Measles Virus Isolates Research Articles

Measles Virus Receptor SLAM (CD150)

Measles virus (MV), a member of the Morbillivirus genus in the Paramyxoviridae family, is an enveloped virus with a nonsegmented negative-strand RNA genome and infects humans and nonhuman primates (Griffin, 2001). Despite the availability of vaccines, MV remains a major cause of childhood mortality, claiming roughly one million lives a year worldwide. The transient immunosuppression that accompanies and follows measles renders the patients susceptible to secondary infections accounting for most of measles-related complications and deaths. MV also causes postinfectious encephalitis, and in rare instances, subacute sclerosing panencephalitis, a persistent infection in the central nervous system. This review is concerned with the identification of a new MV receptor and its implication for understanding the pathology and pathogenesis of MV infection. MV was first isolated in primary human kidney cells inoculated with the blood and throat washings of a child with measles (Enders and Peebles, 1954). This first isolate, the Edmonston strain, was subsequently adapted to chicken embryo fibroblasts and became the progenitor for currently used attenuated vaccines. The Edmonston strain also grows well in continuous cell lines and has become the most extensively studied MV strain in laboratories. Vero cells, an African green monkey kidney cell line, had been commonly used for MV isolation until a decade ago, but several blind passages were usually required before virus propagation and development of cytopathic effect (CPE). Kobune et al. (1990) reported that an Epstein–Barr virus (EBV)-transformed marmoset B cell line B95-8 and its adherent subline B95a were highly sensitive to MV. Furthermore, they showed that MV strains isolated in B95a cells, but not Vero cell-isolated

Measles virus-induced modulation of host-cell gene expression

The influence of measles virus (MV) infection on gene expression by human peripheral blood mononuclear cells (PBMCs) was examined with cDNA microarrays. The mRNA levels of more than 3000 cellular genes were compared between uninfected PBMCs and cells infected with either the Edmonston MV strain or a wild-type MV isolate. The MV-induced upregulation of individual genes identified by microarray analyses was confirmed by RT-PCR. In the present study, a total of 17 genes was found to be upregulated by MV infection. The Edmonston strain grew better in the PBMC cultures than the wild-type MV, and the Edmonston strain was a stronger inducer of the upregulated host cell genes than the wild-type virus. The anti-apoptotic B cell lymphoma 3 (Bcl-3) protein and the transcription factor NF-kappaB p52 subunit were upregulated in infected PBMCs both at the mRNA and at the protein level. Several genes of the interferon system including that for interferon regulatory factor 7 were upregulated by MV. The genes for a number of chaperones, transcription factors and other proteins of the endoplasmic reticulum stress response were also upregulated. These included the gene for the pro-apoptotic and growth arrest-inducing CHOP/GADD153 protein. Thus, the present study demonstrated the activation by MV of cellular mechanisms and pathways that may play a role in the pathogenesis of measles.

Adaptation of wild-type measles virus to tissue culture.

Measles has a host range restricted to humans and monkeys in captivity. Fresh measles virus (MV) isolates replicate readily in several human and simian B-cell lines but need a period of adaptation to other types of cells. The identification of CD46 and CD150 (SLAM) as cellular receptors for MV has helped to clarify certain aspects of the immunobiology of MV infections. We have examined the properties of an MV wild-type strain grown in the epithelial cell line Vero. After adaptation, this virus expressed high levels of both the viral glycoproteins (hemagglutinin and fusion protein) but did not induce fusion (syncytia). No changes in the amino acid sequence were found in either of the viral glycoproteins. Using several approaches, the Vero-adapted virus could not be shown to interact with CD46 either in the initiation or during the course of infection. The presence of human SLAM expressed in the Vero cells rapidly gave rise to fusion and lower yields of infectious virus.

Immune and artificial selection in the haemagglutinin (H) glycoprotein of measles virus.

We present a maximum likelihood (ML) analysis of the selection pressures that have shaped the evolution of the large (L) protein and the haemagglutinin (H) glycoprotein of measles virus (MV). A number of amino acid sites that have potentially been subject to adaptive evolution were identified in the H protein using sequences from every known genotype of MV. All but one of these putative positively selected sites reside within the ectodomain of the H protein, where they often show an association with positions of potential B-cell epitopes and sites known to interact with the CD46 receptor. This suggests that MV may be under pressure from the immune system, albeit relatively weakly, to alter sites within epitopes and hence evade the humoral immune response. The positive selection identified at amino acid 546 was shown to correlate with the passage history of MV isolates in Vero cells. We reveal that Vero cell passaging has the potential to introduce an artificial signal of adaptive evolution through selection for changes that increase affinity for the CD46 receptor.

The cellular receptor for measles virus--elusive no more.

The identity of the measles virus receptor has been controversial. Several years ago CD46 was identified as a cellular receptor for the Edmonston strain of measles virus, but most clinical isolates of measles virus, which are most efficiently isolated in the marmoset B cell line B95a, cannot grow in many CD46+ cell lines. Although some researchers attributed it to post-entry block in viral replication, others believed that there is a receptor other than CD46 for wild-type measles viruses. A new study showed that human signalling lymphocytic activation molecule (SLAM; also known as CDw150) is a cellular receptor for measles virus, including the Edmonston strain. SLAM is expressed on lymphocytes and dendritic cells, and plays an important role in lymphocyte activation. The identification of SLAM as a measles virus receptor nicely explains the pathogenesis of measles virus infection.

Adaptation of wild-type measles virus to CD46 receptor usage.

Vaccine strains of measles virus (MV) use CD46 as receptor and downregulate CD46 from the surface of infected cells. MVs isolated and passaged on B-lymphoid cells (wild-type MVs) seem to use another receptor and do not downregulate CD46. In the present study, we found that isolation of MV on human or marmoset B-lymphoid cells did not alter the MV haemagglutinin (H) protein relative to that in the patient. The wild-type isolates were adapted to the human epithelial HEp-2 cell line or the monkey fibroblast Vero cell line. All HEp-2 cell adapted viruses and 1 out of 4 Vero cell adapted viruses acquired the capacity to use CD46 as receptor, as measured by their ability to infect murine cells expressing human CD46. Adaptation to CD46 receptor usage was coupled to substitution of amino acid 481 of the MV H protein from asparagine to tyrosine but not to CD46 downregulation. The present study demonstrates that CD46 receptor usage can be induced by adaptation of wild-type MV to cells that do not express a wild-type receptor and suggests that a similar mechanism acted on the progenitor viruses of the present MV vaccine strains during their isolation and attenuation.

CDw150(SLAM) Is a Receptor for a Lymphotropic Strain of Measles Virus and May Account for the Immunosuppressive Properties of This Virus

Natural isolates of measles virus readily infect several lymphocyte cell lines. These viruses appear to use a receptor other than CD46, the molecule to which most laboratory strains of virus bind. Methods used to identify and characterize this lymphocyte receptor for measles virus are described in this study. A binding assay with a soluble form of measles virus H protein demonstrated that B-cell lines, activated with Epstein–Barr virus, or T cells, transformed with human T-cell leukemia virus, exhibit this receptor on their cell surfaces. On the other hand, resting lymphocytes, monocytes, or immature leukocytes either failed to express or possessed reduced levels of this receptor. A cDNA library derived from B95-8 marmoset B-cell lines was used to identify this receptor through expression cloning. This molecule was shown to be CDw150, which is also known as the signaling lymphocytic activation molecule (SLAM). When the lymphocyte receptor was expressed in Chinese hamster ovary (CHOP) or human embryonic kidney (293T) cells, these cells became susceptible to lymphotropic as well as laboratory strains of measles virus. Binding assays confirmed that either lymphotropic or laboratory strains of measles virus could adhere to human or marmoset CDw150, but interaction with the mouse homolog was weak. These infections were independent of the presence of CD46 on the host cell surface. Interaction of measles virus with CDw150(SLAM) could explain the immunosuppressive properties of this virus.

UK measles outbreak in non-immune anthroposophic communities: the implications for the elimination of measles from Europe.

We describe the epidemiology of the first nationwide outbreak of measles infection in the UK since the implementation of a mass vaccination campaign. Notifications of infectious diseases, interview and postal questionnaire identified 293 clinical cases, 138 of which were confirmed by salivary IgM, measles virus isolation and PCR. Twelve were epidemiologically linked to confirmed cases. The outbreak began in London, after contact with measles infection probably imported from Italy. Measles genotyping determined by sequence analysis confirmed spread to other unimmunized anthroposophic communities in the north, south west and south coast of England. Only two cases had been vaccinated against measles infection, and 90% of cases were aged under 15 years. Measles virus can selectively target non-immune groups in countries with high vaccine uptake and broader herd immunity. Without harmonization of vaccination policies and uniform high coverage across Europe, the importation and spread of measles virus amongst non-immune groups may prevent the elimination of measles.

SLAM (CDw150) is a cellular receptor for measles virus

Measles virus continues to be a major killer of children, claiming roughly one million lives a year. Measles virus infection causes profound immunosuppression, which makes measles patients susceptible to secondary infections accounting for high morbidity and mortality. The Edmonston strain of measles virus, and vaccine strains derived from it, use as a cellular receptor human CD46 (refs 3, 4), which is expressed on all nucleated cells; however, most clinical isolates of measles virus cannot use CD46 as a receptor. Here we show that human SLAM (signalling lymphocyte-activation molecule; also known as CDw150), a recently discovered membrane glycoprotein expressed on some T and B cells, is a cellular receptor for measles virus, including the Edmonston strain. Transfection with a human SLAM complementary DNA enables non-susceptible cell lines to bind measles virus, support measles virus replication and develop cytopathic effects. The distribution of SLAM on various cell lines is consistent with their susceptibility to clinical isolates of measles virus. The identification of SLAM as a receptor for measles virus opens the way to a better understanding of the pathogenesis of measles virus infection, especially the immunosuppression induced by measles virus.

Sequence analysis of the hemagglutinin gene of measles virus isolates in Denmark 1997-1998: no evidence of persistent circulation of measles virus in DenmarkNote

The hemagglutinin-coding region of 17 virus samples from 12 measles cases in Denmark during 1997-1998 was analysed by partial nucleotide sequencing. The cases appeared as three sporadic cases and two epidemics, both with a limited time course and geographical distribution. The measles strains identified from the three sporadic cases and two epidemics could be allocated to five different previously well-defined sequence groups consistent with the assumption that cases of measles in Denmark are due to repeated introduction from abroad rather than persistent circulation of strains in the population.

Acute renal failure with neurological involvement in adults associated with measles virus isolation

Three people with clinical manifestations of acute renal failure with neurological involvement of unknown cause were admitted to a hospital in Mumbai, India. We describe clinical presentations and investigations of the cause. We analysed case reports and laboratory findings for the patients (age 37-43 years, two men, one woman) that were provided by the clinicians in charge. Serum and cerebrospinal fluid were tested for viral cause by IgM ELISA to Japanese encephalitis, West Nile fever, dengue, and measles. Samples were inoculated in vero-cell culture for virus isolation. The virus isolates were confirmed with indirect immunofluoresence with antimeasles immune sera and mouse monoclonal antibodies to measles HA and F proteins and with neutralisation tests using antimeasles immune sera. Clinical features were fever, vomiting, oliguria or anuria, bilateral facial weakness, impaired hearing, blindness, proximal and distal areflexic limb paralysis, and respiratory paralysis. No patient had a macropapular rash. Blood urea nitrogen (4.64-27.8 mmol/L) and creatinine (601.1-1105.0 micromol/L) were high, and cerebrospinal fluid contained high concentrations of proteins and pleocytosis. Kidney biopsy samples in two patients showed severe interstitial nephritis. IgM antibodies to measles were found in blood and cerebrospinal fluid. Vero-cell cultures from serum and cerebrospinal fluid of one patient and cerebrospinal fluid of two patients, showed cytopathic effects characteristic of measles. Unusual manifestations of acute renal failure with neurological involvement associated with measles virus in adults presenting without rash was confirmed. Our findings may affect the development of measles-elimination programmes.

Genotypic and antigenic characterization of hemagglutinin proteins of African measles virus isolates

A comprehensive phylogenetic study based on the hemagglutinin (H) protein of all known African measles virus (MV) isolates is presented. The study includes 64 new H gene sequences from Ghana, Nigeria and South Africa as well as viruses from Zambia and The Gambia for which only incomplete sequencing data were available and that have previously not been genotyped. The results provide further support to the tentative assignment of the Nigerian and Ghanaian viruses to a new genotype B3 within clade B. A distinct geographic distribution pattern emerged with clade B viruses circulating exclusively in African countries north of the equator. All MV strains from southern Africa grouped in clades A and D with the majority of viruses belonging to genotype D4. The viruses considerably differed by their sensitivity to neutralization by monoclonal antibodies (mAb), but three selected antibodies were sufficient to distinguish between African MVs representing four different genotypes.

Genotyping of measles virus isolates from Central Europe and Russia

Sequence analysis of 285 nucleotides located on the variable part of the N gene was undertaken on measles virus (MV) samples collected from acutely infected patients in Germany, the Czech Republic, Denmark, Poland, and Russia. Two distinct genotypes (C2 and D6) have circulated in Germany between 1993 and 1996. Isolates of genotype C2 were related to strains reported in Germany before 1993. This genotype was also found in the Czech Republic in 1992 and in Denmark in 1997. The occurrence of genotype D6 in Germany is described below for the first time. In 1998, this genotype was identified in Poland. Genotypes C2 and D6 were also reported in Spain and in the United Kingdom between 1992 and 1996. Therefore, it is concluded that these genotypes are widely distributed over Europe. The analysis of the isolates from Russia revealed that genotype A was present in 1988 in the European part of the country and in 1996 in Siberia. An isolate identified in 1997 in Siberia belonged to genotype D6, which had never been found previously in Russia. We also analysed MV obtained from a case of subacute sclerosing panencephalitis (SSPE) in 1995 in Turkey. A comparison of this sequence with published sequences implied that this SSPE case was associated with a new genetic lineage of MV.

Genotyping of measles virus isolates from Central Europe and Russia

Sequence analysis of 285 nucleotides located on the variable part of the N gene was undertaken on measles virus (MV) samples collected from acutely infected patients in Germany, the Czech Republic, Denmark, Poland, and Russia. Two distinct genotypes (C2 and D6) have circulated in Germany between 1993 and 1996. Isolates of genotype C2 were related to strains reported in Germany before 1993. This genotype was also found in the Czech Republic in 1992 and in Denmark in 1997. The occurrence of genotype D6 in Germany is described below for the first time. In 1998, this genotype was identified in Poland. Genotypes C2 and D6 were also reported in Spain and in the United Kingdom between 1992 and 1996. Therefore, it is concluded that these genotypes are widely distributed over Europe. The analysis of the isolates from Russia revealed that genotype A was present in 1988 in the European part of the country and in 1996 in Siberia. An isolate identified in 1997 in Siberia belonged to genotype D6, which had never been found previously in Russia. We also analysed MV obtained from a case of subacute sclerosing panencephalitis (SSPE) in 1995 in Turkey. A comparison of this sequence with published sequences implied that this SSPE case was associated with a new genetic lineage of MV. J. Med. Virol. 58:313–320, 1999. © 1999 Wiley-Liss, Inc.

Molecular epidemiology of Nigerian and Ghanaian measles virus isolates reveals a genotype circulating widely in western and central Africa.

Sub-Saharan Africa is one of the regions of the globe with the highest measles-related morbidity and mortality. Yet only seven virus isolates from this vast region have been phylogenetically characterized on the basis of their nucleoprotein, the last one in 1991. To characterize the prevalent wild-type viruses and to understand their circulation pattern, a large panel (n = 45) of isolates was collected in Ghana and Nigeria in 1997 and 1998. On the basis of their nucleoprotein sequence, the viruses clearly belong to clade B but a reshuffling of the structure of this clade was proposed, tentatively extending the number of genotypes from two to three on the basis of quantitative criteria. The sequences revealed the co-circulation of at least two distinct viruses in the cities of Lagos and Ibadan, suggesting that the number of susceptible individuals seems to be high enough to support endemic circulation of at least two distinct viruses. The endemic co-circulation of several viruses may well be a characteristic of communities with low vaccination rates. One of these viruses was also found in Accra in 1998 as well as in a 1994 case linked to distant Kenya, suggesting that clade B viruses are prevalent in sub-Saharan Africa while non-B viruses seem to dominate the south of Africa.

A single amino acid change in the hemagglutinin protein of measles virus determines its ability to bind CD46 and reveals another receptor on marmoset B cells.

This paper provides evidence for a measles virus receptor other than CD46 on transformed marmoset and human B cells. We first showed that most tissues of marmosets are missing the SCR1 domain of CD46, which is essential for the binding of Edmonston measles virus, a laboratory strain that has been propagated in Vero monkey kidney cells. In spite of this deletion, the common marmoset was shown to be susceptible to infections by wild-type isolates of measles virus, although they did not support Edmonston measles virus production. As one would expect from these results, measles virus could not be propagated in owl monkey or marmoset kidney cell lines, but surprisingly, both a wild-type isolate (Montefiore 89) and the Edmonston laboratory strain of measles virus grew efficiently in B95-8 marmoset B cells. In addition, antibodies directed against CD46 had no effect on wild-type infections of marmoset B cells and only partially inhibited the replication of the Edmonston laboratory strain in the same cells. A direct binding assay with insect cells expressing the hemagglutinin (H) proteins of either the Edmonston or Montefiore 89 measles virus strains was used to probe the receptors on these B cells. Insect cells expressing Edmonston H but not the wild-type H bound to rodent cells with CD46 on their surface. On the other hand, both the Montefiore 89 H and Edmonston H proteins adhered to marmoset and human B cells. Most wild-type H proteins have asparagine residues at position 481 and can be converted to a CD46-binding phenotype by replacement of the residue with tyrosine. Similarly, the Edmonston H protein did not bind CD46 when its Tyr481 was converted to asparagine. However, this mutation did not affect the ability of Edmonston H to bind marmoset and human B cells. The preceding results provide evidence, through the use of a direct binding assay, that a second receptor for measles virus is present on primate B cells.

Isolation of measles virus below 4 months of age during an outbreak in Pune, India

This article reports the isolation of a measles virus from infants under 4 months of age during an outbreak in an orphanage at Pune, India, in 1996. With a total population of 24 orphanages, 6 inmates showed the signs and symptoms of measles; 4 were under 4 months old and the other 2 were 7 and 11 months old, respectively. Bronchopneumonia and weight loss were observed as the main complications of the disease. Isolation of the measles virus was done by obtaining throat swabs, skin scrapings, and blood samples from the victims. Serum testing of the 6 cases showed a positive result for immunoglobulin M (IgM) antibodies. During the same period, mothers of 4 IgM-positive infants were screened for antibodies; 3 mothers were negative for measles-specific antibodies. Because of this, lack of maternal antibodies, malnutrition, and crowding were accounted for the outbreak of measles in the orphanage. These findings reflect a need to assess the prevalence of measles within this age group and develop prevention strategies for measles.

The hemagglutinin of recent measles virus isolates induces cell fusion in a marmoset cell line, but not in other CD46-positive human and monkey cell lines, when expressed together with the F protein.

Measles virus (MV) is efficiently isolated from patients with measles by using B95a cells, a marmoset B cell line. Recent wild-type MV strains isolated using B95a cells did not produce cytopathic effects in any of CD46+ primate cell lines examined (except B95a cells), nor did they induce downregulation of CD46. Transfection of the hemagglutinin (H) and fusion (F) genes of the Edmonston strain of MV produced syncytia in HeLa, Cos and B95a cells. By contrast, the expression of the H gene from the two wild-type strains, together with the F gene of the Edmonston strain, resulted in syncytium production in B95a cells, but not in HeLa and Cos cells. Cocultivation of Cos cells expressing the wild-type H protein and the Edmonston strain F protein with B95a cells, but not with HeLa, Jurkat or BJAB cells, generated large syncytia. The results suggest that these recent MV isolates may use a molecule other than CD46 as the cellular receptor or require another coreceptor to infect cells.

Sequence analysis of the nucleocapsid gene of measles virus isolates from South Africa identifies a new genotype.

Sequence analysis was performed on 20 measles virus (MV) isolates from South Africa, five of which were obtained between 1986 and 1989 and 15 isolates collected during the 1994/95 measles season. A 590 bp fragment of the carboxyl terminus of the nucleocapsid (N) was amplified by PCR and subjected to sequence and phylogenetic analysis. Comparison of the South African MV strains with those previously described revealed that at least two distinct groups of wild-type (wt) MV exist, one of which has been circulating since 1986. The major genotype (I) was represented by the more recent isolates which showed three characteristic amino acid substitutions. Furthermore, three vaccine-like viruses with sequences very similar to the Edmonston wt strain were identified. Phylogenetic analysis of 100 MV strains allowed the assignment of new definitions for MV genotypes and subgroups. Employing these definitions, the majority of South African isolates analysed here formed a new genotype.

Rapid identification of measles virus strains by the heteroduplex mobility assay

The continued endemic presence of measles virus (MV), and the large number of isolates which are made in South Africa each year, demanded the use of a rapid and reliable pre-screening technique to select isolates for molecular epidemiological studies by sequence analysis. The heteroduplex mobility assay (HMA) was used to genetically characterize 47 MV isolates collected from three different provinces in South Africa, made between 1986 and 1995. The carboxyl-terminal 590 nt of the nucleocapsid (N) gene—the most variable region of the genome—was amplified by polymerase chain reaction (PCR) and subsequently subjected to HMA analysis for initial genotyping. The results showed three different patterns of heteroduplex formation by gel electrophoresis, representing two distinct wild-type lineages and one group of vaccine-like viruses. Comparison of HMA results with phylogenetic analysis of sequence data for several of the South African MV strains showed a complete correlation of results. The HMA proved to be a useful tool for screening MV isolates for use in molecular epidemiological studies.