T he effi ciency and persistence of natural protective immunity caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or vaccination are currently unknown. Reinfection cases have been reported in different countries ( 1), but the differentiation between cases of reinfection and viral persistence remains a challenge. The detection of 2 coronavirus disease (COVID-19) episodes >90 days apart and caused by 2 different lineages of SARS-CoV-2 remains the most reliable evidence of reinfection (2). In this article, we describe a reinfection case and highlight details about the genomic features of the 2 COVID-19 episodes. In addition, we demonstrate that the virus in the second episode was related to the emerging variant of interest (VOI) designated as lineage P.2, which is currently circulating throughout Brazil.

BackgroundThe influenza virus can cause seasonal infections with mild to severe symptoms, circulating worldwide, and it can affect people in any age group. Therefore, this infection is a serious public health problem that causes severe illness and death in high-risk populations. Every year, 0.5% of the world’s population is infected by this pathogen. This percentage can increase up to ten times during pandemics. Influenza vaccination is the most effective way to prevent disease. In addition, anti-influenza drugs are essential for prophylactic and therapeutic interventions. The oseltamivir (OST, a neuraminidase inhibitor) is the primary antiviral used in clinics during outbreaks. However, OST resistant viruses may emerge naturally or due to antiviral pressure, with a prevalence of 1–2% worldwide. Thus, the search for new anti-influenza drugs is extremely important. Currently, several groups have been developing studies describing the biotechnological potential of microalgae and cyanobacteria, including antiviral activity of their extracts. In Brazil, this potential is poorly known and explored.MethodsWith the aim of increasing the knowledge on this topic, 38 extracts from microalgae and cyanobacteria isolated from marine and freshwater biomes in Brazil were tested against: cellular toxicity; OST-sensitive and resistant influenza replications; and neuraminidase activity.ResultsFor this purpose, Madin-Darby Canine Kidney (MDCK)-infected cells were treated with 200 μg/mL of each extract. A total of 17 extracts (45%) inhibited influenza A replication, with seven of them resulting in more than 80% inhibition. Moreover, functional assays performed with viral neuraminidase revealed two extracts (from Leptolyngbya sp. and Chlorellaceae) with IC50 mean < 210 μg/mL for influenza A and B, and also OST-sensitive and resistant strains. Furthermore, MDCK cells exposed to 1 mg/mL of all the extracts showed viability higher than 80%.DiscussionOur results suggest that extracts of microalgae and cyanobacteria have promising anti-influenza properties. Further chemical investigation should be conducted to isolate the active compounds for the development of new anti-influenza drugs. The data generated contribute to the knowledge of the biotechnological potential of Brazilian biomes that are still little explored for this purpose.

Influenza A virus (IAV) is the main etiological agent of acute respiratory tract infections. During IAV infection, interferon triggers the overexpression of restriction factors (RFs), the intracellular antiviral branch of the innate immune system. Conversely, severe influenza is associated with an unbalanced pro-inflammatory cytokine release. It is unclear whether other cytokines and chemokines released during IAV infection modulate RFs to control virus replication. Among the molecules enhanced in the infected respiratory tract, ligands of the CCR5 receptor play a key role, as they stimulate the migration of inflammatory cells to the alveoli. We investigated here whether ligands of the CCR5 receptor could enhance RFs to levels able to inhibit IAV replication. For this purpose, the human alveolar basal epithelial cell line (A549) was treated with endogenous (CCL3, CCL4 and CCL5) or exogenous (HIV-1 gp120) ligands prior to IAV infection. The three CC-chemokines tested reduced infectious titers between 30% to 45% upon 24 hours of infection. Eploying RT-PCR, a panel of RF mRNA levels from cells treated with CCR5 agonists was evaluated, which showed that the SAMHD1 expression was up-regulated four times over control upon exposure to CCL3, CCL4 and CCL5. We also found that IAV inhibition by CCL5 was dependent on PKC and that SAMHD1 protein levels were also increased after treatment with CCL5. In functional assays, we observed that the knockdown of SAMHD1 resulted in enhanced IAV replication in A549 cells and abolished both CCL5-mediated inhibition of IAV replication and CCL5-mediated cell death inhibition. Our data show that stimuli unrelated to interferon may trigger the upregulation of SAMHD1 and that this RF may directly interfere with IAV replication in alveolar epithelial cells.

Introduction: The exposure of A(H1N1)pdm09-infected epithelial cells to HIV-1 viral particles, or its gp120, enhanced interferon-induced transmembrane protein (IFITM3) content, a viral restriction factor (VRF), resulting in a decrease in influenza replication. The gp120 binds to CCR5 (R5) or CXCR4 (X4) cell receptors during HIV infection. Then, it is possible that the endogenous ligands of these receptors also modulate the expression of IFITM3 or other cellular factors that restrict viral replication. Objective: We have studied the role of cellular receptors R5 and X4 in modulating VRFs. Methodology: A549 cells (adenocarcinomic human alveolar basal epithelial cells) were treated with 2x effective dose (ED50) of endogenous R5 or X4 receptor agonists, CCL3 (20 ng/ml), CCL4 (10 ng/ml), CCL5 (10 ng/ml) and CXCL12 (100 ng/mL) or exogenous agonists, gp120 Bal-R5, gp120 IIIB-X4 and gp120 mutants (5 µg/mL). The interferon α (10 ng/mL) and oseltamivir (64 nM) were used as control in all assays. Then, 18 h post agonists exposure the cells were infected with the virus A(H3N2) at MOI (multiplicity of infection) 2 for 1 h. 24 hours post infection, the supernatant was harvested and the viral titre assessed by quantifying neuraminidase activity (NA). To evaluate IFITM3 protein levels, A549 cells were exposed to agonists for 18 and 24 h and monolayer was lysed with Laemmli buffer for Western Blot (WB) assay or fixed for indirect immunofluorescence (IFI) assay. In addition, we analyzed other VRFs modulation in A549 18 h post agonists exposure by customized RT 2 Profiler PCR Array. Results: We found that R5 and X4 agonists and gp120 mutants inhibited influenza replication in 54 ± 9%. After 18 and 24 h agonists exposure, we did not observe increase in IFITM3 protein levels through WB and IFI assays. Then, we searched for other VRFs that might be involved in influenza inhibition by a customized RT 2 Profiler PCR Array. We observed an upregulation of SAMHD1 in A549 cells exposed to agonists. SAMDH1 was shown to possess dNTP triphosphohydrolase activity, which was proposed to inhibit HIV-1 replication and the autoimmune response by hydrolyzing cellular dNTPs. However, other groups propose that SAMHD1 possesses RNase activity and that the RNase but not the dNTPase function is essential for HIV-1 restriction. Until now, there are no studies in the literature showing the activity of this protein against influenza replication. Conclusion: Future studies should provide further insights about the role of SAMDH1 in influenza replication inhibition. The aim of this work is to find new strategies of the innate immune system that control important viral infections.

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