Rapid SARS-CoV-2 intra-host and within-household emergence of novel haplotypes

In February 2020, the municipality of Vo’, a small town near Padua (Italy) was quarantined due to the first coronavirus disease 19 (COVID-19)-related death detected in Italy. To investigate the viral prevalence and clinical features, the entire population was swab tested in two sequential surveys. Here we report the analysis of 87 viral genomes, which revealed that the unique ancestor haplotype introduced in Vo’ belongs to lineage B, carrying the mutations G11083T and G26144T. The viral sequences allowed us to investigate the viral evolution while being transmitted within and across households and the effectiveness of the non-pharmaceutical interventions implemented in Vo’. We report, for the first time, evidence that novel viral haplotypes can naturally arise intra-host within an interval as short as two weeks, in approximately 30% of the infected individuals, regardless of symptom severity or immune system deficiencies. Moreover, both phylogenetic and minimum spanning network analyses converge on the hypothesis that the viral sequences evolved from a unique common ancestor haplotype that was carried by an index case. The lockdown extinguished both the viral spread and the emergence of new variant

Mixtures of Chemical Pollutants at European Legislation Safety Concentrations: How Safe Are They?

The risk posed by complex chemical mixtures in the environment to wildlife and humans is increasingly debated, but has been rarely tested under environmentally relevant scenarios. To address this issue, two mixtures of 14 or 19 substances of concern (pesticides, pharmaceuticals, heavy metals, polyaromatic hydrocarbons, a surfactant, and a plasticizer), each present at its safety limit concentration imposed by the European legislation, were prepared and tested for their toxic effects. The effects of the mixtures were assessed in 35 bioassays, based on 11 organisms representing different trophic levels. A consortium of 16 laboratories was involved in performing the bioassays. The mixtures elicited quantifiable toxic effects on some of the test systems employed, including i) changes in marine microbial composition, ii) microalgae toxicity, iii) immobilization in the crustacean Daphnia magna, iv) fish embryo toxicity, v) impaired frog embryo development, and vi) increased expression on oxidative stress-linked reporter genes. Estrogenic activity close to regulatory safety limit concentrations was uncovered by receptor-binding assays. The results highlight the need of precautionary actions on the assessment of chemical mixtures even in cases where individual toxicants are present at seemingly harmless concentration

Mixtures of chemical pollutants at European legislation safety concentrations: how safe are they?

The risk posed by complex chemical mixtures in the environment to wildlife and humans is increasingly debated, but has been rarely tested under environmentally relevant scenarios. To address this issue, two mixtures of 14 or 19 substances of concern (pesticides, pharmaceuticals, heavy metals, polyaromatic hydrocarbons, a surfactant, and a plasticizer), each present at its safety limit concentration imposed by the European legislation, were prepared and tested for their toxic effects. The effects of the mixtures were assessed in 35 bioassays, based on 11 organisms representing different trophic levels. A consortium of 16 laboratories was involved in performing the bioassays. The mixtures elicited quantifiable toxic effects on some of the test systems employed, including i) changes in marine microbial composition, ii) microalgae toxicity, iii) immobilization in the crustacean Daphnia magna, iv) fish embryo toxicity, v) impaired frog embryo development, and vi) increased expression on oxidative stress-linked reporter genes. Estrogenic activity close to regulatory safety limit concentrations was uncovered by receptor-binding assays. The results highlight the need of precautionary actions on the assessment of chemical mixtures even in cases where individual toxicants are present at seemingly harmless concentrations

Relationship between molecular pathogen detection and clinical disease in febrile children across Europe: a multicentre, prospective observational study

BackgroundThe PERFORM study aimed to understand causes of febrile childhood illness by comparing molecular pathogen detection with current clinical practice.MethodsFebrile children and controls were recruited on presentation to hospital in 9 European countries 2016-2020. Each child was assigned a standardized diagnostic category based on retrospective review of local clinical and microbiological data. Subsequently, centralised molecular tests (CMTs) for 19 respiratory and 27 blood pathogens were performed.FindingsOf 4611 febrile children, 643 (14%) were classified as definite bacterial infection (DB), 491 (11%) as definite viral infection (DV), and 3477 (75%) had uncertain aetiology. 1061 controls without infection were recruited. CMTs detected blood bacteria more frequently in DB than DV cases for N. meningitidis (OR: 3.37, 95% CI: 1.92-5.99), S. pneumoniae (OR: 3.89, 95% CI: 2.07-7.59), Group A streptococcus (OR 2.73, 95% CI 1.13-6.09) and E. coli (OR 2.7, 95% CI 1.02-6.71). Respiratory viruses were more common in febrile children than controls, but only influenza A (OR 0.24, 95% CI 0.11-0.46), influenza B (OR 0.12, 95% CI 0.02-0.37) and RSV (OR 0.16, 95% CI: 0.06-0.36) were less common in DB than DV cases. Of 16 blood viruses, enterovirus (OR 0.43, 95% CI 0.23-0.72) and EBV (OR 0.71, 95% CI 0.56-0.90) were detected less often in DB than DV cases. Combined local diagnostics and CMTs respectively detected blood viruses and respiratory viruses in 360 (56%) and 161 (25%) of DB cases, and virus detection ruled-out bacterial infection poorly, with predictive values of 0.64 and 0.68 respectively.InterpretationMost febrile children cannot be conclusively defined as having bacterial or viral infection when molecular tests supplement conventional approaches. Viruses are detected in most patients with bacterial infections, and the clinical value of individual pathogen detection in determining treatment is low. New approaches are needed to help determine which febrile children require antibiotics.FundingEU Horizon 2020 grant 668303

Impact of infection on proteome-wide glycosylation revealed by distinct signatures for bacterial and viral pathogens

Mechanisms of infection and pathogenesis have predominantly been studied based on differential gene or protein expression. Less is known about posttranslational modifications, which are essential for protein functional diversity. We applied an innovative glycoproteomics method to study the systemic proteome-wide glycosylation in response to infection. The protein site-specific glycosylation was characterized in plasma derived from well-defined controls and patients. We found 3862 unique features, of which we identified 463 distinct intact glycopeptides, that could be mapped to more than 30 different proteins. Statistical analyses were used to derive a glycopeptide signature that enabled significant differentiation between patients with a bacterial or viral infection. Furthermore, supported by a machine learning algorithm, we demonstrated the ability to identify the causative pathogens based on the distinctive host blood plasma glycopeptide signatures. These results illustrate that glycoproteomics holds enormous potential as an innovative approach to improve the interpretation of relevant biological changes in response to infection

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

Impairment of Caveolae Formation and T-System Disorganization in Human Muscular Dystrophy with Caveolin-3 Deficiency

Caveolin-3, a muscle specific caveolin-related protein, is the principal structural protein of caveolar membranes. We have recently identified an autosomal dominant form of limb girdle muscular dystrophy (LGMD-1C) that is due to caveolin-3 deficiency and caveolin-3 gene mutations. Here, we studied by electron microscopy, including freeze-fracture and lanthanum staining, the distribution of caveolae and the organization of the T-tubule system in caveolin-3 deficient human muscle fibers. We found a severe impairment of caveolae formation at the muscle cell surface, demonstrating that caveolin-3 is essential for the formation and organization of caveolae in muscle fibers. In addition, we also detected a striking disorganization of the T-system openings at the sub-sarcolemmal level in LGMD-1C muscle fibers. These observations provide new perspectives in our understanding of the role of caveolin-3 in muscle and of the pathogenesis of muscle weakness in caveolin-3 deficient muscle

EU wide campaign exercise on bioassays and chemical mixture effects

Thousands different chemicals are discharged into the environment from agriculture, industry, medical facilities, house-holds. Currently, there is an increasing concern for the environmental impact of mixture of compounds since the additive and eventual synergistic effects are unknown and could produce serious adverse effects. Recently, a document from the European Commission on combination effects of chemicals highlighted the need to ensure that risks associated with chemical mixtures are properly understood and assessed. To address this issue, a joint-effort of 16 European and associated research groups participated to an exercise to test a synthetic reference chemical mixture on the own routine bioassays to investigate the chemical mixtures effects. The reference material included class of pesticides, pharmaceuticals, industrial products, heavy metals and polyaromatic hydrocarbons. The mixures were prepared, each compound at Equivalent Quality Standard (EQS) value, the safety limit concentration allowed by the European Water Framework Directive, (WFD).The bioassays proposed by the groups could cover the entire ecosystem from bacteria to fish as well in vitro assays providing an unique scenario from ecological risk assessment perspective. The results showed that effects were observed at very low concentration on algal-bacteria composition in a marine microcosm, immobilization in crustacean, fish embryo toxicity and frog embryo development. We conclude that some precaution on the chemical mixture assessment should be taken even in case the individual compounds are present at EQS, the safety limit concentration under European legislation

Mixtures of chemical pollutants at European legislation safety concentrations: how safe are they?

The risk posed by complex chemical mixtures in the environment to wildlife and humans is increasingly debated, but has been rarely tested under environmentally relevant scenarios. To address this issue, two mixtures of 14 or 19 substances of concern (pesticides, pharmaceuticals, heavy metals, polyaromatic hydrocarbons, a surfactant and a plasticizer), each present at its safety limit concentration imposed by the European legislation, were prepared and tested for their toxic effects. The effects of the mixtures were assessed in 35 bioassays, based on eleven organisms representing different trophic levels. A consortium of 16 laboratories was involved in performing the bioassays. The mixtures elicited quantifiable toxic effects on some of the test systems employed, including i) changes in marine microbial composition, ii) microalgae toxicity iii) immobilization in the crustacean Daphnia magna, iii) fish embryo toxicity, iv) impaired frog embryo development and v) increased expression on oxidative stress-linked reporter genes. Estrogenic activity close to regulatory safety limit concentrations was uncovered by receptor-binding assays. The results highlight the need of precautionary actions on the assessment of chemical mixtures even in cases where individual toxicants are present at seemingly harmless concentrations.JRC.H.1-Water Resource

TLR4 genotype and environmental LPS mediate RSV bronchiolitis through Th2 polarization

While 30%-70% of RSV-infected infants develop bronchiolitis, 2% require hospitalization. It is not clear why disease severity differs among healthy, full-term infants; however, virus titers, inflammation, and Th2 bias are proposed explanations. While TLR4 is associated with these disease phenotypes, the role of this receptor in respiratory syncytial virus (RSV) pathogenesis is controversial. Here, we evaluated the interaction between TLR4 and environmental factors in RSV disease and defined the immune mediators associated with severe illness. Two independent populations of infants with RSV bronchiolitis revealed that the severity of RSV infection is determined by the TLR4 genotype of the individual and by environmental exposure to LPS. RSV-infected infants with severe disease exhibited a high GATA3/T-bet ratio, which manifested as a high IL-4/IFN-γ ratio in respiratory secretions. The IL-4/IFN-γ ratio present in infants with severe RSV is indicative of Th2 polarization. Murine models of RSV infection confirmed that LPS exposure, Tlr4 genotype, and Th2 polarization influence disease phenotypes. Together, the results of this study identify environmental and genetic factors that influence RSV pathogenesis and reveal that a high IL-4/IFN-γ ratio is associated with severe disease. Moreover, these molecules should be explored as potential targets for therapeutic intervention.Fil: Caballero, Mauricio Tomás. Fundación para la Investigación en Infectología Infantil; ArgentinaFil: Serra, M. Elina. Fundación para la Investigación en Infectología Infantil; ArgentinaFil: Acosta, Patricio Leandro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Investigación en Infectología Infantil; ArgentinaFil: Marzec, Jacqui. Instituto de Efectividad Clínica y Sanitaria; ArgentinaFil: Gibbons, Luz. Institute For Clinical Effectiveness And Health Policy, Ciudad Autonoma de Buenos Aires; ArgentinaFil: Salim, Maximiliano. Hospital Evita Pueblo; Argentina. Fundación para la Investigación en Infectología Infantil; ArgentinaFil: Rodriguez, Andrea. Hospital Mi Pueblo; Argentina. Fundación para la Investigación en Infectología Infantil; ArgentinaFil: Reynaldi, Andrea. Unidad Asistencial "Dr. César Milstein"; Argentina. Fundación para la Investigación en Infectología Infantil; ArgentinaFil: Garcia, Alejandro. Fundación para la Investigación en Infectología Infantil; Argentina. Unidad Asistencial "Dr. César Milstein"; ArgentinaFil: Bado, Daniela. Unidad Asistencial "Dr. César Milstein"; Argentina. Fundación para la Investigación en Infectología Infantil; ArgentinaFil: Buchholz, Ursula J.. National Institute Of Allergy And Infectious Diseases; Estados UnidosFil: Hijano, Diego Raúl. Fundación para la Investigación en Infectología Infantil; Argentina. Vanderbilt University; Estados UnidosFil: Coviello, Silvina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Investigación en Infectología Infantil; ArgentinaFil: Newcomb, Dawn. Vanderbilt University; Estados UnidosFil: Bellabarba, Miguel. Unidad Asistencial "Dr. César Milstein"; Argentina. Fundación para la Investigación en Infectología Infantil; ArgentinaFil: Ferolla, Fausto Martín. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Investigación en Infectología Infantil; ArgentinaFil: Libster, Romina Paula. Fundación para la Investigación en Infectología Infantil; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Vanderbilt University; Estados UnidosFil: Berenstein, Ada. Fundación para la Investigación en Infectología Infantil; ArgentinaFil: Siniawaski, Susana. Fundación para la Investigación en Infectología Infantil; ArgentinaFil: Blumetti, Valeria. Swiss Medical Center; ArgentinaFil: Echavarría, Marcela Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Centro de Educación Medica E Invest.clinicas; ArgentinaFil: Pinto, Leonardo. Fundación para la Investigación en Infectología Infantil; Argentina. Pontificia Universidade Católica do Rio Grande do Sul; BrasilFil: Lawrence, Andrea. Vanderbilt University; Estados UnidosFil: Ossorio, Maria Fabiana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños Pedro Elizalde (ex Casa Cuna); ArgentinaFil: Grosman, Arnoldo. Hospital Espanol; ArgentinaFil: Mateu, Cecilia Gabriela. Fundación para la Investigación en Infectología Infantil; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bayle, Carola. Hospital Nacional Professor Dr. Alejandro Posadas; ArgentinaFil: Dericco, Alejandra. Hospital Nacional Professor Dr. Alejandro Posadas; ArgentinaFil: Pellegrini, Mariana. Hospital Nacional Professor Dr. Alejandro Posadas; ArgentinaFil: Igarza, Ignacio. Hospital Nacional Professor Dr. Alejandro Posadas; ArgentinaFil: Repetto, Horacio A.. Hospital Nacional Professor Dr. Alejandro Posadas; ArgentinaFil: Grimaldi, Luciano Alva. Hospital Zonal General de Agudos Lucio Meléndez; ArgentinaFil: Gudapati, Prathyusha. Vanderbilt University; Estados UnidosFil: Polack, Norberto R.. Unidad Asistencial "Dr. César Milstein"; Argentina. Fundación para la Investigación en Infectología Infantil; ArgentinaFil: Althabe, Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Efectividad Clínica y Sanitaria; ArgentinaFil: Shi, Min. National Institute of Environmental Health Sciences ; Estados UnidosFil: Ferrero, Fernando Claudio. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños Pedro Elizalde (ex Casa Cuna); ArgentinaFil: Bergel, Eduardo. Instituto de Efectividad Clínica y Sanitaria; ArgentinaFil: Stein, Renato T.. Pontificia Universidade Católica do Rio Grande do Sul; BrasilFil: Peebles, R. Stokes. Vanderbilt University; Estados UnidosFil: Boothby, Mark. Vanderbilt University; Estados UnidosFil: Kleeberger, Steven R.. National Institute Of Environmental Health Sciences; Estados UnidosFil: Polack, Fernando Pedro. Fundación para la Investigación en Infectología Infantil; Argentina. Vanderbilt University; Estados Unido