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Presence of oligoclonal immunoglobulin G bands and lack of matrix protein antibodies in cerebrospinal fluids and sera of ferrets with measles virus encephalitis.

Young adult ferrets were immunized with measles vaccine and 5 to 6 weeks later inoculated intracerebrally with Vero cells persistently infected with cell-associated strain D.R. of measles virus isolated from a patient with subacute sclerosing panencephalitis. Of nine ferrets which survived the infection for 3 weeks or longer, five showed neurological signs. At the time of death they had widespread inflammation in their brains, and cell-associated virus was isolated from three ferrets sacrificed from 5 weeks to 7 months after inoculation. Four ferrets did not develop clinical signs, but two of these had mild inflammation in the brain 7 months and 2 1/2 years after inoculation, respectively. Cerebrospinal fluids drawn by cisternal puncture from infected ferrets at the time of sacrifice had neutralizing titers against measles virus similar to the titers found in sera, but antibody against the measles virus matrix protein was not detectable. Cerebrospinal fluid showed increased immunoglobulin G (IgG) and had distinct measles virus-specific oligoclonal IgG bands. The intensity of the bands correlated with the neutralizing titers of the fluids. These results confirm and extend earlier findings and indicate that persistent measles virus infection in ferrets is similar to human subacute sclerosing panencephalitis and can be used to study certain aspects of persistent brain infections leading to subacute encephalitis.

Animals↗

Measles virus synthesizes both leaderless and leader-containing polyadenylated RNAs in vivo.

The minus-sense RNA genome of measles virus serves as a template for synthesizing plus-sense RNAs of genomic length (antigenomes) and subgenomic length [poly(A)+ RNAs]. To elucidate how these different species are produced in vivo, RNA synthesized from the 3'-proximal N gene was characterized by Northern RNA blot and RNase protection analyses. The results showed that measles virus produced three size classes of plus-sense N-containing RNA species corresponding to monocistronic N RNA, bicistronic NP RNA, and antigenomes. Unlike vesicular stomatitis virus, measles virus does not produce a detectable free plus-sense leader RNA. Instead, although antigenomes invariably contain a leader sequence, monocistronic and bicistronic poly(A)+ N-containing RNAs are synthesized either without or with a leader sequence. We cloned and characterized a full-length cDNA representing a product of the latter type of synthesis. mRNAs and antigenomes appeared sequentially and in parallel with leaderless and leader-containing RNAs. These various RNA species accumulated concurrently throughout infection. However, cycloheximide preferentially inhibited accumulation of antigenomes and leader-containing RNA but not leaderless and subgenomic RNAs late in infection, suggesting that synthesis of the former RNA species requires a late protein function or a continuous supply of structural proteins or both. These results reveal a previously undescribed mechanism for RNA synthesis in measles virus.

Animals↗

The molecular length of measles virus RNA and the structural organization of measles nucleocapsids.

Full-length measles virus RNA molecules isolated from purified virions or nucleocapsids and examined by electron microscopy were 5.12(+/- 0.12) micron in length, corresponding to a molecular weight of 5.2 (+/- 0.1) X 10(6). Purified virions examined by negative staining in the electron microscope exhibited a pleomorphic range of particle sizes varying in diameter between 300 nm and 1000 nm. Purified nucleocapsids had dimensions of 21 nm (diameter) X 1254(+/- 7) nm (length) and a central core of diameter about 5 nm. Full-length nucleocapsids were composed of 204 (+/- 3) protein discs. The pitch of the nucleocapsid helix was calculated to be 6.1 nm and the helix angle, alpha, to be 8 degrees 16'. Approximate volume calculations indicate that each enveloped virus particle contains multiple nucleocapsids.

Animals↗

Crohn's disease after in-utero measles virus exposure.

BACKGROUND: An epidemiological association between Crohn's disease and measles virus exposure in early life has been suggested in case-control studies. METHODS: To determine absolute risk estimates for in-utero measles virus exposure and Crohn's disease, maternity charts for all 25000 deliveries at University Hospital, Uppsala, between 1940-49 were reviewed: four cases of measles infection in the mother during pregnancy were identified. The children and two of their mothers were interviewed and case records reviewed. Three offspring had undergone multiple intestinal resections; tissue from these cases were examined by routine histology, and for measles-virus nucleoprotein antigen by immunohistochemistry and immunogold electronmicroscopy. FINDINGS: Three of the four children had Crohn's disease. In each the disease was preceded by recurrent, antibiotic-resistant pneumonia. They had extensive ileal and colonic disease; two patients required intravenous feeding. The only offspring to have had measles as a child did not develop Crohn's disease. Measles virus antigen was detected in foci of granulomatous and lymphocytic inflammation in all children with Crohn's disease. INTERPRETATION: The data indicate that exposure of mothers to measles virus in utero is a risk factor for Crohn's disease in their children. Exposure at this time may lead to persistent infection, or modify the response to infection in later life, leading to persistence of measles virus.

Adult↗

Growth of measles virus in cultures of rat glioma cells.

The interaction of measles virus with RG-6 cells derived from rat glioma was investigated. When a culture of RG-6 cells was infected with measles virus, the synthesis of viral antigens was detected in very few cells, at most 5%. The apparent resistance to measles virus infection was also repeatedly found in all of the subclonal cells derived form RG-6 cells. Although all of the virus-synthesizing cells had the ability to form plaques on Vero cells, they produced only a reduced amount of infectious virus, i.e., 0.1 plaque-forming units per cell. These results imply the existence of some mechanism that regulates growth of measles virus in cultures of RG-6 cells. The transmission of genetic material of measles virus from infected RG-6 cells to Vero cells was not inhibited in the presence of antiviral serum. This fact may provide a basis for interpretation of the persistence of virus, in the presence of antibody, in patients with subacute sclerosing panencephalitis.

Animals↗

Activation of measles virus from silently infected human lymphocytes.

Lymphocytes were incubated with measles virus for 4 days in the absence of a lymphocyte stimulating agent. Such nonstimulated lymphocytes, infected with measles virus, did not express the virus antigens that are detectable by cytotoxic antibodies. Approximately 1 out of 5,000, or even fewer, of such lymphocytes produced virus as demonstrated by the infectious center assay; in the supernate only 10--100 infectious viruses per milliliter were detected. No virus structures could be observed by means of an electron microscope. However, such lymphocytes showed no reaction to phytohemagglutinin (PHA) in terms of DNA synthesis in a subsequent culture in the presence of antibodies against measles to prevent spreading of the infection to other cells. Although stimulation by PHA did not result in a significant increase in [3H]thymidine incorporation, measles virus was activated; 32 h after the addition of PHA nearly 80% of the cells were killed by measles virus antibodies and complement. The number of virus-producing cells increased to approximately 1 in 300 or more, and at 72 h the virus titer in the supernate had risen to 10(6) infectious particles per ml. This reactivation of measles virus was still obtained when PHA was added as late as 8 or more days after the initial infection.

Antigens, Surface↗

Applicability of oral fluid collected onto filter paper for detection and genetic characterization of measles virus strains.

Expansion of measles molecular surveillance to developing countries where measles is endemic will help facilitate measles control. Limited infrastructure in these areas is a barrier to referral of specimens suitable for measles virus (MV) genotyping. In this study, we demonstrate that oral fluid dried onto filter paper can be used for the detection and characterization of MV strains. Using this approach, an MV-positive sample by reverse transcriptase PCR could be obtained from 67% of serologically confirmed acute measles cases. Mimicking certain environmental conditions and duration of transportation established that MV RNA remained detectable and suitable for nucleic acid sequencing in oral fluid spots for at least 1 week. In the context of a measles outbreak in a remote region of the world where infrastructure is poor, oral fluid samples dried onto filter paper and sent to a specialized laboratory for testing will aid in the identification and characterization of the causative MV strain.

Filtration↗

Evaluation of recombinant vaccinia virus--measles vaccines in infant rhesus macaques with preexisting measles antibody.

Immunization of newborn infants with standard measles vaccines is not effective because of the presence of maternal antibody. In this study, newborn rhesus macaques were immunized with recombinant vaccinia viruses expressing measles virus hemagglutinin (H) and fusion (F) proteins, using the replication-competent WR strain of vaccinia virus or the replication-defective MVA strain. The infants were boosted at 2 months and then challenged intranasally with measles virus at 5 months of age. Some of the newborn monkeys received measles immune globulin (MIG) prior to the first immunization, and these infants were compared to additional infants that had maternal measles-neutralizing antibody. In the absence of measles antibody, vaccination with either vector induced neutralizing antibody, cytotoxic T cell (CTL) responses to measles virus and protection from systemic measles infection and skin rash. The infants vaccinated with the MVA vector developed lower measles-neutralizing antibody titers than those vaccinated with the WR vector, and they sustained a transient measles viremia upon challenge. Either maternal antibody or passively transferred MIG blocked the humoral response to vaccination with both WR and MVA, and the frequency of positive CTL responses was reduced. Despite this inhibition of vaccine-induced immunity, there was a reduction in peak viral loads and skin rash after measles virus challenge in many of the infants with preexisting measles antibody. Therefore, vaccination using recombinant vectors such as poxviruses may be able to prevent the severe disease that often accompanies measles in infants.

Animals↗

Measles virus infection of unstimulated blood mononuclear cells in vitro: antigen expression and virus production preferentially in monocytes.

Synthesis of measles virus antigens occurred only in a small percentage of peripheral blood mononuclear cells infected in vitro with measles virus without mitogenic stimulation. The infection cycle was restricted as only low amounts of infectious virus were released but all the major structural viral proteins were present as shown by immunofluorescence with monoclonal antibodies. Cells with viral antigen synthesis were characterized by double labelling and by infecting selectively depleted subpopulations. In 3-day cultures, up to 80% of the cells with measles virus antigen were shown to be monocytes by specific staining with anti-MMA and anti-Leu M3 monoclonal antibodies and up to 40% of the monocytes were infected. Less than 10% of the cells expressing virus antigens carried the lymphocyte marker OKT3, the majority of these cells belonging to the Leu3a (helper) population. Anti-alpha-interferon treatment increased the number of measles-positive cells and the release of infectious virus in preparations enriched for monocytes, but had no significant effect on infection of lymphocytes.

Antibodies, Monoclonal↗

Long untranslated regions of the measles virus M and F genes control virus replication and cytopathogenicity.

Measles is still a major cause of mortality mainly in developing countries. The causative agent, measles virus (MeV), is an enveloped virus having a nonsegmented negative-sense RNA genome, and belongs to the genus Morbillivirus of the family Paramyxoviridae. One feature of the moribillivirus genomes is that the M and F genes have long untranslated regions (UTRs). The M and F mRNAs of MeV have 426-nucleotide-long 3' and 583-nucleotide-long 5' UTRs, respectively. Though these long UTRs occupy as much as approximately 6.4% of the virus genome, their function remains unknown. To elucidate the role of the long UTRs in the context of virus infection, we used the reverse genetics based on the virulent strain of MeV, and generated a series of recombinant viruses having alterations or deletions in the long UTRs. Our results showed that these long UTRs per se were not essential for MeV replication, but that they regulated MeV replication and cytopathogenicity by modulating the productions of the M and F proteins. The long 3' UTR of the M mRNA was shown to have the ability to increase the M protein production, promoting virus replication. On the other hand, the long 5' UTR of the F mRNA was found to possess the capacity to decrease the F protein production, inhibiting virus replication and yet greatly reducing cytopathogenicity. We speculate that the reduction in cytopathogenicity may be advantageous for MeV fitness and survival in nature.

Animals↗

Replication-incompetent adenoviruses as vectors for protective immunization against measles virus infection.

Despite the availability of an efficient live-attenuated measles virus vaccine about 1.5 million annual deaths from measles infections and their complications are recorded worldwide. Particularly in developing countries measles remains an unsolved problem and a new vaccine seems necessary. In our animal model of measles virus infection in rats we studied a candidate vaccine based on infectious replication incompetent (E1A-) adenovirus (RAd) where measles virus genes are expressed under the control of the cytomegalovirus immediate early promoter. The aim of the study was to characterize the cell mediated and humoral immunity after immunization with RAd and its protective effect. After challenge with a lethal dose of measles virus no signs of disease or histopathological changes were detected in RAd68-immunized rats. The elimination of measles virus was successful within a few days. The results demonstrate that defective recombinant adenovirus vectors can induce complete protection from experimental measles infection in rats.

Adenoviridae↗

Measles virus receptors and tropism.

Measles virus (MV) is a member of the Morbillivirus genus in the Paramyxoviridae family. Human signaling lymphocyte activation molecule (SLAM) acts as a cellular receptor for MV. SLAM is expressed on immature thymocytes, activated lymphocytes, macrophages and mature dendritic cells. This distribution of SLAM is in accord with lymphotropism and immunosuppressive nature of MV. Canine distemper and rinderpest viruses, other members of the Morbillivirus genus, also use SLAM as receptors. Laboratory-adapted MV strains often use ubiquitously expressed CD46 as an alternative receptor through the amino acid change(s) in the receptor-binding hemagglutinin. Furthermore, MV can infect various cultured cells, albeit with very low efficiency, via SLAM- and CD46-independent pathway, which may also account for MV infection of SLAM(-) cells in vivo.

Animals↗

Molecular characterization of epitopes on the measles virus hemagglutinin protein.

The measles virus (MV) hemagglutinin (H) gene nucleotide sequences of the LEC-WI strain and 11 branched sequential neutralization escape variants of the strain derived by selection with five monoclonal antibodies (Mabs) were determined by direct analysis of amplified polymerase chain reaction products. The parental LEC-WI strain isolated from a patient with subacute sclerosing panencephalitis exhibited H gene sequence characteristics similar to other persistent virus strains derived from brain materials. Mostly single-point H gene mutations, coding for single amino acid substitutions in the H protein, were found to provide explanations for the resistance to the individual Mabs. Resistance to Mabs 16-CD11 and I-41 resulted from changes of Gly-491 to Asp (or Val) and Phe-552 to Val, respectively. One variant (B89) selected by Mab 16-CD11 had a mutation introduced by a single nucleotide deletion and subsequent nucleotide insertion, which caused a shift in the open reading frame. The epitope of Mab I-29 was assigned to Ser-313 or Gly-314, which were changed to Leu and Arg, respectively. The variants subjected to the Mab I-44 selection exhibited change of Ser-189 to Pro. Radioimmunoprecipitation assay and endoglycosidase H (Endo H) treatment revealed that this change destroyed a potential N-linked glycosylation site, indicating that the carbohydrate chain participates in formation of the epitope or indirectly influences its properties. Resistance to Mab 16-DE6 involved three specific amino acid changes in three different places, Gly-211 to Ser, Gly-388 to Asp, and Ser-532 to Phe or Arg-533 to Gly, reflecting the occurrence of a conformational epitope. In conclusion, this study identifies the precise positions of several critical sites on the MV H protein which react with neutralizing antibodies.

Amino Acid Sequence↗

Factors determining prevalence of maternal antibody to measles virus throughout infancy: a review.

The effectiveness of vaccination against measles, the leading cause of vaccine-preventable deaths in infants globally, is greatly impacted by the level of maternal antibody to measles virus (or "measles maternal antibody"; MMA) during infancy. Variation in the prevalence of maternal antibody to measles virus between infant populations across countries and sociodemographic strata is poorly understood. We reviewed the literature on the prevalence of MMA, focusing on 3 principal determinants: starting level of maternal antibody, placental transfer of maternal antibody, and rate of decay of maternal antibody after birth. Our review identified placental transfer as an important determinant, with greater efficiency found in studies performed in developed countries. Placental transfer was influenced by gestational age, human immunodeficiency virus infection, and malaria. Antibody levels in mothers varied widely between countries, although predictably according to vaccination status within populations. Rates of antibody decay across studies were similar. Future studies should evaluate the utility of the cord blood level of MMA as a predictor of vaccine efficacy in infancy; inclusion of World Health Organization international reference sera will facilitate comparisons. Greater understanding of the determinants of the prevalence of MMA will help national policy makers determine the appropriate age for measles vaccination.

Antibodies, Viral↗

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.

Animals↗

Characterization of a natural mutation in an antigenic site on the fusion protein of measles virus that is involved in neutralization.

Although measles virus is an antigenically monotypic virus, nucleotide sequence analysis of the hemagglutinin and nucleoprotein genes has permitted the differentiation of a number of genotypes. In contrast, the fusion (F) protein is highly conserved; only three amino acid changes have been reported over a 40-year period. We have isolated a measles virus strain which did not react with an anti-F monoclonal antibody (MAb) which we had previously shown to be directed against a dominant antigenic site. This virus strain, Lys-1, had seven amino acid changes compared with the Edmonston strain. We have shown that a single amino acid at position 73 is responsible for its nonreactivity with the anti-F MAb. With the same MAb, antibody-resistant mutants were prepared from the vaccine strain. A single amino acid change at position 73 (R-->W) was observed. The possibility of selecting measles virus variants in vaccinated populations is discussed.

Antibodies, Monoclonal↗

Fine structure of cellular inclusions in measles virus infections.

Cells which are infected with measles virus have been known for some time to contain inclusion material that is distinguishable from normal cellular components by application of traditional staining methods and observation in the light microscope. The fine structure of the inclusion material contained in HeLa cells infected with Edmonston strain of measles virus has been examined in the electron microscope. Two steps have been found necessary in this study: (1) the recognition by phase-contrast microscopy of the living cell of bodies that are defined as inclusion material when the cells are classically stained; and (2) the recognition in the electron microscope of inclusion-body material that had previously been identified in the living cell. The fine structure of the nuclear and cytoplasmic inclusion material in osmium-treated cells was found to consist mainly of randomly arrayed filaments of low electron density. Dense, highly ordered arrays of filaments were found near the center of the nuclear inclusions, sometimes as a two-dimensional, nearly orthogonal arrangement. If the size of the measles virus is taken to be around 100 mmicro in diameter, the strands seen in the inclusions cannot be fully formed virus.

HeLa Cells↗

Molecular analysis of measles virus genome derived from SSPE and acute measles patients in Papua, New Guinea.

A very high annual incidence of 56 per million population below the age of 20 years for subacute sclerosing panencephalitis (SSPE) has been reported from Papua New Guinea (PNG). In a more recent study, we have confirmed this unusual high incidence for Eastern Highlands Province (EHP) of PNG. In the study, it was observed that the vaccination rate among SSPE patients registered at Goroka Base General Hospital (GBGH) in EHP was higher than that of other infants in the province in recent years. To identify the measles virus (MV) responsible for SSPE in EHP, sequence analysis of hypervariable region of the N gene was performed from 13 MV genomes: 2 amplified from clinical specimens of SSPE patients and 11 from acute measles patients. In 2 cases among the 11 with acute measles, nucleotide sequence of the entire H gene derived from isolated viruses was determined. Both nucleotide sequence and phylogenetic tree analyses showed that the amplified MV cDNAs were closely related to one another and belonged to the D3 genotype though they were different from any previously reported MV sequences. No genome sequences of vaccine strains were detected. These findings suggest that the MV strains prevailing in the highlands of PNG belong to genotype D3 of the MV and this wild-type MV rather than the vaccine strains was likely to be responsible for SSPE in these patients.

Acute Disease↗