Co-localization of Middle East respiratory syndrome coronavirus (MERS-CoV) and dipeptidyl peptidase-4 in the respiratory tract and lymphoid tissues of pigs and llamas

This study investigated the co-localization of the Middle East respiratory syndrome coronavirus (MERS-CoV) and its receptor dipeptidyl peptidase-4 (DPP4) by immunohistochemistry (IHC) across respiratory and lymphoid organs of experimentally MERS-CoV infected pigs and llamas. Also, scanning electron microscopy was performed to assess the ciliary integrity of respiratory epithelial cells in both species. In pigs, on day 2 post-inoculation (p.i.), DPP4-MERS-CoV co-localization was detected in medial turbinate epithelium. On day 4 p.i., the virus/receptor co-localized in frontal and medial turbinate epithelial cells in pigs, and epithelial cells distributed unevenly through the whole nasal cavity and in the cervical lymph node in llamas. MERS-CoV viral nucleocapsid was mainly detected in upper respiratory tract sites on days 2 and 4 p.i. in pigs and day 4 p.i. in llamas. No MERS-CoV was detected on day 24 p.i. in any tissue by IHC. While pigs showed severe ciliary loss in the nasal mucosa both on days 2 and 4 p.i. and moderate loss in the trachea on days 4 and 24 p.i., ciliation of respiratory organs in llamas was not significantly affected. Obtained data confirm the role of DPP4 for MERS-CoV entry in respiratory epithelial cells of llamas. Notably, several nasal epithelial cells in pigs were found to express viral antigen but not DPP4, suggesting the possible existence of other molecule/s facilitating virus entry or down regulation of DPP4 upon infection

Experimental infection of dromedaries with Middle East respiratory syndrome-Coronavirus is accompanied by massive ciliary loss and depletion of the cell surface receptor dipeptidyl peptidase 4

Middle East respiratory syndrome (MERS) represents an important respiratory disease accompanied by lethal outcome in one-third of human patients. Recent data indicate that dromedaries represent an important source of infection, although information regarding viral cell tropism and pathogenesis is sparse. In the current study, tissues of eight dromedaries receiving inoculation of MERS-Coronavirus (MERS-CoV) after recombinant Modified-Vaccinia-Virus-Ankara (MVA-S)-vaccination (n = 4), MVA-vaccination (mock vaccination, n = 2) and PBS application (mock vaccination, n = 2), respectively, were investigated. Tissues were analyzed by histology, immunohistochemistry, immunofluorescence, and scanning electron microscopy. MERS-CoV infection in mock-vaccinated dromedaries revealed high numbers of MERS-CoV-nucleocapsid positive cells, T cells, and macrophages within nasal turbinates and trachea at day four post infection. Double immunolabeling demonstrated cytokeratin (CK) 18 expressing epithelial cells to be the prevailing target cell of MERS-CoV, while CK5/6 and CK14 expressing cells did not co-localize with virus. In addition, virus was occasionally detected in macrophages. The acute disease was further accompanied by ciliary loss along with a lack of dipeptidyl peptidase 4 (DPP4), known to mediate virus entry. DPP4 was mainly expressed by human lymphocytes and dromedary monocytes, but overall the expression level was lower in dromedaries. The present study underlines significant species-specific manifestations of MERS and highlights ciliary loss as an important finding in dromedaries. The obtained results promote a better understanding of coronavirus infections, which pose major health challenges.info:eu-repo/semantics/publishedVersio

Generation and characterization of highly purified canine Schwann cells from spinal nerve dorsal roots as potential new candidates for transplantation strategies.

Schwann cells are promising candidates for transplantation strategies in the central nervous system by promoting axonal regeneration. The dog represents a translational model for human spinal cord injury (SCI) for studies with new repair strategies after intervertebral disk herniation (IVDH). To overcome the necessity for an additional surgical procedure, for the first time a protocol for the isolation and purification of canine Schwann cells from spinal nerve biopsies during standard hemilaminectomy in IVDH-affected paraplegic dogs for potential transplantation has been developed. Purity was assessed by flow cytometry. The results were compared with biopsies from dogs without SCI. Within 26 ± 4 days, 90.2 ± 8.8% p75 neurotrophin receptor (p75NTR )-positive cells were achieved in IVDH dogs. The total cell count in acute/subacute and chronic IVDH (acute/subacute: 6.82 ± 6.36 × 106 ; chronic: 2.29 ± 2.00 × 106 ) differed significantly (p = 0.0120) at the potential time point of transplantation. No differences in culture period and purity were detected between dogs with and without IVDH. Despite the small sample size and the altered environment, the isolation of Schwann cells was successful. Negative influences on isolation and purification due to potential pathological changes at the biopsy site of IVDH-diseased dogs were ruled out by comparison of Schwann cell pellets from diseased and control dogs. Finally, the functionality of Schwann cells from dogs with IVDH was outlined in co-culture experiments with canine dorsal root ganglion neurons. In conclusion, nerve root biopsies provide a sufficient number of highly purified and functional Schwann cells within a useful time period for novel therapeutic strategies in dogs with SCI

Polycystic kidneys and GM2 gangliosidosis-like disease in neonatal springboks (Antidorcas marsupialis)

Clinical, gross, histopathologic, electron microscopic findings and enzymatic analysis of 4 captive, juvenile springboks (Antidorcas marsupialis) showing both polycystic kidneys and a storage disease are described. Springbok offspring (4 of 34; 12%) were affected by either one or both disorders in a German zoo within a period of 5 years (2008–2013). Macroscopic findings included bilaterally severely enlarged kidneys displaying numerous cysts in 4 animals and superior brachygnathism in 2 animals. Histopathologically, kidneys of 4 animals displayed cystic dilation of the renal tubules. In addition, abundant cytoplasmic vacuoles with a diameter ranging from 2 to 10 μm in neurons of the central and peripheral nervous system, hepatocytes, thyroid follicular epithelial cells, pancreatic islets of Langerhans and renal tubular cells were found in 2 springbok neonates indicative of an additional storage disease. Ultrastructurally, round electron-lucent vacuoles, up to 4 μm in diameter, were present in neurons. Enzymatic analysis of liver and kidney tissue of 1 affected springbok revealed a reduced activity of total hexosaminidase (Hex) with relatively increased HexA activity at the same level of total Hex, suggesting a hexosaminidase defect. Pedigree analysis suggested a monogenic autosomal recessive inheritance for both diseases. In summary, related springboks showed 2 different changes resembling both polycystic kidney and a GM2 gangliosidosis similar to the human Sandhoff disease. Whether the simultaneous occurrence of these 2 entities represents an incidental finding or has a genetic link needs to be investigated in future studies

Polycystic Kidneys and GM2 Gangliosidosis-Like Disease in Neonatal Springboks (Antidorcas marsupialis)

Clinical, gross, histopathologic, electron microscopic findings and enzymatic analysis of 4 captive, juvenile springboks (Antidorcas marsupialis) showing both polycystic kidneys and a storage disease are described. Springbok offspring (4 of 34; 12%) were affected by either one or both disorders in a German zoo within a period of 5 years (2008-2013). Macroscopic findings included bilaterally severely enlarged kidneys displaying numerous cysts in 4 animals and superior brachygnathism in 2 animals. Histopathologically, kidneys of 4 animals displayed cystic dilation of the renal tubules. In addition, abundant cytoplasmic vacuoles with a diameter ranging from 2 to 10 μm in neurons of the central and peripheral nervous system, hepatocytes, thyroid follicular epithelial cells, pancreatic islets of Langerhans and renal tubular cells were found in 2 springbok neonates indicative of an additional storage disease. Ultrastructurally, round electron-lucent vacuoles, up to 4 μm in diameter, were present in neurons. Enzymatic analysis of liver and kidney tissue of 1 affected springbok revealed a reduced activity of total hexosaminidase (Hex) with relatively increased HexA activity at the same level of total Hex, suggesting a hexosaminidase defect. Pedigree analysis suggested a monogenic autosomal recessive inheritance for both diseases. In summary, related springboks showed 2 different changes resembling both polycystic kidney and a GM2 gangliosidosis similar to the human Sandhoff disease. Whether the simultaneous occurrence of these 2 entities represents an incidental finding or has a genetic link needs to be investigated in future studies