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In December 2019, a new variant coronavirus, SARS-CoV-2, emerged in China, which was initially described as a pneumonia of an unknown agent. The new coronavirus spreads mainly by person-to-person transmission through close contact. The pathophysiology of COVID-19 is related to a complex immune system response that varies between people and, in severe cases of the disease, is characterized by excessive responses called "cytokine storms," which are associated with complications that can lead to a state of hypercoagulation and death. Glucocorticoids and azithromycin are drugs that may be effective in the treatment. This review aims to highlight the clinical findings that demonstrate the effectiveness of glucocorticoid and azithromycin therapy in the treatment of COVID-19. To date, many drugs have been studied for use in combination therapy, and the rapid expansion of knowledge about the virology of SARS-CoV-2 generates a more accurate direction in therapy.

SARS-CoV-2, the causative agent of the ongoing COVID-19, has spread worldwide since it was first identified in November 2019 in Wuhan. Since then, it was already demonstrated an exuberant inflammation, cytokine storm, endothelium dysfunction, platelets hyperactivation and aggregation, following T cell exhaustion leading to severe multi-organ damage and death of COVID-19 patients. Here, we sought to identify molecular biomarkers of disease severity in a Brazilian cohort of COVID-19 patients by measuring the serum levels of endogenous danger signals. Our data revealed that ICU patients that are critically ill, at the early hyperinflammatory phase of COVID-19 (around 12-25 days after hospital admission) display higher serum levels of the classical alarmin HMGB1. Serum levels of HMGB1 were positively correlated with cys-leukotrienes, D-dimer, AST, and ALT. Notably, we verified that HMGB1 levels above 125.4 ng/mL is the cut off that distinguishes the patients that are at higher risk of death. Serum levels of extracellular ATP, PGE2, LTB4, cys-LTs, and tissue factor were also elevated in the serum of ICU patients. In conclusion, we propose that serum levels of HMGB1 serve as prognostic biomarker of risk of death in patients suffering from severe COVID-19.

Vertical transmission of SARS-CoV-2 has already been described, while clinical consequences to the fetus are still under investigation. This article reports a case of systemic fetal inflammatory response and pericardial effusion. As far as is known, this is the first case of fetal/neonatal cardiac complications related to SARS-CoV-2 infection.

BackgroundCoronavirus disease 2019 (COVID-19) can progress to severe pneumonia with respiratory failure and is aggravated by the deregulation of the immune system causing an excessive inflammation including the cytokine storm.MethodsIn this study, we report that severe acutely infected patients have high levels of both type-1 and type-2 cytokines.ResultsOur results show abnormal cytokine levels upon T-cell stimulation, in a nonpolarized profile. Furthermore, our findings indicate that this hyperactive cytokine response is associated with a significantly increased frequency of late-differentiated T cells with particular phenotype of effector exhausted/senescent CD28−CD57+ cells. Of note, we demonstrated for the first time an increased frequency of CD3+CD4+CD28−CD57+ T cells with expression of programmed death 1, one of the hallmarks of T-cell exhaustion.ConclusionsThese findings reveal that COVID-19 is associated with acute immunodeficiency, especially within the CD4+ T-cell compartment, and points to possible mechanisms of loss of clonal repertoire and susceptibility to viral relapse and reinfection events.

BACKGROUND The high demand for adequate material for the gold standard reverse transcription real-time polymerase chain reaction (RT-qPCR)-based diagnosis imposed by the Coronavirus disease 2019 (COVID-19) pandemic, combined with the inherent contamination risks for healthcare workers during nasopharyngeal swab (NP) sample collection and the discomfort it causes patients, brought the need to identify alternative specimens suitable for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). OBJECTIVES The aim of this work was to compare saliva and gingival fluid swabs to NP swabs as specimens for RT-qPCR-based SARS-CoV-2 diagnosis. METHODS We compared gingival fluid swabs (n = 158) and saliva (n = 207) to the rayon-tipped NP swabs obtained from mild-symptomatic and asymptomatic subjects as specimens for RT-qPCR for SARS-CoV-2 detection. FINDINGS When compared to NP swabs, gingival fluid swabs had a concordance rate of 15.4% among positive samples, zero among inconclusive, and 100% among negative ones. For saliva samples, the concordance rate was 67.6% among positive samples, 42.9% among inconclusive, and 96.8% among negative ones. However, the concordance rate between saliva and NP swabs was higher (96.9%) within samples with lower cycle threshold (Ct) values (Ct > 10 ≤ 25). MAIN CONCLUSIONS Our data suggests that whereas gingival fluid swabs are not substitutes for NP swabs, saliva might be considered whenever NP swabs are not available or recommended.

Vaccines are a recommended strategy for controlling influenza A infections in humans and animals. Here, we describe the effects of hydrostatic pressure on the structure, morphology and functional characteristics of avian influenza A H3N8 virus. The effect of hydrostatic pressure for 3 h on H3N8 virus revealed that the particles were resistant to this condition, and the virus displayed only a discrete conformational change. We found that pressure of 3 kbar applied for 6 h was able to inhibit haemagglutination and infectivity while virus replication was no longer observed, suggesting that full virus inactivation occurred at this point. However, the neuraminidase activity was not affected at this approach suggesting the maintenance of neutralizing antibody epitopes in this key antigen. Our data bring important information for the area of structural virology of enveloped particles and support the idea of applying pressure-induced inactivation as a tool for vaccine production.

Secondary bacterial and fungal infections are associated with respiratory viral infections and invasive mechanical ventilation. In Coronavirus disease 2019 (COVID-19), lung injury by SARS-CoV-2 and impaired immune response can provide a favorable environment for microorganism growth and colonization in hospitalized individuals. Recent studies suggest that secondary bacterial pneumonia is a risk factor associated with COVID-19. In Brazil, knowledge about microbiota present in COVID-19 patients is incipient. This work describes the microbiota of 21 COVID-19 patients admitted to intensive care units from two Brazilian centers. We identified respiratory, nosocomial and bacterial pathogens as prevalent microorganisms. Other bacterial opportunistic and commensal species are also represented. Virulence factors of these pathogenic species, metabolic pathways used to evade and modulate immunological processes and the interconnection between bacterial presence and virulence in COVID-19 progression are discussed.Article Summary LineWe identified respiratory, nosocomial and bacterial pathogens as prevalent microorganisms in 21 Brazilian COVID-19 patients admitted to Intensive Care Units. Pathogen virulence factors and immune response evasion metabolic pathways are correlated to COVID-19 severity.

COVID-19 is a disease caused by the novel SARS-CoV-2 coronavirus, originally classified as a severe acute respiratory syndrome coronavirus (SARS-CoV). The most severe cases of COVID-19 can progress to severe pneumonia with respiratory failure, septicemia, multiple organ failure and death. The severity of the disease is aggravated by the deregulation of the immune system causing an excessive initial inflammation including the cytokine storm, compring interleukins characteristic of the T-dependent adaptive response. In the present study we show that severe patients have high levels of T helper type-1 and type-2 cytokines, as well as VGEF. Furthermore, our show abnormal cytokine levels upon T-cell mitogen stimulation, in a non-polarized response profile. This response is not specific, given that the stimulus with the heterologous tuberculin antigen was able to induce high levels of cytokines compared to healthy controls, including the vascular endothelial growth factor VEGF, which promotes neoangiogenesis in physiological and pathophysiological conditions, caused by tissue hypoxia, and involved in a clonal exhaustion program in T cells. This can be decisive given our findings demonstrating for the first time a significantly increased frequency of late-differentiated CD8+ T cells characterized by critically shortened telomeres with particular phenotype (CD57+CD28-) in severe acute COVID-19 infection. These findings reveal that severe COVID-19 is associated with senescence of T cells, especially within the CD8+ T cell compartment and points to possible mechanisms of loss of clonal repertoire and susceptibility to recurrences of COVID-19 symptoms, due to viral relapse and reinfection events.

The novel coronavirus SARS-CoV2 causes COVID-19, a highly pathogenic viral infection threatening millions. The majority of those infected are asymptomatic or mildly symptomatic showing typical clinical signs of common cold. However approximately 20% of the patients can progress to acute respiratory distress syndrome (ARDS) and eventually death in about 5% of cases. Recently, angiotensin-converting enzyme 2 (ACE2) has been shown to be a functional receptor for virus entry into host target cells. The upregulation of ACE2 in patients with comorbidities may represent a propensity for increased viral load and spreading of infection to extrapulmonary tissues. This systemic infection is associated with higher neutrophil to lymphocyte ratio in infected tissues and high levels of pro-inflammatory cytokines leading to an extensive microthrombus formation with multiorgan failure. Herein we investigated whether SARS-CoV2 can stimulate extracellular neutrophils traps (NETs) in a process called NETosis. We demonstrated for the first time that SARS-CoV2 in fact is able to activate NETosis in human neutrophils. Our findings indicated that this process is associated with increased levels of intracellular Reactive Oxygen Species (ROS) in neutrophils. The ROS-NET pathway plays a role in thrombosis formation and our study suggest the importance of this target for therapy approaches against disease.

The novel coronavirus SARS-CoV-2 causes COVID-19, a highly pathogenic viral infection threatening millions. The majority of the individuals infected are asymptomatic or mildly symptomatic showing typical clinical signs of common cold. However, approximately 20% of the patients can progress to acute respiratory distress syndrome (ARDS), evolving to death in about 5% of cases. Recently, angiotensin-converting enzyme 2 (ACE2) has been shown to be a functional receptor for virus entry into host target cells. The upregulation of ACE2 in patients with comorbidities may represent a propensity for increased viral load and spreading of infection to extrapulmonary tissues. This systemic infection is associated with higher neutrophil to lymphocyte ratio in infected tissues and high levels of pro-inflammatory cytokines leading to an extensive microthrombus formation with multiorgan failure. Herein we investigated whether SARS-CoV-2 can stimulate extracellular neutrophils traps (NETs) in a process called NETosis. We demonstrated for the first time that SARS-CoV-2 in fact is able to activate NETosis in human neutrophils. Our findings indicated that this process is associated with increased levels of intracellular Reactive Oxygen Species (ROS) in neutrophils. The ROS-NET pathway plays a role in thrombosis formation and our study suggest the importance of this target for therapy approaches against disease.

Background Avian influenza A viruses can cross naturally into mammals and cause severe diseases, as observed for H5N1. The high lethality of human infections causes major concerns about the real risk of a possible pandemic of severe diseases to which human susceptibility may be high and universal. High hydrostatic pressure (HHP) is a valuable tool for studies regarding the folding of proteins and the assembly of macromolecular structures such as viruses; furthermore, HHP has already been demonstrated to promote viral inactivation. Methods Here, we investigated the structural stability of avian and human influenza viruses using spectroscopic and light-scattering techniques. We found that both particles have similar structural stabilities and that HHP promotes structural changes. Results HHP induced slight structural changes to both human and avian influenza viruses, and these changes were largely reversible when the pressure returned to its initial level. The spectroscopic data showed that H3N2 was more pressure-sensitive than H3N8. Structural changes did not predict changes in protein function, as H3N2 fusion activity was not affected, while H3N8 fusion activity drastically decreased. The fusion activity of H1N1 was also strongly affected by HHP. In all cases, HHP caused inactivation of the different influenza viruses. Conclusions HHP may be a useful tool for vaccine development, as it induces minor and reversible structural changes that may be associated with partial preservation of viral biological activities and may potentiate their immunogenic response while abolishing their infectivity. We also confirmed that, although pressure does not promote drastic changes in viral particle structure, it can distinctly affect viral fusion activity.

Background: Neuraminidase inhibitors (NAIs) are the only class of antivirals in clinical use against influenza virus approved worldwide. However, approximately 1-3% of circulating strains present resistance mutations to oseltamivir (OST), the most used NAI. Therefore, it is important to catalogue new molecules to inhibit influenza virus, especially OST-resistant strains. Natural products from tropical plants used for human consumption represent a worthy class of substances. Their use could be stimulated in resource-limited setting where the access to expensive antiviral therapies is restricted. Methods: We evaluated the anti-influenza virus activity of agathisflavone derived from Anacardium occidentale L. Results: The neuraminidase (NA) activity of wild-type and OST-resistant influenza virus was inhibited by agathisflavone, with IC50 values ranging from 20 to 2.0 µM, respectively. Agathisflavone inhibited influenza virus replication with EC50 of 1.3 µM. Sequential passages of the virus in the presence of agathisflavone revealed the emergence of mutation R249S, A250S and R253Q in the NA gene. These changes are outside the OST binding region, meaning that agathisflavone targets this viral enzyme at a region different than conventional NAIs. Conclusion: Altogether our data suggest that agathisflavone has a promising chemical structure for the development of anti-influenza drugs.

Chagas cardiomyopathy is the most severe clinical manifestation of chronic Chagas disease. The disease affects most of the Latin American countries, being considered one of the leading causes of morbidity and death in the continent. The pathogenesis of Chagas cardiomyopathy is very complex, with mechanisms involving parasite-dependent cytopathy, immune-mediated myocardial damage and neurogenic disturbances. These pathological changes eventually result in cardiac myocyte hypertrophy, arrhythmias, congestive heart failure and stroke during chronic infection phase. Herein, we show that miR-208a, a microRNA that is a key factor in promoting cardiovascular dysfunction during cardiac hypertrophy processes of heart failure, has its circulating levels increased during chronic indeterminate phase when compared to cardiac (CARD) clinical forms in patients with Chagas disease. In contrast, we have not found altered serum levels of miR-34a, a microRNA known to promote pro-apoptotic role in myocardial infarction during degenerative process of cardiac injuries thus indicating intrinsic differences in the nature of the mechanisms underlying the heart failure triggered by Trypanosoma cruzi infection. Our findings support that the chronic indeterminate phase is a progressive phase involved in the genesis of chagasic cardiopathy and point out the use of plasma levels of miR-208a as candidate biomarker in risk-prediction score for the clinical prognosis of Chagas disease.

Influenza virus infections are a serious global health threat, particularly in light of newly emerging strains, such as the avian virus H5N1. In this study, a sample of avian influenza A virus subtype H3N8 inactivated by high hydrostatic pressure was used as a vaccine. Our goal was to study pressurized virus preparations for their ability to induce an immunogenic and protective response when using mice as an animal model. Here, Balb/c mice were treated through the intranasal route with three doses of pressurized virus. After vaccination, the mice were challenged and monitored for virus-specific antibodies (ELISA and neutralization assay), clinical symptoms and death. After immunization, there was an increase of IgG1 and IgG2a in sera and IgA in nasal lavages, which indicated that the serum antibodies were showing neutralizing ability. The viral neutralization assay demonstrated that the produced antibodies were neutralizing. After the challenge, the control group (immunized with saline) showed all measured clinical signs of disease (weight loss, ruffled fur, lethargy and huddling). The vaccinated animals did not develop any clinical signs. The results reveal that the animals were able to produce a satisfactory humoral response after vaccination and protected against the challenge. Our work reaffirms the use of hydrostatic pressure as a means for developing low-cost viral vaccines with good immune response.

Influenza viruses pose a serious global health threat, particularly in light of newly emerging strains, such as the avian influenza H5N1 and H7N9 viruses. Vaccination remains the primary method for preventing acquiring influenza or for avoiding developing serious complications related to the disease. Vaccinations based on inactivated split virus vaccines or on chemically inactivated whole virus have some important drawbacks, including changes in the immunogenic properties of the virus. To induce a greater mucosal immune response, intranasally administered vaccines are highly desired as they not only prevent disease but can also block the infection at its primary site. To avoid these drawbacks, hydrostatic pressure has been used as a potential method for viral inactivation and vaccine production. In this study, we show that hydrostatic pressure inactivates the avian influenza A H3N8 virus, while still maintaining hemagglutinin and neuraminidase functionalities. Challenged vaccinated animals showed no disease signs (ruffled fur, lethargy, weight loss, and huddling). Similarly, these animals showed less Evans Blue dye leakage and lower cell counts in their bronchoalveolar lavage fluid compared with the challenged non-vaccinated group. We found that the whole inactivated particles were capable of generating a neutralizing antibody response in serum, and IgA was also found in nasal mucosa and feces. After the vaccination and challenge we observed Th1/Th2 cytokine secretion with a prevalence of IFN-γ. Our data indicate that the animals present a satisfactory immune response after vaccination and are protected against infection. Our results may pave the way for the development of a novel pressure-based vaccine against influenza virus.

In recent years, many applications in diverse scientific fields with various purposes have examined pressure as a thermodynamic parameter. Pressure studies on viruses have direct biotechnological applications. Currently, most studies that involve viral inactivation by HHP are found in the area of food engineering and focus on the inactivation of foodborne viruses. Nevertheless, studies of viral inactivation for other purposes have also been conducted. HHP has been shown to be efficient in the inactivation of many viruses of clinical importance and the use of HHP approach has been proposed for the development of animal and human vaccines. Several studies have demonstrated that pressure can result in virus inactivation while preserving immunogenic properties. Viruses contain several components that can be susceptible to the effects of pressure. HHP has been a valuable tool for assessing viral structure function relationships because the viral structure is highly dependent on protein-protein interactions. In the case of small icosahedral viruses, incremental increases in pressure produce a progressive decrease in the folding structure when moving from assembled capsids to ribonucleoprotein intermediates (in RNA viruses), free dissociated units (dimers and/or monomers) and denatured monomers. High pressure inactivates enveloped viruses by trapping their particles in a fusion-like intermediate state. The fusogenic state, which is characterized by a smaller viral volume, is the final conformation promoted by HHP, in contrast with the metastable native state, which is characterized by a larger volume. The combined effects of high pressure with other factors, such as low or subzero temperature, pH and agents in sub-denaturing conditions (urea), have been a formidable tool in the assessment of the component’s structure, as well as pathogen inactivation. HHP is a technology for the production of inactivated vaccines that are free of chemicals, safe and capable of inducing strong humoral and cellular immune responses. Here we present a current overview about the pressure-induced viral inactivation and the production of inactivated viral vaccines.

Whole inactivated vaccines (WIVs) possess greater immunogenicity than split or subunit vaccines, and recent studies have demonstrated that WIVs with preserved fusogenic activity are more protective than non-fusogenic WIVs. In this work, we describe the inactivation of human influenza virus X-31 by high hydrostatic pressure (HHP) and analyze the effects on the structure by spectroscopic measurements, light scattering, and electron microscopy. We also investigated the effects of HHP on the glycoprotein activity and fusogenic activity of the viral particles. The electron microscopy data showed pore formation on the viral envelope, but the general morphology was preserved, and small variations were seen in the particle structure. The activity of hemagglutinin (HA) during the process of binding and fusion was affected in a time-dependent manner, but neuraminidase (NA) activity was not affected. Infectious activity ceased after 3 hours of pressurization, and mice were protected from infection after being vaccinated. Our results revealed full viral inactivation with overall preservation of viral structure and maintenance of fusogenic activity, thereby conferring protection against infection. A strong response consisting of serum immunoglobulin IgG1, IgG2a, and serum and mucosal IgA was also detected after vaccination. Thus, our data strongly suggest that applying hydrostatic pressure may be an effective method for developing new vaccines against influenza A as well as other viruses.