Measles Virus Genome Research Articles

Otosclerosis: a measles virus associated inflammatory disease.

The etiology of otosclerosis is still unknown. Immunohistochemical studies have revealed the characteristics of chronic inflammation in the otospongiotic area. Paramyxoviral structures have been identified by electron microscopy and the expression of measles virus antigen has been observed by immunohistochemistry in active otosclerotic tissue. By use of the polymerase chain reaction, measles virus related sequences have been detected in otosclerotic bone tissue but not in control specimens. The aim of our study was i) to detect measles virus genome in affected patients using a more sensitive PCR system and ii) to search for anti-measles virus IgG in the perilymph. In 13 out of 14 specimens of bone fragments from surgically removed stapedes of patients suffering from otosclerosis, measles virus RNA sequences could be detected while other tissues from the same patients and from a negative control group lacked such sequences. Furthermore, IgG anti-measles virus antibodies were detected in the perilymph of 6 patients. Our results support our previously stated hypothesis that otosclerosis is a measles virus associated disease which provokes a local immune response within the inner ear.

Detection of measles virus genome directly from clinical samples by reverse transcriptase-polymerase chain reaction and genetic variability

A simple and sensitive method for the detection of measles virus genome was developed, amplifying the regions encoding the nucleocapsid (N) protein and hemagglutinin (H) protein of measles virus by reverse transcriptase-polymerase chain reaction (RT-PCR). We examined a variety of measles patients: 28 patients with natural infection, 4 with measles encephalitis and 1 with subacute sclerosing panencephalitis (SSPE). In 28 patients with natural measles infection a single step PCR amplyfying the N region resulted in a high detection rate for all plasma samples (28/28) within 3 days of the onset of rash and 80% (20/25) even on day 7 of the onset of rash and later. Within 3 days of the onset of rash, 24/25 (96.0%) of nasopharyngeal secretions (NPS) and 27/28 (96.4%) of peripheral blood mononuclear cells (PBMC) were positive for the N region PCR and the positivity rate of PCR decreased in NPS and PBMC after 7 days of the rash. In acute measles infection, measles genome was detected in all cell fractions, CD4, CD8, B cells, and monocytes/ macrophages by the H gene nested PCR. Measles genome was also detected from cerebrospinal fluids (CSF) in patients with measles encephalitis, SSPE, and acute measles by the H gene nested PCR. PCR products of the N and H regions were sequenced and we confirmed the presence of measles genome. Based on the sequence data, chronological sequence differences were observed over the past 10 years. The sequences obtained from the SSPE patient were closely related to those of the wild viruses that were circulating at the time when the patient initially acquired measles. RT-PCR for NPS, PBMC, CSF, and plasma provides a useful method for the diagnosis of measles and molecular epidemiological study in addition to virus isolation.

Association of measles virus with neurofibrillary tangles in subacute sclerosing panencephalitis: a combined in situ hybridization and immunocytochemical investigation.

Neurofibrillary tangle formation, a cardinal characteristic of Alzheimer's disease, is also a feature of several other neurodegenerative disorders, including subacute sclerosing panencephalitis (SSPE). In the present study the association of measles virus genome with neurofibrillary tangle formation has been studied in five cases of SSPE, using in situ hybridization (measles genome) and immunocytochemistry (tau, ubiquitin and beta/A4 amyloid). In two cases with duration of disease less than one year, neurofibrillary tangle formation was not observed. However, in those cases in which the disease was of several years duration, numerous tau- and ubiquitin-positive neurofibrillary tangles were demonstrated. In the two cases of longest duration, double-labelling techniques demonstrated the frequent association of neurofibrillary tangle formation with neuronal measles virus genome positivity. Immunocytochemistry for beta/A4 amyloid failed to demonstrate amyloid in any of the five cases. These findings support the hypothesis that neurofibrillary tangle formation can occur independently of amyloid formation and that this mechanism may operate in both Alzheimer's disease and in virally-induced disease.

Absence of paramyxovirus RNA in cultures of pagetic bone cells and in pagetic bone.

It has been proposed that Paget's disease of bone is caused by the infection of bone cells with one or several paramyxoviruses. In this study we used the polymerase chain reaction (PCR), which allows the detection of very low levels of a target nucleic acid sequence, to study cultures of pagetic bone cells and samples of pagetic bone. Oligonucleotide primers were designed to flank a sequence of the nucleocapsid genome of measles virus and canine distemper virus (CDV). Within this fragment there were contrasting restriction endonuclease sites specific to measles or CDV that allowed identification of the original template. We were unable to detect paramyxovirus RNA in four strains of human bone cells outgrown from pagetic bone and one strain derived from an uninvolved site of a patient with Paget's disease. Furthermore, paramyxovirus sequences were not detected in cDNA prepared from six samples of pagetic bone biopsies. The work presented here further questions the role of measles and CDV in the abnormal remodeling observed in Paget's disease.

Subacute Measles Encephalitis in the Young Immunocompromised Host: Report of Two Cases Diagnosed by Polymerase Chain Reaction and Treated with Ribavirin and Review of the Literature

Two young patients with subacute measles encephalitis are described: a 20-year-old male hemophiliac infected with human immunodeficiency virus (HIV) and a 4-year-old girl with acute leukemia. Both patients were afebrile and had persistent focal seizures and slurred speech beginning 2 and 7 months, respectively, after the onset of uncomplicated acute measles. The diagnosis of subacute measles encephalitis was established by demonstration of paramyxovirus nucleocapsid on electron microscopy of brain tissue in one case and by detection of measles virus genome with the polymerase chain reaction in both. Treatment of the HIV-infected man with intravenous ribavirin was begun when the patient lost consciousness after several weeks of seizures; he died. The girl with leukemia was treated early after the onset of symptoms and recovered after a 15-week course. Review of 31 previously published cases revealed a typical clinical presentation. Cerebrospinal fluid (CSF) analysis, electroencephalography, measurement of measles antibody in serum and CSF, and computed tomography of the brain were not helpful in the diagnosis of subacute measles encephalitis. In contrast, histologic examination of brain tissue proved useful in establishing the diagnosis. On the basis of our experience and our literature review, we conclude that histologic and polymerase chain reaction studies of brain tissue are required for the early diagnosis of subacute measles encephalitis and that therapy with intravenous ribavirin is effective when administered early.

Measles virus phosphoprotein retains the nucleocapsid protein in the cytoplasm

Measles virus (MV) proteins were efficiently expressed in COS and Vero cells from vectors based on the strong cytomegalovirus enhancer-promoter and the simian virus 40 origin of replication. When expressed alone, nucleocapsid protein (N) migrates predominantly into the nucleus whereas phosphoprotein (P) is located in the cytoplasm. Coexpression of N and P proteins results in retention of the N protein in the cytoplasm, as seen also in infected cells. The retention of N protein is due to specific interactions with the P protein since coexpression of N with either the matrix or the hemagglutinin protein had no effect. Mapping of the regions of NP interactions on P protein revealed that the carboxy-terminal 40% of P was sufficient for specific binding to N; however, the carboxy-terminal 60% of P was required for retention of N in the cytoplasm. Thus, the V and C proteins encoded within the first half of the P gene are not involved in the cytoplasmic retention of N protein. N protein might be fortuitously targeted to the nucleus as a result of its many basic amino acids, presumably destined to interact with the MV genome. However, this set of experiments has allowed to analyze in vivo the interactions between the N and P proteins.

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.

Infectious measles virus from cloned cDNA.

The study of measles virus (MV) and of negative strand RNA viruses in general has been hampered by the lack of an experimental system for genetic manipulation. Here we describe a procedure for generating infectious MV from cloned MV cDNA. First we assembled a genetically marked DNA copy of the MV genome in plasmids, under the control of phage T3 or T7 promoters, allowing production of transcripts almost identical to the MV genome or antigenome. Incubation of these linearized plasmid DNAs with the appropriate phage polymerase and only two ribonucleoside triphosphates yielded committed transcription complexes. Microinjection of these complexes into the cytoplasm of helper cells which provide the proteins necessary for MV genome encapsidation and transcription/replication, reproducibly give rise to lytic MVs. The transcripts of one of these viruses were analysed by sequencing after reverse transcription followed by DNA amplification, and found to contain the genetic tags. The described procedure permits the analysis of a negative strand RNA virus with the same genetic tools previously applicable only to positive strand RNA viruses and retroviruses.

Detection of measles virus genome in blood leucocytes of patients with certain autoimmune diseases.

RNA isolated from lymphocytes of peripheral blood was dot-hybridized to a hybrid plasmid containing specific sequences for measles virus nucleocapsid protein. Viral RNA was detected in the lymphocytes of 28 of 34 (82%) patients with systemic lupus erythematosus (SLE) and of 40 of 68 (59%) patients with chronic glomerulonephritis (CGN), and was not detected in 29 control patients.

A measles virus subgenomic RNA: Structure and generation mechanism

A plaque-derived line of measles virus induced the synthesis of 21S subgenomic RNA in addition to 50S genomic RNA. The nucleotide sequence of the former RNA was determined in comparison with that of the latter. The 21S subgenomic RNA was 2646 nucleotides long and had a copyback structure containing a 50S genomic 5′-end. At the site where copying turns back, there was a 15-nucleotide-long sequence resembling the “measles virus consensus sequence” which is present at the beginning of each gene on the measles virus genome and is presumed to serve as a signal for the initiation of transcription. Recognition of the specific sequence may have been involved in the aberrant replication and may represent an additional and novel copy choice mechanism leading to the generation of subgenomic RNAs.

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.

Sequence variability and function of measles virus 3′ and 5′ ends and intercistronic regions

Sequences critical for the activity of the measles virus (MV) RNA polymerase in transcription and replication were analyzed using a MV genomic cDNA library containing overlapping clones encompassing the entire MV genome. Clones corresponding to the 3′ and 5′ ends of the MV genome were identified and sequenced, and these sequences were confirmed by primer extension experiments. Neither (+) nor (−) strand leader RNAs were detected in MV-infected cell extracts, using high specific activity riboprobes made from these clones. Clones representing each of the MV gene boundaries were also sequenced, and variations including point mutations, insertions, and deletions were noted. Together with the sequence of the MV L gene region, this report completes the sequence determination of the MV genome.

Measles virus L protein evidences elements of ancestral RNA polymerase

We have determined the nucleotide sequence of the measles virus (MV) L gene using a cDNA library encompassing the entire MV genome (J. Crowley et al. (1987) Intervirology, 28, 65–77). The L gene is 6639 nucleotides in length, and contains a single long open reading frame that could code for a protein of 247,611 kDa. Both the L gene and in particular the predicted L protein of MV bear substantial homology to their counterparts in Sendai virus and Newcastle disease virus, suggesting that the multifunctional nature of paramyxovirus L proteins imposes strong evolutionary constraints. The predicted MV L protein also contains distinct elements of a postulated ancestral RNA polymerase.

Multiple viral mutations rather than host factors cause defective measles virus gene expression in a subacute sclerosing panencephalitis cell line.

A measles virus (MV) genome originally derived from brain cells of a subacute sclerosing panencephalitis patient expressed in IP-3-Ca cells an unstable MV matrix protein and was unable to produce virus particles. Transfection of this MV genome into other cell lines did not relieve these defects, showing that they are ultimately encoded by viral mutations. However, these defects were partially relieved in a weakly infectious virus which emerged from IP-3-Ca cells and which produced a matrix protein of intermediate stability. The sequences of several cDNAs related to the unstable and intermediately stable matrix proteins showed many differences in comparison with a stable matrix protein sequence and even appreciable heterogeneity among themselves. Nevertheless, partial restoration of matrix protein stability could be ascribed to a single additional amino acid change. From an examination of additional genes, we estimated that, on average, each MV genome in IP-3-Ca cells differs from the others in 30 to 40 of its 16,000 bases. The role of extreme variability of RNA virus genomes in persistent viral infections is discussed in the context of the pathogenesis of subacute sclerosing panencephalitis and of other human diseases of suspected viral etiology.

Persistent measles virus genome in autoimmune chronic active hepatitis: Cause or coincidence?

A radiolabelled 50-base oligonucleotide complementary with the measles virus gene encoding the nucleocapsid was used as a probe to identify persistent measles virus genome in the lymphocytes from patients with autoimmune chronic active hepatitis (AICAH). Positive hybrids were found in 12 of 18 patients, and correlated strongly with high antibody titers to measles. Among the 45 controls, positive hybrids were found in one patient with measles, 1 of 3 patients with lupus erythematosis and 2 of 4 patients with cryptogenic cirrhosis. Persistence of part of the measles virus genome in AICAH may have important implications in the pathogenesis of the liver disease, and possibly in other disorders such as systemic lupus erythematosis, multiple sclerosis and Paget’s disease where an abnormal immune response to measles has been observed.

PERSISTENT MEASLES VIRUS GENOME IN AUTOIMMUNE CHRONIC ACTIVE HEPATITIS

A radiolabelled 50-base oligonucleotide complementary with the measles virus gene encoding the nucleocapsid was used as a probe to identify persistent measles virus genome in the lymphocytes from patients with autoimmune chronic active hepatitis (AICAH). Positive hybrids were found in 12 of 18 patients, and correlated strongly with high antibody titres to measles. Among the 45 controls, positive hybrids were found in 1 patient with measles, 1 of 3 patients with systemic lupus erythematosus, and 2 of 4 patients with cryptogenic cirrhosis. Persistence of part of the measles virus genome in AICAH may have important implications in the pathogenesis of the liver disease, and possibly in other disorders such as systemic lupus erythematosus, multiple sclerosis, and Paget's disease where an abnormal immune response to measles has been observed.

Altered transcription of a defective measles virus genome derived from a diseased human brain.

Measles virus (MV) is a negative strand RNA virus which usually causes acute disease, but in rare cases its persistence in the human brain induces the lethal disease subacute sclerosing panencephalitis (SSPE). The transcription of MV and of a defective MV derived from autopsy material of a SSPE case was studied in cultured cells. In the lytic infection the levels of the MV mRNAs decreased progressively with the distance of the six cognate genes from the 3' end of the genome, reflecting transcriptional attenuation at every gene junction. Transcripts covering two or three adjacent genes accounted for up to 20% of single gene transcripts; incidentally the MV intergenic transcription signals were found to be less conserved than the analogous signals of other negative strand RNA viruses. Although the analysed SSPE-derived defective MV showed a localized transcription defect at the phosphoprotein--matrix gene junction (substitution of the mRNAs by readthrough transcripts), the corresponding intergenic 'consensus' sequence and the surrounding nucleotides were not altered. This implies that factor(s) involved in the transcription of this defective SSPE virus fail to recognize this particular signal sequence, a constellation which in this and other cases might be causally related to the development of MV persistence.

Molecular cloning of 99% of measles virus genome, positive identification of 5' end clones, and mapping of the L gene region.

Molecular probes specific for paramyxovirus 3' and 5' end sequences are essential for studying viral transcription and replication and the role of defective interfering particles in these processes. We have constructed from measles virus genomic RNA an ordered series of overlapping clones containing over 99% of measles virus genetic information. Clones corresponding to the 3' and 5' ends of the genome were identified by means of a probe made from 'capped' genomic RNA, and their location was confirmed by primer extension experiments. Clones representing the N, P/C, M, F, and H genes were identified by reference to previously characterized clones, and their positions in the genome were defined by restriction analysis and S1 nuclease mapping of intercistronic clones. The L gene region was characterized by site-to-site restriction mapping and measured as 6,550 nucleotides in length.

Accumulated measles virus mutations in a case of subacute sclerosing panencephalitis: Interrupted matrix protein reading frame and transcription alteration

Subacute sclerosing panencephalitis (SSPE) is a fatal disease affecting the human central nervous system several years after acute measles infection. Measles virus (MV) genomes replicating in SSPE brains do not give rise to budding particles and present various defects in gene expression, mostly concerning the matrix (M) protein. For one SSPE case (K), shown previously to be devoid of M protein expression, we examined here in detail the features involved in this defect. In the brain of patient K the normal, monocistronic MV M mRNA was completely substituted by a bicistronic RNA containing the coding sequence of the preceding phosphoprotein (P) gene in addition to the M coding sequence. Analysis of the P-M intercistronic region by direct cDNA sequencing showed that the consensus sequence at this RNA processing site was unaltered but revealed several distant point mutations. cDNA cloning and sequencing of the entire M coding region established that one of the point mutations leads to a stop codon at triplet 12 of the M reading frame. It is unknown whether this defect, explaining by itself the lack of M protein, is related also to the block of M mRNA formation. In addition we note that as much as 1% of the nucleotides different between two overlapping clones from the same brain. This high sequence variability could possibly account for the diversity of defects observed in MV gene expression in SSPE brains and may be a general phenomenon associated with RNA virus persistence.

Transcriptional map of the measles virus genome.

We have constructed a recombinant DNA library of the measles virus genome and identified gene-specific clones containing sequences coding for portions of each of the six viral structural proteins, as well as clones coding for intercistronic sequences. By Northern blotting, we could order the clones according to the pattern of individual gene-specific and readthrough mRNAs. Clones corresponding to the N, P/C and M genes were identified by correlation of the mRNAs with their in vitro translation products; clones corresponding to the H, F and L genes were identified by indirect evidence. The results indicated that the gene order in measles virus is that of a typical paramyxovirus (3'-N,P/C,M,F,H,L-5'), but that the M and F transcripts each contain 1.5 times the coding capacity needed for synthesis of these proteins in vivo.