Probes for INS

Acute kidney injury is common in patients with COVID-19, however mechanisms of kidney injury remain unclear. Since cytokine storm is likely a cause of AKI and glomerular disease, we investigated the two major transcription factors, STAT3 and NF-kB, which are known to be activated by cytokines.This is an observational study of the postmortem kidneys of 50 patients who died with COVID-19 in the Mount Sinai Hospital during the first pandemic surge. All samples were reviewed under light microscopy, electron microscopy, and immunofluorescence by trained renal pathologists. In situ hybridization evaluation for SARS-CoV-2 and immunostaining of transcription factors STAT3 and NF-kB were performed.Consistent with previous findings, acute tubular injury was the major pathological finding, together with global or focal glomerulosclerosis. We were not able to detect SARS-CoV-2 in kidney cells. ACE2 expression was reduced in the tubular cells of patients who died with COVID-19 and did not co-localize with TMPRSS2. SARS-CoV-2 was identified occasionally in the mononuclear cells in the peritubular capillary and interstitium. STAT3 phosphorylation at Tyr705 was increased in 2 cases in the glomeruli and in 3 cases in the tubulointerstitial compartments. Interestingly, STAT3 phosphorylation at Ser727 increased in 9 cases but only in the tubulointerstitial compartment. A significant increase in NF-kB phosphorylation at Ser276 was also found in the tubulointerstitium of the two patients with increased p-STAT3 (Tyr705).Our findings suggest that, instead of tyrosine phosphorylation, serine phosphorylation of STAT3 is commonly activated in the kidney of patients with COVID-19.

Data on the vertical transmission rate of COVID-19 in pregnancy are limited, while data reporting mother-fetal transmission in the second trimester of pregnancy are controversial. We described a case of second trimester twin stillbirth in a woman positive for SARS-CoV-2 in which, despite the absence of respiratory syndrome, placental and fetal markers of infection were detected. The patient developed a clinical chorioamnionitis and spontaneously delivered two stillborn infants. Placental histology and immunohistochemistry demonstrated SARS-CoV-2 infection mostly within the syncytiotrophoblast and the fetal autopsy showed development of interstitial pneumonia. Our findings demonstrate that, in utero vertical transmission is possible, also in asymptomatic SARS-CoV-2 pregnant women and that infection can lead to severe morbidity in the second trimester of pregnancy.

SARS-CoV-2, the causative agent of COVID-19, typically manifests as a respiratory illness although extrapulmonary involvement, such as in the gastrointestinal tract and nervous system, as well as frequent thrombotic events, are increasingly recognised. How this maps onto SARS-CoV-2 organ tropism at the histological level, however, remains unclear. Here, we perform a comprehensive validation of a monoclonal antibody against the SARS-CoV-2 nucleocapsid protein (NP) followed by systematic multisystem organ immunohistochemistry analysis of the viral cellular tropism in tissue from 36 patients, 16 post-mortem cases and 16 biopsies with polymerase chain reaction (PCR)-confirmed SARS-CoV-2 status from the peaks of the pandemic in 2020 and four pre-COVID post-mortem controls. SARS-CoV-2 anti-NP staining in the post-mortem cases revealed broad multiorgan involvement of the respiratory, digestive, haematopoietic, genitourinary and nervous systems, with a typical pattern of staining characterised by punctate paranuclear and apical cytoplasmic labelling. The average time from symptom onset to time of death was shorter in positively versus negatively stained post-mortem cases (mean = 10.3 days versus mean = 20.3 days, p = 0.0416, with no cases showing definitive staining if the interval exceeded 15 days). One striking finding was the widespread presence of SARS-CoV-2 NP in neurons of the myenteric plexus, a site of high ACE-2 expression, the entry receptor for SARS-CoV-2, and one of the earliest affected cells in Parkinson's disease. In the bone marrow, we observed viral SARS-CoV-2 NP within megakaryocytes, key cells in platelet production and thrombus formation. In 15 tracheal biopsies performed in patients requiring ventilation, there was a near complete concordance between immunohistochemistry and PCR swab results. Going forward, our findings have relevance to correlating clinical symptoms to the organ tropism of SARS-CoV-2 in contemporary cases as well as providing insights into potential long-term complications of COVID-19. This article is protected by

Messenger RNA (mRNA) vaccine technology has shown its power in preventing the ongoing COVID-19 pandemic. Two mRNA vaccines targeting the full-length S protein of SARS-CoV-2 have been authorized for emergency use. Recently, we have developed a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor-binding domain (RBD) of SARS-CoV-2 (termed ARCoV), which confers complete protection in mouse model. Herein, we further characterized the protection efficacy of ARCoV in nonhuman primates and the long-term stability under normal refrigerator temperature. Intramuscular immunization of two doses of ARCoV elicited robust neutralizing antibodies as well as cellular response against SARS-CoV-2 in cynomolgus macaques. More importantly, ARCoV vaccination in macaques significantly protected animals from acute lung lesions caused by SARS-CoV-2, and viral replication in lungs and secretion in nasal swabs were completely cleared in all animals immunized with low or high doses of ARCoV. No evidence of antibody-dependent enhancement of infection was observed throughout the study. Finally, extensive stability assays showed that ARCoV can be stored at 2-8 °C for at least 6 months without decrease of immunogenicity. All these promising results strongly support the ongoing clinical trial.

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.

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.

We report the case of a 77-year-old woman affected by coronavirus disease-19 (COVID-19) who developed an occlusive arterial disease of the lower limb requiring a left leg amputation. We studied the mechanisms of vascular damage by SARS-CoV-2 by means of a comprehensive multi-technique in situ analysis on the diseased popliteal arterial district, including immunohistochemistry (IHC), transmission electron microscopy (TEM) and miRNA analysis. At histological analyses, we observed a lymphocytic inflammatory infiltrate, oedema and endothelialitis of adventitial vasa vasorum while the media was normal and the intima had only minor changes. The vasa vasorum expressed the ACE2 receptor and factor VIII; compared with the controls, VEGFR2 staining was reduced. TEM analyses showed endothelial injury and numerous Weibel-Palade bodies in the cytoplasm. No coronavirus particle was seen. IL-6 protein and mRNA, together with miR-155-5p and miRs-27a-5p, which can target IL-6, were significantly increased compared with that in the controls. Our case report suggests an involvement of adventitial artery microcirculation by inflammation in the course of COVID-19. Without evident signs of current infection by SARS-CoV-2, endothelial cells show a spectrum of structural and functional alterations that can fuel the cardiovascular complications observed in people infected with SARS-CoV-2.

Japanese encephalitis virus (JEV), a member of the Flavivirus genus, is an important pathogen that causes human and animal infectious diseases in Asia. So far, no effective antiviral agents are available to treat JEV infection. Here, we found that LDLR is a host factor required for JEV entry. Berbamine significantly decreases the level of LDLR at the plasma membrane by inducing the secretion of LDLR via extracellular vesicles (EVs), thereby inhibiting JEV infection. Mechanistically, berbamine blocks TRPMLs (Ca2+ permeable non-selective cation channels in endosomes and lysosomes) to compromise the endolysosomal trafficking of LDLR. This leads to the increased secretion of LDLR via EVs and the concomitant decrease in its level at the plasma membrane, thereby rendering cells resistant to JEV infection. Berbamine also protects mice from the lethal challenge of JEV. In summary, these results indicate that berbamine is an effective anti-JEV agent by preventing JEV entry.