Functional properties of measles virus proteins derived from a subacute sclerosing panencephalitis patient who received repeated remdesivir treatments.

Subacute sclerosing panencephalitis (SSPE) is a rare but fatal late neurological complication of measles, caused by persistent measles virus (MeV) infection of the central nervous system. There are no drugs approved for the treatment of SSPE. Here, we followed the clinical progression of a 5-year-old SSPE patient after treatment with the nucleoside analog remdesivir, conducted a post-mortem evaluation of the patient's brain, and characterized the MeV detected in the brain. The quality of life of the patient transiently improved after the first two courses of remdesivir, but a third course had no further clinical effect, and the patient eventually succumbed to his condition. Post-mortem evaluation of the brain displayed histopathological changes including loss of neurons and demyelination paired with abundant presence of MeV RNA-positive cells throughout the brain. Next-generation sequencing of RNA isolated from the brain revealed a complete MeV genome with mutations that are typically detected in SSPE, characterized by a hypermutated M gene. Additional mutations were detected in the polymerase (L) gene, which were not associated with resistance to remdesivir. Functional characterization showed that mutations in the F gene led to a hyperfusogenic phenotype predominantly mediated by N465I. Additionally, recombinant wild-type-based MeV with the SSPE-F gene or the F gene with the N465I mutation was no longer lymphotropic but instead efficiently disseminated in neural cultures. Altogether, this case encourages further investigation of remdesivir as a potential treatment of SSPE and highlights the necessity to functionally understand SSPE-causing MeV.IMPORTANCEMeasles virus (MeV) causes acute, systemic disease and remains an important cause of morbidity and mortality in humans. Despite the lack of known entry receptors in the brain, MeV can persistently infect the brain causing the rare but fatal neurological disorder subacute sclerosing panencephalitis (SSPE). SSPE-causing MeVs are characterized by a hypermutated genome and a hyperfusogenic F protein that facilitates the rapid spread of MeV throughout the brain. No treatment against SSPE is available, but the nucleoside analog remdesivir was recently demonstrated to be effective against MeV in vitro. We show that treatment of an SSPE patient with remdesivir led to transient clinical improvement and did not induce viral escape mutants, encouraging the future use of remdesivir in SSPE patients. Functional characterization of the viral proteins sheds light on the shared properties of SSPE-causing MeVs and further contributes to understanding how those viruses cause disease.

M protein of subacute sclerosing panencephalitis virus, synergistically with the F protein, plays a crucial role in viral neuropathogenicity.

Subacute sclerosing panencephalitis (SSPE) is a rare fatal neurodegenerative disease caused by a measles virus (MV) variant, SSPE virus, that accumulates mutations during long-term persistent infection of the central nervous system (CNS). Clusters of mutations identified around the matrix (M) protein in many SSPE viruses suppress productive infectious particle release and accelerate cell-cell fusion, which are features of SSPE viruses. It was reported, however, that these defects of M protein function might not be correlated directly with promotion of neurovirulence, although they might enable establishment of persistent infection. Neuropathogenicity is closely related to the character of the viral fusion (F) protein, and amino acid substitution(s) in the F protein of some SSPE viruses confers F protein hyperfusogenicity, facilitating viral propagation in the CNS through cell-cell fusion and leading to neurovirulence. The F protein of an SSPE virus Kobe-1 strain, however, displayed only moderately enhanced fusion activity and required additional mutations in the M protein for neuropathogenicity in mice. We demonstrated here the mechanism for the M protein of the Kobe-1 strain supporting the fusion activity of the F protein and cooperatively inducing neurovirulence, even though each protein, independently, has no effect on virulence. The occurrence of SSPE has been estimated recently as one in several thousand in children who acquired measles under the age of 5 years, markedly higher than reported previously. The probability of a specific mutation (or mutations) occurring in the F protein conferring hyperfusogenicity and neuropathogenicity might not be sufficient to explain the high frequency of SSPE. The induction of neurovirulence by M protein synergistically with moderately fusogenic F protein could account for the high frequency of SSPE.

CADM1 and CADM2 Trigger Neuropathogenic Measles Virus-Mediated Membrane Fusion by Acting in cis.

Measles virus (MeV), an enveloped RNA virus in the family Paramyxoviridae, is still an important cause of childhood morbidity and mortality worldwide. MeV usually causes acute febrile illness with skin rash, but in rare cases persists in the brain, causing a progressive neurological disorder, subacute sclerosing panencephalitis (SSPE). The disease is fatal, and no effective therapy is currently available. Although transsynaptic cell-to-cell transmission is thought to account for MeV propagation in the brain, neurons do not express the known receptors for MeV. Recent studies have shown that hyperfusogenic changes in the MeV fusion (F) protein play a key role in MeV propagation in the brain. However, how such mutant viruses spread in neurons remains unexplained. Here, we show that cell adhesion molecule 1 (CADM1; also known as IGSF4A, Necl-2, and SynCAM1) and CADM2 (also known as IGSF4D, Necl-3, SynCAM2) are host factors that enable MeV to cause membrane fusion in cells lacking the known receptors and to spread between neurons. During enveloped virus entry, a cellular receptor generally interacts in trans with the attachment protein on the envelope. However, CADM1 and CADM2 interact in cis with the MeV attachment protein on the same cell membrane, causing the fusion protein triggering and membrane fusion. Knockdown of CADM1 and CADM2 inhibits syncytium formation and virus transmission between neurons that are both mediated by hyperfusogenic F proteins. Thus, our results unravel the molecular mechanism (receptor-mimicking cis-acting fusion triggering) by which MeV spreads transsynaptically between neurons, thereby causing SSPE. IMPORTANCE Measles virus (MeV), an enveloped RNA virus, is the causative agent of measles, which is still an important cause of childhood morbidity and mortality worldwide. Persistent MeV infection in the brain causes a fatal progressive neurological disorder, subacute sclerosing panencephalitis (SSPE), several years after acute infection. However, how MeV spreads in neurons, which are mainly affected in SSPE, remains largely unknown. In this study, we demonstrate that cell adhesion molecule 1 (CADM1) and CADM2 are host factors enabling MeV spread between neurons. During enveloped virus entry, a cellular receptor generally interacts in trans with the attachment protein on the viral membrane (envelope). Remarkably, CADM1 and CADM2 interact in cis with the MeV attachment protein on the same membrane, triggering the fusion protein and causing membrane fusion, as viral receptors usually do in trans. Careful screening may lead to more examples of such "receptor-mimicking cis-acting fusion triggering" in other viruses.

Fitness selection of hyperfusogenic measles virus F proteins associated with neuropathogenic phenotypes.

Measles virus (MeV) is resurgent and caused >200,000 deaths in 2019. MeV infection can establish a chronic latent infection of the brain that can recrudesce months to years after recovery from the primary infection. Recrudescent MeV leads to fatal subacute sclerosing panencephalitis (SSPE) or measles inclusion body encephalitis (MIBE) as the virus spreads across multiple brain regions. Most clinical isolates of SSPE/MIBE strains show mutations in the fusion (F) gene that result in a hyperfusogenic phenotype in vitro and allow for efficient spread in primary human neurons. Wild-type MeV receptor-binding protein is indispensable for manifesting these mutant F phenotypes, even though neurons lack canonical MeV receptors (CD150/SLAMF1 or nectin-4). How such hyperfusogenic F mutants are selected and whether they confer a fitness advantage for efficient neuronal spread is unresolved. To better understand the fitness landscape that allows for the selection of such hyperfusogenic F mutants, we conducted a screen of ≥3.1 × 105 MeV-F point mutants in their genomic context. We rescued and amplified our genomic MeV-F mutant libraries in BSR-T7 cells under conditions in which MeV-F-T461I (a known SSPE mutant), but not wild-type MeV, can spread. We recovered known SSPE mutants but also characterized at least 15 hyperfusogenic F mutations with an SSPE phenotype. Structural mapping of these mutants onto the prefusion MeV-F trimer confirm and extend our understanding of the F regulatory domains in MeV-F. Our list of hyperfusogenic F mutants is a valuable resource for future studies into MeV neuropathogenesis and the regulation of paramyxovirus F.

Analysis of a Subacute Sclerosing Panencephalitis Genotype B3 Virus from the 2009-2010 South African Measles Epidemic Shows That Hyperfusogenic F Proteins Contribute to Measles Virus Infection in the Brain.

During a measles virus (MeV) epidemic in 2009 in South Africa, measles inclusion body encephalitis (MIBE) was identified in several HIV-infected patients. Years later, children are presenting with subacute sclerosing panencephalitis (SSPE). To investigate the features of established MeV neuronal infections, viral sequences were analyzed from brain tissue samples of a single SSPE case and compared with MIBE sequences previously obtained from patients infected during the same epidemic. Both the SSPE and the MIBE viruses had amino acid substitutions in the ectodomain of the F protein that confer enhanced fusion properties. Functional analysis of the fusion complexes confirmed that both MIBE and SSPE F protein mutations promoted fusion with less dependence on interaction by the viral receptor-binding protein with known MeV receptors. While the SSPE F required the presence of a homotypic attachment protein, MeV H, in order to fuse, MIBE F did not. Both F proteins had decreased thermal stability compared to that of the corresponding wild-type F protein. Finally, recombinant viruses expressing MIBE or SSPE fusion complexes spread in the absence of known MeV receptors, with MIBE F-bearing viruses causing large syncytia in these cells. Our results suggest that alterations to the MeV fusion complex that promote fusion and cell-to-cell spread in the absence of known MeV receptors is a key property for infection of the brain.IMPORTANCE Measles virus can invade the central nervous system (CNS) and cause severe neurological complications, such as MIBE and SSPE. However, mechanisms by which MeV enters the CNS and triggers the disease remain unclear. We analyzed viruses from brain tissue of individuals with MIBE or SSPE, infected during the same epidemic, after the onset of neurological disease. Our findings indicate that the emergence of hyperfusogenic MeV F proteins is associated with infection of the brain. We also demonstrate that hyperfusogenic F proteins permit MeV to enter cells and spread without the need to engage nectin-4 or CD150, known receptors for MeV that are not present on neural cells.

New Insights into Measles Virus Brain Infections.

Measles virus (MeV) may persist in the brain, causing fatal neurodegenerative diseases, subacute sclerosing panencephalitis, and measles inclusion-body encephalitis. However, the mechanism of MeV propagation in the brain remains unexplained because human neurons affected by the diseases do not express the known receptors for MeV. Recent studies have revealed that certain changes in the ectodomain of the MeV fusion (F) protein play a key role in MeV spread in the brain. These changes destabilize the prefusion form of the F protein and render it hyperfusogenic, which in turn allows the virus to propagate in neurons. Based on crystal structures of the F protein, effective fusion inhibitors could be developed to treat these diseases.

Neuroleptic malignant syndrome as a presenting feature of subacute sclerosing panencephalitis.

Subacute sclerosing panencephalitis (SSPE) is a slowly progressive degenerative disorder caused by measles virus. It is characterised by typical clinical and electrophysiological features in the form of slow myoclonic jerks, with progressive cognitive impairment, visual symptoms, and periodic complexes on EEG, with raised titres of anti-measles antibodies in CSF and serum. Atypical presentations of SSPE have been reported including brainstem involvement, ADEM-like presentation, acute encephalitis, and cerebellar ataxia. Presentation with predominant extrapyramidal features is uncommon. We describe a case of SSPE presenting with extensive rigidity with highly elevated CPK values, mimicking neuroleptic malignant syndrome (NMS) which was most probably due to central dopaminergic blockade induced by the disease process. To our knowledge, this is the first case of SSPE presenting with a NMS-like syndrome.

A Novel Peptide Derived from the Fusion Protein Heptad Repeat Inhibits Replication of Subacute Sclerosing Panencephalitis Virus In Vitro and In Vivo.

Subacute sclerosing panencephalitis (SSPE) is a persistent, progressive, and fatal degenerative disease resulting from persistent measles virus (MV) infection of the central nervous system. Most drugs used to treat SSPE have been reported to have limited effects. Therefore, novel therapeutic strategies are urgently required. The SSPE virus, a variant MV strain, differs virologically from wild-type MV strain. One characteristic of the SSPE virus is its defective production of cell-free virus, which leaves cell-to-cell infection as the major mechanism of viral dissemination. The fusion protein plays an essential role in this cell-to-cell spread. It contains two critical heptad repeat regions that form a six-helix bundle in the trimer similar to most viral fusion proteins. In the case of human immunodeficiency virus type-1 (HIV-1), a synthetic peptide derived from the heptad repeat region of the fusion protein enfuvirtide inhibits viral replication and is clinically approved as an anti-HIV-1 agent. The heptad repeat regions of HIV-1 are structurally and functionally similar to those of the MV fusion protein. We therefore designed novel peptides derived from the fusion protein heptad repeat region of the MV and examined their effects on the measles and SSPE virus replication in vitro and in vivo. Some of these synthetic novel peptides demonstrated high antiviral activity against both the measles (Edmonston strain) and SSPE (Yamagata-1 strain) viruses at nanomolar concentrations with no cytotoxicity in vitro. In particular, intracranial administration of one of the synthetic peptides increased the survival rate from 0% to 67% in an SSPE virus-infected nude mouse model.

Amino acid substitutions in the heptad repeat A and C regions of the F protein responsible for neurovirulence of measles virus Osaka-1 strain from a patient with subacute sclerosing panencephalitis.

Measles virus (MV) is the causative agent of subacute sclerosing panencephalitis (SSPE). We previously reported that the F gene of the SSPE Osaka-2 strain is the major determinant of MV neurovirulence. Because the sites and extents of mutations differ among SSPE strains, it is necessary to determine the mutations responsible for the SSPE-specific phenotypes of individual viral strain. In this study, recombinant viruses containing the envelope-associated genes from the SSPE Osaka-1 strain were generated in the IC323 wild-type MV background. Hamsters inoculated with MV containing the H gene of the Osaka-1 strain displayed hyperactivity and seizures, but usually recovered and survived. Hamsters inoculated with MV containing the F gene of the Osaka-1 strain displayed severe neurologic signs and died. Amino acid substitutions in the heptad repeat A and C regions of the F protein, including a methionine-to-valine substitution at amino acid 94, play major roles in neurovirulence.

Subacute sclerosing panencephalitis and chronic viral encephalitis.

Subacute sclerosing panencephalitis (SSPE) is a chronic infection of the central nervous system associated with the presence of mutant measles virus in the brain. It presents as a progressive, usually fatal disease. The diagnosis is based on clinical criteria and an elevated titer of measles antibodies in the cerebrospinal fluid (CSF). Electroencephalography and imaging studies provide supportive laboratory data. A brain biopsy is indicated only when CSF serology is negative or equivocal in a suspected case to assess the presence of inclusion bodies, measles virus antigens, or viral RNA. Among many drugs and methods tried in the treatment, the highest rate of stabilization or improvement was obtained with intraventricular human lymphoblastoid interferon-α and oral inosiplex. Further research for more available and efficient therapeutic regimens is warranted. Measles and SSPE are preventable by maintenance of high rates of immunization in the population.

Mutant fusion proteins with enhanced fusion activity promote measles virus spread in human neuronal cells and brains of suckling hamsters.

Subacute sclerosing panencephalitis (SSPE) is a fatal degenerative disease caused by persistent measles virus (MV) infection in the central nervous system (CNS). From the genetic study of MV isolates obtained from SSPE patients, it is thought that defects of the matrix (M) protein play a crucial role in MV pathogenicity in the CNS. In this study, we report several notable mutations in the extracellular domain of the MV fusion (F) protein, including those found in multiple SSPE strains. The F proteins with these mutations induced syncytium formation in cells lacking SLAM and nectin 4 (receptors used by wild-type MV), including human neuronal cell lines, when expressed together with the attachment protein hemagglutinin. Moreover, recombinant viruses with these mutations exhibited neurovirulence in suckling hamsters, unlike the parental wild-type MV, and the mortality correlated with their fusion activity. In contrast, the recombinant MV lacking the M protein did not induce syncytia in cells lacking SLAM and nectin 4, although it formed larger syncytia in cells with either of the receptors. Since human neuronal cells are mainly SLAM and nectin 4 negative, fusion-enhancing mutations in the extracellular domain of the F protein may greatly contribute to MV spread via cell-to-cell fusion in the CNS, regardless of defects of the M protein.

Molecular characterization of measles virus strains causing subacute sclerosing panencephalitis in France in 1977 and 2007.

Measles virus strains from two subacute sclerosing panencephalitis (SSPE) cases diagnosed in 1977 (Laine strain) and in 2007 (Hoedts strain) were studied. Phylogenetic analysis based on C-terminal part of the nucleoprotein and the entire H gene showed that Hoedts strain, circulating in France presumably in the 1980s, belonged to genotype C2. However, Laine strain, suspected to have circulated between 1940s and 1960s, could not be assigned to any known measles virus genotypes. Sequences analysis of the Laine strain suggested that it originated from a measles virus that may have circulating at the same period as the Edmonston strain. The analysis of the whole genome of both SSPE strains revealed biased hypermutations in M, F, and H gene. Some of these mutations like the L165P found in the M protein sequence of the Laine strain, the amino acid position 94, where a mutation M94V was found in the F protein sequence of the Hoedts strain are known to play an important role in the glycoprotein interaction and to impair the ability of measles virus strain to produce cell-free infectious viral particles.This is the first study on molecular characterization of the entire coding region of measles virus isolated from SSPE cases in France.

The F gene of the Osaka-2 strain of measles virus derived from a case of subacute sclerosing panencephalitis is a major determinant of neurovirulence.

Measles virus (MV) is the causative agent for acute measles and subacute sclerosing panencephalitis (SSPE). Although numerous mutations have been found in the MV genome of SSPE strains, the mutations responsible for the neurovirulence have not been determined. We previously reported that the SSPE Osaka-2 strain but not the wild-type strains of MV induced acute encephalopathy when they were inoculated intracerebrally into 3-week-old hamsters. The recombinant MV system was adapted for the current study to identify the gene(s) responsible for neurovirulence in our hamster model. Recombinant viruses that contained envelope-associated genes from the Osaka-2 strain were generated on the IC323 wild-type MV background. The recombinant virus containing the M gene alone did not induce neurological disease, whereas the H gene partially contributed to neurovirulence. In sharp contrast, the recombinant virus containing the F gene alone induced lethal encephalopathy. This phenotype was related to the ability of the F protein to induce syncytium formation in Vero cells. Further study indicated that a single T461I substitution in the F protein was sufficient to transform the nonneuropathogenic wild-type MV into a lethal virus for hamsters.

Adult fulminant subacute sclerosing panencephalitis: pathological and molecular studies--a case report.

Subacute sclerosing panencephalitis is an uncommon progressive neurological disorder caused by a persistent defective measles virus, typically affecting children. We describe a case of fulminant subacute sclerosing panencephalitis in a 25-year-old male. Brain tissue biopsy showed histologic evidence of encephalitis with eosinophilic intranuclear inclusion bodies (Cowdry Type A and B), intracytoplasmic inclusion bodies, perivascular lymphoplasmacytic infiltration and gliosis. Immunohistochemical studies were positive using an anti-measles antibody. Reverse transcriptase-PCR detected measles virus RNA and phylogenetic analysis indicated a C2 genotype. The rare adult-onset form is often atypical and difficult to diagnose and should be included in the differential diagnosis of subacute "unexplained" neurological diseases and uncommon infectious disorders.

Atypical subacute sclerosing panencephalitis: case report

Subacute sclerosing panencephalitis (SSPE) is a progressive inflammatory disorder of the central nervous system with both poor prognosis and high mortality. The disease has been related to a persistent and aberrant measles virus infection and no effective treatment has been available. We report a case of SSPE with atypical features including seizures at onset and a fulminant course in a 8 years-old boy who had been previously immunized against measles.

Panencefalite esclerosante subaguda (PES) é uma doença inflamatória e progressiva do sistema nervoso central com prognóstico reservado e alta mortalidade. A doença tem sido relacionada com a infecção persistente e anormal pelo vírus do sarampo e não há tratamento específico disponível. Relatamos um caso de PES com características atípicas representadas por início do quadro com crises convulsivas e apresentação fulminante em menino de 8 anos previamente imunizado contra o vírus do sarampo.

Generation of recombinant adenovirus expressing siRNA against the L mRNA of measles virus and subacute sclerosing panencephalitis virus.

Subacute sclerosing panencephalitis (SSPE) is a fatal neurodegenerative disease caused by prolonged persistent infection of the central nervous system with a measles virus (MV) mutant called SSPE virus. At present, there is no effective treatment to completely cure SSPE and development of a new therapeutic measure(s) against this fatal slow virus infection is needed. We previously reported that replication of MV and SSPE virus was effectively inhibited by small interfering RNA (siRNA), either chemically synthetic or plasmid-driven ones, that were targeted against different sequences of the mRNA for the L protein of MV. In this study, we have generated recombinant adenovirus expressing the siRNAs (rAd-siRNA-MV-L2, -L4 and -L5) and demonstrated that these rAd-siRNAs efficiently inhibited replication of MV and SSPE virus in a dose-dependent manner. Due to their high capacity for gene delivery to nerve cells and the potential to inhibit SSPE virus replication, the rAd-siRNAs could be a good candidate for a novel therapeutic measure against SSPE.

Effect of the alterations in the fusion protein of measles virus isolated from brains of patients with subacute sclerosing panencephalitis on syncytium formation.

Measles virus (MV) is the causative agent of subacute sclerosing panencephalitis (SSPE) and viruses isolated from brains of the patients contain numerous mutations. We have previously demonstrated that the hemagglutinin (H) protein of MV SSPE strains can interact with the signaling lymphocyte activation molecule (SLAM) and an unidentified molecule on Vero cells, but not with CD46, as a receptor. The mechanism by which MV SSPE strains can induce cell-cell fusion in SLAM-negative Vero cells is not understood. We report here on the effect of mutations in the fusion (F) proteins of three MV SSPE strains on syncytium formation. The F proteins of the three SSPE strains were functional and co-expression with H protein from the MV wild-type or SSPE strains in this study induced formation of large syncytia in Vero cells as well as in cell lines expressing SLAM or CD46. Expression of chimeric F proteins of SSPE strains showed that amino acid substitutions in the F protein extracellular as well as cytoplasmic domain contributed to enhanced cell-cell fusion in Vero cells. These findings suggest a common molecular mechanism and a key role of the F protein for syncytium formation in cells expressing an unidentified third receptor for MV.

Subacute sclerosing panencephalitis in Croatia (1994-2004).

We studied five patients with SSPE during a 10-year period (1994-2004). The first clinical symptoms developed at the age of 5-11 years. All patients were vaccinated regularly against measles according to the official immunization schedule. One patient had measles at the age of 18 months. Two of them had a history of morbilliform rash (unrecognized measles) at the age of six and seven months, respectively. In two patients, with no history of measles before vaccination the disease started after varicella infection. Using complement-fixation (F) test and EIA, antibodies to measles virus (MV) were detected in the CSF and sera of all patients. The CF-antibody titers ranged from 1:1024 to 1:65536 in sera and from 1:16 to 1:128 in CSF samples. MV antigen was detected in brain imprints using IFA in two patients. Electron microscopic analysis revealed intranuclear viral inclusions (MV nucleocapsids). Using RT-PCR, viral RNA was found in both patients. Nucleotide sequence analysis showed that the viruses found in the brain tissue belonged to the wild-type MV D6 genotype [7].