Entry, Replication, Immune Evasion, and Neurotoxicity of Synthetically Engineered Bat-Borne Mumps Virus.

Bats harbor a plethora of viruses with an unknown zoonotic potential. In-depth functional characterization of such viruses is often hampered by a lack of virus isolates. The genome of a virus closely related to human mumps viruses (hMuV) was detected in African fruit bats, batMuV. Efforts to characterize batMuV were based on directed expression of the batMuV glycoproteins or use of recombinant chimeric hMuVs harboring batMuV glycoprotein. Although these studies provided initial insights into the functionality of batMuV glycoproteins, the host range, replication competence, immunomodulatory functions, virulence, and zoonotic potential of batMuV remained elusive. Here, we report the successful rescue of recombinant batMuV. BatMuV infects human cells, is largely resistant to the host interferon response, blocks interferon induction and TNF-α activation, and is neurotoxic in rats. Anti-hMuV antibodies efficiently neutralize batMuV. The striking similarities between hMuV and batMuV point at the putative zoonotic potential of batMuV.

Haemagglutinin-neuraminidase from HPIV3 mediates human NK regulation of T cell proliferation via NKp44 and NKp46.

HPIV3 is a respiratory virus causing airway diseases, including pneumonia, croup, and bronchiolitis, during infancy and childhood. Currently there is no effective vaccine or anti-viral therapy for this virus. Studies have suggested that poor T cell proliferation following HPIV3 infection is responsible for impaired immunological memory associated with this virus. We have previously demonstrated that NK cells mediate regulation of T cell proliferation during HPIV3 infection. Here we add to these studies by demonstrating that the regulation of T cell proliferation during HPIV3 infection is mediated via NK receptors NKp44 and NKp46 and involves the surface glycoprotein haemagglutinin-neuraminidase but not the fusion protein of the virus. These studies extend our knowledge of the regulatory repertoire of NK cells and provide mechanistic insights which may explain reoccurring failures of vaccines against this virus.

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.

Sequential conformational changes in the morbillivirus attachment protein initiate the membrane fusion process.

Despite large vaccination campaigns, measles virus (MeV) and canine distemper virus (CDV) cause major morbidity and mortality in humans and animals, respectively. The MeV and CDV cell entry system relies on two interacting envelope glycoproteins: the attachment protein (H), consisting of stalk and head domains, co-operates with the fusion protein (F) to mediate membrane fusion. However, how receptor-binding by the H-protein leads to F-triggering is not fully understood. Here, we report that an anti-CDV-H monoclonal antibody (mAb-1347), which targets the linear H-stalk segment 126-133, potently inhibits membrane fusion without interfering with H receptor-binding or F-interaction. Rather, mAb-1347 blocked the F-triggering function of H-proteins regardless of the presence or absence of the head domains. Remarkably, mAb-1347 binding to headless CDV H, as well as standard and engineered bioactive stalk-elongated CDV H-constructs treated with cells expressing the SLAM receptor, was enhanced. Despite proper cell surface expression, fusion promotion by most H-stalk mutants harboring alanine substitutions in the 126-138 "spacer" section was substantially impaired, consistent with deficient receptor-induced mAb-1347 binding enhancement. However, a previously reported F-triggering defective H-I98A variant still exhibited the receptor-induced "head-stalk" rearrangement. Collectively, our data spotlight a distinct mechanism for morbillivirus membrane fusion activation: prior to receptor contact, at least one of the morbillivirus H-head domains interacts with the membrane-distal "spacer" domain in the H-stalk, leaving the F-binding site located further membrane-proximal in the stalk fully accessible. This "head-to-spacer" interaction conformationally stabilizes H in an auto-repressed state, which enables intracellular H-stalk/F engagement while preventing the inherent H-stalk's bioactivity that may prematurely activate F. Receptor-contact disrupts the "head-to-spacer" interaction, which subsequently "unlocks" the stalk, allowing it to rearrange and trigger F. Overall, our study reveals essential mechanistic requirements governing the activation of the morbillivirus membrane fusion cascade and spotlights the H-stalk "spacer" microdomain as a possible drug target for antiviral therapy.

Functional properties and genetic relatedness of the fusion and hemagglutinin-neuraminidase proteins of a mumps virus-like bat virus.

UNLABELLED: A bat virus with high phylogenetic relatedness to human mumps virus (MuV) was identified recently at the nucleic acid level. We analyzed the functional activities of the hemagglutinin-neuraminidase (HN) and the fusion (F) proteins of the bat virus (batMuV) and compared them to the respective proteins of a human isolate. Transfected cells expressing the F and HN proteins of batMuV were recognized by antibodies directed against these proteins of human MuV, indicating that both viruses are serologically related. Fusion, hemadsorption, and neuraminidase activities were demonstrated for batMuV, and either bat-derived protein could substitute for its human MuV counterpart in inducing syncytium formation when coexpressed in different mammalian cell lines, including chiropteran cells. Cells expressing batMuV glycoproteins were shown to have lower neuraminidase activity. The syncytia were smaller, and they were present in lower numbers than those observed after coexpression of the corresponding glycoproteins of a clinical isolate of MuV (hMuV). The phenotypic differences in the neuraminidase and fusion activity between the glycoproteins of batMuV and hMuV are explained by differences in the expression level of the HN and F proteins of the two viruses. In the case of the F protein, analysis of chimeric proteins revealed that the signal peptide of the bat MuV fusion protein is responsible for the lower surface expression. These results indicate that the surface glycoproteins of batMuV are serologically and functionally related to those of hMuV, raising the possibility of bats as a reservoir for interspecies transmission. IMPORTANCE: The recently described MuV-like bat virus is unique among other recently identified human-like bat-associated viruses because of its high sequence homology (approximately 90% in most genes) to its human counterpart. Although it is not known if humans can be infected by batMuV, the antigenic relatedness between the bat and human forms of the virus suggests that humans carrying neutralizing antibodies against MuV are protected from infection by batMuV. The close functional relationship between MuV and batMuV is demonstrated by cooperation of the respective HN and F proteins to induce syncytium formation in heterologous expression studies. An interesting feature of the glycoproteins of batMuV is the downregulation of the fusion activity by the signal peptide of F, which has not been reported for other paramyxoviruses. These results are important contributions for risk assessment and for a better understanding of the replication strategy of batMuV.

Identification of conformational neutralization sites on the fusion protein of mumps virus.

In spite of the success of the mumps vaccination, recent mumps outbreaks have been reported even among individuals with a history of mumps vaccination. For a better understanding of why the vaccination failed in cases of vaccinees who fell ill during recent mumps outbreaks, the immunological events during infection and/or vaccination should be better defined. In the work presented here we sought to identify new neutralization sites on the mumps virus surface glycoproteins. By using anti-mumps mAbs, three amino acid positions at residues 221, 323 and 373 in the F protein of mumps virus were shown to be located in at least two conformational neutralization epitopes. mAbs that specifically target these sites effectively neutralized mumps virus in vitro. The newly acquired glycosylation site at position 373 or loss of the existing one at position 323 was identified as the mechanism behind the escape from the specific mAbs. Based on the findings of this study, we suggest that the influence of the antigenic structure of the F protein should not be ignored in a thorough investigation of the underlying mechanism of the mumps vaccine failure or when making a strategy for development of a new vaccine.

Genomic diversity of mumps virus and global distribution of the 12 genotypes.

The WHO recently proposed an updated nomenclature for mumps virus (MuV). WHO currently recognizes 12 genotypes of MuV, assigned letters from A to N (excluding E and M), which are based on the nucleotide sequences of small hydrophobic (SH) and haemagglutinin-neuraminidase (HN) genes. A total of 66 MuV genomes are available in GenBank, representing eight of the 12 genotypes. To complete this dataset, whole genomes of seven isolates representing six genotypes (D, H, I, J, K and L) and one unclassified strain were sequenced. SH and HN genes of other representative strains were also sequenced. The degree of genetic divergence, predicted amino acid substitutions in the HN and fusion (F) proteins and geographic distributions of MuV strains were analysed based on the updated dataset. Nucleotide heterogeneity between genotypes reached 20% within the SH gene, with a maximum of 9% within the HN gene. The geographic and chronologic distributions of the 12 genotypes were summarised. This review contributes to our understanding of strain diversity for wild type MuV, and the results support the current WHO nomenclature.

The nature of the N-terminal amino acid residue of HIV-1 RNase H is critical for the stability of reverse transcriptase in viral particles.

UNLABELLED: Reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is synthesized and packaged into the virion as a part of the GagPol polyprotein. Mature RT is released by the action of viral protease. However, unlike other viral proteins, RT is subject to an internal cleavage event leading to the formation of two subunits in the virion: a p66 subunit and a p51 subunit that lacks the RNase H domain. We have previously identified RNase H to be an HIV-1 protein that has the potential to be a substrate for the N-end rule pathway, which is an ubiquitin-dependent proteolytic system in which the identity of the N-terminal amino acid determines the half-life of a protein. Here we examined the importance of the N-terminal amino acid residue of RNase H in the early life cycle of HIV-1. We show that changing this residue to an amino acid structurally different from the conserved residue leads to the degradation of RT and, in some cases, integrase in the virus particle and this abolishes infectivity. Using intravirion complementation and in vitro protease cleavage assays, we show that degradation of RT in RNase H N-terminal mutants occurs in the absence of active viral protease in the virion. Our results also indicate the importance of the RNase H N-terminal residue in the dimerization of RT subunits. IMPORTANCE: HIV-1 proteins are initially made as part of a polyprotein that is cleaved by the viral protease into the proteins that form the virus particle. We were interested in one particular protein, RNase H, that is cleaved from reverse transcriptase. In particular, we found that the first amino acid of RNase H never varied in over 1,850 isolates of HIV-1 that we compared. When we changed the first amino acid, we found that the reverse transcriptase in the virus was degraded. While other studies have implied that the viral protease can degrade mutant RT proteins, we show here that this may not be the case for our mutants. Our results suggest that the presence of active viral protease is not required for the degradation of RT in RNase H N-terminal mutants, suggesting a role for a cellular protease in this process.

Accumulation of defective interfering viral particles in only a few passages in Vero cells attenuates mumps virus neurovirulence.

Immunization programs have implemented live attenuated mumps vaccines which reduced mumps incidence ≥97%. Some of the vaccine strains were abandoned due to unwanted side effects and the genetic marker of attenuation has not been identified so far. Our hypothesis was that non-infectious viral particles, in particular defective interfering particles (DIPs), contribute to neuroattenuation. We showed that non-infectious particles of the mumps vaccine L-Zagreb attenuated neurovirulence of wild type mumps virus 9218/Zg98. Then, we attenuated recent wild type mumps virus MuVi/Zagreb.HRV/28.12 in Vero cells through 16 passages but already the fifth passage (p5) showed accumulation of DIPs and attenuated neurovirulence in a newborn rat model when compared to the second passage (p2). Sequence analysis of the p2 and p5 revealed a single mutation in the 5' untranslated region of the HN gene. Analysis of the expression level of the HN protein showed that this mutation does not affect the expression of the protein. We conclude that the passages of MuVi/Zagreb.HRV/28.12 in Vero cells for only three passages accumulated DIPs which attenuate neurovirulence. These findings reveal DIPs as a very promising and general neuroattenuating factor which should be considered in the rational design of the new mumps vaccine.

Mechanism for active membrane fusion triggering by morbillivirus attachment protein.

The paramyxovirus entry machinery consists of two glycoproteins that tightly cooperate to achieve membrane fusion for cell entry: the tetrameric attachment protein (HN, H, or G, depending on the paramyxovirus genus) and the trimeric fusion protein (F). Here, we explore whether receptor-induced conformational changes within morbillivirus H proteins promote membrane fusion by a mechanism requiring the active destabilization of prefusion F or by the dissociation of prefusion F from intracellularly preformed glycoprotein complexes. To properly probe F conformations, we identified anti-F monoclonal antibodies (MAbs) that recognize conformation-dependent epitopes. Through heat treatment as a surrogate for H-mediated F triggering, we demonstrate with these MAbs that the morbillivirus F trimer contains a sufficiently high inherent activation energy barrier to maintain the metastable prefusion state even in the absence of H. This notion was further validated by exploring the conformational states of destabilized F mutants and stabilized soluble F variants combined with the use of a membrane fusion inhibitor (3g). Taken together, our findings reveal that the morbillivirus H protein must lower the activation energy barrier of metastable prefusion F for fusion triggering.

Respiratory syncytial virus matrix protein induces lung epithelial cell cycle arrest through a p53 dependent pathway.

Respiratory syncytial virus (RSV) is the major cause of viral respiratory infections in children. Our previous study showed that the RSV infection induced lung epithelial cell cycle arrest, which enhanced virus replication. To address the mechanism of RSV-induced cell cycle arrest, we examined the contribution of RSV-matrix (RSV-M) protein. In this report, we show that in both the A549 cell line and primary human bronchial epithelial (PHBE) cells, transfection with RSV-M protein caused the cells to proliferate at a slower rate than in control cells. The cell cycle analysis showed that RSV-M protein induced G1 phase arrest in A549 cells, and G1 and G2/M phase arrest in PHBE cells. Interestingly, RSV-M expression induced p53 and p21 accumulation and decreased phosphorylation of retinoblastoma protein (Rb). Further, induction of cell cycle arrest by RSV-M was not observed in a p53-deficient epithelial cell line (H1299). However, cell cycle arrest was restored after transfection of p53 cDNA into H1299 cells. Taken together, these results indicate that RSV-M protein regulates lung epithelial cell cycle through a p53-dependent pathway, which enhances RSV replication.

Structural rearrangements of the central region of the morbillivirus attachment protein stalk domain trigger F protein refolding for membrane fusion.

It is unknown how receptor binding by the paramyxovirus attachment proteins (HN, H, or G) triggers the fusion (F) protein to fuse with the plasma membrane for cell entry. H-proteins of the morbillivirus genus consist of a stalk ectodomain supporting a cuboidal head; physiological oligomers consist of non-covalent dimer-of-dimers. We report here the successful engineering of intermolecular disulfide bonds within the central region (residues 91-115) of the morbillivirus H-stalk; a sub-domain that also encompasses the putative F-contacting section (residues 111-118). Remarkably, several intersubunit crosslinks abrogated membrane fusion, but bioactivity was restored under reducing conditions. This phenotype extended equally to H proteins derived from virulent and attenuated morbillivirus strains and was independent of the nature of the contacted receptor. Our data reveal that the morbillivirus H-stalk domain is composed of four tightly-packed subunits. Upon receptor binding, these subunits structurally rearrange, possibly inducing conformational changes within the central region of the stalk, which, in turn, promote fusion. Given that the fundamental architecture appears conserved among paramyxovirus attachment protein stalk domains, we predict that these motions may act as a universal paramyxovirus F-triggering mechanism.

One-year follow-up of tick-borne central nervous system infections in childhood.

BACKGROUND: Neurologic sequelae, including cognitive deficits, after childhood tick-borne encephalitis (TBE) and neuroborreliosis (NB) are not well-characterized. These infections are among the most common affecting the central nervous system in children and can be difficult to diagnose due to vague symptomatology. The aim of this study was to investigate long-term (>1 year) consequences of pediatric TBE and NB as well as the value of markers for brain damage and genetic susceptibility. METHODS: From a previous prospective study, children diagnosed with TBE (n = 8) and NB (n = 12) as well as pediatric controls (n = 15) were followed up by clinical examination, semistructured interview and screening for cognitive dysfunction by the Five-to-Fifteen Questionnaire. The follow-up also included detection of serum autoantibodies against the neural proteins; glial fibrillary acidic protein and myelin basic protein, as well as genotyping of a 32 basepair deletion in the chemokine receptor type 5 gene. RESULTS: Children diagnosed with TBE displayed significantly more long-term subjective complaints (ie, fatigue, headache and irritability) compared with the NB and control groups. Significantly higher frequency of disabilities was also detected by the Five-to-Fifteen Questionnaire in the TBE group. Both TBE and NB cause consequences (eg, prolonged convalescence, worries and financial loss) for the families. Markers for genetic susceptibility and brain damage had no prognostic values in this cohort. CONCLUSIONS: Pediatric TBE results in long-lasting residual symptoms and neurologic deficits affecting daily life. Vigilance for TBE-related morbidity among pediatricians and long-term clinical follow-up with assessment of cognitive dysfunctions and appropriate interventions seems reasonable for these children.

Pediatric tick-borne infections of the central nervous system in an endemic region of Sweden: a prospective evaluation of clinical manifestations.

UNLABELLED: Tick-borne encephalitis (TBE) and neuroborreliosis (NB) are well-known central nervous system (CNS) infections in children. Childhood tick-borne CNS infections are generally described as mild conditions. However, this view has recently been challenged, and the natural course, including potential sequelae, has been debated. If the diseases present with nonspecific symptoms and signs, some children may elude diagnosis. This study estimates the incidence of symptomatic tick-borne CNS infections in children under medical care and describes the spectrum of manifestations. One hundred twenty-four children with neurologic symptoms attending the Pediatric Emergency Department were included prospectively. Anti-TBE virus and anti-Borrelia serology results were analyzed together with inflammatory parameters in the blood and cerebrospinal fluid. Nearly one fourth of the children with neurologic symptoms were diagnosed with a tick-borne CNS infection (TBE, n = 10 [8%] and NB, n = 21 [16.8%]). In general, these children displayed an indistinct medical history and presented with nonspecific signs such as malaise/fatigue and headache. Diagnosis was based on analysis of acute and convalescent sera. Blood inflammatory parameters were nonspecific and did not contribute to the diagnostics. CONCLUSION: Pediatric tick-borne CNS infections are unexpectedly common and should be considered in children with unspecific and unexplained acute CNS-related symptoms.

Canine distemper virus infects canine keratinocytes and immune cells by using overlapping and distinct regions located on one side of the attachment protein.

The morbilliviruses measles virus (MeV) and canine distemper virus (CDV) both rely on two surface glycoproteins, the attachment (H) and fusion proteins, to promote fusion activity for viral cell entry. Growing evidence suggests that morbilliviruses infect multiple cell types by binding to distinct host cell surface receptors. Currently, the only known in vivo receptor used by morbilliviruses is CD150/SLAM, a molecule expressed in certain immune cells. Here we investigated the usage of multiple receptors by the highly virulent and demyelinating CDV strain A75/17. We based our study on the assumption that CDV-H may interact with receptors similar to those for MeV, and we conducted systematic alanine-scanning mutagenesis on CDV-H throughout one side of the ß-propeller documented in MeV-H to contain multiple receptor-binding sites. Functional and biochemical assays performed with SLAM-expressing cells and primary canine epithelial keratinocytes identified 11 residues mutation of which selectively abrogated fusion in keratinocytes. Among these, four were identical to amino acids identified in MeV-H as residues contacting a putative receptor expressed in polarized epithelial cells. Strikingly, when mapped on a CDV-H structural model, all residues clustered in or around a recessed groove located on one side of CDV-H. In contrast, reported CDV-H mutants with SLAM-dependent fusion deficiencies were characterized by additional impairments to the promotion of fusion in keratinocytes. Furthermore, upon transfer of residues that selectively impaired fusion induction in keratinocytes into the CDV-H of the vaccine strain, fusion remained largely unaltered. Taken together, our results suggest that a restricted region on one side of CDV-H contains distinct and overlapping sites that control functional interaction with multiple receptors.

Tick-borne encephalitis in childhood: rare or missed?

Retrospective evaluation of medical history and 3635 anti-TBE (tick-borne encephalitis) serologies during the years 2003-2008 indicates that childhood TBE is characterized by vague symptoms. Clinical findings suggest a nonspecific inflammatory disease with restricted encephalitic profile compared with adult TBE. Childhood TBE might elude diagnosis, which is unsatisfactory because of potential long-term consequences.

Identification of key residues in virulent canine distemper virus hemagglutinin that control CD150/SLAM-binding activity.

Morbillivirus cell entry is controlled by hemagglutinin (H), an envelope-anchored viral glycoprotein determining interaction with multiple host cell surface receptors. Subsequent to virus-receptor attachment, H is thought to transduce a signal triggering the viral fusion glycoprotein, which in turn drives virus-cell fusion activity. Cell entry through the universal morbillivirus receptor CD150/SLAM was reported to depend on two nearby microdomains located within the hemagglutinin. Here, we provide evidence that three key residues in the virulent canine distemper virus A75/17 H protein (Y525, D526, and R529), clustering at the rim of a large recessed groove created by beta-propeller blades 4 and 5, control SLAM-binding activity without drastically modulating protein surface expression or SLAM-independent F triggering.

Conserved leucine residue in the head region of morbillivirus fusion protein regulates the large conformational change during fusion activity.

Paramyxovirus cell entry is controlled by the concerted action of two viral envelope glycoproteins, the fusion (F) and the receptor-binding (H) proteins, which together with a cell surface receptor mediate plasma membrane fusion activity. The paramyxovirus F protein belongs to class I viral fusion proteins which typically contain two heptad repeat regions (HR). Particular to paramyxovirus F proteins is a long intervening sequence (IS) located between both HR domains. To investigate the role of the IS domain in regulating fusogenicity, we mutated in the canine distemper virus (CDV) F protein IS domain a highly conserved leucine residue (L372) previously reported to cause a hyperfusogenic phenotype. Beside one F mutant, which elicited significant defects in processing, transport competence, and fusogenicity, all remaining mutants were characterized by enhanced fusion activity despite normal or slightly impaired processing and cell surface targeting. Using anti-CDV-F monoclonal antibodies, modified conformational F states were detected in F mutants compared to the parental protein. Despite these structural differences, coimmunoprecipitation assays did not reveal any drastic modulation in F/H avidity of interaction. However, we found that F mutants had significantly enhanced fusogenicity at low temperature only, suggesting that they folded into conformations requiring less energy to activate fusion. Together, these data provide strong biochemical and functional evidence that the conserved leucine 372 at the base of the HRA coiled-coil of F(wt) controls the stabilization of the prefusogenic state, restraining the conformational switch and thereby preventing extensive cell-cell fusion activity.

Persistence of human parvovirus B19 in multipotent mesenchymal stromal cells expressing the erythrocyte P antigen: implications for transplantation.

Multipotent mesenchymal stromal cells (MSCs) are used to improve the outcome of hematopoietic stem cell transplantation (HCST) and in regenerative medicine. MSCs may harbor persistent viruses that may compromise their clinical benefit, however. Retrospectively screened, 1 of 20 MSCs from healthy donors contained parvovirus B19 (B19) DNA. MSCs express the B19 receptor (P antigen/globoside) and a co-receptor (Ku 80) and can transmit B19 to bone marrow cells in vitro, suggesting that the virus can persist in the marrow stroma of healthy individuals. Two patients undergoing HSCT received the B19-positive MSCs as treatment for graft-versus-host disease; neither developed viremia nor symptomatic B19 infection. These findings demonstrate for the first time that persistent B19 in MSCs can infect hematopoietic stem cells and underscore the importance of monitoring B19 transmission by MSC products.