Mystery of fatal 'staggering disease' unravelled: novel rustrela virus causes severe meningoencephalomyelitis in domestic cats.

'Staggering disease' is a neurological disease entity considered a threat to European domestic cats (Felis catus) for almost five decades. However, its aetiology has remained obscure. Rustrela virus (RusV), a relative of rubella virus, has recently been shown to be associated with encephalitis in a broad range of mammalian hosts. Here, we report the detection of RusV RNA and antigen by metagenomic sequencing, RT-qPCR, in-situ hybridization and immunohistochemistry in brain tissues of 27 out of 29 cats with non-suppurative meningoencephalomyelitis and clinical signs compatible with'staggering disease' from Sweden, Austria, and Germany, but not in non-affected control cats. Screening of possible reservoir hosts in Sweden revealed RusV infection in wood mice (Apodemus sylvaticus). Our work indicates that RusV is the long-sought cause of feline 'staggering disease'. Given its reported broad host spectrum and considerable geographic range, RusV may be the aetiological agent of neuropathologies in further mammals, possibly even including humans.

Structure, function, and evolution of the Orthobunyavirus membrane fusion glycoprotein.

La Crosse virus, responsible for pediatric encephalitis in the United States, and Schmallenberg virus, a highly teratogenic veterinary virus in Europe, belong to the large Orthobunyavirus genus of zoonotic arthropod-borne pathogens distributed worldwide. Viruses in this under-studied genus cause CNS infections or fever with debilitating arthralgia/myalgia syndromes, with no effective treatment. The main surface antigen, glycoprotein Gc (∼1,000 residues), has a variable N-terminal half (GcS) targeted by the patients' antibody response and a conserved C-terminal moiety (GcF) responsible for membrane fusion during cell entry. Here, we report the X-ray structure of post-fusion La Crosse and Schmallenberg virus GcF, revealing the molecular determinants for hairpin formation and trimerization required to drive membrane fusion. We further experimentally confirm the role of residues in the fusion loops and in a vestigial endoplasmic reticulum (ER) translocation sequence at the GcS-GcF junction. The resulting knowledge provides essential molecular underpinnings for future development of potential therapeutic treatments and vaccines.

Vaccine development against Schmallenberg virus: from classical inactivated to modified-live to scaffold particle vaccines.

BACKGROUND: Subsequent to its first detection in 2011, the insect-transmitted bunyavirus Schmallenberg virus (SBV; genus Orthobunyavirus) caused a large-scale epizootic of fetal malformation in the European ruminant population. By now, SBV established an enzootic status in Central Europe with regular wave-like re-emergence, which has prompted intensive research efforts in order to elucidate the pathogenesis and to develop countermeasures. Since different orthobunyaviruses share a very similar structural organization, SBV has become an important model virus to study orthobunyaviruses in general and for the development of vaccines. In this review article, we summarize which vaccine formulations have been tested to prevent SBV infections in livestock animals. MAIN: In a first step, inactivated SBV candidate vaccines were developed, which efficiently protected against an experimental SBV infection. Due to the inability to differentiate infected from vaccinated animals (= DIVA capability), a series of further approaches ranging from modified live, live-vectored, subunit and DNA-mediated vaccine delivery to multimeric antigen-presentation on scaffold particles was developed and evaluated. In short, it was repeatedly demonstrated that the N-terminal half of the glycoprotein Gc, composed of the Gc head and the head-stalk, is highly immunogenic, with a superior immunogenicity of the complete head-stalk domain compared to the Gc head only. Furthermore, in all Gc protein-based vaccine candidates, immunized animals can be readily discriminated from animals infected with the field virus by the absence of antibodies against the viral N-protein. CONCLUSIONS: Using SBV as a model virus, several vaccination-challenge studies in target species underscored the superior performance of antigenic domains compared to linear epitopes regarding their immunogenicity. In addition, it could be shown that holistic approaches combining immunization-challenge infection studies with structural analyses provide essential knowledge required for an improved vaccine design.

Distal femoral arthroplasty for native knee fractures : results from the Australian Orthopaedic Association National Joint Replacement Registry.

AIMS: The aim of this study was to investigate the rate of revision for distal femoral arthroplasty (DFA) performed as a primary procedure for native knee fractures using data from the Australian Orthopaedic Association National Joint Arthroplasty Registry (AOANJRR). METHODS: Data from the AOANJRR were obtained for DFA performed as primary procedures for native knee fractures from 1 September 1999 to 31 December 2020. Pathological fractures and revision for failed internal fixation were excluded. The five prostheses identified were the Global Modular Arthroplasty System, the Modular Arthroplasty System, the Modular Universal Tumour And Revision System, the Orthopaedic Salvage System, and the Segmental System. Patient demographic data (age, sex, and American Society of Anesthesiologists grade) were obtained, where available. Kaplan-Meier estimates of survival were used to determine the rate of revision, and the reasons for revision and mortality data were examined. RESULTS: The AOANJRR identified 153 primary DFAs performed for native knee fractures in 151 patients during the study period, with 63.3% of these (n = 97) performed within the last five years. The median follow-up was 2.1 years (interquartile range 0.8 to 4.4). The patient population was 84.8% female (n = 128), with a mean age of 76.1 years (SD 11.9). The cumulative percent revision rate at three years was 10%. The most common reason for revision was loosening, followed by infection. Patient survival at one year was 87.5%, decreasing to 72.8% at three years postoperatively. CONCLUSION: The use of DFA to treat native knee fractures is increasing, with 63.3% of cases performed within the last five years. While long-term data are not available, the results of this study suggest that DFA may be a reasonable option for elderly patients with native knee fractures where fixation is not feasible, or for whom prolonged non-weightbearing may be detrimental. Cite this article: Bone Joint J 2022;104-B(7):894-901.

Serological screening in wild ruminants in Germany, 2021/2022: No evidence of SARS-CoV-2, bluetongue virus or pestivirus spread but high seroprevalences against Schmallenberg virus.

Wildlife animals may be susceptible to multiple infectious agents of public health or veterinary relevance, thereby potentially forming a reservoir that bears the constant risk of re-introduction into the human or livestock population. Here, we serologically investigated 493 wild ruminant samples collected in the 2021/2022 hunting season in Germany for the presence of antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and four viruses pathogenic to domestic ruminants, namely, the orthobunyavirus Schmallenberg virus (SBV), the reovirus bluetongue virus (BTV) and ruminant pestiviruses like bovine viral diarrhoea virus or border disease virus. The animal species comprised fallow deer, red deer, roe deer, mouflon and wisent. For coronavirus serology, additional 307 fallow, roe and red deer samples collected between 2017 and 2020 at three military training areas were included. While antibodies against SBV could be detected in about 13.6% of the samples collected in 2021/2022, only one fallow deer of unknown age tested positive for anti-BTV antibodies, and all samples reacted negative for antibodies against ruminant pestiviruses. In an ELISA based on the receptor-binding domain (RBD) of SARS-CoV-2, 25 out of 493 (5.1%) samples collected in autumn and winter 2021/2022 scored positive. This sero-reactivity could not be confirmed by the highly specific virus neutralisation test, occurred also in 2017, 2018 and 2019, that is, prior to the human SARS-CoV-2 pandemic, and was likewise observed against the RBD of the related SARS-CoV-1. Therefore, the SARS-CoV-2 sero-reactivity was most likely induced by another hitherto unknown deer virus belonging to the subgenus Sarbecovirus of betacoronaviruses.

Distal Femoral Replacement for Periprosthetic Fractures After TKA: Australian Orthopaedic Association National Joint Replacement Registry Review.

BACKGROUND: Distal femoral replacement (DFR) is a potential treatment option following periprosthetic fracture (PPF) of a total knee arthroplasty (TKA). However, there is limited literature regarding implant survivorship and complication rates. The aim of this study was to examine patient demographics and trends in usage, implant survivorship and modes of failure, and patient mortality following DFR for PPF captured by a national joint replacement registry. METHODS: A retrospective registry review was performed using data from the Australian Orthopaedic Association National Joint Replacement Registry (AOANJRR). In total, 306 DFR were performed for PPF of a known primary TKA. Eighty-five percent of patients were female, and the mean age was 76.4 years. Kaplan-Meier estimates of implant and patient survivorship were performed. RESULTS: The number of DFR performed for PPF has doubled over the past five years. The cumulative percent second revision rate at six years was 12%. The most common indications for revision were infection (37%) and aseptic loosening (33%). Patient survivorship after DFR was 97% and 83% at five and ten years, respectively. CONCLUSION: A national registry review has identified the increasing prevalence of DFR for PPF after primary TKA and demonstrated implant survivorship of 88% at midterm follow-up. Surgeons may consider DFR as an acceptable and durable treatment option. LEVEL OF EVIDENCE: Level III - Case Series.

Development of a Modular Vaccine Platform for Multimeric Antigen Display Using an Orthobunyavirus Model.

Emerging infectious diseases represent an increasing threat to human and animal health. Therefore, safe and effective vaccines that could be available within a short time frame after an outbreak are required for adequate prevention and control. Here, we developed a robust and versatile self-assembling multimeric protein scaffold particle (MPSP) vaccine platform using lumazine synthase (LS) from Aquifex aeolicus. This scaffold allowed the presentation of peptide epitopes by genetic fusion as well as the presentation of large antigens by bacterial superglue-based conjugation to the pre-assembled particle. Using the orthobunyavirus model Schmallenberg virus (SBV) we designed MPSPs presenting major immunogens of SBV and assessed their efficacy in a mouse model as well as in cattle, a target species of SBV. All prototype vaccines conferred protection from viral challenge infection and the multivalent presentation of the selected antigens on the MPSP markedly improved their immunogenicity compared to the monomeric subunits. Even a single shot vaccination protected about 80% of mice from an otherwise lethal dose of SBV. Most importantly, the MPSPs induced a virtually sterile immunity in cattle. Altogether, LS represents a promising platform for modular and rapid vaccine design.

Anti-platelet factor 4 antibodies causing VITT do not cross-react with SARS-CoV-2 spike protein.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe adverse effect of ChAdOx1 nCoV-19 COVID-19 vaccine (Vaxzevria) and Janssen Ad26.COV2.S COVID-19 vaccine, and it is associated with unusual thrombosis. VITT is caused by anti-platelet factor 4 (PF4) antibodies activating platelets through their FcγRIIa receptors. Antibodies that activate platelets through FcγRIIa receptors have also been identified in patients with COVID-19. These findings raise concern that vaccination-induced antibodies against anti-SARS-CoV-2 spike protein cause thrombosis by cross-reacting with PF4. Immunogenic epitopes of PF4 and SARS-CoV-2 spike protein were compared using in silico prediction tools and 3D modeling. The SARS-CoV-2 spike protein and PF4 share at least 1 similar epitope. Reactivity of purified anti-PF4 antibodies from patients with VITT was tested against recombinant SARS-CoV-2 spike protein. However, none of the affinity-purified anti-PF4 antibodies from 14 patients with VITT cross-reacted with SARS-CoV-2 spike protein. Sera from 222 polymerase chain reaction-confirmed patients with COVID-19 from 5 European centers were tested by PF4-heparin enzyme-linked immunosorbent assays and PF4-dependent platelet activation assays. We found anti-PF4 antibodies in sera from 19 (8.6%) of 222 patients with COVID-19. However, only 4 showed weak to moderate platelet activation in the presence of PF4, and none of those patients developed thrombotic complications. Among 10 (4.5%) of 222 patients who had COVID-19 with thrombosis, none showed PF4-dependent platelet-activating antibodies. In conclusion, antibodies against PF4 induced by vaccination do not cross-react with the SARS-CoV-2 spike protein, indicating that the intended vaccine-induced immune response against SARS-CoV-2 spike protein is not the trigger of VITT. PF4-reactive antibodies found in patients with COVID-19 in this study were not associated with thrombotic complications.

Performance of a SARS CoV-2 antibody ELISA based on simultaneous measurement of antibodies against the viral nucleoprotein and receptor-binding domain.

SARS CoV-2 antibody assays measure antibodies against the viral nucleoprotein (NP) or spike protein. The study examined if testing of antibodies against both antigens increases the diagnostic sensitivity. Sera (N=98) from infected individuals were tested with ELISAs based on the NP, receptor-binding domain (RBD), or both proteins. The AUROCs were 0.958 (NP), 0.991 (RBD), and 0.992 (NP/RBD). The RBD- and NP/RBD-based ELISAs showed better performance than the NP-based assay. Simultaneous testing for antibodies against NP and RBD increased the number of true and false positives. If maximum diagnostic sensitivity is required, the NP/RBD-based ELISA is preferable. Otherwise, the RBD-based ELISA is sufficient.

Simultaneous Total Knee Arthroplasty and Proximal Tibiofibular Joint Arthrodesis: A Case Report.

CASE: A 57-year-old man presented with tricompartmental left knee osteoarthritis, as well as proximal tibiofibular joint arthritis and a ganglion cyst. He underwent simultaneous total knee arthroplasty and proximal tibiofibular joint arthrodesis, with an excellent outcome. CONCLUSION: Proximal tibiofibular joint arthritis is uncommon and may be associated with tibiofemoral arthritis, proximal tibiofibular joint instability, and ankylosing spondylitis. We present a case of simultaneous total knee arthroplasty and proximal tibiofibular arthrodesis. This is an effective option for treating patients with dual pathology. The proximal tibiofibular joint should be considered as an uncommon cause of lateral knee pain.

Distal Clavicle Physeal Separation Mimicking an Acromioclavicular Joint Separation: A Case Report.

CASE: A 9-year-old boy sustained a right distal clavicle physeal separation with superior and posterior displacement through the periosteum. He was treated surgically with open reduction, Kirschner wire fixation, and periosteal repair and had an excellent outcome. CONCLUSION: Distal clavicle fractures are rare in children, and acromioclavicular joint (ACJ) separations are exceedingly rare. Differentiating between the 2 is often difficult radiographically and clinically. Our case represents a Type IV distal clavicle fracture but could be confused with an ACJ separation. Surgical treatment was successful.

Differentiation of Antibodies against Selected Simbu Serogroup Viruses by a Glycoprotein Gc-Based Triplex ELISA.

The Simbu serogroup of orthobunyaviruses includes several pathogens of veterinary importance, among them Schmallenberg virus (SBV), Akabane virus (AKAV) and Shuni virus (SHUV). They infect predominantly ruminants and induce severe congenital malformation. In adult animals, the intra vitam diagnostics by direct virus detection is limited to only a few days due to a short-lived viremia. For surveillance purposes the testing for specific antibodies is a superior approach. However, the serological differentiation is hampered by a considerable extent of cross-reactivity, as viruses were assigned into this serogroup based on antigenic relatedness. Here, we established a glycoprotein Gc-based triplex enzyme-linked immunosorbent assay (ELISA) for the detection and differentiation of antibodies against SBV, AKAV, and SHUV. A total of 477 negative samples of various ruminant species, 238 samples positive for SBV-antibodies, 36 positive for AKAV-antibodies and 53 SHUV antibody-positive samples were tested in comparison to neutralization tests. For the newly developed ELISA, overall diagnostic specificities of 84.56%, 94.68% and 89.39% and sensitivities of 89.08%, 69.44% and 84.91% were calculated for SBV, AKAV and SHUV, respectively, with only slight effects of serological cross-reactivity on the diagnostic specificity. Thus, this test system could be used for serological screening in suspected populations or as additional tool during outbreak investigations.

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.

Multi-species ELISA for the detection of antibodies against SARS-CoV-2 in animals.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic with millions of infected humans and hundreds of thousands of fatalities. As the novel disease - referred to as COVID-19 - unfolded, occasional anthropozoonotic infections of animals by owners or caretakers were reported in dogs, felid species and farmed mink. Further species were shown to be susceptible under experimental conditions. The extent of natural infections of animals, however, is still largely unknown. Serological methods will be useful tools for tracing SARS-CoV-2 infections in animals once test systems are evaluated for use in different species. Here, we developed an indirect multi-species ELISA based on the receptor-binding domain (RBD) of SARS-CoV-2. The newly established ELISA was evaluated using 59 sera of infected or vaccinated animals, including ferrets, raccoon dogs, hamsters, rabbits, chickens, cattle and a cat, and a total of 220 antibody-negative sera of the same animal species. Overall, a diagnostic specificity of 100.0% and sensitivity of 98.31% were achieved, and the functionality with every species included in this study could be demonstrated. Hence, a versatile and reliable ELISA protocol was established that enables high-throughput antibody detection in a broad range of animal species, which may be used for outbreak investigations, to assess the seroprevalence in susceptible species or to screen for reservoir or intermediate hosts.

Susceptibility of Raccoon Dogs for Experimental SARS-CoV-2 Infection.

Raccoon dogs might have been intermediate hosts for severe acute respiratory syndrome-associated coronavirus in 2002-2004. We demonstrated susceptibility of raccoon dogs to severe acute respiratory syndrome coronavirus 2 infection and transmission to in-contact animals. Infected animals had no signs of illness. Virus replication and tissue lesions occurred in the nasal conchae.

Bunions: A review of management.

BACKGROUND: Bunions are a common complaint, particularly among older female patients. They are characterised by progressive deformity at the metatarsophalangeal joint, resulting in a painful dorsomedial prominence. This may cause difficulties with shoe wear and contribute to falls in the elderly. OBJECTIVE: The aim of this article is to discuss the aetiology, non-operative and operative management of bunions, as well as indications for referral. DISCUSSION: Initial treatment of symptomatic bunions should be non-operative. Accommodative footwear is important. There is evidence supporting the use of nonsteroidal anti-inflammatory drugs, orthotics, splints/braces and toe spacers. However, these may not provide long-term relief, and referral to an orthopaedic surgeon is recommended if the patient has a painful prominence, has exhausted non-operative treatment and is a suitable operative candidate. Cosmesis alone is not an indication for operative management. Smoking is a relative contraindication to surgery, and cessation is recommended. In paediatric or adolescent patients (juvenile bunion), surgery should be delayed until skeletal maturity.

A Genome-Wide CRISPR-Cas9 Screen Reveals the Requirement of Host Cell Sulfation for Schmallenberg Virus Infection.

Schmallenberg virus (SBV) is an insect-transmitted orthobunyavirus that can cause abortions and congenital malformations in the offspring of ruminants. Even though the two viral surface glycoproteins Gn and Gc are involved in host cell entry, the specific cellular receptors of SBV are currently unknown. Using genome-wide CRISPR-Cas9 forward screening, we identified 3'-phosphoadenosine 5'-phosphosulfate (PAPS) transporter 1 (PAPST1) as an essential factor for SBV infection. PAPST1 is a sulfotransferase involved in heparan sulfate proteoglycan synthesis encoded by the solute carrier family 35 member B2 gene (SLC35B2). SBV cell surface attachment and entry were largely reduced upon the knockout of SLC35B2, whereas the reconstitution of SLC35B2 in these cells fully restored their susceptibility to SBV infection. Furthermore, treatment of cells with heparinase diminished infection with SBV, confirming that heparan sulfate plays an important role in cell attachment and entry, although to various degrees, heparan sulfate was also found to be important to initiate infection by two other bunyaviruses, La Crosse virus and Rift Valley fever virus. Thus, PAPST1-triggered synthesis of cell surface heparan sulfate is required for the efficient replication of SBV and other bunyaviruses.IMPORTANCE SBV is a newly emerging orthobunyavirus (family Peribunyaviridae) that has spread rapidly across Europe since 2011, resulting in substantial economic losses in livestock farming. In this study, we performed unbiased genome-wide CRISPR-Cas9 screening and identified PAPST1, a sulfotransferase encoded by SLC35B2, as a host entry factor for SBV. Consistent with its role in the synthesis of heparan sulfate, we show that this activity is required for efficient infection by SBV. A comparable dependency on heparan sulfate was also observed for La Crosse virus and Rift Valley fever virus, highlighting the importance of heparan sulfate for host cell infection by bunyaviruses. Thus, the present work provides crucial insights into virus-host interactions of important animal and human pathogens.

Particulate multivalent presentation of the receptor binding domain induces protective immune responses against MERS-CoV.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a WHO priority pathogen for which vaccines are urgently needed. Using an immune-focusing approach, we created self-assembling particles multivalently displaying critical regions of the MERS-CoV spike protein ─fusion peptide, heptad repeat 2, and receptor binding domain (RBD) ─ and tested their immunogenicity and protective capacity in rabbits. Using a "plug-and-display" SpyTag/SpyCatcher system, we coupled RBD to lumazine synthase (LS) particles producing multimeric RBD-presenting particles (RBD-LS). RBD-LS vaccination induced antibody responses of high magnitude and quality (avidity, MERS-CoV neutralizing capacity, and mucosal immunity) with cross-clade neutralization. The antibody responses were associated with blocking viral replication and upper and lower respiratory tract protection against MERS-CoV infection in rabbits. This arrayed multivalent presentation of the viral RBD using the antigen-SpyTag/LS-SpyCatcher is a promising MERS-CoV vaccine candidate and this platform may be applied for the rapid development of vaccines against other emerging viruses such as SARS-CoV-2.

Multimeric single-domain antibody complexes protect against bunyavirus infections.

The World Health Organization has included three bunyaviruses posing an increasing threat to human health on the Blueprint list of viruses likely to cause major epidemics and for which no, or insufficient countermeasures exist. Here, we describe a broadly applicable strategy, based on llama-derived single-domain antibodies (VHHs), for the development of bunyavirus biotherapeutics. The method was validated using the zoonotic Rift Valley fever virus (RVFV) and Schmallenberg virus (SBV), an emerging pathogen of ruminants, as model pathogens. VHH building blocks were assembled into highly potent neutralizing complexes using bacterial superglue technology. The multimeric complexes were shown to reduce and prevent virus-induced morbidity and mortality in mice upon prophylactic administration. Bispecific molecules engineered to present two different VHHs fused to an Fc domain were further shown to be effective upon therapeutic administration. The presented VHH-based technology holds great promise for the development of bunyavirus antiviral therapies.

Orthobunyavirus spike architecture and recognition by neutralizing antibodies.

Orthobunyaviruses (OBVs) form a distinct genus of arthropod-borne bunyaviruses that can cause severe disease upon zoonotic transmission to humans. Antigenic drift or genome segment re-assortment have in the past resulted in new pathogenic OBVs, making them potential candidates for causing emerging zoonoses in the future. Low-resolution electron cryo-tomography studies have shown that OBV particles feature prominent trimeric spikes, but their molecular organization remained unknown. Here we report X-ray crystallography studies of four different OBVs showing that the spikes are formed by an N-terminal extension of the fusion glycoprotein Gc. Using Schmallenberg virus, a recently emerged OBV, we also show that the projecting spike is the major target of the neutralizing antibody response, and provide X-ray structures in complex with two protecting antibodies. We further show that immunization of mice with the spike domains elicits virtually sterilizing immunity, providing fundamental knowledge essential in the preparation for potential newly emerging OBV zoonoses.