Flaviviruses, a genus within the Flaviviridae family, encompass major human pathogens yellow fever virus (YFV), dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), Kunjin virus (KUNV), and Japanese encephalitis virus (JEV) [1]. These viruses are enveloped with a positive single-stranded RNA genome, featuring a distinctive translated polyprotein sequence that is divided into ten major proteins. These include three structural proteins: capsid (C), pre-membrane (prM), and envelope, along with seven non-structural proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 [2-6]. While strains within the same flavivirus species are generally conserved, in the case of DENV, four distinct serotypes—DENV-1, DENV-2, DENV-3, and DENV-4—have been identified [2,5].

Flaviviruses are primarily transmitted by arthropods such as mosquitoes and ticks. Since DENV, ZIKV, WNV, and JEV are predominantly spread through mosquito bites, a well-documented correlation exists between mosquito habitats and the incidence of infected individuals [7-10]. Occasionally, flavivirus transmission to humans can also occur via blood transfusions from infected individuals or through sexual contact [11]. Recent years have seen the expansion of tropical and subtropical climates due to global warming, leading to the enlargement of mosquito habitats, which are the primary vectors for flaviviruses. Additionally, advances in transportation have increased international travel and goods movement, facilitating the spread of flaviviruses across countries [7]. These environmental factors have made flaviviruses a persistent threat to human health. Indeed, since the 2010s, widespread outbreaks of YFV, DENV, ZIKV, and JEV have been reported globally, causing considerable casualties and significant social and economic impacts, marking them as a major global health issue [7-11].

Flaviviruses cause a wide range of physiological effects in infected hosts, impacting various systems such as the cardiovascular, neurological, and hepatic systems (Fig. 1). Despite the long-standing recognition of flaviviruses, the pathophysiological mechanisms following infection in humans remain poorly understood. The serious health sequelae and high mortality rates observed during recent flavivirus epidemics have significantly intensified research efforts [12]. Consequently, understanding these physiological consequences is crucial for developing effective diagnostic and therapeutic strategies. Various research approaches, including omics analyses, are widely employed to study these viruses. Transcriptomics, a powerful scientific method, is utilized to study the complete set of RNA molecules, typically employing high-throughput sequencing technologies such as RNA sequencing (RNA-seq) [13-15]. By analyzing vast amounts of RNA, including mRNA (messenger RNA), miRNA (microRNA), and lncRNA (long non-coding RNA), researchers can establish a comprehensive profile of gene activity. Transcriptome analysis involves the identification, quantification, and characterization of RNA transcripts in a biological sample, providing valuable insights into gene expression patterns and the regulatory mechanisms underlying various biological processes [16-21].

Figure 1. The pathogenesis of flavivirus infections. Mosquitoes, the main transmitters of flavivirus, initiate the infection by injecting the virus into humans (left panel). Generally, infection with flavivirus causes mild symptoms such as fever and leads to various physiological changes within the body (middle panel). When symptoms become severe, the infection can affect multiple organs (right panel). ZIKV infection can cause ocular diseases and neurological inflammation, and it can also lead to infection of the placenta. DENV infection can cause damage to endothelial cells, leading to plasma leakage. YFV infection can cause damage to hepatocytes. DENV, YFV and WNV infections can cause acute kidney injury or renal failure.

RNA-seq technologies have significantly advanced our understanding of the pathophysiological impacts of flavivirus infections by providing molecular insights into host-pathogen interactions and host responses at both the cellular and systemic levels. In the field of virology, transcriptome analysis contributes to our understanding of virus virulence, host infection mechanisms, immune responses, and other related aspects [22,23]. Particularly, analyzing the gene expression profiles of viruses enables a comprehensive investigation of the viral infection process. Transcriptome analysis facilitates an in-depth exploration of gene expression patterns and unveils the intricate regulatory mechanisms that govern gene expression. Such insights are crucial for advancing our understanding of biological processes and for gaining critical insights into the mechanisms of disease [24]. Additionally, transcriptome analysis enhances our knowledge of viral infection mechanisms and virus-host interactions. Examining the expression of viral genes and the corresponding host response sheds light on the viral infection process. Transcriptome analysis is also instrumental in discovering new drug development opportunities and treatment strategies, especially in identifying novel therapeutic targets and evaluating drug efficacy through analysis of gene expression patterns.

This review will explore how bulk RNA-seq, single-cell RNA-seq (scRNA-seq), virus-inclusive single-cell RNA-seq (viscRNA-seq) have been utilized in the study of flavivirus infection, host-pathogen interactions, immune responses, and in other relevant research areas (Fig. 2).

Figure 2. Comparative overview of RNA-sequencing (RNA-seq) technologies in flavivirus research. The distinct methodologies and outcomes associated with three different RNA-seq technologies applied in the study of flaviviruses. Bulk RNA-seq, a technique that analyzes the collective gene expression profiles from a population of cells, facilitating the discovery of virus-specific biomarkers. Single-cell RNA sequencing (scRNA-seq), which isolates and characterizes the gene expression of individual cells, revealing the cellular heterogeneity and the diversity of cellular responses to infection. Virus-inclusive single-cell RNA sequencing (viscRNA-seq), which allows for the detailed examination of the transcriptomes of both virus and host cells at the single-cell level, differing responses within a population of infected and uninfected cells.

Research on flavivirus using bulk RNA-seq

Driven by RNA-seq technologies, flavivirus research has witnessed rapid advancements in recent years. These breakthroughs hold immense promise for not only improving our understanding of diverse flaviviruses but also for accelerating the development of effective diagnostics, therapeutics, and preventive strategies against virus-related diseases [13,14]. Current research is focused on deciphering the host's response to flavivirus infection, with the goal of unraveling the complexity of the virus' pathogenic behavior and immune response. Additionally, RNA-seq studies have yielded promising results in identifying therapeutic approaches and vaccine candidates. These advancements are important and serve as a cornerstone for formulating effective treatments, vaccine candidates, and preventive measures against widespread viral threats [25,26]. Table 1 summarizes research on flavivirus using bulk RNA-seq.

Table 1 . Flavivirus research using RNA-seq.

Virus type	Sample type	Collection condition	Repository	Code	Reference
DENV-2	Serum	11 patients at 3 time points post-infection	GEO	GSE152255	[24]
DENV-1,2,3,4	Serum	24 patients	BioProject	PRJNA955953	[25]
DENV-3	Serum	24 patients	BioProject	PRJNA895688	[26]
ZIKV	Cell line	JEG-3 cells at 3 h, 12 h, 24 h post-infection U-251 MG at 24 h post-infection HK-2 cells at 24 h post-infection	-	-	[31]
ZIKV	Primary cell	Human iris pigment epithelial cells at 24 h post-infection	GEO	GSE131605	[34]
ZIKV, KUNV, YFV	Cell line	U87 cells at 24 h post-infection	GEO	GSE232504	[35]

DENV, dengue virus; GEO, gene expression omnibus; KUNV, Kunjin virus; RNA-seq, RNA sequencing; YFV, yellow fever virus; ZIKV, Zika virus.

DENV infections are usually asymptomatic, with only 20%–25% of cases causing symptoms from mild fever to severe dengue [27]. However, the underlying cause (etiology) of severe dengue in natural infections remains unidentified. Hanley et al. [28] employed RNA-seq to analyze the transcriptomic profile of the human immune response to DENV infection using blood samples collected from participants. Their study utilized a partially attenuated challenge virus, rDEN2Δ30. The results revealed that DENV-2 exposure triggered the induction of genes associated with inflammation, including the type I interferon (IFN) pathway and viral restriction pathways. IFN-I plays a crucial role in enhancing antiviral defense and limiting virus replication and spread [29]. These elevated gene expression levels returned to baseline after the virus was cleared from the body. However, the immune regulatory pathways, cell migration, and growth factors in the blood exhibited a decrease to below baseline levels. These results indicate that major rDEN2Δ30 infection induces a variety of gene expression changes, some of which may be related to viral replication or the formation of immune memory, such as inflammatory responses. Additionally, the study examined the impact of specific gene expression changes in dengue-infected patients on clinical responses. Genes regulating inflammatory responses (e.g., IL-6, TNF-α) and interferon signaling pathways (e.g., IFN-α, IFN-β) were activated, along with genes related to T cell and B cell activation (e.g., CD4, CD8, BCR). These changes were closely associated with clinical symptoms such as transient variations in white blood cell counts and mild rashes. Understanding these gene expression changes provides crucial insights into immune responses and informs the development of treatment strategies for dengue infection. These results also correlate with clinical observations of decreased immune regulatory pathways and reduced cell migration and growth factors.

Simultaneous investigation of the host's response to viral infection and its interaction with the virus may improve our understanding of the pathogenesis of dengue fever. Yadav et al. [30] conducted a dual RNA-seq based approach on serum samples from patients infected with dengue fever. Dual RNA-seq is a technique that identifies both host and viral mRNA in a single experiment, allowing the acquisition of early transcriptional signatures that distinguish patients likely to develop severe symptoms from those with mild symptoms. In cell populations with high virus infection rates, an increase in core antiviral and vascular dysfunction genes was observed. These findings emphasize the potential of antiviral genes to induce unique pattern associated with mild disease progression, while increased expression of other gene sets may serve as indicators of severe disease progression. These studies highlight the importance of monitoring early clinical and transcriptomic profiles to facilitating to the treatment of dengue patients.

In 2020, Sarkar et al. [31] linked a recent dengue outbreak in Bangladesh to DENV-3 genotype I, which resembles strains found in China and Thailand, suggesting that this genotype may have been introduced to Bangladesh from these countries. Transcriptomic analysis of dengue patients during this outbreak revealed a significant upregulation of genes associated with the immune system and immunomodulatory proteins, indicating a robust immune response. Additionally, there was a notable increase in the activity of Toll-like receptor (TLR) signaling pathways, which are implicated in the efficient clearance of DENV-infected cells and the activation of both innate and adaptive immune responses. These results provide molecular insights into the physiological outcomes, demonstrating a marked rise in cytokine activity among the dengue patients.

ZIKV exhibits tropism for specific cells and tissues, particularly the brain, kidneys, and placenta, leading to various complications and symptoms upon infection [32-35]. Understanding the molecular characteristics of ZIKV infection in these tissues is crucial for elucidating the underlying pathophysiological mechanisms of its associated organ-specific complications. Chen et al. [36] conducted distinct transcriptome analyses on ZIKV-infected cell lines originating from three different tissues: JEG-3 cells from placental tissue, U-251 MG cells from brain tissue, and HK-2 cells from renal tissue, separately generating RNA-seq data for each. The results demonstrated that following ZIKV infection, interferon production was consistently stimulated across all three cell types, while processes related to metabolism were suppressed. Additionally, distinct immune responses were observed in each type of infected cell. In particular, JEG-3 cells (placental) activated the gonadotropin-releasing hormone (GnRH) signaling pathway, U-251 MG cells (glioblastoma) engaged the mitogen-activated protein kinase (MAPK) signaling pathway, and HK-2 cells (renal) initiated the peroxisome proliferator-activated receptor (PPAR) signaling pathway. Notably, the placenta-derived cell lines exhibited a delayed antiviral interferon response, shedding light on the molecular mechanisms that facilitate ZIKV's rapid replication and vertical transmission within the placenta.

ZIKV infection is also known to cause inflammatory complications such as Guillain-Barré syndrome, encephalitis, and osteomyelitis [37]. Additionally, it is linked to several retinal abnormalities, including microcephaly, choroidal atrophy, changes in the retinal pigment epithelium, and optic nerve abnormalities, all of which can lead to vision impairment [38]. To further explore the interaction between ZIKV and inflammatory ocular conditions, Ryan et al. [39] conducted RNA-seq analysis on human iris pigment epithelial cells infected with ZIKV. This analysis revealed a robust transcriptional response, characterized by enhanced activation of pathogen recognition systems, including retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) and TLR signaling, as well as antiviral and inflammatory cytokine responses mediated by type I and type II interferons (IFN-I and IFN-II) signaling. Note that this transcriptome profiling represents a homogeneous cell type analysis, reflecting the average mechanism of various cellular responses across multiple stages of viral infection.

Transcriptomic studies on a single virus species present challenges in comparing changes in host cell transcriptomes induced by closely related viruses due to variations in experimental conditions [40]. To address this issue, Brand et al. [40] investigated the effects of three types of flaviviruses—KUNV, ZIKV, and YFV—on gene expression levels and splicing in U87 cells. Interestingly, 17 genes associated with nervous system development exhibited significant expression changes during ZIKV infection, with four of these genes significantly upregulated during YFV infection, while no changes were observed in KUNV-infected cells. These findings offer insights into the potential impact of ZIKV on brain development and its role in causing microcephaly.

Research on flavivirus using scRNA-seq

Viruses rely on host cells to acquire essential materials for survival, making intracellular parasitism indispensable. This dependency creates complex interactions between the virus and its host during infection. While conventional RNA-seq studies tend to obscure fundamental heterogeneity by averaging data across cell populations, scRNA-seq enables profiling across the transcriptome at the resolution of individual cells. This provides a powerful tool to explore the transcriptomic heterogeneity of cellular responses [15,41]. scRNA-seq has significantly advanced our understanding of the pathophysiology of flavivirus infections by revealing intricate heterogeneity in immune cell responses. This technique allows for the dissection of complex immune dynamics at a resolution unachievable by traditional bulk RNA-seq methods, making it essential for understanding the interactions between viruses and hosts [42]. Additionally, scRNA-seq elucidates the heterogeneity of host responses and intercellular communication networks [43,44]. These insights facilitate a deeper understanding of the host response, encompassing factors mediated by direct viral infection and the activation of neighboring cells. Table 2 summarizes research on flavivirus using scRNA-seq.

Table 2 . Flavivirus research using scRNA-seq.

Virus type	Sample type	Collection condition	Code	Repository	Reference
DENV-4	PBMC	2 patients at 3 time points	E-MTAB-9467	ArrayExpress	[41]
DENV-1	PBMC	3 patients at 8 time points post-experimental infection 2 patients at 3 time points post-natural infection	GSE154386	GEO	[42]
ZIKV	Cell line	HepG2 cells at 48 h post-infection	GSE147093 & GSE147094	GEO	[43]

DENV, dengue virus; GEO, gene expression omnibus; PBMC, peripheral blood mononuclear cell; scRNA-seq, single-cell RNA sequencing; ZIKV, Zika virus.

For instance, scRNA-seq analysis of peripheral blood mononuclear cell samples collected daily from dengue patients demonstrated how different subpopulations of immune cells respond variably to infection, revealing subsequent physiological consequences [45]. The results revealed significant increases in IFN-I during the early stages of DENV infection in both dengue fever and severe dengue patients. Specifically, there was upregulation of immunoglobulin genes in plasma cells and plasmablasts (PBs), along with enhanced expression of genes associated with tissue residence and skin trafficking in PBs. Additionally, there was increased transcriptional activity of other genes associated with lymphocytes in tissues. Particularly, genes predicted to be associated with severe dengue, such as GYG1, TOR3A, SPON2, GRAP2, and GBP2, exhibited high expression levels in antibody-secreting cells and effector T cells [46]. These findings underscore the potential of scRNA-seq in providing crucial insights into the molecular mechanisms driving physiological responses.

The immunological or molecular characteristics associated with the early stages of viral infection remain unresolved, largely due to the existence of an asymptomatic or incubation period before the onset of symptoms. Consequently, understanding the precise early immunological and molecular features of DENV infection may not only facilitate the development of advanced diagnostic tools but also provide insight into the pathophysiology of infection and disease. Therefore, the study conducted by Waickman et al. [47] utilized the DENV Human Infection Model (DHIM), which exposed individuals to DENV in a controlled environment, allowing the immunological response to infection to be closely monitored before the onset of clinical symptoms. Analysis using scRNA-seq revealed that both experimental and natural DENV-1 infections triggered similar patterns of inflammatory gene activation [47]. However, natural infection resulted in a more significant suppression of genes associated with protein translation and mitochondrial function, particularly within monocytes. The study suggests that although experimental and natural DENV-1 infections trigger similar immune responses, natural infection appears to have a stronger impact on core cellular processes, potentially leading to a more multifaceted antiviral state.

Elucidating the host factors involved in ZIKV replication is important for developing effective treatment strategies. Analysis via scRNA-seq showed that ZIKV infection induces activation of the aryl hydrocarbon receptor (AHR) [48]. Specifically, ZIKV infection promotes the production of kynurenine (Kyn), which activates AHR and limits the production of IFN-I, involved in antiviral immunity. Furthermore, ZIKV-induced AHR activation was found to suppress innate immunity mediated by the promyelocytic leukemia (PML) protein, which restricts ZIKV replication. Viral infection occurs when virus replication can take place even in the presence of an activated host immune system. Recent studies have revealed that ISG15 restricts the replication of DENV and ZIKV through the stabilization of its binding partner, USP18 [49]. The expression of ISG15 is crucial for controlling DENV replication driven by autocrine and paracrine IFN-I signaling. It has been demonstrated that reconstitution of USP18 in ISG15-deficient cells alone restores STAT2 stability and limits viral growth. This underscores the antiviral activity of IFNAR-mediated ISG15 activity. Indeed, these results demonstrate how viruses exploit opportunities to replicate and multiply by circumventing the host cells’ immune response.

Research on flavivirus using viscRNA-seq

Traditional RNA-seq methods face limitations when studying viruses with non-polyadenylated genomes, such as flaviviruses. To overcome these obstacles, the viscRNA-seq platform was developed. This innovative technology provides insights into the complex interactions between host cells and viruses at the single-cell level [44]. Analyzing the complete transcriptome, including both host and viral RNA, allows for a deeper understanding of how cells respond to viral infection. It may also help identify potential therapeutic targets and ultimately develop more effective antiviral strategies. Flavivirus studies performed using viscRNA-seq are represented in Table 3.

Table 3 . Flavivirus research using viscRNA-seq.

Virus type	Sample type	Collection condition	Code	Repository	Reference
DENV-2, ZIKV	Cell line	Huh7 cells at 4 h, 12 h, 24 h, 48 h post-infection	GSE110496	GEO	[45]
DENV	PBMC	6 patients and 4 healthy individuals	GSE116672	GEO	[39]
WNV	Cell line	L929 cells at 24 h post-infection	GSE125241	GEO	[23]

DENV, dengue virus; GEO, gene expression omnibus; PBMC, peripheral blood mononuclear cell; viscRNA-seq, virus-inclusive single-cell RNA sequencing; WNV, West Nile virus; ZIKV, Zika virus.

Analysis of cells infected with DENV and ZIKV using viscRNA-seq revealed the heterogeneity of cells infected by each virus [50]. Several genes, such as ID2 and HSPA5, have been found to play opposing roles in each type of virus infection. Functional loss and gain-of-function screens identified novel proviral (e.g., RPL31, TRAM1, TMED2) and antiviral (e.g., ID2, CTNNB1) factors that regulate DENV infection. Thus, viscRNA-seq has emerged as a powerful method for assessing genome-wide virus-host dynamics at the single-cell level.

The viscRNA-seq platform was employed to identify both proviral and antiviral factors associated with viral loads in cells from patients infected with DENV [51]. This study aimed specifically to detect cells harboring viral RNA in the blood of patients, examine their molecular characteristics to predict the possible progression to severe dengue. A significant upregulation of specific genes was observed in immune cells prior to the onset of severe dengue. These include MX2 in naive B cells, as well as CD32 and IFIT1 in CD14+ CD16+ monocytes. Notably, MX2 induction occurred independently of IRF3 and IRF7, which are typically involved in the interferon response during viral infections. This independence of traditional pathways suggests the presence of an alternative antiviral pathway and MX2 might play is critical for controlling the early stages of DENV infection. Additionally, IFIT1, an interferon-stimulated gene significantly upregulated in the same monocytes, plays a role in inhibiting viral replication and modulating the immune response. Furthermore, CD163, which is known to contribute to the pathology of severe dengue fever in macrophages and CD14+ CD16+ monocytes, was also upregulated. This upregulation is associated with enhanced inflammation and tissue damage, despite its usual role in resolving inflammation by clearing hemoglobin-haptoglobin complexes and promoting anti-inflammatory responses. This finding has clinical implications as CD163 was identified as a potential predictive marker for severe dengue, indicated by the significantly increased levels of serum soluble CD163 in patients with severe dengue [52]. Overall, the viscRNA-seq study sheds light on the molecular mechanisms of DENV infection, uncovering key factors that may serve as prognostic markers for assessing disease severity and potential therapeutic targets.

Neuroinvasive infection caused by WNV can lead to encephalitis and potentially cause persistent neurological disease or death. Recent study on WNV have utilized the modified SMART-seq (Single Molecule Amplification and Real-Time Transcript sequencing) protocol [27]. SMART-seq enables the measurement of mRNA of various lengths by amplifying and sequencing the entire mRNA molecule, thereby providing an accurate measure of mRNA expression even from small cell samples. O'Neal et al. [27] discovered a negative correlation between the number of IFN-stimulated genes and the amount of viral RNA, with a dramatic reduction in expression observed in cells containing high levels of viral RNA. These findings suggest that WNV may suppress cellular antiviral defenses by directly or indirectly interfering with IFN-I signaling pathway, thereby significantly enhancing our understanding of the interactions between viruses and hosts, as well as the mechanisms of viral infection. According to a recent case report, elevated levels of IFN-γ were observed in patients with WNV infection, and the persistence of these elevated levels was associated with prolonged disease duration due to lingering symptoms [53]. This underscores the potential of IFN-γ as a prognostic biomarker for disease progression.

Recent advances in RNA-seq technology have significantly enhanced our understanding of flaviviruses. Studies using RNA-seq have elucidated aspects of viral pathogenesis, the interplay between viral and host immune systems, inflammatory pathways triggered by viral exposure, viral restriction mechanisms, and key pathways involved in disease progression. The continued use of RNA-seq, combined with other methodologies, will further deepen our knowledge of flaviviruses and lead to the development of novel diagnostics and therapeutic approaches.

Single-cell analysis has proven to be a powerful tool for exploring cellular diversity and heterogeneity in host responses to viral infections, enabling detailed transcriptome analyses. These technologies have provided crucial insights into viral genotypes, diversity within hosts, disease severity and progression, cellular heterogeneity in responses, and the impact of viruses on the host immune system. The findings discussed in this review clearly demonstrate how viruses exploit opportunities to circumvent host immune responses to replicate and proliferate, offering important insights into viral interactions and suggesting that scRNA-seq can make significant contributions to understanding virus-host interactions and developing disease management and therapeutic strategies. Moreover, viscRNA-seq and SMART-seq technologies have been instrumental in understanding the interactions between viruses and hosts at the single-cell level. These innovative methods have revealed the complex dynamics between viruses such as DENV, ZIKV, and WNV and host cells, uncovering crucial proviral and antiviral factors involved in infection progression. By identifying diverse responses within infected cells, viscRNA-seq contributes to the discovery of potential therapeutic targets during different viral infections.

Analysis of RNA-seq data has helped bridge the gap between molecular insights and physiological outcomes caused by flavivirus infections. Particularly, DENV can lead to vascular leakage and hemorrhagic manifestations. Transcriptomic studies have shown upregulation of genes involved in vascular permeability and endothelial cell function, such as CD163 and IFIT1, which are linked to severe dengue pathology [51]. ZIKV is known for its neurotropism, causing severe neurological complications such as microcephaly and Guillain-Barré syndrome [38]. Transcriptomic analyses of ZIKV-infected neural cells have revealed the activation of immune response pathways and alterations in genes associated with neural development and function. For instance, studies on human cortical neural progenitors have shown that ZIKV infection leads to the upregulation of TLR3 and other inflammatory pathways, contributing to neuronal damage and developmental defects [35,39]. YFV and DENV can cause significant hepatic dysfunction. RNA-seq data have demonstrated changes in the expression of genes involved in liver metabolism and immune responses. The downregulation of metabolic processes and upregulation of inflammatory cytokines highlight the dual impact of flaviviruses on hepatic function and immune activation [31,39].

While RNA-seq technologies have significantly advanced our understanding of the physiological impacts of flavivirus infections, numerous avenues for future research remain open. An important area is the investigation of long-term physiological effects and the potential for persistent health issues following infection. Studying the chronic impacts on systems such as the cardiovascular and neurological could unveil mechanisms of post-recovery complications and guide the development of supportive care practices. Additionally, identifying specific physiological biomarkers for early detection could transform patient management by enabling early interventions, especially in severe cases. Further research should explore the role of specific physiological pathways affected by flaviviruses, such as inflammation, cell death, and immune responses. These studies could provide new insights into how these viruses manipulate host cellular machinery. Finally, integrating physiological insights gained from RNA-seq with other omics technologies, like proteomics and metabolomics, is essential. This multi-omics approach could offer a more comprehensive understanding of the complex interplay between the virus, host, and environment, potentially leading to the development of more effective therapeutic and diagnostic strategies.

RNA-seq technologies were employed as initial screening tools to provide an unbiased overview of the biological mechanisms impacted by flavivirus infections. While these methodologies are invaluable for identifying broad patterns and novel pathways at a macroscopic level, it is crucial to recognize their preliminary nature in the research continuum. Consequently, to validate the biological relevance and potential therapeutic targets identified through RNA-seq, it is imperative to incorporate preclinical studies into the latter stages of the research process. These subsequent studies would involve more focused experiments on animal models or relevant biological systems to confirm the functionality and physiological impact of the mechanisms uncovered. This step not only enhances the reliability of the findings by providing empirical evidence but also bridges the gap between theoretical discovery and practical application. By integrating preclinical validation, we can ensure that the potential interventions developed based on our transcriptomic insights are both effective and safe, thus advancing closer to clinical applications.

None.

This work was supported by the Research Fund of the Chungnam National University.

The authors declare no conflicts of interest.