Exploring viral infection using single-cell sequencing.

Single-cell sequencing (SCS) has emerged as a valuable tool to study cellular heterogeneity in diverse fields, including virology. By studying the viral and cellular genome and/or transcriptome, the dynamics of viral infection can be investigated at single cell level. Most studies have explored the impact of cell-to-cell variation on the viral life cycle from the point of view of the virus, by analyzing viral sequences, and from the point of view of the cell, mainly by analyzing the cellular host transcriptome. In this review, we will focus on recent studies that use single-cell sequencing to explore viral diversity and cell variability in response to viral replication

The use of single-cell RNA-Seq to understand virus-host interactions.

Single-cell analyses allow uncovering cellular heterogeneity, not only per se, but also in response to viral infection. Similarly, single cell transcriptome analyses (scRNA-Seq) can highlight specific signatures, identifying cell subsets with particular phenotypes, which are relevant in the understanding of virus-host interactions

Clinical metagenomics.

Clinical metagenomic next-generation sequencing (mNGS), the comprehensive analysis of microbial and host genetic material (DNA and RNA) in samples from patients, is rapidly moving from research to clinical laboratories. This emerging approach is changing how physicians diagnose and treat infectious disease, with applications spanning a wide range of areas, including antimicrobial resistance, the microbiome, human host gene expression (transcriptomics) and oncology. Here, we focus on the challenges of implementing mNGS in the clinical laboratory and address potential solutions for maximizing its impact on patient care and public health

Zika virus infection:in vitro models of disease pathogenesis

In this thesis, we established primary and cell line models to study the pathogenesis of the Zika virus. These models are important not only to identify markers of disease pathogenesis but also to screen the antiviral activity of various drugs

Evolution of Two Major Zika Virus Lineages: Implications for Pathology, Immune Response, and Vaccine Development

Zika virus (ZIKV) became a public health emergency of global concern in 2015 due to its rapid expansion from French Polynesia to Brazil, spreading quickly throughout the Americas. Its unexpected correlation to neurological impairments and defects, now known as congenital Zika syndrome, brought on an urgency to characterize the pathology and develop safe, effective vaccines. ZIKV genetic analyses have identified two major lineages, Asian and African, which have undergone substantial changes during the past 50 years. Although ZIKV infections have been circulating throughout Africa and Asia for the later part of the 20th century, the symptoms were mild and not associated with serious pathology until now. ZIKV evolution also took the form of novel modes of transmission, including maternal–fetal transmission, sexual transmission, and transmission through the eye. The African and Asian lineages have demonstrated differential pathogenesis and molecular responses in vitro and in vivo. The limited number of human infections prior to the 21st century restricted ZIKV research to in vitro studies, but current animal studies utilize mice deficient in type I interferon (IFN) signaling in order to invoke enhanced viral pathogenesis. This review examines ZIKV strain differences from an evolutionary perspective, discussing how these differentially impact pathogenesis via host immune responses that modulate IFN signaling, and how these differential effects dictate the future of ZIKV vaccine candidates

Optimization of Recombinant Flavivirus Antigens for Infection Serology: Towards Syndrome-Based Multiplex Tests

Sensitive and specific pathogen detection is an essential prerequisite for the prevention and treatment of infectious diseases. The similarities of clinical symptoms and serological cross-reactivity of viral structural antigens make the diagnosis of flavivirus infection problematic. Therefore, the main aim of this thesis was the development of a non-structural protein 1 (NS1) based serological assay for the diagnosis of flaviviruses. Recombinant NS1 (rNS1) oligomers consistent with the native secreted form of the protein were purified for TBEV, WNV, ZIKV, USUV, and DENV 1-4. The ability of rNS1 proteins to detect specific antibodies was analyzed using sera of immunized mice and well-characterized human sera samples. These antigens were used in a standard ELISA format and shown to be highly sensitive and specific compared to commercial assays. The optimized NS1-based ELISA was used to assess the IgM/IgG responses to WNV and USUV in North-Eastern Italy. The results of the analysis confirmed the area as endemic for USUV and contributed to the characterization of the first human cases of USUV infection in blood donors. The NS1-based ELISA was also applied to 200 sera samples from patients exhibiting febrile illness who visited the University of Maiduguri Teaching Hospital in Nigeria. Only 11 of 200 serum samples were negative for all the flaviviruses tested, while all other samples were positive for at least one pathogen. Molecular analysis confirmed the circulation of flaviviruses in the region, including zika virus. In conclusion, rNS1 represents a valuable option for the serology of flaviviruses with reduced cross-reactivity and high sensitivity