Probes for INS

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.

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.

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.

Liver damage and an exacerbated inflammatory response are hallmarks of Ebola virus (EBOV) infection. Little is known about the intrinsic response to infection in human hepatocytes and their contribution to inflammation. Here, we present an induced pluripotent stem cell (iPSC)-derived hepatocyte-like cell (HLC) platform to define the hepato-intrinsic response to EBOV infection. We used this platform to show robust EBOV infection, with characteristic ultrastructural changes and evidence for viral replication. Transcriptomics analysis revealed a delayed response with minimal early transcriptomic changes, followed by a general downregulation of hepatic function and upregulation of interferon signaling, providing a potential mechanism by which hepatocytes participate in disease severity and liver damage. Using RNA-fluorescence in situ hybridization (FISH), we showed that IFNB1 and CXCL10 were mainly expressed in non-infected bystander cells. We did not observe an inflammatory signature during infection. In conclusion, iPSC-HLCs are an immune competent platform to study responses to EBOV infection.

Mycobacterium bovis is the cause of tuberculosis in most animal species including cattle and is a serious zoonotic pathogen. In man, M. bovis infection can result in disease clinically indistinguishable from that caused by Mycobacterium tuberculosis, the cause of most human tuberculosis. Regardless of host, the typical lesion induced by M. bovis or M. tuberculosis is the tuberculoid granuloma. Tuberculoid granulomas are dynamic structures reflecting the interface between host and pathogen and, therefore, pass through various morphological stages (I to IV). Using a novel in-situ hybridization assay, transcription of various cytokine and chemokine genes was examined qualitatively and quantitatively using image analysis. In experimentally infected cattle, pulmonary granulomas of all stages were examined 150 days after aerosol exposure to M. bovis. Expression of mRNA encoding tumour necrosis factor (TNF)-α, transforming growth factor-β, interferon (IFN)-γ, interleukin (IL)-17A, IL-16, IL-10, CXCL9 and CXCL10 did not differ significantly between granulomas of different stages. However, relative expression of the various cytokines was characteristic of a Th1 response, with high TNF-α and IFN-γ expression and low IL-10 expression. Expression of IL-16 and the chemokines CXCL9 and CXCL10 was high, suggestive of granulomas actively involved in T-cell chemotaxis.

Widespread circulation of SARS-CoV-2 in humans raises the theoretical risk of reverse zoonosis events with wildlife, reintroductions of SARS-CoV-2 into permissive nondomesticated animals. Here we report that North American deer mice (Peromyscus maniculatus) are susceptible to SARS-CoV-2 infection following intranasal exposure to a human isolate, resulting in viral replication in the upper and lower respiratory tract with little or no signs of disease. Further, shed infectious virus is detectable in nasal washes, oropharyngeal and rectal swabs, and viral RNA is detectable in feces and occasionally urine. We further show that deer mice are capable of transmitting SARS-CoV-2 to naïve deer mice through direct contact. The extent to which these observations may translate to wild deer mouse populations remains unclear, and the risk of reverse zoonosis and/or the potential for the establishment of Peromyscus rodents as a North American reservoir for SARS-CoV-2 remains unknown.

-Although primarily considered a respiratory illness, coronavirus disease 2019 (COVID-19) can cause gastrointestinal manifestations. -To evaluate histopathology and in situ hybridization for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in gastrointestinal samples from patients with recent and remote COVID-19. -Patients with positive SARS-CoV-2 nasopharyngeal tests and a gastrointestinal tissue specimen were included. SARS-CoV-2 in situ hybridization (ISH) was performed on each sample. A subset had SARS-CoV-2 next generation sequencing (NGS) performed. -Twenty-five patients met inclusion criteria. Five had positive SARS-CoV-2 nasopharyngeal tests within 7 days of their gastrointestinal procedure. Two were ulcerative colitis patients on steroid therapy who lacked typical COVID-19 symptoms. Their colectomies showed severe ulcerative colitis; one demonstrated SARS-CoV-2 by NGS but a negative ISH. Another had an ischemic colon resected as a complication of the COVID-19 course; however, both ISH and NGS were negative. A fourth had a normal-appearing terminal ileum but positive ISH and NGS. The fifth patient had ileal ulcers with SARS-CoV-2 negativity by both modalities. The remaining 20 patients had positive nasopharyngeal tests an average of 53 days prior to procedure. None of their samples demonstrated SARS-CoV-2 ISH positivity, but one was positive on NGS despite a negative nasopharyngeal test. -Gastrointestinal findings from SARS-CoV-2-infected patients ranged from normal with virus detected by ISH and NGS, to bowel ischemia secondary to systemic viral effects, without evidence of virus in the tissue. No distinct histologic finding was identified in those with gastrointestinal tissue specimens demonstrating SARS-CoV-2 positivity in this cohort.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is transmitted on mink farms between minks and humans in many countries. However, the systemic pathological features of SARS-CoV-2-infected minks are mostly unknown. Here, we demonstrated that minks were largely permissive to SARS-CoV-2, characterized by severe and diffuse alveolar damage, and lasted at least 14 days post inoculation (dpi). We first reported that infected minks displayed multiple organ-system lesions accompanied by an increased inflammatory response and widespread viral distribution in the cardiovascular, hepatobiliary, urinary, endocrine, digestive, and immune systems. The viral protein partially co-localized with activated Mac-2+ macrophages throughout the body. Moreover, we first found that the alterations in lipids and metabolites were correlated with the histological lesions in infected minks, especially at 6 dpi, and were similar to that of patients with severe and fatal COVID-19. Particularly, altered metabolic pathways, abnormal digestion, and absorption of vitamins, lipids, cholesterol, steroids, amino acids, and proteins, consistent with hepatic dysfunction, highlight metabolic and immune dysregulation. Enriched kynurenine in infected minks contributed to significant activation of the kynurenine pathway and was related to macrophage activation. Melatonin, which has significant anti-inflammatory and immunomodulating effects, was significantly downregulated at 6 dpi and displayed potential as a targeted medicine. Our data first illustrate systematic analyses of infected minks to recapitulate those observations in severe and fetal COVID-19 patients, delineating a useful animal model to mimic SARS-CoV-2-induced systematic and severe pathophysiological features and provide a reliable tool for the development of effective and targeted treatment strategies, vaccine research, and potential biomarkers.

The rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created a global health emergency. While most human disease is mild to moderate, some infections lead to a severe disease characterized by acute respiratory distress, hypoxia, anosmia, ageusia, and, in some instances, neurological involvement. Small-animal models reproducing severe disease, including neurological sequela, are needed to characterize the pathophysiological mechanism(s) of disease and to identify medical countermeasures. Transgenic mice expressing the human angiotensin-converting enzyme 2 (hACE2) viral receptor under the control of the K18 promoter develop severe and lethal respiratory disease subsequent to SARS-CoV-2 intranasal challenge when high viral doses are used. Here, we report on SARS-CoV-2 infection of hamsters engineered to express the hACE2 receptor under the control of the K18 promoter. K18-hACE2 hamsters infected with a relatively low dose of 100 or 1,000 PFU of SARS-CoV-2 developed a severe and lethal disease, with most animals succumbing by day 5 postinfection. Hamsters developed severe lesions and inflammation within the upper and lower respiratory system, including infection of the nasal cavities causing marked destruction of the olfactory epithelium as well as severe bronchopneumonia that extended deep into the alveoli. Additionally, SARS-CoV-2 infection spread to the central nervous system (CNS), including the brain stem and spinal cord. Wild-type (WT) hamsters naturally support SARS-CoV-2 infection, with the primary lesions present in the respiratory tract and nasal cavity. Overall, infection in the K18-hACE2 hamsters is more extensive than that in WT hamsters, with more CNS involvement and a lethal outcome. These findings demonstrate the K18-hACE2 hamster model will be valuable for studying SARS-CoV-2. IMPORTANCE The rapid emergence of SARS-CoV-2 has created a global health emergency. While most human SARS-CoV-2 disease is mild, some people develop severe, life-threatening disease. Small-animal models mimicking the severe aspects of human disease are needed to more clearly understand the pathophysiological processes driving this progression. Here, we studied SARS-CoV-2 infection in hamsters engineered to express the human angiotensin-converting enzyme 2 viral receptor under the control of the K18 promoter. SARS-CoV-2 produces a severe and lethal infection in transgenic hamsters that mirrors the most severe aspects of COVID-19 in humans, including respiratory and neurological injury. In contrast to other animal systems, hamsters manifest disease with levels of input virus more consistent with natural human infection. This system will be useful for the study of SARS-CoV-2 disease and the development of drugs targeting this virus.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a historic pandemic of respiratory disease (coronavirus disease 2019 [COVID-19]), and current evidence suggests that severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here, we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2 signaling restricts the viral burden in the lung. We find that a recently developed mouse-adapted SARS-CoV-2 (MA-SARS-CoV-2) strain as well as the emerging B.1.351 variant trigger an inflammatory response in the lung characterized by the expression of proinflammatory cytokines and interferon-stimulated genes. Using intravital antibody labeling, we demonstrate that MA-SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45+ cells into the lung parenchyma that is dominated by monocyte-derived cells. Single-cell RNA sequencing (scRNA-Seq) analysis of lung homogenates identified a hyperinflammatory monocyte profile. We utilize this model to demonstrate that mechanistically, CCR2 signaling promotes the infiltration of classical monocytes into the lung and the expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified a potential CCR2-monocyte axis that is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2 has caused a historic pandemic of respiratory disease (COVID-19), and current evidence suggests that severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here, we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2-dependent infiltration of monocytes restricts the viral burden in the lung. We find that SARS-CoV-2 triggers an inflammatory response in the lung characterized by the expression of proinflammatory cytokines and interferon-stimulated genes. Using RNA sequencing and flow cytometry approaches, we demonstrate that SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45+ cells into the lung parenchyma that is dominated by monocyte-derived cells. Mechanistically, CCR2 signaling promoted the infiltration of classical monocytes into the lung and the expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified that the CCR2 pathway is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection.

During the SARS-CoV-2 pandemic, novel and traditional vaccine strategies have been deployed globally. We investigated whether antibodies stimulated by mRNA vaccination (BNT162b2), including 3rd dose boosting, differ from those generated by infection or adenoviral (ChAdOx1-S and Gam-COVID-Vac) or inactivated viral (BBIBP-CorV) vaccines. We analyzed human lymph nodes after infection or mRNA vaccination for correlates of serological differences. Antibody breadth against viral variants is less after infection compared to all vaccines evaluated, but improves over several months. Viral variant infection elicits variant-specific antibodies, but prior mRNA vaccination imprints serological responses toward Wuhan-Hu-1 rather than variant antigens. In contrast to disrupted germinal centers (GCs) in lymph nodes during infection, mRNA vaccination stimulates robust GCs containing vaccine mRNA and spike antigen up to 8 weeks post-vaccination in some cases. SARS-CoV-2 antibody specificity, breadth and maturation are affected by imprinting from exposure history, and distinct histological and antigenic contexts in infection compared to vaccination.