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BackgroundThe SARS-CoV-2 virus has caused unprecedented mortality since its emergence in late 2019. The continuous evolution of the viral genome through the concerted action of mutational forces has produced distinct variants that became dominant, challenging human immunity and vaccine development.Aim and methodsIn this work, through an integrative genomic approach, we describe the molecular transition of SARS-CoV-2 by analyzing the viral whole genome sequences from 50 critical COVID-19 patients recruited during the first year of the pandemic in Mexico City.ResultsOur results revealed differential levels of the evolutionary forces across the genome and specific mutational processes that have shaped the first two epidemiological waves of the pandemic in Mexico. Through phylogenetic analyses, we observed a genomic transition in the circulating SARS-CoV-2 genomes from several lineages prevalent in the first wave to a dominance of the B.1.1.519 variant (defined by T478K, P681H, and T732A mutations in the spike protein) in the second wave.ConclusionThis work contributes to a better understanding of the evolutionary dynamics and selective pressures that act at the genomic level, the prediction of more accurate variants of clinical significance, and a better comprehension of the molecular mechanisms driving the evolution of SARS-CoV-2 to improve vaccine and drug development.

The study, synthesis, and application of nanomaterials in medicine have grown exponentially in recent years. An example of this is the understanding of how nanomaterials activate or regulate the immune system, particularly macrophages. In this work, nanoparticles were synthesized using Rumex hymenosepalus as a reducing agent (AgRhNPs). According to thermogravimetric analysis, the metal content of nanoparticles is 55.5% by weight. The size of the particles ranges from 5–26 nm, with an average of 11 nm, and they possess an fcc crystalline structure. The presence of extract molecules on the nanomaterial was confirmed by UV-Vis and FTIR. It was found by UPLC-qTOF that the most abundant compounds in Rh extract are flavonols, flavones, isoflavones, chalcones, and anthocyanidins. The viability and apoptosis of the THP-1 cell line were evaluated for AgRhNPs, commercial nanoparticles (AgCNPs), and Rh extract. The results indicate a minimal cytotoxic and apoptotic effect at a concentration of 12.5 μg/mL for both nanoparticles and 25 μg/mL for Rh extract. The interaction of the THP-1 cell line and treatments was used to evaluate the polarization of monocyte subsets in conjunction with an evaluation of CCR2, Tie-2, and Arg-1 expression. The AgRhNPs nanoparticles and Rh extract neither exhibited cytotoxicity in the THP-1 monocyte cell line. Additionally, the treatments mentioned above exhibited anti-inflammatory effects by maintaining the classical monocyte phenotype CD14++CD16, reducing pro-inflammatory interleukin IL-6 production, and increasing IL-4 production.

Dengue and Zika viruses cocirculate annually in endemic areas of Mexico, causing outbreaks of different magnitude and severity every year, suggesting a continuous selection of Flavivirus variants with variable phenotypes of transmissibility and virulence. To evaluate if Flavivirus variants with different phenotypes cocirculate during outbreaks, we isolated dengue and Zika viruses from blood samples of febrile patients from Oaxaca City during the 2016 and 2019 epidemic years. We compared their replication kinetics in human cells, susceptibility to type I interferon antiviral response, and the accumulation of subgenomic RNA on infected cells. We observed correlations between type I interferon susceptibility and subgenomic RNA accumulation, with high hematocrit percentage and thrombocytopenia. Our results suggest that Flaviviruses that cocirculate in Oaxaca, Mexico, have variable sensitivity to the antiviral activity of type I interferons, and this phenotypic trait correlates with the severity of the disease.

Platelets are anucleate cells that have a role in several innate immune functions, including the secretion of proteins with antimicrobial activity. Several studies have demonstrated the ability of platelets to secrete thrombin-induced platelet microbicidal proteins and antimicrobial peptides, like hBD-1. However, the expression and secretion of defensins of the alpha family by platelets have not been fully elucidated. The aim of this study was to characterize the expression of defensin alpha 1 (DEFA1) in human platelets and megakaryocytes. Our data indicate that DEFA1 mRNA and protein are present in peripheral blood platelets and in the megakaryoblastic leukemia cell line (MEG-01). DEFA1 co-localize with α-granules of platelets and MEG-01 cells, and was also detected in cytoplasm of MEG-01 cells. The assay of our in vitro model of platelet-like particles (PLPs) revealed that MEG-01 cells could transfer DEFA1 mRNA to their differentiated PLPs. Furthermore, platelets secreted DEFA1 into the culture medium when activated with thrombin, adenosine diphosphate, and lipopolysaccharide; meanwhile, MEG-01 cells secreted DEFA1 when activated with thrombopoietin. Platelet’s secreted DEFA1 can rebind to platelet’s surface and have antibacterial activity against the gram-negative bacteria Escherichia coli. In summary, our data indicate that both, human platelets and megakaryocytes, can express and secrete DEFA1. These results suggest a new role of platelets and megakaryocytes in the innate immune response.

Despite all successful efforts to develop a COVID-19 vaccine, the need to evaluate alternative antigens to produce next-generation vaccines is imperative to target emerging variants. Thus, the second generation of COVID-19 vaccines employ more than one antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to induce an effective and lasting immune response. Here, we analyzed the combination of two SARS-CoV-2 viral antigens that could elicit a more durable immune response in both T- and B-cells. The nucleocapsid (N) protein, Spike protein S1 domain, and receptor binding domain (RBD) of the SARS-CoV-2 spike surface glycoproteins were expressed and purified in a mammalian expression system, taking into consideration the posttranscriptional modifications and structural characteristics. The immunogenicity of these combined proteins was evaluated in a murine model. Immunization combining S1 or RBD with the N protein induced higher levels of IgG antibodies, increased the percentage of neutralization, and elevated the production of cytokines TNF-α, IFN-γ, and IL-2 compared to the administration of a single antigen. Furthermore, sera from immunized mice recognized alpha and beta variants of SARS-CoV-2, which supports ongoing clinical results on partial protection in vaccinated populations, despite mutations. This study identifies potential antigens for second-generation COVID-19 vaccines.

Dengue virus infection (DENV-2) is transmitted by infected mosquitoes via the skin, where many dermal and epidermal cells are potentially susceptible to infection. Most of the cells in an area of infection will establish an antiviral microenvironment to control viral replication. Although cumulative studies report permissive DENV-2 infection in dendritic cells, keratinocytes, and fibroblasts, among other cells also infected, little information is available regarding cell-to-cell crosstalk and the effect of this on the outcome of the infection. Therefore, our study focused on understanding the contribution of fibroblast and dendritic cell crosstalk to the control or promotion of dengue. Our results suggest that dendritic cells promote an antiviral state over fibroblasts by enhancing the production of type I interferon, but not proinflammatory cytokines. Infected and non-infected fibroblasts promoted partial dendritic cell maturation, and the fibroblast-matured cells were less permissive to infection and showed enhanced type I interferon production. We also observed that the soluble mediators produced by non-infected or Poly (I:C) transfected fibroblasts induced allogenic T cell proliferation, but mediators produced by DENV-2 infected fibroblasts inhibited this phenomenon. Additionally, the effects of fibroblast soluble mediators on CD14+ monocytes were analyzed to assess whether they affected the differentiation of monocyte derived dendritic cells (moDC). Our data showed that mediators produced by infected fibroblasts induced variable levels of monocyte differentiation into dendritic cells, even in the presence of recombinant GM-CSF and IL-4. Cells with dendritic cell-like morphology appeared in the culture; however, flow cytometry analysis showed that the mediators did not fully downregulate CD14 nor did they upregulate CD1a. Our data revealed that fibroblast-dendritic cell crosstalk promoted an antiviral response mediated manly by type I interferons over fibroblasts. Furthermore, the maturation of dendritic cells and T cell proliferation were promoted, which was inhibited by DENV-2-induced mediators. Together, our results suggest that activation of the adaptive immune response is influenced by the crosstalk of skin resident cells and the intensity of innate immune responses established in the microenvironment of the infected skin.

Abstract Background Co-circulation of dengue virus (DENV) and chikungunya virus (CHIKV) is increasing worldwide but information on the viral dynamics and immune response to DENV-CHIKV co-infection, particularly in young infants, is scant. Methods Blood samples were collected from 24 patients, aged 2 months to 82 years, during a CHIKV outbreak in Mexico. DENV and CHIKV were identified by RT-PCR; ELISA was used to detect IgM and IgG antibodies. CHIKV PCR products were cloned, sequenced and subjected to BLAST analysis. To address serological findings, HMEC-1 and Vero cells were inoculated with DENV-1, DENV-2 and CHIKV alone and in combination (DENV-2-CHIKV and DENV-1-CHIKV); viral titers were measured at 24, 48 and 72 h. Results Nine patients (38%) presented co-infection, of who eight were children. None of the patients presented severe illness. Sequence analysis showed that the circulating CHIKV virus belonged to the Asian lineage. Seroconversion to both viruses was only observed in the four patients five years or older, while the five infants under two years of age only seroconverted to CHIKV. Viral titers in the CHIKV mono-infected cells were greater than in the DENV-1 and DENV-2 mono-infected cells. Furthermore, we observed significantly increased CHIKV progeny and reduction of DENV progeny in the co-infected cells. Conclusions In our population, DENV-CHIKV co-infection was not associated with increased clinical severity. Our in vitro assay findings strongly suggest that the lack of DENV IgG conversion in the co-infected infants is due to suppression of DENV replication by the Asian lineage CHIKV. The presence of maternal antibody and immature immune responses in the young infants may also play a role.

BackgroundWhen mosquitoes infected with DENV are feeding, the proboscis must traverse the epidermis several times ("probing") before reaching a blood vessel in the dermis. During this process, the salivary glands release the virus, which is likely to interact first with cells of the various epidermal and dermal layers, cells which could be physiologically relevant to DENV infection and replication in humans. However, important questions are whether more abundant non-hematopoietic cells such as fibroblasts become infected, and whether they play any role in antiviral innate immunity in the very early stages of infection, or even if they might be used by DENV as primary replication cells.Methodology/principal findingsFibroblasts freshly released from healthy skin and infected 12 hours after their isolation show a positive signal for DENV. In addition, when primary skin fibroblast cultures were established and subsequently infected, we showed DENV-2 antigen-positive intracellular signal at 24 hours and 48 hours post-infection. Moreover, the fibroblasts showed productive infection in a conventional plaque assay. The skin fibroblasts infected with DENV-2 underwent potent signaling through both TLR3 and RIG- 1, but not Mda5, triggering up-regulation of IFNβ, TNFα, defensin 5 (HB5) and β defensin 2 (HβD2). In addition, DENV infected fibroblasts showed increased nuclear translocation of interferon (IFN) regulatory factor 3 (IRF3), but not interferon regulatory factor 7 (IRF7), when compared with mock-infected fibroblasts.Conclusions/significanceIn this work, we demonstrated the high susceptibility to DENV infection by primary fibroblasts from normal human skin, both in situ and in vitro. Our results suggest that these cells may contribute to the pro-inflammatory and anti-viral microenvironment in the early stages of interaction with DENV-2. Furthermore, the data suggest that fibroblast may also be used as a primary site of DENV replication and provide viral particles that may contribute to subsequent viral dissemination.

Sepsis is an organic dysfunction that puts at risk the life of patients suffering this disorder due to an exacerbated immunological response to the infection mediated by the host. Platelets have been largely researched on sepsis owing to its role in Disseminated Intracellular Coagulation (DIC) and because thrombocytopenia is an important clinical feature of these patients. Nevertheless, a great number of evidence shows that platelets have also an important role in immunological response since they have pattern recognition receptors, chemokine receptors and granules with stored soluble mediators. In this work, the immunological features of platelets in individuals with sepsis are described. The results show that platelets of these individuals have high levels of surfaces expression of TLR4, CD62P, CD32 and thrombin receptor 1 (PAR-1), these platelets have also greater capability to joinEscherichia coli, and show a different profile of soluble mediators (IL-1β, CD40L and TNF-α). Platelets from patients with sepsis form aggregates with neutrophils in circulation, but are unable to induce the production of reactive oxygen species. This research describes important features of platelets to help the understanding of the immunological role of these cells in sepsis. [ABSTRACT FROM PUBLISHER]

Mosquito-borne flaviviruses are among the most significant arboviral pathogens worldwide. Vaccinations and mosquito population control programs remain the most reliable means for flavivirus disease prevention, and live attenuated viruses remain one of the most attractive flavivirus vaccine platforms. Some live attenuated viruses are capable of infecting principle mosquito vectors, as demonstrated in the laboratory, which in combination with their intrinsic genetic instability could potentially lead to a vaccine virus reversion back to wild-type in nature, followed by introduction and dissemination of potentially dangerous viral strains into new geographic locations. To mitigate this risk we developed a microRNA-targeting approach that selectively restricts replication of flavivirus in the mosquito host. Introduction of sequences complementary to a mosquito-specific mir-184 and mir-275 miRNAs individually or in combination into the 3'NCR and/or ORF region resulted in selective restriction of dengue type 4 virus (DEN4) replication in mosquito cell lines and adult Aedes mosquitos. Moreover a combined targeting of DEN4 genome with mosquito-specific and vertebrate CNS-specific mir-124 miRNA can silence viral replication in two evolutionally distant biological systems: mosquitoes and mouse brains. Thus, this approach can reinforce the safety of newly developed or existing vaccines for use in humans and could provide an additional level of biosafety for laboratories using viruses with altered pathogenic or transmissibility characteristics.