Pulmonary inflammation and viral replication define distinct clinical outcomes in fatal cases of COVID-19.

COVID-19 has affected more than half a billion people worldwide, with more than 6.3 million deaths, but the pathophysiological mechanisms involved in lethal cases and the host determinants that determine the different clinical outcomes are still unclear. In this study, we assessed lung autopsies of 47 COVID-19 patients and examined the inflammatory profiles, viral loads, and inflammasome activation. Additionally, we correlated these factors with the patient's clinical and histopathological conditions. Robust inflammasome activation was detected in the lungs of lethal cases of SARS-CoV-2. Experiments conducted on transgenic mice expressing hACE2 and infected with SARS-CoV-2 showed that Nlrp3-/- mice were protected from disease development and lethality compared to Nlrp3+/+ littermate mice, supporting the involvement of this inflammasome in disease exacerbation. An analysis of gene expression allowed for the classification of COVID-19 patients into two different clusters. Cluster 1 died with higher viral loads and exhibited a reduced inflammatory profile than Cluster 2. Illness time, mechanical ventilation time, pulmonary fibrosis, respiratory functions, histopathological status, thrombosis, viral loads, and inflammasome activation significantly differed between the two clusters. Our data demonstrated two distinct profiles in lethal cases of COVID-19, thus indicating that the balance of viral replication and inflammasome-mediated pulmonary inflammation led to different clinical outcomes. We provide important information to understand clinical variations in severe COVID-19, a process that is critical for decisions between immune-mediated or antiviral-mediated therapies for the treatment of critical cases of COVID-19.

COVID-19-related hyperglycemia is associated with infection of hepatocytes and stimulation of gluconeogenesis.

Occurrence of hyperglycemia upon infection is associated with worse clinical outcome in COVID-19 patients. However, it is still unknown whether SARS-CoV-2 directly triggers hyperglycemia. Herein, we interrogated whether and how SARS-CoV-2 causes hyperglycemia by infecting hepatocytes and increasing glucose production. We performed a retrospective cohort study including patients that were admitted at a hospital with suspicion of COVID-19. Clinical and laboratory data were collected from the chart records and daily blood glucose values were analyzed to test the hypothesis on whether COVID-19 was independently associated with hyperglycemia. Blood glucose was collected from a subgroup of nondiabetic patients to assess pancreatic hormones. Postmortem liver biopsies were collected to assess the presence of SARS-CoV-2 and its transporters in hepatocytes. In human hepatocytes, we studied the mechanistic bases of SARS-CoV-2 entrance and its gluconeogenic effect. SARS-CoV-2 infection was independently associated with hyperglycemia, regardless of diabetic history and beta cell function. We detected replicating viruses in human hepatocytes from postmortem liver biopsies and in primary hepatocytes. We found that SARS-CoV-2 variants infected human hepatocytes in vitro with different susceptibility. SARS-CoV-2 infection in hepatocytes yields the release of new infectious viral particles, though not causing cell damage. We showed that infected hepatocytes increase glucose production and this is associated with induction of PEPCK activity. Furthermore, our results demonstrate that SARS-CoV-2 entry in hepatocytes occurs partially through ACE2- and GRP78-dependent mechanisms. SARS-CoV-2 infects and replicates in hepatocytes and exerts a PEPCK-dependent gluconeogenic effect in these cells that potentially is a key cause of hyperglycemia in infected patients.

Alpha 1-acid glycoprotein is upregulated in severe COVID-19 patients and decreases neutrophil NETs in SARS-CoV-2 infection.

Orosomucoid, or alpha-1 acid glycoprotein (AGP), is a major acute-phase protein expressed in response to systemic injury and inflammation. AGP has been described as an inhibitor of neutrophil migration on sepsis, particularly its immunomodulation effects. AGP's biological functions in coronavirus disease 2019 (COVID-19) are not understood. We sought to investigate the role of AGP in severe COVID-19 infection patients and neutrophils infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Epidemiological data, AGP levels, and other laboratory parameters were measured in blood samples from 56 subjects hospitalized in the ICU with SARS-CoV-2 infection. To evaluate the role of AGP in NETosis in neutrophils, blood samples from health patients were collected, and neutrophils were separated and infected with SARS-CoV-2. Those neutrophils were treated with AGP or vehicle, and NETosis was analyzed by flow cytometry. AGP was upregulated in severe COVID-19 patients (p<0.05). AGP level was positively correlated with IL-6 and C-reactive protein (respectively, p=0.005, p=0.002) and negatively correlated with lactate (p=0.004). AGP treatment downregulated early and late NETosis (respectively, 35.7% and 43.5%) in neutrophils infected with SARS-CoV-2 and up-regulated IL-6 supernatant culture expression (p<0.0001). Our data showed increased AGP in COVID-19 infection and contributed to NETosis regulation and increased IL-6 production, possibly related to the Cytokine storm in COVID-19.

Exploring the impact of high-energy diets on cattle: Insights into subacute rumen acidosis, insulin resistance, and hoof health.

Cattle lameness remains a significant concern, causing economic losses and compromising animal welfare. Claw horn lesions have been identified as a major cause of lameness in dairy cows, but their correlation with high-energy diets and ruminal acidosis remains unclear. Hence, the primary objective of this study was to assess the effects of a high-starch diet and a conventional diet on the rumen environment, acute-phase proteins, and metabolic alterations, with a particular focus on insulin resistance and the consequent implications for the histology of the hooves in Holstein steers. A total of 16 animals were divided into the high-starch (HS; 37% starch) and conventional (CON; 16.8% starch) groups. Glucose tolerance tests (GTT), blood analyses, rumen fluid analyses, and histological evaluations of the hoof tissue were conducted over a 102-d experimental period. The HS group showed a lower ruminal pH than the CON group, and with values indicating SARA. The plasma glucose and IGF-1 concentrations were higher in the HS group, suggesting an anabolic state. Both groups exhibited an increase in the insulin area under the curve (AUC) after the GTT on d 102. Histological analysis of the hooves showed a reduction in the length and width of the epidermal lamella in both groups. We found a significant negative correlation between the insulin AUC and the length and width of the epidermal lamella. Because both groups were similarly affected, the hypothesis that histological alterations were caused by the experimental diets still needs confirmation. Additionally, the development of SARA was not essential for the observed histological changes in the hoof. Further studies are warranted to thoroughly investigate the role of insulin and IGF-1 imbalances in claw health.

CASP4/11 Contributes to NLRP3 Activation and COVID-19 Exacerbation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection triggers activation of the NLRP3 inflammasome, which promotes inflammation and aggravates severe COVID-19. Here, we report that SARS-CoV-2 induces upregulation and activation of human caspase-4/CASP4 (mouse caspase-11/CASP11), and this process contributes to NLRP3 activation. In vivo infections performed in transgenic hACE2 humanized mice, deficient or sufficient for Casp11, indicate that hACE2 Casp11-/- mice were protected from disease development, with the increased pulmonary parenchymal area, reduced clinical score of the disease, and reduced mortality. Assessing human samples from fatal cases of COVID-19, we found that CASP4 was expressed in patient lungs and correlated with the expression of inflammasome components and inflammatory mediators, including CASP1, IL1B, IL18, and IL6. Collectively, our data establish that CASP4/11 promotes NLRP3 activation and disease pathology, revealing a possible target for therapeutic interventions for COVID-19.

Neutrophil virucidal activity against SARS-CoV-2 is mediated by NETs.

BACKGROUND: The inflammation in the lungs and other vital organs in COVID-19 are characterized by the presence of neutrophils and high concentration of neutrophil extracellular traps (NETs), which also seems to mediate host tissue damage. However, it is not known whether NETs could have virucidal activity against SARS-CoV-2. METHODS: We investigated whether NETs could prevent SARS-CoV-2 replication in neutrophils and epithelial cells, and what the consequence of NETs degradation in K18-humanized ACE2 transgenic mice infected with SARS-CoV-2. RESULTS: Here, by immunofluorescence microscopy we observed that viral particles co-localize with NETs in neutrophils isolated from COVID-19 patients or from healthy individuals and infected in vitro. The inhibition of NETs production increased virus replication in neutrophils. In parallel, we observed that NETs inhibited virus abilities to infect and replicate in epithelial cells after 24 h of infection. Degradation of NETs with DNase I prevented their virucidal effect in vitro. Using K18-humanized ACE2 transgenic mice we observed a higher viral load in animals treated with DNase I. On the other hand, the virucidal effect of NETs was not dependent on neutrophil elastase or myeloperoxidase activity. CONCLUSION: Our results provide evidence of the role of NETosis as a mechanism of SARS-CoV-2 viral capture and inhibition.

Enoxaparin improves COVID-19 by reducing Neutrophils Extracellular Traps (NETs) production.

BACKGROUND: COVID-19 causes consequences such as imbalance of the immune system and thrombotic events. During the infection process, NETs in excess induce a pro-inflammatory response and disseminated intravascular coagulation. We evaluated the role of enoxaparin as a potential inhibitor of NETs. METHODS: K18-hACE2 animals infected with the SARS-CoV-2 virus and a group of 23 individuals admitted to the hospital with COVID-19 treated with enoxaparin or without treatment and controls without the disease were included. RESULTS: Enoxaparin decreased the levels of NETs, reduced the signs of the disease and mitigated lung damage in the animals infected with SARS-CoV-2. These effects were partially associated with prevention of SARS-CoV-2 entry and NETs synthesis. Clinical data revealed that treatment with enoxaparin decreased the levels of inflammatory markers, the levels of NETs in isolated neutrophils and the organ dysfunction. CONCLUSION: This study provides evidence for the beneficial effects of enoxaparin in COVID-19 in addition to its anticoagulant role.

Cytokine storm in individuals with severe COVID-19 decreases endothelial cell antioxidant defense via downregulation of the Nrf2 transcriptional factor.

The cytokine storm in SARS-CoV-2 infection contributes to the onset of inflammation and target-organ damage. The endothelium is a key player in COVID-19 pathophysiology and it is an important target for cytokines. Considering that cytokines trigger oxidative stress and negatively impact endothelial cell function, we sought to determine whether serum from individuals with severe COVID-19 decreases endothelial cells' main antioxidant defense, i.e., the antioxidant transcriptional factor Nrf2. Human umbilical vein endothelial cells (HUVECs) were incubated with serum from patients with severe COVID-19 at different time points and the effects on redox balance and Nrf2 activity were determined. Serum from individuals with COVID-19 increased oxidant species, as indicated by higher DHE (dihydroethydine) oxidation, increased protein carbonylation, and induced mitochondrial reactive oxygen species (ROS) generation and dysfunction. Serum from patients with COVID-19, but not serum from healthy individuals, induced cell death and diminished nitric oxide (NO) bioavailability. In parallel, Nrf2 nuclear accumulation and the expression of Nrf2-targeted genes were decreased in endothelial cells exposed to serum from individuals with COVID-19. In addition, these cells exhibited higher expression of Bach-1, a negative regulator of Nrf2 that competes for DNA binding. All events were prevented by tocilizumab, an IL-6 receptor blocker, indicating that IL-6 is key to the impairment of endothelial antioxidant defense. In conclusion, endothelial dysfunction related to SARS-CoV-2 infection is linked to decreased endothelial antioxidant defense via IL-6-dependent mechanisms. Pharmacological activation of Nrf2 may decrease endothelial cell damage in individuals with severe COVID-19.NEW & NOTEWORTHY We demonstrate that endothelial cell dysfunction in SARS-CoV-2-infected individuals is linked to decreased activity of the major antioxidant system regulator, the Nrf2 transcription factor. We provide evidence that this phenomenon relies on IL-6, an important cytokine involved in the pathophysiology of COVID-19. Our data support the view that Nrf2 activation is a potential therapeutical strategy to prevent oxidative stress and vascular inflammation in severe cases of COVID-19.

Targeting neutrophils extracellular traps (NETs) reduces multiple organ injury in a COVID-19 mouse model.

BACKGROUND: COVID-19 is characterized by severe acute lung injury, which is associated with neutrophil infiltration and the release of neutrophil extracellular traps (NETs). COVID-19 treatment options are scarce. Previous work has shown an increase in NETs release in the lung and plasma of COVID-19 patients suggesting that drugs that prevent NETs formation or release could be potential therapeutic approaches for COVID-19 treatment. METHODS: Here, we report the efficacy of NET-degrading DNase I treatment in a murine model of COVID-19. SARS-CoV-2-infected K18-hACE2 mice were performed for clinical sickness scores and lung pathology. Moreover, the levels of NETs were assessed and lung injuries were by histopathology and TUNEL assay. Finally, the injury in the heart and kidney was assessed by histopathology and biochemical-specific markers. RESULTS: DNase I decreased detectable levels of NETs, improved clinical disease, and reduced lung, heart, and kidney injuries in SARS-CoV-2-infected K18-hACE2 mice. Furthermore, our findings indicate a potentially deleterious role for NETs lung tissue in vivo and lung epithelial (A549) cells in vitro, which might explain part of the pathophysiology of severe COVID-19. This deleterious effect was diminished by the treatment with DNase I. CONCLUSIONS: Together, our results support the role of NETs in COVID-19 immunopathology and highlight NETs disruption pharmacological approaches as a potential strategy to ameliorate COVID-19 clinical outcomes.

Gasdermin-D activation by SARS-CoV-2 triggers NET and mediate COVID-19 immunopathology.

BACKGROUND: The release of neutrophil extracellular traps (NETs) is associated with inflammation, coagulopathy, and organ damage found in severe cases of COVID-19. However, the molecular mechanisms underlying the release of NETs in COVID-19 remain unclear. OBJECTIVES: We aim to investigate the role of the Gasdermin-D (GSDMD) pathway on NETs release and the development of organ damage during COVID-19. METHODS: We performed a single-cell transcriptome analysis in public data of bronchoalveolar lavage. Then, we enrolled 63 hospitalized patients with moderate and severe COVID-19. We analyze in blood and lung tissue samples the expression of GSDMD, presence of NETs, and signaling pathways upstreaming. Furthermore, we analyzed the treatment with disulfiram in a mouse model of SARS-CoV-2 infection. RESULTS: We found that the SARS-CoV-2 virus directly activates the pore-forming protein GSDMD that triggers NET production and organ damage in COVID-19. Single-cell transcriptome analysis revealed that the expression of GSDMD and inflammasome-related genes were increased in COVID-19 patients. High expression of active GSDMD associated with NETs structures was found in the lung tissue of COVID-19 patients. Furthermore, we showed that activation of GSDMD in neutrophils requires active caspase1/4 and live SARS-CoV-2, which infects neutrophils. In a mouse model of SARS-CoV-2 infection, the treatment with disulfiram inhibited NETs release and reduced organ damage. CONCLUSION: These results demonstrated that GSDMD-dependent NETosis plays a critical role in COVID-19 immunopathology and suggests GSDMD as a novel potential target for improving the COVID-19 therapeutic strategy.

Celastrol: A lead compound that inhibits SARS-CoV-2 replication, the activity of viral and human cysteine proteases, and virus-induced IL-6 secretion.

The global emergence of coronavirus disease 2019 (COVID-19) has caused substantial human casualties. Clinical manifestations of this disease vary from asymptomatic to lethal, and the symptomatic form can be associated with cytokine storm and hyperinflammation. In face of the urgent demand for effective drugs to treat COVID-19, we have searched for candidate compounds using in silico approach followed by experimental validation. Here we identified celastrol, a pentacyclic triterpene isolated from Tripterygium wilfordii Hook F, as one of the best compounds out of 39 drug candidates. Celastrol reverted the gene expression signature from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected cells and irreversibly inhibited the recombinant forms of the viral and human cysteine proteases involved in virus invasion, such as Mpro (main protease), PLpro (papain-like protease), and recombinant human cathepsin L. Celastrol suppressed SARS-CoV-2 replication in human and monkey cell lines and decreased interleukin-6 (IL-6) secretion in the SARS-CoV-2-infected human cell line. Celastrol acted in a concentration-dependent manner, with undetectable signs of cytotoxicity, and inhibited in vitro replication of the parental and SARS-CoV-2 variant. Therefore, celastrol is a promising lead compound to develop new drug candidates to face COVID-19 due to its ability to suppress SARS-CoV-2 replication and IL-6 production in infected cells.

Silent Infection of B and CD8+ T Lymphocytes by Influenza A Virus in Children with Tonsillar Hypertrophy.

Influenza A viruses (IAVs) cause more than 2 million annual episodes of seasonal acute respiratory infections (ARI) and approximately 500,000 deaths worldwide. Depending on virus strain and host immune status, acute infections by IAV may reach sites other than the respiratory tract. In the present study, IAV RNA and antigens were searched for in tissues of palatine tonsils and adenoids removed from patients without ARI symptoms. A real-time reverse transcriptase PCR (RT-PCR) screening revealed that 8 tissue samples from 7 patients out of 103 were positive for IAV. Positive samples were subjected to next-generation sequencing (NGS) and 3 of 8 tissues yielded complete IAV pH1N1 genomes, whereas in 5 samples, the PB1 gene was not fully assembled. Phylogenetic analysis placed tonsil-derived IAV in clusters clearly segregated from contemporaneous Brazilian viruses. Flow cytometry of dispersed tissue fragments and serial immunohistochemistry of paraffin-embedded sections of naturally infected biopsies indicated that CD20+ B lymphocytes, CD8+ T lymphocytes, and CD11c+ cells are susceptible to IAV infection. We sought to investigate whether these lymphoid tissues could be sites of viral replication and sources of viable virus particles. MDCK cells were inoculated with tissue lysates, enabling recovery of one IAV isolate confirmed by immunofluorescence, reverse transcriptase quantitative PCR (RT-qPCR), and NGS. The data indicate that lymphoid tissues not only harbor expression of IAV proteins but also contain infectious virus. Asymptomatic long-term infection raises the possibility of IAV shedding from tonsils, which may have an impact on host-to-host transmission.IMPORTANCE Influenza A virus (IAV) infections are important threats to human health worldwide. Although extensively studied, some aspects of virus pathogenesis and tissue tropism remain unclear. Here, by different strategies, we describe the asymptomatic infection of human lymphoid organs by IAV in children. Our results indicate that IAV was not only detected and isolated from human tonsils but displayed unique genetic features in comparison with those of contemporaneous IAVs circulating in Brazil and detected in swabs and nasal washes. Inside the tissue microenvironment, immune cells were shown to be carrying IAV antigens, especially B and T CD8+ lymphocytes. Taken together, these results suggest that human lymphoid tissues can be sites of silent IAV infections with possible impact on virus shedding to the population.

Detection of respiratory viruses in primary cholesteatoma tissues.

Cholesteatomas are frequent middle ear benign tumors of unknown etiology. Infectious agents have been considered as possible contributing factors in the pathogenesis of cholesteatomas. Aiming to investigate the presence of respiratory viruses in primary cholesteatoma tissues, 26 formalin-fixed paraffin-embedded primary cholesteatoma tissues obtained from patients seen at the of the Clinical Hospital of the University of São Paulo School of Medicine, in Ribeirão Preto, Brazil were tested by real-time polymerase chain reaction (PCR). Considering the PCR results, 35% of the tissues were positive for human rhinovirus (HRV), 15.3% for human enterovirus (EV), 3.8% for human metapneumovirus (HMPV), and 3.8% for human bocavirus (HBoV). Serial immunohistochemistry for virus antigens and cell surface markers evidenced that the viruses were associated with fibroblasts, dendritic cells, macrophages, B lymphocytes, CD4+ , and CD8+ T lymphocytes. These findings indicate for the first time the presence of active respiratory virus infection in primary cholesteatoma tissues, suggesting that persisting virus infection in the middle could play a role in the pathogenesis and evolution of cholesteatomas.

ACUTE NECROTIZING AND EOSINOPHILIC MYOCARDITIS IN A CHIMPANZEE (PAN TROGLODYTES).

Cardiac disease is of importance in captive chimpanzee (Pan troglodytes) health. Here we report an eosinophilic and necrotizing myocarditis in a 17-y-old chimpanzee with no previous history of cardiac disease that progressed to death within 48 h. Toxic and infectious causes were ruled out. The chimpanzee had eosinophilia at different occasions in previous years. The animal had a severe, diffuse, and acute monophasic necrotizing myocarditis, with a moderate lymphoplasmacytic infiltrate that was rich in eosinophils. Ante- and postmortem investigations are compatible with an unusual eosinophilic myocarditis with clinical evolution and morphology comparable with human eosinophilic myocarditis secondary to hypereosinophilic syndrome.

IL-33 and ST2 as predictors of disease severity in children with viral acute lower respiratory infection.

BACKGROUND: Mechanisms influencing severity of acute lower respiratory infection (ALRI) in children are not established. We aimed to assess the role of inflammatory markers and respiratory viruses in ALRI severity. METHODS: Concentrations of interleukin(IL)-33, soluble suppression of tumorigenicity (sST)2, IL-1ß, tumor necrosis factor α, IL-4, IL-6 and IL- 8 and types of respiratory viruses were evaluated in children at the first and fifth days after hospital admission. Disease severity was defined as need for mechanical ventilation. RESULTS: Seventy-nine children <5 years-old were included; 33(41.8%) received mechanical ventilation. No associations between virus type, viral load or co-detections and severity of disease were observed. Detection of IL-33 and sST2 in nasopharyngeal aspirates (NPA) on admission were associated with higher risk for mechanical ventilation (RR = 2.89 and RR = 4.57, respectively). IL-6 and IL-8 concentrations were higher on Day 5 in mechanically ventilated children. IL-6 NPA concentrations decreased from Day 1 to Day 5 in children who did not receive mechanical ventilation. Increase in sST2 NPA concentrations from Day 1 to Day 5 was associated with longer hospital length of stay (p < 0.01). CONCLUSIONS: An exacerbated local activation of the IL-33/ST2 axis and persistently high sST2 concentrations over time were associated with severity of viral ALRI in children.

Serotonin synthesis protects the mouse colonic crypt from DNA damage and colorectal tumorigenesis.

Serotonin (5-HT) signaling pathways are thought to be involved in colorectal tumorigenesis (CRT), but the role of 5-HT synthesis in the early steps of this process is presently unknown. In this study, we used carcinogen treatment in the tryptophan hydroxylase 1 knockout (Tph1KO) and transgenic (Tph1fl/fl VillinCre ) mouse models defective in 5-HT synthesis to investigate the early mutagenic events associated with CRT. Our observations of the colonic crypt post-treatment followed a timeline designed to understand how disruption of 5-HT synthesis affects the initial steps leading to CRT. We found Tph1KO mice had decreased development of both allograft tumors and colitis-related CRT. Interestingly, carcinogenic exposure alone induced multiple colon tumors and increased cyclooxygenase-2 (Ptgs2) expression in Tph1KO mice. Deletion of interleukin 6 (Il6) in Tph1KO mice confirmed that inflammation was a part of the process. 5-HT deficiency increased colonic DNA damage but inhibited genetic repair of specific carcinogen-related damage, leading to CRT-related inflammatory reactions and dysplasia. To validate a secondary effect of 5-HT deficiency on another DNA repair pathway, we exposed Tph1KO mice to ionizing radiation and found an increase in DNA damage associated with reduced levels of ataxia telangiectasia and Rad3 related (Atr) gene expression in colonocytes. Restoring 5-HT levels with 5-hydroxytryptophan treatment decreased levels of DNA damage and increased Atr expression. Analysis of Tph1fl/fl VillinCre mice with intestine-specific loss of 5-HT synthesis confirmed that DNA repair was tissue specific. In this study, we report a novel protective role for 5-HT synthesis that promotes DNA repair activity during the early stages of colorectal carcinogenesis. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Type C botulism in domestic chickens, dogs and black-pencilled marmoset (Callithrix penicillata) in Minas Gerais, Brazil.

Botulism is a well-known intoxication that affects humans and animals. The disease is endemic in cattle in Brazil and recently emerged as an important disease in commercial laying hens and broiler chickens in Europe. Dogs and other animal species can also be affected. Although antitoxins are commonly administered to humans diagnosed with botulism, in animals this is rarely the case and the treatment of botulism is still based only on support therapy. In the present work, we report an outbreak of type C botulism in Brazil that simultaneously affected domestic chickens, dogs and a black-pencilled marmoset (Callithrix penicillata). The successful use of Clostridium botulinum types C and D antitoxin for the treatment of an affected dog is also described.

Oropouche virus is detected in peripheral blood leukocytes from patients.

Oropouche virus (OROV) is a frequent cause of arboviral febrile disease in the Amazon. The present report describes studies done in two patients, one of them; the first OROV human case acquired outside of the Amazon, which have revealed for the first time the presence of OROV in peripheral blood leukocytes. This novel finding raises important issues regarding pathogenesis of human infections and may offer a new tool, for the rapid diagnosis of this neglected infection. J. Med. Virol. 89:1108-1111, 2017. © 2017 Wiley Periodicals, Inc.

A mimetic peptide of ACE2 protects against SARS-CoV-2 infection and decreases pulmonary inflammation related to COVID-19.

Since human angiotensin-converting enzyme 2 (ACE2) serves as a primary receptor for SARS-CoV-2, characterizing ACE2 regions that allow SARS-CoV-2 to enter human cells is essential for designing peptide-based antiviral blockers and elucidating the pathogenesis of the virus. We identified and synthesized a 25-mer mimetic peptide (encompassing positions 22-46 of the ACE2 alpha-helix α1) implicated in the S1 receptor-binding domain (RBD)-ACE2 interface. The mimetic (wild-type, WT) ACE2 peptide significantly inhibited SARS-CoV-2 infection of human pulmonary Calu-3 cells in vitro. In silico protein modeling predicted that residues F28, K31, F32, F40, and Y41 of the ACE2 alpha-helix α1 are critical for the original, Delta, and Omicron strains of SARS-CoV-2 to establish the Spike RBD-ACE2 interface. Substituting these residues with alanine (A) or aspartic acid (D) abrogated the antiviral protective effect of the peptides, indicating that these positions are critical for viral entry into pulmonary cells. WT ACE2 peptide, but not the A or D mutated peptides, exhibited significant interaction with the SARS-CoV-2 S1 RBD, as shown through molecular dynamics simulations. Through identifying the critical amino acid residues of the ACE2 alpha-helix α1, which is necessary for the Spike RBD-ACE2 interface and mobilized during the in vitro viral infection of cells, we demonstrated that the WT ACE2 peptide protects susceptible K18-hACE2 mice against in vivo SARS-CoV-2 infection and is effective for the treatment of COVID-19.

Breastfeeding by chikungunya virus-infected dams confers resistance to challenge in the offspring.

Vertical transmission of Chikungunya virus (CHIKV) has been reported in humans, but the transmission routes have not been completely understood, and experimental animal models are needed to enable detailed investigation of the transmission and pathogenesis of congenital infections. The intertwining of immune response and virus components at the gestation/breastfeeding interfaces between mother and fetus/newborn may have effects during the offspring development. An experimental model of CHIKV was established by infecting pregnant BALB/c female mice that enabled confirmation that dams inoculated up to the 10th gestational day transmit CHIKV transplacentally to approximately 8.4% of the fetuses, resulting in severe teratogenic effects. CHIKV neutralizing antibodies were detected in sera from adult mice born to healthy females and breastfed by CHIKV-infected dams, while no neutralization was detected in sera from animals born to CHIKV-infected dams. Moreover, adult mice born to healthy dams and cross-fostered for breastfeeding by CHIKV-infected dams were resistant to challenge with CHIKV on the 90th day after birth. The animals also had reduced viral loads in brain and spleen as compared to controls. There was expression of fluorescent CHIKV non-structural protein, and detection of viral RNA by RT-PCR in breast tissue from infected dams. CHIKV RNA and proteins were also detected in breast milk retrieved from the stomachs of recently fed newborns. The experimental results were also complemented by the finding of CHIKV RNA in 6% of colostrum samples from healthy lactating women in a CHIKV-endemic area. Breastfeeding induces immune protection to challenge with CHIKV in mice.