Selection of viral variants during persistent infection of insectivorous bat cells with Middle East respiratory syndrome coronavirus.

Coronaviruses that cause severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) are speculated to have originated in bats. The mechanisms by which these viruses are maintained in individuals or populations of reservoir bats remain an enigma. Mathematical models have predicted long-term persistent infection with low levels of periodic shedding as a likely route for virus maintenance and spillover from bats. In this study, we tested the hypothesis that bat cells and MERS coronavirus (CoV) can co-exist in vitro. To test our hypothesis, we established a long-term coronavirus infection model of bat cells that are persistently infected with MERS-CoV. We infected cells from Eptesicus fuscus with MERS-CoV and maintained them in culture for at least 126 days. We characterized the persistently infected cells by detecting virus particles, protein and transcripts. Basal levels of type I interferon in the long-term infected bat cells were higher, relative to uninfected cells, and disrupting the interferon response in persistently infected bat cells increased virus replication. By sequencing the whole genome of MERS-CoV from persistently infected bat cells, we identified that bat cells repeatedly selected for viral variants that contained mutations in the viral open reading frame 5 (ORF5) protein. Furthermore, bat cells that were persistently infected with ΔORF5 MERS-CoV were resistant to superinfection by wildtype virus, likely due to reduced levels of the virus receptor, dipeptidyl peptidase 4 (DPP4) and higher basal levels of interferon in these cells. In summary, our study provides evidence for a model of coronavirus persistence in bats, along with the establishment of a unique persistently infected cell culture model to study MERS-CoV-bat interactions.

Radial glial elements in the cerebral cortex of the lesser hedgehog tenrec.

We investigated astroglial cells in several areas of the telencephalic cortex of the lesser hedgehog tenrec (Echinops telfairi). Compared to other mammals, the cortex of the tenrec has a relatively large paleocortex and a low encephalization index. We stained sections from tenrec forebrains with structural and functional glia markers focusing on selected cortical areas, the paleocortex, rhinal cortex, neocortex and the dentate gyrus of the hippocampal formation. We found that in all parts of the tenrec forebrain cortex, radial processes exist which are positive for glial fibrillary acidic protein (GFAP) although with differential localization: in the rhinal cortex and neocortical region radial glial fibers are located in the subventricular regions, whereas in the dentate gyrus and paleocortex they appear to arise from the cells in the respective granular layers. The relatively high abundance of the radial fibers in layer III of the paleocortex was very conspicuous. Only few of these radial processes were also co-labeled with doublecortin (DCX), yet most of the DCX-positive cells were negative for GFAP. The GFAP-positive radial fibers were in turn neither positive for glutamine synthetase, nor did they show immunoreactivity for the astroglia-specific water channel aquaporin-4 (AQP4). Star-shaped astrocytes, however, displayed the typical perivascular and subpial expression patterns for AQP4. We conclude that the radial glia in the adult tenrec represents an immature form of astroglia that persists in these animals throughout life.

Influence of feeding habits in the endocrine pancreas of insectivore bat Pteronotus personatus and nectarivore bat Anoura geoffroyi: A comparative stereological and immunohistochemical study.

Pteronotus personatus as an insectivore bat and has a diet that consists of a high protein diet, whereas the diet of Anoura geoffroyi, a predominantly nectarivore bat, is rich in simple sugars like sucrose, glucose and fructose. Considering that diet influences the activation of different pathways, which may influence morphological adaptations in the gastrointestinal system, the aim of this study was to compare the morphology of the endocrine pancreas in P. personatus and A. geoffroyi. For this, histological, stereological and immunohistochemical methods were used. In P. personatus, the average diameter of the pancreatic islet was 40.47µm±13.94, while in A. geoffroyi was 88.16µm±36.40. The total number of pancreatic islets in P. personatus was 26150±2346 and in A. geoffroyi was 15970±1666. In P. personatus, the volume density of the pancreatic islets was 3.4%± 2.6, whereas in A. geoffroyi the volume density was 6.1%±3.7. In addition, the immunodensity of the α, ß and δ cells, in P. personatus was 25.8%±11.9, 35.5%±13.5, 3.9%±0.7, respectively, and in A. geoffroyi was 33.10%±12.7, 55.08%±7.4, 6.2%±4.6, respectively. In conclusion, the results of this study indicate differences in the pancreatic weight/body, weight ratio, diameter and volume density of pancreatic islets and in immunodensity of the ß and α cells between both species, which have different dietary habits.

Nasal anatomy of the non-mammaliaform cynodont Brasilitherium riograndensis (Eucynodontia, Therapsida) reveals new insight into mammalian evolution.

The mammalian nasal cavity is characterized by a unique anatomy with complex internal features. The evolution of turbinals was correlated with endothermic and macrosmatic adaptations in therapsids and in early mammals, which is still apparent in their twofold function (warming and moistening of air, olfaction). Fossil evidence for the transformation from the nonmammalian to the mammalian nasal cavity pattern has been poor and inadequate. Ossification of the cartilaginous nasal capsule and turbinals seems to be a feature that occurred only very late in synapsid evolution but delicate ethmoidal bones are rarely preserved. Here we provide the first µCT investigation of the nasal cavity of the advanced non-mammaliaform cynodont Brasilitherium riograndensis from the Late Triassic of Southern Brazil, a member of the sister-group of mammaliaforms, in order to elucidate a critical anatomical transition in early mammalian evolution. Brasilitherium riograndensis already had at least partially ossified turbinals as remnants of the nasoturbinal and the first ethmoturbinal are preserved. The posterior nasal septum is partly ossified and contributes to a mesethmoid. The nasal cavity is posteriorly expanded and forms a distinctive pars posterior (ethmoidal recess) that is ventrally separated from the nasopharyngeal duct by a distinct lamina terminalis. Thus, our observations clearly demonstrate that principal features of the mammalian nasal cavity were already present in the sister-group of mammaliaforms.

Brain volume of the newly-discovered species Rhynchocyon udzungwensis (Mammalia: Afrotheria: Macroscelidea): implications for encephalization in sengis.

The Gray-faced Sengi (Rhynchocyon udzungwensis) is a newly-discovered species of sengi (elephant-shrew) and is the largest known extant representative of the order Macroscelidea. The discovery of R. udzungwensis provides an opportunity to investigate the scaling relationship between brain size and body size within Macroscelidea, and to compare this allometry among insectivorous species of Afrotheria and other eutherian insectivores. We performed a spin-echo magnetic resonance imaging (MRI) scan on a preserved adult specimen of R. udzungwensis using a 7-Tesla high-field MR imaging system. The brain was manually segmented and its volume was compiled into a dataset containing previously-published allometric data on 56 other species of insectivore-grade mammals including representatives of Afrotheria, Soricomorpha and Erinaceomorpha. Results of log-linear regression indicate that R. udzungwensis exhibits a brain size that is consistent with the allometric trend described by other members of its order. Inter-specific comparisons indicate that macroscelideans as a group have relatively large brains when compared with similarly-sized terrestrial mammals that also share a similar diet. This high degree of encephalization within sengis remains robust whether sengis are compared with closely-related insectivorous afrotheres, or with more-distantly-related insectivorous laurasiatheres.

Comparative morphology of the Papillae Linguales and their connective tissue cores in the tongue of the greater japanese shrew-mole, Urotrichus talpoides.

The external morphology of the papillae linguales (papillae filiformes, papillae fungiformes and papillae vallatae) and their connective tissue cores (CTCs) of the greater Japanese shrew-mole (Urotrichus talpoides) were analysed by optical and scanning electron microscopy. Papillae filiformes were distributed over the dorsal surface of the apex linguae, and on the rostral and caudal regions of the corpus linguae but were less numerous in the mid-region. They were absent from the radix linguae. A pair of oval papillae vallatae was situated at the border between the corpus linguae and the radix linguae. Papillae foliatae were absent. The epithelial surface of each papilla filiformis consisted of a circular concavity, a ring-like wall and either a single thumb-like process or 2-3 slender pointed processes, depending on their location. The morphology of the CTCs of the papillae filiformes also varied regionally. The papillae linguales of the Japanese shrew-mole were morphologically similar to those of other Talpidae and Soricidae, including the common shrew, particularly with respect to the papillae filiformes in the mid- and caudal regions of the corpus linguae.

Venomous mammals: a review.

The occurrence of venom in mammals has long been considered of minor importance, but recent fossil discoveries and advances in experimental techniques have cast new light into this subject. Mammalian venoms form a heterogeneous group having different compositions and modes of action and are present in three classes of mammals, Insectivora, Monotremata, and Chiroptera. A fourth order, Primates, is proposed to have venomous representatives. In this review we highlight recent advances in the field while summarizing biochemical characteristics of these secretions and their effects upon humans and other animals. Historical aspects of venom discovery and evolutionary hypothesis regarding their origin are also discussed.

Amygdalar connections in the lesser hedgehog tenrec.

The present study analyses the overall extrinsic connectivity of the non-olfactory amygdala (Ay) in the lesser hedgehog tenrec. The data were obtained from tracer injections into the lateral and intermediate portions of the Ay as well as several non-amygdalar brain regions. Both the solitary and the parabrachial nucleus receive descending projections from the central nucleus of the Ay, but only the parabrachial nucleus appears to project to the Ay. There is one prominent region in the ventromedial hypothalamus connected reciprocally with the medial and central Ay. Amygdalar afferents clearly arise from the dorsomedial thalamus, the subparafascicular nuclei and the medial geniculate complex (GM). Similar to other subprimate species, the latter projections originate in the dorsal and most caudal geniculate portions and terminate in the dorsolateral Ay. Unusual is the presence of amygdalo-projecting cells in the marginal geniculate zone and their virtual absence in the medial GM. As in other species, amygdalo-striatal projections mainly originate in the basolateral Ay and terminate predominantly in the ventral striatum. Given the poor differentiation of the tenrec's neocortex, there is a remarkable similarity with regard to the amygdalo-cortical connectivity between tenrec and rat, particularly as to prefrontal, limbic and somatosensorimotor areas as well as the rhinal cortex throughout its length. The tenrec's isocortex dorsomedial to the caudal rhinal cortex, on the other hand, may not be connected with the Ay. An absence of such connections is expected for primary auditory and visual fields, but it is unusual for their secondary fields.

Volumetric analysis of the African elephant ventricular system.

This study used magnetic resonance imaging (MRI) to determine the volume of the ventricular system in the brain of three adult male African elephants (Loxodonta africana). The ventricular system of the elephant has a volume of ∼240 mL, an order of magnitude larger than that seen in the adult human. Despite this large size, allometric analysis indicates that the volume of the ventricles in the elephant is what one would expect for a mammal with an ∼5 kg brain. Interestingly, our comparison with other mammals revealed that primates appear to have small relative ventricular volumes, and that megachiropterans and microchiropterans follow different scaling rules when comparing ventricular volume to brain mass indicating separate phylogenetic histories. The current study provides context for one aspect of the elephant brain in the broader picture of mammalian brain evolution.

A quill vibrating mechanism for a sounding apparatus in the streaked tenrec (Hemicentetes semispinosus).

The streaked tenrec (Hemicentetes semispinosus) is equipped with a quill vibrating mechanism on the dorsal side of the caudal trunk that has evolved as an extraordinary sounding apparatus for communication. An arrangement of 15 or 16 light-brown quills was observed. Thickened cutaneous muscles were confirmed beneath quills. We named this structure the "quill vibrator disc" (QVD). The QVD was 16.8 mm long and 8.55 mm wide in a typical adult. Longitudinal musculature symmetrical about the sagittal plane was developed in the QVD. Myocytes were found immunohistochemically to contain mainly fast myosin but not slow myosin. These findings indicate that the QVD is a specialized apparatus in the cutaneous muscle that contributes to the vibration of quills and to the production of sound for communication.

Aggrecan-based extracellular matrix shows unique cortical features and conserved subcortical principles of mammalian brain organization in the Madagascan lesser hedgehog tenrec (Echinops telfairi Martin, 1838).

The Madagascan tenrecs (Afrotheria), an ancient mammalian clade, are characterized by unique brain anatomy. Striking features are an expanded paleocortex but a small and poorly differentiated neocortex devoid of a distinct granular layer IV. To investigate the organization of cortical areas we analyzed extracellular matrix components in perineuronal nets (PNs) using antibodies to aggrecan, lectin staining and hyaluronan-binding protein. Selected subcortical regions were studied to correlate the cortical patterns with features in evolutionary conserved systems. In the neocortex, paleocortex and hippocampus PNs were associated with nonpyramidal neurons. Quantitative analysis in the cerebral cortex revealed area-specific proportions and laminar distribution patterns of neurons ensheathed by PNs. Cortical PNs showed divergent structural phenotypes. Diffuse PNs forming a cotton wool-like perisomatic rim were characteristic of the paleocortex. These PNs were associated with a dense pericellular plexus of calretinin-immunoreactive fibres. Clearly contoured PNs were devoid of a calretinin-positive plexus and predominated in the neocortex and hippocampus. The organization of the extracellular matrix in subcortical nuclei followed the widely distributed mammalian type. We conclude that molecular properties of the aggrecan-based extracellular matrix are conserved during evolution of mammals; however, the matrix scaffold is adapted to specific wiring patterns of cortical and subcortical neuronal networks.

Tracing thalamo-cortical connections in tenrecA further attempt to characterize poorly differentiated neocortical regions, particularly the motor cortex.

The hedgehog tenrec (Afrosoricidae) has a very poorly differentiated neocortex. Previously its primary sensory regions have been characterized with hodological and electrophysiological techniques. Unlike the marsupial opossum the tenrec may also have a separate motor area as far as there are cortico-spinal cells located rostral to the primary somatosensory cortex. However, not knowing its thalamic input it may be premature to correlate this area with the true (mirror-image-like) primary motor cortex in higher mammals. For this reason the tenrec's thalamo-cortical connections were studied following tracer injections into various neocortical regions. The main sensory areas were confirmed by their afferents from the principal thalamic nuclei. The dorsal lateral geniculate nucleus, in addition, was connected with the retrosplenial area and a rostromedial visual region. Unlike the somatosensory cortex the presumed motor area did not receive afferents from the ventrobasal thalamus but fibers from the cerebello-thalamic target regions. These projections, however, were not restricted to the motor area, but involved the entire somatosensorimotor field as well as adjacent regions. The projections appeared similar to those arising in the rat thalamic ventromedial nucleus known to have a supporting function rather than a specific motor task. The question was raised whether the input from the basal ganglia might play a crucial role in the evolution of the mammalian motor cortex? Certainly, in the tenrec, the poor differentiation of the motor cortex coincides with the virtual absence of an entopeduncular projection to the ventrolateral thalamus.

The presence and absence of prosencephalic cell groups relaying striatal information to the medial and lateral thalamus in tenrec.

Although there are remarkable differences regarding the output organization of basal ganglia between mammals and non-mammals, mammalian species with poorly differentiated brain have scarcely been investigated in this respect. The aim of the present study was to identify the pallidal neurons giving rise to thalamic projections in the Madagascar lesser hedgehog tenrec (Afrotheria). Following tracer injections into the thalamus, retrogradely labelled neurons were found in the depth of the olfactory tubercle (particularly the hilus of the Callejal islands and the insula magna), in subdivisions of the diagonal band complex, the peripeduncular region and the thalamic reticular nucleus. No labelled cells were seen in the globus pallidus. Pallidal neurons were tentatively identified on the basis of their striatal afferents revealed hodologically using anterograde axonal tracer substances and immunohistochemically with antibodies against enkephalin and substance P. The data showed that the tenrec's medial thalamus received prominent projections from ventral pallidal cells as well as from a few neurons within and ventral to the cerebral peduncle. The only regions projecting to the lateral thalamus appeared to be the thalamic reticular nucleus (RTh) and the dorsal peripeduncular nucleus (PpD). On the basis of immunohistochemical data and the topography of its thalamic projections, the PpD was considered to be an equivalent to the pregeniculate nucleus in other mammals. There was no evidence of entopeduncular (internal pallidal) neurons being present within the RTh/PpD complex, neuropils of which did not stain for enkephalin and substance P. The ventrolateral portion of RTh, the only region eventually receiving a striatal input, projected to the caudolateral rather than the rostrolateral thalamus. Thus, the striatopallidal output organization in the tenrec appeared similar, in many respects, to the output organization in non-mammals. This paper considers the failure to identify entopeduncular neurons projecting to the rostrolateral thalamus in a mammal with a little differentiated cerebral cortex, and also stresses the discrepancy between this absence and the presence of a distinct external pallidal segment (globus pallidus).

The semifossorial function of the forelimb in the common rice tenrec (Oryzorictes hova) and the streaked tenrec (Hemicentetes hemispinosus).

The forelimb muscles of the two semifossorial species of Tenrecidae (Oryzoryctinae: common rice tenrec; and Tenrecinae: streaked tenrec) were compared macroscopically with those of the unspecialized terrestrial-arboreal species, the Talazac long-tailed tenrec. The structure of the hand was also observed using three-dimensional reconstructed images from computed tomography data. The two semifossorial species had similar muscle weight ratios in the lateral and long heads of M. triceps brachii and M. teres major. A similar hand skeleton structure (in which the second, third and fourth metacarpals and phalanges act as a digging apparatus) was observed in both species. Our observations confirm that both these species have muscular-skeletal adaptations supporting fossorial locomotion. As each species belongs to a monophyletic subfamily within the Tenrecidae isolated in Madagascar, such semifossorial adaptations are assumed to have evolved convergently.