Dengue NS1 interaction with lipids alters its pathogenic effects on monocyte derived macrophages.

BACKGROUND: While dengue NS1 antigen has been shown to be associated with disease pathogenesis in some studies, it has not been linked in other studies, with the reasons remaining unclear. NS1 antigen levels in acute dengue are often associated with increased disease severity, but there has been a wide variation in results based on past dengue infection and infecting dengue virus (DENV) serotype. As NS1 engages with many host lipids, we hypothesize that the type of NS1-lipid interactions alters its pathogenicity. METHODS: Primary human monocyte derived macrophages (MDMs) were co-cultured with NS1 alone or with HDL, LDL, LPS and/or platelet activating factor (PAF) from individuals with a history of past dengue fever (DF = 8) or dengue haemorrhagic fever (DHF = 8). IL-1ß levels were measured in culture supernatants, and gene expression analysis carried out in MDMs. Monocyte subpopulations were assessed by flow cytometry. Hierarchical cluster analysis with Euclidean distance calculations were used to differentiate clusters. Differentially expressed variables were extracted and a classifier model was developed to differentiate between past DF and DHF. RESULTS: Significantly higher levels of IL-1ß were seen in culture supernatants when NS1 was co-cultured with LDL (p = 0.01, median = 45.69 pg/ml), but lower levels when NS1 was co-cultured with HDL (p = 0.05, median = 4.617 pg/ml). MDMs of those with past DHF produced higher levels of IL-1ß when NS1 was co-cultured with PAF (p = 0.02). MDMs of individuals with past DHF, were significantly more likely to down-regulate RPLP2 gene expression when macrophages were co-cultured with either PAF alone, or NS1 combined with PAF, or NS1 combined with LDL. When NS1 was co-cultured with PAF, HDL or LDL two clusters were detected based on IL10 expression, but these did not differentiate those with past DF or DHF. CONCLUSIONS: As RPLP2 is important in DENV replication, regulating cellular stress responses and immune responses and IL-10 is associated with severe disease, it would be important to further explore how differential expression of RPLP2 and IL-10 could lead to disease pathogenesis based on NS1 and lipid interactions.

Genomic Surveillance and Evolutionary Dynamics of Influenza A Virus in Sri Lanka.

Background: Influenza A has been named as a priority pathogen by the WHO due to the potential to cause pandemics. Genomic sequencing of influenza strains is important to understand the evolution of the influenza strains and also to select the appropriate influenza vaccines to be used in the different influenza seasons in Sri Lanka. Therefore, we sought to understand the molecular epidemiology of the influenza viruses in the Western Province of Sri Lanka, including mutational analysis to investigate the evolutionary dynamics. Methodology: A total of 349 individuals presenting with fever and respiratory symptoms were enrolled in this study from November 2022 to May 2024. Nasopharyngeal and oropharyngeal specimens were collected and screened using quantitative PCR to detect Influenza A, Influenza B, and SARS-CoV-2. Subtyping and genomic sequencing was carried out on influenza A strains using Oxford Nanopore Technology. Results: Influenza A was detected in 49 (14 %) patients, influenza B in 20 (5.7%) and SARS-CoV-2 in 41 (11.7%). Co-infections were observed in five participants. The phylogenetic analysis assigned the H1N1 HA gene sequences within the 6B.1A.5a.2a clade. The HA gene of the H1N1 sequences in 2023 were assigned as belonging to the subclades C.1, C.1.2, and C.1.8, while the 2024 sequences were assigned to subclades C.1.8 and C.1.9. The H3N2 sequences from 2023 were assigned to the 3C.2a1b.2a.2a.1b clade and subclade G.1.1.2, while the 2024 sequences were assigned to the 3C.2a1b.2a.2a.3a.1 clade and subclade J.2. The K54Q, A186T, Q189E, E224A, R259K, K308R, I418V, and X215A amino acid substitutions were seen in the H1N1 in the 2023 and 2024 sequences. The 2024 H1N1 sequences additionally exhibited further substitutions, such as V47I, I96T, T120A, A139D, G339X, K156X, and T278S. Conclusion: In this first study using genomic sequencing to characterize the influenza A strains in Sri Lanka, which showed different influenza A viruses circulating in an 18-month period. As the Sri Lankan strains also had certain mutations of unknown significance, it would be important to continue detailed surveillance of the influenza strains in Sri Lanka to choose the most suitable vaccines for the population and the timing of vaccine administration.

Dengue NS1 interaction with lipids alters its pathogenic effects on monocyte derived macrophages.

Background: While dengue NS1 antigen has been shown to be associated with disease pathogenesis in some studies, it has not been linked in other studies, with the reasons remaining unclear. NS1 antigen levels in acute dengue are often associated with increased disease severity, but there have been a wide variation in results based on past dengue infection and infecting dengue virus (DENV) serotype. As NS1 engages with many host lipids, we hypothesize that the type of NS1-lipid interactions alters its pathogenicity. Methods: Primary human monocyte derived macrophages (MDMs) were co-cultured with NS1 alone or with HDL, LDL, LPS and/or platelet activating factor (PAF) from individuals with a history of past dengue fever (DF=8) or dengue haemorrhagic fever (DHF=8). IL-1ß levels were measured in culture supernatants, and gene expression analysis carried out in MDMs. Monocyte subpopulations were assessed by flow cytometry. Hierarchical cluster analysis with Euclidean distance calculations were used to differentiate clusters. Differentially expressed variables were extracted and a classifier model was developed to differentiate between past DF and DHF. Results: Significantly higher levels of IL-1ß were seen in culture supernatants when NS1 was co-cultured with LDL (p=0.01), but with lower levels with HDL (p=0.05). MDMs of those past DHF produced more IL-1ß when NS1 with PAF (p=0.02). MDMs of individuals with past DHF, were significantly more likely to down-regulate RPLP2 gene expression when macrophages were co-cultured with either PAF alone, or NS1 combined with PAF, or NS1 combined with LDL. When NS1 was co-cultured with PAF, HDL or LDL two clusters were detected based on IL10 expression, but these did not differentiate those with past DF or DHF. Conclusions: As RPLP2 is important in DENV replication and in regulating cellular stress responses and immune responses and IL-10 is associated with severe disease, it would be important to further explore how differential expression of RPLP2 and IL-10 could lead to disease pathogenesis based on NS1 and lipid interactions.

Simultaneous co-circulation of two genotypes of dengue virus serotype 3 causing a large outbreak in Sri Lanka in year 2023.

As many other countries, Sri Lanka experienced a marked rise in the number of dengue cases in 2023, with an unusual pattern of disease epidemiology. This rise coincided with the emergence of dengue virus (DENV) serotype 3 in Sri Lanka as the predominant serotype after 2009. Interestingly, a discrepancy between NS1 rapid antigen test positivity and quantitative real time PCR positivity was observed, with 50% of NS1 positive samples being negative by molecular diagnostics. Following sequencing of the DENV-3 strains in 2023, we identified two DENV-3 genotypes (I and III) co-circulating. While DENV-3 genotype III was detected by the modified CDC DENV-3 primers, genotype I evaded detection due to key mutations at forward and reverse primer binding sites. The co-circulation of multiple genotypes associated with an increase in cases highlights the importance of continuous surveillance of DENVs to identify mutations resulting in non-detection by diagnostics and differences in virulence.

Integrative temporal multi-omics reveals uncoupling of transcriptome and proteome during human T cell activation.

Engagement of the T cell receptor (TCR) triggers molecular reprogramming leading to the acquisition of specialized effector functions by CD4 helper and CD8 cytotoxic T cells. While transcription factors, chemokines, and cytokines are known drivers in this process, the temporal proteomic and transcriptomic changes that regulate different stages of human primary T cell activation remain to be elucidated. Here, we report an integrative temporal proteomic and transcriptomic analysis of primary human CD4 and CD8 T cells following ex vivo stimulation with anti-CD3/CD28 beads, which revealed major transcriptome-proteome uncoupling. The early activation phase in both CD4 and CD8 T cells was associated with transient downregulation of the mRNA transcripts and protein of the central glucose transport GLUT1. In the proliferation phase, CD4 and CD8 T cells became transcriptionally more divergent while their proteome became more similar. In addition to the kinetics of proteome-transcriptome correlation, this study unveils selective transcriptional and translational metabolic reprogramming governing CD4 and CD8 T cell responses to TCR stimulation. This temporal transcriptome/proteome map of human T cell activation provides a reference map exploitable for future discovery of biomarkers and candidates targeting T cell responses.