Spatiotemporal analysis of SARS-CoV-2 infection reveals an expansive wave of monocyte-derived macrophages associated with vascular damage and virus clearance in hamster lungs.

IMPORTANCE: We present the first study of the 3D kinetics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the early host response in a large lung volume using a combination of tissue imaging and transcriptomics. This approach allowed us to make a number of important findings: Spatially restricted antiviral response is shown, including the formation of monocytic macrophage clusters and upregulation of the major histocompatibility complex II in infected epithelial cells. The monocyte-derived macrophages are linked to SARS-CoV-2 clearance, and the appearance of these cells is associated with post-infection endothelial damage; thus, we shed light on the role of these cells in infected tissue. An early onset of tissue repair occurring simultaneously with inflammatory and necrotizing processes provides the basis for longer-term alterations in the lungs.

Halogenated Rocaglate Derivatives: Pan-antiviral Agents against Hepatitis E Virus and Emerging Viruses.

The synthesis of a library of halogenated rocaglate derivatives belonging to the flavagline class of natural products, of which silvestrol is the most prominent example, is reported. Their antiviral activity and cytotoxicity profile against a wide range of pathogenic viruses, including hepatitis E, Chikungunya, Rift Valley Fever virus and SARS-CoV-2, were determined. The incorporation of halogen substituents at positions 4', 6 and 8 was shown to have a significant effect on the antiviral activity of rocaglates, some of which even showed enhanced activity compared to CR-31-B and silvestrol.

Associations between gait performance and pain intensity, psychosocial factors, executive functions as well as prefrontal cortex activity in chronic low back pain patients: A cross-sectional fNIRS study.

Introduction: Activities of daily living, such as walking, are impaired in chronic low back pain (CLBP) patients compared to healthy individuals. Thereby, pain intensity, psychosocial factors, cognitive functioning and prefrontal cortex (PFC) activity during walking might be related to gait performance during single and dual task walking (STW, DTW). However, to the best of our knowledge, these associations have not yet been explored in a large sample of CLBP patients. Method: Gait kinematics (inertial measurement units) and PFC activity (functional near-infrared spectroscopy) during STW and DTW were measured in 108 CLBP patients (79 females, 29 males). Additionally, pain intensity, kinesiophobia, pain coping strategies, depression and executive functioning were quantified and correlation coefficients were calculated to determine the associations between parameters. Results: The gait parameters showed small correlations with acute pain intensity, pain coping strategies and depression. Stride length and velocity during STW and DTW were (slightly to moderately) positively correlated with executive function test performance. Specific small to moderate correlations were found between the gait parameters and dorsolateral PFC activity during STW and DTW. Conclusion: Patients with higher acute pain intensity and better coping skills demonstrated slower and less variable gait, which might reflect a pain minimization strategy. Psychosocial factors seem to play no or only a minor role, while good executive functions might be a prerequisite for a better gait performance in CLBP patients. The specific associations between gait parameters and PFC activity during walking indicate that the availability and utilization of brain resources are crucial for a good gait performance.

Compellingly high SARS-CoV-2 susceptibility of Golden Syrian hamsters suggests multiple zoonotic infections of pet hamsters during the COVID-19 pandemic.

Golden Syrian hamsters (Mesocricetus auratus) are used as a research model for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). Millions of Golden Syrian hamsters are also kept as pets in close contact to humans. To determine the minimum infective dose (MID) for assessing the zoonotic transmission risk, and to define the optimal infection dose for experimental studies, we orotracheally inoculated hamsters with SARS-CoV-2 doses from 1 * 105 to 1 * 10-4 tissue culture infectious dose 50 (TCID50). Body weight and virus shedding were monitored daily. 1 * 10-3 TCID50 was defined as the MID, and this was still sufficient to induce virus shedding at levels up to 102.75 TCID50/ml, equaling the estimated MID for humans. Virological and histological data revealed 1 * 102 TCID50 as the optimal dose for experimental infections. This compelling high susceptibility leading to productive infections in Golden Syrian hamsters must be considered as a potential source of SARS-CoV-2 infection for humans that come into close contact with pet hamsters.

The Waiting Room Assessment to Virtual Emergency Department pathway: Initiating video-based telemedicine in the pediatric emergency department.

The COVID-19 pandemic has presented pediatric emergency departments with unique challenges, resulting in a heightened demand for adapted clinical pathways. In response to this need, the Montreal Children's Hospital pediatric emergency department introduced the WAVE (Waiting Room Assessment to Virtual Emergency Department) pathway, a video-based telemedicine pathway for selected non-critical patients, aiming to reduce safety issues related to emergency department overcrowding, while providing timely care to all children presenting and registering at our emergency department. The objective of the WAVE pilot phase was to evaluate the feasibility and acceptability of telemedicine in our pediatric emergency department, which was previously unfamiliar with this mode of care delivery. During the six-week, three-evening per week deployment, we conducted 18 five-hour telemedicine shifts. In total, 27 patients participated in the WAVE pathway. Results from this pilot phase met four of five a priori feasibility and acceptability criteria. Overall, participating families were satisfied with this novel care pathway and reported no disruptive technological barriers.

Curing Cats with Feline Infectious Peritonitis with an Oral Multi-Component Drug Containing GS-441524.

Feline infectious peritonitis (FIP) caused by feline coronavirus (FCoV) is a common dis-ease in cats, fatal if untreated, and no effective treatment is currently legally available. The aim of this study was to evaluate efficacy and toxicity of the multi-component drug Xraphconn® in vitro and as oral treatment in cats with spontaneous FIP by examining survival rate, development of clinical and laboratory parameters, viral loads, anti-FCoV antibodies, and adverse effects. Mass spectrometry and nuclear magnetic resonance identified GS-441524 as an active component of Xraphconn®. Eighteen cats with FIP were prospectively followed up while being treated orally for 84 days. Values of key parameters on each examination day were compared to values before treatment initiation using linear mixed-effect models. Xraphconn® displayed high virucidal activity in cell culture. All cats recovered with dramatic improvement of clinical and laboratory parameters and massive reduction in viral loads within the first few days of treatment without serious adverse effects. Oral treatment with Xraphconn® containing GS-441524 was highly effective for FIP without causing serious adverse effects. This drug is an excellent option for the oral treatment of FIP and should be trialed as potential effective treatment option for other severe coronavirus-associated diseases across species.

Impact of animal saliva on the performance of rapid antigen tests for detection of SARS-CoV-2 (wildtype and variants B.1.1.7 and B.1.351).

SARS-CoV-2 infects several animal species and SARS-CoV-2 variants of concern (VOCs) may even show (as in humans) enhanced inter- and intra-species transmission rates. We correlated sensitivity data of SARS-CoV-2 rapid antigen tests (RATs) to viral RNA genome equivalents analyzed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Further, we checked their suitability for testing animals by assessing saliva and VOC effects. Viral loads up to 2 logs (RNA copy number) under the hypothetical SARS-CoV-2 infectivity threshold were detected by most analyzed RATs. However, while saliva from various animal species showed generally no adverse effects on the RATs' analytical sensitivities, the detection of VOCs B.1.1.7 and B.1.351 was in some RATs inferior to non-VOC viruses.

In Vivo Characterization of a Bank Vole-Derived Cowpox Virus Isolate in Natural Hosts and the Rat Model.

Cowpox virus (CPXV) belongs to the genus Orthopoxvirus in the Poxviridae family and is endemic in western Eurasia. Based on seroprevalence studies in different voles from continental Europe and UK, voles are suspected to be the major reservoir host. Recently, a CPXV was isolated from a bank vole (Myodes glareolus) in Germany that showed a high genetic similarity to another isolate originating from a Cotton-top tamarin (Saguinus oedipus). Here we characterize this first bank vole-derived CPXV isolate in comparison to the related tamarin-derived isolate. Both isolates grouped genetically within the provisionally called CPXV-like 3 clade. Previous phylogenetic analysis indicated that CPXV is polyphyletic and CPXV-like 3 clade represents probably a different species if categorized by the rules used for other orthopoxviruses. Experimental infection studies with bank voles, common voles (Microtusarvalis) and Wistar rats showed very clear differences. The bank vole isolate was avirulent in both common voles and Wistar rats with seroconversion seen only in the rats. In contrast, inoculated bank voles exhibited viral shedding and seroconversion for both tested CPXV isolates. In addition, bank voles infected with the tamarin-derived isolate experienced a marked weight loss. Our findings allow for the conclusion that CPXV isolates might differ in their replication capacity in different vole species and rats depending on their original host. Moreover, the results indicate host-specific differences concerning CPXV-specific virulence. Further experiments are needed to identify individual virulence and host factors involved in the susceptibility and outcome of CPXV-infections in the different reservoir hosts.

What a Difference a Gene Makes: Identification of Virulence Factors of Cowpox Virus.

Cowpox virus (CPXV) is a zoonotic orthopoxvirus (OPV) that causes spillover infections from its animal hosts to humans. In 2009, several human CPXV cases occurred through transmission from pet rats. An isolate from a diseased rat, RatPox09, exhibited significantly increased virulence in Wistar rats and caused high mortality compared to that caused by the mildly virulent laboratory strain Brighton Red (BR). The RatPox09 genome encodes four genes which are absent in the BR genome. We hypothesized that their gene products could be major factors influencing the high virulence of RatPox09. To address this hypothesis, we employed several BR-RatPox09 chimeric viruses. Using Red-mediated mutagenesis, we generated BR-based knock-in mutants with single or multiple insertions of the respective RatPox09 genes. High-throughput sequencing was used to verify the genomic integrity of all recombinant viruses, and transcriptomic analyses confirmed that the expression profiles of the genes that were adjacent to the modified ones were unaltered. While the in vitro growth kinetics were comparable to those of BR and RatPox09, we discovered that a knock-in BR mutant containing the four RatPox09-specific genes was as virulent as the RatPox09 isolate, causing death in over 75% of infected Wistar rats. Unexpectedly, the insertion of gCPXV0030 (g7tGP) alone into the BR genome resulted in significantly higher clinical scores and lower survival rates matching the rate for rats infected with RatPox09. The insertion of gCPXV0284, encoding the BTB (broad-complex, tramtrack, and bric-à-brac) domain protein D7L, also increased the virulence of BR, while the other two open reading frames failed to rescue virulence independently. In summary, our results confirmed our hypothesis that a relatively small set of four genes can contribute significantly to CPXV virulence in the natural rat animal model.IMPORTANCE With the cessation of vaccination against smallpox and its assumed cross-protectivity against other OPV infections, waning immunity could open up new niches for related poxviruses. Therefore, the identification of virulence mechanisms in CPXV is of general interest. Here, we aimed to identify virulence markers in an experimental rodent CPXV infection model using bacterial artificial chromosome (BAC)-based virus recombineering. We focused our work on the recent zoonotic CPXV isolate RatPox09, which is highly pathogenic in Wistar rats, unlike the avirulent BR reference strain. In several animal studies, we were able to identify a novel set of CPXV virulence genes. Two of the identified virulence genes, encoding a putative BTB/POZ protein (CPXVD7L) and a B22R-family protein (CPXV7tGP), respectively, have not yet been described to be involved in CPXV virulence. Our results also show that single genes can significantly affect virulence, thus facilitating adaptation to other hosts.

Molecular Detection and Characterization of the First Cowpox Virus Isolate Derived from a Bank Vole.

Cowpox virus (CPXV) is a zoonotic orthopoxvirus (OPV) that infects a wide range of mammals. CPXV-specific DNA and antibodies were detected in different vole species, such as common voles (Microtus arvalis) and bank voles (Myodes glareolus). Therefore, voles are the putative main reservoir host of CPXV. However, CPXV was up to now only isolated from common voles. Here we report the detection and isolation of a bank vole-derived CPXV strain (GerMygEK 938/17) resulting from a large-scale screening of bank voles collected in Thuringia, Germany, during 2017 and 2018. Phylogenetic analysis using the complete viral genome sequence indicated a high similarity of the novel strain to CPXV clade 3 and to OPV "Abatino" but also to Ectromeliavirus (ECTV) strains. Phenotypic characterization of CPXV GerMygEK 938/17 using inoculation of embryonated chicken eggs displayed hemorrhagic pock lesions on the chorioallantoic membrane that are typical for CPXV but not for ECTV. CPXV GerMygEK 938/17 replicated in vole-derived kidney cell lines but at lower level than on Vero76 cell line. In conclusion, the first bank vole-derived CPXV isolate provides new insights into the genetic variability of CPXV in the putative reservoir host and is a valuable tool for further studies about CPXV-host interaction and molecular evolution of OPV.

Common vole (Microtus arvalis) and bank vole (Myodes glareolus) derived permanent cell lines differ in their susceptibility and replication kinetics of animal and zoonotic viruses.

Pathogenesis and reservoir host adaptation of animal and zoonotic viruses are poorly understood due to missing adequate cell culture and animal models. The bank vole (Myodes glareolus) and common vole (Microtus arvalis) serve as hosts for a variety of zoonotic pathogens. For a better understanding of virus association to a putative animal host, we generated two novel cell lines from bank voles of different evolutionary lineages and two common vole cell lines and assayed their susceptibility, replication and cytopathogenic effect (CPE) formation for rodent-borne, suspected to be rodent-associated or viruses with no obvious rodent association. Already established bank vole cell line BVK168, used as control, was susceptible to almost all viruses tested and efficiently produced infectious virus for almost all of them. The Puumala orthohantavirus strain Vranica/Hällnäs showed efficient replication in a new bank vole kidney cell line, but not in the other four bank and common vole cell lines. Tula orthohantavirus replicated in the kidney cell line of common voles, but was hampered in its replication in the other cell lines. Several zoonotic viruses, such as Cowpox virus, Vaccinia virus, Rift Valley fever virus, and Encephalomyocarditis virus 1 replicated in all cell lines with CPE formation. West Nile virus, Usutu virus, Sindbis virus and Tick-borne encephalitis virus replicated only in a part of the cell lines, perhaps indicating cell line specific factors involved in replication. Rodent specific viruses differed in their replication potential: Murine gammaherpesvirus-68 replicated in the four tested vole cell lines, whereas murine norovirus failed to infect almost all cell lines. Schmallenberg virus and Foot-and-mouth disease virus replicated in some of the cell lines, although these viruses have never been associated to rodents. In conclusion, these newly developed cell lines may represent useful tools to study virus-cell interactions and to identify and characterize host cell factors involved in replication of rodent associated viruses.

Anthocyanin synthesis and light utilisation can be enhanced by reflective mulch - Visualisation of light penetration into a tree canopy.

With the increasing use of hailnets and decrease in light availability in the ripening period of apple fruit, insufficient light exposure often causes poor colouration viz anthocyanin synthesis on certain parts of the fruit and on certain fruit within the tree. The aim of this study was to investigate the potential of improving anthocyanin synthesis, in terms of fruit colouration, the major incentive for a consumer. Therefore, the reflective ground cover Lumilys® was spread between 'Braeburn Mariri Red' apple rows under a crystalline hailnet seven weeks prior to harvest and colour measured on 240 attached apple fruit. The reflective ground cover increased the reflected light by 1.6 to 3.9 times 1.0 m above ground. The improved light utilization led to an improved peel colouration, especially on the shaded side of the apple fruit and fruit in the lower inner part of the canopy, A coloured visualization from orange (high light intensity), yellow (medium) to green (low light intensity) as a result of the individual PAR measurements every 20 cm inside the canopy showed how the reflective mulch influences the light penetration into the different parts of the tree canopy.

Impact of Different Aortic Entry Tear Sites on Early Outcomes and Long-Term Survival in Patients with Stanford A Acute Aortic Dissection.

BACKGROUND: Stanford A acute aortic dissection (AAD) is a life-threatening emergency. The aim of this study was to compare the impact of three different aortic entry tear sites on early outcomes and long-term survival of patients with Stanford A AAD. METHODS: From January 2006 to April 2015, a total of 240 consecutive patients with diagnosed Stanford A AAD underwent emergent, isolated surgical aortic repair in our center. Patients were divided into three groups comprising isolated ascending aorta, proximal aortic arch, and distal aortic arch entry tear site and were followed up for up to 9 years. RESULTS: Thirty-day mortality as well as major cerebrovascular events were significantly different between the three groups (p = 0.007 and p = 0.048, respectively). Overall cumulative short- and long-term survival of all patients revealed significant differences (Log-Rank p = 0.002), whereas survival of all patients free from major cerebrovascular events was similar (Log-Rank p = 0.780). Subgroup analysis of short- and long-term survival of patients showed significant differences in terms of men (Log-Rank p = 0.043), women (Log-Rank p = 0.004), patients over 65 years of age (Log-Rank p = 0.007), and hypertensive patients (Log-Rank p = 0.003). Kaplan-Meier survival estimation plots significantly showed poorest survival for distal aortic arch entry tear site group. CONCLUSION: The location of the primary entry tear in patients with Stanford A AAD significantly influences early outcomes, short- and long-term survival of patients, whereas survival of patients free from major cerebrovascular events showed similar results among the three groups. Distal aortic entry tear site showed poorest outcomes and survival.

Experimental Cowpox Virus (CPXV) Infections of Bank Voles: Exceptional Clinical Resistance and Variable Reservoir Competence.

Cowpox virus (CPXV) is a zoonotic virus and endemic in wild rodent populations in Eurasia. Serological surveys in Europe have reported high prevalence in different vole and mouse species. Here, we report on experimental CPXV infections of bank voles (Myodes glareolus) from different evolutionary lineages with a spectrum of CPXV strains. All bank voles, independently of lineage, sex and age, were resistant to clinical signs following CPXV inoculation, and no virus shedding was detected in nasal or buccal swabs. In-contact control animals became only rarely infected. However, depending on the CPXV strain used, inoculated animals seroconverted and viral DNA could be detected preferentially in the upper respiratory tract. The highest antibody titers and virus DNA loads in the lungs were detected after inoculation with two strains from Britain and Finland. We conclude from our experiments that the role of bank voles as an efficient and exclusive CPXV reservoir seems questionable, and that CPXV may be maintained in most regions by other hosts, including other vole species. Further investigations are needed to identify factors that allow and modulate CPXV maintenance in bank voles and other potential reservoirs, which may also influence spill-over infections to accidental hosts.