Recombinant T-cell epitope conjugation: A new approach for Dermatophagoides hypoallergen design.

BACKGROUND: Allergen-specific immunotherapy (AIT) is the only clinical approach that can potentially cure some allergic diseases by inducing immunological tolerance. Dermatophagoides pteronyssinus is considered as the most important source of mite allergens worldwide, with high sensitization rates for the major allergens Der p 1, Der p 2 and Der p 23. The aim of this work is to generate a hypoallergenic hybrid molecule containing T-cell epitopes from these three major allergens. METHODS: The hybrid protein termed Der p 2231 containing T-cell epitopes was purified by affinity chromatography. The human IgE reactivity was verified by comparing those with the parental allergens. The hybrid was also characterized immunologically through an in vivo mice model. RESULTS: The hybrid rDer p 2231 stimulated in peripheral blood mononuclear cells (PBMCs) isolated from allergic patients with higher levels of IL- 2, IL-10, IL-15 and IFN-γ, as well as lower levels of IL-4, IL-5, IL-13, TNF-α and GM-CSF. The use of hybrid molecules as a therapeutic model in D. pteronyssinus allergic mice led to the reduction of IgE production and lower eosinophilic peroxidase activity in the airways. We found increased levels of IgG antibodies that blocked the IgE binding to the parental allergens in the serum of allergic patients. Furthermore, the stimulation of splenocytes from mice treated with rDer p 2231 induced higher levels of IL-10 and IFN-γ and decreased the secretion of IL-4 and IL-5, when compared with parental allergens and D. pteronyssinus extract. CONCLUSIONS: rDer p 2231 has the potential to be used in AIT in patients co-sensitized with D. pteronyssinus major allergens, once it was able to reduce IgE production, inducing allergen-specific blocking antibodies, restoring and balancing Th1/Th2 immune responses, and inducing regulatory T-cells.

The hybrid protein BTH2 suppresses allergic airway inflammation in a murine model of HDM-specific immunotherapy.

BACKGROUND: Allergen-specific immunotherapy (AIT) is the only disease-modifying treatment approach to change disease-causing allergens. Hypoallergenic derivatives show promise as potential therapeutics, amongst which BTH2 was designed to induce tolerance against Blomia tropicalis allergy. Our aim was to investigate the hypoallergenicity and immunoregulatory activity of BTH2 in vitro and its therapeutic potential in a mouse model of AIT. METHODS: Recombinant Blo t 5 and Blo t 21 allergens and their hybrid derivatives (BTH1 and BTH2) were expressed and purified. IgE binding capacity was tested by ELISA using sera from Brazilian, Colombian, and Ecuadorian subjects. Secretion of cytokines in supernatants from human cell cultures was measured following stimulation with the four recombinants and controls. The capacity of BTH2 to ameliorate allergic airway inflammation induced by B. tropicalis extract was evaluated in a murine model of AIT. RESULTS: rBlo t 5 and rBlo t 21 were identified as major allergens in Latin American patients, and BTH2 had the lowest IgE binding. In vitro stimulation of human cells induced greater levels of IL-10 and IFN-γ and reduced the secretion of Th2 cytokines. BTH2 ameliorated allergic airway inflammation in B. tropicalis-challenged A/J mice, as evidenced by the histopathological and humoral biomarkers: decreased Th2 cytokines and cellular infiltration (especially eosinophils), lower activity of eosinophil peroxidase, an increase in IgG blocking antibodies and strong reduction of mucus production by goblet cells. CONCLUSIONS: Our study shows that BTH2 represents a promising candidate for the treatment of B. tropicalis allergy with hypoallergenic, immune regulatory and therapeutic properties. Further pre-clinical studies are required in murine models of chronic asthma to further address the efficacy and safety of BTH2 as a vaccine against B. tropicalis-induced allergy.

Structural factors associated with SARS-CoV-2 infection risk in an urban slum setting in Salvador, Brazil: A cross-sectional survey.

BACKGROUND: The structural environment of urban slums, including physical, demographic, and socioeconomic attributes, renders inhabitants more vulnerable to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. Yet, little is known about the specific determinants that contribute to high transmission within these communities. We therefore aimed to investigate SARS-CoV-2 seroprevalence in an urban slum in Brazil. METHODS AND FINDINGS: We performed a cross-sectional serosurvey of an established cohort of 2,041 urban slum residents from the city of Salvador, Brazil between November 2020 and February 2021, following the first Coronavirus Disease 2019 (COVID-19) pandemic wave in the country and during the onset of the second wave. The median age in this population was 29 years (interquartile range [IQR] 16 to 44); most participants reported their ethnicity as Black (51.5%) or Brown (41.7%), and 58.5% were female. The median size of participating households was 3 (IQR 2 to 4), with a median daily per capita income of 2.32 (IQR 0.33-5.15) US Dollars. The main outcome measure was presence of IgG against the SARS-CoV-2 spike protein. We implemented multilevel models with random intercepts for each household to estimate seroprevalence and associated risk factors, adjusting for the sensitivity and specificity of the assay, and the age and gender distribution of our study population. We identified high seroprevalence (47.9%, 95% confidence interval [CI] 44.2% to 52.1%), particularly among female residents (50.3% [95% CI 46.3% to 54.8%] versus 44.6% [95% CI 40.1% to 49.4%] among male residents, p < 0.01) and among children (54.4% [95% CI 49.6% to 59.3%] versus 45.4% [95% CI 41.5% to 49.7%] among adults, p < 0.01). Adults residing in households with children were more likely to be seropositive (48.6% [95% CI 44.8% to 52.3%] versus 40.7% [95% CI 37.2% to 44.3%], p < 0.01). Women who were unemployed and living below the poverty threshold (daily per capita household income <$1.25) were more likely to be seropositive compared to men with the same employment and income status (53.9% [95% CI 47.0% to 60.6%] versus 32.9% [95% CI 23.2% to 44.3%], p < 0.01). Participation in the study was voluntary, which may limit the generalizability of our findings. CONCLUSIONS: Prior to the peak of the second wave of the COVID-19 pandemic, cumulative incidence as assessed by serology approached 50% in a Brazilian urban slum population. In contrast to observations from industrialized countries, SARS-CoV-2 incidence was highest among children, as well as women living in extreme poverty. These findings emphasize the need for targeted interventions that provide safe environments for children and mitigate the structural risks posed by crowding and poverty for the most vulnerable residents of urban slum communities.

A hybrid of two major Blomia tropicalis allergens as an allergy vaccine candidate.

INTRODUCTION: Allergen-specific immunotherapy (AIT) represents a curative approach for treating allergies. In the tropical and subtropical regions of the world, Blomia tropicalis (Blo t 5 and Blo t 21) is the likely dominant source of indoor allergens. AIM: To generate a hypoallergenic Blo t 5/Blo t 21 hybrid molecule that can treat allergies caused by B tropicalis. METHODS: Using in silico design of B tropicalis hybrid proteins, we chose two hybrid proteins for heterologous expression. Wild-type Blo t 5/Blo t 21 hybrid molecule and a hypoallergenic version, termed BTH1 and BTH2, respectively, were purified by ion exchange and size exclusion chromatography and characterized by physicochemical, as well as in vitro and in vivo immunological, experiments. RESULTS: BTH1, BTH2 and the parental allergens were purified to homogeneity and characterized in detail. BTH2 displayed the lowest IgE reactivity that induced basophil degranulation using sera from allergic rhinitis and asthmatic patients. BTH2 essentially presented the same endolysosomal degradation pattern as the shortened rBlo t 5 and showed a higher resistance towards degradation than the full-length Blo t 5. In vivo immunization of mice with BTH2 led to the production of IgG antibodies that competed with human IgE for allergen binding. Stimulation of splenocytes from BTH2-immunized mice produced higher levels of IL-10 and decreased secretion of IL-4 and IL-5. In addition, BTH2 stimulated T-cell proliferation in PBMCs isolated from allergic patients, with secretion of higher levels of IL-10 and lower levels of IL-5 and IL-13, when compared to parental allergens. CONCLUSIONS AND CLINICAL RELEVANCE: BTH2 is a promising hybrid vaccine candidate for immunotherapy of Blomia allergy. However, further pre-clinical studies addressing its efficacy and safety are needed.

Therapeutic potential of a novel hybrid protein: Mitigating allergy and airway remodeling in chronic asthma models induced by Dermatophagoides pteronyssinus.

BACKGROUND: The house-dust mite Dermatophagoides pteronyssinus is a key trigger of allergic asthma. Therefore, it is essential to develop new vaccines that can alter inflammatory processes and airway remodeling. The goal of this study was to test the hypoallergenic and immunogenic characteristics of the hypoallergen rDer p 2231 in a murine model of chronic asthma induced by D. pteronyssinus. METHODS: For this, we measured the levels of IgE, IgG1, IgG2a, and cytokines produced by mice receiving the rDer p 2231 protein. Histopathological parameters of the chronic inflammatory response were also investigated by assessing inflammation and airway remodeling. RESULTS: rDer p 2231 given as a therapeutic vaccine, led to a reduction in the production of IgE, eosinophils, and neutrophils, a lower activity of eosinophilic peroxidase in the airways, and an increase in the production of IgG1 and IgG2a antibodies. IgG antibodies blocked IgE binding to parental allergens in sera from atopic patients. Splenocytes, BALF, and lung from mice treated with rDer p 2231 secreted higher levels of Th1 and regulatory cytokines, as well as reduced levels of Th2 cytokines. Histopathological investigation of the lower airways demonstrated reductions in the thickness of the bronchiolar smooth muscle layer, in the subepithelial fibrosis, and in the goblet cells hyperplasia. CONCLUSIONS: Our preclinical studies suggest that rDer p 2231 is a promising candidate for the treatment of D. pteronyssinus allergy, as the hypoallergen has demonstrated the ability to reduce IgE production, induce specific blocking antibodies, restore and balance Th1/Th2 immune responses, and significantly reduce airway remodeling factors. However, additional clinical studies are needed to more accurately assess the efficacy and safety of rDer p 2231 as a vaccine against D. pteronyssinus-induced allergy.

Overestimation of Severe Acute Respiratory Syndrome Coronavirus 2 Household Transmission in Settings of High Community Transmission: Insights From an Informal Settlement Community in Salvador, Brazil.

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant has spread globally. However, the contribution of community versus household transmission to the overall risk of infection remains unclear. Methods: Between November 2021 and March 2022, we conducted an active case-finding study in an urban informal settlement with biweekly visits across 1174 households with 3364 residents. Individuals displaying coronavirus disease 2019 (COVID-19)-related symptoms were identified, interviewed along with household contacts, and defined as index and secondary cases based on reverse-transcription polymerase chain reaction (RT-PCR) and symptom onset. Results: In 61 households, we detected a total of 94 RT-PCR-positive cases. Of 69 sequenced samples, 67 cases (97.1%) were attributed to the Omicron BA.1* variant. Among 35 of their households, the secondary attack rate was 50.0% (95% confidence interval [CI], 37.0%-63.0%). Women (relative risk [RR], 1.6 [95% CI, .9-2.7]), older individuals (median difference, 15 [95% CI, 2-21] years), and those reporting symptoms (RR, 1.73 [95% CI, 1.0-3.0]) had a significantly increased risk for SARS-CoV-2 secondary infection. Genomic analysis revealed substantial acquisition of viruses from the community even among households with other SARS-CoV-2 infections. After excluding community acquisition, we estimated a household secondary attack rate of 24.2% (95% CI, 11.9%-40.9%). Conclusions: These findings underscore the ongoing risk of community acquisition of SARS-CoV-2 among households with current infections. The observed high attack rate necessitates swift booster vaccination, rapid testing availability, and therapeutic options to mitigate the severe outcomes of COVID-19.

Extensive transmission of SARS-CoV-2 BQ.1* variant in a population with high levels of hybrid immunity: A prevalence survey.

OBJECTIVES: The SARS-CoV-2 BQ.1* variant rapidly spread globally in late 2022, posing a challenge due to its increased immune evasion. METHODS: We conducted a prevalence survey in Brazil from November 16 to December 22, 2022, as part of a cohort study. We conducted interviews and collected nasal samples for reverse transcription-polymerase chain reaction (RT-PCR) testing and whole-genome sequencing. Cumulative incidence was estimated using RT-PCR positivity, cycle threshold values, and external data on the dynamics of RT-PCR positivity following infection. RESULTS: Among 535 participants, 54% had documented SARS-CoV-2 exposure before this outbreak and 74% had received COVID-19 vaccination. In this study, 14.8% tested positive for SARS-CoV-2, with BQ.1* identified in 90.7% of cases. Using case data and cycle threshold values, cumulative incidence was estimated at 56% (95% confidence interval, 36-88%). Of the 79 positive participants, 48.1% had a symptomatic illness, with a lower proportion fulfilling the World Health Organization COVID-19 case definition compared to prior Omicron waves. No participants required medical attention. CONCLUSIONS: Despite high population-level hybrid immunity, the BQ.1* variant attacked 56% of our population. Lower disease severity was associated with BQ.1* compared to prior Omicron variants. Hybrid immunity may provide protection against future SARS-CoV-2 variants but in this case was not able to prevent widespread transmission.

Serum 25-hydroxyvitamin D deficiency is associated with atopy, and sex may be an effect modifier of its association with asthma in teenagers from northeast Brazil.

OBJECTIVE: Sufficient vitamin D (25-hydroxyvitamin D [25(OH)D]) serum levels are associated with decreased asthma symptoms. Our aim was to investigate associations between vitamin D and atopy, asthma, asthma severity, and asthma phenotypes in Brazilian teenagers. METHODS: This cross-sectional study involved 942 individuals (11-19 years old) engaged in an asthma cohort. The ISAAC questionnaire was employed to diagnosis asthma and asthma severity. Serum allergen-specific immunoglobulin E (sIgE) was measured by ImmunoCap and serum 25(OH)D was measured by ELISA. We calculated the correlation between sIgE and 25(OH)D. We used multivariate logistic regression analysis to assess associations of interest. RESULTS: We found that 25(OH)D deficiency was positively associated with atopy (OR 1.45, confidence interval [CI] 1.05-2.00) and high levels of this vitamin negatively correlated with sIgE to Dermatophagoides pteronyssinus (r = -0.11, p = 0.019). The average 25(OH)D serum level was 27.0 ± 9.5 ng/ml; 366 individuals (38.8%) had a sufficient level. There was no association between 25(OH)D and asthma, asthma severity or asthma phenotypes in the population. However, sex was a possible effect modifier of the association between vitamin D and asthma: insufficiency in asthmatic women (86%) was higher than in asthmatic men (42%), and there was an association between insufficient vitamin D levels and greater asthma risk only in women (OR = 3.06, 95% CI 1.16-8.07). CONCLUSION: We have shown that vitamin D deficiency was associated with greater risk of atopy in both sexes and vitamin D insufficiency was associated with asthma only in women. There was no association between vitamin D levels and asthma phenotypes or asthma severity.

Reliable estimation of SARS-CoV-2 anti-spike protein IgG titers from single dilution optical density values in serologic surveys.

Reliable and scalable seroepidemiology methods are needed to estimate SARS-CoV-2 incidence and monitor the dynamics of population-level immunity as the pandemic evolves. We aimed to evaluate the reliability of SARS-CoV-2 normalized ELISA optical density (nOD) at a single dilution compared to titers derived from serial dilutions. We conducted serial serosurveys within a community-based cohort in Salvador, Brazil. Anti-S IgG ELISA (Euroimmun AG) was performed with 5 serial 3-fold dilutions of paired sera from 54 participants. Changes in nOD reliably predicted increases and decreases in titers (98.1% agreement, κ = 95.8%). Fitting the relationship between nOD and interpolated titers to a log-log curve yields highly accurate predictions of titers (r2 = 0.995) and changes in titers (r2 = 0.975), using only 1 to 2 dilutions. This approach can significantly reduce the time, labor and resources needed for large-scale serosurveys to ascertain population-level changes in exposure and immunity.

Toxocara canis extract fractions promote mainly the production of Th1 and regulatory cytokines by human leukocytes in vitro.

Helminths possibly down-modulate immune responses to airborne allergens through the induction of a regulatory network. The identification of helminths bioactive molecules is highly desirable, given their immunomodulatory potential which could be used in immunotherapies for allergy and autoimmune diseases. To investigate the immunoregulatory potential of the adult Toxocara canis crude extract and ten protein fractions of its extract, human peripheral blood mononuclear cells (PBMC) from 10 allergic and 9 non-allergic individuals were cultivated, in vitro, in the presence or absence of these antigens, and their supernatants were evaluated for cytokine production (TGF-ß, IL-10, IL-12, TNF-α, IL-6, IL-5, IL13, and IL-17). To determine the cell viability, the PBMC were cultivated for 24 h in the presence of the antigens and, following, they were subjected to a cytotoxicity assay. The viability of the PBMC was not affected by incubation with the T. canis antigens. As some fractions stimulated the production of immunoregulatory (TGF-ß and/or IL-10), IL-12 and Th1 (TNF-α) cytokines, without stimulating Th2 cytokines (IL-5 and IL13) and IL-17, it was proposed that they would be potential candidates for further studies, especially involving the purification and characterization of specific proteins, which could be tested separately to evaluate their specific role as adjuvants in immunotherapy for inflammatory diseases.

Structural factors contributing to SARS-CoV-2 infection risk in the urban slum setting.

BACKGROUND: The structural environment of urban slums, including physical, demographic and socioeconomic attributes, renders inhabitants more vulnerable to SARS-CoV-2 infection. Yet, little is known about the specific determinants that contribute to high transmission within these communities. METHODS AND FINDINGS: We performed a serosurvey of an established cohort of 2,035 urban slum residents from the city of Salvador, Brazil between November 2020 and February 2021, following the first COVID-19 pandemic wave in the country. We identified high SARS-CoV-2 seroprevalence (46.4%, 95% confidence interval [CI] 44.3-48.6%), particularly among female residents (48.7% [95% CI 45.9-51.6%] vs. 43.2% [95% CI 39.8-46.6%] among male residents), and among children (56.5% [95% CI 52.3-60.5%] vs. 42.4% [95% CI 39.9-45.0%] among adults). In multivariable models that accounted for household-level clustering, the odds ratio for SARS-CoV-2 seropositivity among children was 1.96 (95% CI 1.42-2.72) compared to adults aged 30-44 years. Adults residing in households with children were more likely to be seropositive; this effect was particularly prominent among individuals with age 30-44 and 60 years or more. Women living below the poverty threshold (daily per capita household income <$1.25) and those who were unemployed were more likely to be seropositive. CONCLUSIONS: During a single wave of the COVID-19 pandemic, cumulative incidence as assessed by serology approached 50% in a Brazilian urban slum population. In contrast to observations from industrialized countries, SARS-CoV-2 incidence was highest among children, as well as women living in extreme poverty. These findings emphasize the need for targeted interventions that provide safe environments for children and mitigate the structural risks posed by crowding and poverty for the most vulnerable residents of urban slum communities.

En route to personalized medicine: uncovering distinct IgE reactivity pattern to house dust mite components in Brazilian and Austrian allergic patients.

AIM: Molecular sensitization profile analyses of allergic individuals to the house dust mites (HDM) Blomia tropicalis and Dermatophagoides pteronyssinus from Brazil and Austria, in the attempt to comprehend the individual contribution of the molecular components in the diagnosis of HDM allergy. METHODOLOGY: These analyses were made using a new in vitro multiplex allergen assay which allows simultaneous measurement of specific IgE against the whole allergen extract as well its components. RESULTS AND CONCLUSION: The data showed that in Brazil the inclusion of the molecular components Blo t 5 and/or Blo t 21 major allergens and Blo t 2 can increase the sensitivity and specificity of the assay for the diagnosis of allergy to B. tropicalis, using matrix-based methodologies. Also we highlighted, for the first time, the importance of Blo t 2 analysis for a sensitive diagnosis, since some individuals were sensitized only to this molecular component. Regarding the sensitization profile of individuals sensitized to D. pteronyssinus, we point out the importance of analyzing the molecular components Der p23 and Der p 7, in addition to Der p 1 and Der p 2 for an accurate diagnosis based on matrices.

WSB1 and IL21R Genetic Variants Are Involved in Th2 Immune Responses to Ascaris lumbricoides.

Genetic and epigenetic factors are considered to be critical for host-parasite interactions. There are limited data on the role of such factors during human infections with Ascaris lumbricoides. Here, we describe the potential role of genetic factors as determinants of the Th2 immune response to A. lumbricoides in Brazilian children. Stool samples were collected from the children to detect A. lumbricoides by microscopy and peripheral blood leukocytes (PBLs) were cultured in whole blood cultures for detection of cytokines (IL-5, IL-10, and IL-13) in vitro. Levels of anti-A. lumbricoides IgE and IgG4 were measured in plasma. DNA was extracted from PBLs and genotyped using Illumina 2.5 Human Omni Beadchip. Candidate genes associated with A. lumbricoides responses were identified and SNVs in these selected genes associated with the Th2 immune response to A. lumbricoides. Haplotype, gene expression, and epigenetic analyses were done to identify potential associations with Th2 immune responses. GWAS on samples from 1,189 children identified WSB1 as a candidate gene, and IL-21R was selected as a biologically relevant linked gene for further analysis. Variants in WSB1 and IL21R were associated with markers of Th2 immune responses: increased A. lumbricoides-specific IgE and IL-5/IL-13 by PBLs from infected compared to uninfected individuals. In infected children, WSB1 but not IL21R gene expression was suppressed and increased methylation was observed in the WSB1 promoter region. This is the first study to show an association between genetic variants in WSB1 and IL21R and Th2 immune responses during A. lumbricoides infections in children. WSB1/IL21R pathways could provide a potential target for the treatment of Th2-mediated diseases.

First genome sequencing of SARS-CoV-2 recovered from an infected cat and its owner in Latin America.

An 11-year-old male mixed-breed cat, with exclusively indoor life, presented 3 cough episodes after the owners tested positive by RT-PCR for SARS-CoV-2. The house is inhabited by 5 people (3 adults and 2 children), and 2 of the adults have shown mild symptoms associated with throat discomfort. The cat was vaccinated, had no history of any previous disease, and tested negative for feline coronavirus (FCoV), feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV). Rectal sample collected from the cat was positive for SARS-CoV-2 by RT-PCR. Viral genome sequences recovered from human and cat samples showed an average 99.4% sequence identity. This is the first report of genome sequences of SARS-CoV-2 recovered from a cat and its owner in Latin America.

Recurrent COVID-19 including evidence of reinfection and enhanced severity in thirty Brazilian healthcare workers.

BACKGROUND: There is growing concern about individuals reported to suffer repeat COVID-19 disease episodes, these in a small number of cases characterised as de novo infections with distinct sequences, indicative of insufficient protective immunity even in the short term. METHODS: Observational case series and case-control studies reporting 33 cases of recurrent, symptomatic, qRT-PCR positive COVID-19. Recurrent disease was defined as symptomatic recurrence after symptom-free clinical recovery, with release from isolation >14 days from the beginning of symptoms confirmed by qRT-PCR. The case control study-design compared this group of patients with a control group of 62 patients randomly selected from the same COVID-19 database. RESULTS: Of 33 recurrent COVID-19 patients, 26 were female and 30 were HCW. Mean time to recurrence was 50.5 days which was associated with being a HCW (OR 36.4 (p <0.0001)), and blood type A (OR 4.8 (p = 0.002)). SARS-CoV-2 antibodies were signifcantly lower in recurrent patients after initial COVID-19  (2.4 ±â€¯0.610; p<0.0001) and after recurrence (6.4 ±â€¯11.34; p = 0.007).  Virus genome sequencing identified reinfection by a different isolate in one patient. CONCLUSIONS: This is the first detailed case series showing COVID-19 recurrence with qRT-PCR positivity. For one individual detection of phylogenetically distinct genomic sequences in the first and second episodes confirmed bona fide renfection, but in most cases the data do not formally distinguish between reinfection and re-emergence of a chronic infection reservoir. These episodes were significantly associated with reduced Ab response during initial disease and argue the need for ongoing vigilance without an assumption of protection after a first episode.

High-Quality Resolution of the Outbreak-Related Zika Virus Genome and Discovery of New Viruses Using Ion Torrent-Based Metatranscriptomics.

Arboviruses, including the Zika virus, have recently emerged as one of the most important threats to human health. The use of metagenomics-based approaches has already proven valuable to aid surveillance of arboviral infections, and the ability to reconstruct complete viral genomes from metatranscriptomics data is key to the development of new control strategies for these diseases. Herein, we used RNA-based metatranscriptomics associated with Ion Torrent deep sequencing to allow for the high-quality reconstitution of an outbreak-related Zika virus (ZIKV) genome (10,739 nt), with extended 5'-UTR and 3'-UTR regions, using a newly-implemented bioinformatics approach. Besides allowing for the assembly of one of the largest complete ZIKV genomes to date, our strategy also yielded high-quality complete genomes of two arthropod-infecting viruses co-infecting C6/36 cell lines, namely: Alphamesonivirus 1 strain Salvador (20,194 nt) and Aedes albopictus totivirus-like (4618 nt); the latter likely represents a new viral species. Altogether, our results demonstrate that our bioinformatics approach associated with Ion Torrent sequencing allows for the high-quality reconstruction of known and unknown viral genomes, overcoming the main limitation of RNA deep sequencing for virus identification.

Genetic polymorphisms in vitamin D pathway influence 25(OH)D levels and are associated with atopy and asthma.

BACKGROUND: Vitamin D deficiency or insufficiency, has been associated with atopy and lack of asthma control. Our objective was to investigate associations between variants in genes of vitamin D pathway with serum levels of 25-hydroxyvitamin D (25(OH)D), atopy, asthma and asthma severity in teenagers from Northeast Brazil. METHODS: This is a cross sectional study nested in a cohort population of asthma. 25(OH)D was quantified from 968 of 11-17 years old individuals by ELISA. Asthma diagnosis was obtained by using the ISAAC Phase III questionnaire. Specific IgE was determined by ImmunoCAP; genotyping was performed using the 2.5 HumanOmni Biochip from Illumina. Statistical analyses were performed in PLINK 1.07 and SPSS 22.1. RESULTS: After quality control, 104 Single Nucleotides Variants (SNVs) in vitamin D pathway genes, typed in 792 individuals, were included in the analysis. The allele A of rs10875694 on VDR was positively associated with atopy (OR = 1.35; 95% CI 1.01-1.81). The allele C of rs9279 on VDR, was negatively associated with asthma risk (OR = 0.66; 95% CI 0.45-0.97), vitamin D insufficiency (OR = 0.78; 95% CI 0.70-0.96) and higher VDR expression. Two variants in VDR were associated with asthma severity, the allele A of rs2189480 (OR = 0.34; 95% CI 0.13-0.89) and the allele G of rs4328262 (OR = 3.18; 95% CI 1.09-9.28). The combination of variants in CYP2R1 and CYP24A1 (GAC, to rs10500804, rs12794714 and rs3886163, respectively) was negatively associated with vitamin D production (ß = - 1.24; 95% CI - 2.42 to - 0.06). CONCLUSIONS: Genetic variants in the vitamin D pathway affect vitamin D serum levels and, thus, atopy and asthma.

Ion torrent-based nasopharyngeal swab metatranscriptomics in COVID-19.

Herein, we describe the detection of a SARS-CoV-2 genome through metatranscriptome next-generation sequencing directly from the nasopharyngeal swab of a suspected case of local transmission of Covid-19, in Brazil. Depletion of human ribosomal RNA and use of an optimized in-house developed bioinformatics strategy contributed to successful detection of the virus.