The "Investigating and translating genomic evidence for public health response to SARS-CoV-2 (INSIDE SARS-CoV-2)" project - Network of excellence. Commentary.

The "Investigating and translating genomic evidence for public health response to SARS-CoV-2 (INSIDE SARS-CoV-2)" project is part of the initiative "Joint science and technology cooperation call for joint project proposals for the years 2021-2023" promoted by the Italian Ministry of Foreign Affairs and International Cooperation (MAECI) and the Republic of India. To start the project activities, the pandemic response and the epidemiological situation in Italy and in India, together with the genomic surveillance strategies for SARS-CoV-2 virus in the two countries, are here described.

Single cell genomics based insights into the impact of cell-type specific microbial internalization on disease severity.

Host-microbe interactions are complex and ever-changing, especially during infections, which can significantly impact human physiology in both health and disease by influencing metabolic and immune functions. Infections caused by pathogens such as bacteria, viruses, fungi, and parasites are the leading cause of global mortality. Microbes have evolved various immune evasion strategies to survive within their hosts, which presents a multifaceted challenge for detection. Intracellular microbes, in particular, target specific cell types for survival and replication and are influenced by factors such as functional roles, nutrient availability, immune evasion, and replication opportunities. Identifying intracellular microbes can be difficult because of the limitations of traditional culture-based methods. However, advancements in integrated host microbiome single-cell genomics and transcriptomics provide a promising basis for personalized treatment strategies. Understanding host-microbiota interactions at the cellular level may elucidate disease mechanisms and microbial pathogenesis, leading to targeted therapies. This article focuses on how intracellular microbes reside in specific cell types, modulating functions through persistence strategies to evade host immunity and prolong colonization. An improved understanding of the persistent intracellular microbe-induced differential disease outcomes can enhance diagnostics, therapeutics, and preventive measures.

Reduced protein-coding transcript diversity in severe dengue emphasises the role of alternative splicing.

Dengue fever, a neglected tropical arboviral disease, has emerged as a global health concern in the past decade. Necessitating a nuanced comprehension of the intricate dynamics of host-virus interactions influencing disease severity, we analysed transcriptomic patterns using bulk RNA-seq from 112 age- and gender-matched NS1 antigen-confirmed hospital-admitted dengue patients with varying severity. Severe cases exhibited reduced platelet count, increased lymphocytosis, and neutropenia, indicating a dysregulated immune response. Using bulk RNA-seq, our analysis revealed a minimal overlap between the differentially expressed gene and transcript isoform, with a distinct expression pattern across the disease severity. Severe patients showed enrichment in retained intron and nonsense-mediated decay transcript biotypes, suggesting altered splicing efficiency. Furthermore, an up-regulated programmed cell death, a haemolytic response, and an impaired interferon and antiviral response at the transcript level were observed. We also identified the potential involvement of the RBM39 gene among others in the innate immune response during dengue viral pathogenesis, warranting further investigation. These findings provide valuable insights into potential therapeutic targets, underscoring the importance of exploring transcriptomic landscapes between different disease sub-phenotypes in infectious diseases.

Generation of an induced pluripotent stem cell line IGIBi18-A from an Indian patient with Rubinstein Taybi Syndrome.

Rubinstein Taybi Syndrome (RSTS) is a rare genetic disorder which is caused by mutations in either CREBBP or EP300. RSTS with mutations in CREBBP is known as RSTS-1. We have generated an induced pluripotent stem cell (iPSC) line, IGIBi018-A from an Indian RSTS-patient using the episomal reprogramming method. The CREBBP gene in the patient harbours a nonsense mutation at position NM_004380.3(c.6876 del C). IGIBi018-A iPSC showed expression of pluripotent stem cell markers, has a normal karyotype and could be differentiated into three germ layers. This iPSC line will help to explore the role of CREBBP in RSTS associated developmental defects.

Protocol to decode the role of transcriptionally active microbes in SARS-CoV-2-positive patients using an RNA-seq-based approach.

The elucidation of the role of microorganisms in human infections has been hindered by difficulties using conventional culture-based techniques. Here, we present a protocol for the investigation of transcriptionally active microbes (TAMs) using an RNA sequencing (RNA-seq)-based approach. We describe the steps for RNA isolation, viral genome sequencing, RNA-seq library preparation, and metatranscriptomic and transcriptomic analysis. This protocol permits a comprehensive evaluation of TAMs' contributions to the differential severity of infectious diseases, with a particular focus on diseases such as COVID-19. For complete details on the use and execution of this protocol, please refer to Devi et al.1.

Integrative analysis discovers Imidurea as dual multitargeted inhibitor of CD69, CD40, SHP2, lysozyme, GATA3, cCBL, and S-cysteinase from SARS-CoV-2 and M. tuberculosis.

Two of the deadliest infectious diseases, COVID-19 and tuberculosis (TB), have combined to establish a worldwide pandemic, wreaking havoc on economies and claiming countless lives. The optimised, multitargeted medications may diminish resistance and counter them together. Based on computational expression studies, 183 genes were co-expressed in COVID-19 and TB blood samples. We used the multisampling screening algorithms on the top ten co-expressed genes (CD40, SHP2, Lysozyme, GATA3, cCBL, SIVmac239 Nef, CD69, S-adenosylhomocysteinase, Chemokine Receptor-7, and Membrane Protein). Imidurea is a multitargeted inhibitor for COVID-19 and TB, as confirmed by extensive screening and post-filtering utilising MM\GBSA algorithms. Imidurea has shown docking and MM\GBSA scores of -8.21 to -4.75 Kcal/mol and -64.16 to -29.38 Kcal/mol, respectively. The DFT, pharmacokinetics, and interaction patterns suggest that Imidurea may be a drug candidate, and all ten complexes were tested for stability and bond strength using 100 ns for all MD atoms. The modelling findings showed the complex's repurposing potential, with a cumulative deviation and fluctuation of <2 Å and significant intermolecular interaction, which validated the possibilities. Finally, an inhibition test was performed to confirm our in-silico findings on SARS-CoV-2 Delta variant infection, which was suppressed by adding imidurea to Vero E6 cells after infection.

RNA editing in host lncRNAs as potential modulator in SARS-CoV-2 variants-host immune response dynamics.

Both host and viral RNA editing plays a crucial role in host's response to infection, yet our understanding of host RNA editing remains limited. In this study of in-house generated RNA sequencing (RNA-seq) data of 211 hospitalized COVID-19 patients with PreVOC, Delta, and Omicron variants, we observed a significant differential editing frequency and patterns in long non-coding RNAs (lncRNAs), with Delta group displaying lower RNA editing compared to PreVOC/Omicron patients. Notably, multiple transcripts of UGDH-AS1 and NEAT1 exhibited high editing frequencies. Expression of ADAR1/APOBEC3A/APOBEC3G and differential abundance of repeats were possible modulators of differential editing across patient groups. We observed a shift in crucial infection-related pathways wherein the pathways were downregulated in Delta compared to PreVOC and Omicron. Our genomics-based evidence suggests that lncRNA editing influences stability, miRNA binding, and expression of both lncRNA and target genes. Overall, the study highlights the role of lncRNAs and how editing within host lncRNAs modulates the disease severity.

The Effective Inhibitory Concentration of Interferon-ß Correlates with Infectivity and Replication Fitness of SARS-CoV-2 Variants.

India saw a spike in COVID-19 cases in early 2023, and this wave of infection was attributed to XBB sublineages of SARS-CoV-2 Omicron variant. The impact of XBB wave was significantly shorter with low burden of severe cases or hospitalization as compared with previous SARS-CoV-2 variants of concern. Although a combination of old and new mutations in the spike region of XBB.1.16 variant led to a drastic reduction in the ability of antibodies from prior immunity to neutralize this virus, additional nonspike mutations suggested a possible change in its ability to suppress innate immune responses. In this study, we tested the sensitivity of Delta, BA.2.75, and XBB.1.16 variants to interferon-ß (IFN-ß) treatment and found that XBB.1.16 variant was most sensitive to IFN-ß. We next tested the ability of serum antibodies from healthy individuals to neutralize XBB.1.16. We showed that most of the individuals with hybrid immunity maintained a low but significant level of neutralizing antibodies to XBB.1.16 variant. Therefore, our observations indicated that both hybrid immunity because of natural infection and enhanced sensitivity to IFNs may have contributed to the low impact of XBB.1.16 infections in India.

Uncovering the diversity of pathogenic invaders: insights into protozoa, fungi, and worm infections.

Post COVID-19, there has been renewed interest in understanding the pathogens challenging the human health and evaluate our preparedness towards dealing with health challenges in future. In this endeavour, it is not only the bacteria and the viruses, but a greater community of pathogens. Such pathogenic microorganisms, include protozoa, fungi and worms, which establish a distinct variety of disease-causing agents with the capability to impact the host's well-being as well as the equity of ecosystem. This review summarises the peculiar characteristics and pathogenic mechanisms utilized by these disease-causing organisms. It features their role in causing infection in the concerned host and emphasizes the need for further research. Understanding the layers of pathogenesis encompassing the concerned infectious microbes will help expand targeted inferences with relation to the cause of the infection. This would strengthen and augment benefit to the host's health along with the maintenance of ecosystem network, exhibiting host-pathogen interaction cycle. This would be key to discover the layers underlying differential disease severities in response to similar/same pathogen infection.

Cell-specific housekeeping role of lncRNAs in COVID-19-infected and recovered patients.

A plethora of studies have demonstrated the roles of lncRNAs in modulating disease severity and outcomes during infection. However, the spatio-temporal expression of these lncRNAs is poorly understood. In this study, we used single-cell RNA-seq to understand the spatio-temporal expression dynamics of lncRNAs across healthy, SARS-CoV-2-infected, and recovered individuals and their functional role in modulating the disease and recovery. We identified 203 differentially expressed lncRNAs, including cell type-specific ones like MALAT1, NEAT1, ZFAS1, SNHG7, SNHG8, and SNHG25 modulating immune function in classical monocyte, NK T, proliferating NK, plasmablast, naive, and activated B/T cells. Interestingly, we found invariant lncRNAs (no significant change in expression across conditions) regulating essential housekeeping functions (for example, HOTAIR, NRAV, SNHG27, SNHG28, and UCA1) in infected and recovered individuals. Despite similar repeat element abundance, variant lncRNAs displayed higher Alu content, suggesting increased interactions with proximal and distal genes, crucial for immune response modulation. The comparable repeat abundance but distinct expression levels of variant and invariant lncRNAs highlight the significance of investigating the regulatory mechanisms of invariant lncRNAs. Overall, this study offers new insights into the spatio-temporal expression patterns and functional roles of lncRNAs in SARS-CoV-2-infected and recovered individuals while highlighting the importance of invariant lncRNAs in the disease context.

Understanding bacterial pathogenicity: a closer look at the journey of harmful microbes.

Bacteria are the most prevalent form of microorganisms and are classified into two categories based on their mode of existence: intracellular and extracellular. While most bacteria are beneficial to human health, others are pathogenic and can cause mild to severe infections. These bacteria use various mechanisms to evade host immunity and cause diseases in humans. The susceptibility of a host to bacterial infection depends on the effectiveness of the immune system, overall health, and genetic factors. Malnutrition, chronic illnesses, and age-related vulnerabilities are the additional confounders to disease severity phenotypes. The impact of bacterial pathogens on public health includes the transmission of these pathogens from healthcare facilities, which contributes to increased morbidity and mortality. To identify the most significant threats to public health, it is crucial to understand the global burden of common bacterial pathogens and their pathogenicity. This knowledge is required to improve immunization rates, improve the effectiveness of vaccines, and consider the impact of antimicrobial resistance when assessing the situation. Many bacteria have developed antimicrobial resistance, which has significant implications for infectious diseases and favors the survival of resilient microorganisms. This review emphasizes the significance of understanding the bacterial pathogens that cause this health threat on a global scale.

Biofertilizer and biocontrol properties of Stenotrophomonas maltophilia BCM emphasize its potential application for sustainable agriculture.

Introduction: Microbial biofertilizers or biocontrol agents are potential sustainable approaches to overcome the limitations of conventional agricultural practice. However, the limited catalog of microbial candidates for diversified crops creates hurdles in successfully implementing sustainable agriculture for increasing global/local populations. The present study aimed to explore the wheat rhizosphere microbiota for microbial strains with a biofertilizer and biocontrol potential. Methods: Using a microbial culturing-based approach, 12 unique microbial isolates were identified and screened for biofertilizer/biocontrol potential using genomics and physiological experimentations. Results and discussion: Molecular, physiological, and phylogenetic characterization identified Stenotrophomonas maltophilia BCM as a potential microbial candidate for sustainable agriculture. Stenotrophomonas maltophilia BCM was identified as a coccus-shaped gram-negative microbe having optimal growth at 37°C in a partially alkaline environment (pH 8.0) with a proliferation time of ~67 minutes. The stress response physiology of Stenotrophomonas maltophilia BCM indicates its successful survival in dynamic environmental conditions. It significantly increased (P <0.05) the wheat seed germination percentage in the presence of phytopathogens and saline conditions. Genomic characterization decoded the presence of genes involved in plant growth promotion, nutrient assimilation, and antimicrobial activity. Experimental evidence also correlates with genomic insights to explain the potential of Stenotrophomonas maltophilia BCM as a potential biofertilizer and biocontrol agent. With these properties, Stenotrophomonas maltophilia BCM could sustainably promote wheat production to ensure food security for the increasing population, especially in native wheat-consuming areas.

Dynamics of Whole Transcriptome Analysis (WTA) and Surface markers expression (AbSeq) in Immune Cells of COVID-19 Patients and Recovered captured through Single Cell Genomics.

Introduction: Single-cell multi-omics studies, such as multidimensional transcriptomics (whole transcriptomic analysis, WTA), and surface marker analysis (antibody sequencing, AbSeq), have turned out to be valuable techniques that offer inaccessible possibilities for single-cell profiling of mRNA, lncRNA, and proteins. Methods: We used this technique to understand the dynamics of mRNA and protein-level differences in healthy, COVID-19-infected and recovered individuals using peripheral blood mononuclear cells (PBMCs). Our results demonstrate that compared to mRNA expression, protein abundance is a better indicator of the disease state. Results: We demonstrate that compared to mRNA expression, protein abundance is a better indicator of the disease state. We observed high levels of cell identity and regulatory markers, CD3E, CD4, CD8A, CD5, CD7, GITR, and KLRB1 in healthy individuals, whereas markers related to cell activation, CD38, CD28, CD69, CD62L, CD14, and CD16 elevated in the SARS-CoV-2 infected patients at both WTA and AbSeq levels. Curiously, in recovered individuals, there was a high expression of cytokine and chemokine receptors (CCR5, CCR7, CCR4, CXCR3, and PTGRD2). We also observed variations in the expression of markers within cell populations under different states. Discussion: Furthermore, our study emphasizes the significance of employing an oligo-based method (AbSeq) that can help in diagnosis, prognosis, and protection from disease/s by identifying cell surface markers that are unique to different cell types or states. It also allows simultaneous study of a vast array of markers, surpassing the constraints of techniques like FACS to query the vast repertoire of proteins.

An interpretative review of the wastewater-based surveillance of the SARS-CoV-2: where do we stand on its presence and concern?

Wastewater-based epidemiology (WBE) has been used for monitoring infectious diseases like polio, hepatitis, etc. since the 1940s. It is also being used for tracking the SARS-CoV-2 at the population level. This article aims to compile and assess the information for the qualitative and quantitative detection of the SARS-CoV-2 in wastewater. Based on the globally published studies, we highlight the importance of monitoring SARS-CoV-2 presence/detection in the wastewater and concurrently emphasize the development of early surveillance techniques. SARS-CoV-2 RNA sheds in the human feces, saliva, sputum and mucus that ultimately reaches to the wastewater and brings viral RNA into it. For the detection of the virus in the wastewater, different detection techniques have been optimized and are in use. These are based on serological, biosensor, targeted PCR, and next generation sequencing for whole genome sequencing or targeted amplicon sequencing. The presence of the SARS-CoV-2 RNA in wastewater could be used as a potential tool for early detection and devising the strategies for eradication of the virus before it is spread in the community. Additionally, with the right and timely understanding of viral behavior in the environment, an accurate and instructive model that leverages WBE-derived data may be created. This might help with the creation of technological tools and doable plans of action to lessen the negative effects of current viral epidemics or future potential outbreaks on public health and the economy. Further work toward whether presence of viral load correlates with its ability to induce infection, still needs evidence. The current increasing incidences of JN.1 variant is a case in point for continued early detection and surveillance, including wastewater.

Suppressed transcript diversity and immune response in COVID-19 ICU patients: a longitudinal study.

Understanding the dynamic changes in gene expression during Acute Respiratory Distress Syndrome (ARDS) progression in post-acute infection patients is crucial for unraveling the underlying mechanisms. Study investigates the longitudinal changes in gene/transcript expression patterns in hospital-admitted severe COVID-19 patients with ARDS post-acute SARS-CoV-2 infection. Blood samples were collected at three time points and patients were stratified into severe and mild ARDS, based on their oxygenation saturation (SpO2/FiO2) kinetics over 7 d. Decline in transcript diversity was observed over time, particularly in patients with higher severity, indicating dysregulated transcriptional landscape. Comparing gene/transcript-level analyses highlighted a rather limited overlap. With disease progression, a transition towards an inflammatory state was evident. Strong association was found between antibody response and disease severity, characterized by decreased antibody response and activated B cell population in severe cases. Bayesian network analysis identified various factors associated with disease progression and severity, viz. humoral response, TLR signaling, inflammatory response, interferon response, and effector T cell abundance. The findings highlight dynamic gene/transcript expression changes during ARDS progression, impact on tissue oxygenation and elucidate disease pathogenesis.

Dual RNA-Seq reveals transcriptionally active microbes (TAMs) dynamics in the serum of dengue patients associated with disease severity.

Introduction: Dengue virus (DENV) is a flavivirus that has emerged as a global health threat, characterized by either asymptomatic or mild self-limiting febrile illness, but a subset of DENV outbreaks have been associated with severe disease. Studies have looked into the host immune response and dengue viral load during infection. However, it remains unknown how the active microbial isolates modulate the dengue viral infection. In this study, we demonstrate the significance of in-depth analysis of microbiota composition in the serum samples of dengue-infected patients. Materials and methods: RNA was extracted from the serum samples collected from 24 dengue positive patients. The human mapped reads generated through RNA-Sequencing (RNA-Seq) were removed, while the unmapped (non-human) reads were employed for microbial taxonomic classification using Kraken2 and Bracken2. Further, we assessed the initial blood parameters analyzing the complete blood count (CBC) profile of the patients. Results: Findings revealed differential abundance of commensals and pathogenic microbes in the early febrile period of hospitalized dengue patients, segregated into, High Viral Reads (HVR) and Low Viral Reads (LVR). The Campylobacter genus was abundant in the HVR whereas Lactobacillus dominated the LVR patients. At species level, the microbiota of HVR exhibited higher abundance of unique potential opportunistic microbes, compared to the commensal microbes' enrichment in the LVR patients'. We hypothesize that the DENV might alter the microbiota composition as observed by the increase in preponderance of opportunistic pathogens and an absence of commensals in the HVR. The presence of commensals in the LVR might explain, i) overall lower dengue viral reads compared to the HVR, and ii) shift in lymphocytes (high) and neutrophils (low) counts; resulting in a comparatively milder clinical manifestation in this group. Our findings may help in understanding the co-infection aspect that will be important to develop dengue therapeutics and vaccines. Discussion: This study highlights the potential of the unexplored roles of the TAMs in modulating the dengue disease severity using the metatranscriptomic sequencing. This study serves to enhance our understanding of the distinctive microbial and hematologic signatures in the early infection stage that differentiate patients with high viral reads patients from those with low dengue viral reads.

From bench to bedside: potential of translational research in COVID-19 and beyond.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease 2019 (COVID-19) have been around for more than 3 years now. However, due to constant viral evolution, novel variants are emerging, leaving old treatment protocols redundant. As treatment options dwindle, infection rates continue to rise and seasonal infection surges become progressively common across the world, rapid solutions are required. With genomic and proteomic methods generating enormous amounts of data to expand our understanding of SARS-CoV-2 biology, there is an urgent requirement for the development of novel therapeutic methods that can allow translational research to flourish. In this review, we highlight the current state of COVID-19 in the world and the effects of post-infection sequelae. We present the contribution of translational research in COVID-19, with various current and novel therapeutic approaches, including antivirals, monoclonal antibodies and vaccines, as well as alternate treatment methods such as immunomodulators, currently being studied and reiterate the importance of translational research in the development of various strategies to contain COVID-19.

Co-evolution of SARS-CoV-2 variants and host immune response trajectories underlie COVID-19 pandemic to epidemic transition.

COVID-19 pandemic saw emergence of multiple SAR-CoV-2 variants. Exacerbated risk of severe outcome and hospital admissions led us to comprehend differential host-immune kinetics associated with SARS-CoV-2 variants. Longitudinal investigation was conducted through different time periods of Pre-VOC and VOCs (Delta & Omicron) mapping host transcriptome features. Robust antiviral type-1 interferon response marked Omicron infection, which was largely missing during Pre-VOC and Delta waves. SARS-CoV-2-host protein-protein interactions and docking complexes highlighted N protein to interact with HNRNPA1 in Pre-VOC, demonstrating its functional role for enhanced viral replication. Omicron revealed enhanced binding efficiency of LARP1 to N protein, probably potentiating antiviral effects of LARP1. Differential expression of zinc finger protein genes, especially in Omicron, mechanistically support induction of strong IFN (Interferon) response, thereby strengthening early viral clearance. Study highlights eventual adaptation of host to immune activation patterns that interrupt virus evolution with enhanced immune-evasion mutations and counteraction mechanisms, delimiting the next phase of COVID-19 pandemic.

Single-cell RNA-Seq reveals intracellular microbial diversity within immune cells during SARS-CoV-2 infection and recovery.

Intracellular microorganisms, like viruses, bacteria, and fungi, pose challenges in detection due to their non-culturable forms. Transcriptomic analysis at cellular level enables exploration of distributions and the impact of these microorganisms on host cells, a domain that remains underexplored because of methodological limitations. Single-cell technology shows promise in addressing this by capturing polyadenine-tailed transcripts, because recent studies confirmed polyadenylation in microbial transcriptomes. We utilized single-cell RNA-seq from PBMCs to probe intracellular microbes in healthy, SARS-CoV-2-positive, and recovered individuals. Among 76 bacterial species detected, 16 showed significant abundance differences. Buchnera aphidicola, Streptomyces clavuligerus, and Ehrlichia canis emerged significantly in memory-B, Naïve-T, and Treg cells. Staphylococcus aureus, Mycoplasma mycoides, Leptospira interrogans, and others displayed elevated levels in SARS-CoV-2-positive patients, suggesting possible disease association. This highlights the strength of single-cell technology in revealing potential microorganism's cell-specific functions. Further research is essential for functional understanding of their cell-specific abundance across physiological states.