Genome Tunisia Project: paving the way for precision medicine in North Africa.

BACKGROUND: Key discoveries and innovations in the field of human genetics have led to the foundation of molecular and personalized medicine. Here, we present the Genome Tunisia Project, a two-phased initiative (2022-2035) which aims to deliver the reference sequence of the Tunisian Genome and to support the implementation of personalized medicine in Tunisia, a North African country that represents a central hub of population admixture and human migration between African, European, and Asian populations. The main goal of this initiative is to develop a healthcare system capable of incorporating omics data for use in routine medical practice, enabling medical doctors to better prevent, diagnose, and treat patients. METHODS: A multidisciplinary partnership involving Tunisian experts from different institutions has come to discern all requirements that would be of high priority to fulfill the project's goals. One of the most urgent priorities is to determine the reference sequence of the Tunisian Genome. In addition, extensive situation analysis and revision of the education programs, community awareness, appropriate infrastructure including sequencing platforms and biobanking, as well as ethical and regulatory frameworks, have been undertaken towards building sufficient capacity to integrate personalized medicine into the Tunisian healthcare system. RESULTS: In the framework of this project, an ecosystem with all engaged stakeholders has been implemented including healthcare providers, clinicians, researchers, pharmacists, bioinformaticians, industry, policymakers, and advocacy groups. This initiative will also help to reinforce research and innovation capacities in the field of genomics and to strengthen discoverability in the health sector. CONCLUSIONS: Genome Tunisia is the first initiative in North Africa that seeks to demonstrate the major impact that can be achieved by Human Genome Projects in low- and middle-income countries to strengthen research and to improve disease management and treatment outcomes, thereby reducing the social and economic burden on healthcare systems. Sharing this experience within the African scientific community is a chance to turn a major challenge into an opportunity for dissemination and outreach. Additional efforts are now being made to advance personalized medicine in patient care by educating consumers and providers, accelerating research and innovation, and supporting necessary changes in policy and regulation.

miRNAs mediate the impact of smoking on dental pulp stem cells via the p53 pathway.

Cigarette smoke changes the genomic and epigenomic imprint of cells. In this study, we investigated the biological consequences of extended cigarette smoke exposure on dental pulp stem cells (DPSCs) and the potential roles of miRNAs. DPSCs were treated with various doses of cigarette smoke condensate (CSC) for up to 6 weeks. Cell proliferation, survival, migration, and differentiation were evaluated. Cytokine and miRNA expression were profiled. The results showed that extended exposure to CSC significantly impaired the regenerative capacity of the DPSCs. Bioinformatic analysis showed that the cell cycle pathway, cancer pathways (small cell lung cancer, pancreatic, colorectal, and prostate cancer), and pathways for TNF, TGF-ß, p53, PI3K-Akt, mTOR, and ErbB signal transduction, were associated with altered miRNA profiles. In particular, 3 miRNAs has-miR-26a-5p, has-miR-26b-5p, and has-miR-29b-3p fine-tune the p53 and cell cycle signaling pathways to regulate DPSC cellular activities. The work indicated that miRNAs are promising targets to modulate stem cell regeneration and understanding miRNA-targeted genes and their associated pathways in smoking individuals have significant implications for disease control and prevention.

Infectivity of three Mayaro Virus geographic isolates in human cell lines.

Mayaro virus (MAYV) is an emergent arthropod-borne virus that causes an acute febrile illness accompanied by arthralgia, similar to chikungunya virus. Increasing urbanization of MAYV outbreaks in the Americas has led to concerns for geographic expansion and spillover. Given the potential importance of this pathogen, we sought to fill critical gaps in knowledge regarding MAYV infectivity and geographic variation. This study describes the cytopathogenicity of MAYV in human dermal fibroblasts, human skeletal muscle satellite cells, human embryonic kidney cells (HEK), peripherally derived human macrophages, and Vero cells. We found that regional differences between these viruses do not affect replication kinetics, with high titers peaking at 37 h post infection. MAYV-U, did however, cause the most cytopathic effect in a time-dependent manner. Compared to the other two prototypic isolates, MAYV-U harbors unique mutations in the E2 protein, D60G and S205F, that are likely to interact with the host cell receptor and could affect infectivity. We further demonstrate that pre-treatment of cells with interferon-ß inhibited viral replication in a dose-dependent manner. Together, these findings advance our understanding of MAYV infection of human target cells and provide initial data regarding variation according to geography.

Everglades virus evolution: Genome sequence analysis of the envelope 1 protein reveals recent mutation and divergence in South Florida wetlands.

Everglades virus (EVEV) is a subtype (II) of Venezuelan equine encephalitis virus (VEEV), endemic in southern Florida, USA. EVEV has caused clinical encephalitis in humans, and antibodies have been found in a variety of wild and domesticated mammals. Over 29,000 Culex cedecei females, the main vector of EVEV, were collected in 2017 from Big Cypress and Fakahatchee Strand Preserves in Florida and pool-screened for the presence of EVEV using reverse transcription real-time polymerase chain reaction. The entire 1 E1 protein gene was successfully sequenced from fifteen positive pools. Phylogenetic analysis showed that isolates clustered, based on the location of sampling, into two monophyletic clades that diverged in 2009. Structural analyses revealed two mutations of interest, A116V and H441R, which were shared among all isolates obtained after its first isolation of EVEV in 1963, possibly reflecting adaptation to a new host. Alterations of the Everglades ecosystem may have contributed to the evolution of EVEV and its geographic compartmentalization. This is the first report that shows in detail the evolution of EVEV in South Florida. This zoonotic pathogen warrants inclusion into routine surveillance given the high natural infection rate in the vectors. Invasive species, increasing urbanization, the Everglades restoration, and modifications to the ecosystem due to climate change and habitat fragmentation in South Florida may increase rates of EVEV spillover to the human population.

The Spike Mutants Website: A Worldwide Used Resource against SARS-CoV-2.

A large number of SARS-CoV-2 mutations in a short period of time has driven scientific research related to vaccines, new drugs, and antibodies to combat the new variants of the virus. Herein, we present a web portal containing the structural information, the tridimensional coordinates, and the molecular dynamics trajectories of the SARS-CoV-2 spike protein and its main variants. The Spike Mutants website can serve as a rapid online tool for investigating the impact of novel mutations on virus fitness. Taking into account the high variability of SARS-CoV-2, this application can help the scientific community when prioritizing molecules for experimental assays, thus, accelerating the identification of promising drug candidates for COVID-19 treatment. Below we describe the main features of the platform and illustrate the possible applications for speeding up the drug discovery process and hypothesize new effective strategies to overcome the recurrent mutations in SARS-CoV-2 genome.

Structural Evolution of Delta (B.1.617.2) and Omicron (BA.1) Spike Glycoproteins.

The vast amount of epidemiologic and genomic data that were gathered as a global response to the COVID-19 pandemic that was caused by SARS-CoV-2 offer a unique opportunity to shed light on the structural evolution of coronaviruses and in particular on the spike (S) glycoprotein, which mediates virus entry into the host cell by binding to the human ACE2 receptor. Herein, we carry out an investigation into the dynamic properties of the S glycoprotein, focusing on the much more transmissible Delta and Omicron variants. Notwithstanding the great number of mutations that have accumulated, particularly in the Omicron S glycoprotein, our data clearly showed the conservation of some structural and dynamic elements, such as the global motion of the receptor binding domain (RBD). However, our studies also revealed structural and dynamic alterations that were concentrated in the aa 627-635 region, on a small region of the receptor binding motif (aa 483-485), and the so-called "fusion-peptide proximal region". In particular, these last two S regions are known to be involved in the human receptor ACE2 recognition and membrane fusion. Our structural evidence, therefore, is likely involved in the observed different transmissibility of these S mutants. Finally, we highlighted the role of glycans in the increased RBD flexibility of the monomer in the up conformation of Omicron.

Evidence of SARS-CoV-2 symptomatic reinfection in four healthcare professionals from the same hospital despite the presence of antibodies.

OBJECTIVES: Since the onset of the COVID-19 pandemic, cases of reinfection with SARS-CoV-2 have been reported, raising additional public health concerns. SARS-CoV-2 reinfection was assessed in healthcare workers (HCWs) in Tunisia because they are at the greatest exposure to infection by different variants. METHODS: We conducted whole-genome sequencing of the viral RNA from clinical specimens collected during the initial infection and the suspected reinfection from 4 HCWs, who were working at the Habib Bourguiba University Hospital (Sfax, Tunisia) and retested positive for SARS-CoV-2 through reverse transcriptase-polymerase chain reaction (RT-PCR) after recovery from a first infection. A total of 8 viral RNAs from the patients' respiratory specimens were obtained, which allowed us to characterize the differences between viral genomes from initial infection and positive retest. The serology status for total Ig, IgG, and IgM against SARS-CoV-2 was also determined and followed after the first infection. RESULTS: We confirmed through whole-genome sequencing of the viral samples that all 4 cases experienced a reinfection event. The interval between the 2 infection events ranged between 45 and 141 days, and symptoms were milder in the second infection for 2 patients and more severe for the remaining 2 patients. Reinfection occurred in all 4 patients despite the presence of antibodies in 3 of them. CONCLUSION: This study adds to the rapidly growing evidence of COVID-19 reinfection, where viral sequences were used to confirm infection by distinct isolates of SARS-CoV-2 in HCWs. These findings suggest that individuals who are exposed to different SARS-CoV-2 variants might not acquire sufficiently protective immunity through natural infection and emphasize the necessity of their vaccination and the regular follow-up of their immune status both in quantitative and qualitative terms.

Contribution of Nanotechnologies to Vaccine Development and Drug Delivery against Respiratory Viruses.

According to the Center for Disease Control and Prevention (CDC), the coronavirus disease 2019, a respiratory viral illness linked to significant morbidity, mortality, production loss, and severe economic depression, was the third-largest cause of death in 2020. Respiratory viruses such as influenza, respiratory syncytial virus, SARS-CoV-2, and adenovirus, are among the most common causes of respiratory illness in humans, spreading as pandemics or epidemics throughout all continents. Nanotechnologies are particles in the nanometer range made from various compositions. They can be lipid-based, polymer-based, protein-based, or inorganic in nature, but they are all bioinspired and virus-like. In this review, we aimed to present a short review of the different nanoparticles currently studied, in particular those which led to publications in the field of respiratory viruses. We evaluated those which could be beneficial for respiratory disease-based viruses; those which already have contributed, such as lipid nanoparticles in the context of COVID-19; and those which will contribute in the future either as vaccines or antiviral drug delivery systems. We present a short assessment based on a critical selection of evidence indicating nanotechnology's promise in the prevention and treatment of respiratory infections.

Phylodynamic and phylogeographic analysis of the complete genome of the West Nile virus lineage 2 (WNV-2) in the Mediterranean basin.

BACKGROUND: The West Nile virus is a highly contagious agent for a wide range of hosts. Its spread in the Mediterranean region raises several questions about its origin and the risk factors underlying the virus's dispersal. MATERIALS AND METHODS: The present study aims to reconstruct the temporal and spatial phylodynamics of West Nile virus lineage 2 in the Mediterranean region using 75 complete genome sequences from different host species retrieved from international databases. RESULTS: This data set suggests that current strains of WNV-2 began spreading in South Africa or nearby regions in the early twentieth century, and it migrated northwards via at least one route crossing the Mediterranean to reach Hungary in the early 2000s, before spreading throughout Europe. Another introduction event, according to the data set collected and analyses performed, is inferred to have occurred in around 1978. Migratory birds constitute, among others, additional risk factors that enhance the geographical transmission of the infection. CONCLUSION: Our data underline the importance of the spatial-temporal tracking of migratory birds and phylodynamic reconstruction in setting up an efficient surveillance system for emerging and reemerging zoonoses in the Mediterranean region.

Altered Local Interactions and Long-Range Communications in UK Variant (B.1.1.7) Spike Glycoprotein.

The COVID-19 pandemic is caused by SARS-CoV-2. Currently, most of the research efforts towards the development of vaccines and antibodies against SARS-CoV-2 were mainly focused on the spike (S) protein, which mediates virus entry into the host cell by binding to ACE2. As the virus SARS-CoV-2 continues to spread globally, variants have emerged, characterized by multiple mutations of the S glycoprotein. Herein, we employed microsecond-long molecular dynamics simulations to study the impact of the mutations of the S glycoprotein in SARS-CoV-2 Variant of Concern 202012/01 (B.1.1.7), termed the "UK variant", in comparison with the wild type, with the aim to decipher the structural basis of the reported increased infectivity and virulence. The simulations provided insights on the different dynamics of UK and wild-type S glycoprotein, regarding in particular the Receptor Binding Domain (RBD). In addition, we investigated the role of glycans in modulating the conformational transitions of the RBD. The overall results showed that the UK mutant experiences higher flexibility in the RBD with respect to wild type; this behavior might be correlated with the increased transmission reported for this variant. Our work also adds useful structural information on antigenic "hotspots" and epitopes targeted by neutralizing antibodies.

SARS-CoV-2 tracking in Tunisia through next-generation sequencing: lessons for the future.

In this study, data available from GISAID on the whole-genome sequences of SARS-CoV-2 variants circulating in Tunisia were analyzed, and the prevalences of those variants in Tunisia were compared to their prevalences in other North African countries and around the world. Our results show new mutations and different prevalences of some lineages. In particular, new sets of mutations were identified in the spike protein of the virus during the analysis of 85 Tunisian samples, and the lineage B1.160 was found to be the most prevalent (18%) lineage in Tunisia. The prevalence of this lineage in Tunisia was significantly higher than its prevalence worldwide and in samples from neighboring countries (3%). This preliminary study shows the importance of tracking virus variants by next-generation sequencing in order to assess the dynamics of the COVID-19 pandemic and the impact of vaccination on the evolution of the virus.

Multiple Recombination Events and Strong Purifying Selection at the Origin of SARS-CoV-2 Spike Glycoprotein Increased Correlated Dynamic Movements.

Our evolutionary and structural analyses revealed that the severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) spike gene is a complex mosaic resulting from several recombination events. Additionally, the fixation of variants has mainly been driven by purifying selection, suggesting the presence of conserved structural features. Our dynamic simulations identified two main long-range covariant dynamic movements of the novel glycoprotein, and showed that, as a result of the evolutionary duality, they are preserved. The first movement involves the receptor binding domain with the N-terminal domain and the C-terminal domain 2 and is maintained across human, bat and pangolin coronaviruses. The second is a complex network of long-range dynamics specific to SARS-CoV-2 involving the novel PRRA and the conserved KR*SF cleavage sites, as well as conserved segments in C-terminal domain 3. These movements, essential for host cell binding, are maintained by hinges conserved across human, bat, and pangolin coronaviruses glycoproteins. The hinges, located around Threonine 333 and Proline 527 within the N-terminal domain and C-terminal domain 2, represent candidate targets for the future development of novel pan-coronavirus inhibitors. In summary, we show that while recombination created a new configuration that increased the covariant dynamic movements of the SARS-CoV-2 glycoprotein, negative selection preserved its inter-domain structure throughout evolution in different hosts and inter-species transmissions.

New Insights into the Effect of Residue Mutations on the Rotavirus VP1 Function Using Molecular Dynamic Simulations.

Rotavirus group A remains a major cause of diarrhea in infants and young children worldwide. The permanent emergence of new genotypes puts the potential effectiveness of vaccines under serious questions. Thirteen VP1 structures with mutations mapping to the RNA entry site were analyzed using molecular dynamics simulations, and the results were combined with the experimental findings reported previously. The results revealed structural fluctuations in the protein-protein recognition sites and in the bottleneck of the RNA entry site that may affect the interaction of different proteins and delay the initiation of the viral replication, respectively. Altogether, the structural analysis of VP1 in the region crucial for the initiation of the viral replication, mainly the bottleneck site, may boost efforts to develop antivirals, as they might complement the available vaccines.

Association of cytokine Th2 gene polymorphisms with autoimmune thyroid diseases in Tunisian population.

Autoimmune thyroid diseases (AITD) including Graves' disease (GD) and Hashimoto's thyroiditis (HT) are complex genetic diseases. Th2 cytokines act on the development of AITD. This study was conducted on Tunisian patients with AITD to investigate the association of Th2 cytokine gene polymorphisms and haplotype combination with GD or HT risk. A total of 156 controls, 160 patients with HT and 88 patients with GD were genotyped for IL-4 rs2243250, IL-5 rs2069812, IL-6 rs1800796 and IL-13 rs1800925 polymorphisms by PCR-RFLP. The AITD risk was assessed by a logistic regression analysis using the SNP stats statistical program. False-positive report probability (FPRP) was estimated to evaluate significant findings. IL-13 rs1800925 was associated with GD, after adjustment for age and gender, in codominant, dominant and allele genetic models (p = .0072; p = .0018; p = .012, respectively). Significant association of the IL-6 rs1800796C/G genotype with GD was also detected (p = .025). Furthermore, increased risk of HT was still found for IL-13 rs1800925T allele (p = .039, OR = 1.39) and for IL-4 rs2243250T/T genotype both in codominant (p = .033, OR = 2.59) and recessive (p = .011, OR = 2.73) models after adjustment for age and gender. Interestingly, haplotype analysis performed on the IL-4, IL-5 and IL-13 genes revealed a high risk of HT with CTT haplotype (p = .008, OR = 2.12). However, the CCT haplotype is a protective factor (OR = 0.36). Patients carrying the CT haplotype with only one minor allele had a moderate risk of HT (OR = 1.56). The FPRP analysis showed that the association of IL-13 rs1800925 polymorphism with GD and HT and the association of CTT haplotype with HT were noteworthy. In conclusion, the IL-4, IL-5, IL-6 and IL-13 polymorphism may play a role in susceptibility to GD and HT in the Tunisian population. Furthermore, gene-gene interaction between the IL-4, IL-5 and IL-13 significantly increases the risk of AITD. Further studies with larger numbers of individuals are needed to confirm the results.

Coding-Gene Coevolution Analysis of Rotavirus Proteins: A Bioinformatics and Statistical Approach.

Rotavirus remains a major cause of diarrhea in infants and young children worldwide. The permanent emergence of new genotypes puts the potential effectiveness of vaccines under serious question. The distribution of unusual genotypes subject to viral fitness is influenced by interactions among viral proteins. The present work aimed at analyzing the genetic constellation and the coevolution of rotavirus coding genes for the available rotavirus genotypes. Seventy-two full genome sequences of different genetic constellations were analyzed using a genetic algorithm. The results revealed an extensive genome-wide covariance network among the 12 viral proteins. Altogether, the emergence of new genotypes represents a challenge to the outcome and success of vaccination and the coevolutionary analysis of rotavirus proteins may boost efforts to better understand the interaction networks of proteins during viral replication/transcription.

WDR73-related galloway mowat syndrome with collapsing glomerulopathy.

Galloway-Mowat syndrome (GAMOS [MIM 251300]) is a rare autosomal recessive disorder that manifests as a combination of nephrotic syndrome, brain abnormalities and developmental delay. It is a clinically and genetically heterogeneous disease. The WDR73 variations are associated with GAMOS1. Here we report two consanguineous families affected by GAMOS1. In the first family, three sisters are affected and in the second family, only one index case is identified. They all show a nephrotic syndrome, a neurological involvement and a collapsing glomerulopathy. The analysis of mutations of WDR73 revealed a new homozygous missense mutation NM_032856.3 c.293T > C; p.(Leu98Pro) in two patients from the first family, and a new homozygous missense mutation NM_032856.3: c.767G > A; p.(Arg256Gln) in the second one. This study extended the clinical and molecular spectrum of GAMOS1 with other cases associated with collapsing glomerulopathy and two novel WDR73 variations that are most likely pathogenic.

Culture conditions improvement of Crassostrea gigas using a potential probiotic Bacillus sp strain.

It is well demonstrated that some probiotics improve rearing water quality and thereby have beneficial effects on reared organisms. We conducted this study to determine the effect of Bacillus consortium on Crassostrea gigas reared in contemned seawater with indigo dye priory treated with Bacillus or no treated. This effect was studied by assessing hemocytes death using flow cytometry analysis. We found that the percentage of decolorization of indigo dye in polluted seawater in presence of C. gigas increased from 41% to 90% when using Bacillus consortium. In these conditions, the hemocytes mortality of reared C. gigas decreased from 87% to 56%. We have demonstrated also that seawater contemned with priory treated indigo with Bacillus consortium is less toxic than seawater contemned with the no treated indigo. The percentage of hemocytes death is 81% for the contemned seawater with indigo and 56% for no contemned seawater. This consortium shows a protector effect of C. gigas against Vibrio harveyi contemning reared seawater.

Clinical surveillance for human astrovirus in Monastir region, Tunisia.

BACKGROUND/AIMS: Astroviruses (AstVs) are enteric viruses that can cause gastroenteritis in children. This study is part of monitoring the circulation of astroviruses in children hospitalized and/or outpatients for acute gastroenteritis at the primary care center of Ouerdanine or at the Pediatric Department of the University Hospital Fattouma-Bourguiba (Monastir, Tunisia). The aims of our study were to know the prevalence of human astrovirus in clinical samples of children, characterize the strains and evaluate the infectivity of isolated strains on cell culture. METHODS: Fifty stool samples were collected from children under five years old in the region of Monastir (Tunisia) from October 2010 to June 2011. All specimens were subjected to RT-PCR amplification followed by sequencing and phylogenetic analysis. RESULTS: The study shows a low prevalence of astrovirus (4 %) in children. The two positive samples obtained were HAstV type 3. Samples that were RT-PCR positive were cultured in CaCO-2 cells and the presence of infectious viral particles was confirmed. The phylogenetic analysis shows that the different HAstV-3 strains isolated in Tunisia are grouped into two clusters. The first cluster includes strains obtained in 2004, which belong to lineage HAstV-3a, while strains isolated in 2010 belong to lineage HAstV-3c. CONCLUSIONS: This study is part of monitoring the circulation of astroviruses in children younger than five years old from Monastir region, Tunisia. The results show low prevalence (4 %). All genotyped samples belonged to lineage HAstV-3c, which could be presently emerging. Two different lineages have been isolated in Tunisia: HAstV-3a in 2004 and HAstV-3c in 2010.

Electrochemical impedance immunosensor for rapid detection of stressed pathogenic Staphylococcus aureus bacteria.

In this work, we report the adaptation of bacteria to stress conditions that induce instability of their cultural, morphological, and enzymatic characters, on which the identification of pathogenic bacteria is based. These can raise serious issues during the characterization of bacteria. The timely detection of pathogens is also a subject of great importance. For this reason, our objective is oriented towards developing an immunosensing system for rapid detection and quantification of Staphylococcus aureus. Polyclonal anti-S. aureus are immobilized onto modified gold electrode by self-assembled molecular monolayer (SAM) method. The electrochemical performances of the developed immunosensor were evaluated by impedance spectroscopy through the monitoring of the charge transfer resistance at the modified solid/liquid interface using ferri-/ferrocyanide as redox probe. The developed immunosensor was applied to detect stressed and resuscitate bacteria. As a result, a stable and reproducible immunosensor with sensitivity of 15 kΩ/decade and a detection limit of 10 CFU/mL was obtained for the S. aureus concentrations ranging from 10(1) to 10(7) CFU/mL. A low deviation in the immunosensor response (±10 %) was signed when it is exposed to stressed and not stressed bacteria.