Development and validation of LC-MS/MS methods for the simultaneous quantification of sofosbuvir and its major metabolite (GS-331007) in blood plasma and cerebrospinal and seminal fluid: Application to a pilot clinical trial with a focus on Zika.

Zika still poses a threat to global health owing to its association with serious neurological conditions and the absence of a vaccine and treatment. Sofosbuvir, an anti-hepatitis C drug, has shown anti-Zika effects in animal and cell models. Thus, this study aimed to develop and validate novel LC-MS/MS methods for the quantification of sofosbuvir and its major metabolite (GS-331007) in human plasma and cerebrospinal (CSF) and seminal fluid (SF), and apply the methods to a pilot clinical trial. The samples were prepared by liquid-liquid extraction and separated using isocratic mode on Gemini C18 columns. Analytical detection was performed using a triple quadrupole mass spectrometer equipped with an electrospray ionization source. The validated ranges for sofosbuvir were 0.5-2,000 ng/mL (plasma) and 0.5-100 ng/mL (CSF and SF), while for the metabolite they were 2.0-2,000 ng/mL (plasma), 5.0-200 ng/mL (CSF) and 10-1,500 ng/mL (SF). The intra-day and inter-day accuracies (90.8-113.8%) and precisions (1.4-14.8%) were within the acceptance range. The developed methods fulfilled all validation parameters concerning selectivity, matrix effect, carryover, linearity, dilution integrity, precision, accuracy and stability, confirming the suitability of the method for the analysis of clinical samples.

Monkeypox (Mpox) virus isolation and ultrastructural characterisation from a Brazilian human sample case.

BACKGROUND: According to the last 2023 Monkeypox (Mpox) Outbreak Global Map from the Centres for Disease Control and Prevention (CDC), more than 100 countries with no Mpox infection report cases. Brazil stands out in this group and is the second country with the highest number of cases in the last outbreak. OBJECTIVE: To contribute to knowledge of the virus infection effects in a cellular model, which is important for diagnosis infections not yet included in a provider´s differential diagnosis and for developing viral inhibition strategies. METHODS: We describe a virus isolation protocol for a human clinical sample from a patient from Brazil, the viral growth in a cell model through plaque forming units (PFU) assay, reverse transcriptase polymerase chain reaction (RT-PCR) and transmission electron microscopy (TEM). FINDINGS: We follow the viral isolation in Vero cell culture from a Mpox positive clinically diagnosed sample and show the infection effects on cellular structures using a TEM. MAIN CONCLUSIONS: Understanding the impact of viral growth on cellular structures and its replication kinetics may offer better strategies for the development of new drugs with antiviral properties.

A Biosafety Level 2 Mouse Model for Studying Betacoronavirus-Induced Acute Lung Damage and Systemic Manifestations.

The emergence of life-threatening zoonotic diseases caused by betacoronaviruses, including the ongoing coronavirus disease 19 (COVID-19) pandemic, has highlighted the need for developing preclinical models mirroring respiratory and systemic pathophysiological manifestations seen in infected humans. Here, we showed that C57BL/6J wild-type mice intranasally inoculated with the murine betacoronavirus murine hepatitis coronavirus 3 (MHV-3) develop a robust inflammatory response leading to acute lung injuries, including alveolar edema, hemorrhage, and fibrin thrombi. Although such histopathological changes seemed to resolve as the infection advanced, they efficiently impaired respiratory function, as the infected mice displayed restricted lung distention and increased respiratory frequency and ventilation. Following respiratory manifestation, the MHV-3 infection became systemic, and a high virus burden could be detected in multiple organs along with morphological changes. The systemic manifestation of MHV-3 infection was also marked by a sharp drop in the number of circulating platelets and lymphocytes, besides the augmented concentration of the proinflammatory cytokines interleukin 1 beta (IL-1ß), IL-6, IL-12, gamma interferon (IFN-γ), and tumor necrosis factor (TNF), thereby mirroring some clinical features observed in moderate and severe cases of COVID-19. Importantly, both respiratory and systemic changes triggered by MHV-3 infection were greatly prevented by blocking TNF signaling, either via genetic or pharmacologic approaches. In line with this, TNF blockage also diminished the infection-mediated release of proinflammatory cytokines and virus replication of human epithelial lung cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Collectively, results show that MHV-3 respiratory infection leads to a large range of clinical manifestations in mice and may constitute an attractive, lower-cost, biosafety level 2 (BSL2) in vivo platform for evaluating the respiratory and multiorgan involvement of betacoronavirus infections. IMPORTANCE Mouse models have long been used as valuable in vivo platforms to investigate the pathogenesis of viral infections and effective countermeasures. The natural resistance of mice to the novel betacoronavirus SARS-CoV-2, the causative agent of COVID-19, has launched a race toward the characterization of SARS-CoV-2 infection in other animals (e.g., hamsters, cats, ferrets, bats, and monkeys), as well as adaptation of the mouse model, by modifying either the host or the virus. In the present study, we utilized a natural pathogen of mice, MHV, as a prototype to model betacoronavirus-induced acute lung injure and multiorgan involvement under biosafety level 2 conditions. We showed that C57BL/6J mice intranasally inoculated with MHV-3 develops severe disease, which includes acute lung damage and respiratory distress that precede systemic inflammation and death. Accordingly, the proposed animal model may provide a useful tool for studies regarding betacoronavirus respiratory infection and related diseases.

Fluorine Atoms on C6H5-Corrole Affect the Interaction with Mpro and PLpro Proteases of SARS-CoV-2: Molecular Docking and 2D-QSAR Approaches.

The chymotrypsin-like cysteine protease (3CLpro, also known as main protease-Mpro) and papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been used as the main targets for screening potential synthetic inhibitors for posterior in vitro evaluation of the most promising compounds. In this sense, the present work reports for the first time the evaluation of the interaction between Mpro/PLpro with a series of 17 porphyrin analogues-corrole (C1), meso-aryl-corrole (C2), and 15 fluorinated-meso-aryl-corrole derivatives (C3-C17) via molecular docking calculations. The impact of fluorine atoms on meso-aryl-corrole structure was also evaluated in terms of binding affinity and physical-chemical properties by two-dimensional quantitative structure-activity relationship (2D-QSAR). The presence of phenyl moieties increased the binding capacity of corrole for both proteases and depending on the position of fluorine atoms might impact positively or negatively the binding capacity. For Mpro the para-fluorine atoms might decrease drastically the binding capacity, while for PLpro there was a certain increase in the binding affinity of fluorinated-corroles with the increase of fluorine atoms into meso-aryl-corrole structure mainly from tri-fluorinated insertions. The 2D-QSAR models indicated two separated regions of higher and lower affinity for Mpro:C1-C17 based on dual electronic parameters (σI and σR), as well as one model was obtained with a correlation between the docking score value of Mpro:C2-C17 and the corresponding 13C nuclear magnetic resonance (NMR) chemical shifts of the sp2 carbon atoms (δC-1 and δC-2) of C2-C17. Overall, the fluorinated-meso-aryl-corrole derivatives showed favorable in silico parameters as potential synthetic compounds for future in vitro assays on the inhibition of SARS-CoV-2 replication.

The Role of Pyrazolopyridine Derivatives on Different Steps of Herpes Simplex Virus Type-1 In Vitro Replicative Cycle.

Herpes simplex virus type-1 (HSV-1) infection causes several disorders, and acyclovir is used as a reference compound. However, resistant strains are commonly observed. Herein, we investigate the effects of N-heterocyclic compounds (pyrazolopyridine derivatives), named ARA-04, ARA-05, and AM-57, on HSV-1 in vitro replication. We show that the 50% effective concentration (EC50) values of the compounds ARA-04, ARA-05, and AM-57 were 1.00 ± 0.10, 1.00 ± 0.05, and 0.70 ± 0.10 µM, respectively. These compounds presented high 50% cytotoxic concentration (CC50) values, which resulted in a selective index (SI) of 1000, 1000, and 857.1 for ARA-04, ARA-05, and AM-57, respectively. To gain insight into which step of the HSV-1 replication cycle these molecules would impair, we performed adsorption and penetration inhibition assays and time-of-addition experiments. Our results indicated that ARA-04 and ARA-05 affected viral adsorption, while AM-57 interfered with the virus replication during its α- and γ-phases and decreased ICP27 content during initial and late events of HSV-1 replication. In addition, we also observed that AM-57 caused a strong decrease in viral gD content, which was reinforced by in silico calculations that suggested AM-57 interacts preferentially with the viral complex between a general transcription factor and virion protein (TFIIBc-VP16). In contrast, ARA-04 and ARA-05 interact preferentially in the proteins responsible for the viral adsorption process (nectin-1 and glycoprotein). Thus, our results suggest that the 1H-pyrazolo[3,4-b]pyridine derivatives inhibit the HSV-1 replicative cycle with a novel mechanism of action, and its scaffold can be used as a template for the synthesis of promising new molecules with antiviral effects, including to reinforce the presented data herein for a limited number of molecules.

Genetic Evidence and Host Immune Response in Persons Reinfected with SARS-CoV-2, Brazil.

The dynamics underlying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfection remain poorly understood. We identified a small cluster of patients in Brazil who experienced 2 episodes of coronavirus disease (COVID-19) in March and late May 2020. In the first episode, patients manifested an enhanced innate response compared with healthy persons, but neutralizing humoral immunity was not fully achieved. The second episode was associated with different SARS-CoV-2 strains, higher viral loads, and clinical symptoms. Our finding that persons with mild COVID-19 may have controlled SARS-CoV-2 replication without developing detectable humoral immunity suggests that reinfection is more frequent than supposed, but this hypothesis is not well documented.

In vitro antiviral activity of the anti-HCV drugs daclatasvir and sofosbuvir against SARS-CoV-2, the aetiological agent of COVID-19.

BACKGROUND: Current approaches of drug repurposing against COVID-19 have not proven overwhelmingly successful and the SARS-CoV-2 pandemic continues to cause major global mortality. SARS-CoV-2 nsp12, its RNA polymerase, shares homology in the nucleotide uptake channel with the HCV orthologue enzyme NS5B. Besides, HCV enzyme NS5A has pleiotropic activities, such as RNA binding, that are shared with various SARS-CoV-2 proteins. Thus, anti-HCV NS5B and NS5A inhibitors, like sofosbuvir and daclatasvir, respectively, could be endowed with anti-SARS-CoV-2 activity. METHODS: SARS-CoV-2-infected Vero cells, HuH-7 cells, Calu-3 cells, neural stem cells and monocytes were used to investigate the effects of daclatasvir and sofosbuvir. In silico and cell-free based assays were performed with SARS-CoV-2 RNA and nsp12 to better comprehend the mechanism of inhibition of the investigated compounds. A physiologically based pharmacokinetic model was generated to estimate daclatasvir's dose and schedule to maximize the probability of success for COVID-19. RESULTS: Daclatasvir inhibited SARS-CoV-2 replication in Vero, HuH-7 and Calu-3 cells, with potencies of 0.8, 0.6 and 1.1 µM, respectively. Although less potent than daclatasvir, sofosbuvir alone and combined with daclatasvir inhibited replication in Calu-3 cells. Sofosbuvir and daclatasvir prevented virus-induced neuronal apoptosis and release of cytokine storm-related inflammatory mediators, respectively. Sofosbuvir inhibited RNA synthesis by chain termination and daclatasvir targeted the folding of secondary RNA structures in the SARS-CoV-2 genome. Concentrations required for partial daclatasvir in vitro activity are achieved in plasma at Cmax after administration of the approved dose to humans. CONCLUSIONS: Daclatasvir, alone or in combination with sofosbuvir, at higher doses than used against HCV, may be further fostered as an anti-COVID-19 therapy.

Chemistry and anti-herpes simplex virus type 1 evaluation of 4-substituted-1H-1,2,3-triazole-nitroxyl-linked hybrids.

HSV disease is distributed worldwide. Anti-herpesvirus drugs are a problem in clinical settings, particularly in immunocompromised individuals undergoing herpes simplex virus type 1 infection. In this work, 4-substituted-1,2,3-1H-1,2,3-triazole linked nitroxyl radical derived from TEMPOL were synthesized, and their ability to inhibit the in vitro replication of HSV-1 was evaluated. The nitroxide derivatives were characterized by infrared spectroscopy and elemental analysis, and three of them had their crystal structures determined by single-crystal X-ray diffraction. Four hybrid molecules showed important anti-HSV-1 activity with IC50 values ranged from 0.80 to 1.32 µM. In particular, one of the nitroxide derivatives was more active than Acyclovir (IC50 = 0.99 µM). All compounds tested were more selective inhibitors than the reference antiviral drug. Among them, two compounds were 4.5 (IC50 0.80 µM; selectivity index CC50/IC50 3886) and 7.7 times (IC50 1.10 µM; selectivity index CC50/IC50 6698) more selective than acyclovir (IC50 0.99 µM; selectivity index CC50/IC50: 869). These nitroxide derivatives may be elected as leading compounds due to their antiherpetic activities and good selectivity.

Increased expression of CDKN1A/p21 in HIV-1 controllers is correlated with upregulation of ZC3H12A/MCPIP1.

BACKGROUND: Some multifunctional cellular proteins, as the monocyte chemotactic protein-induced protein 1 (ZC3H12A/MCPIP1) and the cyclin-dependent kinase inhibitor CDKN1A/p21, are able to modulate the cellular susceptibility to the human immunodeficiency virus type 1 (HIV-1). Several studies showed that CDKN1A/p21 is expressed at high levels ex vivo in cells from individuals who naturally control HIV-1 replication (HIC) and a recent study supports a coordinate regulation of ZC3H12A/MCPIP1 and CDKN1A/p21 transcripts in a model of renal carcinoma cells. Here, we explored the potential associations between mRNA expression of ZC3H12A/MCPIP1 and CDKN1A/p21 in HIC sustaining undetectable (elite controllers-EC) or low (viremic controllers-VC) viral loads. RESULTS: We found a selective upregulation of ZC3H12A/MCPIP1 and CDKN1A/p21 mRNA levels in PBMC from HIC compared with both ART-suppressed and HIV-negative control groups (P≤ 0.02) and higher MCPIP1 and p21 proteins levels in HIC than in HIV-1 negative subjects. There was a moderate positive correlation (r ≥ 0.57; P ≤ 0.014) between expressions of both transcripts in HIC and in HIC combined with control groups. We found positive correlations between the mRNA level of CDKN1A/p21 with activated CD4+ T cells levels in HIC (r ≥ 0.53; P ≤ 0.017) and between the mRNA levels of both CDKN1A/p21 (r = 0.74; P = 0.005) and ZC3H12A/MCPIP1 (r = 0.58; P = 0.040) with plasmatic levels of sCD14 in EC. Reanalysis of published transcriptomic data confirmed the positive association between ZC3H12A/MCPIP1 and CDKN1A/p21 mRNA levels in CD4+ T cells and monocytes from disparate cohorts of HIC and other HIV-positive control groups. CONCLUSIONS: These data show for the first time the simultaneous upregulation of ZC3H12A/MCPIP1 and CDKN1A/p21 transcripts in the setting of natural suppression of HIV-1 replication in vivo and the positive correlation of the expression of these cellular factors in disparate cohorts of HIV-positive individuals. The existence of a common regulatory pathway connecting ZC3H12A/MCPIP1 and CDKN1A/p21 could have a synergistic effect on HIV-1 replication control and pharmacological manipulation of these multifunctional host factors may open novel therapeutic perspectives to prevent HIV-1 replication and disease progression.

Atazanavir, Alone or in Combination with Ritonavir, Inhibits SARS-CoV-2 Replication and Proinflammatory Cytokine Production.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is already responsible for far more deaths than previous pathogenic coronaviruses (CoVs) from 2002 and 2012. The identification of clinically approved drugs to be repurposed to combat 2019 CoV disease (COVID-19) would allow the rapid implementation of potentially life-saving procedures. The major protease (Mpro) of SARS-CoV-2 is considered a promising target, based on previous results from related CoVs with lopinavir (LPV), an HIV protease inhibitor. However, limited evidence exists for other clinically approved antiretroviral protease inhibitors. Extensive use of atazanavir (ATV) as antiretroviral and previous evidence suggesting its bioavailability within the respiratory tract prompted us to study this molecule against SARS-CoV-2. Our results show that ATV docks in the active site of SARS-CoV-2 Mpro with greater strength than LPV, blocking Mpro activity. We confirmed that ATV inhibits SARS-CoV-2 replication, alone or in combination with ritonavir (RTV) in Vero cells and a human pulmonary epithelial cell line. ATV/RTV also impaired virus-induced enhancement of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) levels. Together, our data strongly suggest that ATV and ATV/RTV should be considered among the candidate repurposed drugs undergoing clinical trials in the fight against COVID-19.

Differential Shedding and Antibody Kinetics of Zika and Chikungunya Viruses, Brazil.

In seroconversion panels obtained from patients from Brazil, diagnostic testing for Zika virus infection was improved by combining multiple antibody isotypes, techniques, and antigens, but sensitivity remained suboptimal. In contrast, chikungunya virus diagnostic testing was unambiguous. Recurrent recent arbovirus infections suggested by serologic data and unspecific symptoms highlight the need for exhaustive virologic testing.

Beyond Members of the Flaviviridae Family, Sofosbuvir Also Inhibits Chikungunya Virus Replication.

Chikungunya virus (CHIKV) causes a febrile disease associated with chronic arthralgia, which may progress to neurological impairment. Chikungunya fever (CF) is an ongoing public health problem in tropical and subtropical regions of the world, where control of the CHIKV vector, Aedes mosquitos, has failed. As there is no vaccine or specific treatment for CHIKV, patients receive only palliative care to alleviate pain and arthralgia. Thus, drug repurposing is necessary to identify antivirals against CHIKV. CHIKV RNA polymerase is similar to the orthologue enzyme of other positive-sense RNA viruses, such as members of the Flaviviridae family. Among the Flaviviridae, not only is hepatitis C virus RNA polymerase susceptible to sofosbuvir, a clinically approved nucleotide analogue, but so is dengue, Zika, and yellow fever virus replication. Here, we found that sofosbuvir was three times more selective in inhibiting CHIKV production in human hepatoma cells than ribavirin, a pan-antiviral drug. Although CHIKV replication in human induced pluripotent stem cell-derived astrocytes was less susceptible to sofosbuvir than were hepatoma cells, sofosbuvir nevertheless impaired virus production and cell death in a multiplicity of infection-dependent manner. Sofosbuvir also exhibited antiviral activity in vivo by preventing CHIKV-induced paw edema in adult mice at a dose of 20 mg/kg of body weight/day and prevented mortality in a neonate mouse model at 40- and 80-mg/kg/day doses. Our data demonstrate that a prototypic alphavirus, CHIKV, is also susceptible to sofosbuvir. As sofosbuvir is a clinically approved drug, our findings could pave the way to it becoming a therapeutic option against CF.

Influenza virus RNA polymerase may be activated inside the virion.

Influenza A and B virions are packaged with their polymerases to catalyse RNA-dependent RNA polymerase activity. Since there is no evidence to rule in or out the permissiveness of influenza virions to triphosphate ribonucleotides, we functionally evaluated this. We found the means to stimulate influenza A and B RNA polymerase activity inside the virion, called natural endogenous RNA polymerase (NERP) activity. Stimulation of NERP activity increased up to 3 log10 viral RNA content, allowing the detection of influenza virus in otherwise undetectable clinical samples. NERP activation also improved our capacity to sequence misidentified regions of the influenza genome from clinical samples. By treating the samples with the ribavirin triphosphate we inhibited NERP activity, which confirms our hypothesis and highlights that this assay could be used to screen antiviral drugs. Altogether, our data show that NERP activity could be explored to increase molecular diagnostic sensitivity and/or to develop antiviral screening assays.

An observational clinical case of Zika virus-associated neurological disease is associated with primary IgG response and enhanced TNF levels.

Descriptive clinical data help to reveal factors that may provoke Zika virus (ZIKV) neuropathology. The case of a 24-year-old female with a ZIKV-associated severe acute neurological disorder was studied. The levels of ZIKV in the cerebrospinal fluid (CSF) were 50 times higher than the levels in other compartments. An acute anti-flavivirus IgG, together with enhanced TNF-alpha levels, may have contributed to ZIKV invasion in the CSF, whereas the unbiased genome sequencing [obtained by next-generation sequencing (NGS)] of the CSF revealed that no virus mutations were associated with the anatomic compartments (CSF, serum, saliva and urine).

Immunogenicity and sustainability of the immune response in Brazilian HIV-1-infected individuals vaccinated with inactivated triple influenza vaccine.

HIV-infected individuals have a higher risk of serious illnesses following infection by infection with influenza. Although anti-influenza vaccination is recommended, immunosuppression may limit their response to active immunization. We followed-up a cohort of HIV-infected individuals vaccinated against influenza to assess the immunogenicity and sustainability of the immune response to vaccination. Individuals were vaccinated 2011 with inactivated triple influenza vaccine (TIV), and they had received in 2010 the monovalent anti-A(H1N1)pdm09 vaccine. The sustainability of the immune response to A(H1N1)pdm09 at 12 months after monovalent vaccination fell, both in individuals given two single or two double doses. For these individuals, A(H1N1)pdm09 component from TIV acted as a booster, raising around 40% the number of seroprotected individuals. Almost 70% of the HIV-infected individuals were already seroprotected to A/H3N2 at baseline. Again, TIV boosted over 90% the seroprotection to A/H3N2. Anti-A/H3N2 titers dropped by 20% at 6 months after vaccination. Pre-vaccination seroprotection rate to influenza B (victoria lineage) was the lowest among those tested, seroconversion rates were higher after vaccination. Seroconversion/protection after TIV vaccination did not differ significantly across categories of clinical and demographic variables. Anti-influenza responses in Brazilian HIV-infected individuals reflected both the previous history of virus circulation in Brazil and vaccination.

1,2,3-Triazolyl-4-oxoquinolines: A feasible beginning for promising chemical structures to inhibit oseltamivir-resistant influenza A and B viruses.

We described the synthesis of a new congener series of 1,2,3-triazolyl-4-oxoquinolines and evaluated their ability to inhibit oseltamivir (OST)-resistant influenza strains. Oxoquinoline derivative 1i was the most potent compound within this series, inhibiting 94% of wild-type (WT) influenza neuraminidase (NA) activity. Compound 1i inhibited influenza virus replication with an EC50 of 0.2µM with less cytotoxicity than OST, and also inhibited different OST-resistant NAs. These results suggest that 1,2,3-triazolyl-4-oxoquinolines represent promising lead molecules for further anti-influenza drug design.

Oseltamivir-resistant influenza A(H1N1)pdm2009 strains found in Brazil are endowed with permissive mutations, which compensate the loss of fitness imposed by antiviral resistance.

The 2009 pandemic influenza A virus outbreak led to the systematic use of the neuraminidase (NA) inhibitor oseltamivir (OST). Consequently, OST-resistant strains, carrying the mutation H275Y, emerged in the years after the pandemics, with a prevalence of 1-2%. Currently, OST-resistant strains have been found in community settings, in untreated individuals. To spread in community settings, H275Y mutants must contain additional mutations, collectively called permissive mutations. We display the permissive mutations in NA of OST-resistant A(H1N1)pdm09 virus found in Brazilian community settings. The NAs from 2013 are phylogenetically distinct from those of 2012, indicating a tendency of positive selection of NAs with better fitness. Some previously predicted permissive mutations, such as V241I and N369K, found in different countries, were also detected in Brazil. Importantly, the change D344N, also predicted to compensate loss of fitness imposed by H275Y mutation, was found in Brazil, but not in other countries in 2013. Our results reinforce the notion that OST-resistant A(H1N1)pdm09 strains with compensatory mutations may arise in an independent fashion, with samples being identified in different states of Brazil and in different countries. Systematic circulation of these viral strains may jeopardise the use of the first line of anti-influenza drugs in the future.

The effects of neurotrophins and the neuropeptides VIP and PACAP on HIV-1 infection: histories with opposite ends.

The nerve growth factor (NGF) and other neurotrophins, and the neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) are largely present in human tissue and can exert modulatory activities on nervous, endocrine and immune system functions. NGF, VIP and PACAP receptors are expressed systemically in organisms, and thus these mediators exhibit pleiotropic natures. The human immunodeficiency virus type 1 (HIV-1), the causal agent of the acquired immunodeficiency syndrome (AIDS), infects immune cells, and its replication is modulated by a number of endogenous factors that interact with HIV-1-infected cells. NGF, VIP and PACAP can also affect HIV-1 virus particle production upon binding to their receptors on the membranes of infected cells, which triggers cell signaling pathways that modify the HIV-1 replicative cycle. These molecules exert opposite effects on HIV-1 replication, as NGF and other neurotrophins enhance and VIP and PACAP reduce viral production in HIV-1-infected human primary macrophages. The understanding of AIDS pathogenesis should consider the mechanisms by which the replication of HIV-1, a pathogen that causes chronic morbidity, is influenced by neurotrophins, VIP and PACAP, i.e. molecules that exert a broad spectrum of physiological activities on the neuroimmunoendocrine axis. In this review, we will present the main effects of these two groups of mediators on the HIV-1 replicative cycle, as well as the mechanisms that underlie their abilities to modulate HIV-1 production in infected immune cells, and discuss the possible repercussion of the cross talk between NGF and both neuropeptides on the pathogenesis of HIV-1 infection.

Molecular findings from influenza A(H1N1)pdm09 detected in patients from a Brazilian equatorial region during the pandemic period.

After the World Health Organization officially declared the end of the first pandemic of the XXI century in August 2010, the influenza A(H1N1)pdm09 virus has been disseminated in the human population. In spite of its sustained circulation, very little on phylogenetic data or oseltamivir (OST) resistance is available for the virus in equatorial regions of South America. In order to shed more light on this topic, we analysed the haemagglutinin (HA) and neuraminidase (NA) genes of influenza A(H1N1)pdm09 positive samples collected during the pandemic period in the Pernambuco (PE), a northeastern Brazilian state. Complete HA sequences were compared and amino acid changes were related to clinical outcome. In addition, the H275Y substitution in NA, associated with OST resistance, was investigated by pyrosequencing. Samples from PE were grouped in phylogenetic clades 6 and 7, being clustered together with sequences from South and Southeast Brazil. The D222N/G HA gene mutation, associated with severity, was found in one deceased patient that was pregnant. Additionally, the HA mutation K308E, which appeared in Brazil in 2010 and was only detected worldwide the following year, was identified in samples from hospitalised cases. The resistance marker H275Y was not identified in samples tested. However, broader studies are needed to establish the real frequency of resistance in this Brazilian region.

Extracellular vesicles from primary human macrophages stimulated with VIP or PACAP mediate anti-SARS-CoV-2 activities in monocytes through NF-κB signaling pathway.

Infection by SARS-CoV-2 is associated with uncontrolled inflammatory response during COVID-19 severe disease, in which monocytes are one of the main sources of pro-inflammatory mediators leading to acute respiratory distress syndrome. Extracellular vesicles (EVs) from different cells play important roles during SARS-CoV-2 infection, but investigations describing the involvement of EVs from primary human monocyte-derived macrophages (MDM) on the regulation of this infection are not available. Here, we describe the effects of EVs released by MDM stimulated with the neuropeptides VIP and PACAP on SARS-CoV-2-infected monocytes. MDM-derived EVs were isolated by differential centrifugation of medium collected from cells cultured for 24 h in serum-reduced conditions. Based on morphological properties, we distinguished two subpopulations of MDM-EVs, namely large (LEV) and small EVs (SEV). We found that MDM-derived EVs stimulated with the neuropeptides inhibited SARS-CoV-2 RNA synthesis/replication in monocytes, protected these cells from virus-induced cytopathic effects and reduced the production of pro-inflammatory mediators. In addition, EVs derived from VIP- and PACAP-treated MDM prevented the SARS-CoV-2-induced NF-κB activation. Overall, our findings suggest that MDM-EVs are endowed with immunoregulatory properties that might contribute to the antiviral and anti-inflammatory responses in SARS-CoV-2-infected monocytes and expand our knowledge of EV effects during COVID-19 pathogenesis.