Measles Virus RNA Research Articles

Combination therapy with intraventricular interferon-α and ribavirin for subacute sclerosing panencephalitis and monitoring measles virus RNA by quantitative PCR assay

Subacute sclerosing panencephalitis (SSPE) is a degenerative disease of the central nervous system that leads to death within a few years. Recently, it has been reported that combination therapy with intraparenchymal interferon-α (INF-α) and intraventricular ribavirin is effective. An 11-year-old SSPE patient whose clinical symptoms progressed rapidly, was treated first with intraventricular INF-α and then with combined intraventricular INF-α and ribavirin therapy. To monitor viral load over the course of the therapy, measles virus RNA was quantified using a real-time polymerase chain reaction assay. Measles virus RNA decreased rapidly after the INF-α therapy was started, paralleling the decrease in the measles antibody titer in the cerebrospinal fluid and the improvement in the neurological disability. After intraventricular ribavirin was combined with INF-α therapy, no further improvement was observed. The neurological disability gradually progressed, although the amount of virus RNA remained low.

Use of RT-PCR on oral fluid samples to assist the identification of measles cases during an outbreak.

This study investigated the occurrence of mild modified measles cases during an outbreak in Niterói, RJ, Brazil by using RT-PCR on oral fluid samples. From August to December 1997 a total of 76 patients with rash were seen at the study sites. Confirmed diagnosis by serology was achieved in 47 cases: measles (39.5%), rubella (13.2%), HHV-6 (3.9%), human parvovirus B19 (3.9%), dengue fever (3%). For 19 of the 29 patients without a conclusive diagnosis paired serum and saliva samples were available for further tests. In four of them, measles virus RNA was detected by RT-PCR in saliva samples in the absence of specific IgM in serum samples. Vaccination histories obtained from three of the RT-PCR positive cases showed that individuals previously immunized can still be infected and contribute to the circulation of measles virus. This study demonstrated the usefulness of RT-PCR on non-invasive clinical samples for the investigation of measles cases.

The CXC Chemokine GCP-2/CXCL6 Is Predominantly Induced in Mesenchymal Cells by Interleukin-1β and Is Down-Regulated by Interferon-γ: Comparison with Interleukin-8/CXCL8

Human granulocyte chemotactic protein-2 (GCP-2)/CXCL6 is a CXC chemokine that functionally uses both of the IL-8/CXCL8 receptors to chemoattract neutrophils but that is structurally most related to epithelial cell–derived neutrophil attractant-78 (ENA-78)/CXCL5. This study provides the first evidence that GCP-2 protein is, compared with IL-8, weakly produced by some sarcoma, but less by carcinoma cells, and is tightly regulated in normal mesenchymal cells. IL-1β was the predominant GCP-2 inducer in fibroblasts, chondrocytes, and endothelial cells, whereas IL-8 was equally well up-regulated in these cells by TNF-α, measles virus, or double-stranded RNA (dsRNA). In contrast, lipopolysaccharide (LPS) was a relatively better stimulus for GCP-2 versus IL-8 in fibroblasts. IFN-γ down-regulated the GCP-2 production in fibroblasts induced by IL-1β, TNF-α, LPS, or dsRNA. The kinetics of GCP-2 induction by IL-1β, LPS, or dsRNA in fibroblasts differed from those of IL-8. Freshly isolated peripheral blood mononuclear leukocytes, which are a good source of IL-8 and ENA-78, failed to produce GCP-2. However, lung macrophages and blood monocyte–derived macrophages produced GCP-2 in response to LPS. Quantitatively, secretion of GCP-2 always remained inferior to that of IL-8, despite the fact that the ELISA recognized all posttranslationally modified GCP-2 isoforms. The expression of GCP-2 was confirmed in vivo by immunohistochemistry. The patterns of producer cell types, inducers and kinetics and the quantities of GCP-2 produced, suggest a unique role for GCP-2 in physiologic and pathologic processes.

Subacute measles encephalitis in an immunocompetent adult

Subacute sclerosing panencephalitis (SSPE) and subacute measles encephalitis (SME) are both rare complications of measles virus infection. SSPE typically affects immunocompetent children, has an insidious onset and follows a steadily progressive course. SME mainly occurs in immunosuppressed children and has a rapidly progressive course. We describe a 43 year old immunocompetent man who presented with a rapidly progressive fatal encephalopathy. Histological examination of the brain showed a meningoencephalitis with inclusion bodies. Complement fixing antibody to measles virus was present in his serum and CSF. Measles virus RNA was found in the brain, spinal cord and eye, but not in the CSF. Analysis of the nucleoprotein gene isolated from this patient did not show similarity to SSPE strains of the measles virus. This patient demonstrates that subacute encephalitis secondary to measles virus infection can develop in an immunocompetent adult host.

Efficiency of measles virus entry and dissemination through different receptors.

The efficiency with which different measles virus (MV) strains enter cells through the immune cell-specific protein SLAM (CD150) or other receptors, including the ubiquitous protein CD46, may influence their pathogenicity. We compared the cell entry efficiency of recombinant MV differing only in their attachment protein hemagglutinin (H). We constructed these viruses with an additional gene expressing an autofluorescent reporter protein to allow direct detection of every infected cell. A virus with a wild-type H protein entered cells through SLAM two to three times more efficiently than a virus with the H protein of the attenuated strain Edmonston, whereas cell entry efficiency through CD46 was lower. However, these subtle differences were amplified at the cell fusion stage because the wild-type H protein failed to fuse CD46-expressing cells. We also proved formally that a mutation in H protein residue 481 (asparagine to tyrosine) results in improved CD46-specific entry. To define the selective pressure exerted on that codon, we monitored its evolution in different H protein backgrounds and found that several passages in CD46-expressing Vero cells were necessary to shift it in the majority of the MV RNA. To verify the importance of these observations for human infections, we examined MV entry into peripheral blood mononuclear cells and observed that viruses with asparagine 481 H proteins infect these cells more efficiently.

Detection of measles virus RNA in whole blood stored on filter paper

The purpose of this study was to evaluate the use of dried blood spots stored on filter paper as a means to provide specimens for virologic surveillance for measles virus (MV) in situations when the reverse cold chain is not available. Two single-step RT-PCR assays were evaluated for sensitivity of detection of MV nucleoprotein gene RNA. The more sensitive assay was then used to assess the stability of MV RNA in dried whole blood stored on filter paper. MV RNA was found to be stable in dried blood spots for up to 2 months at room temperature or 1 month at 37 degrees C. As few as 100 infected human peripheral blood mononuclear cells (PBMC) per blood spot could be detected using a single-step RT-PCR reaction and ethidium bromide detection. MV RNA was also detected in dried blood spots obtained from rhesus macaques after challenge with wild-type MV. In the rhesus samples, the single-step RT-PCR reaction could detect approximately 10(3) TCID(50) per blood spot, while nested PCR detected 3 TCID(50) per blood spot. The results of this laboratory-based study suggest that the use of dried blood spots stored on filter has the potential to improve virologic surveillance for MV in some areas, and they emphasize the need for continued testing under field conditions.

Restriction of measles virus RNA synthesis by a mouse host cell line: trans-complementation by polymerase components or a human cellular factor(s).

The mouse epithelial MODE-K cell line expressing human CD46 or CD150 cellular receptors was found to be nonpermissive for measles virus (MV) replication. The virus binding and membrane fusion steps were unimpaired, but only very limited amounts of virus protein and RNA synthesized were detected after the infection. In a minigenome chloramphenicol acetyltransferase assay, MODE-K cells were as able as the permissive HeLa cells in supporting MV polymerase activity. The restriction phenotype of MODE-K cells could be alleviated by providing, in trans, either N-P-L or N-P functional protein complexes but not by P-L complexes or individual N, P, and L proteins. Several human x mouse (HeLa x MODE-K) somatic hybrid clones expressing human CD46 were isolated and found to be either nonpermissive or permissive according to their human chromosomal contents. The MV-restricted phenotype exhibited by the MODE-K cell line suggests that a cellular factor(s) can control MV transcription, possibly by stabilizing the incoming virus polymerase templates.

Sequence analysis of measles virus nucleocapsid transcripts in patients with Paget's disease.

It has been debated for almost 30 years whether Paget's disease of bone results from paramyxoviral infection of osteoclasts (OCs). Paramyxoviral-like nuclear inclusions are found in OCs from patients with Paget's disease, and measles virus (MV) or canine distemper virus (CDV) messenger RNA (mRNA) transcripts have been detected by in situ hybridization in bone cells from pagetic lesions. Furthermore, immunocytochemical studies have shown the presence of several paramyxoviral species in OCs from patients with Paget's disease. However, others have been unable to detect paramyxoviral transcripts in bone samples from patients with Paget's disease or marrow cultures from involved sites of patients with Paget's disease. Furthermore, no one has been able to isolate an infectious virus from pagetic bone samples or marrow cells from patients with Paget's disease, and a full-length viral gene has not been sequenced from pagetic samples. In this study, we have obtained the full-length sequence for the MV nucleocapsid (MVNP) gene in bone marrow from an involved site from a patient with Paget's disease and more than 700 base pairs (bps) of MVNP sequence in 3 other patients with Paget's disease. These sequences were undetectable in four normal marrow samples studied simultaneously. The sequences from the patients contained multiple mutations that differed from the Edmonston strain MVNP gene. These findings are consistent with the presence of a chronic MV infection in affected sites from these patients with Paget's disease.

Persistent measles virus infection and otosclerosis.

The cause of otosclerosis is still unknown. Recently, measles virus involvement has been implicated. The aim of this study was to analyze the presence of measles virus RNA within the otosclerotic focus and to evaluate the perilymphatic antibody pattern. Bone and perilymph specimens from 40 patients with the spontaneous form of otosclerosis and from control patients were investigated by reverse transcription polymerase chain reaction (RT-PCR), Western blot techniques, and cell culture. By the use of RT-PCR, measles virus RNA could be detected in 32 patients, but not in controls. Analysis of perilymph revealed the presence of antibodies to N, F1, and M measles virus proteins in all cases, and antibodies against H protein in 2 additional cases. In preosteoblasts cultured from otosclerotic bone chips, no measles virus RNA could be amplified. We conclude that the spontaneous form of otosclerosis is, in the vast majority of cases, a measles virus-associated disease of the otic capsule.

Prolonged measles virus shedding in human immunodeficiency virus-infected children, detected by reverse transcriptase-polymerase chain reaction.

A reverse transcriptase-polymerase chain reaction assay was used to detect measles virus RNA in peripheral blood mononuclear cells, urine, and nasopharyngeal specimens from Zambian children during hospitalization and approximately 1-2 months after discharge. Of 47 children, 29 (61.7%) had prolonged measles virus shedding, as defined by detection of measles virus RNA in > or =1 specimen obtained 30-61 days after rash onset. Ten (90.9%) of 11 human immunodeficiency virus (HIV)-infected children had prolonged measles virus shedding, compared with 19 (52.8%) of 36 HIV-uninfected children (P=.02). Prolonged measles virus shedding did not correlate with levels of measles virus-specific antibody. HIV-infected children with measles may have a prolonged infectious period that potentially enhances measles virus transmission and hinders measles control.

Investigation of optimal specimen type and sampling time for detection of measles virus RNA during a measles epidemic.

At various times postonset of rash, 74 patients positive for measles virus-specific immunoglobulin M provided samples for detection of measles virus RNA by a reverse transcriptase PCR. Of lymphocytes, urine, throat swab, and serum specimens, throat swab specimens were optimal for detection of measles virus RNA during the first 2 weeks after the rash.

Persistent Measles Virus Infection as a Possible Cause of Otosclerosis: State of the Art

The etiopathogenesis of otosclerosis is still largely unexplained and remains controversial. Morphologic examinations have shown the presence of a chronic inflammation in otosclerotic tissue. Among the proposed explanations for this inflammation are an immunologic reaction against collagen, mutations of collagen gene 1A1, and a viral infection. In this paper, we focus on the role of measles virus in otosclerosis, and we review the current literature, devoting particular attention to a suspected paramyxoviral etiopathogenesis in Paget's disease. Our examination of footplate fragments by reverse transcription polymerase chain reaction testing in 95 patients with otosclerosis revealed the presence of measles virus RNA in 83% of cases. Quantification of measles virus immunoglobulin G (IgG) in otosclerosis patients indicated that the ratio of antimeasles virus IgG in total IgG was higher in perilymph than in serum. Furthermore, an almost identical incidence of otosclerosis and measles virus-caused mortality in women suggests that women are more susceptible to measles virus infection. Finally, since the introduction of the measles virus vaccination program in Europe, there has been a decline in the incidence of otosclerosis. Moreover, the average age of patients at diagnosis and surgery at our hospital has increased to 54 years. Our findings, when they are considered along with findings regarding the presence of paramyxoviral RNA in Paget's disease, support the hypothesis that measles virus is involved in the etiopathogenesis of otosclerosis.

No evidence of measles virus in stapes samples from patients with otosclerosis.

Otosclerosis is a localized bone dystrophy of unknown etiology mainly involving the stapes. The hypothesis of a persistent infection by the measles virus was based on the inconstant detection of the virus by various methods, including reverse transcription-PCR (RT-PCR) of patients' stapes samples. The aim of this work was to investigate the presence of the measles virus in stapedial otosclerosis foci by different sensitive methods. Pathologic stapes samples were obtained from 35 patients suffering from otosclerosis. Measles virus detection was performed by (i) cocultures of Vero cells and primary cell cultures of bone samples (n = 7), (ii) immunofluorescence study of these cocultures (n = 3), and (iii) RT-PCR on RNA directly obtained from fresh frozen samples (n = 28) and on RNA extracted from the primary cell cultures (n = 2). Viral genomic regions coding for N (nucleoprotein) and M (matrix) proteins were separately amplified. PCR sensitivity was optimized on the measles virus Edmonston strain. Glyceraldehyde-3-phosphate dehydrogenase mRNA was used as a marker of total RNA recovery. PCR products were tested by Southern blot hybridization technique to improve sensitivity and specificity. PCRs amplifying the M and the N protein genes were able to detect the control measles virus RNA at titers as low as 0.1 and 0.01 50% tissue culture infective dose, respectively. With these highly sensitive methods, we could not evidence the presence of the measles virus in any of our bone samples or primary bone cell cultures. Our results do not confirm the hypothesis of persistent measles virus infection in otosclerosis.

Requirements for measles virus induction of RANTES chemokine in human astrocytoma-derived U373 cells.

Interferons and chemokines play a critical role in regulating the host response to viral infection. Measles virus, a member of the Paramyxoviridae family, induces RANTES expression by astrocytes. We have examined the mechanism of this induction in U373 cells derived from a human astrocytoma. RANTES was induced in a dose- and time-dependent manner by measles virus infection. Inhibition of receptor binding by the anti-CD46 antibody TRA-2.10 and of virus-membrane fusion by the tripeptide X-Phe-Phe-Gly reduced RANTES expression. Formalin-inactivated virus, which can bind but not fuse, and extensively UV-irradiated virus, which can bind and fuse, were both ineffective. Therefore, virus binding to the cellular receptor CD46 and subsequent membrane fusion were necessary, but not sufficient, to induce RANTES. UV irradiation of virus for less than 10 min proportionally inhibited viral transcription and RANTES expression. RANTES induction was decreased in infected cells treated with ribavirin, which inhibits measles virus transcription. However, RANTES mRNA was superinduced by measles virus in the presence of cycloheximide. These data suggest that partial transcription of the viral genome is sufficient and necessary for RANTES induction, whereas viral protein synthesis and replication are not required. This hypothesis was supported by the fact that RANTES was induced through transient expression of the measles virus nucleocapsid gene but not by measles genes encoding P or L proteins or by leader RNA in A549 cells. Thus, transcription of specific portions of measles virus RNA, such as the nucleocapsid gene, appears able to generate the specific signaling required to induce RANTES gene expression.

Differential induction of monocyte chemotactic protein-3 in mononuclear leukocytes and fibroblasts by interferon-alpha/beta and interferon-gamma reveals MCP-3 heterogeneity.

Monocyte chemotactic protein-3 (MCP-3) is a pluripotent CC chemokine, attracting most leukocytic cell types. With the use of a sensitive and specific ELISA, MCP-3 was found to be inducible in fibroblasts and peripheral blood mononuclear cells (PBMC) by cytokines and cytokine inducers. MCP-3 production levels (1-10 ng/ml) were tenfold lower compared to those of MCP-1. In diploid fibroblasts, synergistic induction of MCP-3, but not of MCP-1, mRNA and protein was observed by combined treatment with IL-1beta and IFN-gamma. In PBMC, IFN-alpha and IFN-beta (but not IFN-gamma), as well as measles virus and double-stranded RNA, were potent inducers of MCP-3, which suggests a role for this chemokine in an early stage of viral infections. In contrast, endotoxin failed to induce MCP-3 production in fibroblasts and PBMC. Purification of MCP-3 from PBMC revealed biochemical heterogeneity. In monocyte chemotaxis and calcium mobilization assays, pure 11-kDa MCP-3 from PBMC showed similar potencies as MCP-3 from tumor cells. It was concluded that the induction of MCP-3 by IFN is regulated differently in fibroblasts and PBMC. In view of the multiple target cells for MCP-3, local and strictly regulated chemokine production might be important to conduct selectively the immune response in infection or inflammation.

Clinicopathological study on liver dysfunction in measles.

We analyzed the clinical course of eight patients with liver dysfunction in measles. All of the patients showed an elevation of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH), but no jaundice. These levels returned to normal about 3 weeks after the onset of the rash. A percutaneous liver biopsy was done in two cases. Histological examination showed slight necrosis of liver cells but no significant changes in portal area. On electron microscopy, virus particles were not detected. We detected measles virus RNA in the liver specimen by RT-PCR, which suggests that the measles virus affects liver cells directly in measles.

Measles virus RNA is not detected in inflammatory bowel disease using hybrid capture and reverse transcription followed by the polymerase chain reaction

Recent epidemiological and immunohistochemical studies have indicated a possible link between measles virus and inflammatory bowel disease (IBD). The aim of this study was to use a sensitive and robust method for the detection of measles virus RNA in IBD and control clinical samples. Peripheral blood mononuclear cells and intestinal resection tissue from IBD and control patients were studied. Two methods were used to determine the presence of measles virus RNA: hybrid capture, using measles virus-specific oligonucleotides linked to paramagnetic solid-phase supports, was carried out on total cellular RNA to enrich for measles virus RNA sequences. Reverse transcription followed by the polymerase chain reaction (RT-PCR) using rTth DNA polymerase was employed for amplification of measles virus N-gene sequences amongst the enriched species. Total RNA was also used for RT-PCR of a housekeeping mRNA species to assess RNA quality. RT-PCR for another region of the measles genome (the haemagglutinin (H) gene) was also undertaken in order to confirm the results obtained using N-gene primers for analysis of these samples. None of the samples were positive for measles N- or H-gene RNA using RT-PCR. Positive control samples confirmed the sensitivity of the methods employed. These results show that either measles virus RNA was not present in the samples, or was present below the sensitivity limits known to have been achieved.

Measles virus RNA is not detected in inflammatory bowel disease using hybrid capture and reverse transcription followed by the polymerase chain reaction

Recent epidemiological and immunohistochemical studies have indicated a possible link between measles virus and inflammatory bowel disease (IBD). The aim of this study was to use a sensitive and robust method for the detection of measles virus RNA in IBD and control clinical samples. Peripheral blood mononuclear cells and intestinal resection tissue from IBD and control patients were studied. Two methods were used to determine the presence of measles virus RNA: hybrid capture, using measles virus-specific oligonucleotides linked to paramagnetic solid-phase supports, was carried out on total cellular RNA to enrich for measles virus RNA sequences. Reverse transcription followed by the polymerase chain reaction (RT-PCR) using rTth DNA polymerase was employed for amplification of measles virus N-gene sequences amongst the enriched species. Total RNA was also used for RT-PCR of a housekeeping mRNA species to assess RNA quality. RT-PCR for another region of the measles genome (the haemagglutinin (H) gene) was also undertaken in order to confirm the results obtained using N-gene primers for analysis of these samples. None of the samples were positive for measles N- or H-gene RNA using RT-PCR. Positive control samples confirmed the sensitivity of the methods employed. These results show that either measles virus RNA was not present in the samples, or was present below the sensitivity limits known to have been achieved. J. Med. Virol. 55:305–311, 1998. © 1998 Wiley-Liss, Inc.

Absence of detectable measles virus genome sequence in inflammatory bowel disease tissues and peripheral blood lymphocytes

A highly sensitive measles-specific RT-PCR-nested PCR system was established, which consistently amplified measles virus genome sequence from control samples containing as little as 5.5 x 10(-3) pfu per reaction. This method failed to detect the presence of measles virus in 93 colonoscopic biopsies and 31 peripheral blood lymphocyte preparations, examined and obtained from patients with inflammatory bowel disease (IBD) and noninflammatory controls. All patients had detectable levels of serum neutralization antibody against measles virus. Each biopsy was estimated to have about one million cells, based on the amplification of the beta actin gene. The assay was calibrated by use of a known number of lymphocytes. The method applied was able to amplify measles virus RNA from a nucleic acid mixture equivalent to 18 cells derived from subacute sclerosing panencephalitis (SSPE) brain material. The level of measles RNA present, if any, in the biopsies is therefore at least 50,000-fold less than in SSPE.

Leukaemia inhibitory factor mRNA is expressed in the brains of patients with subacute sclerosing panencephalitis

We have examined the in situ transcription of leukaemia inhibitory factor (LIF) in brain tissue from 3 cases of subacute sclerosing panencephalitis (SSPE) and in 2 non-neurological control brains. This has been compared with expression of interleukin 2 (IL-2), interleukin 6 (IL-6) and tumour necrosis factor beta (TNF beta) in the same tissues. All of the cytokines in the study were expressed in cells in the inflammatory infiltrate as well as in glial cells. LIF mRNA was also found to be expressed in neurons, in foci where these cells were also virally infected. No hybridization was found with any of the probes in areas of SSPE brain, which were negative for measles virus RNA or in the non-neurological control cases, although expression was demonstrated in the latter by use of reverse transcription-polymerase chain reaction (RT-PCR). Differentiated, cultured human neuronal cells were also positive, by RT-PCR, for LIF. This is the first demonstration of LIF expression in human brain and the results suggest that this cytokine is up-regulated, in several cell types, including neurons, following virus infection.