LitCovid-sentences  

PMC:7537531 / 1138-15002 JSONTXT 9 Projects

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Id Subject Object Predicate Lexical cue
T11 0-15 Sentence denotes 1 INTRODUCTION
T12 16-210 Sentence denotes The rapid worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has caused an unparalleled crisis and requires urgent testing of prophylactic and therapeutic measures.
T13 211-404 Sentence denotes SARS‐CoV‐2 likely emerged from a yet unknown animal reservoir; its rapid spread can be attributed to efficient replication in the upper respiratory tract and robust human‐to‐human transmission.
T14 405-535 Sentence denotes This characteristic clearly distinguishes this novel coronavirus from the related SARS‐CoV‐1 and MERS‐CoV (Andersen et al., 2020).
T15 536-660 Sentence denotes Interestingly, SARS‐CoV‐2 human‐to‐animal transmissions have also been reported (Halfmann et al., 2020; Leroy et al., 2020).
T16 661-751 Sentence denotes Currently, we find ourselves racing for efficacious therapies and protective vaccinations.
T17 752-924 Sentence denotes In this context, small animal models are indispensable as pre‐clinical studies in animals are of critical importance for basic and applied research on SARS‐CoV‐2 infection.
T18 925-1231 Sentence denotes Several SARS‐CoV‐2 animal models have been investigated, including non‐human primates, ferrets, transgenic mice, cats and hamsters, (Chan et al., 2020; Dhama et al., 2020; Imai et al., 2020; Lakdawala & Menachery, 2020; Osterrieder et al., 2020; Sia et al., 2020; Trimpert et al., 2020; Yuan et al., 2020).
T19 1232-1561 Sentence denotes However, in silico models with respect to potential usage of the entry receptor by the spike glycoprotein (S) of SARS‐CoV‐2 suggest susceptibility of Chinese hamsters (Cricetulus griseus) (Luan et al., 2020; Luan et al., 2020) and a previous study in Chinese hamsters demonstrated susceptibility to SARS‐CoV‐1 (Luo et al., 2007).
T20 1562-1708 Sentence denotes We here determined SARS‐CoV‐2 susceptibility of Chinese hamsters and established this species as novel small animal model for SARS‐CoV‐2 research.
T21 1710-1734 Sentence denotes 2 MATERIALS AND METHODS
T22 1736-1758 Sentence denotes 2.1 Viruses and cells
T23 1759-1872 Sentence denotes The SARS‐CoV‐2 München isolate (BetaCoV/Germany/BavPat1/2020) (Wölfel et al., 2020) was used for virus challenge.
T24 1873-2201 Sentence denotes Stocks were propagated and titrated on Vero E6 cells (ATCC CRL‐1586) in minimal essential medium (MEM; PAN Biotech, Aidenbach, Germany) supplemented with 10% foetal bovine serum (PAN Biotech), 100 IU/ml penicillin G and 100 µg/ml streptomycin (Carl Roth, Karlsruhe, Germany) and stored at −80°C prior to experimental infections.
T25 2203-2250 Sentence denotes 2.2 Experimental infection of Chinese hamsters
T26 2251-2448 Sentence denotes 24 five‐ to seven‐week‐old female and male Chinese hamsters (Cricetulus griseus) were obtained via the German pet trade and kept in groups of six animals in enriched, individually ventilated cages.
T27 2449-2601 Sentence denotes The hamsters had ad libidum access to food and water, and were allowed acclimatization to these conditions for seven days prior to SARS‐CoV‐2 infection.
T28 2602-2725 Sentence denotes Cage temperatures and relative humidities were recorded daily and ranged from 23 to 24°C and from 50% to 65%, respectively.
T29 2726-2956 Sentence denotes The animals were randomly distributed into two groups: SARS‐CoV‐2‐infected (n = 12; 1 × 105 plaque‐forming units (pfu) in a volume of 30 µl) and mock‐infected (n = 12; 30 µl cell culture supernatant from uninfected Vero E6 cells).
T30 2957-3044 Sentence denotes The infection was performed exactly as previously described (Osterrieder et al., 2020).
T31 3045-3230 Sentence denotes Baseline body weights of all hamsters and body temperatures of at least three hamsters per group were measured before infection and then recorded daily throughout the 14‐day experiment.
T32 3231-3324 Sentence denotes Moreover, clinical signs of all animals were monitored twice daily throughout the experiment.
T33 3325-3514 Sentence denotes On days 2, 3, 5 and 14 post‐infection (dpi), three randomly assigned hamsters of each group were euthanized by exsanguination under general anaesthesia as described (Nakamura et al., 2017).
T34 3515-3658 Sentence denotes For virus titrations and RT‐qPCR and/or histopathological examinations, blood, bucco‐laryngeal swabs and lungs (left and right) were collected.
T35 3660-3705 Sentence denotes 2.3 Histopathology and in situ hybridization
T36 3706-3798 Sentence denotes For histopathology, lungs were processed as previously described (Osterrieder et al., 2020).
T37 3799-3961 Sentence denotes In brief, the left lung lobe was carefully removed and immersion fixed in buffered 4% formalin, pH 7.0, for 48 hr, embedded in paraffin and cut at 2 μm thickness.
T38 3962-4118 Sentence denotes Sections were stained with haematoxylin and eosin (HE) followed by blinded microscopic evaluation by board certified veterinary pathologists (K.D., A.D.G.).
T39 4119-4321 Sentence denotes For in situ hybridization (ISH), the ViewRNA™ ISH Tissue Assay Kit (Invitrogen by Thermo Fisher Scientific, Darmstadt, Germany) was used following the manufacturer's instructions with minor adjustments.
T40 4322-4467 Sentence denotes A probe for the detection of the N gene RNA of SARS‐CoV‐2 (NCBI database NC_045512.2, nucleotides 28,274–29,533, assay ID: VPNKRHM) was employed.
T41 4468-4596 Sentence denotes Next, lung sections of 2 μm thickness were mounted on adhesive glass slides, dewaxed in xylol and dehydrated in graded ethanols.
T42 4597-4687 Sentence denotes Tissues were incubated at 95°C for 10 min, and subsequently, protease digested for 20 min.
T43 4688-4809 Sentence denotes Sections were fixed with 4% paraformaldehyde dissolved in phosphate‐buffered saline (PBS) and hybridized with the probes.
T44 4810-4939 Sentence denotes Amplifier and label probe hybridizations were performed according to the manufacturer's instructions using fast red as chromogen.
T45 4940-5117 Sentence denotes Sections were counterstained with haematoxylin for 45 s, washed in tap water for 5 min and mounted with Roti®‐Mount Fluor‐Care DAPI (4, 6‐diaminidino‐2‐phenylindole; Carl Roth).
T46 5118-5213 Sentence denotes For negative and morphologically intact controls, lungs from uninfected hamsters were included.
T47 5214-5333 Sentence denotes An irrelevant probe for the detection of streptococcal pneumolysin was used as a control for sequence‐specific binding.
T48 5334-5561 Sentence denotes HE‐stained and ISH slides were analysed and photographs were taken using an Olympus BX41 microscope with a DP80 Microscope Digital Camera and the cellSens™ Imaging Software, Version 1.18 (Olympus Corporation, Münster, Germany).
T49 5562-5793 Sentence denotes For overviews, slides were automatically digitized using the Aperio CS2 slide scanner (Leica Biosystems Imaging Inc., Vista, CA, USA) followed by image file generation using the Image Scope Software (Leica Biosystems Imaging Inc.).
T50 5795-5845 Sentence denotes 2.4 Virus titrations, RNA extractions and RT‐qPCR
T51 5846-5980 Sentence denotes Virus titres from 50 mg lung tissue were assessed by serial dilution of tissue homogenates and subsequent titrations on Vero E6 cells.
T52 5981-6069 Sentence denotes 3 dpi, cells were formalin‐fixed, stained with crystal violet, and plaques were counted.
T53 6070-6406 Sentence denotes RNA was extracted from tissue samples, blood and tracheal swabs using the innuPREP Virus RNA kit (Analytic Jena, Jena, Germany) and quantified with a one‐step RT‐qPCR reaction (NEB Luna Universal Probe One‐Step RT‐qPCR kit; New England Biolabs, Ipswitch, MA, USA) and previously published TaqMan primers and probe (Corman et al., 2020).
T54 6408-6433 Sentence denotes 2.5 Statistical analyses
T55 6434-6473 Sentence denotes Data were analysed with GraphPad PRISM.
T56 6474-6610 Sentence denotes We used Mann–Whitney U tests to compare body weights and body temperatures in mock‐infected versus SARS‐CoV‐2 infected Chinese hamsters.
T57 6611-6677 Sentence denotes Data were considered significant if p < .05 (*p < .05; **p < .01).
T58 6679-6704 Sentence denotes 3 RESULTS AND DISCUSSION
T59 6705-6884 Sentence denotes The study, which was approved by the Landesamt für Gesundheit und Soziales in Berlin, Germany (permit # 0086/20), involved 24 female and male Chinese hamsters of 5–7 weeks of age.
T60 6885-7410 Sentence denotes Hamsters were randomly assigned to two groups (SARS‐CoV‐2‐infected, n = 12; mock‐infected, n = 12) Upon intranasal infection with 1x105 plaque‐forming units (pfu) of a human SARS‐CoV‐2 isolate, we observed clinical symptoms including transient but significant body weight loss and subtle drops in body temperatures at early time points after infection, a course of disease comparable to that in Syrian hamsters (Figure 1) (Chan et al., 2020; Imai et al., 2020; Kreye et al., 2020; Osterrieder et al., 2020; Sia et al., 2020).
T61 7411-7662 Sentence denotes Differences in body weights between infected and mock‐infected animals were significant at 1–3 and at 5 days post‐infection (dpi), infected hamsters did not reach pre‐infection body weights before 14 dpi when the experiment was terminated (Figure 1a).
T62 7663-7888 Sentence denotes However, the largest difference in mean body temperatures of infected compared to mock‐infected animals was only −0.6°C at 3 dpi (Figure 1b), suggesting that this parameter may not be useful to assess the status of infection.
T63 7889-8058 Sentence denotes We determined virus replication and virus spread by measuring RNA copy numbers in the lungs, bucco‐laryngeal swabs and whole blood samples of all hamsters (Figure 1c–e).
T64 8059-8436 Sentence denotes Figure 1 Body weight changes, body temperatures and virus loads of mock‐infected and SARS‐CoV‐2‐infected Chinese hamsters. (a) Individual relative body weights of mock‐infected (green) and SARS‐CoV‐2‐infected (red) hamsters over the course of 14 days after infection. p‐values indicate significant differences (Mann–Whitney U test). (b) Temperature changes (as means with SD).
T65 8437-8695 Sentence denotes Virus loads were determined from total RNA in (c) 2.5 mg homogenized right cranial lung lobes, in (d) bucco‐laryngeal swabs and (e) 2.5 µl of whole blood samples by RT‐qPCR In addition, we determined viral titres in the lungs of infected animals (Figure S1).
T66 8696-8888 Sentence denotes Our assays confirmed high RNA copy numbers and corresponding viral titres at early time points after infection followed by a rapid but incomplete clearance of the virus infection until 14 dpi.
T67 8889-9128 Sentence denotes With titres of up to 5 × 107 pfu per 50 mg lung tissue and viral RNA copies in the range of up to 1 × 107 per 2.5 mg lung tissue at early time points after infection, we concluded that Chinese hamsters are highly susceptible to SARS‐CoV‐2.
T68 9129-9344 Sentence denotes On the other hand, virus loads in blood samples were below 1 × 102 RNA copies per 2.5 µl of whole blood (15.6 ± 29.8 SD) at all time points, indicating lack of significant viremia and systemic infection (Figure 1e).
T69 9345-9451 Sentence denotes These observations match findings from other animal models for emerging coronaviruses (Yuan et al., 2020).
T70 9452-9713 Sentence denotes Lung histopathology revealed tissue damage and inflammation principally similar to what has been described in Syrian hamsters (Figure 2) (Chan et al., 2020; Gruber et al., 2020; Imai et al., 2020; Kreye et al., 2020; Osterrieder et al., 2020; Sia et al., 2020).
T71 9714-9842 Sentence denotes However, the course of bronchitis and pneumonia was in general milder than in Syrian hamsters, and pneumonia was more prolonged.
T72 9843-10041 Sentence denotes Specifically, prominent acute alveolar damage was present until 14 dpi with persistence of viral RNA (Figures 1c, 2g‐i and S2), indicating overall slower recovery than described for Syrian hamsters.
T73 10042-10223 Sentence denotes Similarly, viral RNA was localized by in situ hybridization to bronchial epithelial cells, alveolar epithelial cells type‐I and type‐II and alveolar macrophages (Figure S2, arrows).
T74 10224-10321 Sentence denotes Figure 2 Histopathological assessment of mock‐infected and SARS‐CoV‐2‐infected Chinese hamsters.
T75 10322-10535 Sentence denotes Histopathology of haematoxylin–eosin‐stained lung sections from SARS‐CoV‐2‐infected Chinese hamsters revealed suppurative bronchitis (a, b; arrow: neutrophils) and necrosuppurative pneumonia (c, d) at 2 and 3 dpi.
T76 10536-10642 Sentence denotes At 5 dpi (e, f), additional hyperplasia of alveolar epithelial cells (AEC)‐type II was salient (f, arrow).
T77 10643-10754 Sentence denotes Tissue damage, cell influx and hyperplasia of AEC‐II (h, arrow) were milder but still present at 14 dpi (g, h).
T78 10755-10884 Sentence denotes Of note, acute alveolar damage (I, arrows) was multifocally distributed throughout the lungs across all time points investigated.
T79 10885-10952 Sentence denotes None of these lesions was detected in mock‐infected animals (j, k).
T80 10953-10958 Sentence denotes Bars:
T81 10959-11302 Sentence denotes 1 mm (a, c, e, g, j) or 50 µm (b, d, f, h, i, k) In agreement with the clinical course of disease, virological and histopathological results confirm that this animal model withstands the comparison with the Syrian hamster model with the advantage of a prolonged period of significantly reduced body weights following infection with SARS‐CoV‐2.
T82 11303-11510 Sentence denotes Moreover, the considerably smaller size of the Chinese hamster offers an important practical advantage when compared to the rather large Syrian hamster and allows husbandry in smaller cages and lager groups.
T83 11511-11801 Sentence denotes Additionally, the Chinese hamster genome, transcriptome and translatome are well characterized and a variety of molecular tools is available for this species as Chinese hamster ovary (CHO) cells are widely used in biopharmaceutical research and industry (Matasci et al., 2008; Walsh, 2018).
T84 11802-12054 Sentence denotes At the same time, the usefulness of any model depends on fundamental knowledge of species‐specific similarities to and differences from the human disease, as well as other confounding factors, including age and comorbidities (Metersky & Waterer, 2020).
T85 12055-12272 Sentence denotes Because of the heterogeneous disease phenotype in humans, there is a need for different SARS‐CoV‐2 animal models that allow evaluation of vaccines and antiviral therapies for different subsets of the human population.
T86 12273-12462 Sentence denotes Taken together, our results suggest the suitability of the Chinese hamster small animal model to study SARS‐CoV‐2 infections, with body weight losses as a robust clinical disease parameter.
T87 12463-12603 Sentence denotes As an established model for SARS‐CoV‐1 infection, the Chinese hamster may also allow comparison between experimental coronavirus infections.
T88 12604-12815 Sentence denotes Moreover, our findings highlight the possibility of comparing, in future studies, the effects of preventive as well as therapeutic measures to restrain infection and viral replication in different animal models.
T89 12817-12837 Sentence denotes CONFLICT OF INTEREST
T90 12838-12881 Sentence denotes The authors declare no competing interests.
T91 12883-12903 Sentence denotes AUTHOR CONTRIBUTIONS
T92 12904-12949 Sentence denotes NO and JT were involved in conceptualization.
T93 12950-13009 Sentence denotes LDB, DV, KD, AA, ADG and JT were involved in investigation.
T94 13010-13063 Sentence denotes LDB, DV, KD, ADG, NO and JT were involved in writing.
T95 13064-13160 Sentence denotes All authors were involved in editing and had the opportunity to comment on the draft manuscript.
T96 13162-13178 Sentence denotes ETHICAL APPROVAL
T97 13179-13385 Sentence denotes The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to, and the appropriate ethical review committee approval has been received.
T98 13386-13624 Sentence denotes Animal experimentations were approved by the Landesamt für Gesundheit und Soziales in Berlin, Germany (AUP number 0086/20) and performed in compliance with relevant national and international guidelines for care and humane use of animals.
T99 13625-13793 Sentence denotes All in vitro and animal work was conducted under appropriate biosafety conditions in the BSL‐3 facility at the Institute of Virology, Freie Universität Berlin, Germany.
T100 13795-13817 Sentence denotes Supporting information
T101 13818-13827 Sentence denotes Fig S1‐S2
T102 13828-13864 Sentence denotes Click here for additional data file.