Immunological relationships between phocid and canine distemper virus studied with monoclonal antibodies.

The immunological relationships between distemper viruses, isolated from a seal and mink in Denmark and from a dog in Greenland, were investigated with 39 previously developed monoclonal antibodies (MAbs) directed against four major structural proteins of canine distemper virus (CDV). They were also investigated with 16 newly developed MAbs directed against the fusion (F) and large glycoprotein (named H in analogy with measles virus) of phocid distemper virus (PDV) isolated from a harbour seal (Phoca vitulina). These MAbs were reacted with the three different isolated viruses and with the LEC strain of measles virus, in ELISA and immunofluorescence tests. In addition, immunoprecipitation tests were carried out with some of the cross-reacting antibodies. All 55 MAbs reacted identically with distemper virus isolated from seals or mink. When the MAbs produced against CDV were tested, 37 of 39 antibodies reacted with a virus isolated from a sled dog diseased in an outbreak of distemper in Greenland prior to the epizootic among seals in the North Sea. Of the 39 antibodies, 25 reacted with PDV and distemper virus isolated from mink. Of these antibodies, only three of the nine antibodies directed against the H protein of CDV cross-reacted with PDV and distemper virus from mink. Eleven MAbs, reacting with six epitopes of the H protein of PDV, were produced. All 11 antibodies reacted with distemper virus from mink, two of the antibodies reacted with CDV and none reacted with measles virus. All five antibodies reacting with three different epitopes of the F protein of PDV reacted with distemper virus from mink and CDV. Of these five antibodies three, directed against two epitopes, reacted with measles virus. Of the two envelope proteins, the H protein shows pronounced immunological differences between PDV and CDV. In contrast, immunologically the F protein appears to be well conserved among morbilliviruses. It is concluded that the virus causing the epizootic in seals in the North Sea in 1988 may have infected mink on land, or, alternatively, the virus in the sea may have originated from virus-infected mink.

Localization of nucleocapsid associated polypeptides in measles virus-infected cells by immunogold labelling after resin embedding.

The nucleo-, phospho- and matrix protein of measles virus were localized at high resolution within infected cells by use of post-embedding immunogold labelling techniques. In general, labelling with monospecific antibodies as well as with a polyvalent rabbit anti-measles hyperimmune antiserum revealed measles virus polypeptides to be distributed non-randomly within infected cells with the label largely confined to specific sites, namely inclusions of nucleocapsids and assembled virus structures at the plasma membrane. Immunogold double labelling indicated that the phosphoprotein strictly co-localized with the nucleoprotein in cytoplasmic inclusions of nucleocapsids and in budding virions, whereas intranuclear inclusions of nucleocapsids were devoid of phosphoprotein labelling. Antibodies to the matrix protein clearly labelled assembled virus structures at the plasma membrane but exhibited no significant cytoplasmic or intranuclear reaction. The data indicate that the composition of nucleocapsids varies with the cellular compartment with which they are associated, supporting the view of a rapid assembly of paramyxovirus nucleocapsid polypeptides, and emphasize the proposed selective role of the matrix protein in virus assembly and budding at the plasma membrane.

Protein antigen-monoclonal antibody contact sites investigated by limited proteolysis of monoclonal antibody-bound antigen: protein "footprinting".

This study describes the use of limited proteolysis of monoclonal antibody (mAb)-bound antigens in the analysis of the two measles virus surface glycoproteins. This approach is dubbed protein "footprinting" in analogy with DNA "footprinting." Protein footprinting was superior to competitive-binding assays and as good as in vitro mAb-selected variant analysis in differentiating among mAbs with various specificities to a given protein. Proteolytic digestion of the antigen prior to mAb binding drastically reduced mAb binding resulting in poor differentiation among mAbs. In contrast, protein footprinting showed that some mAbs retained the ability to immunoprecipitate such fragments. Thus footprinting could be used for localization of mAb epitopes on a protein and proved also to be an effective means of distinguishing among mAb-selected variants differing in single epitopes. Conformational changes caused by heat-denaturation or the binding of anti-antibody to an antigen-antibody complex could also be detected by footprinting.

Fusion glycoprotein of measles virus: nucleotide sequence of the gene and comparison with other paramyxoviruses.

The sequence of the fusion (F) glycoprotein mRNA of the Hallé strain of measles virus was determined from a cDNA clone representing the entire length of the mRNA. It contained 2384 nucleotides, excluding poly(A), with a 5' consensus sequence typical of paramyxoviruses and a 3' terminus found in measles virus mRNAs. The coding sequence was preceded by an unusually long (580 nucleotide) 5' non-translated region, which contained 44% cytosine. The longest open reading frame coded for a polypeptide of 553 amino acids with a predicted molecular weight of 59.84 K. Comparison of the sequence with that of the Edmonston strain of measles virus showed that the gene is highly conserved. No amino acid differences were observed between the two strains. The F polypeptide had three regions of high hydrophobicity: an N-terminal signal peptide, the N-terminus of F1 and a C-terminal membrane-spanning region. The four potential asparagine-linked glycosylation sites (one in the signal peptide) were all in the F2 subunit. Comparison of the measles virus F amino acid sequence with other paramyxoviruses revealed homologies with these viruses. Certain regions such as the N terminus of F1 and ten cysteine residues which probably impose structural restraints were highly conserved.

Non-infectious morphologically altered nucleocapsids of measles virus from persistently infected cells.

Persistent measles virus infection of human HEp-2 or L-41 cells was accompanied by pronounced structural and functional changes of isolated intracellular viral nucleocapsids (NCs). The bulk of persistent NCs possessed altered conformation and a "string-of-beads" appearance, contained substantial amounts of subgenomic size RNAs, exhibited reduced transcriptase activity in vitro and lacked infectivity on transfection of susceptible cells. Immunogold staining revealed negligible binding of anti-P protein monoclonal antibodies to the "string-of-beads" type NCs, thus suggesting their non-functional state.

Fuzzy material surrounding measles virus nucleocapsids identified as matrix protein. Brief report.

Measles virus grown in Vero cell cultures was examined at the ultrastructural level after immunoperoxidase staining with antiserum against the matrix protein. The antiserum clearly preferentially labeled the fuzzy material surrounding cytoplasmic nucleocapsids, but not the nucleocapsids themselves.

Sequence analysis of the P and C protein genes of human parainfluenza virus type 3: patterns of amino acid sequence homology among paramyxovirus proteins.

The complete nucleotide sequence of the P + C mRNA of human parainfluenza virus type 3 (PF3) was determined by sequencing cDNA, viral genomic RNA and mRNA. The P + C mRNA is 2009 nucleotides in length, exclusive of poly(A), and contains two overlapping open reading frames (ORFs). The P + C mRNA encodes two proteins, the 602 amino acid nucleocapsid phosphoprotein P and the 199 amino acid non-structural protein C. Peptide mapping confirmed that the two proteins are unrelated. Hybrid-arrest translation experiments assigned each of the two proteins to its respective ORF. These studies showed that the coding strategy of the PF3 P + C mRNA is similar to that of Sendai virus. Amino acid sequence alignment showed that the P and C proteins of PF3 and Sendai virus represent homologous pairs. However, these homologies are represented by high contents of accepted amino acid substitutions and by similarity in hydropathy profiles rather than by high contents of exact amino acid matches. Homology with the P and C proteins of measles, canine distemper and respiratory syncytial viruses was at the threshold of significance. The patterns of amino acid sequence homology among the paramyxovirus HN, F, NP, P and C proteins are compared.

Circulating immune complexes in patients with subacute sclerosing panencephalitis.

Levels of immune complexes (ICs) in serum and cerebrospinal fluid (CSF) specimens from subacute sclerosing panencephalitis (SSPE) patients were measured using a solid-phase C1q radioimmunoassay. Single or serial serum specimens were available from 19 patients, while serial CSF specimens were available from two patients. ICs isolated from one CSF specimen by C1q immobilized to Affi-Gel were analyzed for measles virus antigens by binding of measles virus-specific antisera and by polyacrylamide gel electrophoresis followed by Western blotting. Of 78 serum specimens analyzed, 36 (46%) were positive for ICs. When the 8 patients with 3 or more serum specimens were analyzed, 6 had fluctuating levels of ICs. Two of 5 CSF specimens obtained from a patient during acute disease onset were IC-positive, while a second patient exhibited a rapid increase and decrease of IC levels in 14 CSF specimens obtained during an acute disease exacerbation. Composition analysis of ICs isolated from one of the CSF specimens revealed the presence of antigens corresponding in size to measles virus polymerase, nucleoprotein, and possibly, hemagglutinin polypeptides. These results show that SSPE patients frequently have ICs, that IC levels fluctuate in both serum and CSF, and that the ICs are at least partially composed of measles virus antigens.

Analogous amino acid sequences in myelin proteolipid and viral proteins.

Computer analysis of the intrinsic membrane protein, myelin proteolipid, shows strong sequence similarities between the putative extramembrane segments of the proteolipid protein and a number of viral proteins, several of which infect humans. These similarities are even more striking than those reported previously between viral proteins and the encephalitogenic myelin basic protein (MBP). These findings, along with other reports of molecular mimicry by viruses, suggest that immunological cross-reactions between virus-induced antibodies or T-cells and analogous antigenic determinants (epitopes) in myelin proteolipid could be involved in the pathophysiology of multiple sclerosis or post-infectious demyelinating syndromes.

[Characteristics of the ribonucleoprotein of the measles virus persisting in a human cell culture]. / Kharakteristika ribonukleoproteida virusa kori, persistiruiushchego v kul'ture kletok cheloveka.

The properties of virus-specific RNPs recovered from human HEp-2 and L-41 cells chronically infected with measles virus were studied in comparison with those of RNPs formed in acute infection of L-41 cells. The persisting RNP was shown to contain nucleoprotein not differing in the electrophoretic mobility from the same protein of measles virus virions, and RNA in the persisting RNP was found to be insensitive to the action of RN-ase. RNP from chronically infected cells had a changed ultrastructure and conformation as compared with RNP of the original virus and, unlike the latter had no infectivity upon transfection of the sensitive cells by calcium-phosphate precipitation. No differences in relationships of RNP with the cytoskeleton of the infected cells in the acute and chronic infection were observed.

Isolation of measles virus polypeptides from infected brain tissue by affinity chromatography.

A method has been developed to isolate measles virus proteins from infected hamster brain tissue. Suckling hamsters inoculated intracerebrally with the HBS strain of measles virus were used in these studies. Viral proteins were isolated from infected brain lysates by affinity chromatography on Sepharose beads coupled with IgG from rabbit hyperimmune anti-measles serum. The eluted proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), electrophoretically transferred onto blotting matrix, and immunolabelled with anti-measles antibodies. Individual viral proteins were identified by labelling with monoclonal or monospecific antibodies. All viral proteins except the fusion (F1) protein were identifiable on the immunoblots in relative amounts comparable to purified virions. In addition, a second phosphoprotein (P) band not found in purified virions was present in infected brains and cell cultures infected with HBS or LEC strains of virus. This method should be useful for isolating small quantities of viral proteins from large amounts of tissue, and should make possible the characterization of measles virus proteins in persistently infected CNS tissue.

Characteristics of fresh isolates of wild measles virus.

Characteristics of fresh isolates of wild measles virus were studied by immunofluorescence using monoclonal antibodies, SDS-polyacrylamide gel electrophoresis, and electron microscopy. By the immunofluorescence test, hemagglutinin, nucleocapsid-associated phosphorylated, and fusion proteins were found only in the cytoplasm of cells infected with the virus at a low passage level. Nucleocapsid (NP) and membrane (M) proteins were found in both the cytoplasm and nuclei. Since the M protein is present only in the cytoplasm of cells infected with the laboratory strain of measles virus, this intranuclear M protein may be specific to the wild virus. At an increased passage level, the intranuclear M protein became undetectable, but the NP protein was still present in the nuclei. SDS-polyacrylamide and electron microscopy showed that wild measles viruses were shown to have characteristics similar to those of laboratory strains.

Subacute sclerosing panencephalitis: measles virus matrix protein nucleic acid sequences detected by in situ hybridization.

Subacute sclerosing panencephalitis (SSPE) is characterized by a hyperimmune state toward the polypeptides of measles virus except the matrix (M) protein. Using cloned (3H)-labeled complementary DNA probes for in situ hybridization, we found the M protein and nucleocapsid (NP) protein nucleotide sequences in glial cells and neurons of cryostat sections from two SSPE brains. In one SSPE brain, M protein was lacking, but the other measles polypeptides were present. IgG and IgM antibodies eluted from that brain lacked antibodies to M protein, but antibodies to other measles polypeptides were present. In SSPE brain, the viral M-protein defect is not a deletion of the M gene, but rather a block in gene expression.

Characterization of major structural proteins of measles virus with monoclonal antibodies.

We have prepared and characterized monoclonal antibodies against five major structural proteins, i.e. the HA, P, NP, F and M proteins, of measles virus. At least three non-overlapping antigenic sites were delineated on the HA protein, three on the P, four on the NP, four on the F and five on the M proteins by competitive binding assays. Antigenic sites on the HA and F proteins roughly represented functional domains defined by serological tests. The reactivity of monoclonal antibodies with various measles virus strains including those from subacute sclerosing panencephalitis (SSPE) and other members of the morbillivirus family was studied by immunofluorescence. A monoclonal antibody or set of monoclonal antibodies to each of the antigenic sites showed a characteristic pattern of cross-reactivity with heterologous strains. The HA and NP proteins were antigenically the most variable, followed by the F and M proteins, while the P protein was relatively stable. None of the 14 anti-M monoclonal antibodies reacted with non-virus-producing SSPE cells, strongly suggesting the absence of M protein in these cells.

A double labeling technique for performing immunocytochemistry and in situ hybridization in virus infected cell cultures and tissues.

This report describes a combined immunocytochemical and in situ hybridization procedure which allows visualization of cellular or viral antigens and viral RNA in the same cell. Cultures infected with visna or measles virus were fixed in periodate-lysine-paraformaldehyde-glutaraldehyde, stained by the avidin-biotin-peroxidase technique using antibodies to viral or cellular proteins and then incubated with radiolabeled specific DNA probes (in situ hybridization). The immunoperoxidase stain was preserved through the hybridization procedure. Nonspecific 'sticking' of probes over peroxidase stained cells was prevented by incorporation of 0.1% Triton X-100 into the hybridization solution and the post-hybridization washes. The in situ hybridization signal (silver grains/cell) on peroxidase-stained cells was reduced relative to hybridization with unstained cells. The double labeling technique was also applied to sections of paraffin-embedded tissues from a sheep infected with visna virus and mice infected with the HNT strain of measles virus. Visna virus RNA was detected in immunocytochemically identified macrophages in the synovium. A greater number of these cells had viral RNA than had viral protein. In measles virus-infected brains viral RNA was detected only in cells with viral protein. This technique provides a new approach to the study of viral pathogenesis by: identifying the types of cells which are infected in the host and identifying points of blockade in the virus life cycle during persistent infections.

Measles virus: conditions for the propagation and purification of infectious virus in high yield.

Tissue culture conditions for the efficient propagation of cell-free measles virus, and a novel method for the purification of infectious virus in milligram quantities are described in this report. Infected suspension cultures of HeLa cells incubated at 32.5 degrees C yielded virus titers approaching 10(8) PFU/ml, 30-50% of which was cell-free. After concentration by ultrafiltration and sedimentation, infectious virus was separated from host cell membranes and proteins by density equilibrium centrifugation in gradients of colloidal silica. Residual contaminants and silica particles were removed by chromatographic elution through agarose gel. This protocol achieved a approximately equal to 1400 fold purification of virus which retained approximately equal to 75% infectivity while yielding approximately equal to 1.5 mg of viral protein from each liter of infected cell culture medium. Electron microscopy of the purified virus revealed only intact particles having the morphological characteristics of the paramyxoviruses. Serological studies confirmed the purified material to be antigenically reactive measles virus. SDS-PAGE analysis of the virus preparation identified eight polypeptide species as described by others. Seven of these are virus-specified structural proteins corresponding to the L, H, P, NP, F1, M, and F2 polypeptides. The eighth major structural protein was defined as host cell derived actin.

Recognition of host-membrane antigens in the envelope of measles virions.

Trypsin- and acetone-treated virions from either of two strains of measles virus grown in Vero cells stimulated the production in guinea pigs of (i) virus-specific antibodies to polypeptide (P) of molecular weight 70,000 (70K) and to the portion of the HA spike embedded in the viral envelope, but not to M protein, and (ii) antibodies to two host cell membrane antigens which were identified as glycoproteins with molecular weights of 108K and 104K. These host cell antigens were present in increased amounts in infected cells and were intimately associated with the virus. Untreated measles virions grown in Vero cells also stimulated the production of antibody to the 108K glycoprotein. The host polypeptides were less antigenic in virus derived from HEp2 cells, which apparently contained less of these antigens.

Oligoclonal IgG bands with and without measles antibody activity in sera of patients with subacute sclerosing panencephalitis (SSPE).

Oligoclonal IgG bands from SSPE sera were isolated by combination of Protein A-Sepharose 4B column and preparative isoelectric focusing gel procedures. Each eluted fraction, when examined in analytic IEF, showed two or three individual bands with isoelectric points close to one another, compared to approximately fifteen IgG bands seen in whole serum. When the bands were tested for measles antibody activity in immunofixation with measles virus followed by peroxidase staining, the bands eluted in pH region 8.5 to 9.3 were found to be measles specific, whereas those in pH 7.0 to 8.4 lacked significant measles activity. When eluted fractions containing groups of bands were absorbed with measles virus, the bands in pH region 8.5 to 9.3 were removed, whereas those in pH 7.0 to 8.4 region remained unchanged; this indicated that a number of oligoclonal IgG bands without measles virus activities are present in SSPE. The bands lacking measles-specific activity may be synthesized against other infectious agents or they may represent nonspecific activation of B cell clones.

A study of phosphorylation of the measles membrane protein.

A polypeptide (designated X) which migrates with a mobility similar to the membrane protein (M) of measles virus has been found in virus-infected cells. This polypeptide appears to be phosphorylated. However, limited proteolysis has shown that this protein is not a phosphorylated form of the M protein, but appears related to the P protein of measles virus.