3D imaging of SARS-CoV-2 infected hamster lungs by X-ray phase contrast tomography enables drug testing.

X-ray Phase Contrast Tomography (XPCT) based on wavefield propagation has been established as a high resolution three-dimensional (3D) imaging modality, suitable to reconstruct the intricate structure of soft tissues, and the corresponding pathological alterations. However, for biomedical research, more is needed than 3D visualisation and rendering of the cytoarchitecture in a few selected cases. First, the throughput needs to be increased to cover a statistically relevant number of samples. Second, the cytoarchitecture has to be quantified in terms of morphometric parameters, independent of visual impression. Third, dimensionality reduction and classification are required for identification of effects and interpretation of results. To address these challenges, we here design and implement a novel integrated and high throughput XPCT imaging and analysis workflow for 3D histology, pathohistology and drug testing. Our approach uses semi-automated data acquisition, reconstruction and statistical quantification. We demonstrate its capability for the example of lung pathohistology in Covid-19. Using a small animal model, different Covid-19 drug candidates are administered after infection and tested in view of restoration of the physiological cytoarchitecture, specifically the alveolar morphology. To this end, we then use morphometric parameter determination followed by a dimensionality reduction and classification based on optimal transport. This approach allows efficient discrimination between physiological and pathological lung structure, thereby providing quantitative insights into the pathological progression and partial recovery due to drug treatment. Finally, we stress that the XPCT image chain implemented here only used synchrotron radiation for validation, while the data used for analysis was recorded with laboratory µ CT radiation, more easily accessible for pre-clinical research.

Distribution and suitability of pulmonary surfactants as a vehicle for topically applied antibodies in healthy and SARS-CoV-2 infected rodent lungs.

The use of natural pulmonary surfactants (PS) as a drug delivery vehicle for biologics is a more recent therapeutic modality. Herein, we tested different contents of PS regarding their physicochemical properties under stress conditions. The PS content of 12.25 mg/ml (Formulation B) showed desired properties such as an isotonic osmolality ∼300 mOsm/kg and an acceptable viscosity of 8.61 cSt, being lower than in commercially available PS solutions. Formulation B passed the specifications of surface lowering capacities of >80 % total lung capacity and physiologically desired formulation properties were independent of the antibody used in the composition. The identified formulation showed the capability of significantly increasing the oxygen saturation in ex vivo isolated perfused rat lungs, compared to a control and up to 30 min post lavage. In the in vivo setting, we showed that intratracheal administration of a human mAB with and without pulmonary surfactant led to higher amounts of delivered antibody within the alveolar tissue compared to intravenous administration. The antibody with the PS formulation remained longer in the alveolar tissues than the antibody without the PS formulation. Further, SARS-CoV-2 infected Golden Syrian hamsters showed that the intranasally applied antibody reached the site of infection in the alveoli and could be detected in the alveolar region 24 h after the last administration. With this work, we demonstrated that pulmonary surfactants can be used as a pulmonary drug delivery mechanism for antibodies and may subsequently improve the antibody efficacy by increasing the residence time at the desired site of action in the alveolar tissue.

SARS-CoV and SARS-CoV -2 cross-reactive antibodies in domestic animals and wildlife in Nigeria suggest circulation of sarbecoviruses.

Anthropogenic exposure of domestic animals, as well as wildlife, can result in zoonotic transmission events with known and unknown pathogens including sarbecoviruses. During the COVID-19 pandemic, SARS-CoV-2 infections in animals, most likely resulting from spill-over from humans, have been documented worldwide. However, only limited information is available for Africa. The anthropozoonotic transmission from humans to animals, followed by further inter- and intraspecies propagation may contribute to viral evolution, and thereby subsequently alter the epidemiological patterns of transmission. To shed light on the possible role of domestic animals and wildlife in the ecology and epidemiology of sarbecoviruses in Nigeria, and to analyze the possible circulation of other, undiscovered, but potentially zoonotic sarbecoviruses in animals, we tested 504 serum samples from dogs, rabbits, bats, and pangolins collected between December 2020 and April 2022. The samples were analyzed using an indirect multi-species enzyme-linked immunosorbent assay (ELISA) based on the receptor binding domain (RBD) of SARS-CoV and SARS-CoV -2, respectively. ELISA reactive sera were further analyzed by highly specific virus neutralization test and indirect immunofluorescence assay for confirmation of the presence of antibodies. In this study, we found SARS-CoV reactive antibodies in 16 (11.5%) dogs, 7 (2.97%) rabbits, 2 (7.7%) pangolins and SARS-CoV-2 reactive antibodies in 20 (13.4%) dogs, 6 (2.5%) rabbits and 2 (7.7%) pangolins, respectively. Interestingly, 2 (2.3%) bat samples were positive only for SARS-CoV RBD reactive antibodies. These serological findings of SARS-CoV and/or SARS-CoV-2 infections in both domestic animals and wildlife indicates exposure to sarbecoviruses and requires further One Health-oriented research on the potential reservoir role that different species might play in the ecology and epidemiology of coronaviruses at the human-animal interface.

Spatiotemporal analysis of SARS-CoV-2 infection reveals an expansive wave of monocyte-derived macrophages associated with vascular damage and virus clearance in hamster lungs.

IMPORTANCE: We present the first study of the 3D kinetics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the early host response in a large lung volume using a combination of tissue imaging and transcriptomics. This approach allowed us to make a number of important findings: Spatially restricted antiviral response is shown, including the formation of monocytic macrophage clusters and upregulation of the major histocompatibility complex II in infected epithelial cells. The monocyte-derived macrophages are linked to SARS-CoV-2 clearance, and the appearance of these cells is associated with post-infection endothelial damage; thus, we shed light on the role of these cells in infected tissue. An early onset of tissue repair occurring simultaneously with inflammatory and necrotizing processes provides the basis for longer-term alterations in the lungs.

Annual (2023) taxonomic update of RNA-directed RNA polymerase-encoding negative-sense RNA viruses (realm Riboviria: kingdom Orthornavirae: phylum Negarnaviricota).

In April 2023, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by one new family, 14 new genera, and 140 new species. Two genera and 538 species were renamed. One species was moved, and four were abolished. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

SARS-CoV-2 induced changes in the glycosylation pattern in the respiratory tract of Golden Syrian hamsters.

Even after more than two years of intensive research, not all of the pathophysiological processes of Coronavirus Disease 2019 (COVID-19), induced by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, have been fully elucidated. The initial virus-host interaction at the respiratory epithelium plays a crucial role in the course and progression of the infection, and is highly dependent on the glycosylation pattern of the host cell and of the secreted mucins. Glycans are polysaccharides that can be attached to proteins and thereby add to their stability and functionality. Lectins are glycan-binding proteins that recognize specific glycan motifs, and lectin histochemistry is a suitable tool to visualize and examine glycosylation pattern changes in tissues. In this study we used lectins with different glycan-specificities for the visualization of glycosylation pattern changes in the respiratory tract of SARS-CoV-2 infected Golden Syrian hamsters. While some lectins (LEL, STL) enable the visualization of the damage to alveolar type 1 pneumocytes, other lectins, e.g., GSLI, visualized the loss and subsequent hyperplasia of type 2 pneumocytes. UEAI staining was co-localized with KI67, a proliferation marker. Double staining of lectins LEL, STL and WGA with specific immune cell markers (Iba1, CD68) showed co-localization and the dominant infiltration of monocyte-derived macrophages into infected alveolar tissue. The elucidation of the glycosylation pattern of the respiratory tract cells in uninfected and infected Golden Syrian hamsters revealed physiological and pathological aspects of the disease that may open new possibilities for therapeutic development.

Discrimination of Classical and Atypical BSE by a Distinct Immunohistochemical PrPSc Profile.

Bovine spongiform encephalopathy (BSE) belongs to the group of transmissible spongiform encephalopathies and is associated with the accumulation of a pathological isoform of the host-encoded glycoprotein, designated prion protein (PrPSc). Classical BSE (C-type) and two atypical BSE forms (L- and H-type) are known, and can be discriminated by biochemical characteristics. The goal of our study was to identify type-specific PrPSc profiles by using Immunohistochemistry. In our study, brain samples from 21 cattle, intracerebrally inoculated with C-, H-, and L-type BSE, were used. In addition, the corresponding samples from three orally C-type BSE infected animals were also included. From all animals, a lesion and PrPSc-profiles of six brain regions were determined. The lesion profile and the neuroanatomical distribution of PrPSc was highly consistent between the groups, but the immunohistochemical analysis revealed a distinct PrPSc profile for the different BSE-types, which included both the topographic and cellular pattern of PrPSc. This qualitative and quantitative analysis of PrPSc affected structures sheds new light into the pathogenesis of the different BSE types. Furthermore, immunohistochemical characterization is supported as an additional diagnostic tool in BSE surveillance programs, especially when only formalin-fixed tissue samples are available.

2022 taxonomic update of phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales.

In March 2022, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by two new families (bunyaviral Discoviridae and Tulasviridae), 41 new genera, and 98 new species. Three hundred forty-nine species were renamed and/or moved. The accidentally misspelled names of seven species were corrected. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

Baseline of Physiological Body Temperature and Hematological Parameters in Captive Rousettus aegyptiacus and Eidolon helvum Fruit Bats.

The discovery of bats as reservoir hosts for a number of highly pathogenic zoonotic agents has led to an increasing interest of infectious disease research in experimental studies with bats. Therefore, we established breeding colonies of Rousettus aegyptiacus and Eidolon helvum fruit bats, which both have been identified as reservoir hosts for relevant zoonotic disease agents, such as Marburg virus and Lagos bat virus. Since 2013, individuals of both species have been recruited to the Friedrich-Loeffler-Institut (FLI) from zoological gardens in Europe, to where these species had been introduced from the wild several decades ago. The aviaries have been designed according to national recommendations published by the Federal Ministry of Agriculture. Under these conditions, both species have been reproducing for years. To better understand the physiology of these animals, and to generate baseline knowledge for infection experiments, we monitored the body core temperatures of R. aegyptiacus bats in the aviaries, and found a circadian variation between 34°C and 41.5°C. We also determined the hematological parameters of both species, and detected specific differences between both bat species. For values of clinical chemistry, no correlation to age or sex was observed. However, species-specific differences were detected since ALT, BUN and CREA were found to be significantly higher in R. aegyptiacus and GLU and TP were significantly higher in E. helvum bats. A higher hematocrit, hemoglobin and red blood cell level was observed in subadult R. aegyptiacus, with hemoglobin and red blood cells also being significantly increased compared to E. helvum. Lymphocytes were found to be the dominant white blood cells in both species and are higher in female E. helvum. Neutrophil granulocytes were significantly higher in E. helvum bats. This underlines the necessity to define baseline profiles for each bat species prior to their use in experimental challenge.

Landscape and age dynamics of immune cells in the Egyptian rousette bat.

Bats harbor high-impact zoonotic viruses often in the absence of disease manifestation. This restriction and disease tolerance possibly rely on specific immunological features. In-depth molecular characterization of cellular immunity and imprinting of age on leukocyte compartments remained unexplored in bats. We employ single-cell RNA sequencing (scRNA-seq) and establish immunostaining panels to characterize the immune cell landscape in juvenile, subadult, and adult Egyptian rousette bats (ERBs). Transcriptomic and flow cytometry data reveal conserved subsets and substantial enrichments of CD79a+ B cells and CD11b+ T cells in juvenile animals, whereas neutrophils, CD206+ myeloid cells, and CD3+ T cells dominate as bats reach adulthood. Despite differing frequencies, phagocytosis of circulating and tissue-resident myeloid cells and proliferation of peripheral and splenic lymphocytes are analogous in juvenile and adult ERBs. We provide a comprehensive map of the immune landscape in ERBs and show age-imprinted resilience progression and find that variability in cellular immunity only partly recapitulates mammalian archetypes.

Compellingly high SARS-CoV-2 susceptibility of Golden Syrian hamsters suggests multiple zoonotic infections of pet hamsters during the COVID-19 pandemic.

Golden Syrian hamsters (Mesocricetus auratus) are used as a research model for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). Millions of Golden Syrian hamsters are also kept as pets in close contact to humans. To determine the minimum infective dose (MID) for assessing the zoonotic transmission risk, and to define the optimal infection dose for experimental studies, we orotracheally inoculated hamsters with SARS-CoV-2 doses from 1 * 105 to 1 * 10-4 tissue culture infectious dose 50 (TCID50). Body weight and virus shedding were monitored daily. 1 * 10-3 TCID50 was defined as the MID, and this was still sufficient to induce virus shedding at levels up to 102.75 TCID50/ml, equaling the estimated MID for humans. Virological and histological data revealed 1 * 102 TCID50 as the optimal dose for experimental infections. This compelling high susceptibility leading to productive infections in Golden Syrian hamsters must be considered as a potential source of SARS-CoV-2 infection for humans that come into close contact with pet hamsters.

3D Ex vivo tissue platforms to investigate the early phases of influenza a virus- and SARS-CoV-2-induced respiratory diseases.

Pandemic outbreaks of viruses such as influenza virus or SARS-CoV-2 are associated with high morbidity and mortality and thus pose a massive threat to global health and economics. Physiologically relevant models are needed to study the viral life cycle, describe the pathophysiological consequences of viral infection, and explore possible drug targets and treatment options. While simple cell culture-based models do not reflect the tissue environment and systemic responses, animal models are linked with huge direct and indirect costs and ethical questions. Ex vivo platforms based on tissue explants have been introduced as suitable platforms to bridge the gap between cell culture and animal models. We established a murine lung tissue explant platform for two respiratory viruses, influenza A virus (IAV) and SARS-CoV-2. We observed efficient viral replication, associated with the release of inflammatory cytokines and the induction of an antiviral interferon response, comparable to ex vivo infection in human lung explants. Endolysosomal entry could be confirmed as a potential host target for pharmacological intervention, and the potential repurposing potentials of fluoxetine and interferons for host-directed therapy previously seen in vitro could be recapitulated in the ex vivo model.

Serological Hendra Virus Diagnostics Using an Indirect ELISA-Based DIVA Approach with Recombinant Hendra G and N Proteins.

Since the identification of Hendra virus (HeV) infections in horses in Australia in 1994, more than 80 outbreaks in horses have been reported, and four out of seven spillover infections in humans had a fatal outcome. With the availability of a subunit vaccine based on the HeV-Glycoprotein (HeV-G), there is a need to serologically Differentiate the Infected from the Vaccinated Animals (DIVA). We developed an indirect ELISA using HeV-G expressed in Leishmania tarentolae and HeV-Nucleoprotein (HeV-N) expressed in recombinant baculovirus-infected insect cells as antigens. During evaluation, we tested panels of sera from naïve, vaccinated and infected horses that either originated from a Hendra-virus free region, or had been pre-tested in validated diagnostic tests. Our data confirm the reliability of this approach, as HeV-N-specific antibodies were only detected in sera from infected horses, while HeV-G-specific antibodies were detected in infected and vaccinated horses with a high level of specificity and sensitivity. Given the excellent correlation of data obtained for German and Australian HeV-negative horses, we assume that this test can be applied for the testing of horse serum samples from a variety of geographical regions.

Strain Typing of Classical Scrapie and Bovine Spongiform Encephalopathy (BSE) by Using Ovine PrP (ARQ/ARQ) Overexpressing Transgenic Mice.

Transmissible spongiform encephalopathies (TSE), caused by abnormal prion protein (PrPSc), affect many species. The most classical scrapie isolates harbor mixtures of strains in different proportions. While the characterization of isolates has evolved from using wild-type mice to transgenic mice, no standardization is established yet. Here, we investigated the incubation period, lesion profile and PrPSc profile induced by well-defined sheep scrapie isolates, bovine spongiform encephalopathy (BSE) and ovine BSE after intracerebral inoculation into two lines of ovine PrP (both ARQ/ARQ) overexpressing transgenic mice (Tgshp IX and Tgshp XI). All isolates were transmitted to both mouse models with an attack rate of almost 100%, but genotype-dependent differences became obvious between the ARQ and VRQ isolates. Surprisingly, BSE induced a much longer incubation period in Tgshp XI compared to Tgshp IX. In contrast to the histopathological lesion profiles, the immunohistochemical PrPSc profiles revealed discriminating patterns in certain brain regions in both models with clear differentiation of both BSE isolates from scrapie. These data provide the basis for the use of Tgshp IX and XI mice in the characterization of TSE isolates. Furthermore, the results enable a deeper appreciation of TSE strain diversity using ovine PrP overexpressing transgenic mice as a biological prion strain typing approach.

Inhibitors of dihydroorotate dehydrogenase cooperate with molnupiravir and N4-hydroxycytidine to suppress SARS-CoV-2 replication.

The nucleoside analog N4-hydroxycytidine (NHC) is the active metabolite of the prodrug molnupiravir, which has been approved for the treatment of COVID-19. SARS-CoV-2 incorporates NHC into its RNA, resulting in defective virus genomes. Likewise, inhibitors of dihydroorotate dehydrogenase (DHODH) reduce virus yield upon infection, by suppressing the cellular synthesis of pyrimidines. Here, we show that NHC and DHODH inhibitors strongly synergize in the inhibition of SARS-CoV-2 replication in vitro. We propose that the lack of available pyrimidine nucleotides upon DHODH inhibition increases the incorporation of NHC into nascent viral RNA. This concept is supported by the rescue of virus replication upon addition of pyrimidine nucleosides to the media. DHODH inhibitors increased the antiviral efficiency of molnupiravir not only in organoids of human lung, but also in Syrian Gold hamsters and in K18-hACE2 mice. Combining molnupiravir with DHODH inhibitors may thus improve available therapy options for COVID-19.

A multiplex assay for the detection of antibodies to relevant swine pathogens in serum.

Livestock industry supports the livelihood of around 1.3 billion people in the world, with swine industry contributing with 30% of total livestock production worldwide. To maintain and guarantee this production, a pivotal point according to the OIE is addressing potential biohazards. To control them, permanent sero-surveillance is crucial to achieve more focused veterinary public health intervention and prevention strategies, to break the chains of transmission, and to enable fast responses against outbreaks. Within this context, multiplex assays are powerful tools with the potential to simplify surveillance programs, since they reduce time, labour, and variability within analysis. In the present work, we developed a multiplex bead-based assay for the detection of specific antibodies to six relevant pathogens affecting swine: ASFV, CSFV, PRRSV, SIV, TB and HEV. The most immunogenic target antigen of each pathogen was selected as the target protein to coat different microsphere regions in order to develop this multiplex assay. A total of 1544 serum samples from experimental infections as well as field samples were included in the analysis. The 6-plex assay exhibited credible diagnostic parameters with sensitivities ranging from 87.0% to 97.5% and specificities ranging from 87.9% to 100.0%, demonstrating it to be a potential high throughput tool for surveillance of infectious diseases in swine.

Selection and stability validation of reference gene candidates for transcriptional analysis in Rousettus aegyptiacus.

Bats are the only mammals capable of powered flight and their body temperature can reach up to 42 °C during flight. Additionally, bats display robust type I IFN interferon (IFN-I) responses and some species constitutively express IFN-α. Reference genes with stable expression under temperature oscillations and IFN-I release are therefore critical for normalization of quantitative reverse-transcription polymerase chain reaction (qRT-PCR) data in bats. The expression stability of reference genes in Rousettus aegyptiacus remains elusive, although this species is frequently used in the infection research. We selected ACTB, EEF1A1, GAPDH and PGK1 as candidate reference genes and evaluated their expression stability in various tissues and cells from this model bat species upon IFN-I treatment at 35 °C, 37 °C and 40 °C by qRT-PCR. We employed two statistical algorithms, BestKeeper and NormFinder, and found that EEF1A1 exhibited the highest expression stability under all tested conditions. ACTB and GAPDH displayed unstable expression upon temperature change and IFN-I treatment, respectively. By normalizing to EEF1A1, we uncovered that GAPDH expression was significantly induced by IFN-I in R. aegyptiacus. Our study identifies EEF1A1 as the most suitable reference gene for qRT-PCR studies upon temperature changes and IFN-I treatment and unveils the induction of GAPDH expression by IFN-I in R. aegyptiacus. These findings are pertinent to other bat species and may be relevant for non-volant mammals that show physiological fluctuations of core body temperature.

Prion Infectivity and PrPBSE in the Peripheral and Central Nervous System of Cattle 8 Months Post Oral BSE Challenge.

After oral exposure of cattle with classical bovine spongiform encephalopathy (C-BSE), the infectious agent ascends from the gut to the central nervous system (CNS) primarily via the autonomic nervous system. However, the timeline of this progression has thus far remained widely undetermined. Previous studies were focused on later time points after oral exposure of animals that were already 4 to 6 months old when challenged. In contrast, in this present study, we have orally inoculated 4 to 6 weeks old unweaned calves with high doses of BSE to identify any possible BSE infectivity and/or PrPBSE in peripheral nervous tissues during the first eight months post-inoculation (mpi). For the detection of BSE infectivity, we used a bovine PrP transgenic mouse bioassay, while PrPBSE depositions were analyzed by immunohistochemistry (IHC) and by protein misfolding cyclic amplification (PMCA). We were able to show that as early as 8 mpi the thoracic spinal cord as well as the parasympathetic nodal ganglion of these animals contained PrPBSE and BSE infectivity. This shows that the centripetal prion spread starts early after challenge at least in this age group, which represents an essential piece of information for the risk assessments for food, feed, and pharmaceutical products produced from young calves.

Circulation of Nor98 Atypical Scrapie in Portuguese Sheep Confirmed by Transmission of Isolates into Transgenic Ovine ARQ-PrP Mice.

Portugal was among the first European countries to report cases of Atypical Scrapie (ASc), the dominant form of Transmissible Spongiform Encephalopathy (TSE) in Portuguese small ruminants. Although the diagnostic phenotypes observed in Portuguese ASc cases seem identical to those described for Nor98, unequivocal identification requires TSE strain-typing using murine bioassays. In this regard, we initiated characterization of ASc isolates from sheep either homozygous for the ARQ genotype or the classical scrapie-resistant ARR genotype. Isolates from such genotypes were transmitted to TgshpXI mice expressing ovine PrPARQ. Mean incubation periods were 414 ± 58 and 483 ± 107 days in mice inoculated with AL141RQ/AF141RQ and AL141RR/AL141RR sheep isolates, respectively. Both isolates produced lesion profiles similar to French ASc Nor98 'discordant cases', where vacuolation was observed in the hippocampus (G6), cerebral cortex at the thalamus (G8) level, cerebellar white matter (W1) and cerebral peduncles (W3). Immunohistochemical PrPSc deposition was observed in the hippocampus, cerebellar cortex, cerebellar white matter and cerebral peduncles in the form of aggregates and fine granules. These findings were consistent with previously reported cases of ASc Nor98 transmitted to transgenic TgshpXI mice, confirming that the ASc strain present in Portuguese sheep corresponds to ASc Nor98.