Detection of SARS-CoV-2 RNA in a Zoo-Kept Red Fox (Vulpes vulpes).

Many different animal species are susceptible to SARS-CoV-2, including a few Canidae (domestic dog and raccoon dog). So far, only experimental evidence is available concerning SARS-CoV-2 infections in red foxes (Vulpes vulpes). This is the first report of SARS-CoV-2 RNA detection in a sample from a red fox. The RT-qPCR-positive fox was zoo-kept together with another fox and two bears in the Swiss Canton of Zurich. Combined material from a conjunctival and nasal swab collected for canine distemper virus diagnostics tested positive for SARS-CoV-2 RNA with Ct values of 36.9 (E gene assay) and 35.7 (RdRp gene assay). The sample was analysed for SARS-CoV-2 within a research project testing residual routine diagnostic samples from different animal species submitted between spring 2020 and December 2022 to improve knowledge on SARS-CoV-2 infections within different animal species and investigate their potential role in a One Health context. Within this project, 246 samples from 153 different animals from Swiss zoos and other wild animal species all tested SARS-CoV-2 RT-qPCR and/or serologically negative so far, except for the reported fox. The source of SARS-CoV-2 in the fox is unknown. The fox disappeared within the naturally structured enclosure, and the cadaver was not found. No further control measures were undertaken.

Ancestral allele of DNA polymerase gamma modifies antiviral tolerance.

Mitochondria are critical modulators of antiviral tolerance through the release of mitochondrial RNA and DNA (mtDNA and mtRNA) fragments into the cytoplasm after infection, activating virus sensors and type-I interferon (IFN-I) response1-4. The relevance of these mechanisms for mitochondrial diseases remains understudied. Here we investigated mitochondrial recessive ataxia syndrome (MIRAS), which is caused by a common European founder mutation in DNA polymerase gamma (POLG1)5. Patients homozygous for the MIRAS variant p.W748S show exceptionally variable ages of onset and symptoms5, indicating that unknown modifying factors contribute to disease manifestation. We report that the mtDNA replicase POLG1 has a role in antiviral defence mechanisms to double-stranded DNA and positive-strand RNA virus infections (HSV-1, TBEV and SARS-CoV-2), and its p.W748S variant dampens innate immune responses. Our patient and knock-in mouse data show that p.W748S compromises mtDNA replisome stability, causing mtDNA depletion, aggravated by virus infection. Low mtDNA and mtRNA release into the cytoplasm and a slow IFN response in MIRAS offer viruses an early replicative advantage, leading to an augmented pro-inflammatory response, a subacute loss of GABAergic neurons and liver inflammation and necrosis. A population databank of around 300,000 Finnish individuals6 demonstrates enrichment of immunodeficient traits in carriers of the POLG1 p.W748S mutation. Our evidence suggests that POLG1 defects compromise antiviral tolerance, triggering epilepsy and liver disease. The finding has important implications for the mitochondrial disease spectrum, including epilepsy, ataxia and parkinsonism.

Concerns about the histological assessment in a mouse model of human celiac disease.

Commentary on: Abadie V et al. IL­15, gluten and HLA­DQ8 drive tissue destruction in coeliac disease. Nature. 2020; 578: 600­604

The effect of molnupiravir and nirmatrelvir on SARS-CoV-2 genome diversity in infected and immune suppressed mice.

Objectives: Immunocompromised individuals are susceptible to severe COVID-19 and potentially contribute to the emergence of variants with altered pathogenicity due to persistent infection. This study investigated the impact of immunosuppression on SARS-CoV-2 infection in k18-hACE2 mice and the effectiveness of antiviral treatments in this context. Methods: Mice were immunosuppressed using cyclophosphamide and infected with a B lineage of SARS-CoV-2. Molnupiravir and nirmatrelvir, alone and in combination, were administered and viral load and viral sequence diversity was assessed. Results: Treatment of infected but immune compromised mice with both compounds either singly or in combination resulted in decreased viral loads and pathological changes compared to untreated animals. Treatment also abrogated infection of neuronal tissue. However, no consistent changes in the viral consensus sequence were observed, except for the emergence of the S:H655Y mutation. Molnupiravir, but not nirmatrelvir or immunosuppression alone, increased the transition/transversion (Ts/Tv) ratio, representative of A>G and C>U mutations and this increase was not altered by the co-administration of nirmatrelvir with molnupiravir.Notably, immunosuppression itself did not appear to promote the emergence of mutational characteristic of variants of concern (VOCs). Conclusions: Further investigations are warranted to fully understand the role of immunocompromised individuals in VOC development and to inform optimised public health strategies. It is more likely that immunodeficiency promotes viral persistence but does not necessarily lead to substantial consensus-level changes in the absence of antiviral selection pressure. Consistent with mechanisms of action, molnupiravir showed a stronger mutagenic effect than nirmatrelvir in this model.

Nanobody engineering for SARS-CoV-2 neutralization and detection.

In response to the ongoing COVID-19 pandemic, the quest for coronavirus inhibitors has inspired research on a variety of small proteins beyond conventional antibodies, including robust single-domain antibody fragments, i.e., "nanobodies." Here, we explore the potential of nanobody engineering in the development of antivirals and diagnostic tools. Through fusion of nanobody domains that target distinct binding sites, we engineered multimodular nanobody constructs that neutralize wild-type SARS-CoV-2 and the Alpha and Delta variants at high potency, with IC50 values as low as 50 pM. Despite simultaneous binding to distinct epitopes, Beta and Omicron variants were more resistant to neutralization by the multimodular nanobodies, which highlights the importance of accounting for antigenic drift in the design of biologics. To further explore the applications of nanobody engineering in outbreak management, we present an assay based on fusions of nanobodies with fragments of NanoLuc luciferase that can detect sub-nanomolar quantities of the SARS-CoV-2 spike protein in a single step. Our work showcases the potential of nanobody engineering to combat emerging infectious diseases. IMPORTANCE: Nanobodies, small protein binders derived from the camelid antibody, are highly potent inhibitors of respiratory viruses that offer several advantages over conventional antibodies as candidates for specific therapies, including high stability and low production costs. In this work, we leverage the unique properties of nanobodies and apply them as building blocks for new therapeutic and diagnostic tools. We report ultra-potent SARS-CoV-2 inhibition by engineered nanobodies comprising multiple modules in structure-guided combinations and develop nanobodies that carry signal molecules, allowing rapid detection of the SARS-CoV-2 spike protein. Our results highlight the potential of engineered nanobodies in the development of effective countermeasures, both therapeutic and diagnostic, to manage outbreaks of emerging viruses.

Hypoxia inducible factors inhibit respiratory syncytial virus infection by modulation of nucleolin expression.

Respiratory syncytial virus (RSV) is a global healthcare problem, causing respiratory illness in young children and elderly individuals. Our knowledge of the host pathways that define susceptibility to infection and disease severity are limited. Hypoxia inducible factors (HIFs) define metabolic responses to low oxygen and regulate inflammatory responses in the lower respiratory tract. We demonstrate a role for HIFs to suppress RSV entry and RNA replication. We show that hypoxia and HIF prolyl-hydroxylase inhibitors reduce the expression of the RSV entry receptor nucleolin and inhibit viral cell-cell fusion. We identify a HIF regulated microRNA, miR-494, that regulates nucleolin expression. In RSV-infected mice, treatment with the clinically approved HIF prolyl-hydroxylase inhibitor, Daprodustat, reduced the level of infectious virus and infiltrating monocytes and neutrophils in the lung. This study highlights a role for HIF-signalling to limit multiple aspects of RSV infection and associated inflammation and informs future therapeutic approaches for this respiratory pathogen.

Malignant catarrhal fever in a goat: manifestation of virus-induced erythema multiforme.

Malignant catarrhal fever (MCF), caused by ovine herpesvirus 2 (OvHV2; Orthoherpesviridae, Macavirus ovinegamma2), has sheep as natural hosts. OvHV2 is an important macavirus globally that induces fatal disease in dead-end hosts. Goats, which can be infected subclinically with OvHV2, rarely develop MCF. A 28-wk-old female goat was presented with fever and multifocal crusty skin lesions. Histologic examination of a skin biopsy suggested erythema multiforme (EM), with pyoderma and dermal vasculitis. The doe was euthanized and subjected to postmortem and histologic examination. MCF was suspected and PCR assays for macaviruses were performed, followed by immunohistochemistry (IHC) for OvHV2 latency-associated nuclear antigen (oLANA), RNA in situ hybridization for Ov2.5 mRNA, and IHC to characterize infiltrating leukocytes. The main postmortem finding was severe multifocal ulcerative dermatitis with macrophage- and T cell-mediated arteritis. The latter was also detected in kidney, spleen, heart, and intestinal wall. The PCR assay detected high loads of OvHV2 in tissues. OvHV2 oLANA and Ov2.5 mRNA were expressed within the lesions in leukocytes, endothelial cells, fibroblasts, and/or keratinocytes. Our case confirms that MCF can initially manifest clinically as a skin disease in goats and as EM with confirmed viral etiology.

Verminous pneumonia in European hedgehogs (Erinaceus europaeus).

The European hedgehog (Erinaceus europaeus) is a common wildlife species in European countries. Populations are declining due to anthropogenic factors and natural diseases. Verminous pneumonia has been observed as a frequent infectious disease in hedgehogs submitted for diagnostic postmortem examination. This prompted the present in-depth investigation on the lungs of 27 necropsied hedgehogs with confirmed lungworm infections, with or without antiparasitic treatment prior to death. The histological and/or parasitic (fecal samples) examination identified Capillaria aerophila infection in most animals (82%). The parasites were found free in the airway lumen and/or within the airway epithelium, from the larynx to bronchioles. Embedded worms and eggs were associated with epithelial hyperplasia or metaplasia, and long-term inflammation. More than half of the animals (59%) carried Crenosoma striatum, and 41% had a coinfection. C striatum adults were predominantly found free in the lumen of bronchi and bronchioles, and larvae were occasionally seen in granulomas in the pulmonary interstitium, the liver, and the intestine. Independent of the parasite species, a lymphoplasmacytic peribronchitis and, less frequently, interstitial infiltration of eosinophils, neutrophils, and macrophages as well as pneumocyte type II hyperplasia was seen. Interestingly, the extent of pneumonia was not correlated with age, respiratory clinical signs, antiparasitic treatment, or single or coinfection. Verminous pneumonia appeared to be the cause of death in over 25% of the animals, indicating that these parasites not only coexist with hedgehogs but can also be a primary pathogen in this species.

A Multiplex RT-PCR Method for the Detection of Reptarenavirus Infection.

Reptarenaviruses cause Boid Inclusion Body Disease (BIBD), a fatal disease of boid snakes with an economic and ecological impact, as it affects both captive and wild constrictor snakes. The clinical picture of BIBD is highly variable but often only limited. Intracytoplasmic inclusion bodies (IB), which develop in most cell types including blood cells, are the pathognomonic hallmark of BIBD; their detection represents the diagnostic gold standard of the disease. However, IBs are not consistently present in clinically healthy reptarenavirus carriers, which can, if undetected, lead to and maintain the spread of the disease within and between snake populations. Sensitive viral detection tools are required for screening and control purposes; however, the genetic diversity of reptarenaviruses hampers the reverse transcription (RT) PCR-based diagnostics. Here, we describe a multiplex RT-PCR approach for the molecular diagnosis of reptarenavirus infection in blood samples. The method allows the detection of a wide range of reptarenaviruses with the detection limit reaching 40 copies per microliter of blood. Using 245 blood samples with a reference RT-PCR result, we show that the technique performs as well as the segment-specific RT-PCRs in our earlier studies. It can identify virus carriers and serve to limit reptarenavirus spreading in captive snake collections.

Chemoprophylactic Assessment of Combined Intranasal SARS-CoV-2 Polymerase and Exonuclease Inhibition in Syrian Golden Hamsters.

Pibrentasvir (PIB) has been demonstrated to block exonuclease activity of the SARS-CoV-2 polymerase, protecting favipiravir (FVP) and remdesivir (RDV) from post-incorporation excision and eliciting antiviral synergy in vitro. The present study investigated the chemoprophylactic efficacy of PIB, FVP, RDV, FVP with PIB, or RDV with PIB dosed intranasally twice a day, using a Syrian golden hamster contact transmission model. Compared to the saline control, viral RNA levels were significantly lower in throat swabs in FVP (day 7), RDV (day 3, 5, 7), and RDV+PIB (day 3, 5) treatment groups. Similarly, findings were evident for nasal turbinate after PIB and RDV treatment, and lungs after PIB, FVP, and FVP+PIB treatment at day 7. Lung viral RNA levels after RDV and RDV+PIB treatment were only detectable in two animals per group, but the overall difference was not statistically significant. In situ examination of the lungs confirmed SARS-CoV-2 infection in all animals, except for one in each of the RDV and RDV+PIB treatment groups, which tested negative in all virus detection approaches. Overall, prevention of transmission was observed in most animals treated with RDV, while other agents reduced the viral load following contact transmission. No benefit of combining FVP or RDV with PIB was observed.

The Stereotypic Response of the Pulmonary Vasculature to Respiratory Viral Infections: Findings in Mouse Models of SARS-CoV-2, Influenza A and Gammaherpesvirus Infections.

The respiratory system is the main target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 19 (COVID-19) where acute respiratory distress syndrome is considered the leading cause of death. Changes in pulmonary blood vessels, among which an endothelialitis/endotheliitis has been particularly emphasized, have been suggested to play a central role in the development of acute lung injury. Similar vascular changes are also observed in animal models of COVID-19. The present study aimed to determine whether the latter are specific for SARS-CoV-2 infection, investigating the vascular response in the lungs of mice infected with SARS-CoV-2 and other respiratory viruses (influenza A and murine gammaherpesvirus) by in situ approaches (histology, immunohistology, morphometry) combined with RNA sequencing and bioinformatic analysis. Non-selective recruitment of monocytes and T and B cells from larger muscular veins and arteries was observed with all viruses, matched by a comparable transcriptional response. There was no evidence of endothelial cell infection in any of the models. Both the morphological investigation and the transcriptomics approach support the interpretation that the lung vasculature in mice mounts a stereotypic response to alveolar and respiratory epithelial damage. This may have implications for the treatment and management of respiratory disease in humans.

Evaluation of Nafamostat as Chemoprophylaxis for SARS-CoV-2 Infection in Hamsters.

The successful development of a chemoprophylaxis against SARS-CoV-2 could provide a tool for infection prevention that is implementable alongside vaccination programmes. Nafamostat is a serine protease inhibitor that inhibits SARS-CoV-2 entry in vitro, but it has not been characterised for chemoprophylaxis in animal models. Clinically, nafamostat is limited to intravenous delivery and has an extremely short plasma half-life. This study sought to determine whether intranasal dosing of nafamostat at 5 mg/kg twice daily was able to prevent the airborne transmission of SARS-CoV-2 from infected to uninfected Syrian Golden hamsters. SARS-CoV-2 RNA was detectable in the throat swabs of the water-treated control group 4 days after cohabitation with a SARS-CoV-2 inoculated hamster. However, throat swabs from the intranasal nafamostat-treated hamsters remained SARS-CoV-2 RNA negative for the full 4 days of cohabitation. Significantly lower SARS-CoV-2 RNA concentrations were seen in the nasal turbinates of the nafamostat-treated group compared to the control (p = 0.001). A plaque assay quantified a significantly lower concentration of infectious SARS-CoV-2 in the lungs of the nafamostat-treated group compared to the control (p = 0.035). When taken collectively with the pathological changes observed in the lungs and nasal mucosa, these data are strongly supportive of the utility of intranasally delivered nafamostat for the prevention of SARS-CoV-2 infection.

Complete Protection from SARS-CoV-2 Lung Infection in Mice Through Combined Intranasal Delivery of PIKfyve Kinase and TMPRSS2 Protease Inhibitors.

Emerging variants of concern of SARS-CoV-2 can significantly reduce the prophylactic and therapeutic efficacy of vaccines and neutralizing antibodies due to mutations in the viral genome. Targeting cell host factors required for infection provides a complementary strategy to overcome this problem since the host genome is less susceptible to variation during the life span of infection. The enzymatic activities of the endosomal PIKfyve phosphoinositide kinase and the serine protease TMPRSS2 are essential to meditate infection in two complementary viral entry pathways. Simultaneous inhibition in cultured cells of their enzymatic activities with the small molecule inhibitors apilimod dimesylate and nafamostat mesylate synergistically prevent viral entry and infection of native SARS-CoV-2 and vesicular stomatitis virus (VSV)-SARS-CoV-2 chimeras expressing the SARS-CoV-2 surface spike (S) protein and of variants of concern. We now report prophylactic prevention of lung infection in mice intranasally infected with SARS-CoV-2 beta by combined intranasal delivery of very low doses of apilimod dimesylate and nafamostat mesylate, in a formulation that is stable for over 3 months at room temperature. Administration of these drugs up to 6 hours post infection did not inhibit infection of the lungs but substantially reduced death of infected airway epithelial cells. The efficiency and simplicity of formulation of the drug combination suggests its suitability as prophylactic or therapeutic treatment against SARS-CoV-2 infection in households, point of care facilities, and under conditions where refrigeration would not be readily available.

Amino acids 1811-1960 of myosin heavy chain 9 is involved in murine gammaherpesvirus 68 infection.

Myosin heavy chain 9 (MYH9) has been identified as a crucial factor in gammaherpesvirus infection. Murine gammaherpesvirus 68 (MHV-68) was used as an appropriate viral model for investigating gammaherpesviruses in vivo and developing antiviral treatments. However, the roles of MYH9 in MHV-68 infection have not been documented. In the study, the relationship between the expression of MYH9 and MHV-68 infection and MYH9 as the antiviral target were analyzed. The results revealed that MYH9 was enriched on the cell surface and co-localized with MHV-68 upon viral infection. Knocking down MYH9 with siRNA or using the specific inhibitor of MYH9 activity, Blebbistatin, resulted in the decreasing of MHV-68 infection. Furthermore, polyclonal antibodies against MYH9 reduced infection by approximately 74% at a dose of 100 µg/ml. The study determined that MYH9 contributes to MHV-68 infection by interacting with viral glycoprotein 150 (gp150) in the BHK-21 cell membrane. The specific region of MYH9, amino acids 1811-1960 (C-150), was identified as the key domain involved in the interaction with MHV-68 gp150 and was found to inhibit MHV-68 infection. Moreover, C-150 was also shown to decrease HSV-1 infection in Vero cells by approximately 73%. Both C-150 and Blebbistatin were found to inhibit MHV-68 replication and reduce histopathological lesions in vivo in C57BL/6J mice. Taken together, these findings suggested that MYH9 is crucial for MHV-68 infection through its interaction with viral gp150 and that C-150 may be a promising antiviral target for inhibiting MHV-68 infection in vitro and in vivo.

Autosomal recessive hyposegmentation of granulocytes in Australian Shepherd Dogs indicates a role for LMBR1L in myeloid leukocytes.

Pelger-Huët anomaly (PHA) in humans is an autosomal dominant hematological phenotype without major clinical consequences. PHA involves a characteristic hyposegmentation of granulocytes (HG). Human PHA is caused by heterozygous loss of function variants in the LBR gene encoding lamin receptor B. Bi-allelic variants and complete deficiency of LBR cause the much more severe Greenberg skeletal dysplasia which is lethal in utero and characterized by massive skeletal malformation and gross fetal hydrops. HG phenotypes have also been described in domestic animals and homology to human PHA has been claimed in the literature. We studied a litter of Australian Shepherd Dogs with four stillborn puppies in which both parents had an HG phenotype. Linkage analysis excluded LBR as responsible gene for the stillborn puppies. We then investigated the HG phenotype in Australian Shepherd Dogs independently of the prenatal lethality. Genome-wide association mapped the HG locus to chromosome 27 and established an autosomal recessive mode of inheritance. Whole genome sequencing identified a splice site variant in LMBR1L, c.191+1G>A, as most likely causal variant for the HG phenotype. The mutant allele abrogates the expression of the longer X2 isoform but does not affect transcripts encoding the shorter X1 isoform of the LMBR1L protein. The homozygous mutant LMBR1L genotype associated with HG is common in Australian Shepherd Dogs and was found in 39 of 300 genotyped dogs (13%). Our results point to a previously unsuspected function of LMBR1L in the myeloid lineage of leukocytes.

Reptarenavirus S Segment RNA Levels Correlate with the Presence of Inclusion Bodies and the Number of L Segments in Snakes with Reptarenavirus Infection-Lessons Learned from a Large Breeding Colony.

Reptarenaviruses cause boid inclusion body disease (BIBD), a fatal disease particularly impacting captive boa constrictor collections. The development of cytoplasmic inclusion bodies (IBs) comprising reptarenavirus nucleoprotein (NP) in many cell types of affected snakes is characteristic of BIBD. However, snakes can harbor reptarenaviruses without showing IBs, hence representing carriers and a potential source of transmission. The RNA genome of reptarenaviruses comprises a small (S) and a large (L) segment, and the snakes with BIBD commonly carry a swarm of reptarenavirus segments. To design sensitive and reliable tools for the diagnosis of reptarenavirus infection in snake colonies, we used metatranscriptomics to determine the reptarenavirus segments present in a large boa constrictor breeding colony. The analysis identified one reptarenavirus S segment and three L segments in the colony. The sequence data served to design real-time reverse transcription-PCR (RT-PCR) targeting the found S segment. This allowed us to identify all infected animals and to quantify the S segment RNA levels, which we found to correlate with the presence of IBs. We further found a positive correlation between the number of L segments and the S segment RNA level, which could suggest that L segment excess also contributes to the IB formation. Information on cohousing of the snakes showed a clear association of reptarenavirus infection with cohousing in general and cohousing with infected animals. Information on breeding and offspring confirmed that vertical transmission occurred. Furthermore, our data suggest that some animals might be able to clear the infection or at least exhibit transient or intermittent viremia. IMPORTANCE Boid inclusion body disease (BIBD) is caused by reptarenavirus infection, and while reptarenavirus nucleoprotein is the main component of the inclusion bodies (IBs) characteristic of BIBD, not all reptarenavirus-infected snakes demonstrate IBs in their cells. Identification of infected individuals is critical for controlling the spread of the disease; however, the genetic divergence of reptarenaviruses complicates reverse transcription-PCR (RT-PCR)-based diagnostics. Here, we tested a next-generation-sequencing-based approach to establish a tailored "colony-specific" set of diagnostic tools for the detection of reptarenavirus small (S) and large (L) genome segments. With this approach, we could demonstrate that an S-segment-specific RT-PCR is highly effective in identifying the infected individuals. We further found the S segment RNA level to positively correlate with the presence of IBs and the number of L segments, which could direct future studies to identify the BIBD pathogenetic mechanisms.

Intranasal administration of adenoviral vaccines expressing SARS-CoV-2 spike protein improves vaccine immunity in mouse models.

The ongoing SARS-CoV-2 pandemic is controlled but not halted by public health measures and mass vaccination strategies which have exclusively relied on intramuscular vaccines. Intranasal vaccines can prime or recruit to the respiratory epithelium mucosal immune cells capable of preventing infection. Here we report a comprehensive series of studies on this concept using various mouse models, including HLA class II-humanized transgenic strains. We found that a single intranasal (i.n.) dose of serotype-5 adenoviral vectors expressing either the receptor binding domain (Ad5-RBD) or the complete ectodomain (Ad5-S) of the SARS-CoV-2 spike protein was effective in inducing i) serum and bronchoalveolar lavage (BAL) anti-spike IgA and IgG, ii) robust SARS-CoV-2-neutralizing activity in the serum and BAL, iii) rigorous spike-directed T helper 1 cell/cytotoxic T cell immunity, and iv) protection of mice from a challenge with the SARS-CoV-2 beta variant. Intramuscular (i.m.) Ad5-RBD or Ad5-S administration did not induce serum or BAL IgA, and resulted in lower neutralizing titers in the serum. Moreover, prior immunity induced by an intramuscular mRNA vaccine could be potently enhanced and modulated towards a mucosal IgA response by an i.n. Ad5-S booster. Notably, Ad5 DNA was found in the liver or spleen after i.m. but not i.n. administration, indicating a lack of systemic spread of the vaccine vector, which has been associated with a risk of thrombotic thrombocytopenia. Unlike in otherwise genetically identical HLA-DQ6 mice, in HLA-DQ8 mice Ad5-RBD vaccine was inferior to Ad5-S, suggesting that the RBD fragment does not contain a sufficient collection of helper-T cell epitopes to constitute an optimal vaccine antigen. Our data add to previous promising preclinical results on intranasal SARS-CoV-2 vaccination and support the potential of this approach to elicit mucosal immunity for preventing transmission of SARS-CoV-2.

Intranasal trimeric sherpabody inhibits SARS-CoV-2 including recent immunoevasive Omicron subvariants.

The emergence of increasingly immunoevasive SARS-CoV-2 variants emphasizes the need for prophylactic strategies to complement vaccination in fighting the COVID-19 pandemic. Intranasal administration of neutralizing antibodies has shown encouraging protective potential but there remains a need for SARS-CoV-2 blocking agents that are less vulnerable to mutational viral variation and more economical to produce in large scale. Here we describe TriSb92, a highly manufacturable and stable trimeric antibody-mimetic sherpabody targeted against a conserved region of the viral spike glycoprotein. TriSb92 potently neutralizes SARS-CoV-2, including the latest Omicron variants like BF.7, XBB, and BQ.1.1. In female Balb/c mice intranasal administration of just 5 or 50 micrograms of TriSb92 as early as 8 h before but also 4 h after SARS-CoV-2 challenge can protect from infection. Cryo-EM and biochemical studies reveal triggering of a conformational shift in the spike trimer as the inhibitory mechanism of TriSb92. The potency and robust biochemical properties of TriSb92 together with its resistance against viral sequence evolution suggest that TriSb92 could be useful as a nasal spray for protecting susceptible individuals from SARS-CoV-2 infection.

Effect of Cumulative Ionizing Radiation on Flexural Strength, Flexural Modulus, and Elasticity Modulus of Dentin in Unerupted Human Third Molars.

Purpose: This in vitro study aimed to investigate the changes in mechanical properties in dentin of third molars after radiation therapy using variable doses and frequencies. Methods and Materials: Rectangular cross sectioned dentin hemisections (N = 60, n = 15 per group; >7 × 4 × 1.2 mm) were prepared using extracted third molars. After cleansing and storage in artificial saliva, random distribution was performed to 2 irradiation settings, namely AB or CD (A, 30 single doses of irradiation [2 Gy each] for 6 weeks; B, control group of A; C, 3 single doses of irradiation [9 Gy each]; and D, control group of C). Various parameters (fracture strength/maximal force, flexural strength, and elasticity modulus) were assessed using a universal Testing Machine (ZwickRoell). The effect of irradiation on dentin morphology was evaluated by histology, scanning electron microscopy, and immunohistochemistry. Statistical analysis was performed using 2-way analysis of variance and paired and unpaired t tests at a significance level of 5%. Results: Significance could be found considering the maximal force applied to failure when the irradiated groups were compared with their control groups (A/B, P < .0001; C/D, P = .008). Flexural strength was significantly higher in the irradiated group A compared with control group B (P < .001) and for the irradiated groups A and C (P = .022) compared with each other. Cumulative radiation with low irradiation doses (30 single doses; 2 Gy) and single irradiation with high doses (3 single doses; 9 Gy) make the tooth substance more prone to fracture, lowering the maximal force. The flexural strength decreases when cumulative irradiation is applied, but not after single irradiation. The elasticity modulus showed no alteration after irradiation treatment. Conclusions: Irradiation therapy affects the prospective adhesion of dentin and the bond strength of future restorations, potentially leading to an increased risk of tooth fracture and retention loss in dental reconstructions.

Epididymectomy as a novel surgical procedure; application in the domestic cat.

Feline overpopulation raises issues concerning health, ecology, economy, and ethics. Procedures to limit overpopulation should carefully address animal welfare, efficiency, costs, and feasibility. Vasectomy in unowned cats is suggested as preferable to standard neutering as it maintains male sexual behaviour which may induce ovulation and pseudopregnancy in intact females and may prevent immigration of other males. Vasectomy is not performed routinely because it is fastidious, time consuming and requires more material than standard neutering. We describe epididymectomy as an alternative. In a first experiment, we analysed semen, testosterone, behaviour and pain in six experimental cats before and after epididymectomy, and after castration two months later. Excised tissues were analysed histologically. Testosterone concentrations did not differ significantly between intact and epididymectomised animals but were significantly different after castration. Sexual behaviour and testicular spermatogenesis persisted after epididymectomy, but with a marked drop in the semen count after 7 days. The Glasgow pain scores did not differ significantly after epididymectomy and castration. In a subsequent experiment, 20 privately owned cats were epididymectomised and castrated immediately afterwards, to analyse the learning curve and perioperative complications. The time required for an epididymectomy was significantly shorter than for castration. The study confirms that epididymectomy is quicker and less invasive than castration, it is associated with minimal risks and post-operative pain while easy to learn and inexpensive. Further field studies are required to test its efficiency for feline feral population control or in other species such as in bears, lions or deer, where infertility is required and castration not wanted.