SARS-CoV-2 Infection Remodels the Phenotype and Promotes Angiogenesis of Primary Human Lung Endothelial Cells
Caccuri, F;Bugatti, A;Zani, A;De Palma, A;Di Silvestre, D;Manocha, E;Filippini, F;Messali, S;Chiodelli, P;Campisi, G;Fiorentini, S;Facchetti, F;Mauri, P;Caruso, A;
| DOI: 10.3390/microorganisms9071438
SARS-CoV-2-associated acute respiratory distress syndrome (ARDS) and acute lung injury are life-threatening manifestations of severe viral infection. The pathogenic mechanisms that lead to respiratory complications, such as endothelialitis, intussusceptive angiogenesis, and vascular leakage remain unclear. In this study, by using an immunofluorescence assay and in situ RNA-hybridization, we demonstrate the capability of SARS-CoV-2 to infect human primary lung microvascular endothelial cells (HL-mECs) in the absence of cytopathic effects and release of infectious particles. Preliminary data point to the role of integrins in SARS-CoV-2 entry into HL-mECs in the absence of detectable ACE2 expression. Following infection, HL-mECs were found to release a plethora of pro-inflammatory and pro-angiogenic molecules, as assessed by microarray analyses. This conditioned microenvironment stimulated HL-mECs to acquire an angiogenic phenotype. Proteome analysis confirmed a remodeling of SARS-CoV-2-infected HL-mECs to inflammatory and angiogenic responses and highlighted the expression of antiviral molecules as annexin A6 and MX1. These results support the hypothesis of a direct role of SARS-CoV-2-infected HL-mECs in sustaining vascular dysfunction during the early phases of infection. The construction of virus-host interactomes will be instrumental to identify potential therapeutic targets for COVID-19 aimed to inhibit HL-mEC-sustained inflammation and angiogenesis upon SARS-CoV-2 infection.
Japanese encephalitis virus manipulates lysosomes membrane for RNA replication and utilizes autophagy components for intracellular growth
Xu, Q;Huang, L;Xing, J;Zhang, J;Li, H;Liu, L;Hu, C;Liao, M;Yue, J;Qi, W;
PMID: 33725516 | DOI: 10.1016/j.vetmic.2021.109025
Japanese encephalitis virus is absolutely dependent on their host cells and has evolved various strategies to manipulate the cellular secretory pathways for viral replication. However, how cellular secretory pathways are hijacked, and the origin of the viral vesicles remains elusive during JEV replication. Here we show how JEV manipulates multiple components of the cellular secretory pathway, including autophagic machinery, to generate a superior environment for genome replication. We utilized double-strand RNA antibodies to label JEV RNA complex seeking the viral replication compartments and found that JEV genome replication takes place in lysosomes (LAMP1), not in autophagosomes (LC3). Subsequently, in situ hybridization results showed that viral RNAs (vRNAs) of JEV strongly colocalized with LAMP1. What surprised us was that JEV vRNAs markedly colocalized with LC3, indicating that autophagy plays an active role in JEV replication. Interestingly, we found that JEV utilized autophagic components for intracellular growth in an autophagy-dependent manner and the fusion of autophagosome-lysosome plays a positive role in JEV post-RNA replication processes. Collectively, our findings demonstrate that JEV can manipulate cellular secretory pathway to form genome replication organelles and exploit autophagy components for intracellular growth, providing new insights into the life cycle of JEV and uncovering an attractive target for antiviral drugs.
The Skin as a critical window in unveiling the pathophysiologic principles of COVID-19
Magro, C;Nuovo, G;Mulvey, J;Laurence, J;Harp, J;Neil Crowson, A;
| DOI: 10.1016/j.clindermatol.2021.07.001
The severe acute respiratory distress syndrome-associated coronavirus-2 (SARS-CoV-2), the etiologic agent of Coronavirus disease 2019 (COVID-19), is a single-stranded RNA virus whose sequence is known. COVID-19 is associated with a heterogeneous clinical phenotype ranging from asymptomatic to fatal disease. It appears that access to nasopharyngeal respiratory epithelia expressing angiotensin-converting enzyme (ACE) 2, the receptor for SARS CoV-2, is followed by viral replication in the pulmonary alveolar septal capillary bed. We have shown in prior studies that incomplete viral particles, termed pseudovirions, dock to deep subcutaneous and other vascular beds potentially contributing to the prothrombotic state and systemic complement activation that characterizes severe and critical COVID-19. A variety of skin rashes have been described in the setting of SARS-CoV-2 infection and more recently, following COVID-19 vaccination. The vaccines deliver a laboratory synthesized mRNA that encodes a protein that is identical to the spike glycoprotein of SARS-COV-2 allowing the production of immunogenic spike glycoprotein that will then elicit T cell and B cell adaptive immune responses. In this paper we review an array of cutaneous manifestations of COVID-19 that provide an opportunity to study critical pathophysiologic mechanisms that underlie all clinical facets of COVID-19 ranging from asymptomatic/mild to severe and critical COVID-19. We classify cutaneous COVID-19 according to underlying pathophysiologic principles. In this regard we propose two main pathways: 1) complement mediated thrombotic vascular injury syndromes deploying the alternative and mannan binding lectin pathways in the setting of severe and critical COVID-19 and 2) the robust T cell and type I interferon driven inflammatory and humoral driven immune complex mediated vasculitic cutaneous reactions seen with mild and moderate COVID-19. Novel data on cutaneous vaccine reactions are presented that manifest a clinical and morphologic parallel with similar eruptions seen in patients suffering from mild and moderate COVID-19 and in most cases represent systemic eczematoid hypersensitivity reactions to a putative vaccine based antigen. Finally, we show for the first time the localization of human synthesized spike glycoprotein following the COVID-19 vaccine to the cutaneous and subcutaneous vasculature confirming the ability of SARS CoV-2 spike glycoprotein to bind endothelium in the absence of intact virus.
Forebrain neural precursor cells are differentially vulnerable to Zika virus infection
Shelton, SM;Soucy, AR;Kurzion, R;Zeldich, E;Connor, JH;Haydar, TF;
PMID: 34272257 | DOI: 10.1523/ENEURO.0108-21.2021
Prenatal exposure to Zika virus (ZIKV) can result in microencephaly and congenital Zika syndrome, though some brain cells and structures are spared by the virus for unknown reasons. Here, a novel murine model of fetal ZIKV infection incorporating intraventricular infection and cell type specific in utero electroporation was used to identify the time course of ZIKV infection and to determine the identity of cells that are initially infected or spared during neocortical neurogenesis. In vivo time course studies revealed the presence of ZIKV in apical radial glial cells (aRGCs) at early time points following virus exposure, while basal intermediate progenitor cells (bIPCs) became maximally (ZIKV+) after 3 days of virus exposure. ZIKV-infected fetal brains exhibited microencephaly as early as one day following infection, regardless of developmental age. This change in brain size was caused in part by apoptosis and reduced proliferation that persisted until birth. While 60% of aRGC basal fibers were perturbed during infection, 40% retained normal morphology, indicating that aRGCs are not uniformly vulnerable to ZIKV infection. To investigate this heterogeneous vulnerability, we performed genetic fate mapping using cell type-specific probes derived from a mouse E15.5 neocortical wall single cell RNA-Seq dataset. The results indicate that one class of aRGCs preferentially express the putative ZIKV entry receptor AXL, and that these cells are more vulnerable to ZIKV infection than other aRGC subtypes with low AXL expression. Together, these data uncover crucial temporal and cellular details of ZIKV fetal brain infection for prevention strategies and for management of congenital Zika syndrome.Significance StatementThe transcriptional signatures of neural precursor cells were utilized for the first time to test Zika virus susceptibility in a direct fetal brain infection model. This novel methodology allowed for elucidation of time point specific differences in neural precursor cell susceptibility that have been debated in the field. Additionally, elucidation of cell morphological features using in utero electroporation revealed substantial but incomplete interruption of basal fibers, a finding that implies interference with neuronal migration. The model presented here, allows for assessment of pre-natal development after exposure to a variety of viruses. The improved specificity of apical radial glial cell labeling afforded by the cell-specific labeling tools uncover functional differences between apical radial glial cell types that will have important implications for children exposed to ZIKV as well as for understanding corticogenesis.
ORAL SECONDARY SYPHILIS IN PEOPLE LIVING WITH HIV: A 16-YEAR EXPERIENCE IN MEXICO CITY
Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology
Anaya-Saavedra, G;Castillejos-García, I;Maldonado-Mendoza, J;Ramírez-Amador, V;
| DOI: 10.1016/j.oooo.2021.03.041
Background The increase in syphilis rates worldwide, particularly in people living with HIV (PLWH), as well as the challenging diagnosis that secondary syphilis represents, make essential the accurate recognition of its manifestations, particularly in easy-access sites like the oral mucosa. Objective To describe the clinicopathologic spectrum of oral secondary syphilis (OSS) in PLWH. Methods A cross-sectional and descriptive study that included PLWH with OSS from 3 HIV referral centers in Mexico City (2004-2020). Demographic and clinical data were obtained. A comprehensive oral examination was done. OSS was diagnosed following established criteria. Histopathologic/cytological procedures were performed to rule out specific oral lesions. In all patients, Venereal Disease Research Laboratory tests were assessed and, if possible, a confirmatory fluorescent treponemal antibody test or biopsy was performed. Statistical analysis was performed using SPSS v25. Results Forty-seven PLWH with OSS (97.8% male, median age: 32 years, 63.8% with acquired immunodeficiency syndrome) were included. Thirty-five were receiving combination antiretroviral therapy (74.5%; median of 1146 [Q1-Q3: 337.5-1971] days) with a median CD4+ count of 385 (Q1-Q3: 223-664) cells/mm3 and a Log10 HIV viral load of 4.1 (Q1-Q3: 3.7-5.3) copies/mL. Forty had a complete clinical-serological diagnosis (85.1%; 17 had histopathologic confirmation) and 7 had a clinical-histopathologic diagnosis. Twenty-nine individuals presented 1 lesion (61.7%), and mucous patch was the most common type mainly on oropharyngeal mucosa, followed by ulcers and macular lesions. Ten patients presented maculopapular dermatosis (21.3%). Conclusions In PLWH, oral lesions, particularly mucous patch and/or ulcers on the oral and oropharyngeal mucosa, must alert specialists to consider a diagnosis of syphilis and perform a comprehensive panel of confirmatory tests.
Virchows Archiv : an international journal of pathology
Zito Marino, F;De Cristofaro, T;Varriale, M;Zannini, G;Ronchi, A;La Mantia, E;Campobasso, CP;De Micco, F;Mascolo, P;Municinò, M;Municinò, E;Vestini, F;Pinto, O;Moccia, M;De Stefano, N;Nappi, O;Sementa, C;Zotti, G;Pianese, L;Giordano, C;Franco, R;
PMID: 35103846 | DOI: 10.1007/s00428-021-03262-8
Post-mortem examination plays a pivotal role in understanding the pathobiology of the SARS-CoV-2; thus, the optimization of virus detection on the post-mortem formalin-fixed paraffin-embedded (FFPE) tissue is needed. Different techniques are available for the identification of the SARS-CoV-2, including reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), in situ hybridization (ISH), and electron microscopy. The main goal of this study is to compare ISH versus RT-PCR to detect SARS-CoV-2 on post-mortem lung samples of positive deceased subjects. A total of 27 samples were analyzed by RT-PCR targeting different viral RNA sequences of SARS-CoV-2, including envelope (E), nucleocapsid (N), spike (S), and open reading frame (ORF1ab) genes and ISH targeting S and Orf1ab. All 27 cases showed the N gene amplification, 22 out of 27 the E gene amplification, 26 out of 27 the S gene amplification, and only 6 the ORF1ab gene amplification. The S ISH was positive only in 12 out of 26 cases positive by RT-PCR. The S ISH positive cases with strong and diffuse staining showed a correlation with low values of the number of the amplification cycles by S RT-PCR suggesting that ISH is a sensitive assay mainly in cases carrying high levels of S RNA. In conclusion, our findings demonstrated that ISH assay has lower sensitivity to detect SARS-CoV-2 in FFPE compared to RT-PCR; however, it is able to localize the virus in the cellular context since it preserves the morphology.
Usefulness of high-risk human papillomavirus mRNA silver in situ hybridization diagnostic assay in oropharyngeal squamous cell carcinomas
Pathology, research and practice
Gale, N;Poljak, M;Volavšek, M;Hošnjak, L;Velkavrh, D;Bolha, L;Komloš, KF;Strojan, P;Aničin, A;Zidar, N;
PMID: 34455364 | DOI: 10.1016/j.prp.2021.153585
The transcriptional activity of high-risk human papillomaviruses (HR-HPV) within oropharyngeal squamous cell carcinomas (OPSCC) has been linked to improved survival of patients. HR-HPV mRNA silver in situ hybridization (SISH) was evaluated on a cohort of OPSCC and compared with viral HPV DNA tests and p16 expression. Clinical outcomes of HPV-driven OPSCC and non-HPV related OPSCC were also studied.We evaluated 67 OPSCC and 3 papillomas, obtained from 62 patients, for detection of HR-HPV DNA by PCR tests. The positive samples were additionally studied by the SISH method using three probes of HPV16, HPV18, and HP33, and for p16 expression detected by immunohistochemistry. SISH assays were evaluated for the presence/number and intensity of signals in cancer cells. Prognostic significance of HPV status in our cohort was evaluated with univariate and multivariate statistics.According to the HR-HPV PCR tests, 46 (69%) OPSCC cases were HPV positive, while three papillomas were negative. Of total 46 HPV-positive OPSCCs, 43 cases were also SISH-positive, while p16 overexpression was found in 45 of 46 HPV positive OPSCC cases. In OPSCC specimens, the sensitivity and specificity of the combined SISH probes (HPV16 and 33) were both 100.00%, when compared to HPV PCR. HPV positivity of the tumors appeared significant for predicting progression-free survival, cause specific survival and overall survival in a multivariate setting.The recently developed mRNA SISH methodology can detect HPV-driven OPSCCs without any additional test in 79% of cases. Positive SISH signals enable the visualization of viral transcripts required to recognize clinically relevant HPV infection. However, rare and tiny signals require an experienced pathologist to establish a consensus interpretation of results. The currently applied HR-HPV mRNA SISH analysis may serve as a groundwork for additional studies.
Endothelial cell infection and dysfunction, immune activation in severe COVID-19
Qin, Z;Liu, F;Blair, R;Wang, C;Yang, H;Mudd, J;Currey, JM;Iwanaga, N;He, J;Mi, R;Han, K;Midkiff, CC;Alam, MA;Aktas, BH;Heide, RSV;Veazey, R;Piedimonte, G;Maness, NJ;Ergün, S;Mauvais-Jarvis, F;Rappaport, J;Kolls, JK;Qin, X;
PMID: 34335981 | DOI: 10.7150/thno.61810
Rationale: Pulmonary vascular endotheliitis, perivascular inflammation, and immune activation are observed in COVID-19 patients. While the initial SARS-CoV-2 infection mainly infects lung epithelial cells, whether it also infects endothelial cells (ECs) and to what extent SARS-CoV-2-mediated pulmonary vascular endotheliitis is associated with immune activation remain to be determined. Methods: To address these questions, we studied SARS-CoV-2-infected K18-hACE2 (K18) mice, a severe COVID-19 mouse model, as well as lung samples from SARS-CoV-2-infected nonhuman primates (NHP) and patient deceased from COVID-19. We used immunostaining, RNAscope, and electron microscopy to analyze the organs collected from animals and patient. We conducted bulk and single cell (sc) RNA-seq analyses, and cytokine profiling of lungs or serum of the severe COVID-19 mice. Results: We show that SARS-CoV-2-infected K18 mice develop severe COVID-19, including progressive body weight loss and fatality at 7 days, severe lung interstitial inflammation, edema, hemorrhage, perivascular inflammation, systemic lymphocytopenia, and eosinopenia. Body weight loss in K18 mice correlated with the severity of pneumonia, but not with brain infection. We also observed endothelial activation and dysfunction in pulmonary vessels evidenced by the up-regulation of VCAM1 and ICAM1 and the downregulation of VE-cadherin. We detected SARS-CoV-2 in capillary ECs, activation and adhesion of platelets and immune cells to the vascular wall of the alveolar septa, and increased complement deposition in the lungs, in both COVID-19-murine and NHP models. We also revealed that pathways of coagulation, complement, K-ras signaling, and genes of ICAM1 and VCAM1 related to EC dysfunction and injury were upregulated, and were associated with massive immune activation in the lung and circulation. Conclusion: Together, our results indicate that SARS-CoV-2 causes endotheliitis via both infection and infection-mediated immune activation, which may contribute to the pathogenesis of severe COVID-19 disease.
Tissue-Based SARS-Cov-2 Detection in Fatal COVID-19 Infections: Sustained Direct Viral-Induced Damage is Not Necessary to Drive Disease Progression
El Jamal, SM;Pujadas, E;Ramos, I;Bryce, C;Grimes, ZM;Amanat, F;Tsankova, NM;Mussa, Z;Olson, S;Salem, F;Miorin, L;Aydillo, T;Schotsaert, M;Albrecht, RA;Liu, WC;Marjanovic, N;Francoeur, N;Sebra, R;Sealfon, SC;García-Sastre, A;Fowkes, M;Cordon-Cardo, C;Westra, WH;
PMID: 33961839 | DOI: 10.1016/j.humpath.2021.04.012
Coronavirus disease 2019 (COVID-19) is an ongoing pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although viral infection is known to trigger inflammatory processes contributing to tissue injury and organ failure, it is unclear whether direct viral damage is needed to sustain cellular injury. An understanding of pathogenic mechanisms has been handicapped by the absence of optimized methods to visualize the presence and distribution of SARS-CoV-2 in damaged tissues. We first developed a positive control cell line (Vero E6) to validate SARS-CoV-2 detection assays. We then evaluated multiple organs (lungs, kidneys, heart, liver, brain, intestines, lymph nodes and spleen) from fourteen COVID-19 autopsy cases using immunohistochemistry (IHC) for the spike and the nucleoprotein proteins, and RNA in-situ hybridization (RNA ISH) for the spike protein mRNA. Tissue detection assays were compared with quantitative PCR (qPCR)-based detection. SARS-CoV-2 was histologically detected in the Vero E6 positive cell line control, 1 of 14 (7%) lungs, and none (0%) of the other 59 organs. There was perfect concordance between the IHC and RNA ISH results. qPCR confirmed high viral load in the SARS-CoV-2 ISH-positive lung tissue, and absent or low viral load in all ISH-negative tissues. In patients who die of COVID-19-related organ failure, SARS-CoV-2 is largely not detectable using tissue-based assays. Even in lungs showing widespread injury, SARS-CoV-2 viral RNA or proteins were detected in only a small minority of cases. This observation supports the concept that viral infection is primarily a trigger for multiple organ pathogenic pro-inflammatory responses. Direct viral tissue damage is a transient phenomenon that is generally not sustained throughout disease progression.
Braspenning, SE;Verjans, GMGM;Mehraban, T;Messaoudi, I;Depledge, DP;Ouwendijk, WJD;
PMID: 34807956 | DOI: 10.1371/journal.ppat.1010084
Primary infection with varicella-zoster virus (VZV) causes varicella and the establishment of lifelong latency in sensory ganglion neurons. In one-third of infected individuals VZV reactivates from latency to cause herpes zoster, often complicated by difficult-to-treat chronic pain. Experimental infection of non-human primates with simian varicella virus (SVV) recapitulates most features of human VZV disease, thereby providing the opportunity to study the pathogenesis of varicella and herpes zoster in vivo. However, compared to VZV, the transcriptome and the full coding potential of SVV remains incompletely understood. Here, we performed direct long-read RNA sequencing to annotate the SVV transcriptome in lytically SVV-infected African green monkey (AGM) and rhesus macaque (RM) kidney epithelial cells. We refined structures of canonical SVV transcripts and uncovered numerous RNA isoforms, splicing events, fusion transcripts and non-coding RNAs, mostly unique to SVV. We verified the expression of canonical and newly identified SVV transcripts in vivo, using lung samples from acutely SVV-infected cynomolgus macaques. Expression of selected transcript isoforms, including those located in the unique left-end of the SVV genome, was confirmed by reverse transcription PCR. Finally, we performed detailed characterization of the SVV homologue of the VZV latency-associated transcript (VLT), located antisense to ORF61. Analogous to VZV VLT, SVV VLT is multiply spliced and numerous isoforms are generated using alternative transcription start sites and extensive splicing. Conversely, low level expression of a single spliced SVV VLT isoform defines in vivo latency. Notably, the genomic location of VLT core exons is highly conserved between SVV and VZV. This work thus highlights the complexity of lytic SVV gene expression and provides new insights into the molecular biology underlying lytic and latent SVV infection. The identification of the SVV VLT homolog further underlines the value of the SVV non-human primate model to develop new strategies for prevention of herpes zoster.
Dowall, S;Salguero, FJ;Wiblin, N;Fotheringham, S;Hatch, G;Parks, S;Gowan, K;Harris, D;Carnell, O;Fell, R;Watson, R;Graham, V;Gooch, K;Hall, Y;Mizen, S;Hewson, R;
PMID: 34835057 | DOI: 10.3390/v13112251
The global pandemic of coronavirus disease (COVID-19) caused by infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to an international thrust to study pathogenesis and evaluate interventions. Experimental infection of hamsters and the resulting respiratory disease is one of the preferred animal models since clinical signs of disease and virus shedding are similar to more severe cases of human COVID-19. The main route of challenge has been direct inoculation of the virus via the intranasal route. To resemble the natural infection, we designed a bespoke natural transmission cage system to assess whether recipient animals housed in physically separate adjacent cages could become infected from a challenged donor animal in a central cage, with equal airflow across the two side cages. To optimise viral shedding in the donor animals, a low and moderate challenge dose were compared after direct intranasal challenge, but similar viral shedding responses were observed and no discernible difference in kinetics. The results from our natural transmission set-up demonstrate that most recipient hamsters are infected within the system developed, with variation in the kinetics and levels of disease between individual animals. Common clinical outputs used for the assessment in directly-challenged hamsters, such as weight loss, are less obvious in hamsters who become infected from naturally acquiring the infection. The results demonstrate the utility of a natural transmission model for further work on assessing the differences between virus strains and evaluating interventions using a challenge system which more closely resembles human infection.
Porcine Epidemic Diarrhea Virus Induces Vero Cell Apoptosis via the p53-PUMA Signaling Pathway
Yang, L;Wang, C;Shu, J;Feng, H;He, Y;Chen, J;Shu, J;
PMID: 34202551 | DOI: 10.3390/v13071218
Porcine Epidemic Diarrhea Virus (PEDV) is the causative agent of swine epidemic diarrhea. In order to study the pathogenic mechanism of PEDV, PEDV was inoculated into Vero cells cultured in vitro, and the total RNA of Vero cells was extracted to construct a library for Illumina high-throughput sequencing and screening of differentially expressed genes (p < 0.05). Five differentially expressed genes for qRT-PCR verification analysis were randomly selected, and the verification results were consistent with the transcriptome sequencing results. The Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway enrichment analysis was performed on the differentially expressed genes screened above. The results showed that the target gene annotations of differentially expressed genes in the African green monkey genome were mainly enriched in the TNF signaling pathway, the P53 signaling pathway, the Jak-STAT signaling pathway, the MAPK signaling pathway, and immune inflammation. In addition, it has been reported that Puma can promote apoptosis and is a key mediator of P53-dependent and non-dependent apoptosis pathways. However, there is no report that PEDV infection can activate Puma and induce apoptosis in a P53-dependent pathway. It was found by flow cytometry that PEDV infection induced apoptosis, and by Western Blotting detection, PEDV infection significantly increased the expression of p53, BAX, and Puma apoptosis-related proteins. Treatment Vero cells with the p53 inhibitor, PFT-α, could significantly inhibit PEDV-induced apoptosis. Studies have shown that PEDV infection can activate Puma and induce apoptosis in a P53-dependent pathway. These findings provide data support for further elucidating the pathogenic mechanism of PEDV and developing an effective vaccine against PEDV.