody response were used both as priming and stimulating antigens. None of the peptides was able to elicit a secondary response when MV-primed LN cells were stimulated in vitro. However, each peptide primed T cells for a secondary challenge with purified, inactivated MV, which was demonstrated by proliferation and a delayed-type hypersensitivity assay and also by transfer experiments with peptide-primed cells.

HLA class II-restricted presentation of cytoplasmic measles virus antigens to cytotoxic T cells.

To analyze the nature of the HLA class II-restricted cytotoxic T-lymphocyte (CTL) response to measles virus, murine fibroblasts were transfected with expressible cDNA clones for human HLA-DR antigen and for measles virus matrix or nucleocapsid proteins. DR-positive murine fibroblasts transfected with measles virus matrix or nucleocapsid genes were lysed by class II-restricted measles virus-specific CTL lines. Lysis was as efficient as with infected autologous B-cell lines, even though the measles virus cytoplasmic proteins were undetectable by antibodies in the transfected target cells. These results demonstrate that cytoplasmic viral antigens can be presented to CTL in the context of HLA class II antigens and that measles virus matrix and nucleocapsid proteins contribute to class II-restricted measles virus-specific CTL responses. These results also show that endogenously synthesized measles virus proteins can be efficiently presented by class II antigens. The implications of these findings for measles virus pathogenesis and for antigen processing are discussed.

Induction of measles virus-specific human cytotoxic T cells by purified measles virus nucleocapsid and hemagglutinin polypeptides.

The measles virus polypeptide specificity of human measles virus-specific, HLA class II restricted cytotoxic T cells have been examined. Measles virus-specific CTL have been generated using purified measles virus nucleocapsid and hemagglutinin polypeptides during a primary, in vitro stimulation of bulk cultures. Both the purified preparations of measles virus nucleocapsid and hemagglutinin polypeptides were effective in stimulating a measles virus-specific CTL response. The measles virus nucleocapsid-induced CTL response could be blocked by an anti-HLA class II monoclonal antibody but not an anti-HLA class I antiserum. Moreover, considerably less measles virus nucleocapsid was required to stimulate a comparable CTL response than the measles virus hemagglutinin which suggests that the CTL response to measles virus may be skewed towards internal viral determinants of measles virus. These studies indicate that both internal and external components of measles virus are effective in inducing measles virus-specific CTL. The recognition of internal viral components may represent an important part of the T cell mediated immune response to viruses.

Immunoassays for measles virus nucleocapsid antigen: effect of antigen-antibody complexes.

An inhibition radioimmunoassay and a two-site immunoradiometric (four-layer) assay for the quantification of measles virus nucleocapsid antigen were established and their sensitivities compared. Both assays exhibited threshold sensitivities of from 1 to 10 ng nucleocapsid antigen per ml. Concentrations of reagents used in the assays were shown to be an important factor determining the sensitivities of the assays. Purified mumps or parainfluenza 2 virus nucleocapsids did not compete with measles virus nucleocapsids in the inhibition assay but some degree of cross-reactivity with canine distemper virus nucleocapsid was observed. Pretreatment of virus-infected cells with detergent had a significant effect on the amount of antigen detectable by the assays. SDS greatly decreased the reactivity of measles virus nucleocapsid but Triton X-100 and, to a larger degree, sodium deoxycholate released antigen from the cells in quantities greater than that detected when no detergent pretreatment was employed. Complexing of antibodies to measles virus nucleocapsids in vitro decreased dramatically the threshold sensitivity of the nucleocapsid assays. The antigen-antibody complexes could be disrupted and the components separated but the conditions employed destroyed 90% of the antigenic reactivity of the nucleocapsid and also had a deleterious effect on the antibody. Nucleocapsid antigen was readily detected in brains from hamsters with acute measles encephalitis. The assays, however, were not sensitive enough for routine detection of antigen in brains from hamsters with chronic measles encephalitis.

Measles virus gene expression in subacute sclerosing panencephalitis.

RNA was extracted from the diseased brain of a case of human subacute sclerosing panencephalitis (SSPE) and analysed for the expression of measles-specific RNA. Measles virus-specific mRNAs were present, but the amount of matrix (M) protein mRNA was greatly reduced in comparison to lytically infected cells and phospho- (P) protein mRNA was hardly detectable whereas the level of the corresponding intermediate-sized (is-) RNA was greatly increased. RNA obtained from the human brain was also translated in vitro and measles virus nucleocapsid and P protein was produced. However, in marked contrast to control reactions M protein was not detected in the products formed by translation in vitro. These results indicate an impaired measles virus M protein mRNA synthesis in infected brain tissue.

Host cell factors involved in the production of slowly sedimenting nucleocapsids in measles virus-infected cells.

A decrease in the sedimentation rates of the measles virus nucleocapsid, and the RNA contained within, were observed during acute measles virus infection when the growth conditions of Vero cells were altered. The change in sedimentation rates of virus nucleocapsids in these experiments was apparently due to the physiological state of the cell and was independent of the history of the measles virus used for infection since: (i) the same virus stock was used to infect cells from which nucleocapsids were prepared, (ii) nucleocapsid sedimentation rates were rapid when Vero cells freshly revived from liquid nitrogen were infected, but nucleocapsid profiles showed no decrease in the amount of slowly sedimenting material using the same cells and changing the virus preparation used for infection. Frequent cell splittings and numerous medium changes were among the growth factors which appeared to correlate to slowly sedimenting particle production. Changes in the amount of self-complementarity of the measles virus RNA were also observed under these conditions.

Transmembrane communication in cells chronically infected with measles virus.

The transmembrane association of the measles virus hemagglutinin and hemolysin surface proteins with intracellular viral antigens was studied. Rabbit antisera monospecific for measles virus matrix and nucleocapsid proteins and a human antiserum containing specificities for both the hemagglutinin and hemolysin proteins were used to study the co-capping of these proteins in human Lu 106 cell-line, chronically infected with measles virus. Capping of the surface-associated envelope components was accompanied by co-capping of the matrix and nucleocapsid proteins, the latter being localized mainly within the inclusions. This demonstrated transmembrane communication between surface-associated envelope components and the intracellular measles virus matrix and nucleocapsid proteins. The results demonstrated the existence of a linkage between viral inclusions and viral proteins associated with cell membranes. In the presence of cytochalasin B (1--2 micrograms/ml), co-capping of the matrix protein was unchanged or slightly enhanced, whereas co-capping of the nucleocapsid protein decreased, indicating that actin filaments may mediate the communication between viral nucleocapsids and the cell membrane.

Circular and elongated linear forms of measles virus nucleocapsid.

PARAMYXOVIRUSES are assuming increasing importance as possible causative agents of several human diseases1,2. To date little is known about their mode of replication, although it has been suggested that the nucleocapsid replicates without extensive dissociation of the protein from the nucleic acid component3. Previous reports on the structure of nucleocapsids extracted from virions or infected cells, with or without further purification, have shown by electron microscopy that the molecules are linear, and although short pieces can be found, the majority are about 1.0 µm long4. We report here the occurrence of rare circular forms of measles nucleocapsid as well as linear molecules of greater than normal length.

Measles antibodies and immunoglobulins in sera from patients with subacute sclerosing panencephalitis (SSPE) and from an infected ferret

Cellulose acetate electrophoresis of sera from 2 patients with SSPE revealed a slowmoving and a fast-moving gamma-globulin band. The IgG fractions from sera were isolated by the combination of starch block electrophoresis, Sephadex gel filtration and isoelectric focussing techniques. The purity of each protein was tested by immunoelectrophoresis using a potent goat anti-whole human serum. The isolated IgG preparations were tested in HI, CF and N tests against measles virus envelope, measles virus nucleocapsid and whole measles virus, respectively. The results showed that isolated IgG fractions, having different electrophoretic mobilities, contained high titers of HI, CF and N antibodies, and these titers were proportional to the protein concentration of IgG. Although both IgG fractions appeared to show an increase of K-type chain, neither fraction was devoid of L-type chain. These results indicate that the homogeneous IgG bands observed in the SSPE serum were not monoclonal as they failed to show selective increase of one type of light chain and uneven distribution of measles antibody activity. These findings were compared with the results from a ferret, which developed encephalitis 2 months after intracerebral inoculation with cell-associated measles virus derived from the brain of an SSPE patient. The electrophoretic pattern of the ferret serum was similar to that of the human. High titers of measles HI and N antibodies were seen in the isolated slow- and fast-moving IgG's. The similarities observed in the human and ferret IgG's, with respect to homogeneous bands in electrophoresis and measles HI and H antibody activities, suggest that the ferret may be a useful animal model for studying the immune mechanism of SSPE.

Determination of measles virus-specific nucleocapsid antibodies by means of counterimmunoelectrophoresis.

A counterimmunoelectrophoretic method for quantitative determination of antibodies against measles virus nucleocapsid is described. Unfractionated material from measles virus infected cell cultures was used as antigen, diluted to give only a nucleocapsid-specific precipitate. Purified nucleocapsid and a rabbit hyperimmune serum against this antigen was used to establish the specificity of the test. The measles virus specific antibody titres determined by the method were comparable to those found in nucleocapsid complement fixation tests. It is concluded that the technique described offers a simple and sensitive method to determine nucleocapsid specific antibodies.

Characteristics of the Release of Measles Virus from Latently Infected Cells after Co-Cultivation with BSC-1 Cells

The events during the release of measles virus from latently infected hamster cells after co-cultivation with BSC-1 cells were studied by immunofluorescence and electron microscopy techniques. Before co-cultivation, measles virus antigens (nucleocapsid) were distributed in an apparently random fashion throughout the cytoplasm. Six hours after co-cultivation with BSC-1 cells, the measles virus nucleocapsid became aggregated in close proximity to the nuclear membrane. These antigens then diffused towards the cell periphery, and progeny virus was observed budding from the cell surface by 16 h after co-cultivation. Fusion of the BSC-1 cells to the latently infected hamster cells was necessary for virus release to occur, and an intact, viable BSC-1 cell was also required. A possible mechanism for the block of virus replication in the latently infected cells is discussed.