No abstract

Since its first identification in Scotland, over 1000 cases of unexplained pediatric hepatitis in children have been reported worldwide, including 278 cases in the UK 1 . Here we report investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator subjects, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in liver, blood, plasma or stool from 27/28 cases. We found low levels of Adenovirus (HAdV) and Human Herpesvirus 6B (HHV-6B), in 23/31 and 16/23 respectively of the cases tested. In contrast, AAV2 was infrequently detected at low titre in blood or liver from control children with HAdV, even when profoundly immunosuppressed.AAV2, HAdV and HHV-6 phylogeny excluded 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 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.

Background The extent to which children and adolescents contribute to SARS-CoV-2 transmission remains not fully understood. Novel high-capacity testing methods may provide real-time epidemiological data in educational settings helping to establish a rational approach to prevent and minimize SARS-CoV-2 transmission. We investigated whether pooling of samples for SARS-CoV-2 detection by RT-qPCR is a sensitive and feasible high-capacity diagnostic strategy for surveillance of SARS-CoV-2 infections in schools. Methods In this study, students and school staff of 14 educational facilities in Germany were tested sequentially between November 9 and December 23, 2020, two or three times per week for at least three consecutive weeks. Participants were randomized for evaluation of two different age adjusted swab sampling methods (oropharyngeal swabs or buccal swabs compared to saliva swabs using a ‘lolli method’). Swabs were collected and pooled for SARS-CoV-2 RT-qPCR. Individuals of positive pooled tests were retested by RT-qPCR the same or the following day. Positive individuals were quarantined while the SARS-CoV-2 negative individuals remained in class with continued pooled RT-qPCR surveillance. The study is registered with the German Clinical Trials register (registration number: DRKS00023911). Findings 5,537 individuals were eligible and 3970 participants were enroled and included in the analysis. In students, a total of 21,978 swabs were taken and combined in 2218 pooled RT-qPCR tests. We detected 41 positive pooled tests (1·8%) leading to 36 SARS-CoV-2 cases among students which could be identified by individual re-testing. The cumulative 3-week incidence for primary schools was 564/100,000 (6/1064, additionally 1 infection detected in week 4) and 1249/100,000 (29/2322) for secondary schools. In secondary schools, there was no difference in the number of SARS-CoV-2 positive students identified from pooled oropharyngeal swabs compared to those identified from pooled saliva samples (lolli method) (14 vs. 15 cases; 1·3% vs. 1·3%; OR 1.1; 95%-CI 0·5–2·5). A single secondary school accounted for 17 of 36 cases (47%) indicating a high burden of asymptomatic prevalent SARS-CoV-2 cases in the respective school and community. Interpretation In educational settings, SARS-CoV-2 screening by RT-qPCR-based pooled testing with easily obtainable saliva samples is a feasible method to detect incident cases and observe transmission dynamics. Funding Federal Ministry of education and research (BMBF; Project B-FAST in “NaFoUniMedCovid19”; registration number: 01KX2021).

Our approach aims to optimize postscreening target validation strategies using viral vector-driven RNA interference (RNAi) cell models. The RNAiONE validation platform is an array of plasmid-based expression vectors that each drives tandem expression of the gene of interest (GOI) with one small hairpin RNA (shRNA) from a set of computed candidate sequences. The best-performing shRNA (>85% silencing efficiency) is then integrated in an inducible, all-in-one lentiviral vector to transduce pharmacologically relevant cell types that endogenously express the GOI. VariCHECK is used subsequently to combine the inducible knockdown with an equally inducible rescue of the GOI for ON-target phenotype verification. The complete RNAiONE-VariCHECK system relies on three key elements to ensure high predictability: (1) maximized silencing efficiencies by a focused shRNA validation process, (2) homogeneity of the RNAi cell pools by application of sophisticated viral vector technologies, and (3) exploiting the advantages of inducible expression systems. By using a reversible expression system, our strategy adds critical information to hot candidates from RNAi screens and avoids potential side effects that may be caused by other, irreversible genomic manipulation methods such as transcription activator-like effector nucleases (TALEN) or clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas). This approach will add credibility to top-hit screening candidates and protect researchers from costly misinterpretations early in the preclinical drug development process.

ImportanceCardiac dysfunction and myocarditis have emerged as serious complications of multisystem inflammatory syndrome in children (MIS-C) and vaccines against SARS-CoV-2. Understanding the role of autoantibodies in these conditions is essential for guiding MIS-C management and vaccination strategies in children.ObjectiveTo investigate the presence of anticardiac autoantibodies in MIS-C or COVID-19 vaccine-induced myocarditis.Design, Setting, and ParticipantsThis diagnostic study included children with acute MIS-C or acute vaccine myocarditis, adults with myocarditis or inflammatory cardiomyopathy, healthy children prior to the COVID-19 pandemic, and healthy COVID-19 vaccinated adults. Participants were recruited into research studies in the US, United Kingdom, and Austria starting January 2021. Immunoglobulin G (IgG), IgM, and IgA anticardiac autoantibodies were identified with immunofluorescence staining of left ventricular myocardial tissue from 2 human donors treated with sera from patients and controls. Secondary antibodies were fluorescein isothiocyanate–conjugated antihuman IgG, IgM, and IgA. Images were taken for detection of specific IgG, IgM, and IgA deposits and measurement of fluorescein isothiocyanate fluorescence intensity. Data were analyzed through March 10, 2023.Main Outcomes and MeasuresIgG, IgM and IgA antibody binding to cardiac tissue.ResultsBy cohort, there were a total of 10 children with MIS-C (median [IQR] age, 10 [13-14] years; 6 male), 10 with vaccine myocarditis (median age, 15 [14-16] years; 10 male), 8 adults with myocarditis or inflammatory cardiomyopathy (median age, 55 [46-63] years; 6 male), 10 healthy pediatric controls (median age, 8 [13-14] years; 5 male), and 10 healthy vaccinated adults (all older than 21 years, 5 male). No antibody binding above background was observed in human cardiac tissue treated with sera from pediatric patients with MIS-C or vaccine myocarditis. One of the 8 adult patients with myocarditis or cardiomyopathy had positive IgG staining with raised fluorescence intensity (median [IQR] intensity, 11 060 [10 223-11 858] AU). There were no significant differences in median fluorescence intensity in all other patient cohorts compared with controls for IgG (MIS-C, 6033 [5834-6756] AU; vaccine myocarditis, 6392 [5710-6836] AU; adult myocarditis or inflammatory cardiomyopathy, 5688 [5277-5990] AU; healthy pediatric controls, 6235 [5924-6708] AU; healthy vaccinated adults, 7000 [6423-7739] AU), IgM (MIS-C, 3354 [3110-4043] AU; vaccine myocarditis, 3843 [3288-4748] AU; healthy pediatric controls, 3436 [3313-4237] AU; healthy vaccinated adults, 3543 [2997-4607] AU) and IgA (MIS-C, 3559 [2788-4466] AU; vaccine myocarditis, 4389 [2393-4780] AU; healthy pediatric controls, 3436 [2425-4077] AU; healthy vaccinated adults, 4561 [3164-6309] AU).Conclusions and RelevanceThis etiological diagnostic study found no evidence of antibodies from MIS-C and COVID-19 vaccine myocarditis serum binding cardiac tissue, suggesting that the cardiac pathology in both conditions is unlikely to be driven by direct anticardiac antibody–mediated mechanisms.