Intra-host evolution during SARS-CoV-2 prolonged infection.

Long-term infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents a challenge to virus dispersion and the control of coronavirus disease 2019 (COVID-19) pandemic. The reason why some people have prolonged infection and how the virus persists for so long are still not fully understood. Recent studies suggested that the accumulation of intra-host single nucleotide variants (iSNVs) over the course of the infection might play an important role in persistence as well as emergence of mutations of concern. For this reason, we aimed to investigate the intra-host evolution of SARS-CoV-2 during prolonged infection. Thirty-three patients who remained reverse transcription polymerase chain reaction (RT-PCR) positive in the nasopharynx for on average 18 days from the symptoms onset were included in this study. Whole-genome sequences were obtained for each patient at two different time points. Phylogenetic, populational, and computational analyses of viral sequences were consistent with prolonged infection without evidence of coinfection in our cohort. We observed an elevated within-host genomic diversity at the second time point samples positively correlated with cycle threshold (Ct) values (lower viral load). Direct transmission was also confirmed in a small cluster of healthcare professionals that shared the same workplace by the presence of common iSNVs. A differential accumulation of missense variants between the time points was detected targeting crucial structural and non-structural proteins such as Spike and helicase. Interestingly, longitudinal acquisition of iSNVs in Spike protein coincided in many cases with SARS-CoV-2 reactive and predicted T cell epitopes. We observed a distinguishing pattern of mutations over the course of the infection mainly driven by increasing A→U and decreasing G→A signatures. G→A mutations may be associated with RNA-editing enzyme activities; therefore, the mutational profiles observed in our analysis were suggestive of innate immune mechanisms of the host cell defense. Therefore, we unveiled a dynamic and complex landscape of host and pathogen interaction during prolonged infection of SARS-CoV-2, suggesting that the host's innate immunity shapes the increase of intra-host diversity. Our findings may also shed light on possible mechanisms underlying the emergence and spread of new variants resistant to the host immune response as recently observed in COVID-19 pandemic.

In-depth characterization of congenital Zika syndrome in immunocompetent mice: Antibody-dependent enhancement and an antiviral peptide therapy.

BACKGROUND: Zika virus (ZIKV) infection during pregnancy may cause major congenital defects, including microcephaly, ocular, articular and muscle abnormalities, which are collectively defined as Congenital Zika Syndrome. Here, we performed an in-depth characterization of the effects of congenital ZIKV infection (CZI) in immunocompetent mice. METHODS: Pregnant dams were inoculated with ZIKV on embryonic day 5.5 in the presence or absence of a sub-neutralizing dose of a pan-flavivirus monoclonal antibody (4G2) to evaluate the potential role of antibody-dependent enhancement phenomenon (ADE) during short and long outcomes of CZI. FINDINGS: ZIKV infection induced maternal immune activation (MIA), which was associated with occurrence of foetal abnormalities and death. Therapeutic administration of AH-D antiviral peptide during the early stages of pregnancy prevented ZIKV replication and death of offspring. In the post-natal period, CZI was associated with a decrease in whole brain volume, ophthalmologic abnormalities, changes in testicular morphology, and disruption in bone microarchitecture. Some alterations were enhanced in the presence of 4G2 antibody. INTERPRETATION: Our results reveal that early maternal ZIKV infection causes several birth defects in immunocompetent mice, which can be potentiated by ADE phenomenon and are associated with MIA. Additionally, antiviral treatment with AH-D peptide may be beneficial during early maternal ZIKV infection. FUND: This work was supported by the Brazilian National Science Council (CNPq, Brazil), Minas Gerais Foundation for Science (FAPEMIG), Funding Authority for Studies and Projects (FINEP), Coordination of Superior Level Staff Improvement (CAPES), National Research Foundation of Singapore and Centre for Precision Biology at Nanyang Technological University.

Phosphatidyl Inositol 3 Kinase-Gamma Balances Antiviral and Inflammatory Responses During Influenza A H1N1 Infection: From Murine Model to Genetic Association in Patients.

Influenza A virus (IAV) infection causes severe pulmonary disease characterized by intense leukocyte infiltration. Phosphoinositide-3 kinases (PI3Ks) are central signaling enzymes, involved in cell growth, survival, and migration. Class IB PI3K or phosphatidyl inositol 3 kinase-gamma (PI3Kγ), mainly expressed by leukocytes, is involved in cell migration during inflammation. Here, we investigated the contribution of PI3Kγ for the inflammatory and antiviral responses to IAV. PI3Kγ knockout (KO) mice were highly susceptible to lethality following infection with influenza A/WSN/33 H1N1. In the early time points of infection, infiltration of neutrophils was higher than WT mice whereas type-I and type-III IFN expression and p38 activation were reduced in PI3Kγ KO mice resulting in higher viral loads when compared with WT mice. Blockade of p38 in WT macrophages infected with IAV reduced levels of interferon-stimulated gene 15 protein to those induced in PI3Kγ KO macrophages, suggesting that p38 is downstream of antiviral responses mediated by PI3Kγ. PI3Kγ KO-derived fibroblasts or macrophages showed reduced type-I IFN transcription and altered pro-inflammatory cytokines suggesting a cell autonomous imbalance between inflammatory and antiviral responses. Seven days after IAV infection, there were reduced infiltration of natural killer cells and CD8+ T lymphocytes, increased concentration of inflammatory cytokines in bronchoalveolar fluid, reduced numbers of resolving macrophages, and IL-10 levels in PI3Kγ KO. This imbalanced environment in PI3Kγ KO-infected mice culminated in enhanced lung neutrophil infiltration, reactive oxygen species release, and lung damage that together with the increased viral loads, contributed to higher mortality in PI3Kγ KO mice compared with WT mice. In humans, we tested the genetic association of disease severity in influenza A/H1N1pdm09-infected patients with three potentially functional PIK3CG single-nucleotide polymorphisms (SNPs), rs1129293, rs17847825, and rs2230460. We observed that SNPs rs17847825 and rs2230460 (A and T alleles, respectively) were significantly associated with protection from severe disease using the recessive model in patients infected with influenza A(H1N1)pdm09. Altogether, our results suggest that PI3Kγ is crucial in balancing antiviral and inflammatory responses to IAV infection.

In Vitro TNF-α Inhibitory Activity of Brazilian Plants and Anti-Inflammatory Effect of Stryphnodendron adstringens in an Acute Arthritis Model.

Stryphnodendron species, popularly named "barbatimão," are traditionally used in Brazil as anti-inflammatory agents. This study aimed to investigate the effect of barbatimão and 11 other species on the production of tumor necrosis factor-alpha (TNF-α) in lipopolysaccharide- (LPS-) stimulated THP-1 cells, as well as their anti-arthritis activity. The extracts of Stryphnodendron adstringens, Stryphnodendron obovatum, Campomanesia lineatifolia, and Terminalia glabrescens promoted a concentration-dependent inhibition of TNF-α. Mice injected with LPS in the knee joint were treated per os with fractions from the selected extracts. Both the organic (SAO) and the aqueous (SAA) fractions of S. adstringens promoted a dose-dependent reduction of leukocyte migration and neutrophil accumulation into the joint, but none of them reduced CXCL1 concentration in the periarticular tissue. In contrast, treatment with C. lineatifolia and T. glabrescens fractions did not ameliorate the inflammatory parameters. Analyses of SAO by Ultra Performance Liquid Chromatography (UPLC) coupled to electrospray ionization mass spectrometry (ESI-MS) led to the identification of gallic acid along with 11 prodelphinidins, characterized as monomers and dimers of the B-type. Our findings contribute to some extent to corroborating the traditional use of S. adstringens as an anti-inflammatory agent. This activity is probably related to a decrease of leukocyte migration into the inflammatory site. Polyphenols like gallic acid and prodelphinidins, identified in the active fraction, may contribute to the observed activity.

Antigen production using heterologous expression of dengue virus-2 non-structural protein 1 (NS1) in Nicotiana tabacum (Havana) for immunodiagnostic purposes.

KEY MESSAGE: Expression of dengue-2 virus NS1 protein in Nicotiana tabacum plants for development of dengue immunodiagnostic kits. Dengue is one of the most important diseases caused by arboviruses in the world. A significant increase in its geographical distribution has been noticed over the last 20 years, with continuous transmission of several serotypes and emergence of the hemorrhagic fever in areas where the disease was previously not prevalent. Although the methodological processes for dengue diagnosis are in deep development and improvement, a limitation for the realization of dengue diagnostic tests is the difficulty of large-scale production of the antigen to be used in diagnostic tests. Due to this demand, the purpose of this study was to obtain the non-structural protein 1 (NS1) from dengue-2 serotype by heterologous expression in Nicotiana tabacum (Havana). After confirmation of the NS1 protein gene integration in the plant genome, the heterologous protein was characterized using SDS-PAGE and immunoblotting. In an immunoenzymatic test, the recombinant NS1 protein presents an antigen potential for development of dengue immunodiagnostic kits.

A model of DENV-3 infection that recapitulates severe disease and highlights the importance of IFN-γ in host resistance to infection.

There are few animal models of dengue infection, especially in immunocompetent mice. Here, we describe alterations found in adult immunocompetent mice inoculated with an adapted Dengue virus (DENV-3) strain. Infection of mice with the adapted DENV-3 caused inoculum-dependent lethality that was preceded by several hematological and biochemical changes and increased virus dissemination, features consistent with severe disease manifestation in humans. IFN-γ expression increased after DENV-3 infection of WT mice and this was preceded by increase in expression of IL-12 and IL-18. In DENV-3-inoculated IFN-γ(-/-) mice, there was enhanced lethality, which was preceded by severe disease manifestation and virus replication. Lack of IFN-γ production was associated with diminished NO-synthase 2 (NOS2) expression and higher susceptibility of NOS2(-/-) mice to DENV-3 infection. Therefore, mechanisms of protection to DENV-3 infection rely on IFN-γ-NOS2-NO-dependent control of viral replication and of disease severity, a pathway showed to be relevant for resistance to DENV infection in other experimental and clinical settings. Thus, the model of DENV-3 infection in immunocompetent mice described here represents a significant advance in animal models of severe dengue disease and may provide an important tool to the elucidation of immunopathogenesis of disease and of protective mechanisms associated with infection.

Monitoring the bacterial community dynamics in a petroleum refinery wastewater membrane bioreactor fed with a high phenolic load.

The phenolic compounds are a major contaminant class often found in industrial wastewaters and the biological treatment is an alternative tool commonly employed for their removal. In this sense, monitoring microbial community dynamics is crucial for a successful wastewater treatment. This work aimed to monitor the structure and activity of the bacterial community during the operation of a laboratory-scale continuous submerged membrane bioreactor (SMBR), using PCR and RT-PCR followed by Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA libraries. Multivariate analyses carried out using DGGE profiles showed significant changes in the total and metabolically active dominant community members during the 4-week treatment period, explained mainly by phenol and ammonium input. Gene libraries were assembled using 16S rDNA and 16S rRNA PCR products from the fourth week of treatment. Sequencing and phylogenetic analyses of clones from 16S rDNA library revealed a high diversity of taxa for the total bacterial community, with predominance of Thauera genus (ca. 50%). On the other hand, a lower diversity was found for metabolically active bacteria, which were mostly represented by members of Betaproteobacteria (Thauera and Comamonas), suggesting that these groups have a relevant role in the phenol degradation during the final phase of the SMBR operation.