RTN3 and RTN4: Architects of SARS-CoV-2 replication organelles.

SARS-CoV-2 depends on host proteins for successful replication. In this issue, Williams et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202203060) report that the ER membrane-modulating proteins RTN3 and RTN4 are required for the formation of SARS-CoV-2 replication organelles via direct interaction with viral proteins NSP3 and NSP4

Relevance of the regional lymph node in scrapie pathogenesis after peripheral infection of hamsters

<p>Abstract</p> <p>Background</p> <p>The exact role of the lymphoreticular system in the spread of peripheral prion infections to the central nervous system still needs further elucidation. Against this background, the influence of the regional lymph node <it>(Ln. popliteus</it>) on the pathogenesis of scrapie was monitored in a hamster model of prion infection <it>via </it>the footpad.</p> <p>Methods</p> <p>Surgical lymphadenectomy was carried out at different time points after infection, or prior to inoculation, in order to elucidate the impact of the lymph node on lethal neuroinvasion.</p> <p>Results</p> <p>The <it>Ln. popliteus </it>did not show an influence on pathogenesis when a high dose of infectivity was administered. However, it was found to modulate the interval of time until the development of terminal scrapie in a subset of animals lymphadenectomized after low-dose infection. In additon, lymphadenectomy performed four weeks before inoculation prevented cerebral PrP^TSEdeposition and development of disease during the period of observation (314 days) in the majority of hamsters challenged with a very low dose of scrapie agent.</p> <p>Conclusion</p> <p>Our findings suggest the regional lymph node as a potentially facilitating or even essential factor for invasion of the brain after peripheral challenge with low doses of infectious scrapie agent. The invasive <it>in vivo </it>approach pursued in this study may be applied also to other animal species for further elucidating the involvement of lymphoid tissue in the pathogenesis of experimental and natural TSEs.</p

Video-based sleepiness measurement under the conditions of the maintenance of wakefulness test

Quantifikation von Tagesschläfrigkeit ist in der schlafmedizinischen Diagnostik und Unfallprävention von Interesse. Neben Schlafmangel können unterschiedliche Erkrankungen Tagesschläfrigkeit hervorrufen. Die Methoden zur Feststellung von Tagesschläfrigkeit unterscheiden sich durch individuelle Stärken und Schwächen. Die Polysomnographie (PSG), welche Elektroenzephalographie mit anderen Messwerten vereint, gilt als Referenzmethode der schlafmedizinischen Diagnostik und wird unter standardisierten Bedingungen durchgeführt. Der Einsatz der PSG im Straßenverkehr wird durch ihre hohe Komplexität limitiert, es kommen daher pragmatische, videobasierte Verfahren zum Einsatz, bei denen eine Aufzeichnung des Auges zur Schläfrigkeitsbestimmung genutzt wird. Der Einsatz videobasierter Schläfrigkeitsmessung durch augenbezogene Parameter in der Schlafmedizin wurde bei Kratzel et al. 2021 erprobt. Die dort gesammelten Daten werden in der vorliegenden Arbeit einer weiteren Analyse unterzogen. Die Daten stammen von 30 Patientinnen und Patienten, bei denen je vier (insgesamt 120) PSG-Aufzeichnungen mit paralleler videometrischer Augenparameter-Aufzeichnung unter den Bedingungen des Multiplen Wachbleibetests (MWT) erfolgten. In der vorliegenden Arbeit werden der lidschlussabhängige Parameter Mean blink duration (MBD) sowie der Pupillendurchmesser der Patientinnen und Patienten während der PSG-Aufzeichnungen untersucht und es wird geprüft, ob diese geeignet sind, den Einschlafzeitpunkt im Vergleich zur PSG zu bestimmen. Der Anteil unter der Grenzwertoptimierungskurve für die Erkennung von Schlaf liegt bei dem Parameter MBD bei 85%, ein Grenzwert von 12 Sekunden für das Feststellen von Schlaf ist für diesen Parameter optimal. Mit diesem Grenzwert werden Einschlafereignisse unter Bedingungen des MWT mit einer Sensitivität von 78% und einer Spezifität von 79% erkannt. Die Schlaflatenz (SL), bestimmt durch MBD, ist stark mit der SL, gemessen mit der Referenzmethode PSG, korreliert (mit Rho = 0.78; p<0.05), das 95% Konfidenzintervall der mit MBD gemessenen SL beträgt ±17.1 Minuten. Die Einschlafereignisse sind nicht mit veränderten Pupillenoszillationen assoziiert. Der lidschlussabhängige Parameter MBD ist ähnlich gut für die Feststellung von Schlaf unter den Bedingungen des MWT geeignet wie der bei Kratzel et al. 2021 dargestellte Parameter Percentage of eyelid closure (PERCLOS). Beide Parameter könnten unterstützend für die Feststellung von Schlaf unter den Bedingungen des MWT eingesetzt werden, sie können die PSG jedoch nicht ersetzen. Die Auswertung von Pupillenoszillationen scheint zur Feststellung von Schlaf unter den Bedingungen des MWT ungeeignet.The quantification of daytime sleepiness is important for diagnostics in sleep medicine and in the prevention of accidents. Daytime sleepiness can be caused by a lack of sleep or by underlying diseases. Several methods are available for detecting sleepiness, which present individual strengths and weaknesses and can be selected according to the specific requirements of the application. The polysomnography (PSG), combining electroencepalography with other physiological signal, is used as a reference in medical diagnostics and is usually applied under standardised conditions. The high complexity of the PSG limits its use in the prevention of traffic accidents and necessitate the use of other more convenient methods, which rely on videometric, ocular parameters. The application of videobased sleepiness detection using ocular parameters has been examined in Kratzel et al. 2021. The data recorded in this previous study undergoes further analyses in the current paper. The data was recorded in 30 patients. In each of the patients, four recordings of PSG and videometric recordings of eye signals took place in parallel, under the conditions of the maintenance of wakefulness test (MWT). The current paper focuses on the eyelid-based parameter mean blink duration (MBD) and the analysis of the pupil diameter, and investigates whether these parameters are suitable for the detection of sleep under the conditions of the MWT. The area under the receiver operator characteristics curve for the detection of sleep is at 85% when considering the parameter MBD, and a cutoff value of 12 seconds for the detection of sleep using MBD was found to be ideal. Using this cutoff value under the condition of the MWT, the occurrence of sleep is correctly detected with a sensitivity of 78% and a specificity of 79%. The sleep latency measured with MBD is highly correlated to the sleep latency with the PSG reference (rho = 0.78; p < 0.05), the 95% confidence interval of the MBD measured sleep latency is ±17.1 minutes. Sleep onset is not linked to the oscillation behaviour of the pupil diameter. The eyelid-dependent parameter MBD is comparable to the parameter percentage of eyelid closure (PERCLOS) which was investigated in Kratzel et al. 2021. Both parameters can be used in support of the PSG to detect sleep under the conditions of the MWT, but cannot replace the PSG. Pupil diameter oscillations are not suitable for detecting sleep under the conditions of the MWT

Establishment of well-differentiated camelid airway cultures to study Middle East respiratory syndrome coronavirus.

In 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in Saudi Arabia and was mostly associated with severe respiratory illness in humans. Dromedary camels are the zoonotic reservoir for MERS-CoV. To investigate the biology of MERS-CoV in camelids, we developed a well-differentiated airway epithelial cell (AEC) culture model for Llama glama and Camelus bactrianus. Histological characterization revealed progressive epithelial cellular differentiation with well-resemblance to autologous ex vivo tissues. We demonstrate that MERS-CoV displays a divergent cell tropism and replication kinetics profile in both AEC models. Furthermore, we observed that in the camelid AEC models MERS-CoV replication can be inhibited by both type I and III interferons (IFNs). In conclusion, we successfully established camelid AEC cultures that recapitulate the in vivo airway epithelium and reflect MERS-CoV infection in vivo. In combination with human AEC cultures, this system allows detailed characterization of the molecular basis of MERS-CoV cross-species transmission in respiratory epithelium

A safe, effective and adaptable live-attenuated SARS-CoV-2 vaccine to reduce disease and transmission using one-to-stop genome modifications.

Approved vaccines are effective against severe COVID-19, but broader immunity is needed against new variants and transmission. Therefore, we developed genome-modified live-attenuated vaccines (LAV) by recoding the SARS-CoV-2 genome, including 'one-to-stop' (OTS) codons, disabling Nsp1 translational repression and removing ORF6, 7ab and 8 to boost host immune responses, as well as the spike polybasic cleavage site to optimize the safety profile. The resulting OTS-modified SARS-CoV-2 LAVs, designated as OTS-206 and OTS-228, are genetically stable and can be intranasally administered, while being adjustable and sustainable regarding the level of attenuation. OTS-228 exhibits an optimal safety profile in preclinical animal models, with no side effects or detectable transmission. A single-dose vaccination induces a sterilizing immunity in vivo against homologous WT SARS-CoV-2 challenge infection and a broad protection against Omicron BA.2, BA.5 and XBB.1.5, with reduced transmission. Finally, this promising LAV approach could be applicable to other emerging viruses

Possible Case of Maternal Transmission of Feline Spongiform Encephalopathy in a Captive Cheetah

Feline spongiform encephalopathy (FSE) is considered to be related to bovine spongiform encephalopathy (BSE) and has been reported in domestic cats as well as in captive wild cats including cheetahs, first in the United Kingdom (UK) and then in other European countries. In France, several cases were described in cheetahs either imported from UK or born in France. Here we report details of two other FSE cases in captive cheetah including a 2nd case of FSE in a cheetah born in France, most likely due to maternal transmission. Complete prion protein immunohistochemical study on both brains and peripheral organs showed the close likeness between the two cases. In addition, transmission studies to the TgOvPrP4 mouse line were also performed, for comparison with the transmission of cattle BSE. The TgOvPrP4 mouse brains infected with cattle BSE and cheetah FSE revealed similar vacuolar lesion profiles, PrPd brain mapping with occurrence of typical florid plaques. Collectively, these data indicate that they harbor the same strain of agent as the cattle BSE agent. This new observation may have some impact on our knowledge of vertical transmission of BSE agent-linked TSEs such as in housecat FSE, or vCJD

Characterization of a rat osteotomy model with impaired healing

<p>Abstract</p> <p>Background</p> <p>Delayed union or nonunion are frequent and feared complications in fracture treatment. Animal models of impaired bone healing are rare. Moreover, specific descriptions are limited although understanding of the biological course of pathogenesis of fracture nonunion is essential for therapeutic approaches.</p> <p>Methods</p> <p>A rat tibial osteotomy model with subsequent intramedullary stabilization was performed. The healing progress of the osteotomy model was compared to a previously described closed fracture model. Histological analyses, biomechanical testing and radiological screening were undertaken during the observation period of 84 days (d) to verify the status of the healing process. In this context, particular attention was paid to a comparison of bone slices by histological and immunohistological (IHC) methods at early points in time, <it>i.e</it>. at 5 and 10 d post bone defect.</p> <p>Results</p> <p>In contrast to the closed fracture technique osteotomy led to delayed union or nonunion until 84 d post intervention. The dimensions of whole reactive callus and the amounts of vessels in defined regions of the callus differed significantly between osteotomized and fractured animals at 10 d post surgery. A lower fraction of newly formed bone and cartilaginous tissue was obvious during this period in osteotomized animals and more inflammatory cells were observed in the callus. Newly formed bone tissue accumulated slowly on the anterior tibial side with both techniques. New formation of reparative cartilage was obviously inhibited on the anterior side, the surgical approach side, in osteotomized animals only.</p> <p>Conclusion</p> <p>Tibial osteotomy with intramedullary stabilisation in rats leads to pronounced delayed union and nonunion until 84 d post intervention. The early onset of this delay can already be detected histologically within 10 d post surgery. Moreover, the osteotomy technique is associated with cellular and vascular signs of persistent inflammation within the first 10 d after bone defect and may be a contributory factor to impaired healing. The model would be excellent to test agents to promote fracture healing.</p

The spike gene is a major determinant for the SARS-CoV-2 Omicron-BA.1 phenotype.

Variant of concern (VOC) Omicron-BA.1 has achieved global predominance in early 2022. Therefore, surveillance and comprehensive characterization of Omicron-BA.1 in advanced primary cell culture systems and animal models are urgently needed. Here, we characterize Omicron-BA.1 and recombinant Omicron-BA.1 spike gene mutants in comparison with VOC Delta in well-differentiated primary human nasal and bronchial epithelial cells in vitro, followed by in vivo fitness characterization in hamsters, ferrets and hACE2-expressing mice, and immunized hACE2-mice. We demonstrate a spike-mediated enhancement of early replication of Omicron-BA.1 in nasal epithelial cultures, but limited replication in bronchial epithelial cultures. In hamsters, Delta shows dominance over Omicron-BA.1, and in ferrets Omicron-BA.1 infection is abortive. In hACE2-knock-in mice, Delta and a Delta spike clone also show dominance over Omicron-BA.1 and an Omicron-BA.1 spike clone, respectively. Interestingly, in naïve K18-hACE2 mice, we observe Delta spike-mediated increased replication and pathogenicity and Omicron-BA.1 spike-mediated reduced replication and pathogenicity, suggesting that the spike gene is a major determinant of replication and pathogenicity. Finally, the Omicron-BA.1 spike clone is less well-controlled by mRNA-vaccination in K18-hACE2-mice and becomes more competitive compared to the progenitor and Delta spike clones, suggesting that spike gene-mediated immune evasion is another important factor that led to Omicron-BA.1 dominance

The spike gene is a major determinant for the SARS-CoV-2 Omicron-BA. 1 phenotype

Variant of concern (VOC) Omicron-BA.1 has achieved global predominance in early 2022. Therefore, surveillance and comprehensive characterization of Omicron-BA.1 in advanced primary cell culture systems and animal models are urgently needed. Here, we characterize Omicron-BA.1 and recombinant Omicron-BA.1 spike gene mutants in comparison with VOC Delta in well-differentiated primary human nasal and bronchial epithelial cells in vitro, followed by in vivo fitness characterization in hamsters, ferrets and hACE2-expressing mice, and immunized hACE2-mice. We demonstrate a spike-mediated enhancement of early replication of Omicron-BA.1 in nasal epithelial cultures, but limited replication in bronchial epithelial cultures. In hamsters, Delta shows dominance over Omicron-BA.1, and in ferrets Omicron-BA.1 infection is abortive. In hACE2-knock-in mice, Delta and a Delta spike clone also show dominance over Omicron-BA.1 and an Omicron-BA.1 spike clone, respectively. Interestingly, in naïve K18-hACE2 mice, we observe Delta spike-mediated increased replication and pathogenicity and Omicron-BA.1 spike-mediated reduced replication and pathogenicity, suggesting that the spike gene is a major determinant of replication and pathogenicity. Finally, the Omicron-BA.1 spike clone is less well-controlled by mRNA-vaccination in K18-hACE2-mice and becomes more competitive compared to the progenitor and Delta spike clones, suggesting that spike gene-mediated immune evasion is another important factor that led to Omicron-BA.1 dominance