We characterized the complete genome of a novel dog circovirus (DogCV) from the liver of a dog with severe hemorrhagic gastroenteritis, vasculitis, and granulomatous lymphadenitis. DogCV was detected by PCR in fecal samples from 19/168 (11.3%) dogs with diarrhea and 14/204 (6.9%) healthy dogs and in blood from 19/409 (3.3%) of dogs with thrombocytopenia and neutropenia, fever of unknown origin, or past tick bite. Co-infection with other canine pathogens was detected for 13/19 (68%) DogCV-positive dogs with diarrhea. DogCV capsid proteins from different dogs varied by up to 8%. In situ hybridization and transmission electron microscopy detected DogCV in the lymph nodes and spleens of 4 dogs with vascular compromise and histiocytic inflammation. The detection of a circovirus in tissues of dogs expands the known tropism of these viruses to a second mammalian host. Our results indicate that circovirus, alone or in co-infection with other pathogens, might contribute to illness and death in dogs.

Bovine papillomaviruses (BPV1/BPV2) have long been associated with equine sarcoids; deciphering their contribution has been difficult due to their ubiquitous presence on skin and in the environment, as well as the lack of decent techniques to interrogate their role in pathogenesis. We have developed and characterized an in situ hybridization (ISH) assay that uses a pool of probes complementary to portions of the E5, E6, and E7 genes. This assay is highly sensitive for direct visualization of viral transcript and nucleic acid in routinely processed histopathologic samples. We demonstrate here the visualization of BPV nucleic acid in 18 of 18 equine sarcoids, whereas no detectable viral DNA was present in 15 of 15 nonsarcoid controls by this technique. In nearly 90% (16/18) of the sarcoids, 50% or more of the fibroblastic cell nuclei distributed throughout the neoplasm had detectable hybridization. In the remaining 2 cases, fewer than half of the fibroblastic cells contained detectable hybridization, but viral nucleic acid was also detected in epithelial cells of the sebaceous glands, hair follicles and epidermis. A sensitive ISH assay is an indispensable addition to the molecular methods used to detect viral nucleic acid in tissue. We have used this technique to determine the specific cellular localization and distribution of BPV in a subset of equine sarcoids.

Equus caballus papillomavirus 2 (EcPV2) has been proposed as an etiologic agent for genital squamous cell carcinoma (SCC), the most common malignant tumor of the horse penis. EcPV2 is commonly detected by polymerase chain reaction (PCR) on normal horse genitalia; therefore, unraveling the virus' role in oncogenic transformation requires other methods of detection. In this study, a highly sensitive multiple-probe chromogenic in situ hybridization (ISH) technique was designed to recognize the E6/E7 oncogenes of EcPV2. ISH demonstrated abundant virus within 6 of 13 penile and preputial SCCs, whereas evidence of solar damage was found in 6 cases that were negative for EcPV2 by ISH. The ISH technique is valuable for studies of pathogenesis, since it demonstrates for the first time that the vast majority of neoplastic cells contain virus. Moreover, hybridization was present in all metastases examined, implying stability of E6/E7 expression in these clonal populations of neoplastic cells. This study contributes to the accumulating evidence for a causal role of EcPV2 in a subset of genital SCCs in horses.

A fundamental challenge in studying principles of organization used by the olfactory system to encode odor concentration information has been identifying comprehensive sets of activated odorant receptors (ORs) across a broad concentration range inside freely behaving animals. In mammals, this has recently become feasible with high-throughput sequencing-based methods that identify populations of activated ORs in vivo. In this study, we characterized the mouse OR repertoires activated by the two odorants, acetophenone and 2,5-dihydro-2,4,5trimethylthiazoline, from 0.01% to 100% (v/v) as starting concentrations using phosphorylated ribosomal protein S6 capture followed by RNA-Seq. We found Olfr923 to be one of the most sensitive ORs that is enriched by acetophenone. Using a mouse line that genetically labels Olfr923-positive axons, we provided evidence that acetophenone activates the Olfr923 glomeruli in the olfactory bulb. Through molecular dynamics stimulations, we identified amino acid residues in the Olfr923 binding cavity that facilitate acetophenone binding. This study sheds light on the active process by which unique OR repertoires may collectively facilitate the discrimination of odorant concentrations. 4 Significance Statement The ability of animals to discriminate odors over a range of odor concentrations while recognizing concentration-invariant odor identity presents an encoding challenge for the olfactory system. To further our understanding on how animals sense odors at different concentrations, it is important to describe how odor concentration information is represented at the receptor level. Here, we establish a sensitive in vivo approach to screen populations of odorant receptors enriched in the odor-activated sensory neurons in mice. We identified comprehensive lists of enriched odorant receptors against a 10,000-fold concentration range for two odorants. Describing the concentration-dependent activation for unique populations of odorant receptors is fundamental for future studies in determining how individual odorant receptors contribute to olfactory sensitivity and odor intensity coding.

Directed differentiation of human pluripotent stem cells (hPSCs) has enabled the generation of specific neuronal subtypes that approximate the intended primary mammalian cells on both the RNA and protein levels. These cells offer unique opportunities, including insights into mechanistic understanding of the early driving events in neurodegenerative disease, replacement of degenerating cell populations, and compound identification and evaluation in the context of precision medicine. However, whether the derived neurons indeed recapitulate the physiological features of the desired bona fide neuronal subgroups remains an unanswered question and one important for validating stem cell models as accurate functional representations of the primary cell types. Here, we purified both hPSC-derived and primary mouse spinal motor neurons in parallel and used extracellular multi-electrode array (MEA) recording to compare the pharmacological sensitivity of neuronal excitability and network function. We observed similar effects for most receptor and channel agonists and antagonists, supporting the consistency between human PSC-derived and mouse primary spinal motor neuron models from a physiological perspective.

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SUMMARY High-throughput physiological assays lose single-cell resolution, precluding subtype-specific analyses of activation mechanism and drug effects. We demonstrate APPOINT (automated physiological phenotyping of individual neuronal types), a physiological assay platform combining calcium imaging, robotic liquid handling, and automated analysis to generate physiological activation profiles of single neurons at large scale. Using unbiased techniques, we quantify responses to sequential stimuli, enabling subgroup identification by physiology and probing of distinct mechanisms of neuronal activation within subgroups. Using APPOINT, we quantify primary sensory neuron activation by metabotropic receptor agonists and identify potential contributors to pain signaling. We expand the role of neuroimmune interactions by showing that human serum directly activates sensory neurons, elucidating a new potential pain mechanism. Finally, we apply APPOINT to develop a high-throughput, all-optical approach for quantification of activation threshold and pharmacologically validate contributions of ion channel families to optical activation.

Sensory gene families are of special interest for both what they can tell us about molecular evolution and what they imply as mediators of social communication. The vomeronasal type-1 receptors (V1Rs) have often been hypothesized as playing a fundamental role in driving or maintaining species boundaries given their likely function as mediators of intraspecific mate choice, particularly in nocturnal mammals. Here, we employ a comparative genomic approach for revealing patterns of V1R evolution within primates, with a special focus on the small-bodied nocturnal mouse and dwarf lemurs of Madagascar (genera Microcebus and Cheirogaleus, respectively). By doubling the existing genomic resources for strepsirrhine primates (i.e. the lemurs and lorises), we find that the highly speciose and morphologically cryptic mouse lemurs have experienced an elaborate proliferation of V1Rs that we argue is functionally related to their capacity for rapid lineage diversification. Contrary to a previous study that found equivalent degrees of V1R diversity in diurnal and nocturnal lemurs, our study finds a strong correlation between nocturnality and V1R elaboration, with nocturnal lemurs showing elaborate V1R repertoires and diurnal lemurs showing less diverse repertoires. Recognized subfamilies among V1Rs show unique signatures of diversifying positive selection, as might be expected if they have each evolved to respond to specific stimuli. Furthermore, a detailed syntenic comparison of mouse lemurs with mouse (genus Mus) and other mammalian outgroups shows that orthologous mammalian subfamilies, predicted to be of ancient origin, tend to cluster in a densely populated region across syntenic chromosomes that we refer to as a V1R “hotspot.”