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Supplementary Table from Differential Survival and Therapy Benefit of Patients with Breast Cancer Are Characterized by Distinct Epithelial and Immune Cell Microenvironments

Supplementary Data from Differential Survival and Therapy Benefit of Patients with Breast Cancer Are Characterized by Distinct Epithelial and Immune Cell Microenvironments

Purpose:Extensive work in preclinical models has shown that microenvironmental cells influence many aspects of cancer cell behavior, including metastatic potential and their sensitivity to therapeutics. In the human setting, this behavior is mainly correlated with the presence of immune cells. Here, in addition to T cells, B cells, macrophages, and mast cells, we identified the relevance of nonimmune cell types for breast cancer survival and therapy benefit, including fibroblasts, myoepithelial cells, muscle cells, endothelial cells, and seven distinct epithelial cell types.Experimental Design:Using single-cell sequencing data, we generated reference profiles for all these cell types. We used these reference profiles in deconvolution algorithms to optimally detangle the cellular composition of more than 3,500 primary breast tumors of patients that were enrolled in the SCAN-B and MATADOR clinical trials, and for which bulk mRNA sequencing data were available.Results:This large data set enables us to identify and subsequently validate the cellular composition of microenvironments that distinguish differential survival and treatment benefit for different treatment regimens in patients with primary breast cancer. In addition to immune cells, we have identified that survival and therapy benefit are characterized by various contributions of distinct epithelial cell types.Conclusions:From our study, we conclude that differential survival and therapy benefit of patients with breast cancer are characterized by distinct microenvironments that include specific populations of immune and epithelial cells.

The BRCA1 tumor suppressor gene encodes a multidomain protein for which several functions have been described. These include a key role in homologous recombination repair (HRR) of DNA double-strand breaks, which is shared with two other high-risk hereditary breast cancer suppressors, BRCA2 and PALB2. Although both BRCA1 and BRCA2 interact with PALB2, BRCA1 missense variants affecting its PALB2-interacting coiled-coil domain are considered variants of uncertain clinical significance (VUS). Using genetically engineered mice, we show here that a BRCA1 coiled-coil domain VUS, Brca1 p.L1363P, disrupts the interaction with PALB2 and leads to embryonic lethality. Brca1 p.L1363P led to a similar acceleration in the development of Trp53-deficient mammary tumors as Brca1 loss, but the tumors showed distinct histopathologic features, with more stable DNA copy number profiles in Brca1 p.L1363P tumors. Nevertheless, Brca1 p.L1363P mammary tumors were HRR incompetent and responsive to cisplatin and PARP inhibition. Overall, these results provide the first direct evidence that a BRCA1 missense variant outside of the RING and BRCT domains increases the risk of breast cancer.Significance:These findings reveal the importance of a patient-derived BRCA1 coiled-coil domain sequence variant in embryonic development, mammary tumor suppression, and therapy response.See related commentary by Mishra et al., p. 6080

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Supplementary Figure from Differential Survival and Therapy Benefit of Patients with Breast Cancer Are Characterized by Distinct Epithelial and Immune Cell Microenvironments

The heterogeneity of colorectal cancer (CRC) contributes to substantial differences in patient response to standard therapies. The consensus molecular subtypes (CMS) of CRC is the most widely-used gene expression-based classification and has contributed to a better understanding of disease heterogeneity and prognosis. Nevertheless, CMS intratumoral heterogeneity restricts its clinical application, stressing the necessity of further characterizing the composition and architecture of CRC. Here, we used Spatial Transcriptomics (ST) in combination with single-cell RNA sequencing (scRNA-seq) to decipher the spatially resolved cellular and molecular composition of CRC. In addition to mapping the intratumoral heterogeneity of CMS and their microenvironment, we identified cell communication events in the tumor-stroma interface of CMS2 carcinomas. This includes tumor growth-inhibiting as well as -activating signatures, such as the potential regulation of the ETV4 transcriptional activity by DCN or the PLAU-PLAUR ligand-receptor interaction. Our data show the power of ST to bring the CMS-based classification of CRC to another level and thereby gain useful molecular insights for personalized therapy.

The BRCA1 tumor suppressor gene encodes a multidomain protein for which several functions have been described. These include a key role in homologous recombination repair (HRR) of DNA double-strand breaks, which is shared with two other high-risk hereditary breast cancer suppressors, BRCA2 and PALB2. Although both BRCA1 and BRCA2 interact with PALB2, BRCA1 missense variants affecting its PALB2-interacting coiled-coil domain are considered variants of uncertain clinical significance (VUS). Using genetically engineered mice, we show here that a BRCA1 coiled-coil domain VUS, Brca1 p.L1363P, disrupts the interaction with PALB2 and leads to embryonic lethality. Brca1 p.L1363P led to a similar acceleration in the development of Trp53-deficient mammary tumors as Brca1 loss, but the tumors showed distinct histopathologic features, with more stable DNA copy number profiles in Brca1 p.L1363P tumors. Nevertheless, Brca1 p.L1363P mammary tumors were HRR incompetent and responsive to cisplatin and PARP inhibition. Overall, these results provide the first direct evidence that a BRCA1 missense variant outside of the RING and BRCT domains increases the risk of breast cancer. Significance: These findings reveal the importance of a patient-derived BRCA1 coiled-coil domain sequence variant in embryonic development, mammary tumor suppression, and therapy response. See related commentary by Mishra et al., p. 6080

(1) Background: an increasing number of breast cancer patients develop lethal brain metastases (BM). The complete removal of these tumors by surgery becomes complicated when cells infiltrate into the brain parenchyma. However, little is known about the nature of these invading cells in breast cancer brain metastasis (BCBM). (2) Methods: we use intravital microscopy through a cranial window to study the behavior of invading cells in a mouse model of BCBM. (3) Results: we demonstrate that BCBM cells that escape from the metastatic mass and infiltrate into brain parenchyma undergo epithelial-to-mesenchymal transition (EMT). Moreover, cells undergoing EMT revert to an epithelial state when growing tumor masses in the brain. Lastly, through multiplex immunohistochemistry, we confirm the presence of these infiltrative cells in EMT in patient samples. (4) Conclusions: together, our data identify the critical role of EMT in the invasive behavior of BCBM, which warrants further consideration to target those cells when treating BCBM.

The basement membrane (BM) around tumor lobes forms a barrier to prevent cancer cells from invading the surrounding tissue. Although myoepithelial cells are key producers of the healthy mammary epithelium BM, they are nearly absent in mammary tumors. To study the origin and dynamics of the BM, we developed and imaged a laminin beta1-Dendra2 mouse model. We show that the turnover of laminin beta1 is faster in the BMs that surround the tumor lobes than in the BMs that surround the healthy epithelium. Moreover, we find that epithelial cancer cells and tumor-infiltrating endothelial cells synthesize laminin beta1 and that this production is temporarily and locally heterogeneous, leading to local discontinuity of the BM laminin beta1. Collectively, our data draw a new paradigm for tumor BM turnover in which the disassembly happens at a constant rate, and a local misbalance of compensating production leads to reduction or even complete disappearance of the BM.

Purpose: Extensive work in preclinical models has shown that microenvironmental cells influence many aspects of cancer cell behavior, including metastatic potential and their sensitivity to therapeutics. In the human setting, this behavior is mainly correlated with the presence of immune cells. Here, in addition to T cells, B cells, macrophages, and mast cells, we identified the relevance of nonimmune cell types for breast cancer survival and therapy benefit, including fibroblasts, myoepithelial cells, muscle cells, endothelial cells, and seven distinct epithelial cell types. Experimental Design: Using single-cell sequencing data, we generated reference profiles for all these cell types. We used these reference profiles in deconvolution algorithms to optimally detangle the cellular composition of more than 3,500 primary breast tumors of patients that were enrolled in the SCAN-B and MATADOR clinical trials, and for which bulk mRNA sequencing data were available. Results: This large data set enables us to identify and subsequently validate the cellular composition of microenvironments that distinguish differential survival and treatment benefit for different treatment regimens in patients with primary breast cancer. In addition to immune cells, we have identified that survival and therapy benefit are characterized by various contributions of distinct epithelial cell types. Conclusions: From our study, we conclude that differential survival and therapy benefit of patients with breast cancer are characterized by distinct microenvironments that include specific populations of immune and epithelial cells.

An increasing number of breast cancer patients develop brain metastases (BM). Standard-of-care treatments are largely inefficient, and breast cancer brain metastasis (BCBM) patients are considered untreatable. Immunotherapies are not successfully employed in BCBM, in part because breast cancer is a "cold" tumor and also because the brain tissue has a unique immune landscape. Here, we generate and characterize immunocompetent models of BCBM derived from PyMT and Neu mammary tumors to test how harnessing the pro-senescence properties of doxorubicin can be used to prime the specific immune BCBM microenvironment. We reveal that BCBM senescent cells, induced by doxorubicin, trigger the recruitment of PD1-expressing T cells to the brain. Importantly, we demonstrate that induction of senescence with doxorubicin improves the efficacy of immunotherapy with anti-PD1 in BCBM in a CD8 T cell-dependent manner, thereby providing an optimized strategy to introduce immune-based treatments in this lethal disease. In addition, our BCBM models can be used for pre-clinical testing of other therapeutic strategies in the future.

Thyrotropin receptor (TSHR), sodium iodide symporter (NIS), pendrin, and thyroid peroxidase (TPO) are essential for the uptake of iodine by follicular thyroid cells. The aim of this study was to establish immunohistochemistry (IHC) protocols for TSHR, NIS, pendrin, and TPO in canine tissues and characterize their expression in organoids derived from canine follicular cell thyroid carcinoma (FTC) and in the respective primary tumors. This constitutes a fundamental step to establish organoids as a model to study the uptake of iodine in canine FTC. Commercially available antibodies directed against human proteins were selected. Antibody specificity was confirmed by western blot using lysates of the HTori-3 human thyroid cell line and healthy canine thyroid gland. IHC was validated using HTori-3 cells and a set of canine normal tissues including healthy thyroid gland. The expression of TSHR, NIS, pendrin, and TPO was evaluated in 3 organoid lines derived from FTC and respective primary tumors. All 4 antibodies produced specific bands by western blot and cytoplasmic labeling in follicular cells by IHC in both human HTori-3 cells and canine thyroid gland. NIS also showed basolateral membrane immunolabeling in follicular cells. All 4 proteins were highly expressed in organoids derived from FTC. The expression was similar or higher compared to the primary tumors. The results of this study characterize organoids derived from canine FTC as a suitable in vitro model to investigate iodine uptake, opening new research possibilities in the field of canine thyroid cancer therapy.

We report necropsy findings in a captive 60-year-old female greater flamingo (Phoenicopterus roseus) that died suddenly following rupture of a pulmonary artery aneurysm. Histologically, there was focally extensive, intramural granulomatous inflammation with intralesional fungal hyphae, and adjacent severe mixed-cell inflammation and acute haemorrhage at the rupture site. Aspergillus fumigatus was identified as the aetiological agent following DNA PCR amplification and sequencing from paraffin-embedded pulmonary artery tissue sections. The most likely explanation is that this lesion was a consequence of haematogenous spread, secondary to mycotic pneumonia or aerosacculitis, following aspiration of A. fumigatus conidiospores. However, no further fungal-related lesions were observed on gross or histopathological examination.

GM1-gangliosidosis is caused by a reduced activity of β-galactosidase (Glb1), resulting in intralysosomal accumulations of GM1. The aim of this study was to reveal the pathogenic mechanisms of GM1-gangliosidosis in a new Glb1 knockout mouse model. Glb1−/− mice were analyzed clinically, histologically, immunohistochemically, electrophysiologically and biochemically. Morphological lesions in the central nervous system were already observed in two-month-old mice, whereas functional deficits, including ataxia and tremor, did not start before 3.5-months of age. This was most likely due to a reduced membrane resistance as a compensatory mechanism. Swollen neurons exhibited intralysosomal storage of lipids extending into axons and amyloid precursor protein positive spheroids. Additionally, axons showed a higher kinesin and lower dynein immunoreactivity compared to wildtype controls. Glb1−/− mice also demonstrated loss of phosphorylated neurofilament positive axons and a mild increase in non-phosphorylated neurofilament positive axons. Moreover, marked astrogliosis and microgliosis were found, but no demyelination. In addition to the main storage material GM1, GA1, sphingomyelin, phosphatidylcholine and phosphatidylserine were elevated in the brain. In summary, the current Glb1−/− mice exhibit a so far undescribed axonopathy and a reduced membrane resistance to compensate the functional effects of structural changes. They can be used for detailed examinations of axon–glial interactions and therapy trials of lysosomal storage diseases.

GM1-gangliosidosis is caused by a reduced activity of β-galactosidase (Glb1), resulting in intralysosomal accumulations of GM1. The aim of this study was to reveal the pathogenic mechanisms of GM1-gangliosidosis in a new Glb1 knockout mouse model. Glb1−/− mice were analyzed clinically, histologically, immunohistochemically, electrophysiologically and biochemically. Morphological lesions in the central nervous system were already observed in two-month-old mice, whereas functional deficits, including ataxia and tremor, did not start before 3.5-months of age. This was most likely due to a reduced membrane resistance as a compensatory mechanism. Swollen neurons exhibited intralysosomal storage of lipids extending into axons and amyloid precursor protein positive spheroids. Additionally, axons showed a higher kinesin and lower dynein immunoreactivity compared to wildtype controls. Glb1−/− mice also demonstrated loss of phosphorylated neurofilament positive axons and a mild increase in non-phosphorylated neurofilament positive axons. Moreover, marked astrogliosis and microgliosis were found, but no demyelination. In addition to the main storage material GM1, GA1, sphingomyelin, phosphatidylcholine and phosphatidylserine were elevated in the brain. In summary, the current Glb1−/− mice exhibit a so far undescribed axonopathy and a reduced membrane resistance to compensate the functional effects of structural changes. They can be used for detailed examinations of axon–glial interactions and therapy trials of lysosomal storage diseases.

As sufficient extracellular arginine is crucial for T cell function, depletion of extracellular arginine by elevated arginase 1 (Arg1) activity has emerged as a hallmark immunosuppressive mechanism. However, the potential cell-autonomous roles of arginases in T cells have remained unexplored. Here, we show that the arginase isoform expressed by T cells, the mitochondrial Arg2, is a cell-intrinsic regulator of CD8(+) T cell activity. Both germline Arg2 deletion and adoptive transfer of Arg2(–/–) CD8(+) T cells significantly reduced tumor growth in preclinical cancer models by enhancing CD8(+) T cell activation, effector function, and persistence. Transcriptomic, proteomic, and high-dimensional flow cytometry characterization revealed a CD8(+) T cell–intrinsic role of Arg2 in modulating T cell activation, antitumor cytoxicity, and memory formation, independently of extracellular arginine availability. Furthermore, specific deletion of Arg2 in CD8(+) T cells strongly synergized with PD-1 blockade for the control of tumor growth and animal survival. These observations, coupled with the finding that pharmacologic arginase inhibition accelerates activation of ex vivo human T cells, unveil Arg2 as a potentially new therapeutic target for T cell–based cancer immunotherapies.

A missense variant in the autophagy-related ATG4D-gene has been associated with a progressive degenerative neurological disease in Lagotto Romagnolo (LR) dogs. In addition to neural lesions, affected dogs show an extraneural histopathological phenotype characterized by severe cytoplasmic vacuolization, a finding not previously linked with disturbed autophagy in animals. Here we aimed at testing the hypothesis that autophagy is altered in the affected dogs, at reporting the histopathology of extraneural tissues and at excluding lysosomal storage diseases. Basal and starvation-induced autophagy were monitored by Western blotting and immunofluorescence of microtubule associated protein 1A/B light chain3 (LC3) in fibroblasts from 2 affected dogs. The extraneural findings of 9 euthanized LRs and skin biopsies from 4 living affected LRs were examined by light microscopy, electron microscopy, and immunohistochemistry (IHC), using antibodies against autophagosomal membranes (LC3), autophagic cargo (p62), and lysosomal membranes (LAMP2). Biochemical screening of urine and fibroblasts of 2 affected dogs was performed. Under basal conditions, the affected fibroblasts contained significantly more LC3-II and LC3-positive vesicles than did the controls. Morphologically, several cells, including serous secretory epithelium, endothelial cells, pericytes, plasma cells, and macrophages, contained cytoplasmic vacuoles with an ultrastructure resembling enlarged amphisomes, endosomes, or multivesicular bodies. IHC showed strong membranous LAMP2 positivity only in sweat glands. The results show that basal but not induced autophagy is altered in affected fibroblasts. The ultrastructure of affected cells is compatible with altered autophagic and endo-lysosomal vesicular traffic. The findings in this spontaneous disease provide insight into possible tissue-specific roles of basal autophagy.

The manner in which laboratory rodents are housed is driven by economics (minimal use of space and resources), ergonomics (ease of handling and visibility of animals), hygiene, and standardization (reduction of variation). This has resulted in housing conditions that lack sensory and motor stimulation and restrict the expression of species-typical behavior. In mice, such housing conditions have been associated with indicators of impaired welfare, including abnormal repetitive behavior (stereotypies, compulsive behavior), enhanced anxiety and stress reactivity, and thermal stress. However, due to concerns that more complex environmental conditions might increase variation in experimental results, there has been considerable resistance to the implementation of environmental enrichment beyond the provision of nesting material. Here, using 96 C57BL/6 and SWISS female mice, respectively, we systematically varied environmental enrichment across four levels spanning the range of common enrichment strategies: (1) bedding alone; (2) bedding + nesting material; (3) deeper bedding + nesting material + shelter + increased vertical space; and (4) semi-naturalistic conditions, including weekly changes of enrichment items. We studied how these different forms of environmental enrichment affected measures of animal welfare, including home-cage behavior (time-budget and stereotypic behavior), anxiety (open field behavior, elevated plus-maze behavior), growth (food and water intake, body mass), stress physiology (glucocorticoid metabolites in fecal boluses and adrenal mass), brain function (recurrent perseveration in a two-choice guessing task) and emotional valence (judgment bias). Our results highlight the difficulty in making general recommendations across common strains of mice and for selecting enrichment strategies within specific strains. Overall, the greatest benefit was observed in animals housed with the greatest degree of enrichment. Thus, in the super-enriched housing condition, stereotypic behavior, behavioral measures of anxiety, growth and stress physiology varied in a manner consistent with improved animal welfare compared to the other housing conditions with less enrichment. Similar to other studies, we found no evidence, in the measures assessed here, that environmental enrichment increased variation in experimental results.

In situ hybridization (ISH) is a technique to determine potential correlations between viruses and lesions. The aim of the study was to compare ISH techniques for the detection of various viruses in different tissues. Tested RNA viruses include atypical porcine pestivirus (APPV) in the cerebellum of pigs, equine and bovine hepacivirus (EqHV, BovHepV) in the liver of horses and cattle, respectively, and Schmallenberg virus (SBV) in the cerebrum of goats. Examined DNA viruses comprise canine bocavirus 2 (CBoV-2) in the intestine of dogs, porcine bocavirus (PBoV) in the spinal cord of pigs and porcine circovirus 2 (PCV-2) in cerebrum, lymph node, and lung of pigs. ISH with self-designed digoxigenin-labelled RNA probes revealed a positive signal for SBV, CBoV-2, and PCV-2, whereas it was lacking for APPV, BovHepV, EqHV, and PBoV. Commercially produced digoxigenin-labelled DNA probes detected CBoV-2 and PCV-2, but failed to detect PBoV. ISH with a commercially available fluorescent ISH (FISH)-RNA probe mix identified nucleic acids of all tested viruses. The detection rate and the cell-associated positive area using the FISH-RNA probe mix was highest compared to the results using other probes and protocols, representing a major benefit of this method. Nevertheless, there are differences in costs and procedure time.

To the Editor: In 2013, a 6-week-old female piglet kept in a flatdeck cage had coughing, growth retardation, and diarrhea and was taken to a local veterinarian in Hannover, Germany; the piglet was euthanized. After necropsy at the University of Veterinary Medicine in Hannover, histologic investigation found interstitial pneumonia; a mild, multifocal, lymphohistiocytic panencephalitis that affected the cerebrum and cerebellum, including brain stem and medulla oblongata; and a mild, multifocal, lymphohistiocytic panmyelitis. Results from screening for typical neurotropic viruses (classical swine fever virus, suid herpesvirus 1, rabies virus, teschovirus, porcine enterovirus 8, 9, and 10) were negative; Mycoplasma hyorhinis was detected by multiplex PCR (Institute of Virology, University of Veterinary Medicine Hannover) within the lung and pulmonary lymph nodes. Cerebral tissue from the pig was processed for viral metagenomics by random RNA and DNA virus screening and next-generation sequencing (NGS) with the 454 sequencing platform (GS Junior; Roche, Basel, Switzerland), as described (1), and 21,359 reads were obtained. Analysis by using blastn and blastx (2) showed 10 reads had >97% nt identity with porcine bocavirus (PBoV) KU14. No other viral sequences were detected. By using primers based on sequence data of the PBoV, partially overlapping PCR amplicons were obtained to confirm and extend the NGS data of the isolate, which was named PBoV S1142/13 (1; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KU311698","term_id":"1040287233","term_text":"KU311698"}}KU311698). A total of 2,176 nt of PBoV S1142/13 were obtained, consisting of the partial nucleoprotein (NP) 1 and the nearly complete viral protein (VP) 1 gene. By using MAFFT version 7 (http://mafft.cbrc.jp/alignment/server/), we aligned the nearly complete VP1 gene of PBoV S1142/13 with various closely related members of the genus Bocaparvovirus and built a maximum-likelihood tree by using the general time reversible plus invariable sites plus gamma distribution method, as determined by jModelTest 2.0 (3) and default parameters in MEGA6.06 (4). Results confirmed that PBoV S1142/13 was most closely related to PBoV KU14 (Figure, panel A). The partial genome of PBoV S1142/13 differed at 8 nt positions from PBoV KU14, resulting in 99.6% nt identity. Of these nucleotide differences, 4 resulted in an amino acid difference, including position 2733 (T→C on the basis of PBoV KU14 as a reference genome), which is part of the NP1 stopcodon of PBoV KU14. These results indicate that the stopcodon was located 39 nt farther downstream than for PBoV KU14. The other 3 aa differences were present in the VP1 protein; each of these differences was within the same group of amino acids as those detected in PBoV KU14. For further substantiation of a potential cause-effect relationship of histologic (Figure, panel B) and NGS results, we performed fluorescent in situ hybridization (FISH) on formalin-fixed, paraffin-embedded central nervous system (CNS) sections of the diseased animal and of a control pig with no CNS lesions. We used an RNA probe specific for the obtained NP1 and VP1 sequences covering 1,153 nt (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s protocol, with minor variations (ViewRNA ISH Tissue 1-Plex Assay Kit and ViewRNA Chromogenic Signal Amplification Kit, Affymetrix). A probe specific for porcine ubiquitin (Sus scrofa ubiquitin C; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"XM_005657305","term_id":"545868586","term_text":"XM_005657305"}}XM_005657305; nt 2–890) served as a positive control. The spinal cord of the diseased pig showed diffuse intracytoplasmic and intranuclear PBoV-specific signals within scattered neurons adjacent to the histologically detected inflammatory lesions (Figure, panel C). The negative control and the nonprobe incubation lacked PBoV-specific signals. The porcine ubiquitin probe provided a strong intracellular and extracellular staining within the CNS of both pigs. Figure Phylogenetic analysis and staining of porcine bocavirus (PBoV) from the spinal cord of a diseased pig, Hannover, Germany. A) Phylogenetic relationship of PBoV isolate S1142/13 (bold) with other bocaviruses. The nucleotide sequence of the nearly complete ... PBoV (genus Bocaparvovirus, family Parvoviridae) was first described in 2009 as porcine boca-like virus in pigs in Sweden with postweaning multisystemic wasting syndrome (5). PBoV is usually involved in respiratory and intestinal diseases in pigs (5) but has not been detected in the CNS. In the pig in our study, the lack of detection of other viral sequences by using NGS indicates the potential role of PBoV as a pathogen that triggers encephalomyelitis. FISH substantiated the NGS results and revealed neuronal intracytoplasmic and intranuclear PBoV-specific signals adjacent to the lesion, indicating intraneuronal transcription and replication (6). Nevertheless, a potential synergistic effect of M. hyorhinis on the PBoV pathogenesis cannot be ruled out. Similarly, co-infection of M. hyorhinis and porcine circovirus type 2 has been associated with enhanced inflammatory lesions in the lungs of pigs (7). The CNS tropism of PBoV S1142/13 could result from various factors, including specific amino acid changes that enable the virus to pass the blood–brain barrier and infect neurons. Additional studies are necessary to elucidate a possible role of the amino acid differences between PBoV S1142/13 and PBoV KU14 in the tropism of these viruses. Human bocavirus has recently been found in the cerebrospinal fluid of patients having encephalitis (8), and related human parvovirus 4 (9) and human parvovirus B19 (10) have been reported in human encephalitis. The correlation of PBoV-specific signals by using FISH for histologic detection of encephalomyelitis assigns PBoV a potential role in provoking CNS lesions. PBoV should be considered as a cause of encephalomyelitis but needs further investigation.

Hepatitis C virus (HCV) displays a restricted host species tropism and only humans and chimpanzees are susceptible to infection. A robust immunocompetent animal model is still lacking, hampering mechanistic analysis of virus pathogenesis, immune control, and prophylactic vaccine development. The closest homolog of HCV is the equine nonprimate hepacivirus (NPHV), which shares similar features with HCV and thus represents an animal model to study hepacivirus infections in their natural hosts. We aimed to dissect equine immune responses after experimental NPHV infection and conducted challenge experiments to investigate immune protection against secondary NPHV infections. Horses were i.v. injected with NPHV containing plasma. Flow cytometric analysis was used to monitor immune cell frequencies and activation status. All infected horses became viremic after 1 or 2 wk and viremia could be detected in two horses for several weeks followed by a delayed seroconversion and viral clearance. Histopathological examinations of liver biopsies revealed mild, periportally accentuated infiltrations of lymphocytes, macrophages, and plasma cells with some horses displaying subclinical signs of hepatitis. Following viral challenge, an activation of equine immune responses was observed. Importantly, after a primary NPHV infection, horses were protected against rechallenge with the homologous as well as a distinct isolate with only minute amounts of circulating virus being detectable.

Highlights • An outbreak of fatal enteritis occurred in a dog litter. • Major known causes of enteritis of young dogs were excluded. • A novel canine bocavirus 2 strain was detected by random PCR and NGS. • CaBoV-2 was detected in the intestinal tract and lymphoid tissue by ISH. • No additional cases were identified by a small retrospective analysis., Bocaviruses are small non-enveloped viruses with a linear ssDNA genome, that belong to the genus Bocaparvovirus of the subfamiliy Parvovirinae. Bocavirus infections are associated with a wide spectrum of disease in humans and various mammalian species. Here we describe a fatal enteritis associated with infection with a novel strain of canine bocavirus 2 (CaBoV-2), that occurred in a litter of German wirehaired pointers. Necropsy performed on three puppies revealed an enteritis reminiscent of canine parvovirus associated enteritis, accompanied with signs of lymphocytolytic disease in bone marrow, spleen, lymph nodes and thymus. While other major causes of enteritis of young dogs, including canine parvovirus, were excluded, by random PCR in combination with next-generation sequencing, a novel CaBoV-2 strain was detected. Phylogenetic analysis of the genome of this novel canine bocavirus strain indicated that this virus was indeed most closely related to group 2 canine bocaviruses. Infection with canine bocavirus was confirmed by in situ hybridization, which revealed the presence of CaBoV-2 nucleic acid in the intestinal tract and lymphoid tissues of the dogs. In a small-scale retrospective analysis concerning the role of CaBoV-2 no additional cases were identified. The findings of this study provide novel insights into the pathogenicity of canine bocaviruses.

Bunyaviruses have evolved a variety of strategies to counteract the antiviral defence systems of mammalian cells. Here we show that the NSs protein of Schmallenberg virus (SBV) induces the degradation of the RPB1 subunit of RNA polymerase II and consequently inhibits global cellular protein synthesis and the antiviral response. In addition, we show that the SBV NSs protein enhances apoptosis in vitro and possibly in vivo, suggesting that this protein could be involved in SBV pathogenesis in different ways.

Schmallenberg virus (SBV), an arbovirus within the family Bunyaviridae, represents a ruminant pathogen that has caused epidemic abortion and birth of malformed or stillborn animals in many European countries since August 2011. Histological and immunohistochemical analysis of peripheral tissues of SBV-infected animals, including lymphoid tissues, endocrine organs and tissues of the gastrointestinal, urogenital and respiratory system, were analyzed in order to elucidate the occurrence of SBV-associated changes and the presence of viral antigens and RNA. Twenty calves and 12 lambs as well as age-matched controls were included in this study. Significant muscular hypoplasia with fatty replacement was noted in affected calves and lambs. In addition, hepatocellular degeneration with lymphohistiocytic inflammation, interstitial fibrosis and biliary hyperplasia was detected in calves. All animals lacked SBV-positive cells in the peripheral organs. These observations resemble those found in Akabane virus- and Cache Valley virus-infected animals and support the occurrence of few residual lesions in peripheral organs following SBV infection.

A 2-year-old female boxer dog was presented with a vaginal serosanguineous discharge not associated with oestrus. There was a friable mass occupying the upper caudal part of the vagina. Cytological and histological examination revealed a monomorphic population of neoplastic round cells consistent with canine transmissible venereal tumour (TVT). In addition, Leishmania spp. amastigotes were found within the neoplastic tissue. In order to characterize whether the amastigotes were present inside macrophages and/or neoplastic cells, a co-localization study using cell- and pathogen-specific markers was performed. To detect Leishmania spp. a 5.8S ribosomal RNA (rRNA) parasite-specific sequence was used for in-situ hybridization and Mac387 was used as a macrophage marker for immunohistochemistry. Leishmania spp. rRNA was detected inside Mac387(+) macrophages and within the cytoplasm of some neoplastic cells. DNA isolation and polymerase chain reaction using specific primers and sequencing analysis identified the organism as Leishmania infantum (syn. Leishmania chagasi). This is the first report describing infection of tumour cells by L. infantum in a genital TVT from an asymptomatic bitch. Transplantation of Leishmania-laden neoplastic cells could represent an alternative route of venereal transmission of leishmaniasis among dogs.

Although pilots are often supported by signage, markings and lighting when taxiing on the airport surface, navigation and monitoring remain workload intense tasks even in good weather conditions. Radio communication is near capacity limits on many airports today resulting in waiting times and delay. Apart from well-known safety issues this also constitutes a negative impact on the environment. The European aviation research program SESAR addressed this problem and came up with a solution: A new surface traffic management concept proposes the automated use of Airfield Ground Lighting with individually switched green taxiway centerline lights indicating the path to be followed. This paper presents official validation results indicating that SESAR developed a safer, quicker and greener surface traffic management concept.

Serial passage of viruses in cell culture has been traditionally used to attenuate virulence and identify determinants of viral pathogenesis. In a previous study, we found that a strain of Schmallenberg virus (SBV) serially passaged in tissue culture (termed SBVp32) unexpectedly displayed increased pathogenicity in suckling mice compared to wild-type SBV. In this study, we mapped the determinants of SBVp32 virulence to the viral genome M segment. SBVp32 virulence is associated with the capacity of this virus to reach high titers in the brains of experimentally infected suckling mice. We also found that the Gc glycoprotein, encoded by the M segment of SBVp32, facilitates host cell protein shutoff in vitro . Interestingly, while the M segment of SBVp32 is a virulence factor, we found that the S segment of the same virus confers by itself an attenuated phenotype to wild-type SBV, as it has lost the ability to block the innate immune system of the host. Single mutations present in the Gc glycoprotein of SBVp32 are sufficient to compensate for both the attenuated phenotype of the SBVp32 S segment and the attenuated phenotype of NSs deletion mutants. Our data also indicate that the SBVp32 M segment does not act as an interferon (IFN) antagonist. Therefore, SBV mutants can retain pathogenicity even when they are unable to fully control the production of IFN by infected cells. Overall, this study suggests that the viral glycoprotein of orthobunyaviruses can compensate, at least in part, for the function of NSs. In addition, we also provide evidence that the induction of total cellular protein shutoff by SBV is determined by multiple viral proteins, while the ability to control the production of IFN maps to the NSs protein. IMPORTANCE The identification of viral determinants of pathogenesis is key to the development of prophylactic and intervention measures. In this study, we found that the bunyavirus Gc glycoprotein is a virulence factor. Importantly, we show that mutations in the Gc glycoprotein can restore the pathogenicity of attenuated mutants resulting from deletions or mutations in the nonstructural protein NSs. Our findings highlight the fact that careful consideration should be taken when designing live attenuated vaccines based on deletions of nonstructural proteins since single mutations in the viral glycoproteins appear to revert attenuated mutants to virulent phenotypes.

Clinical, pathological and genetic examination revealed an as yet uncharacterized juvenile-onset neuroaxonal dystrophy (NAD) in Spanish water dogs. Affected dogs presented with various neurological deficits including gait abnormalities and behavioral deficits. Histopathology demonstrated spheroid formation accentuated in the grey matter of the cerebral hemispheres, the cerebellum, the brain stem and in the sensory pathways of the spinal cord. Iron accumulation was absent. Ultrastructurally spheroids contained predominantly closely packed vesicles with a double-layered membrane, which were characterized as autophagosomes using immunohistochemistry. The family history of the four affected dogs suggested an autosomal recessive inheritance. SNP genotyping showed a single genomic region of extended homozygosity of 4.5 Mb in the four cases on CFA 8. Linkage analysis revealed a maximal parametric LOD score of 2.5 at this region. By whole genome re-sequencing of one affected dog, a perfectly associated, single, non-synonymous coding variant in the canine tectonin beta-propeller repeat-containing protein 2 (TECPR2) gene affecting a highly conserved region was detected (c.4009C>T or p.R1337W). This canine NAD form displays etiologic parallels to an inherited TECPR2 associated type of human hereditary spastic paraparesis (HSP). In contrast to the canine NAD, the spinal cord lesions in most types of human HSP involve the sensory and the motor pathways. Furthermore, the canine NAD form reveals similarities to cases of human NAD defined by widespread spheroid formation without iron accumulation in the basal ganglia. Thus TECPR2 should also be considered as candidate gene for human NAD. Immunohistochemistry and the ultrastructural findings further support the assumption, that TECPR2 regulates autophagosome accumulation in the autophagic pathways. Consequently, this report provides the first genetic characterization of juvenile canine NAD, describes the histopathological features associated with the TECPR2 mutation and provides evidence to emphasize the association between failure of autophagy and neurodegeneration.

The order Nidovirales contains large, enveloped viruses with a non-segmented positive-stranded RNA genome. Nidoviruses have been detected in man and various animal species, but, to date, there have been no reports of nidovirus in reptiles. In the present study, we describe the detection, characterization, phylogenetic analyses and disease association of a novel divergent nidovirus in the lung of an Indian python (Python molurus) with necrotizing pneumonia. Characterization of the partial genome (>33 000 nt) of this virus revealed several genetic features that are distinct from other nidoviruses, including a very large polyprotein 1a, a putative ribosomal frameshift signal that was identical to the frameshift signal of astroviruses and retroviruses and an accessory ORF that showed some similarity with the haemagglutinin–neuraminidase of paramyxoviruses. Analysis of genome organization and phylogenetic analysis of polyprotein 1ab suggests that this virus belongs to the subfamily Torovirinae. Results of this study provide novel insights into the genetic diversity within the order Nidovirales.

Hepatitis C virus (HCV) has a very narrow species and tissue tropism and efficiently replicates only in humans and the chimpanzee. Recently, several studies identified close relatives to HCV in different animal species. Among these novel viruses, the nonprimate hepaciviruses (NPHV) that infect horses are the closest relatives of HCV described to date. In this study, we analyzed the NPHV prevalence in northern Germany and characterized the clinical course of infection and viral tissue tropism to explore the relevance of HCV-related horse viruses as a model for HCV infection. We found that approximately 31.4% of 433 horses were seropositive for antibodies (Abs) against NPHV and approximately 2.5% carried viral RNA. Liver function analyses revealed no indication for hepatic impairment in 7 of 11 horses. However, serum gamma-glutamyl transferase (GGT) concentrations were mildly elevated in 3 horses, and 1 horse displayed even highly elevated GGT levels. Furthermore, we observed that NPHV infection could be cleared in individual horses with a simultaneous emergence of nonstructural (NS)3-specific Abs and transient elevation of serum levels of liver-specific enzymes indicative for a hepatic inflammation. In other individual horses, chronic infections could be observed with the copresence of viral RNA and NS3-specific Abs for over 6 months. For the determination of viral tissue tropism, we analyzed different organs and tissues of 1 NPHV-positive horse using quantitative real-time polymerase chain reaction and fluorescent in situ hydridization and detected NPHV RNA mainly in the liver and at lower amounts in other organs. Conclusion: Similar to HCV infections in humans, this work demonstrates acute and chronic stages of NPHV infection in horses with viral RNA detectable predominantly within the liver. (Hepatology 2015;61:448-459)

Schmallenberg virus (SBV) is an orthobunyavirus of the family Bunyaviridae that is associated with stillbirth and malformations in ruminants. The infection has been identified in many European countries since August 2011. The present study investigated retrospectively the occurrence of SBV infection in ruminants using immunohistochemistry and in-situ hybridization in brain tissues archived between 1961 and 2010 (112 cattle, 57 sheep, 16 goats and 27 wild ruminants). Eighty-five animals with inflammatory brain lesions and 47 animals with malformations were included. Due to the lack of SBV protein and RNA detection, SBV appears to have been introduced recently into Northern parts of Europe from tropical or subtropical regions.

Schmallenberg virus (SBV) is an emerging orthobunyavirus of ruminants associated with outbreaks of congenital malformations in aborted and stillborn animals. Since its discovery in November 2011, SBV has spread very rapidly to many European countries. Here, we developed molecular and serological tools, and an experimental in vivo model as a platform to study SBV pathogenesis, tropism and virus-host cell interactions. Using a synthetic biology approach, we developed a reverse genetics system for the rapid rescue and genetic manipulation of SBV. We showed that SBV has a wide tropism in cell culture and “synthetic” SBV replicates in vitro as efficiently as wild type virus. We developed an experimental mouse model to study SBV infection and showed that this virus replicates abundantly in neurons where it causes cerebral malacia and vacuolation of the cerebral cortex. These virus-induced acute lesions are useful in understanding the progression from vacuolation to porencephaly and extensive tissue destruction, often observed in aborted lambs and calves in naturally occurring Schmallenberg cases. Indeed, we detected high levels of SBV antigens in the neurons of the gray matter of brain and spinal cord of naturally affected lambs and calves, suggesting that muscular hypoplasia observed in SBV-infected lambs is mostly secondary to central nervous system damage. Finally, we investigated the molecular determinants of SBV virulence. Interestingly, we found a biological SBV clone that after passage in cell culture displays increased virulence in mice. We also found that a SBV deletion mutant of the non-structural NSs protein (SBVΔNSs) is less virulent in mice than wild type SBV. Attenuation of SBV virulence depends on the inability of SBVΔNSs to block IFN synthesis in virus infected cells. In conclusion, this work provides a useful experimental framework to study the biology and pathogenesis of SBV., Author Summary Schmallenberg virus (SBV) was discovered in Germany (near the town of Schmallenberg) in November 2011 and since then has been found to be the cause of malformations and stillbirths in ruminants. SBV has spread very rapidly to many European countries including the Netherlands, Belgium, France and the United Kingdom. Very little is known about the biological properties of this virus and there is no vaccine available. In this study (i) we developed an approach (called reverse genetics) that allows the recovery of “synthetic” SBV under laboratory conditions; (ii) we developed a mouse model of infection for SBV; (iii) we showed that SBV replicates in neurons of experimentally infected mice similar to naturally infected lambs and calves; (iv) we developed viral mutants that are not as pathogenic as the original virus due to the inability to counteract the host cell defenses; and v) we identified mutations that are associated with increased virulence. This work provides the experimental tools to understand how this newly emerged virus causes disease in ruminants. In addition, it will now be possible to manipulate the SBV genome in order to develop highly effective vaccines.

To the Editor: In 2011, a new virus spread throughout ruminant populations in the Netherlands, Germany, and other European countries (1). Infected dairy cattle exhibited fever, reduced milk yield, loss of appetite, and diarrhea. Weeks after these signs appeared, epidemic abortions; births of malformed or stillborn animals; and perinatal deaths of calves, lambs, and goat kids were reported. Metagenomic analysis identified a novel orthobunyavirus (family Bunyaviridae), termed Schmallenberg virus (SBV), which is closely related to Akabane and Shamonda viruses (1). These arthropod-borne viruses are well-recognized ruminant pathogens in Africa, Asia, and Oceania; fetal infection is associated with an arthrogryposis–hydranencephaly syndrome. Previous reports about SBV have addressed clinical signs (2), serologic findings (3), pathologic findings (4), and SBV RNA distribution in organs of malformed calves (5) detected by real-time quantitative reverse transcription PCR (qRT-PCR). However, pathogenesis and viral cell tropism are largely unknown. Therefore, our aims were to establish an in situ hybridization method (ISH) to detect SBV mRNA, to evaluate the usefulness of this method as a complementary diagnostic tool, and to further analyze SBV pathogenesis. The in situ probe was generated according to established protocols (6). The SBV qRT-PCR product and primers used for diagnostics (5) were used to amplify an 88-bp segment of the nucleoprotein, encoded by the small segment of the SBV genome. The amplicon was cloned into a pCR4-TOPO vector (Invitrogen, Darmstadt, Germany) and sequenced. For generation of a digoxigenin-labeled antisense probe to detect viral mRNA, we used M13 reverse and SBV-S-469R primers (5). Specificity of the probe was ensured by distinct ISH signals in the brains of SBV-positive (as determined by qRT-PCR) animals (6). No reaction was detected in SBV-negative (as determined by qRT-PCR) goats, sheep, and calves; in ruminants with various non–SBV-associated nervous system lesions; or in the brain of a mouse that was experimentally infected with Akabane virus. Thereafter, we investigated SBV mRNA distribution in the central nervous system (CNS) of 82 naturally infected ruminants (46 lambs, 2 goat kids, and 34 calves), all of which had previously been found by qRT-PCR to be positive for SBV. In addition, ISH was used to examine the following tissues from 10 of these animals (4 lambs, 1 goat kid, and 5 calves): placenta, muscle, eye, heart, aorta, lung, trachea, liver, kidney, spleen, small and large intestine, mesenteric and pulmonary lymph nodes, thymus, adrenal gland, testis, and uterus. SBV mRNA was found in varying amounts, predominantly in neurons (Figure) of the cerebrum, cerebellum, brain stem, medulla oblongata, and spinal cord of 7 lambs, 1 goat kid, and 2 calves. Randomly distributed clusters of SBV-positive neurons were frequently found in small ruminants, whereas only single positive cells were found in both calves. SBV mRNA was not found in any peripheral organ. The Technical Appendix provides an overview of ISH results in relation to malformations and inflammatory CNS lesions. Histologic examination identified encephalitis, characterized by lymphohistiocytic, perivascular cuffs, in 11 (9 lambs, 1 goat kid, and 1 calf) of the 82 animals. Among animals that were positive according to ISH, all small ruminants showed inflammation, whereas both calves lacked inflammatory changes. Figure In situ detection by using Nomarski differential interference contrast microscopy of Schmallenberg virus mRNA in neurons in the medulla of a neonatal sheep for which Schmallenberg virus infection was confirmed by ... The most frequent macroscopic lesions or conditions in animals that were SBV positive by qRT-PCR, especially calves, were arthrogryposis, brachygnathia inferior, torticollis, kyphosis, lordosis, scoliosis, and muscle hypoplasia (especially in calves) (4). The predominant CNS lesions or conditions were cerebellar and cerebral hypoplasia, hydranencephaly, porencephaly, hydrocephalus, and micromyelia. Among the 8 small ruminants that were SBV positive by ISH, hydrocephalus was found in 5, cerebellar hypoplasia in 5, hydranencephaly in 1, and arthrogryposis in 6. For the 2 calves that were SBV positive, micromyelia and arthrogryposis were found in both and cerebellar hypoplasia in 1. This study indicates that neurons are the predominant target in SBV-infected neonates. Surprisingly, only 10 of 82 ruminants were positive for SBV by qRT-PCR and ISH. This discrepancy could result from low SBV mRNA copy numbers per cell, which are detectable by qRT-PCR but not ISH, and/or from reduced viral load in individual cells because of the long time between assumed infection in early gestation and time of examination after birth (≈17 weeks in sheep and 27 weeks in calves). The latter explanation is indicated by the high incidence of malformations (7) typical for teratogenic insults and might especially apply to calves. Detection of CNS inflammation in all small ruminants that were SBV positive according to ISH indicates a cause-and-effect association between high SBV mRNA copy numbers per cell and lymphohistiocytic immune response. Furthermore, low numbers of infected cells and/or reduced replication as assumed for animals that were positive by qRT-PCR but negative by ISH might explain the absence of inflammation in the calf brains. The 3 tested animals in which inflammation was found but that were SBV negative by ISH could represent an advanced resolving lesion after viral clearance or reduction. In situ detection of SBV mRNA represents a suitable way to study SBV pathogenesis, especially in the active phase of infection, and might enable identification of SBV as the causative agent in cases of CNS inflammation of unknown etiology. Technical Appendix: Absolute numbers and percentages of animals that were positive for Schmallenberg virus according to in situ hybridization in relation to central nervous system inflammation and occurrence of skeletal and brain malformations. Data refer to absolute numbers of 82 small and large ruminants that were positive for Schmallenberg virus by real-time quantitative reverse transcription PCR. Click here to view.(91K, pdf)