Wide occurrence of measles virus subgenomic RNAs in attenuated live-virus vaccines.

Nine measles vaccine preparations, including four different viral strains, provided by eight different manufacturers were analysed by Northern blot for the nature of their nucleocapsid RNAs. Out of nine preparations, six were shown to contain subgenomic RNAs, along with the full length genomic RNA. Presence or absence of the subgenomic RNAs correlated strictly with the viral strains used. The role of the defective interfering particles in measles virus vaccine attenuation, and in its seroconversion efficacy upon vaccination, as well as the potential hazard of the presence of defective interfering particles in live-virus vaccine preparations, is discussed.

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.

Postmortem detection of measles virus in non-neural tissues in subacute sclerosing panencephalitis.

Subacute sclerosing panencephalitis, a rare, progressive, fatal central nervous system disease of children, is caused by measles virus. Clinical signs occur months to several years after recovery from acute measles infection. It is not known where the virus persists while the disease is inapparent. Involvement of organs outside the central nervous system has rarely been documented. To search for possible peripheral reservoirs of measles virus we used in situ hybridization to probe for measles virus RNA and immunocytochemical studies to localize measles virus antigens ina variety of organs taken at autopsy from confirmed cases of subacute sclerosing panencephalitis. Seven of 9 cadavers were found to contain measles virus RNA or antigens, or both, in at least one location outside the central nervous system. These sites included lymphoid organs such as thymus, spleen, lymph nodes, and tonsil, suggesting a role for lymphocytes in disease pathogenesis. Virus was also detected in kidney, lung, and glandular tissues such as pancreas, adrenal, and pituitary. These reservoirs may provide the antigenic stimulus leading to the elevated response characteristic for subacute sclerosing panencephalitis.

Characterization of a halothane-resistant strain of measles virus.

A strain of measles virus (MVr) whose replication demonstrated increased resistance to halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) exposure compared with the susceptible parental strain (MVs) is described. After exposure to a 1.2% halothane concentration, substantial amounts of the measles virus H protein were detected in MVr-infected Vero cell lysates by immunoprecipitation and polyacrylamide gel electrophoresis or by quantitative immunofluorescence staining. The protein was barely detectable in identically treated MVs-infected lysates, however. The recovery of all other measles virus proteins studied was the same in MVr- and MVs-infected cells at this anesthetic concentration. Thus, the altered expression of a single gene product appears to be responsible for the observed halothane resistance.

Generation of measles virus defective interfering particles and their presence in a preparation of attenuated live-virus vaccine.

By starting from a thrice-purified wild-type measles virus plaque, the generation of detectable subgenomic RNAs was achieved within a series of five serial infections of Vero cells. The evolution of these subgenomic RNAs was followed for seven serial passages and ended with the preparation of a highly interfering viral stock. On the other hand, the detection of discrete subgenomic RNAs was achieved during the first infection of Vero cells with at least one of three measles virus vaccine preparations tested. These subgenomic RNAs, which interfered very efficiently with the replication of the endogenous standard genomes upon vaccine infection but showed a moderate interfering activity with a standard virus stock derived by plaque purification from the vaccine preparation, resulted from the presence of defective interfering particles in the vaccine preparation. The relevance of this finding for the attenuation, stability, and potential capacity for persistent infection of such a vaccine is discussed.

[Analysis of the possible mechanisms for the persistence of the vaccinal strain of the measles virus in a human cell culture]. / Analiz vozmozhnykh mekhanizmov persistentsii vaktsinnogo shtamma virusa kori v kul'ture kletok cheloveka.

The mechanisms (factors) of the measles virus vaccine L-16 strain persistence in HEp-2 cell culture were analysed. Among the known mechanisms, most likely is the reduction of the cell-destroying properties of the persisting virus due to mutations in nucleoprotein gene manifested by changes of the isoelectric point of NP protein and temperature sensitivity of its synthesis.

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.

Measles virus persistence in a hamster brain cell line: cyclical fluctuation of viral expression during serial passages.

Measles virus persistence in a hamster brain cell line was examined for the appearance of viral antigens and for the synthesis of infectious virus, viral RNA, and virus-specific proteins during 19 serial passages. Cyclical fluctuation was detected at all levels of measles virus replication. After reaching the maximal activity at the passages 9 to 11 viral synthesis diminished rapidly, being lowest after 2 to 3 subsequent passages. No release of infectious virus was detected at the passages 14 and 15 and only 20% of the cells contained measles virus antigens when tested by immunofluorescence. After the nonproductive phase, the cells released virus again and the number of antigen-positive cells increased. The alternation in the amount of the measles virus-specific proteins and viral RNA correlated directly with the parameters mentioned above. No apparent defects in the synthesis of individual viral proteins were observed.

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.

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.

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.

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.

[RNA analysis of the measles virus in a human cell culture of the chronic infection]. / Analiz RNK virusa kori v kul'ture kletok cheloveka pri khronicheskoi infektsii.

Synthesis of virus-specific RNAs in human HEP-2 and L-41 cells chronically infected with measles virus was studied in comparison with synthesis of viral RNA in acutely infected L-41 cells. The RNA, a component of RNP isolated from chronically infected cells, was shown to be represented mainly by "minus" chains and to contain 23-25% "plus"-RNA. It was demonstrated by blotting hybridization that 1 species of genomic RNA with a molecular weight of 5 megadaltons was synthesized in acute infection whereas in chronically infected cells a small amount of subgenomic RNAs was additionally detected in RNP. The level of virus genome transcription in chronically infected cells was 7-8 fold lower than that in acute infection. The RNA-transcriptase activity of RNP isolated from chronically infected HEP-2 and L-41 cells was also lower than RNP activity from acutely infected L-41 cells. The observed features of virus-specific RNA synthesis in chronically infected cells seem to be likely to play a role in the maintenance of virus persistence.

Measles encephalitis in rodents: defective expression of viral proteins.

Synthesis of measles virus proteins in rodent brains and Vero cell cultures infected with the hamster neurotropic (HNT), and for comparison the LEC strain, was studied by use of monoclonal antibodies against five structural components. In the brains of HNT-infected adult BALB/C mice two proteins, the nucleocapsid (NP) and phosphoprotein (P) were detected. Suckling hamster brains in addition expressed demonstrable hemagglutinin (HA) protein. In cell cultures all structural components except the matrix (M) protein were detected. In contrast, all five proteins were found in LEC strain-infected suckling hamster brains and cell cultures. The restriction in HNT viral replication observed may be caused by a primary defectiveness in M-protein expression, but the possibility that this restriction is secondary to cellular suppression remains to be explored. Minimal inflammation was seen in the brains of HNT-infected adult mice and viral antigen was primarily located in the cerebral cortex. A selective necrosis of the pyramidal cell layer of the hippocampus was observed. This change did not seem to correlate with virus replication.

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.