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Abstract Live-Attenuated Vaccines (LAVs) stimulate robust mucosal and cellular responses and have the potential to protect against Respiratory Syncytial Virus (RSV) and Human Metapneumovirus (HMPV), the main etiologic agents of viral bronchiolitis and pneumonia in children. We inserted the RSV-F gene into an HMPV-based LAV (Metavac®) we previously validated for the protection of mice against HMPV challenge, and rescued a replicative recombinant virus (Metavac®-RSV), exposing both RSV- and HMPV-F proteins at the virion surface and expressing them in reconstructed human airway epithelium models. When administered to BALB/c mice by the intranasal route, bivalent Metavac®-RSV demonstrated its capacity to replicate with reduced lung inflammatory score and to protect against both RSV and lethal HMPV challenges in vaccinated mice while inducing strong IgG and broad RSV and HMPV neutralizing antibody responses. Altogether, our results showed the versatility of the Metavac® platform and suggested that Metavac®-RSV is a promising mucosal bivalent LAV candidate to prevent pneumovirus-induced diseases.

Abstract Acute respiratory infections, a large part being of viral origin, constitute a major public health issue. To propose alternative and/or new therapeutic approaches, it is necessary to increase our knowledge about the interactions between respiratory viruses and their primary cellular targets using the most biologically relevant experimental models. In this study, we used RNAseq to characterize and compare the transcriptomic signature of infection induced by different major respiratory viruses (Influenza viruses, hRSV and hMPV) in a model of reconstituted human airway epithelia. Our results confirm the importance of several cellular pathways commonly or specifically induced by these respiratory viruses, such as the innate immune response or antiviral defense. A very interesting common feature revealed by the global virogenomic signature shared between hRSV, hMPV and influenza viruses is the global downregulation of cilium-related gene expression, in good agreement with experimental evaluation of mucociliary clearance. Beyond providing new information about respiratory virus/host interactions, our study also underlines the interest of using biologically relevant experimental models to study human respiratory viruses.

Abstract: Few studies have investigated the simultaneous effect of individual and contextual factors on the occurrences of anemia. This study aims to evaluate the variability of children’s hemoglobin levels from municipalities in social vulnerability and its association with factors of individual and municipal nature. This is a cross-sectional, multi-center study, with children data (12-59 months) collected from 48 municipalities of the Southern region of Brazil, that were included in the Brazil Without Poverty Plan. Individuals’ data were collected using a structured questionnaire, and secondary and ecological data of children’s municipalities were collected via national surveys and institutional websites. The outcome was defined as the hemoglobin level obtained by HemoCue. A multilevel analysis was performed using Generalized Linear Models for Location Scale and Shape using R, with a 5% significance level. A total of 1,501 children were evaluated. The mean hemoglobin level was 12.8g/dL (95%CI: 12.7-12.8), with significant variability between municipalities. Lower values of hemoglobin were observed in children who lived in municipalities with a higher urbanization rate and a lower number of Community Health Agents, in relation to the reference categories. At the individual level, lower hemoglobin values were identified for children under 24 months, not enrolled at daycares, who were beneficiaries of the conditional cash transfer program and diagnosed with underweight. The results shed light on important factors at the municipal and the individual levels that were associated to the hemoglobin levels of children living in municipalities in social vulnerability.

Summary: In the current COVID-19 pandemic context, proposing and validating effective treatments represents a major challenge. However, the scarcity of biologically relevant pre-clinical models of SARS-CoV-2 infection imposes a significant barrier for scientific and medical progress, including the rapid transition of potentially effective treatments to the clinical setting. We use reconstituted human airway epithelia to isolate and then characterize the viral infection kinetics, tissue-level remodeling of the cellular ultrastructure, and transcriptional early immune signatures induced by SARS-CoV-2 in a physiologically relevant model. Our results emphasize distinctive transcriptional immune signatures between nasal and bronchial HAE, both in terms of kinetics and intensity, hence suggesting putative intrinsic differences in the early response to SARS-CoV-2 infection. Most important, we provide evidence in human-derived tissues on the antiviral efficacy of remdesivir monotherapy and explore the potential of the remdesivir-diltiazem combination as an option worthy of further investigation to respond to the still-unmet COVID-19 medical need.

The development of a live-attenuated vaccine (LAV) for the prevention of human metapneumovirus (HMPV) infection is often hampered by the lack of highly efficient and scalable cell-based production systems that support eventual global vaccine production. Avian cell lines cultivated in suspension compete with traditional cell platforms used for viral vaccine manufacture. We investigated whether the DuckCelt®-T17 avian cell line (Vaxxel), previously described as an efficient production system for several influenza strains, could also be used to produce a new HMPV LAV candidate (Metavac®, SH gene-deleted A1/C-85473 HMPV). To that end, we characterized the operational parameters of MOI, cell density, and trypsin addition to achieve the optimal production of Metavac®, and demonstrated that the DuckCelt®-T17 cell line is permissive and well-adapted to the production of the wild-type A1/C-85473 HMPV and the Metavac® vaccine candidate. Moreover, our results confirmed that the LAV candidate produced in DuckCelt®-T17 cells conserves its advantageous replication properties in LLC-MK2 and 3D-reconstituted human airway epithelium models, and its capacity to induce efficient neutralizing antibodies in a BALB/c mouse model. Our results suggest that the DuckCelt®-T17 avian cell line is a very promising platform for the scalable in-suspension serum-free production of the HMPV-based LAV candidate Metavac®.

NOD-like receptor protein 3 (NLRP3) inflammasome activation triggers caspase-1 activation-induced maturation of interleukin (IL)-1β and IL-18 and therefore is important for the development of the host defense against various RNA viral diseases. However, the implication of this protein complex in human metapneumovirus (HMPV) disease has not been fully studied. Herein, we report that NLRP3 inflammasome plays a detrimental role during HMPV infection because NLRP3 inflammasome inhibition protected mice from mortality and reduced weight loss and inflammation without impacting viral replication. We also demonstrate that NLRP3 inflammasome exerts its deleterious effect via IL-1β production since we observed reduced mortality, weight loss and inflammation in IL-1β-deficient (IL-1β-/-) mice, as compared to wild-type animals during HMPV infection. Moreover, the effect on these evaluated parameters was not different in IL-1β-/- and wild-type mice treated with an NLRP3 inflammasome inhibitor. The production of IL-1β was also abrogated in bone marrow derived macrophages deficient for NLRP3. Finally, we show that small hydrophobic protein-deleted recombinant HMPV (HMPV ΔSH) failed to activate caspase-1, which is responsible for IL-1β cleavage and maturation. Furthermore, HMPV ΔSH-infected mice had less weight loss, showed no mortality and reduced inflammation, as compared to wild-type HMPV-infected mice. Thus, NLRP3 inflammasome activation seems to be triggered by HMPV SH protein in HMPV disease. In summary, once activated by the HMPV SH protein, NLRP3 inflammasome promotes the maturation of IL-1β, which exacerbates HMPV-induced inflammation. Therefore, the blockade of IL-1β production by using NLRP3 inflammasome inhibitors might be a novel potential strategy for the therapy and prevention of HMPV infection.

An increasing amount of evidence indicates a relatively high prevalence of superinfections associated with coronavirus disease 2019 (COVID-19), including invasive aspergillosis, but the underlying mechanisms remain to be characterized. In the present study, to better understand the biological impact of superinfection, we determine and compare the host transcriptional response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) versus Aspergillus superinfection, using a model of reconstituted human airway epithelium. Our analyses reveal that both simple infection and superinfection induce strong deregulation of core components of innate immune and inflammatory responses, with a stronger response to superinfection in the bronchial epithelial model compared to its nasal counterpart. Our results also highlight unique transcriptional footprints of SARS-CoV-2 Aspergillus superinfection, such as an imbalanced type I/type III IFN, and an induction of several monocyte and neutrophil associated chemokines, that could be useful for the understanding of Aspergillus-associated COVID-19 and also the management of severe forms of aspergillosis in this specific context.

Human metapneumovirus (HMPV) is a major pediatric respiratory pathogen with currently no specific treatment or licensed vaccine. Different strategies to prevent this infection have been evaluated, including live-attenuated vaccines (LAV) based on SH and/or G protein deletions. This approach showed promising outcomes but has not been evaluated further using different viral strains. In that regard, we previously showed that different HMPV strains harbor distinct in vitro fusogenic and in vivo pathogenic phenotypes, possibly influencing the selection of vaccine strains. In this study, we investigated the putative contribution of the low conserved SH or G accessory proteins in such strain-dependent phenotypes and generated recombinant wild type (WT) and SH- or G-deleted viruses derived from two different patient-derived HMPV strains, A1/C-85473 and B2/CAN98-75. The ΔSH and ΔG deletions led to different strain-specific phenotypes in both LLC-MK2 cell and reconstituted human airway epithelium models. More interestingly, the ΔG-85473 and especially ΔSH-C-85473 recombinant viruses conferred significant protection against HMPV challenge and induced immunogenicity against a heterologous strain. In conclusion, our results show that the viral genetic backbone should be considered in the design of live-attenuated HMPV vaccines, and that a SH-deleted virus based on the A1/C-85473 HMPV strain could be a promising LAV candidate as it is both attenuated and protective in mice while being efficiently produced in a cell-based system.

Human metapneumovirus (HMPV) is an important cause of acute respiratory tract infections (ARTI) in children, elderly individuals and immunocompromised patients. In vitro, different HMPV strains can induce variable cytopathic effects ranging from large multinucleated syncytia to focal cell rounding. In this study, we investigated the impact of different in vitro phenotypes of two HMPV strains on viral replication and disease severity in a BALB/c mouse model. We first generated two recombinant GFP-expressing HMPV viruses: C-85473, a syncytium-inducing strain (rC-85473) belonging to the A1 subtype and CAN98-75, a focal cell rounding-inducing strain (rCAN98-75) of the B2 subtype. We subsequently exchanged the F genes of both strains to create the chimeric viruses rC-85473_F and rCAN98-75_F. We demonstrated that the F protein was the sole protein responsible for the syncytium phenotype and that viruses carrying a syncytium-inducing F protein replicated to significantly higher titers in vitro. In vivo, however, the virulence and replicative capacity of the different HMPV strains did not appear to be solely dependent on the F gene but also on the viral background, with the strains containing the C-85473 background inducing more weight loss as well as increased lung viral titers, pro-inflammatory cytokines and inflammation than strains containing the CAN98-75 background. In conclusion, the F protein is the main determinant of syncytium formation and replication kinetics in vitro, although it is not the only factor implicated in HMPV disease severity in mice.

ABSTRACT During their nuclear replication stage, influenza viruses hijack the host splicing machinery to process some of their RNA segments, the M and NS segments. In this review, we provide an overview of the current knowledge gathered on this interplay between influenza viruses and the cellular spliceosome, with a particular focus on influenza A viruses (IAV). These viruses have developed accurate regulation mechanisms to reassign the host spliceosome to alter host cellular expression and enable an optimal expression of specific spliced viral products throughout infection. Moreover, IAV segments undergoing splicing display high levels of similarity with human consensus splice sites and their viral transcripts show noteworthy secondary structures. Sequence alignments and consensus analyses, along with recently published studies, suggest both conservation and evolution of viral splice site sequences and structure for improved adaptation to the host. Altogether, these results emphasize the ability of IAV to be well adapted to the host’s splicing machinery, and further investigations may contribute to a better understanding of splicing regulation with regard to viral replication, host range, and pathogenesis.

Abstract Objective: To estimate the prevalence of vitamin A deficiency (VAD) in children and associated risk factors. Design: Analysis of data from a cross-sectional multicentre study performed in the primary care units of the municipalities from January to June 2015. The children’s legal guardians answered a socio-economic questionnaire, and the children’s blood samples were obtained by venipuncture. Plasma retinol was determined by HPLC. Plasma retinol values of

This study aimed to verify the association between the consumption of omega-3-rich (n-3) fish and depressive symptoms in older adults living in Southern Brazil. This is a cross-sectional analysis with data from the second wave of the EpiFloripa Aging cohort study (2013/2014) including 1,130 individuals aged 60 years or older. The presence of depressive symptoms was measured by the 15-items Geriatric Depression Scale (GDS-15), and the consumption of n-3-rich fish by a question of weekly frequency. The minimum set of variables for adjustment was defined using directed acyclic graph (DAG). Poisson regression with robust error variance was applied (adjusted by Model 1: demographic and socioeconomic variables, Model 2: added behavioral variables, Model 3: added health variables). We identified the prevalence of depressive symptoms in 19% of older adults and 51.8% reported eating n-3-rich fish once a week. Models 1 and 3 showed an inverse association between n-3-rich fish and depressive symptoms. However, the association was reduced when behavioral factors (leisure-time physical activity) were included in Model 2. These findings suggest that n-3-rich fish intake tends to be associated with depressive symptoms in older adults. However, other factors, such as physical exercise, are as pivotal as n-3 fatty acids in preventing the development of depressive symptoms.

Abstract Background Older adults are one of the most susceptible populations to depression, especially those living in low- and middle-income countries. As well, they are also considering a risk group for vitamin D deficiency. Low serum vitamin D has been associated with an increased risk of brain neuropsychiatry disorders. We aimed to investigate the association between serum 25-hydroxycholecalciferol concentrations and depressive symptoms in adults aged 60 years and over from southern Brazil. Methods A cross-sectional analysis was performed using data collected during 2013–2014 from the populational-based longitudinal EpiFloripa Aging Study (n = 1197). Serum 25-hydroxycholecalciferol concentrations were analyzed and classified according to the Endocrine Society reference values [sufficiency (≥ 30 ng/mL), insufficiency (21–29 ng/mL), and deficiency (≤ 20 ng/mL)]. Depressive symptoms were evaluated using the Geriatric Depression Scale (15-item GDS). Logistic regression was performed to assess depressive symptoms in each vitamin D category. The analysis was adjusted for sex, age, skin color, family income, leisure-time physical activities, social or religious groups attendance, morbidities, cognitive impairment, and dependence in activities of daily living. Results A total of 557 participants with complete data for exposure and outcome were enrolled in the analysis. Most of the sample participants were female (63.1%), age-range 60–69 years (42.2%), white skin color (85.1%), and vitamin D serum level samples were collected in autumn (50.7%). Depressive symptoms were present in 15.8% of the participants, and the prevalence was higher in individuals classified as deficient in vitamin D (23.2, 95% confidence interval [CI] = 15.6;32.9) and insufficiency (17.2, 95%CI = 11.0;25.9). The crude analysis showed that vitamin D deficient participants had 3.08 (CI = 1.53;6.20) times higher odds to present depressive symptoms compared to vitamin D sufficiency. After adjusting, the association was maintained [OR 2.27 (95%CI = 1.05;4.94). Conclusions Serum 25-hydroxycholecalciferol deficiency was positively associated with depressive symptoms in older adults from southern Brazil.

Since the COVID-19 outbreak, studies across diverse countries have strongly pointed toward the emergence of a mental health crisis, with a dramatic increase in the prevalence of depressive psychopathology and suicidal tendencies. Vitamin D deficiency has been associated with an increased risk of mental health problems as well as individual responses to stress. Studies have discussed the relationship between low serum vitamin D concentrations and depressive symptoms, suggesting that maintaining adequate concentrations of serum vitamin D seems to have a protective effect against it. Vitamin D was found to contribute to improved serotonergic neurotransmission in the experimental model of depression by regulating serotonin metabolism. The signaling of 1,25-dihydroxyvitamin D3, the active form of vitamin D, through vitamin D receptor (VDR) induces the expression of the gene of tryptophan hydroxylase 2 (TPH2), influences the expression of serotonin reuptake transporter (SERT) as well as the levels of monoamine oxidase-A (MAO-A), the enzyme responsible for serotonin catabolism. Vitamin D also presents a relevant link with chronobiological interplay, which could influence the development of depressive symptoms when unbalance between light-dark cycles occurs. In this Perspective, we discussed the significant role of vitamin D in the elevation of stress-related depressive symptoms during the COVID-19 pandemic. It is suggested that vitamin D monitoring and, when deficiency is detected, supplementation could be considered as an important healthcare measure while lockdown and social isolation procedures last during the COVID-19 pandemic.

Objetivo: Investigar a associação entre a presença de sintomas de ansiedade e estado nutricional, estimado pelo índice de massa corporal (IMC), em idosos residentes de Florianópolis/SC. Métodos: Estudo transversal realizado com 146 idosos frequentadores do Núcleo de Estudos da Terceira Idade (NETI) – UFSC/Florianópolis. Para avaliar presença de sintomas de ansiedade, aplicou-se o Geriatric Anxiety Inventory (GAI). O estado nutricional foi avaliado por meio do IMC, classificado de acordo com os parâmetros da World Health Organization (WHO, 2008) e de Lipschitz (1994). Para a análise estatística, utilizou-se o software Stata 11.0. Testes qui-quadrado e correlação de Pearson foram usados para aferir a relação entre IMC e presença de ansiedade. Utilizando regressão logística, verificou-se o risco para ansiedade segundo as categorias de IMC. Foi considerado P10, classificando-os como ansiosos. Houve associação da presença de sintomas de ansiedade com o IMC quando usados os critérios de classificação l da WHO (β=13,09; P=0,004), mas não quando usados os critérios de Lipschitz (β=3,78; P=0,151). Quando aplicada a regressão logística bruta, houve maior risco para ansiedade quando IMC ≥ 30 (OR=2,67; IC95% 1,2375 – 5,7621; P=0,012). No modelo ajustado pela idade, o risco aumentou (OR=2,91; IC95% 1,3257 – 6,4047; P=0,008). Conclusão: Existe relação entre a presença de ansiedade e obesidade em idosos. Idosos obesos têm quase 3 vezes mais risco de serem ansiosos que as demais categorias de IMC. DOI: 10.12957/demetra.2017.28085

Identificar os fatores associados à introdução de alimentos não recomendados no primeiro ano de vida, entre crianças residentes em municípios de baixo nível socioeconômico. Estudo multicêntrico transversal com 1.567 crianças de 12 a 59 meses de idade residentes em 48 municípios participantes do plano Brasil Sem Miséria da Região Sul do Brasil. Aplicou-se questionário estruturado aos responsáveis pelas crianças para a obtenção das informações sociodemográficas e idade na qual alimentos não recomendados foram introduzidos pela primeira vez na alimentação complementar. A prevalência de introdução de açúcar antes dos quatro meses de idade da criança foi de 35,5% (n = 497; IC95%: 33,1-38,0). As prevalências de introdução de biscoito doce/salgado, queijo petit suisse e gelatina antes do sexto mês de vida da criança foram de 20,4% (n = 287; IC95%: 18,3-22,3), 24,8% (n = 349; IC95%: 22,4-27,1) e 13,8% (n = 192; IC95%: 12,0-15,7), respectivamente. Identificou-se associação entre a menor escolaridade materna (RP = 1,25; IC95%: 1,03-1,51) e a menor renda mensal familiar (RP = 1,22; IC95%: 1,01-1,48) com a introdução de alimentos não recomendados. Verificou-se a introdução de alimentos não recomendados no primeiro ano de vida entre crianças residentes em municípios de alta vulnerabilidade socioeconômica da Região Sul do Brasil, e esta prática associou-se à menor escolaridade materna e menor renda familiar mensal.

Introdução: A ação da Tropa de Choque caracteriza-se pela rapidez na dispersão do grupamento através da força e potência muscular, entretanto sua preparação caracteriza-se pela especificidade aeróbica. Objetivo: O objetivo deste trabalho foi analisar características antropométricas, força muscular e desempenho físico aeróbico e anaeróbico avaliados em soldados da Tropa de Choque do Exército e comparar com resultados da literatura obtidos em atletas do futebol americano, rúgbi e cabo de guerra. Métodos: Foram avaliados 24 soldados quanto às características antropométricas, potência anaeróbica (Teste de Wingate e salto vertical), força de preensão manual, força de membros superiores no supino e membros inferiores no agachamento através de uma Repetição Máxima (1RM), teste de corrida de 12 min e força muscular localizada avaliados pelo Teste de Avaliação Física (TAF). As variáveis foram analisadas através da estatística descritiva (média ± desvio-padrão). Resultados: Em relação às características antropométricas, foram as médias encontradas foram 178 ± 5 cm para estatura; 79,9 ± 7,8 kg para massa corporal total; e 68,60 ± 4,9 kg para massa magra. O pico de potência foi de 773,0 ± 124,8 W; a potência média, 604,5 ± 69,7 W; e a distância no salto vertical, 55 ± 6 cm. A força de preensão manual foi de 60,8 ± 15,2 kgf (dominante). No supino, a média foi de 75,6 ± 20,1 kg e no agachamento 126,4 ± 19,8 kg para os testes de 1RM. Todos os resultados do TAF atingiram os conceitos máximos exigidos. Conclusão: Os participantes do estudo apresentaram condicionamento aeróbico e resistência muscular localizada de acordo com as exigências do TAF, porém possuem características antropométricas, níveis de força muscular e potência anaeróbica inferiores aos atletas dos esportes considerados como referência.

A ação da Tropa de Choque caracteriza-se pela rapidez na dispersão do grupamento através da força e potência muscular, entretanto sua preparação caracteriza-se pela especificidade aeróbica. O objetivo deste trabalho foi analisar características antropométricas, força muscular e desempenho físico aeróbico e anaeróbico avaliados em soldados da Tropa de Choque do Exército com resultados da literatura obtidos com atletas do futebol americano, rúgbi e cabo de guerra. Foram avaliados 24 soldados quanto às características antropométricas, potência anaeróbica (Teste de Wingate e salto vertical), força de preensão manual, força de membros superiores no supino e membros inferiores no agachamento através de uma Repetição Máxima (1RM) e Teste de corrida de 12 min e força muscular localizada obtidos no Teste de Aptidão Física (TAF). Os valores médios das características antropométricas foram: estatura (178 ± 5 cm), massa corporal total (79,9 ± 7,8 kg), massa magra (68,60 ± 4,9 kg), percentual de massa gorda (13,8 ± 3,6 %). O pico de potência foi de 773,0 ± 124,8 W, potência média de 604,5 ± 69,7 W, trabalho total de 18.136 ± 2.091 J e Índice de Fadiga de 58,4 ± 10,5 %. A distância no salto vertical foi de 55 ± 6 cm. A força de preensão manual foi de 60,8 ± 15, 2 kgf (dominante) e 56,2 ± 14,7 kgf (não-dominante). No supino foi obtido 75,6 ± 20,1 kg e no agachamento 126,4 ± 19,8 kg. Todos os resultados do TAF atingiram os conceitos máximos exigidos. O condicionamento aeróbico e a resistência muscular localizada dos soldados da Tropa de Choque estão de acordo com as exigências do TAF, porém possuem características antropométricas, níveis de força muscular e potência anaeróbica inferiores aos atletas dos esportes analisados.

A osteogênese imperfeita (OI) é uma doença genética caracterizada por fragilidade e deformidades ósseas, fraturas de repetição e baixa estatura. Esses desfechos se devem a mutações genéticas que ocasionam a deficiência na produção de colágeno tipo I. Apresentamos aqui o caso de um paciente de 9 anos, com diagnóstico de OI tipo IV, que interna para realização de novo ciclo de pamidronato dissódico. O caso demonstra a importância do tratamento nutricional da OI, que aborda principalmente a ingestão de cálcio e vitamina D, evitando seus fatores antinutricionais.

Few studies have investigated the simultaneous effect of individual and contextual factors on the occurrences of anemia. This study aims to evaluate the variability of children's hemoglobin levels from municipalities in social vulnerability and its association with factors of individual and municipal nature. This is a cross-sectional, multi-center study, with children data (12-59 months) collected from 48 municipalities of the Southern region of Brazil, that were included in the Brazil Without Poverty Plan. Individuals' data were collected using a structured questionnaire, and secondary and ecological data of children's municipalities were collected via national surveys and institutional websites. The outcome was defined as the hemoglobin level obtained by HemoCue. A multilevel analysis was performed using Generalized Linear Models for Location Scale and Shape using R, with a 5% significance level. A total of 1,501 children were evaluated. The mean hemoglobin level was 12.8g/dL (95%CI: 12.7-12.8), with significant variability between municipalities. Lower values of hemoglobin were observed in children who lived in municipalities with a higher urbanization rate and a lower number of Community Health Agents, in relation to the reference categories. At the individual level, lower hemoglobin values were identified for children under 24 months, not enrolled at daycares, who were beneficiaries of the conditional cash transfer program and diagnosed with underweight. The results shed light on important factors at the municipal and the individual levels that were associated to the hemoglobin levels of children living in municipalities in social vulnerability.