High genetic variability of Schmallenberg virus M-segment leads to efficient immune escape from neutralizing antibodies.

Schmallenberg virus (SBV) is the cause of severe fetal malformations when immunologically naïve pregnant ruminants are infected. In those malformed fetuses, a "hot-spot"-region of high genetic variability within the N-terminal region of the viral envelope protein Gc has been observed previously, and this region co-localizes with a known key immunogenic domain. We studied a series of M-segments of those SBV variants from malformed fetuses with point mutations, insertions or large in-frame deletions of up to 612 nucleotides. Furthermore, a unique cell-culture isolate from a malformed fetus with large in-frame deletions within the M-segment was analyzed. Each Gc-protein with amino acid deletions within the "hot spot" of mutations failed to react with any neutralizing anti-SBV monoclonal antibodies or a domain specific antiserum. In addition, in vitro virus replication of the natural deletion variant could not be markedly reduced by neutralizing monoclonal antibodies or antisera from the field. The large-deletion variant of SBV that could be isolated in cell culture was highly attenuated with an impaired in vivo replication following the inoculation of sheep. In conclusion, the observed amino acid sequence mutations within the N-terminal main immunogenic domain of glycoprotein Gc result in an efficient immune evasion from neutralizing antibodies in the special environment of a developing fetus. These SBV-variants were never detected as circulating viruses, and therefore should be considered to be dead-end virus variants, which are not able to spread further. The observations described here may be transferred to other orthobunyaviruses, particularly those of the Simbu serogroup that have been shown to infect fetuses. Importantly, such mutant strains should not be included in attempts to trace the spatial-temporal evolution of orthobunyaviruses in molecular-epidemiolocal approaches during outbreak investigations.

The role of N-linked glycosylation in proteolytic processing and cell surface transport of the Cedar virus fusion protein.

BACKGROUND: N-linked glycans on viral glycoproteins have been shown to be important for protein expression, processing and intracellular transport. The fusion glycoprotein F of Cedar virus (CedV) contains six potential N-glycosylation sites. FINDINGS: To investigate their impact on cell surface transport, proteolytic cleavage and biological activity, we disrupted the consensus sequences by conservative mutations (Asn to Gln) and found that five of the six potential N-glycosylation sites are actually utilized. The individual removal of N-glycan g1 (N66), g2 (N79) and g3 (N98) in the CedV F2 subunit had no or only little effect on cell surface transport, proteolytic cleavage and fusion activity of CedV F. Interestingly, removal of N-linked glycan g6 (N463) in the F1 subunit resulted in reduced cell surface expression but slightly increased fusogenicity upon co-expression with the CedV receptor-binding protein G. Most prominent effects however were observed for the disruption of N-glycosylation motif g4 (N413), which significantly impaired the transport of CedV F to the cell surface, thereby also affecting proteolytic cleavage and fusion activity. CONCLUSIONS: Our findings indicate that the individual N-linked modifications, with the exception of glycan g4, are dispensable for processing of CedV F protein in transfection experiments. However, removal of g4 led to a phenotype that was strongly impaired concerning cell surface expression and proteolytic activation.

The amino terminal subdomain of glycoprotein Gc of Schmallenberg virus: disulfide bonding and structural determinants of neutralization.

Orthobunyaviruses are enveloped viruses that can cause human and animal diseases. A novel and major member is the Schmallenberg virus (SBV), the etiological agent of an emerging disease of ruminants that has been spreading all over Europe since 2011. The glycoproteins Gn and Gc of orthobunyaviruses mediate the viral entry, and specifically Gc is a major target for the humoral immune response. For example, the N terminal subdomain of the SBV glycoprotein Gc is targeted by neutralizing monoclonal antibodies that recognize conformational epitopes. Here, we determined the structural features of the N terminus of Gc, and analysed its interaction with monoclonal antibodies. We were able to demonstrate that one of two N-glycosylation sites is essential for secretion and interaction with a subset of Gc-specific monoclonal antibodies. Furthermore, four disulfide bonds (S-S) were identified and the deletion of the third S-S blocked reactivity with another subset of mAbs with virus-neutralizing and non-neutralizing activity. The mutagenesis of the N-glycosylation sites and the disulfide bonds strongly indicated the independent folding of two subdomains within the SBV Gc N terminus. Further, the epitopes recognized by a panel of mAbs could be grouped into two clusters, as revealed by fine mapping using chimeric proteins. Combining the disulfide bonding and epitope mapping allowed us to generate a structural model of the SBV Gc N-terminus. This novel information about the role and structure of the amino terminal region of SBV Gc is of general relevance for the design of antivirals and vaccines against this virus.

Deletion mutants of Schmallenberg virus are avirulent and protect from virus challenge.

UNLABELLED: Since its emergence, Schmallenberg virus (SBV), a novel insect-transmitted orthobunyavirus which predominantly infects ruminants, has caused a large epidemic in European livestock. Newly developed inactivated vaccines are available, but highly efficacious and safe live vaccines are still not available. Here, the properties of novel recombinant SBV mutants lacking the nonstructural protein NSs (rSBVΔNSs) or NSm (rSBVΔNSm) or both of these proteins (rSBVΔNSs/ΔNSm) were tested in vitro and in vivo in type I interferon receptor knockout mice (IFNAR(-/-)) and in a vaccination/challenge trial in cattle. As for other bunyaviruses, both nonstructural proteins of SBV are not essential for viral growth in vitro. In interferon-defective BHK-21 cells, rSBVΔNSs and rSBVΔNSm replicated to levels comparable to that of the parental rSBV; the double mutant virus, however, showed a mild growth defect, resulting in lower final virus titers. Additionally, both mutants with an NSs deletion induced high levels of interferon and showed a marked growth defect in interferon-competent sheep SFT-R cells. Nevertheless, in IFNAR(-/-) mice, all mutants were virulent, with the highest mortality rate for rSBVΔNSs and a reduced virulence for the NSm-deleted virus. In cattle, SBV lacking NSm caused viremia and seroconversion comparable to those caused by the wild-type virus, while the NSs and the combined NSs/NSm deletion mutant induced no detectable virus replication or clinical disease after immunization. Furthermore, three out of four cattle immunized once with the NSs deletion mutant and all animals vaccinated with the virus lacking both nonstructural proteins were fully protected against a challenge infection. Therefore, the double deletion mutant will provide the basis for further developments of safe and efficacious modified live SBV vaccines which could be also a model for other viruses of the Simbu serogroup and related orthobunyaviruses. IMPORTANCE: SBV induces only mild clinical signs in adult ruminants but causes severe fetal malformation and, thereby, can have an important impact on animal welfare and production. As SBV is an insect-transmitted pathogen, vaccination will be one of the most important aspects of disease control. Here, mutant viruses lacking one or two proteins that essentially contribute to viral pathogenicity were tested as modified live vaccines in cattle. It could be demonstrated that a novel recombinant double deletion mutant is a safe and efficacious vaccine candidate. This is the first description of a putative modified live vaccine for the complete genus Orthobunyavirus, and in addition, such a vaccine type has never been tested in cattle for any virus of the entire family Bunyaviridae. Therefore, the described vaccine also represents the first model for a broad range of related viruses and is of high importance to the field.

Autoimmune pancreatitis in MRL/Mp mice is a T cell-mediated disease responsive to cyclosporine A and rapamycin treatment.

BACKGROUND: Autoimmune pancreatitis (AIP) in humans invariably responds to steroid treatment, but little is known about the underlying pathogenesis and the benefits of alternative treatments. OBJECTIVE: To study the pathogenesis, and the efficacy of alternative immunosuppressant agents in the MRL/Mp mouse model of AIP. DESIGN: MRL/Mp mice were pretreated for 4 weeks with polyinosinic:polycytidylic acid to induce AIP. Pancreatic sections of mice genetically deleted for CTLA-4 were analysed. Blockage of CTLA-4 was achieved by intraperitoneal antibody treatment with 2 µg/g anti-mouse-CD152. Subsequent therapeutic studies were performed for a period of 4 weeks using cyclosporine A (40 µg/g), rapamycin (1 µg/g) or azathioprine (15 µg/g). RESULTS: Blockage of CTLA-4 in MRL/Mp mice suppressed regulatory T cell (Treg) function and raised the effector T cell (Teff) response with subsequent histomorphological organ destruction, indicating that AIP is a T cell-driven disease. Using an established histopathological score, we found that dexamethasone, cyclosporine A and rapamycin, but less so azathioprine, reduced pancreatic damage. However, the beneficial effects of cyclosporine A and rapamycin were achieved via different mechanisms: cyclosporine A inhibited Teff activation and proliferation whereas rapamycin led to selective expansion of Tregs which subsequently suppressed the Teff response. CONCLUSIONS: The calcineurin inhibitor cyclosporine A and the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, improve the course of AIP in MRL/Mp mice via different mechanisms. These findings further support the concept of autoreactive T cells as key players in the pathogenesis of AIP and suggest that cyclosporine A and rapamycin should be considered for treatment of AIP in humans.

Role of endoplasmic reticulum stress and protein misfolding in disorders of the liver and pancreas.

The endoplasmic reticulum (ER) is the site of synthesis and folding of membrane and secretory proteins. The fraction of protein passing through the ER represents a large proportion of the total protein in the cell. Protein folding, glycosylation, sorting and transport are essential tasks of the ER and a compromised ER folding network has been recognized to be a key component in the disease pathogenicity of common neurodegenerative, metabolic and malignant diseases. On the other hand, the ER protein folding machinery also holds significant potential for therapeutic interventions. Many causes can lead to ER stress. A disturbed calcium homeostasis, the generation of reactive oxygen species (ROS) and a persistent overload of misfolded proteins within the ER can drive the course of adisease. In this review the role of ER-stress in diseases of the liver and pancreas will be examined using pancreatitis and Wilson´s disease as examples. Potential therapeutic targets in ER-stress pathways will also be discussed.

Schmallenberg virus non-structural protein NSm: Intracellular distribution and role of non-hydrophobic domains.

Schmallenberg virus (SBV) induces fetal malformation, abortions and stillbirth in ruminants. While the non-structural protein NSs is a major virulence factor, the biological function of NSm, the second non-structural protein which consists of three hydrophobic transmembrane (I, III, V) and two non-hydrophobic regions (II, IV), is still unknown. Here, a series of NSm mutants displaying deletions of nearly the entire NSm or of the non-hydrophobic domains was generated and the intracellular distribution of NSm was assessed. SBV-NSm is dispensable for the generation of infectious virus and mutants lacking domains II - V showed growth properties similar to the wild-type virus. In addition, a comparable intracellular distribution of SBV-NSm was observed in mammalian cells infected with domain II mutants or wild-type virus. In both cases, NSm co-localized with the glycoprotein Gc in the Golgi compartment. However, domain IV-deletion mutants showed an altered distribution pattern and no co-localization of NSm and Gc.

Schmallenberg virus-two years of experiences.

In autumn 2011, a novel species of the genus Orthobunyavirus of the Simbu serogroup was discovered close to the German/Dutch border and named Schmallenberg virus (SBV). Since then, SBV has caused a large epidemic in European livestock. Like other viruses of the Simbu serogroup, SBV is transmitted by insect vectors. Adult ruminants may show a mild transient disease, while an infection during a critical period of pregnancy can lead to severe congenital malformation, premature birth or stillbirth. The current knowledge about the virus, its diagnosis, the spread of the epidemic, the impact and the possibilities for preventing infections with SBV is described and discussed.