Zika Virus Impairs Neurogenesis and Synaptogenesis Pathways in Human Neural Stem Cells and Neurons

Growing evidences have associated Zika virus (ZIKV) infection with congenital malformations, including microcephaly. Nonetheless, signaling mechanisms that promote the disease outcome are far from being understood, affecting the development of suitable therapeutics. In this study, we applied shotgun mass spectrometry (MS)-based proteomics combined with cell biology approaches to characterize altered molecular pathways on human neuroprogenitor cells (NPC) and neurons derived from induced pluripotent stem cells infected by ZIKV-BR strain, obtained from the 2015 Brazilian outbreak. Furthermore, ZIKV-BR infected NPCs showed unique alteration of pathways involved in neurological diseases, cell death, survival and embryonic development compared to ZIKV-AF, showing a human adaptation of the Brazilian viral strain. Besides, infected neurons differentiated from NPC presented an impairment of neurogenesis and synaptogenesis processes. Taken together, these data explain that CNS developmental arrest observed in Congenital Zika Syndrome is beyond neuronal cell death

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Evaluation of the susceptibility of respiratory syncytial virus escape mutants to Palivizumab using in vitro microneutralization test.

O Vírus Sincicial respiratório (HRSV) é um patógeno de grande importância para crianças. É o maior causador de infecções respiratórias agudas e também um dos principais causadores de internações e mortes de crianças menores de 5 anos. Cepas de HRSV com mutações no sítio de ligação do Palivizumab vêm apresentando resistência ao medicamento. Pouco se sabe sobre a prevalência destas mutações em amostras clínicas, apesar de sua potencial importância na patogênese viral. Além disso, existem poucos dados sobre a evolução molecular da proteína F do HRSV, sobretudo no Brasil. O presente estudo tem como objetivo caracterizar fenotipicamente através de ensaios de microneutralização in vitro, cepas oriundas de crianças não tratadas com Palivizumab circulantes em 2013 apresentando mutações na proteína F, mais especificamente relacionadas a potencial resistência nos epítopos de ligação do Palivizumab. Como resultado deste trabalho todas as amostras testadas foram neutralizadas pelo Palivizumab, concluindo-se que as mutações encontradas não conferem resistência ao monoclonal.Respiratory Syncytial Virus (HRSV) is a pathogen of great importance for children. It is the major cause of acute respiratory infections and also one of the main causes of hospitalizations and deaths of children under 5 years. Strains of HRSV with mutations in the binding site of Palivizumab have been showing resistance to the drug. Little is known about the prevalence of these mutations in clinical samples, despite their potential importance in viral pathology. In addition, there is little data on the molecular evolution of HRSV F protein, especially in Brazil. The present study aims to characterize phenotypically by in vitro microneutralization assays, strains from children not treated with Palivizumab circulating in 2013 showing mutations in F protein, more specifically related to potential resistance in the binding epitopes of Palivizumab. As result of this study all samples tested were neutralized by Palivizumab, concluding that the mutations found did not confer resistance to the monoclonal

Infection of human B lymphocytes by respiratory syncytial virus

O vírus sincicial respiratório humano (HRSV) é frequente causa de infecção respiratória aguda (IRA) em crianças. Em estudo anterior detectamos HRSV (por RTPCR em tempo real) em 15% das crianças com hipertrofia amigdaliana crônica sem sintomas de IRA, indicando que essas crianças podem ser portadoras silenciosas e saudáveis. Dados preliminares do laboratório incluem a detecção de proteínas estruturais do HRSV em células linfomononucleares de tonsilas e de linfonodos, incluindo linfócitos T CD4+ e B. O presente estudo visou a investigar os efeitos da infecção por HRSV especificamente em linfócitos B. Nosso objetivo foi analisar o impacto do HRSV na viabilidade celular e funcionalidade de linfócitos B infectados com HRSV. Confirmamos a presença de infecção natural em linfócitos B de tonsilas humanas, inclusive com formação de estruturas indicativas de corpos de inclusão. HRSV também infecta in vitro linfócitos B purificados de tonsilas humanas, também com formação de corpos de inclusão. Em seguida, estudamos a infecção por HRSV na linhagem de linfócitos B Jeko-1, e constatamos que ela é susceptível ao HRSV, com a presença de estruturas semelhantes a corpos de inclusão. A porcentagem de Jeko-1 infectada por HRSV determinada por citometria de fluxo 24h e 240h após a infecção foi de 40% e 4%, respectivamente. A infecção por RSV não reduziu significantemente a viabilidade de células Jeko-1. Os dados indicam que embora haja acúmulo de RNA viral nas células, isso não se reflete em aumento correspondente no sobrenadante. De modo inusitado, a dosagem por real-time PCR dos RNAs mensageiros virais mostrou ausência quase completa de RNAm para a proteína L. A secreção de citocinas no sobrenadante de células Jeko-1 infectadas HRSV mostrou ser diferente de células inoculadas com mock, especialmente evidenciado por aumento na secreção de IL-6, IL-10 e TNF-&alpha; nas células infectadas. O presente modelo in vitro de infecção de linhagem de célula B por HRSV ratifica a capacidade do HRSV de infectar linfócitos B, com formação de fábricas virais, porém com permissividade comprometida, favorecendo uma infecção pouco produtiva e de longa duração.Human respiratory syncytial virus (HRSV) is a frequent cause of acute respiratory infection (ARI) in children. In a previous study, we detected HRSV (by realtime RT-PCR) in 15% of children with chronic tonsillar hypertrophy without ARI symptoms, indicating that these children may be silent healthy carriers. Preliminary laboratory data evidenced detection of HRSV structural proteins in lymphomononuclear cells of tonsils and lymph nodes, including CD4+ T and B lymphocytes. The present study aimed to investigate the effects of HRSV infection specifically on B lymphocytes, the impact of HRSV on viability and functionality of HRSV-infected B lymphocytes. We confirmed the presence of natural infection in human tonsil B lymphocytes, including formation of structures indicative of inclusion bodies. HRSV also infects in vitro purified B lymphocytes from human tonsils, also with formation of inclusion bodies. Next, we studied the HRSV infection in the Jeko-1 B lymphocyte lineage, and found that it is susceptible to HRSV, with the presence of inclusion body-like structures. The percentage of HRSV-infected Jeko-1 determined by flow cytometry 24h and 240h after infection was 40% and 4%, respectively. RSV infection did not significantly reduce the viability of Jeko-1 cells. The data indicate that although there is accumulation of viral RNA in the cells, this is not reflected in a corresponding increase of virus RNA progeny in the supernatant. Unexpectedly, the real-time PCR measurement of viral messenger RNAs showed almost complete absence of RNAm for the L protein. The secretion of cytokines in the supernatant of HRSV infected Jeko-1 cells showed to be different from mock inoculated cells, especially evidenced by increased secretion of IL-6, IL-10 and TNF-&alpha; in infected cells. The present in vitro model of B cell lineage infection by HRSV confirms the ability of HRSV to infect B lymphocytes, with the formation of viral factories, but with compromised permissiveness, favoring an unproductive and long-lasting infection

Correction: Laboratory strains of Aedes aegypti are competent to Brazilian Zika virus.

[This corrects the article DOI: 10.1371/journal.pone.0171951.]

Laboratory strains of <i>Aedes aegypti</i> are competent to Brazilian Zika virus

<div><p>The Zika virus outbreaks are unprecedented human threat in relation to congenital malformations and neurological/autoimmune complications. Since this virus has high potential to spread in regions presenting the vectors, improvement in mosquito control is a top priority. Thus, <i>Aedes aegypti</i> laboratory strains will be fundamental to support studies in different research fields implicated on Zika-mosquito interactions which are the basis for the development of innovative control methods. In this sense, our aim was to determine the main infection aspects of a Brazilian Zika strain in reference <i>Aedes aegypti</i> laboratory mosquitoes. We orally exposed Rockefeller, Higgs and Rexville mosquitoes to the Brazilian ZIKV (ZIKV^BR) and qRT-PCR was applied to determine the infection, dissemination and detection rates of ZIKV in the collected saliva as well as viral levels in mosquito tissues. The three strains sustain the virus development but Higgs showed significantly lower viral loads in bodies at 14 days post-infection (dpi) and the lowest prevalences in bodies and heads. The Rockefeller strain was the most susceptible at 7 dpi but similar dissemination rates were observed at 14 dpi. Although variations exist, the ZIKV^BR RNA shows detectable levels in saliva of the three strains at 14 dpi but is only detected in Rockefeller at 7 dpi. Moreover, saliva samples from the three strains were confirmed to be infectious when intrathoracically injected into mosquitoes. The ZIKV^BR kinetics was monitored in Rockefeller mosquitoes and virus could be identified in the heads at 4 dpi but was more consistently detected late in infection. Our study presents the first evaluation on how Brazilian Zika virus behaves in reference <i>Aedes aegypti</i> strains and shed light on how the infection evolves over time. Vector competence and hallmarks of the ZIKV^BR development were revealed in laboratory mosquitoes, providing additional information to accelerate studies focused on ZIKV-mosquito interactions.</p></div

ZIKV^BR kinetics in bodies and heads of <i>Ae</i>. <i>aegypti</i> from the ROCK strain.

<p>The infection rate and viral levels per tissue were individually recorded at 1, 4, 7, 11 and 14 days following a ZIKV-infected blood meal (dpi). Solid circles and open triangles represent each body and head sample, respectively, from the ROCK females. Solid (body) and dashed (head) blue lines indicate the viral infection progression (mean levels) during the time-course experiment. Dashed blue bars indicate early (1 to 4 dpi) and late (7 to 14 dpi) infection stages in heads in which a significant difference in infection prevalence was observed between the two stages using Fisher’s exact test (*, 1 dpi x 7 dpi—p = 0.0031; 1 dpi x 11 dpi—p = 0.0007; 1dpi x 14 dpi—p<0.0001; 4 dpi x 7 dpi—p = 0.0198; 4 dpi x 11 dpi—p = 0.0055; 4 dpi x 14 dpi—p = 0.0001). The dashed grey line demonstrates the detection limit.</p

ZIKV^BR infection rates and viral levels in bodies and heads from <i>Ae</i>. <i>aegypti</i> laboratory strains.

<p>The infection rate and viral levels per tissue were individually recorded in ROCK, HWE and RED females at 7 and 14 days following a ZIKV-infected blood meal (dpi). (<b>A)</b> Viral prevalence and infection levels in the bodies. Each body sample is represented by a solid circle. The infection prevalences between HWE x ROCK and HWE x RED at 7 (*p = 0.0197) and 14 dpi (*p = 0.0436) were significant different by Fisher’s exact test. The viral infection levels of the HWE were significantly lower in the bodies at 14 dpi compared to the ROCK (***p<0.001) or RED (**p<0.01) by two-way ANOVA with Bonferroni post-tests. (<b>B)</b> Viral prevalence and infection levels in the heads. Individual heads are indicated by open triangles. The infection prevalences between HWE x ROCK (**p = 0.0033) at 7 dpi and HWE x ROCK and HWE x RED (**p = 0.0033) at 14 dpi were significantly different by Fisher’s exact test. Black bars indicate the mean viral copy numbers and the dashed grey line demonstrates the detection limit.</p