Viral mumps: Increasing occurrences in the vaccinated population.

Before the introduction of the vaccine, mumps was the most common salivary gland disease and was one of the most common infectious diseases in children globally. Following the introduction of the mumps vaccine in 1967, the disease was almost nonexistent in the United States and was only found to occur in nonvaccinated patients, and even then, it did not present in epidemic portions because of the extent of vaccination in the population at large. Beginning in the early 2000s, viral mumps began to present itself in vaccinated populations, and currently, outbreaks are continuing to increase in number. This article presents information on the various outbreaks, a review of the virus and the disease, including symptoms and comorbidities, and new recommendations for management. Dental practitioners should be aware of the increasing incidence and prevalence of this disease, be able to recognize it, and make appropriate referrals for management.

Investigation of the thermal shift assay and its power to predict protein and virus stabilizing conditions.

Protein thermal shift assay (TSA) has been extensively used in investigation of protein stabilization (for protein biopharmaceutics stabilization, protein crystallization studies or screening of recombinant proteins) and drug discovery (screening of ligands or inhibitors). This work aimed to analyze thermal shift assay results in comparison to protein polymerization (multimerization and aggregation) propensity and test the most stabilizing formulations for their stabilization effect on enveloped viruses. Influence of protein concentration, buffer pH and molarity was tested on three proteins (immunoglobulin G, ovalbumin, and albumin) and results showed that each of these factors has an impact on determined shift in protein melting point Tm, and the impact was similar for all three proteins. In case of ovalbumin, molecular dynamics simulations were performed with the goal to understanding molecular basis of protein's thermal stability dependence on pH. Effect of three denaturing agents in a wide concentration range on Tm showed nicely that chemical denaturation occurs only at the highest concentrations. Results showed similar effect on Tm for most formulations on different proteins. Most successful formulations were tested for enveloped virus stabilizing potential using cell culture infectivity assay (CCID50) and results showed lack of correlation with TSA results. Only weak correlation of Tm shift and protein polymerization measured by SEC-HPLC was obtained, meaning that polymerization cannot be predicted from Tm shifts.

Recombinant mumps viruses expressing the batMuV fusion glycoprotein are highly fusion active and neurovirulent.

A recent study reported the detection of a bat-derived virus (BatPV/Epo_spe/AR1/DCR/2009, batMuV) with phylogenetic relatedness to human mumps virus (hMuV). Since all efforts to isolate infectious batMuV have reportedly failed, we generated recombinant mumps viruses (rMuVs) in which the open reading frames (ORFs) of the fusion (F) and haemagglutinin-neuraminidase (HN) glycoproteins of an hMuV strain were replaced by the corresponding ORFs of batMuV. The batMuV F and HN proteins were successfully incorporated into viral particles and the resultant chimeric virus was able to mediate infection of Vero cells. Distinct differences were observed between the fusogenicity of rMuVs expressing one or both batMuV glycoproteins: viruses expressing batMuV F were highly fusogenic, regardless of the origin of HN. In contrast, rMuVs expressing human F and bat-derived HN proteins were less fusogenic compared to hMuV. The growth kinetics of chimeric MuVs expressing batMuV HN in combination with either hMuV or batMuV F were similar to that of the backbone virus, whereas a delay in virus replication was obtained for rMuVs harbouring batMuV F and hMuV HN. Replacement of the hMuV F and HN genes or the HN gene alone by the corresponding batMuV genes led to a slight reduction in neurovirulence of the highly neurovirulent backbone strain. Neutralizing antibodies inhibited infection mediated by all recombinant viruses generated. Furthermore, group IV anti-MuV antibodies inhibited the neuraminidase activity of bat-derived HN. Our study reports the successful generation of chimeric MuVs expressing the F and HN proteins of batMuV, providing a means for further examination of this novel batMuV.

Molecular biology, pathogenesis and pathology of mumps virus.

Mumps is caused by the mumps virus (MuV), a member of the Paramyxoviridae family of enveloped, non-segmented, negative-sense RNA viruses. Mumps is characterized by painful inflammatory symptoms, such as parotitis and orchitis. The virus is highly neurotropic, with laboratory evidence of central nervous system (CNS) infection in approximately half of cases. Symptomatic CNS infection occurs less frequently; nonetheless, prior to the introduction of routine vaccination, MuV was a leading cause of aseptic meningitis and viral encephalitis in many developed countries. Despite being one of the oldest recognized diseases, with a worldwide distribution, surprisingly little attention has been given to its study. Cases of aseptic meningitis associated with some vaccine strains and a global resurgence of cases, including in highly vaccinated populations, has renewed interest in the virus, particularly in its pathogenesis and the need for development of clinically relevant models of disease. In this review we summarize the current state of knowledge on the virus, its pathogenesis and its clinical and pathological outcomes.

The innate antiviral factor APOBEC3G targets replication of measles, mumps and respiratory syncytial viruses.

The cytidine deaminase APOBEC3G (apolipoprotein B mRNA-editing enzyme-catalytic polypeptide 3G; A3G) exerts antiviral activity against retroviruses, hepatitis B virus, adeno-associated virus and transposable elements. We assessed whether the negative-strand RNA viruses measles, mumps and respiratory syncytial might be affected by A3G, and found that their infectivity was reduced by 1-2 logs (90-99 %) in A3G overexpressing Vero cells, and in T-cell lines expressing A3G at physiological levels. Viral RNA was co-precipitated with HA-tagged A3G and could be amplified by RT-PCR. Interestingly, A3G reduced viral transcription and protein expression in infected cells by 50-70 %, and caused an increased mutation frequency of 0.95 mutations per 1000 nt in comparison to the background level of 0.22/1000. The observed mutations were not specific for A3G [cytidine to uridine (C→U) or guanine to adenine (G→A) hypermutations], nor specific for ADAR (adenosine deaminase acting on RNA, A→G and U→C transitions, with preference for next neighbour-nucleotides U = A>C>G). In addition, A3G mutants with inactivated catalytic deaminase (H257R and E259Q) were inhibitory, indicating that the deaminase activity is not required for the observed antiviral activity. In combination, impaired transcription and increased mutation frequencies are sufficient to cause the observed reduction in viral infectivity and eliminate virus replication within a few passages in A3G-expressing cells.

Rescue of wild-type mumps virus from a strain associated with recent outbreaks helps to define the role of the SH ORF in the pathogenesis of mumps virus.

Mumps virus (MuV) causes acute infections in humans. In recent years, MuV has caused epidemics among highly vaccinated populations. The largest outbreak in the U.S. in the past 20 years occurred in 2005-2006 with over 5000 reported cases in which the majority of the cases was in vaccinated young adults. We sequenced the complete genome of a representative strain from the epidemic (MuV-IA). MuV-IA is a member of genotype G, the same genotype of MuV that was associated with the outbreak in the UK in 2004-2005. We constructed a reverse genetics system for MuV-IA (rMuV-IA), and rescued a virus lacking the open reading frame (ORF) of the SH gene (rMuV∆SH). rMuV∆SH infection in L929 cells induced increased NF-κB activation, TNF-α production and apoptosis compared to rMuV-IA. rMuV∆SH was attenuated in an animal model. These results indicated that the SH ORF of MuV plays a significant role in interfering with TNF-α signaling and viral pathogenesis during virus infection.

Modulation of apoptosis by V protein mumps virus.

BACKGROUND: The Urabe AM9 vaccine strain of mumps virus contains two variants of V protein: VWT (of HN-A1081 viral population) and VGly (of HN-G1081). The V protein is a promoting factor of viral replication by blocking the IFN antiviral pathway. FINDINGS: We studied the relationship between V protein variants and IFN-α2b-induced apoptosis. V proteins decrease activation of the extrinsic IFN-α2b-induced apoptotic pathway monitored by the caspase 8 activity, being the effect greater with the VWT protein. Both V proteins decrease the activity of caspase 9 of the intrinsic apoptotic pathway. In a system without IFN, the VWT and VGly proteins expression promotes activation of caspases 3 and 7. However, when the cellular system was stimulated with IFN-α, this activity decreased partially. TUNEL assay shows that for treatment with IFN-α and ibuprofen of cervical adenocarcinoma cells there is nuclear DNA fragmentation but the V protein expression reduces this process. CONCLUSIONS: The reduction in the levels of caspases and DNA fragmentation, suggesting that V protein, particularly VWT protein of Urabe AM9 vaccine strain, modulates apoptosis. In addition, the VWT protein shows a protective role for cell proliferation in the presence of antiproliferative signals.

Use of attenuated paramyxoviruses for cancer therapy.

Paramyxoviruses, measles virus (MV), mumps virus (MuV) and Newcastle disease virus (NDV), are well known for causing measles and mumps in humans and Newcastle disease in birds. These viruses have been tamed (attenuated) and successfully used as vaccines to immunize their hosts. Remarkably, pathogenic MuV and vaccine strains of MuV, MV and NDV efficiently infect and kill cancer cells and are consequently being investigated as novel cancer therapies (oncolytic virotherapy). Phase I/II clinical trials have shown promise but treatment efficacy needs to be enhanced. Technologies being developed to increase treatment efficacy include: virotherapy in combination with immunosuppressive drugs (cyclophosphamide); retargeting of viruses to specific tumor types or tumor vasculature; using infected cell carriers to protect and deliver the virus to tumors; and genetic manipulation of the virus to increase viral spread and/or express transgenes during viral replication. Transgenes have enabled noninvasive imaging or tracking of viral gene expression and enhancement of tumor destruction.

Mumps virus small hydrophobic protein targets ataxin-1 ubiquitin-like interacting protein (ubiquilin 4).

The small hydrophobic (SH) protein of mumps virus has been reported to interfere with innate immunity by inhibiting tumour necrosis factor alpha-mediated apoptosis. In a yeast two-hybrid screen we have identified the ataxin-1 ubiquitin-like interacting protein (A1Up) as a cellular target of the SH protein. A1Up contains an amino-terminal ubiquitin-like (UbL) domain, a carboxy-terminal ubiquitin-associated (UbA) domain and two stress-inducible heat shock chaperonin-binding (Sti1) motifs. This places it within the ubiquitin-like protein family that is involved in proteasome-mediated activities. Co-immunoprecipitation confirmed the binding of SH and A1Up and demonstrates that a truncated protein fragment corresponding to aa 136-270 of A1Up, which represents the first Sti1-like repeat and an adjacent hydrophobic region, was sufficient for interaction, whereas neither the UbL nor the UbA domains were required for interaction. The ectopic expression of A1Up leads to a redistribution of SH to punctate structures that co-localize with the 20S proteasome in transfected or infected mammalian cells.

Single amino acid changes in the mumps virus haemagglutinin-neuraminidase and polymerase proteins are associated with neuroattenuation.

It has previously been shown that three amino acid changes, one each in the fusion (F; Ala/Thr-91-->Thr), haemagglutinin-neuraminidase (HN; Ser-466-->Asn) and polymerase (L; Ile-736-->Val) proteins, are associated with attenuation of a neurovirulent clinical isolate of mumps virus (88-1961) following serial passage in vitro. Here, using full-length cDNA plasmid clones and site-directed mutagenesis, it was shown that the single amino acid change in the HN protein and to a lesser extent, the change in the L protein, resulted in neuroattenuation, as assessed in rats. The combination of both amino acid changes caused neuroattenuation of the virus to levels previously reported for the clinical isolate following attenuation in vitro. The amino acid change in the F protein, despite having a dramatic effect on protein function in vitro, was previously shown to not be involved in the observed neuroattenuation, highlighting the importance of conducting confirmatory in vivo studies. This report provides additional supporting evidence for the role of the HN protein as a virulence factor and, as far as is known, is the first report to associate an amino acid change in the L protein with mumps virus neuroattenuation.

A single nucleotide change in the mumps virus F gene affects virus fusogenicity in vitro and virulence in vivo.

Mumps virus is highly neurotropic, with evidence of infection of the central nervous system in more than half of clinical cases. In the prevaccine era, mumps was a major cause of viral meningitis in most developed countries. Despite efforts to attenuate the virus, some mumps vaccines have retained virulence properties and have caused aseptic meningitis in vaccinees, resulting in public resistance to vaccination in some countries. Ensuring the safety of mumps vaccines is an important public health objective, as the need for robust immunization programs has been made clear by the recent resurgence of mumps outbreaks worldwide, including the United States, which in 2006 experienced its largest mumps outbreak in 20 years. To better understand the molecular basis of mumps virus attenuation, the authors developed two infectious full-length cDNA clones for a highly neurovirulent strain of mumps virus. The clones differed at only one site, possessing either an A or G at nucleotide position 271 in the F gene, to represent the heterogeneity identified in the original virulent clinical isolate. In comparison to the clinical isolate, virus rescued from the A-variant cDNA clone grew to higher cumulative titers in vitro but exhibited similar cytopathic effects in vitro and virulence in vivo. In contrast, virus rescued from the G-variant cDNA clone, in comparison to the clinical isolate and the A-variant, was more fusogenic in vitro but replicated to lower cumulative titers and was less neurovirulent in vivo. These data suggest that nucleotide position 271 in the F gene plays a significant role in virus pathogenesis. This infectious clone system will serve as a key tool for further examination of the molecular basis for mumps virus neurovirulence and neuroattenuation.

The F gene of rodent brain-adapted mumps virus is a major determinant of neurovirulence.

Prior to the introduction of live-attenuated vaccines, mumps virus (MuV) was the leading cause of virus-induced meningitis. Although vaccination has been effective at controlling the disease, the use of insufficiently attenuated strains has been associated with high rates of aseptic meningitis in vaccinees. The molecular basis of MuV attenuation is poorly understood, and no reliable molecular markers of virulence have been identified. In this study, reverse genetics has been used to identify molecular determinants of MuV neuropathogenesis. Recombinant viruses, containing the envelope-associated genes from the Kilham (MuV(KH)) rodent brain-adapted strain of MuV, were generated in the Jeryl Lynn 5 (MuV(JL5)) vaccine strain background. The syncytium phenotypes of the recombinant viruses on Vero cells differed depending on the source of the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins, with heterologous combinations showing either an increase or a decrease in the level of cell fusion compared to that of the homologous parental combinations. This was confirmed by transiently cotransfecting eukaryotic F and HN glycoprotein expression constructs. A Lewis rat model that discriminates between neurovirulent and nonneurovirulent MuV strains based on the extent of hydrocephalus induced in the rat brain after intracerebral inoculation was used to assess the phenotype of the recombinant viruses. Expression of the matrix (M), small hydrophobic (SH), or HN gene in isolation did not confer a neurovirulent phenotype. Expression of the F gene of the neurovirulent strain alone was sufficient to induce significant levels of hydrocephalus. Coexpression of the homologous HN gene led to a marginal increase in the level of hydrocephalus.

Structural characterization of mumps virus fusion protein core.

The fusion proteins of enveloped viruses mediating the fusion between the viral and cellular membranes comprise two discontinuous heptad repeat (HR) domains located at the ectodomain of the enveloped glycoproteins. The crystal structure of the fusion protein core of Mumps virus (MuV) was determined at 2.2 A resolution. The complex is a six-helix bundle in which three HR1 peptides form a central highly hydrophobic coiled-coil and three HR2 peptides pack against the hydrophobic grooves on the surface of central coiled-coil in an oblique antiparallel manner. Fusion core of MuV, like those of simian virus 5 and human respiratory syncytium virus, forms typical 3-4-4-4-3 spacing. The similar characterization in HR1 regions, as well as the existence of O-X-O motif in extended regions of HR2 helix, suggests a basic rule for the formation of the fusion core of viral fusion proteins.

Two clones obtained from Urabe AM9 mumps virus vaccine differ in their replicative efficiency in neuroblastoma cells.

A high rate of post-vaccinal aseptic meningitis for Urabe AM9 mumps virus strain is well documented. This strain is composed of two virus variants differing at the nt 1081 (A/G) region in the hemagglutinin-neuraminidase (HN) gene. An association of HN-A(1081) variant with neurovirulence has been proposed. In order to test for neurotropism we isolated the HN-A(1081) and HN-G(1081) virus variants from Urabe AM9 mumps virus vaccine. Sequential passages were performed in monkey kidney Vero cells and human neuroblastoma SH-SY5Y cells. Viral replication was determined by conventional and real-time RT-PCR. The results show that clone HN-A(1081) can replicate efficiently in both cell types. However, a defective replication of clone HN-G(1081), lacking its genetic marker, was observed after the third passage in neuroblastoma cells. Kinetics assays showed that clone HN-A(1081) replicates faster than clone HN-G(1081). Viral clones were also inoculated into the brains of newborn rats. Clone HN-A(1081) replicated 14 times, while clone HN-G(1081) merely duplicated its level over the initial inoculum. These results suggest that there is a selective replication of HN-A(1081) mumps virus variants in cells of nervous origin.

Characterization of mumps virus strains with varying neurovirulence.

Mumps virus strains isolated during an epidemic in Lithuania in 1998 - 2000 were studied. Viruses of the neurovirulent C1 and non-neurovirulent C2 small hydrophobic (SH) genotype variant were sequenced for the haemagglutinin-neuraminidase (HN) and fusion (F) protein genes. Amino acid differences between C1 and C2 strains were found for both proteins. Two amino acid differences were of potential importance for the non-neurovirulent phenotype of the C2 virus. Four of 5 C2 strains exhibited the amino acid arginine instead of lysine at position 335 of the HN protein, and the amino acid phenylalanine was found instead of serine at amino acid position 195 of the F protein. Amino acid differences at these positions have previously been reported to associate with a change in neurovirulence and fusion activity. In addition, the HN gene of the neurovirulent Kilham strain of genotype A was sequenced. The deduced amino acid sequence showed different amino acids compared to both genotypes A and C on some positions. Notably, amino acid differences located in previously identified neutralizing epitopes were found at positions 266, 354 and 356 of the HN protein compared to other genotype A strains. The amino acid differences between Kilham virus strain and other genotype A strains and the similarity of the Kilham HN protein (7 positions) to neurovirulent genotype C strains on some amino acid positions may indicate a possible role for this protein in mumps virus neurovirulence.

Mumps virus V protein antagonizes interferon without the complete degradation of STAT1.

Mumps virus (MuV) has been shown to antagonize the antiviral effects of interferon (IFN) through proteasome-mediated complete degradation of STAT1 by using the viral V protein (T. Kubota et al., Biochem. Biophys. Res. Commun. 283:255-259, 2001). However, we found that MuV could inhibit IFN signaling and the generation of a subsequent antiviral state long before the complete degradation of cellular STAT1 in infected cells. In MuV-infected cells, nuclear translocation and phosphorylation of STAT1 and STAT2 tyrosine residue (Y) at 701 and 689, respectively, by IFN-beta were significantly inhibited but the phosphorylation of Jak1 and Tyk2 was not inhibited. The transiently expressed MuV V protein also inhibited IFN-beta-induced Y701-STAT1 and Y689-STAT2 phosphorylation, suggesting that the V protein could block IFN-beta-induced signal transduction without the aid of other viral components. Finally, a substitution of an alanine residue in place of a cysteine residue in the C-terminal V-unique region known to be required for STAT1 degradation and inhibition of anti-IFN signaling resulted in the loss of V protein function to inhibit the Y701-STAT1 and Y689-STAT2 phosphorylation.

STAT3 ubiquitylation and degradation by mumps virus suppress cytokine and oncogene signaling.

Mumps virus is a common infectious agent of humans, causing parotitis, meningitis, encephalitis, and orchitis. Like other paramyxoviruses in the genus Rubulavirus, mumps virus catalyzes the proteasomal degradation of cellular STAT1 protein, a means for escaping antiviral responses initiated by alpha/beta and gamma interferons. We demonstrate that mumps virus also eliminates cellular STAT3, a protein that mediates transcriptional responses to cytokines, growth factors, nonreceptor tyrosine kinases, and a variety of oncogenic stimuli. STAT1 and STAT3 are independently targeted by a single mumps virus protein, called V, that assembles STAT-directed ubiquitylation complexes from cellular components, including STAT1, STAT2, STAT3, DDB1, and Cullin4A. Consequently, mumps virus V protein prevents responses to interleukin-6 and v-Src signals and can induce apoptosis in STAT3-dependent multiple myeloma cells and transformed murine fibroblasts. These findings demonstrate a unique cytokine and oncogene evasion property of mumps virus that provides a molecular basis for its observed oncolytic properties.

Localization of a new neutralizing epitope on the mumps virus hemagglutinin-neuraminidase protein.

Four protein fragments which span the entire hemagglutinin-neuraminidase protein (HN) of mumps virus were expressed in HeLa cells and cell extracts were tested for their capability to induce neutralizing antibodies in mice. Fragment HN3 (aa 213-372) was able to induce the production of hemagglutination-inhibiting and neutralizing antibodies. When a subfragment of HN3, the synthetic peptide NSTLGVKSAREF (aa 329-340 of HN) was used for immunization, hemagglutination-inhibiting and neutralizing antibodies against mumps wild type virus but not against the Urabe Am9 vaccine virus were raised. The peptide could, therefore, contain a new epitope, which may be critical for protective host humoral immune response.

Experimental schizencephaly induced by Kilham strain of mumps virus: pathogenesis of cleft formation.

The pathogenesis of cleft formation in schizencephaly was analyzed by examining the brain lesions produced by the infection of the Kilham strain of mumps virus during the period of neuronal migration in hamsters. Mumps virus antigen was detected in the neuroepithelial cells within the ventricular zone, the choroid plexus in the lateral ventricles, and vimentin-immunoreactive radial glial fibers. The main pathological findings were cerebral hemorrhage, neuronal necrosis, microsulci on the cerebral cortex and cleft formation through the entire thickness of the cerebral mantle. The clefts seen in these experiments were lined by embryonal elements such as neuroepithelial cells and germinal cells. Based on these results and the original definition by Yakovlev and Wadsworth, the following two conclusions were suggested. First, the mumps virus localized to the neuroepithelial cells within the ventricular zone and the radial glial fibers may induce a destructive process and subsequent anomalous neuronal migration, resulting in cleft formation. Second, the formation of the ventricular cleft extending to the pial surface, which should be complete before the cortical infolding appears, is necessary in order to produce the characteristic cleft in schizencephaly which is associated with the pial-ependymal seam.