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Importance: Mesial temporal lobe epilepsy (MTLE) is the most common focal epilepsy subtype and is often refractory to antiseizure medications. While most patients with MTLE do not have pathogenic germline genetic variants, the contribution of postzygotic (ie, somatic) variants in the brain is unknown., Objective: To test the association between pathogenic somatic variants in the hippocampus and MTLE., Design, Setting, and Participants: This case-control genetic association study analyzed the DNA derived from hippocampal tissue of neurosurgically treated patients with MTLE and age-matched and sex-matched neurotypical controls. Participants treated at level 4 epilepsy centers were enrolled from 1988 through 2019, and clinical data were collected retrospectively. Whole-exome and gene-panel sequencing (each genomic region sequenced more than 500 times on average) were used to identify candidate pathogenic somatic variants. A subset of novel variants was functionally evaluated using cellular and molecular assays. Patients with nonlesional and lesional (mesial temporal sclerosis, focal cortical dysplasia, and low-grade epilepsy-associated tumors) drug-resistant MTLE who underwent anterior medial temporal lobectomy were eligible. All patients with available frozen tissue and appropriate consents were included. Control brain tissue was obtained from neurotypical donors at brain banks. Data were analyzed from June 2020 to August 2022., Exposures: Drug-resistant MTLE., Main Outcomes and Measures: Presence and abundance of pathogenic somatic variants in the hippocampus vs the unaffected temporal neocortex., Results: Of 105 included patients with MTLE, 53 (50.5%) were female, and the median (IQR) age was 32 (26-44) years; of 30 neurotypical controls, 11 (36.7%) were female, and the median (IQR) age was 37 (18-53) years. Eleven pathogenic somatic variants enriched in the hippocampus relative to the unaffected temporal neocortex (median [IQR] variant allele frequency, 1.92 [1.5-2.7] vs 0.3 [0-0.9]; P = .01) were detected in patients with MTLE but not in controls. Ten of these variants were in PTPN11, SOS1, KRAS, BRAF, and NF1, all predicted to constitutively activate Ras/Raf/mitogen-activated protein kinase (MAPK) signaling. Immunohistochemical studies of variant-positive hippocampal tissue demonstrated increased Erk1/2 phosphorylation, indicative of Ras/Raf/MAPK activation, predominantly in glial cells. Molecular assays showed abnormal liquid-liquid phase separation for the PTPN11 variants as a possible dominant gain-of-function mechanism., Conclusions and Relevance: Hippocampal somatic variants, particularly those activating Ras/Raf/MAPK signaling, may contribute to the pathogenesis of sporadic, drug-resistant MTLE. These findings may provide a novel genetic mechanism and highlight new therapeutic targets for this common indication for epilepsy surgery.

T cell exhaustion is a hallmark of hepatitis C virus (HCV) infection and limits protective immunity in chronic viral infections and cancer. Limited knowledge exists of the initial viral and immune dynamics that characterise exhaustion in humans. We studied longitudinal blood samples from a unique cohort of individuals with primary infection using single-cell multi-omics to identify the functions and phenotypes of HCV-specific CD8 + T cells. Early elevated IFN-γ response against the transmitted virus is associated with the rate of immune escape, larger clonal expansion, and early onset of exhaustion. Irrespective of disease outcome, we find heterogeneous subsets of progenitors of exhaustion, based on the level of PD-1 expression and loss of AP-1 transcription factors. Intra-clonal analysis shows distinct trajectories with multiple fates and evolutionary plasticity of precursor cells. These findings challenge the current paradigm on the contribution of CD8 + T cells to HCV disease outcome and provide data for future studies on T cell differentiation in human infections., (© 2022. The Author(s).)

Tuberous sclerosis complex (TSC) is a multi-system genetic disorder. Most patients have germline mutations in TSC1 or TSC2 but, 10%-15% patients do not have TSC1/TSC2 mutations detected on routine clinical genetic testing. We investigated the contribution of low-level mosaic TSC1/TSC2 mutations in unsolved sporadic patients and families with TSC. Thirty-one sporadic TSC patients negative on routine testing and eight families with suspected parental mosaicism were sequenced using deep panel sequencing followed by droplet digital polymerase chain reaction. Pathogenic variants were found in 22/31 (71%) unsolved sporadic patients, 16 were mosaic (median variant allele fraction [VAF] 6.8% in blood) and 6 had missed germline mutations. Parental mosaicism was detected in 5/8 families (median VAF 1% in blood). Clinical testing laboratories typically only report pathogenic variants with allele fractions above 10%. Our findings highlight the critical need to change laboratory practice by implementing higher sensitivity assays to improve diagnostic yield, inform patient management and guide reproductive counseling., (© 2022 The Authors. Human Mutation published by Wiley Periodicals LLC.)

Viral whole-genome sequencing (WGS) provides critical insight into the transmission and evolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Long-read sequencing devices from Oxford Nanopore Technologies (ONT) promise significant improvements in turnaround time, portability and cost, compared to established short-read sequencing platforms for viral WGS (e.g., Illumina). However, adoption of ONT sequencing for SARS-CoV-2 surveillance has been limited due to common concerns around sequencing accuracy. To address this, here we perform viral WGS with ONT and Illumina platforms on 157 matched SARS-CoV-2-positive patient specimens and synthetic RNA controls, enabling rigorous evaluation of analytical performance. We report that, despite the elevated error rates observed in ONT sequencing reads, highly accurate consensus-level sequence determination was achieved, with single nucleotide variants (SNVs) detected at >99% sensitivity and >99% precision above a minimum ~60-fold coverage depth, thereby ensuring suitability for SARS-CoV-2 genome analysis. ONT sequencing also identified a surprising diversity of structural variation within SARS-CoV-2 specimens that were supported by evidence from short-read sequencing on matched samples. However, ONT sequencing failed to accurately detect short indels and variants at low read-count frequencies. This systematic evaluation of analytical performance for SARS-CoV-2 WGS will facilitate widespread adoption of ONT sequencing within local, national and international COVID-19 public health initiatives.

Cytotoxic CD8 + T cells are key for immune protection against viral infections. The breadth and cross-reactivity of these responses are important against rapidly mutating RNA viruses, such as dengue (DENV), yet how viral diversity affect T cell responses and their cross-reactivity against multiple variants of the virus remains poorly defined. In this study, an integrated analysis was performed to map experimentally validated CD8 + T cell epitopes onto the distribution of DENV genome sequences across the 4 serotypes worldwide. Despite the higher viral diversity observed within HLA-I restricted epitopes, mapping of 609 experimentally validated epitopes sequences on 3985 full-length viral genomes revealed 19 highly conserved epitopes across the four serotypes within the immunogenic regions of NS3, NS4B and NS5. These conserved epitopes were associated with a higher magnitude of IFN-γ response when compared to non-conserved epitopes and were restricted to 13 HLA class I genotypes, hence providing high coverage among human populations. Phylogeographic analyses showed that these epitopes are largely conserved in most of the endemic regions of the world, and with only some of these epitopes presenting distinct mutated variants circulating in South America and Asia.This study provides evidence for the existence of highly immunogenic and conserved epitopes across serotypes, which may impact design of new universal T-cell-inducing vaccine candidates that minimise detrimental effects of viral diversification and at the same time induce responses to a broad human population.

Current methods for dengue virus (DENV) genome amplification, amplify parts of the genome in at least 5 overlapping segments and then combine the output to characterize a full genome. This process is laborious, costly and requires at least 10 primers per serotype, thus increasing the likelihood of PCR bias. We introduce an assay to amplify near full-length dengue virus genomes as intact molecules, sequence these amplicons with third generation "nanopore" technology without fragmenting and use the sequence data to differentiate within-host viral variants with a bioinformatics tool (Nano-Q). The new assay successfully generated near full-length amplicons from DENV serotypes 1, 2 and 3 samples which were sequenced with nanopore technology. Consensus DENV sequences generated by nanopore sequencing had over 99.5% pairwise sequence similarity to Illumina generated counterparts provided the coverage was > 100 with both platforms. Maximum likelihood phylogenetic trees generated from nanopore consensus sequences were able to reproduce the exact trees made from Illumina sequencing with a conservative 99% bootstrapping threshold (after 1000 replicates and 10% burn-in). Pairwise genetic distances of within host variants identified from the Nano-Q tool were less than that of between host variants, thus enabling the phylogenetic segregation of variants from the same host.

Invariant natural killer T (iNKT) cells are capable of rapid activation and production of cytokines upon recognition of antigenic lipids presented by CD1d molecules. They have been shown to play a significant role in many viral infections and were observed to be highly activated in patients with acute dengue infection. In order to characterize further their role in dengue infection, we investigated the proportion of iNKT cells and their phenotype in adult patients with acute dengue infection. The functionality of iNKT cells in patients was investigated by both interferon (IFN)-γ and interleukin (IL)-4 ex-vivo enzyme-linked immunospot (ELISPOT) assays following stimulation with alpha-galactosyl-ceramide (αGalCer). We found that circulating iNKT cell proportions were significantly higher (P = 0·03) in patients with acute dengue when compared to healthy individuals and were predominantly of the CD4(+) subset. iNKT cells of patients with acute dengue had reduced proportions expressing CD8α and CD161 when compared to healthy individuals. The iNKT cells of patients were highly activated and iNKT activation correlated significantly with dengue virus-specific immunoglobulin (Ig)G antibody levels. iNKT cells expressing Bcl-6 (P = 0·0003) and both Bcl-6 and inducible T cell co-stimulator (ICOS) (P = 0·006) were increased significantly in patients when compared to healthy individuals. Therefore, our data suggest that in acute dengue infection there is an expansion of highly activated CD4(+) iNKT cells, with reduced expression of CD161 markers., (© 2016 British Society for Immunology.)

Both dengue NS1 antigen and serum interleukin (IL)-10 levels have been shown to associate with severe clinical disease in acute dengue infection, and IL-10 has also been shown to suppress dengue-specific T cell responses. Therefore, we proceeded to investigate the mechanisms by which dengue NS1 contributes to disease pathogenesis and if it is associated with altered IL-10 production. Serum IL-10 and dengue NS1 antigen levels were assessed serially in 36 adult Sri Lankan individuals with acute dengue infection. We found that the serum IL-10 levels correlated positively with dengue NS1 antigen levels (Spearman's r = 0·47, P < 0·0001), and NS1 also correlated with annexin V expression by T cells in acute dengue (Spearman's r = 0·63, P = 0·001). However, NS1 levels did not associate with the functionality of T cell responses or with expression of co-stimulatory molecules. Therefore, we further assessed the effect of dengue NS1 on monocytes and T cells by co-culturing primary monocytes and peripheral blood mononuclear cells (PBMC), with varying concentrations of NS1 for up to 96 h. Monocytes co-cultured with NS1 produced high levels of IL-10, with the highest levels seen at 24 h, and then declined gradually. Therefore, our data show that dengue NS1 appears to contribute to pathogenesis of dengue infection by inducing IL-10 production by monocytes., (© 2016 British Society for Immunology.)

Background: Although antibody responses to dengue virus (DENV) in naturally infected individuals have been extensively studied, the functionality of DENV specific memory T cell responses in relation to clinical disease severity is incompletely understood., Methodology/principal Findings: Using ex vivo IFNγ ELISpot assays, and by determining cytokines produced in ELISpot supernatants, we investigated the functionality of DENV-specific memory T cell responses in a large cohort of individuals from Sri Lanka (n=338), who were naturally infected and were either hospitalized due to dengue or had mild or sub clinical dengue infection. We found that T cells of individuals with both past mild or sub clinical dengue infection and who were hospitalized produced multiple cytokines when stimulated with DENV-NS3 peptides. However, while DENV-NS3 specific T cells of those with mild/sub clinical dengue infection were more likely to produce only granzyme B (p=0.02), those who were hospitalized were more likely to produce both TNFα and IFNγ (p=0.03) or TNFα alone. We have also investigated the usefulness of a novel T cell based assay, which can be used to determine the past infecting DENV serotype. 92.4% of DENV seropositive individuals responded to at least one DENV serotype of this assay and none of the seronegatives responded. Individuals who were seronegative, but had received the Japanese encephalitis vaccine too made no responses, suggesting that the peptides used in this assay did not cross react with the Japanese encephalitis virus., Conclusions/significance: The types of cytokines produced by DENV-specific memory T cells appear to influence the outcome of clinical disease severity. The novel T cell based assay, is likely to be useful in determining the past infecting DENV serotype in immune-epidemiological studies and also in dengue vaccine trials.

Background: Early detection of complications significantly reduces dengue associated mortality and morbidity. We set out to determine if the NS1 rapid antigen detection test could be used as a point of care test to predict severe disease., Methods: 186 adult patients with confirmed dengue were enrolled during day 3-8 of illness. Clinical and laboratory parameters were recorded during the course of the illness and NS1 antigen levels were determined using both the Panbio dengue early ELISA (Panbio, Australia) and a NS1 rapid antigen detection kit (SD Bioline, South Korea)., Results: 59.1% of patients presented to hospital on day 5-6 of illness when NS1 antigen positivity was significantly (p = 0.008) associated with severe dengue (odds ratio 3.0, 95% CI 1.39 to 6.47) and the NS1 antigen levels were significantly higher (p = 0.03) in those who went on to develop shock. Serum NS1 antigen levels significantly (p < 0.0001) and inversely correlated with the total white cell counts and lymphocyte counts. The bedside NS1 test showed comparable sensitivity (97.4%) and specificity (93.7%) to the laboratory NS1 test in our setting and cohort., Conclusion: NS1 antigen positivity is associated with a higher risk of developing severe dengue especially when positive beyond day 5 of illness in our cohort, and while further validation studies are required, the test can therefore potentially be used as a bedside point of care test as a warning sign of severe dengue.