Lee, D;Helal, Z;Kim, J;Hunt, A;Barbieri, A;Tocco, N;Frasca, S;Kerr, K;Hyeon, J;Chung, D;Risatti, G;
| DOI: 10.3390/v13112141
We report the first detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a 3-month-old dog in Connecticut that died suddenly and was submitted to the state veterinary diagnostic laboratory for postmortem examination. Viral RNA was detected in multiple organs of the dog by reverse transcription real time-PCR (RT-qPCR). Negative and positive sense strands of viral RNA were visualized by in situ hybridization using RNAscope technology. Complete genome sequencing and phylogenetic analysis of the hCoV-19/USA/CT-CVMDL-Dog-1/2021 (CT_Dog/2021) virus were conducted to identify the origin and lineage of the virus. The CT_Dog/2021 virus belonged to the GH/B1.2. genetic lineage and was genetically similar to SARS-CoV-2 identified in humans in the U.S. during the winter of 2020-2021. However, it was not related to other SARS-CoV-2 variants identified from companion animals in the U.S. It contained both the D614G in spike and P323L in nsp12 substitutions, which have become the dominant mutations in the United States. The continued sporadic detections of SARS-CoV-2 in companion animals warrant public health concerns about the zoonotic potential of SARS-CoV-2 and enhance our collective understanding of the epidemiology of the virus.
Journal of clinical pathology
Humphries, MP;Bingham, V;Abdullah Sidi, F;Craig, S;Lara, B;El-Daly, H;O'Doherty, N;Maxwell, P;Lewis, C;McQuaid, S;Lyness, J;James, J;Snead, DRJ;Salto-Tellez, M;
PMID: 36717223 | DOI: 10.1136/jcp-2022-208525
Interrogation of immune response in autopsy material from patients with SARS-CoV-2 is potentially significant. We aim to describe a validated protocol for the exploration of the molecular physiopathology of SARS-CoV-2 pulmonary disease using multiplex immunofluorescence (mIF).The application of validated assays for the detection of SARS-CoV-2 in tissues, originally developed in our laboratory in the context of oncology, was used to map the topography and complexity of the adaptive immune response at protein and mRNA levels.SARS-CoV-2 is detectable in situ by protein or mRNA, with a sensitivity that could be in part related to disease stage. In formalin-fixed, paraffin-embedded pneumonia material, multiplex immunofluorescent panels are robust, reliable and quantifiable and can detect topographic variations in inflammation related to pathological processes.Clinical autopsies have relevance in understanding diseases of unknown/complex pathophysiology. In particular, autopsy materials are suitable for the detection of SARS-CoV-2 and for the topographic description of the complex tissue-based immune response using mIF.
Choudhary, S;Kanevsky, I;Yildiz, S;Sellers, RS;Swanson, KA;Franks, T;Rathnasinghe, R;Munoz-Moreno, R;Jangra, S;Gonzalez, O;Meade, P;Coskran, T;Qian, J;Lanz, TA;Johnson, JG;Tierney, CA;Smith, JD;Tompkins, K;Illenberger, A;Corts, P;Ciolino, T;Dormitzer, PR;Dick, EJ;Shivanna, V;Hall-Ursone, S;Cole, J;Kaushal, D;Fontenot, JA;Martinez-Romero, C;McMahon, M;Krammer, F;Schotsaert, M;García-Sastre, A;
PMID: 35128980 | DOI: 10.1177/01926233211072767
Coronavirus disease 2019 (COVID-19) in humans has a wide range of presentations, ranging from asymptomatic or mild symptoms to severe illness. Suitable animal models mimicking varying degrees of clinical disease manifestations could expedite development of therapeutics and vaccines for COVID-19. Here we demonstrate that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection resulted in subclinical disease in rhesus macaques with mild pneumonia and clinical disease in Syrian hamsters with severe pneumonia. SARS-CoV-2 infection was confirmed by formalin-fixed, paraffin-embedded (FFPE) polymerase chain reaction (PCR), immunohistochemistry, or in situ hybridization. Replicating virus in the lungs was identified using in situ hybridization or virus plaque forming assays. Viral encephalitis, reported in some COVID-19 patients, was identified in one macaque and was confirmed with immunohistochemistry. There was no evidence of encephalitis in hamsters. Severity and distribution of lung inflammation were substantially more in hamsters compared with macaques and exhibited vascular changes and virus-induced cytopathic changes as seen in COVID-19 patients. Neither the hamster nor macaque models demonstrated evidence for multisystemic inflammatory syndrome (MIS). Data presented here demonstrate that macaques may be appropriate for mechanistic studies of mild asymptomatic COVID-19 pneumonia and COVID-19-associated encephalitis, whereas Syrian hamsters may be more suited to study severe COVID-19 pneumonia.
Keeler AM, Sapp E, Chase K, Sottosanti E, Danielson E, Pfister E, Stoica L, DiFiglia M, Aronin N, Sena-Esteves M.
PMID: 27689620 | DOI: 10.3233/JHD-160215
Abstract
BACKGROUND:
The genetic mutation in Huntington's disease (HD) is a CAG repeat expansion in the coding region of the huntingtin (Htt) gene. RNAi strategies have proven effective in substantially down-regulating Htt mRNA in the striatum through delivery of siRNAs or viral vectors based on whole tissue assays, but the extent of htt mRNA lowering in individual neurons is unknown.
OBJECTIVE:
Here we characterize the effect of an AAV9-GFP-miRHtt vector on Htt mRNA levels in striatal neurons of Q140/Q140 knock-in mice.
METHODS:
HD mice received bilateral striatal injections of AAV9-GFP-miRHtt or AAV9-GFP at 6 or 12 weeks and striata were evaluated at 6 months of age for levels of Htt mRNA and protein and for mRNA signal within striatal neurons using RNAscope multiplex fluorescence in situ hybridization.
RESULTS:
Compared to controls, the striatum of 6-month old mice treated at 6 or 12 weeks of age with AAV9-GFP-miRHtt showed a reduction of 40-50% in Htt mRNA and lowering of 25-40% in protein levels. The number of Htt mRNA foci in medium spiny neurons (MSNs) of untreated Q140/Q140 mice varied widely per cell (0 to 34 per cell), with ∼10% of MSNs devoid of foci. AAV9-GFP-miRHtt treatment shifted the distribution toward lower numbers and the percentage of cells without foci increased to 14-20%. The average number of Htt mRNA foci per MSN was reduced by 43%.
CONCLUSIONS:
The findings here show that intrastriatal infusion of an AAV9-GFP-miRHtt vector lowers mRNA expression of Htt in striatum by ∼50%, through a partial reduction in the number of copies of mutant Htt mRNAs per cell. These findings demonstrate at the neuronal level the variable levels of Htt mRNA expression in MSNs and the neuronal heterogeneity of RNAi dependent Htt mRNA knockdown.
Diffuse trophoblast damage is the hallmark of SARS-CoV-2-associated fetal demise
Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc
Garrido-Pontnou, M;Navarro, A;Camacho, J;Crispi, F;Alguacil-Guillén, M;Moreno-Baró, A;Hernandez-Losa, J;Sesé, M;Ramón Y Cajal, S;Garcia Ruíz, I;Serrano, B;Garcia-Aguilar, P;Suy, A;Ferreres, JC;Nadal, A;
PMID: 34006935 | DOI: 10.1038/s41379-021-00827-5
Placental pathology in SARS-CoV-2-infected pregnancies seems rather unspecific. However, the identification of the placental lesions due to SARS-CoV-2 infection would be a significant advance in order to improve the management of these pregnancies and to identify the mechanisms involved in a possible vertical transmission. The pathological findings in placentas delivered from 198 SARS-CoV-2-positive pregnant women were investigated for the presence of lesions associated with placental SARS-CoV-2 infection. SARS-CoV-2 infection was investigated in placental tissues through immunohistochemistry, and positive cases were further confirmed by in situ hybridization. SARS-CoV-2 infection was also investigated by RT-PCR in 33 cases, including all the immunohistochemically positive cases. Nine cases were SARS-CoV-2-positive by immunohistochemistry, in situ hybridization, and RT-PCR. These placentas showed lesions characterized by villous trophoblast necrosis with intervillous space collapse and variable amounts of mixed intervillous inflammatory infiltrate and perivillous fibrinoid deposition. Such lesions ranged from focal to massively widespread in five cases, resulting in intrauterine fetal death. Two of the stillborn fetuses showed some evidence of SARS-CoV-2 positivity. The remaining 189 placentas did not show similar lesions. The strong association between trophoblastic damage and placenta SARS-CoV-2 infection suggests that this lesion is a specific marker of SARS-CoV-2 infection in placenta. Diffuse trophoblastic damage, massively affecting chorionic villous tissue, can result in fetal death associated with COVID-19 disease.
Glycated ACE2 receptor in diabetes: open door for SARS-COV-2 entry in cardiomyocyte
Cardiovascular diabetology
D'Onofrio, N;Scisciola, L;Sardu, C;Trotta, MC;De Feo, M;Maiello, C;Mascolo, P;De Micco, F;Turriziani, F;Municinò, E;Monetti, P;Lombardi, A;Napolitano, MG;Marino, FZ;Ronchi, A;Grimaldi, V;Hermenean, A;Rizzo, MR;Barbieri, M;Franco, R;Campobasso, CP;Napoli, C;Municinò, M;Paolisso, G;Balestrieri, ML;Marfella, R;
PMID: 33962629 | DOI: 10.1186/s12933-021-01286-7
About 50% of hospitalized coronavirus disease 2019 (COVID-19) patients with diabetes mellitus (DM) developed myocardial damage. The mechanisms of direct SARS-CoV-2 cardiomyocyte infection include viral invasion via ACE2-Spike glycoprotein-binding. In DM patients, the impact of glycation of ACE2 on cardiomyocyte invasion by SARS-CoV-2 can be of high importance. To evaluate the presence of SARS-CoV-2 in cardiomyocytes from heart autopsy of DM cases compared to Non-DM; to investigate the role of DM in SARS-COV-2 entry in cardiomyocytes. We evaluated consecutive autopsy cases, deceased for COVID-19, from Italy between Apr 30, 2020 and Jan 18, 2021. We evaluated SARS-CoV-2 in cardiomyocytes, expression of ACE2 (total and glycosylated form), and transmembrane protease serine protease-2 (TMPRSS2) protein. In order to study the role of diabetes on cardiomyocyte alterations, independently of COVID-19, we investigated ACE2, glycosylated ACE2, and TMPRSS2 proteins in cardiomyocytes from DM and Non-DM explanted-hearts. Finally, to investigate the effects of DM on ACE2 protein modification, an in vitro glycation study of recombinant human ACE2 (hACE2) was performed to evaluate the effects on binding to SARS-CoV-2 Spike protein. The authors included cardiac tissue from 97 autopsies. DM was diagnosed in 37 patients (38%). Fourth-seven out of 97 autopsies (48%) had SARS-CoV-2 RNA in cardiomyocytes. Thirty out of 37 DM autopsy cases (81%) and 17 out of 60 Non-DM autopsy cases (28%) had SARS-CoV-2 RNA in cardiomyocytes. Total ACE2, glycosylated ACE2, and TMPRSS2 protein expressions were higher in cardiomyocytes from autopsied and explanted hearts of DM than Non-DM. In vitro exposure of monomeric hACE2 to 120 mM glucose for 12 days led to non-enzymatic glycation of four lysine residues in the neck domain affecting the protein oligomerization. The upregulation of ACE2 expression (total and glycosylated forms) in DM cardiomyocytes, along with non-enzymatic glycation, could increase the susceptibility to COVID-19 infection in DM patients by favouring the cellular entry of SARS-CoV2.
Bräuninger, H;Stoffers, B;Fitzek, ADE;Meißner, K;Aleshcheva, G;Schweizer, M;Weimann, J;Rotter, B;Warnke, S;Edler, C;Braun, F;Roedl, K;Scherschel, K;Escher, F;Kluge, S;Huber, TB;Ondruschka, B;Schultheiss, HP;Kirchhof, P;Blankenberg, S;Püschel, K;Westermann, D;Lindner, D;
PMID: 34647998 | DOI: 10.1093/cvr/cvab322
Cardiac involvement in COVID-19 is associated with adverse outcome. However, it is unclear whether cell specific consequences are associated with cardiac SARS-CoV-2 infection. Therefore, we investigated heart tissue utilizing in situ hybridization, immunohistochemistry and RNA-sequencing in consecutive autopsy cases to quantify virus load and characterize cardiac involvement in COVID-19.In this study, 95 SARS-CoV-2-positive autopsy cases were included. A relevant SARS-CoV-2 virus load in the cardiac tissue was detected in 41/95 deceased (43%). MACE-RNA-sequencing was performed to identify molecular pathomechanisms caused by the infection of the heart. A signature matrix was generated based on the single-cell dataset "Heart Cell Atlas" and used for digital cytometry on the MACE-RNA-sequencing data. Thus, immune cell fractions were estimated and revealed no difference in immune cell numbers in cases with and without cardiac infection. This result was confirmed by quantitative immunohistological diagnosis.MACE-RNA-sequencing revealed 19 differentially expressed genes (DEGs) with a q-value <0.05 (e.g. up: IFI44L, IFT3, TRIM25; down: NPPB, MB, MYPN). The upregulated DEGs were linked to interferon pathways and originate predominantly from endothelial cells. In contrast, the downregulated DEGs originate predominately from cardiomyocytes. Immunofluorescent staining showed viral protein in cells positive for the endothelial marker ICAM1 but rarely in cardiomyocytes. The GO term analysis revealed that downregulated GO terms were linked to cardiomyocyte structure, whereas upregulated GO terms were linked to anti-virus immune response.This study reveals, that cardiac infection induced transcriptomic alterations mainly linked to immune response and destruction of cardiomyocytes. While endothelial cells are primarily targeted by the virus, we suggest cardiomyocyte-destruction by paracrine effects. Increased pro-inflammatory gene expression was detected in SARS-CoV-2-infected cardiac tissue but no increased SARS-CoV-2 associated immune cell infiltration was observed.Cardiac injury can be documented in COVID-19, regardless the direct cardiac virus infection and is known to be associated with outcome. However, the direct virus infection of the myocardium leads to transcriptomic alterations and might therefore additionally contribute to pathophysiological processes in COVID-19. Therefore, consequences of cardiac virus infection need to be investigated in future studies, since they might also contribute to long-term effects in case of survival.
Tissue factor upregulation is associated with SARS-CoV-2 in the lungs of COVID-19 patients
Journal of thrombosis and haemostasis : JTH
Subrahmanian, S;Borczuk, A;Salvatore, S;Fung, KM;Merrill, JT;Laurence, J;Ahamed, J;
PMID: 34236752 | DOI: 10.1111/jth.15451
A substantial proportion of patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop severe/critical coronavirus disease 2019 (COVID-19) characterized by acute respiratory distress syndrome (ARDS) with thrombosis.We tested the hypothesis that SARS-CoV-2--induced upregulation of tissue factor (TF) expression may be responsible for thrombus formation in COVID-19.We compared autopsy lung tissues from 11 patients with COVID-19--associated ARDS with samples from 6 patients with ARDS from other causes (non-COVID-19 ARDS) and 11 normal control lungs.Dual RNA in situ hybridization for SARS-CoV-2 and TF identified sporadic clustered SARS-CoV-2 with prominent co-localization of SARS-CoV-2 and TF RNA. TF expression was 2-fold higher in COVID-19 than in non-COVID-19 ARDS lungs (P = .017) and correlated with the intensity of SARS-CoV-2 staining (R2 = .36, P = .04). By immunofluorescence, TF protein expression was 2.1-fold higher in COVID-19 versus non-COVID-19 ARDS lungs (P = .0048) and 11-fold (P < .001) higher than control lungs. Fibrin thrombi and thrombi positive for platelet factor 4 (PF4) were found in close proximity to regions expressing TF in COVID-19 ARDS lung, and correlated with TF expression (fibrin, R2 = .52, P < .001; PF4, R2 = .59, P < .001).These data suggest that upregulation of TF expression is associated with thrombus formation in COVID-19 lungs and could be a key therapeutic target. Correlation of TF expression with SARS-CoV-2 in lungs of COVID-19 patients also raises the possibility of direct TF induction by the virus.
SARS-CoV-2 crosses the blood-brain barrier accompanied with basement membrane disruption without tight junctions alteration
Signal transduction and targeted therapy
Zhang, L;Zhou, L;Bao, L;Liu, J;Zhu, H;Lv, Q;Liu, R;Chen, W;Tong, W;Wei, Q;Xu, Y;Deng, W;Gao, H;Xue, J;Song, Z;Yu, P;Han, Y;Zhang, Y;Sun, X;Yu, X;Qin, C;
PMID: 34489403 | DOI: 10.1038/s41392-021-00719-9
SARS-CoV-2 has been reported to show a capacity for invading the brains of humans and model animals. However, it remains unclear whether and how SARS-CoV-2 crosses the blood-brain barrier (BBB). Herein, SARS-CoV-2 RNA was occasionally detected in the vascular wall and perivascular space, as well as in brain microvascular endothelial cells (BMECs) in the infected K18-hACE2 transgenic mice. Moreover, the permeability of the infected vessel was increased. Furthermore, disintegrity of BBB was discovered in the infected hamsters by administration of Evans blue. Interestingly, the expression of claudin5, ZO-1, occludin and the ultrastructure of tight junctions (TJs) showed unchanged, whereas, the basement membrane was disrupted in the infected animals. Using an in vitro BBB model that comprises primary BMECs with astrocytes, SARS-CoV-2 was found to infect and cross through the BMECs. Consistent with in vivo experiments, the expression of MMP9 was increased and collagen IV was decreased while the markers for TJs were not altered in the SARS-CoV-2-infected BMECs. Besides, inflammatory responses including vasculitis, glial activation, and upregulated inflammatory factors occurred after SARS-CoV-2 infection. Overall, our results provide evidence supporting that SARS-CoV-2 can cross the BBB in a transcellular pathway accompanied with basement membrane disrupted without obvious alteration of TJs.
Acta neuropathologica communications
Bauer, S;Chen, CY;Jonson, M;Kaczmarczyk, L;Magadi, SS;Jackson, WS;
PMID: 36670467 | DOI: 10.1186/s40478-022-01500-x
Although Huntington's disease (HD) is classically defined by the selective vulnerability of striatal projection neurons, there is increasing evidence that cerebellar degeneration modulates clinical symptoms. However, little is known about cell type-specific responses of cerebellar neurons in HD. To dissect early disease mechanisms in the cerebellum and cerebrum, we analyzed translatomes of neuronal cell types from both regions in a new HD mouse model. For this, HdhQ200 knock-in mice were backcrossed with the calm 129S4 strain, to constrain experimental noise caused by variable hyperactivity of mice in a C57BL/6 background. Behavioral and neuropathological characterization showed that these S4-HdhQ200 mice had very mild behavioral abnormalities starting around 12 months of age that remained mild up to 18 months. By 9 months, we observed abundant Huntingtin-positive neuronal intranuclear inclusions (NIIs) in the striatum and cerebellum. The translatome analysis of GABAergic cells of the cerebrum further confirmed changes typical of HD-induced striatal pathology. Surprisingly, we observed the strongest response with 626 differentially expressed genes in glutamatergic neurons of the cerebellum, a population consisting primarily of granule cells, commonly considered disease resistant. Our findings suggest vesicular fusion and exocytosis, as well as differentiation-related pathways are affected in these neurons. Furthermore, increased expression of cyclin D1 (Ccnd1) in the granular layer and upregulated expression of polycomb group complex protein genes and cell cycle regulators Cbx2, Cbx4 and Cbx8 point to a putative role of aberrant cell cycle regulation in cerebellar granule cells in early disease.
Human Type II Taste Cells Express ACE2 and are Infected by SARS-CoV-2
The American journal of pathology
Doyle, ME;Appleton, A;Liu, QR;Yao, Q;Mazucanti, CH;Egan, JM;
PMID: 34102107 | DOI: 10.1016/j.ajpath.2021.05.010
Chemosensory changes are well-reported symptoms of SARS-CoV-2 infection. The virus targets cells for entry by binding of its spike protein to cell-surface angiotensin-converting enzyme- 2 (ACE2). It was not known whether ACE2 is expressed on taste receptor cells (TRCs) nor if TRCs are infected directly. Using an in-situ hybridization (ISH) probe and an antibody specific to ACE2, ACE2 is present on a subpopulation of TRCs, namely, Type II cells in taste buds in taste papillae. Fungiform papillae (FP) of a SARS-CoV-2+ patient exhibiting symptoms of COVID-19, including taste changes, were biopsied. Based on ISH, replicating SARS-CoV-2 was present in Type II cells. Therefore, taste Type II cells provide a potential portal for viral entry that predicts vulnerabilities to SARS-CoV-2 in the oral cavity. The continuity and cell turnover of the patient's FP taste stem cell layer were disrupted during infection and had not completely recovered 6 weeks post symptom onset. Another patient suffering post-COVID-19 taste disturbances also had disrupted stem cells. These results demonstrate the possibility that novel and sudden taste changes frequently reported in COVID-19 may be the result of direct infection of taste papillae by SARS-CoV-2. This may result in impaired taste receptor stem cell activity and suggest more work is needed to understand the acute and post-acute dynamics of viral kinetics in the human taste bud.
Robustelli Test, E;Sena, P;Locatelli, AG;Carugno, A;di Mercurio, M;Moggio, E;Gambini, DM;Arosio, MEG;Callegaro, A;Morotti, D;Gianatti, A;Vezzoli, P;
PMID: 34989043 | DOI: 10.1111/pde.14903
Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, an increasing number of chilblain-like lesions (ChLL) have been increasingly reported worldwide. To date, the causal link between ChLL and SARS-CoV-2 infection has not been unequivocally established.In this case series, we present demographic, clinical, laboratory, and histopathological information regarding 27 young patients with a clinical diagnosis of ChLL who referred to the Dermatology Unit of Papa Giovanni XXIII Hospital, Bergamo, Italy, from 1 April 2020 to 1 June 2020.The mean age was 14.2 years, and 21 patients (78%) experienced mild systemic symptoms a median of 28 days before the onset of cutaneous lesions. ChLL mostly involved the feet (20 patients - 74%). Among acral lesions, we identified three different clinical patterns: (i) chilblains in 20 patients (74%); (ii) fixed erythematous macules in 4 children (15%); (iii) erythrocyanosis in 3 female patients (11%). Blood examinations and viral serologies, including parvovirus B19, cytomegalovirus (CMV), Epstein-Barr virus (EBV), and coxsackievirus were normal in all. Three patients (11%) underwent nasopharyngeal swab for RT-PCR for SARS-CoV-2 showing only 1 positive. Histopathological examinations of 7 skin biopsies confirmed the clinical diagnosis of chilblains; vessel thrombi were observed only in 1 case. Our findings failed to demonstrate the direct presence of SARS-CoV-2 RNA in skin biopsies, both with real-time polymerase chain reaction (RT-PCR) and RNAscope in situ hybridization (ISH).Limited number of cases, unavailability of laboratory confirmation of COVID-19 in all patients, potential methodological weakness, and latency of skin biopsies in comparison to cutaneous lesions onset.These observations may support the hypothesis of an inflammatory pathogenesis rather than the presence of peripheral viral particles. Although, we could not exclude an early phase of viral endothelial damage followed by an IFN-I or complement-mediated inflammatory phase. Further observations on a large number of patients are needed to confirm this hypothesis.
