The importance of paying attention to the role of lipid-lowering drugs in controlling dengue virus infection

Abstract

The Flaviviridae family includes the dengue virus (DENV). About half of the world’s population is in danger because of the estimated 390 million infections and 96 million symptomatic cases that occur each year. An effective treatment for dengue fever (DF) does not yet exist. Therefore, a better knowledge of how viral proteins and virus-targeted medicines may exert distinct functions depending on the exact cellular region addressed may aid in creating much-needed antiviral medications. Lipids facilitate the coordination of many viral replication phases, from entrance to dissemination. In addition, flaviviruses masterfully plan a significant rearrangement of the host cell’s lipid metabolism to foster the growth of new viruses. Recent research has consistently shown the significance of certain lipid classes in flavivirus infections. For instance, in DENV-infected cells, overall cellular cholesterol (CHO) levels are only a little altered, and DENV replication is significantly reduced when CHO metabolism is inhibited. Moreover, statins significantly decrease DENV serotype 2 (DENV-2) titers, indicating that CHO is a prerequisite for the dengue viral cycle. Furthermore, many Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors are now being evaluated in human research. A new pharmacological target for the management of high CHO is PCSK9. Moreover, suppression of PCSK9 has been proposed as a possible defense against DENV. Numerous studies have generally recommended the use of lipid-lowering medications to suppress the DENV. As a result, we have investigated the DENV and popular treatment techniques in this research. We have also examined how lipid metabolism, cellular lipids, and lipid receptors affect DENV replication regulation. Lastly, we have looked at how different lipid-lowering medications affect the DENV. This article also discusses the treatment method’s future based on its benefits and drawbacks.

Graphical abstract

Introduction

The dengue virus (DENV) is the cause of dengue, a viral illness that is spread to people via mosquito bite. DENV has become a primary worldwide public health concern in recent years, despite being a tropical sickness often disregarded. Dengue fever (DF) has become an epidemic in the last few decades, with the number of recorded cases skyrocketing from 505,430 in 2000 to an estimated 5.2 million in 2019. In 2023, more than 80 nations across all World Health Organization (WHO) regions were hit by the DF pandemic. With almost 6.5 million cases and over 7300 dengue-related fatalities documented since the beginning of 2023, the combination of continued transmission and an unexpected surge in dengue infections has reached a historic level [1]. This prevalent acute systemic viral illness spread by mosquitoes is attributed to DENV, a positive-sense, single-stranded RNA virus belonging to the Orthoflavivirus genus (Family Flaviviridae). Every year, dengue causes around 400 million illnesses worldwide, 500,000 hospital admissions, and 24,000 fatalities (mainly in children) [2].

In addition to DENV, a large number of other flaviviruses can cause severe illnesses in humans, including tick-borne encephalitis virus (TBEV), yellow fever virus (YFV), West Nile virus (WNV), and Japanese encephalitis virus (JEV). There is presently no antiflavivirus treatment that works well. Due to the requirement to simultaneously immunize against all four DENV serotypes and induce long-lasting protection against them, the development of DENV vaccines has proven difficult; an individual with incomplete immunization may become sensitized to dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), a potentially fatal illness [3, 4]. Primary DENV infection results in the development of long-term host immunity that is uniquely targeted against the particular serotype while providing short-term protection against other serotypes. Follow-up infection with a distinct serotype may result in antibody-dependent amplification, raising the likelihood of severe dengue syndrome [5].

Although the majority of DENV afflicted have either minor symptoms or no symptoms at all, around 5% experience severe illness and need hospitalization [6]. Improved monitoring systems and focused research are crucial elements in preventing and managing dengue. The prevention and management of DENV transmission also still depend on suppressing the mosquito population via various vector control measures, including active and ongoing monitoring of cases and vectors, since the dengue vaccine is yet insufficiently effective [7]. Thus, there is an urgent need to discover safe and efficient treatments for illnesses brought on by DENV.

The cellular lipid membrane serves as the first line of defense against viral infection of the host cell and is also a critical point of the first viral interaction. Viruses may sometimes use lipids as viral receptors. During much of their life cycle, viruses rely heavily on lipid rafts for efficient infection. It has been shown that various viruses utilize diverse strategies to modify the lipid raft to attach, internalize, fuse membranes, replicate their genomes, assemble, and release their genetic material [8]. In addition, cholesterol (CHO) serves as a precursor for important signaling molecules and is a necessary component of mammalian cell membranes [9]. It has been shown that CHO and/or viral entry and/or morphogenesis are related to various viruses. Therefore, the lipid metabolic pathways and the combination of membranes may be precisely targeted to hinder the viral replication phases as a basis for antiviral treatment [10]. Furthermore, targeted CHO reduction reduces viral success because CHO is critical for DENV entrance. Additionally, flaviviruses have shown that they may affect cellular CHO concentrations in order to enhance viral reproduction [11, 12]. Additionally, DENV replication is impacted by fatty acid (FA) metabolism in DENV-infected tissues and the modulation of FA synthase (FASN) to influence FA synthesis [13, 14].

Lipid-lowering medications are an option among Food and Drug Administration (FDA)-approved treatments for flavivirus infections. For example, lovastatin (LOV) has shown antiviral activity both in vivo and in vitro; hence, its inexpensive and safe nature has been suggested as a potential treatment option. Nevertheless, no evidence of a beneficial effect on any clinical symptoms of DENV or viremia has been found in adult patients treated with LOV [15]. Lipid-lowering drugs are essential for treating viral illnesses like DENV and preventing consequences. Consequently, it is crucial to comprehend the critical pathways associated with cellular lipids to modulate the virus replication cycle to develop DENV treatment methods. The present study has concentrated on the examination of the impact of lipids and lipid-lowering medications on DENV.

Characterization of dengue virus

Human characteristics affecting dengue transmission include age, population density, socioeconomic status, behavior, and mobility. The presence of both natural and artificial mosquito breeding grounds, which are often linked to human activity, is essential for the transmission and spread of dengue. Even in non-endemic locations where no prior dengue cases have been reported, the growth of metropolitan areas, deforestation, and human mobility via travel, urbanization, and trade may contribute to increased dengue transmission. The temperature and the quality of the water may have an impact on the mosquito’s life cycle [16].

Once within the host cell, the virus’s protein synthesis machinery starts translating its approximately 11 kb genome, producing structural and non-structural (NS) viral proteins. The DENV genome produces seven NS proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) that aid in replication in addition to the three structural proteins (Capsid, Membrane, and Envelope). Other structural proteins make up the virion envelope; a complex is formed by the interaction of the capsid protein and the RNA genome [17]. The outcome of DENV infections may be significantly influenced by the virus’s cell and tissue tropism. The lack of a suitable animal illness model significantly limits our study’s comprehension of the function of DENV tropism. The pathogenic consequences of DENV infection in these systems, the tropism of DENV for cells of the relevant systems, and the significance of these events for the general pathogenesis of DENV infection will all be discussed [18]. The DENV’s genetic makeup and structure are illustrated in Fig. 1. Multiple factors, including the virus itself, the host’s genes, and the immune system, interact in a complicated way to cause DF. Significant variables that determine disease susceptibility include host factors such as ADE, autoimmunity, memory cross-reactive T cells, anti-DENV NS1 antibodies, and genetics. The pathophysiology of severe dengue was thought to be caused by the NS1 protein and anti-DENV NS1 antibodies. Multiple infections with distinct DENV serotypes may modify the cytokine response of cross-reactive CD4 + T cells, which in turn may increase levels of pro-inflammatory cytokines that contribute to an immunological response that is harmful. Increased vascular permeability and malfunctioning of vascular endothelial cells occur as a consequence of immune cell cytokine release in Fcγ receptor (FcγR)-mediated antibody-dependent enhancement (ADE). The severity of DF is determined by viral factors, such as the virus’s genetic diversity and subgenomic flavivirus RNA (sfRNA), which inhibits the host’s immune response. Autoantibodies against DENV NS1 antigen, DENV prM, and E proteins may be produced during dengue infection. These autoantibodies can then cross-react with other self-antigens, including plasminogen, integrin, and platelet cells. In addition to viral factors, the pathophysiology of DENV infection is influenced by many host genetic variables and gene polymorphisms [19].

Fig. 1

Examination of the DENV’s genetic makeup and structure. The 3′untranslated region (UTR), open reading frame (ORF), and 5′UTR make up DENV’s genome. After the ORF is translated into a polyprotein, it is sequence-processed to yield three structural proteins (C, E, and M) and seven NS proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5)

By attaching E to cellular receptors, a process known as receptor-mediated endocytosis, DENV penetrates host cells. A DENV enters human and mosquito cells by clathrin-mediated endocytosis. As DENV particles diffuse over the cell surface, they go toward an already-existing pit coated with clathrin. With the aid of dynamin, the clathrin-coated pit changes into a clathrin-coated vesicle. The virus-containing vesicle is then moved to early endosomes, which mature into late endosomes. When the endosome internalizes and becomes acidic due to the viral and vesicular membranes fusing owing to conformational changes in the E protein, the nucleocapsid is liberated [20]. After nucleocapsid production, it buds into the ER lumen and picks up the lipid bilayer and viral E and prM proteins. The newly formed particles follow the secretory pathway. When prM in the trans-Golgi network is proteolyzed by furin, causing homodimerization and rearrangement of E, mature viral particles are created. Viruses secreted are composed of a mixture of immature, partially mature, and ultimately mature particles [20, 21]. Life cycle of DENV are illustrated in Fig. 2.

Fig. 2

Life cycle of DENV. The virus may enter a cell and initiate the DENV replication cycle by binding to Fc receptors on the target host cell or other host cell receptors by the Fc portion of the virus-antibody immunological complex. DENV enters the cell by attaching itself to host cell receptors. During internalization, receptor-mediated endocytosis forms an early endosome. The uncoating of the DNA occurs when the pH inside the early endosome lowers. As a result of conformational changes, the nucleocapsid is released into the cytoplasm. The dissolution of the nucleocapsid enables viral RNA assembly in the cytoplasm. Viral RNA is translated and then translocates into the Endoplasmic Reticulum (ER), where it is broken down into a single polyprotein by both host and viral proteases. Additionally, as a result of a translation switch, antisense viral RNA is used to transcribe viral RNA. The nucleocapsid is formed when the capsid protein encases the freshly produced viral RNA. Viruses gather on the surface of the ER. When immature viral particles are exposed to furin protease in the trans-Golgi network, acidity induces conformational changes that result in the production of mature viral particles. When mature virus particles are exocytosed into the extracellular matrix, their replication cycle is complete [17]

Dengue virus treatment

Important cytokines that play a crucial role in the pathogenesis of dengue include interferon-gamma (IFN-γ), tumor necrosis factor (TNF), and interleukin (IL)-10. Travelers are more vulnerable to developing DF, and the severity of the illness is often linked to the CD8 + T cell response. Death rates may rise to 50% in situations as severe as DHF/DSS if suitable care is not received. DF can lead to several complications, including neuromuscular problems, neurological manifestations like encephalopathy, encephalitis, cerebral venous thrombosis, myelitis and posterior reversible encephalopathy syndrome, and immune-mediated neurological syndromes like acute disseminated encephalomyelitis, mononeuropathy, acute transverse myelitis, Guillain–Barre syndrome, and myelitis [22, 23]. Therapeutic strategies usually include pursuing therapeutic goals, monitoring disease activity using composite indicators, and delivering intravenous fluids based on symptomatology. Since there is currently no specific antiviral therapy for dengue, early on, supportive care—especially hydration—is crucial [22].

Some seaweed-derived polysaccharide compounds have shown antiviral efficacy against all infectious serotypes of DENV, including dL-galactan hybrid C2S-3, carrageenan G3d, Caulerpa cupressoides, and curdlan. Researchers work by blocking the interaction of the virus with the host at receptor sites. Likewise, combinatorial pharmacological regimens have shown that ribavirin, a vaccine candidate, reduces DENV activity in host cells. Ribavirin is used to stop the creation of RNA and viral mRNA capping. As a result, ribavirin functions as a prodrug that, upon metabolism, mimics purine RNA nucleotides. It also functions as a nucleoside analog. In this state, it obstructs the metabolism of RNA necessary for viral replication. Ribavirin’s efficacy as a prodrug against dengue has been studied using many approaches. Researchers were shown anti-DENV properties in experiments, including glycyrrhizin derivatives, nucleoside adenosine, NITD008, and uridine analog 6-aziridine. These compounds function by altering and preventing DNA and corresponding protein production [24].

Researchers demonstrated JNJ-1802, a potent DENV inhibitor that blocks the NS3–NS4B interaction within the viral replication complex. JNJ-1802 has picomolar to low nanomolar in vitro antiviral activity, a high barrier to resistance, and good in vivo efficacy in mice against infection with any of the four DENV serotypes. Finally, research indicates that the small-molecule inhibitor JNJ-1802 is very effective in shielding non-human primates from DENV-1 or DENV-2 viral infection. The successful completion of a phase I first-in-human clinical study with healthy volunteers has shown the safety and well-tolerated character of JNJ-1802. These findings support the ongoing clinical development of JNJ-1802, a first-class antiviral drug against dengue that is now advancing in clinical studies for the treatment and prevention of dengue [25].

Surender Rawat et al. [26] examined how DENV-2 affected naïve neutrophil effector capabilities and how its secretome affected various immune cells. These phenotypically changed neutrophils exhibit diminished phagocytic capability and delayed apoptosis via the NF-κB and phosphatidylinositol 3-kinase (PI3K) pathways. The release of double-stranded DNA produced by DENV-2 was dramatically decreased when myeloperoxidase and PAD4 inhibitors were administered to neutrophils before DENV-2 incubation. This suggests that myeloperoxidase and PAD4 were involved in the activation of neutrophils and the release of double-stranded DNA at an early stage. The researchers further note that via binding TNF-α to TNF receptor 1/2 receptors, DENV-2-stimulated neutrophil secretome had a substantial impact on platelet activation, viral infection, and naïve neutrophil survival. Moreover, endothelial cells may experience inhibitions in their ability to increase and mend wounds and endothelial cell death while being incubated with the DENV-2-stimulated neutrophil secretome. These effects may lead to the malfunctioning of the endothelium barrier. In summary, the interaction between neutrophils and DENV-2 regulates the phenotype of neutrophils and the release of prosurvival and antiviral secretomes, which might have dual effects during dengue pathogenesis [26].

It is critically necessary to find an anti-DENV drug that inhibits both DENV replication and cytokine release. The anti-inflammatory, anti-human immunodeficiency viruses (HIV), and anti-tumor chemical cepharanthine (CEP), which was isolated from Stephania cepharantha Hayata, was examined in this research to see whether it may prevent DENV infection. All DENV-1, 2, 3, and 4 serotypes showed a substantial reduction in viral output and viral E protein after CEP therapy. Furthermore, DENV-infected A549 cells’ production of the proinflammatory cytokine IL-6 was decreased by CEP therapy. When combined, CEP selectively inhibits DENV infection during the early stages of viral replication and promotes the release of proinflammatory cytokines. As such, it shows promise for future development as an anti-DENV therapy [27]. Table 1 summarizes the types of treatment that can be used for DENV.

Table 1 Dengue virus treatment methods

Lipid metabolism in dengue virus

Usually able to permeate the mitochondrial membrane, citrate serves as the primary carbon source for producing FAs or CHO. It is cleaved into acetyl-CoA, which is then carboxylated by acetyl-CoA carboxylase (ACC) to produce malonyl-CoA. However, from cytosolic acetyl-CoA and malonyl-CoA, FASN catalyzes the synthesis of palmitic acid (C16:0). After that, the palmitic acid may be treated further and employed to make cell membranes, stored in liquid drops, or palmitoylate viral and host proteins. In terms of sterol biosynthesis, acetyl-CoA is converted into acetoacetyl-CoA by two units of acetyl-CoA processing, and this then enters the metabolic pathway of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase to produce CHO [29]. Furthermore, catabolic beta-oxidation may digest FAs and produce large quantities of ATP. Since they make up the majority of viral membranes, CHO and FA are necessary for viral replication. Therefore, it has been shown that the metabolism of these lipids is essential for the replication of different viruses. The FASN enzyme, which regulates FA production and may limit viral replication, is a crucial component of this process. Certain viruses may even use this process by making FASN more expressed and active [30].

Lipid metabolism is necessary for flaviviruses to finish their replication cycle in the following ways: (A) Firstly, during the process of viral entry, the lipid bilayers of the flavivirus envelope that are extracted from the ER membrane take part in the attachment, binding, and fusing of the virus. (b) Secondly, an increase in FA and CHO synthesis facilitates the development of replicative complexes (RCs) that invade the ER membrane, where the virus replicates and translates; (c) the flavivirus assembly. The virions’ construction is completed when the nucleocapsid breaks through the ER membrane. Through the exocytic pathway, the virions are sent to the Golgi complex, where they mature and are freed from the infected cell [31].

Several cell types need CHO and FAs for the DENV replicative cycle. For example, it has been shown that lipid rafts, which are membrane microdomains rich in CHO and sphingolipids, are essential for DENV entry into macrophages regardless of the presence of aiding antibodies. Several cellular components known as DENV receptors are either transported to or reside in lipid rafts after DENV binding. Furthermore, undamaged lipid rafts and CHO are necessary for the cellular signaling initiated in human macrophages during the early stages of DENV infection. This signaling involves the activation of MAPKs like JNK. Early on, following Huh-7 cell infection, researchers demonstrated an increase in total CHO and the formation of lipid rafts. This increase is associated with a decrease in the phosphorylation level of HMGCR and an increase in the quantity of low-density lipoprotein receptors (LDLR) expressed on the surface of infected cells. Huh-7 cells infected with Vesicular Stomatitis Virus (VSV) exhibited no alterations as a control. Early after infection, researchers suggested that DENV infection elevates intracellular CHO levels by increasing HMG-CoA reductase enzymatic activity and modulating LDL particle absorption [32].

Researchers showed that genes related to lipogenesis, lipolysis, and FA β-oxidation showed altered transcription and expression after DENV infection. Using either siRNA or orlistat to interfere with FASN had a considerable impact on the production of viruses; the latter had a half maximal effective concentration (EC₅₀) value of 10.07 μM 24 h after infection. On the other hand, NS protein expression remained unaltered primarily. The use of drugs reduced the viral titer by up to 3Log10 but had no discernible effect on the expression of the NS protein DENV, suggesting that FASN works by regulating virion formation [33].

Beyond their shared lipid composition, the dengue NS1 protein and HDL lipoprotein have many other commonalities. Based on plasmon resonance assay results (NS1 = 47.02 nM and HDL = 18.71 nM), Alcala et al. discovered that soluble DENV NS1 protein can directly bind to the primary HDL receptor, the Scavenger Receptor class B, member 1 (SRB1). When antibodies or HDL competition are used to block the SRB1 in human-derived liver cells, the quantity of NS1 seen within the cells is significantly reduced [34]. The researchers detailed the connection between the key HDL protein, apolipoprotein A1 (ApoA1), and the DENV NS1. Noninfected cell membranes have an increase in lipid rafts, which is enhanced after subsequent DENV infections due to the DENV NS1 protein. DENV is unable to bind to cell surfaces when ApoA1-mediated lipid raft depletion is applied. Furthermore, ApoA1 may inhibit NS1-mediated increase of DENV infection and counteract NS1-induced cell activation. In addition, the DENV NS1 protein directly binds to the Apo-A1 protein moiety of HDL via nonpolar interactions. This interaction appears to change the vulnerability of membranes to viral infection and changes the ways in which DENV triggers immune response evasion mechanisms [35]. There are new ways that NS1 could contribute to dengue pathogenesis, such as increased virus replication, cytokine storm, and thrombocytopenia. Its similarities to HDL in terms of lipid composition and receptor usage, as well as its ability to bind to HDL particles or its protein moiety Apo-A1, are intriguing. In the long run, they may also help shed light on why dengue patients have abnormalities in lipid homeostasis. [36, 37].

A possible mechanism of Wolbachia-mediated viral blocking in insects is the competition between viruses and Wolbachia for host lipids. Researchers discovered metabolic markers of intracellular activities brought on by infection, but they did not find much evidence to suggest that Wolbachia and the virus directly competed for host lipids. Lipid profiles of mosquitoes with dual infection match those of mosquitoes with DENV3 mono-infection, indicating that virus-driven modulation predominates over Wolbachia-driven modulation. Interestingly, cardiolipins may host DENV3 and Wolbachia replication if necessary metabolic enzymes are knocked down. These results characterize the metabolic relationship between Wolbachia and DENV3 as indirectly antagonistic, as opposed to directly competing, and open up new study directions regarding molecular interactions between mosquitoes and viruses [38].

The genus Wolbachia, which contains the wMel and wAlbB strains of insect endosymbiotic bacteria, is used as a biocontrol agent to lower the prevalence of viral infections like dengue that are carried by Aedes aegypti (Ae. Aegypti). Robson K. Loterio et al. developed a panel of Ae. aegypti-derived cell lines that were infected with either the antiviral strains wMel and wAlbB or the non-antiviral Wolbachia strain wPip to understand better the morphological changes in host cells that are specifically brought about by antiviral strains. Antiviral strains were often found to be fully encased in the host ER membrane, in contrast to wPip bacteria, which clustered autonomously in the cytoplasm of the host cell. ER-derived Lipid droplets (LDs) increased when wMel- and wAlbB-infected cell lines and mosquito tissues were compared to those infected with wPip or Wolbachia-free controls. Inhibiting FASN, which is required for triacylglycerol synthesis, significantly restored ER-associated DENV replication and slowed the progression of LD in cells colonized by wMel. All of this suggests that Wolbachia strains with antiviral properties might specifically alter the lipid content of the ER to inhibit the formation of DENV replication complexes. Determining the antiviral mechanisms of Wolbachia will contribute to the continued usage and efficacy of this widely deployed biocontrol agent [39].

Vacuole Membrane Protein 1 (VMP1) and Transmembrane Protein 41B (TMEM41B) are two ER-associated lipid scramblases involved in cellular lipid metabolism and autophagosome production. Another newly verified host component needed by coronaviruses and flaviviruses is TMEM41B. Researchers have confirmed that VMP1 and TMEM41B are necessary to host dependence factors for human coronavirus OC43 (HCoV-OC43) and all four DENV serotypes, but not for CHIKV. Yousefi et al. [40] found lower levels of DENV infections in TMEM41B- and VMP1-deficient cells, although HCoV-OC43 could not reproduce completely in these cell lines. An elevation of the innate immune dsRNA sensors, RIG-I, and MDA5 followed these infections. However, neither the downstream effector TBK1 activation nor the production of IFN-β was induced by this increase. Despite modest levels of DENV replication, the infected TMEM41B-deficient cells did not display typical DENV replication organelles. This suggests that rather than being a cause of reduced DENV infection, the overexpression of dsRNA sensors is most likely the outcome of abnormal viral replication. Surprisingly, researchers found that although TMEM41B deficiency still inhibits DENV replication, exogenous FA supplementation may partially restore this effect, not HCoV-OC43. In contrast, the VMP1 loss cannot be recovered by metabolite treatment. By examining the reported phenotypes, researchers found that VMP1- and TMEM41B-deficient cells exhibited higher levels of damaged mitochondria. This was especially true for VMP1 deficiency, which results in severe dysregulations of mitochondrial beta-oxidation. Using a metabolomic profiling approach, researchers discovered that TMEM41B- and VMP1-deficient cells had distinct global dysregulations of the cellular metabolome, particularly the lipidome. To facilitate the reproduction of coronaviruses and flaviviruses, the study’s findings highlight the crucial roles that TMEM41B and VMP1 play in controlling several cellular processes, including lipid mobilization, mitochondrial beta-oxidation, and global metabolic regulation [40].

Role of the lipid drop in dengue virus

LDs, which were once thought to be inert organelles with specific, restricted activities, are now considered to be dynamic, multipurpose structures inside the cell. Their crucial function as significant energy reserves in the form of lipids that can be digested to fulfill cellular energy needs has been highlighted by recent studies. Their remarkable dynamism is emphasized by their capacity to interact with a wide range of cellular organelles, including the mitochondria, which produce energy from tiny LDs, and the ER, which is the location of LD creation. Beyond their role in the bioenergetics of cells, LDs have been linked to viral infections. There is evidence that viruses can co-opt LDs to promote their infection cycle. Recent findings further emphasize the function of LDs in regulating the host’s immunological response. Changes in LD levels during viral infections indicate their possible role in the pathophysiology of illness, maybe by using LD lipids as precursors to produce proinflammatory mediators [41].

The number of LDs per cell is increased by dengue infection, and DENV replication is significantly decreased by pharmacological suppression of LD production. Furthermore, researchers have shown that the surface of LDs accumulates the viral capsid protein in infected cells. Researchers developed a reporter DENV and used it to manipulate infected clones to determine the molecular mechanisms behind capsid protein attachment to LDs. The growth of DENV infectious particles and the accumulation of capsid protein on LDs have been shown to depend on specific amino acids on the α2 helix, which is vital to the capsid protein. According to research, LDs help viruses replicate by acting as a platform for producing nucleocapsids during encapsidation. Researchers showed how the DENV uses cellular organelles to coordinate several viral life cycle phases [42].

LDs are typical organelles that produce lipid mediators, which regulate inflammatory and immunological responses. Researchers have shown that when Enterobacter cloacae, Sindbis, and DENV attack Ae. aegypti Aag2 cells, an immune-responsive cell lineage, they acquire LDs. High transcript levels of genes linked to lipid storage and LD biogenesis were found in a microarray study of Aag2 challenged with E. cloacae or infected with the DENV, which was correlated with the higher numbers of LDs in those circumstances. Similar to this, after a blood meal, LDs build up in mosquito midgut cells in response to the Sindbis virus and Serratia marcescens or when the natural microbiota multiplies. Additionally, the number of LDs in the midgut is increased by constitutive activation of the Toll and IMD pathways by the knockdown of their respective negative modulators (Cactus and Caspar). Researchers suggested a function for LDs in mosquito immunity. They demonstrated for the first time an infection-induced buildup of LDs in response to both bacterial and viral infections in Ae. Aegypti. These results provide new avenues for investigating the immunological reactions of insects linked to lipid metabolism. Likewise, LDs accumulate in mosquito midgut cells after a blood meal or in response to Serratia marcescens and Sindbis virus. These processes are caused by the local microbiota multiplying. Furthermore, the number of LDs in the midgut increases under constitutive activation of the Toll and IMD pathways by downregulating the respective negative modulators, Cactus and Caspar. Ferreira Barletta et al.’s study revealed for the first time that Ae. Aegypti may develop LDs in response to bacterial and viral infections, and scientists hypothesize that LDs may play a role in mosquito immunity. These results provide new avenues for investigating immune responses related to insect lipid metabolism [43].

Moreover, platelets exposed to DENV in vitro aggregate with monocytes and initiate the production of LD and the release of CXCL8/IL-8, IL-10, CCL2, and PGE2. PGE2 secretion by platelet–monocyte complexes is inhibited by pharmacologic suppression of LD biogenesis, although CXCL8/IL-8 release remains unaffected. Researchers investigated the underlying processes and found that platelet-produced MIF is partly responsible for LD formation in monocytes exposed to DENV-activated platelets. Furthermore, LD formation is increased in monocytes with adherent platelets, indicating that platelet adhesion plays a significant role in platelet-mediated regulation of lipid metabolism in monocytes, independent of paracrine signaling [44].

When DENV infection occurs, triglycerides and LDs are processed by autophagy to liberate free FAs. Consequently, there is a rise in cellular β-oxidation, leading to the production of ATP. To replicate DENV efficiently, several procedures are necessary. Significantly, exogenous FAs can replace autophagy’s need for DENV replication. These results shed light on autophagy’s role in DENV infection and reveal a mechanism by which viruses may alter cellular lipid metabolism to promote replication [45]. The structure and function of LDs are illustrated in Fig. 3.

Fig. 3

Lipid droplets (LDs) function in viral infections. LDs and lipid metabolism play a crucial role in the positive strand ( +) RNA viral cycle, including that of flaviviruses. The process of triggering the NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome, which cleaves the Sterol regulatory element-binding proteins (SREBPs) and activates the lipogenesis genes and structural LDs proteins, including PLIN3/TIP 47, is what causes the induction of LD during viral infection. Following LD buildup, it may be a substrate for DENV assembly or viral replication. LDs are depleted by DENV-induced lipophagy, which supplies energy for viral replication [46, 47]

Role of the cholesterol in dengue virus

Recent research has shown that the production and transportation of CHO are essential in controlling how the host reacts to many viruses in the Flaviviridae family, including the hepatitis C virus (HCV). Research indicates that the CHO pathway is crucial for transmitting various flaviviruses, including WNV, JEV, and DENV [38, 48,49,50]. Though the precise mechanism is yet unclear, the metabolism of CHO is necessary for the entry and growth of the Flavivirus and the host’s immune system’s response to the virus [8, 10]. It was shown that siRNAs targeting Mevalonate (diphospho) decarboxylase (MVD) suppressed DENV multiplication in both naïve A549 cells and a stable A549 subgenomic Renilla replicon cell line (Rluc-replicon) after DENV-2 New Guinea C (NGC) live virus infection. Modulating either endogenous CHO synthesis or exogenous CHO absorption may influence DENV replication, according to the researchers. Researchers discovered that MVD knockdown might limit DENV replication after evaluating a library of siRNAs targeting critical nodes of the CHO biosynthesis pathway. Genetic data shows that MVD might be a potential target in the CHO production pathway for small molecule treatments. Knocking down MVD can block both a subgenomic replicon and dengue live virus infection [51].

Researchers indicated that employing the CHO transport (RCT) inhibitor U18666A to interfere with the infected cells’ CHO consumption impacts DENV infection. It was shown that the two mechanisms causing the antiviral effect were the delayed viral trafficking in the CHO-loaded late endosomes/lysosomes and the prevention of de novo sterol synthesis in treated infected cells. Furthermore, researchers discovered that the antiviral drug U18666A and the FA synthase FASN inhibitor C75 had an additive effect, suggesting that the DENV relies on the host’s CHO and FA synthesis for efficient reproduction [52].

In mosquito C6/36 cells, monkey Vero cells, and human endothelial-like ECV304 cells, the entrance of DENV-1 and DENV-2 was observed to occur independently of plasma membrane CHO reduction by CHO sequestration using methyl-beta cyclodextrin (MCD), nystatin, or filipin therapy [32, 53,54,55]. Likewise, DENV-1 and DENV-2 did not need CHO in the target cell membrane, as shown by the infection of C6/36 cells that had had their CHO levels reduced by repeated passages in media containing delipidated serum [56]. In contrast, other research using MCD or filipin to treat human monocytes or mouse neuroblastoma cells revealed that DENV-2 infection required cellular CHO [53, 57].

Currently, it is unclear whether variations might be caused by the kind of cell or the circumstances under which the membrane CHO concentration was changed during therapy. Regarding the importance of CHO as a component of the virion, preliminary research showed that pretreating viral suspensions with MCD before infection significantly reduced the infectivity of DENV-2 [58]. This led us to explore the effects of CHO content on the infectivity of the four DENV serotypes using MCD or nystatin, as well as the mechanism of virion infectivity inactivation after treatment with these CHO-binding medications [11]. Researchers showed that virion suspensions treated with MCD in the presence of bovine serum partly recovered infectivity while CHO-extracting medicines similarly inactivated all four DENV serotypes. The inactivating effect of MCD could not be reversed by adding serum or external water-soluble CHO after MCD therapy. The addition of additional CHO to the virion therapy also had a virucidal impact. The uptake and binding of CHO-deficient DENV into the host cell were unaffected. However, the retention of capsid protein in cells infected with MCD-inactivated-DENV virions indicated that the subsequent step of virion uncoating and release of nucleocapsids to the cytoplasm was prevented. After that, infection was almost entirely suppressed since cells infected with virions treated with MCD failed to produce viral RNA and viral proteins. According to these results, researchers demonstrated that DENV entrance fusion relies heavily on envelope CHO [11].

FcγR is known to attach to lipid rafts when IgG binds, and this relationship is necessary for effective signaling. Lipid rafts are tiny (10–200 nm), highly dynamic areas of the plasma membrane that are abundant in sphingolipids and CHO. They are distinguished by the fact that they may serve as docking sites for several intracellular signaling proteins and that they remain insoluble after being extracted using a cold detergent. Furthermore, lipid rafts are a significant route of viral invasion in cells, and some proteins associated with rafts also serve as viral receptors [59, 60]. In the absence of antibodies, CHO is necessary for DENV to infect human macrophages because medications like methyl-β-cyclodextrin (MβCD), which remove CHO from plasma membranes, prevent this process from happening. Additionally, DF and the more severe strains of the virus known as DHF/DSS are caused by DENV. DHF/DSS development is thought to be at risk due to secondary infections that have a different serotype than the original infection. The ADE hypothesis is one reason for the higher risk of developing DHF/DSS after heterologous secondary infections. According to this theory, the newly discovered serotype-infecting virus would combine with pre-existing non-neutralizing antibodies to create immunological complexes, which will increase the virus’s ability to infect macrophages and other cells that express the FcγR. When DENVs infect macrophages by ADE infection, lipid rafts and CHO are present on the plasma membrane. Therefore, the low-affinity connections between DENV immune complexes and FcγRII may never become sufficiently stable without lipid rafts, resulting in complex dissociation. Furthermore, complicated internalization-related signaling cascades won’t activate if receptor clustering inside the lipid raft is inhibited. These two pathways might account for the observed decrease in ADE in U937 cells after lipid raft rupture since they are not mutually exclusive [61].

In a separate investigation, researchers investigated the correlation between the development of severe dengue and total CHO, High-Density Lipoprotein Cholesterol (HDL-C), and Low-Density Lipoprotein Cholesterol (LDL-C) by analyzing data from a prospective hospital-based study of pediatric dengue in Managua, Nicaragua. Regardless of the categorization method, CHO varied throughout the disease outcome and declined during the illness. Patients with dengue were shown to have lower levels of LDL-C than HDL-C when compared to patients with other febrile diseases and between patients with severe and moderate dengue episodes. Lower LDL-C and total blood CHO levels at presentation were linked in multivariate models to a later risk of developing DHF/DSS (as classified by the WHO in 1997). Researchers suggested that LDL-C lowering should be considered a possible predictive biomarker panel for severe dengue since it is most likely the cause of the declines in total blood CHO levels among dengue-positive patients [62].

For the cells of Ae. aegypti to maintain CHO homeostasis, sterol carrier protein 2 (SCP-2) is necessary. SCP-2 is an essential host factor for DENV production in mosquito Aag2 cells. In mosquito cells, but not in human cells, treatment with N-(4-((4-(3,4-dichlorophenyl)-1,3-thiazol-2-yl)amino)phenyl)acetamide hydrobromide, a known inhibitor of SCP-2, or SCP-2 knockdown dramatically decreased the production of the virus. The common name for this minuscule molecule is SCPI-1. The intracellular distribution of CHO in mosquito cells was altered by treatment with SCP-2 inhibitors, suggesting a possible involvement of the SCP-2-mediated CHO trafficking pathway in DENV viral production. Comparing the effects of SCP-2 on human and mosquito cells, it is discovered that treating both cell lines with SCPI-1 reduces CHO. Nevertheless, this reduction in CHO only results in a drop in the viral titer in the mosquito’s host cells, presumably due to a more severe effect on perinuclear CHO storages in mosquito cells that weren’t there in human cells. Since SCP-2 did not stop the development of another encapsulated RNA virus in mosquito cells, it does not have a broad anti-cellular or antiviral impact. According to the results of the researchers’ cell culture, SCP-2 could represent a constraint on the ability of mosquitoes to transmit dengue [63].

Role of the lipid receptors in dengue virus

The purpose of the researchers was to ascertain if the human hepatoma cell line (Huh7) internalizes DENV-2 when apolipoproteins A1 (apoA1), B (apoB), or E (apoE) are present. Researchers demonstrated that the first attachment of DENV-2 to the cell surface was improved by apoA1 and apoB, indicating their function in DENV internalization and infectivity [64]. DENV adhesion to the cell surface is inhibited by ApoA1-mediated lipid raft depletion. Moreover, ApoA1 may stop NS1-mediated increase of DENV infection and counteract NS1-induced cell activation. Furthermore, lipid raft depletion may be mediated by ApoA1 mimic peptide 4F to inhibit DENV infection. When combined, the study’s findings point to the possibility of using reverse RCT-based treatments to treat dengue. These findings need to spur research on the significance of RCT in DENV infection in vivo [35].

An alternative investigation used DENV to examine the unfolded protein response (UPR) and the sterol-regulatory-element-binding protein-2 (SREBP-2) pathway regarding the early expansion and restructuring of the ER after infection. Using mouse embryonic fibroblast cells deficient in XBP1 and ATF6, researchers reveal that ER rearrangement occurs early after DENV infection and that this process is independent of the UPR. Subsequently, it is shown by researchers that SREBP-2 upregulates and activates 3-hydroxy-3-methylglutaryl-Coenzyme-A reductase, the rate-limiting enzyme in the CHO synthesis pathway, regardless of the ER enlargement. Moreover, this ER rearrangement is not stopped by giving the CHO-inhibiting drug LOV to DENV-infected cells. Using the translation elongation inhibitor cycloheximide and the transcription inhibitor actinomycin D, researchers show that host transcription is not necessary for ER expansion and rearrangement but rather for de novo viral protein synthesis. Finally, the researchers show that LDs are reabsorbed into the ER due to viral infection. Researchers’ findings suggest that viral protein expression drives the modification of the cell’s intracellular membrane architecture early after DENV-2 infection and does not need the activation of the SREBP-2 and UPR pathways. This result opens the door to further research on creating cubic membranes and membrane rearrangements caused by viruses [65].

The large endo/lysosomal membrane protein (MP) Niemann-Pick C1 (NPC1), which is involved in cellular RCT, is a crucial intracellular receptor for viral infection. The controlled release of CHO from lysosomes depends on the ubiquitous housekeeping protein NPC1. When it is lacking in humans, a deadly lysosomal storage disorder known as Niemann-Pick type C sickness results. NPC1 is a crucial viral receptor and host element for filovirus entrance, infection, and pathogenesis. NPC1 doesn’t need to operate in the cell for filovirus entrance. In addition, blocking NPC1 stops the African swine fever virus from entering the body and growing via disrupting CHO homeostasis. Additionally, the cell entrance of quasi-enveloped hepatitis A and hepatitis E viruses has been linked to NPC1. NPC1 controls CHO levels, which are necessary for the effective release of reovirus cores into the cytoplasm [66].

Researchers tried to infect Aedes cells in a model system that expressed human NPC1 (hNPC1) with a recombinant vesicular stomatitis virus that expressed the glycoprotein of the Ebola virus (EBOV). No appreciable rise in infection was seen in the hNPC1-expressing cells when compared to the control cells, indicating that host factors other than NPC1 determine mosquito cells’ susceptibility to filovirus infection and that human NPC1 expression is insufficient to support filovirus infection [67]. The Ae. Aegypti vector restricts infection by activating the Toll, JAK/STAT, and RNAi pathways in response to DENV exposure. According to the DENV infection-responsive transcriptome study, members of the NPC1 and myeloid differentiation 2-related lipid recognition protein (ML) families exhibit greater mRNA abundances following DENV infection. It has been shown that the lipid-binding proteins encoded by these genes participate in host–pathogen interactions in other species. RNA interference (RNAi)-mediated gene silencing of an ML and a member of the NPC1 gene family significantly enhanced resistance to DENV in mosquito midguts, revealing roles for these genes as DENV agonists in both laboratory strain and field-derived Ae. aegypti. Studies on gene expression have shown that members of the ML and NPC1 families may be involved in virus-cell entry and replication and promote viral infection by altering the mosquito’s immunological competence [68].

During infection, there was an extracellular fragment of LRP-1, the protein associated with LDLR. It has been suggested in earlier research that LRP-1 controls CHO homeostasis. Researchers postulated that DENV alters the expression of the LRP-1 protein to preserve intracellular CHO obtained from the host, thus promoting viral replication in membrane-associated replication compartments. Consequently, LRP-1 protein expression was decreased by stimuli that are present during flavivirus infection. Maya O. Tree and colleagues also discovered that DENV viral RNA and intracellular CHO were elevated by dsRNA suppression of LRP-1. Moreover, infection was decreased by intracellular lipid depletion. All of these findings point to the possibility that DENV decreases LRP-1 protein production, maybe via regulated intramembrane proteolysis (RIP), to raise intracellular CHO and promote Ae. aegypti replication [12]. DENV may employ the LDL receptor gene family, widely expressed in humans and mosquitoes, to gain access to cells. Nuclear localization signals (NLS) motifs and lysine-based LDL receptor ligands are in the dengue capsid and envelope proteins. On the cell surface, synthetic peptides of DENV proteins that may represent binding sites for LDL receptors colocalize with Apo E and LDL. The widespread expression of LDL receptors in humans and other animals may significantly increase the infectivity of viruses. While the details of DENV’s cell entrance procedures are still being worked out, researchers offer another fascinating potential for the molecular mechanics of viral entry [69].

DENV virions are bound by LDLR-related protein-1 (LRP1) via interaction with the viral envelope glycoprotein DIII. The purified receptor significantly inhibits DENV infection at 5 × 10⁻⁸ mol/L, and a natural ligand of LRP1 likewise blocks the envelope protein’s interaction with LRP1. 100 times less infectious virus is produced when LRP1 is depleted than controls. According to the researchers’s findings, LRP1 is an additional DENV receptor, making it a desirable target to assess in the quest to create potent antiviral medications to combat DENV [70]. In Table 2, we summarize the role of lipid types in DENV.

Table 2 The role of the lipid metabolism, lipid raft, and lipid receptors in DENV

Lipid-lowering drugs in dengue virus

Drug repurposing is a promising strategy in antiviral medicine that aims to find new uses for licensed drugs. It may reduce the time and expense associated with traditional drug development. Emergent viral infections may also be treated with this method. Notably, repurposing drugs is a strategy that shows promise. atorvastatin (ATV) and ezetimibe, two FDA-approved drugs for decreasing CHO, have shown encouraging antiviral characteristics against a range of viruses, including HIV-1, hepatitis B virus (HBV), and flaviviruses. These medications interfere with the production or absorption of CHO by disturbing the lipid rafts and replicative complex formation [50].

Hepatocytes produce CHO complexly, and some combinations may hurt the synthesis or distribution of CHO in cells. By preventing desmosterol Δ24-reductase and cell CHO transit, U18666A inhibits the production of CHO. Because of the linked diethylaminoethyl chain of the 3-hydroxyl, U18666A is a cationic amphiphile. Other cationic amphiphiles with radically different structures may also inhibit the outflow of CHO from lysosomes. Initially, the mechanism of inhibition was not evident because of the structural variations. In 1989, it was shown that U18666A also functions in another way, namely by preventing the transit of LDL-derived CHO from lysosomes where it had been deposited, causing a state similar to NPC1 deficiency. The late endosomal and lysosomal protein NPC1 is required to enter the EBOV, and U18666A causes symptoms that are in line with NPC1 abnormalities. Among U18666A’s alleged effects is the inhibition of viral replication [10]. U18666A was used as a tool by researchers to investigate the function of CHO during DENV infection. Mee Kian Poh et al. discovered that when cells were treated with U18666A, virus particles were confined in the Lamp-1 positive late endosome/lysosome compartment. Additionally, the administration of U18666A had an impact on viral replication. Moreover, when C75, a FASN inhibitor, was combined with U81666A, researchers saw an additional antiviral effect, indicating the involvement of FA and CHO in DENV [52].

It is critically necessary to find an antiviral medication that works against all four DENV serotypes to prevent and treat this illness. In the early stages of disease, lowering the viral load may help patients become less susceptible to DHF or DSS. This study looked at the antiviral effects of five commonly used protease inhibitors on DENV infection since both viral and host proteases may play a role in effective viral replication. Previous research has shown that the serine protease inhibitor AEBSF (4-(2-aminoethyl) benzene sulfonyl fluoride) inhibits the protease activity of DENV NS3. The results showed that AEBSF significantly and dose-dependently decreased DENV protein production and genome replication. AEBSF inhibited the expression of many genes, including LDLR, HMGCR, and HMGCS. Moreover, AEBSF significantly decreased intracellular CHO synthesis and HMGCR activity after DENV infection. All four DENV serotypes and three cell lines validated AEBSF’s anti-DENV activity. These results imply that AEBSF uses host and viral protease activity to prevent DENV infection [71].

It has been shown that bovine lactoferrin (bLF), which is found in milk, inhibits a number of viral infections. bLF dramatically reduced the four DENV serotypes’ ability to infect Vero cells. In the time-of-drug addition test, adding bLF simultaneously as or before the virus attached significantly reduced the amount of DENV-2 infection. Moreover, Chen et al. discovered that bLF interacts with LDLR, heparan sulfate (HS), and dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) to prevent DENV-2 from attaching to the cellular membrane. Furthermore, in a suckling mouse challenge paradigm, bLF reduces morbidity and suppresses DENV-2 infection. Researchers validated the hypothesis that bLF, by binding to putative DENV receptors, may prevent DENV infection [72].

Additionally, cyclovirobuxine D (Cvb D) protects neonatal mice from lethal DENV infection and inhibits DENV replication in vitro in a dose-dependent manner. The expression of genes associated with the cellular CHO pathway is regulated by Cvb D mechanistically. As a result, Cvb D inhibits viral-dependent autophagy, activates mTOR, and increases cellular CHO synthesis and accumulation. Cvb D inhibits the lysosome transcription factor TFEB’s nuclear translocation, although it does not prevent the start of autophagy [73].

Some plants used in Southeast Asian and other traditional medicines contain the alkaloid berberine (BBR), which has been shown to have antiviral properties. It was demonstrated that BBR has cellular effects that raise the amount of cellular DENV E protein without also affecting DENV nonstructural proteins, indicating a potential impact on the production or egress of viral particles. Although it was shown that BBR activated ERK1/2, this did not lead to flaws in the processes of viral egress. BBR’s main impact on the generation of viruses was probably caused by its activation of AMPK and interference with lipid metabolism. These findings together imply that BBR affects DENV infection in two ways and that it may 1 day be developed as an anti-DENV antiviral [74].

Statin as antivirals in dengue virus

Before presenting statins as potentially valuable drugs for treating viral infections, their antiviral properties must be carefully investigated. Despite significant disparities in the data, a sizable body of research from both clinical and experimental studies suggests that statins may help treat viral infections due to their immunomodulatory properties and ability to stop viral reproduction. Examples include preventing adhesion molecules on T cells from interacting with antigen-presenting cells (APCs), suppressing the expression of virus-specific cell surface receptors on T lymphocytes, such as C–C chemokine receptor type 5 (CCR5), and altering the secretion of proteins and cytokines, such as RANTES. Nonetheless, vital data indicates that statins could have direct antiviral effects. CHO and isoprenoids need mevalonic acid as a precursor, and statins prevent this from happening by blocking the action of HMG-CoA reductase. Hence, statins inhibit the intracellular manufacture of CHO, an essential component that supports the viral infection cycle. Moreover, statins reduce the availability of geranylgeranyl pyrophosphate (GGP) and farnesylpyrophosphate isoprenoids, which are necessary for the prenylation of proteins such as Rho and Ras GTPases. It is noteworthy that these proteins have several functions in intracellular signaling networks, some of which regulate the viral life cycle [75].

Because statins may reduce CHO and have pleiotropic effects on inflammation and oxidative stress, they benefit cardiovascular disorders. Statins affect immune response at several levels, such as cytokine generation, antigen presentation, and immune cell adhesion and migration. Additionally, they improve endothelial integrity, function, and nitric oxide bioavailability while reestablishing the vascular redox balance via the reduction of reactive oxygen species and the increase of antioxidants. The majority of these effects rely on the down-regulation of redox-sensitive proinflammatory transcriptional factors like NF-κB as a result of statin-mediated reduction of isoprenoids’ synthesis, which is an essential component of small GTPases (including Ras, Rho, and Rac). Additionally, ATV 40 mg significantly improved symptoms in statin-naïve hospitalized patients for seasonal influenza compared to placebo in a recently completed RCT (ClinicalTrials.gov number, NCT02056340). There is further evidence linking the use of outpatient statins to decrease the severity of illness in individuals admitted during the 2009 H1N1 pandemic [76,77,78].

ATV, fluvastatin, LOV, pitavastatin, pravastatin, rosuvastatin, and simvastatin are the seven statins presently on the market. Even though these medications are usually well tolerated, skeletal muscle anomalies such as severe, fatal rhabdomyolysis or myalgia may happen. Drug interactions and other factors that raise statin concentrations may increase the chance of these side effects. The pharmacokinetic profile of statins determines how drugs interact with one another. Cytochrome P450 (CYP) 3A metabolizes Simvastatin, LOV, and ATV, while this CYP does not metabolize the other statins. Organic anion transporter polypeptide 1B1, an uptake transporter found in hepatocyte membranes, is a substrate of all statins and may potentially account for specific drug-drug interactions [79]. Statin use upon hospital admission did not lessen the severity of dengue in adult dengue patients who presented with hyperlipidemia. Nonetheless, statin usage in the past did not raise the risk of increasing liver inflammation, which supports the safety of continued statin use in dengue patients [80].

LOV was shown to be safe and well-tolerated in adult dengue patients in a different trial. However, researchers could not find any indication of a favorable impact on dengue viremia or clinical symptoms, even though the trial was not designed to examine effectiveness. It’s safe to keep people on established statin treatment if they have dengue [81]. Treatment of DENV-infected cultures with LOV has been shown to impact viral assembly. Researchers assessed the effects of LOV on the viremia levels and survival rate of AG129 mice infected with DENV-2. The results demonstrated that the quantity of dosages given and the time of therapy both affect how LOV affects viremia. Due to a delay in the disease’s course, researchers saw a considerable rise in the survival rate in both schemes. However, since this treatment plan raises viremia and researchers are unsure how this increase would impact disease development in people, the data obtained in the post-treatment scheme need to be taken cautiously [82].

LOV therapy was tested in a randomized, double-masked, placebo-controlled experiment to demonstrate how well it treats dengue. There are no data to support the sample size estimate in this exploratory safety investigation. Based on clinical judgment and practical considerations, a target sample size of 300 patients was selected for the second phase, with enrollment occurring across two dengue seasons. About 10% and 30% of participants, respectively, suffered at least one significant adverse event or adverse incident in a prior randomized dengue study. Researchers have 80% power to identify a 12% (from 10 to 22%) or 16% (from 30 to 46%) increase in the frequency of adverse events with 300 patients. Moreover, this sample size guarantees some power to investigate the effectiveness of statins [83].

In a different research, CHO production was genetically (HMGCR RNAi) and pharmaceutically (fluvastatin, ATV, LOV, pravastatin, and simvastatin treatment) reduced in both uninfected and DENV-2-infected hepatoma Huh 7 cells. The researchers evaluated lipid levels, DENV titer, and the profile of cellular antiviral expression. Following a 48-h course of treatment with 10 µM fluvastatin, 10 µM ATV, 20 µM LOV, and 20 µM simvastatin—which, in turn, achieved 70, 70, 65, and 55% inhibition of DENV-2—DENV-infected cells showed a drop in their DENV titer, measured in plaque forming units, in comparison to the untreated cells. Moreover, the cytopathic effect was reduced in the DENV-infected cells treated with statins. Statins simultaneously reduced CHO levels at 48 h, except for DENV-2-infected cells. The production of CHO was genetically blocked using HMGCR siRNA RNA interference. Consequently, there was no discernible decline in CHO levels at 48 h after infection, although there was a little drop in the DENV-2 titer. In addition, researchers discovered that in every experimental scenario, DENV-2 infection raised intracellular CHO levels. Differential expression patterns for the antiviral genes under research were found when DENV-2 infection, statin treatment, and HMGCR siRNA were utilized to examine the cellular antiviral responses in infected, uninfected, treated, and untreated Huh7 cells. Genes linked to cellular immune responses and pro-inflammatory processes were expressed less often in all downregulating CHO medications that were tested. Researchers found that the downregulation of DENV-2 infectious particle number mediated by statins is partly mediated by modulating the cellular antiviral profile and is independent of CHO levels [84].

By preventing endocytosis via the NPC1-Like 1 (NPC1L1) receptor, which is present on the membranes of hepatocytes and enterocytes, the FDA-approved drug ezetimibe reduces the absorption of CHO. Researchers demonstrated that when Huh-7 cells are infected with DENV, there is a surface increase in NPC1L1, which is associated with higher levels of CHO. Even at 50 μM, blocking NPC1L1 with ezetimibe does not change DENV binding or entry into Huh-7 cells; however, it does reduce total cellular CHO, the proportion of infected cells, viral yield, viral RNA, and protein synthesis. Ezetimibe also stopped the development of DENV replicative complexes and the build-up of LDs. These results demonstrate that ezetimibe is a very effective medicine for suppressing DENV infection and support the idea that CHO is a critical target for blocking viral infection [85].

Researchers assessed LOV’s possible antiviral efficacy against DENV infection of endothelium and epithelial cells. LOV reduced the virus output before and after viral injection (80% for HMECs and 25% for VERO cells). On the other hand, LOV therapy after injection resulted in a notable 2-to ninefold increase in virus-positive RNA but only a 13–23% rise in viral protein. There was a small decrease in viral titer in the cells and an increase in DENV genomic RNA and protein. Scientists implied that LOV has a more significant impact on viral assembly than on replication, leading to viral genomic RNA and proteins within cells that do not assemble according to standard protocol [86].

It is currently unknown how DENV’s NS3 gets nuclear imported, despite recent evidence that it is found in the nucleus of infected cells. Researchers found that Ivermectin (IVM) blocks the Importin α/β1 pathway, preventing NS3 from localizing to the nucleus. ATV can alter the nuclear transport of DENV-2’s NS5 polymerase and NS3 protease, according to research by Selvin Noé Palacios-Rápalo et al. However, they also discovered that although ATV and IVM treatments modify nuclear pore complex (NPC) proteins, their combination decreased DENV infection in vivo and in vitro. Thus, researchers deduced that this drug’s additional antiviral impact is ATV transport inhibition and proposed a possible anti-DENV treatment in conjunction with IVM [87].

Researchers draw attention to the role that neutrophils play in developing dengue pathogenesis, particularly in cases of DSS, by carrying overexpressed matrix metalloproteinases (MMPs). The expression patterns of MMP-1, MMP-2, MMP-9, and MMP-14 are markedly increased with NS1 exposure to neutrophils, and this may be the cause of vascular permeability and the subsequent development of DSS. The expression patterns of MMP-2 and MMP-14, which indicate protective effects, have been markedly reversed by ATV. Moreover, ATV reversed the dose-dependent release of cytokines IL-6 that NS1 had caused. Neutrophil NS1 activation dramatically increases the VEGF expression profile, which may contribute to endothelial dysfunction. Additionally, neutrophils may produce VEGF growth factors, which may then contribute to endothelial dysfunctions that result in DSS [88].

Anti-PCSK9 in dengue virus

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a relatively new focus when lowering LDL-C. The pharmaceutical decrease of circulating PCSK9 facilitates the removal of LDL-C from circulation. Mechanisms mediated by LDL receptors are used to accomplish this. Currently licensed as injectable lipid-lowering treatments (LLT) is the clinical usage of fully humanized monoclonal antibodies (mAbs) targeting PCSK9, evolocumab, and alirocumab, as well as inclisiran, a synthetic small interfering RNA (siRNA) that suppresses the translation of PCSK9 mRNA. According to European (ESC/EAS) and American (ACC/AHA) guidelines, adult patients with familial hypercholesterolemia (FH) or established Atherosclerotic cardiovascular disease (ASCVD) who need additional LDL-C lowering to lower the risk of myocardial infarction stroke, and coronary revascularization should receive PCSK9-targeted therapy as additional lipid-lowering agents [89].

The cyclase-associated protein-1 (CAP-1), a newly discovered PCSK9 binding partner, is thought to be essential for PCSK9-mediated LDLR degradation. In a caveolin-dependent process, the LDLR/PCSK9/CAP-1 complex leads to lysosomal degradation when the PCSK9 catalytic domain binds to LDLR and the PCSK9 CHRD interacts with CAP-1 [90]. The various cellular mechanisms through which PCSK9 is involved suggest that this protein can regulate plasma lipids via targeting other LDLRs, including the very low-density lipoprotein (VLDL) receptor, apoE receptor 2 (apoER2), and LRP1. At least in specific tissues, it’s conceivable that PCSK9 contact with these receptors does not result in their destruction [91,92,93]. PCSK9, when combined with its inhibitory prodomain, is released into the bloodstream as a dormant enzyme. The strong mRNA expression in liver hepatocytes and its location on chromosome 1p32 made it easy for us to identify three patient families with the PCSK9 mutations S127R or F216L. This locus is related to FH alongside LDLR and APOB. Mice given a CHO-rich diet had downregulated levels of Pcsk9 and Ldlr, whereas LDLR degradation was caused by PCSK9 overexpression. So, researchers showed that PCSK9’s bioactivity is modulated by gain-of-function (GOF) and loss-of-function variants; specifically, PCSK9 binds the LDLR nonenzymatically to trigger its destruction in endosomes and lysosomes. In addition to its activities in controlling hypercholesterolemia and atherosclerosis, vascular inflammation, viral infections, and immunological checkpoint regulation in cancer, PCSK9 is known to have a significant role in targeting additional receptors for degradation [94].

Additionally, the researchers found that dietary CHO dramatically downregulated PCSK9, whereas SREBP1a and SREBP2 significantly increased it, indicating that PCSK9 is a CHO-regulated gene. Horton and colleagues subsequently confirmed this significant discovery and demonstrated that statins may increase PCSK9 transcription. Interestingly, PCSK9 and LDLR mRNA levels were positively regulated by both CHO deficiency and statin treatment. PCSK9 was also able to effectively degrade LDLR protein, which may account for some human mutations that have been linked to hypercholesterolemia. Therefore, PCSK9 GOF mutations increased the amount of LDLR degradation that PCSK9 produced [95]. The structure and function of PCSK9 are illustrated in Fig. 4.

Fig. 4

Suppression of LDLR recycling by PCSK9. The LDLR is bound to the cell surface by PCSK9. The LDLR’s epidermal growth factor repeats the catalytic domain of PCSK9 binds A. Through clathrin-mediated endocytosis, the LDLR: PCSK9 complex is taken within the cell. In the sorting endosome, PCSK9 stays attached to the LDLR because of an extra electrostatic contact at acidic pH between the C-terminal domain of PCSK9 and the ligand-binding domain of the LDLR. This prevents the LDLR from achieving a closed conformation, which ultimately leads to degradation rather than recycling. Ectodomain cleavage of the extended LDLR by a cysteine cathepsin in the sorting endosome seems to be the mechanism by which the LDLR fails to recycle. In preparation for destruction in the endosomal/lysosomal tract, the vesicular portion of the sorting endosome will include the cleaved LDLR ectodomain. Consequently, the plasma membrane has a reduced supply of LDLR for the binding and clearance of LDL-C [100, 101]

A novel family of drugs known as PCSK9 inhibitors is gaining prominence in the fight against high LDL-C levels. Both the US and EU have authorized the use of two PCSK9 inhibitors, alirocumab and evolocumab, to treat hypercholesterolemia. Serum LDL-C levels may be dramatically decreased by inhibiting PCSK9 and increasing the recycling of LDL receptors [96, 97]. Reducing the production of PCSK9 is another strategy. A new anti-sense medicine called Inclisiran is based on tiny interfering RNA. There is a drop in LDL-C levels and an increase in hepatocyte recycling and membrane expression of LDL receptors when inclisiran binds to the messenger RNA (mRNA) precursor of PCSK9. This suppresses the PCSK9 gene expression. Adults with ASCVD or heterozygous familial hypercholesterolaemia who have an LDL-C level of 100 mg/dl and who do not achieve target LDL-C levels with statin and ezetimibe or who do not take statin or ezetimibe because of intolerance or contraindication, should be considered for addition therapy with this innovative CHO-lowering drug [98]. As a replacement for protein-based LLT, researchers created a DNA-encoded mAb (DMAb) targeting PCSK9 (daPCSK9) and studied its expression and efficacy. By day 7, wild-type mice had significantly reduced levels of total CHO (by 10.3%) and non-HDL-C by 28.6% after a single intramuscular injection of mouse daPCSK9 had produced expression in vivo for more than 42 days. The expression of DMAb increased with each subsequent dose of the plasmid, reaching 7.5 μg/mL on day 62. Whether used alone or in conjunction with existing LDL-lowering therapies, daPCSK9 treatments have the potential to provide a new, easy, less frequent, and cost-effective method of decreasing LDL-C [99].

Among its several functional pathways that affect homeostasis in vivo, PCSK9 promotes thrombosis, apoptosis, pyroptosis, autophagy suppression, platelet activation, immune response stimulation, and lipid metabolism regulation [102]. PCSK9 has a unique immunological role in promoting the maturation of dendritic cells brought on by oxidized LDL and activating T lymphocytes originating from human blood and atherosclerotic plaque [103]. PCSK9 in similar ex vivo settings, where atherosclerotic plaques’ T cells and DCs are studied, and patients exhibiting signs of potentially deadly cardiovascular disease (CVD) are surgically treated. Immunological studies revealed that oxLDL promoted DC maturation and PCSK9 expression in DCs [104]. Researchers showed that not only did inhibiting PCSK9 promote an anti-inflammatory phenotype, but it also blocked DCs from activating T cells produced by OxLDL and turned back the polarization to Th1 and Th17 cells that OxLDL caused [105].

Additionally, researchers observed that PCSK9 silencing, with mock silence as control, inhibited proinflammatory cytokines such as TNF-α, IL-1β, and IL-6. Conversely, T regulatory cells, TGF-β, and IL-10 were all activated. Among these basic mechanisms was miRNA. PCSK silencing decreased let-7c and miR-27a levels, but OxLDL up let-7 c, miR-27a, miR-27b, and miR-185 levels. Researchers showed that the effect of statins on mi-RNA was marginally different from that of PCSK9-inhibition [106].

When compared to a placebo, PCSK9 inhibition was linked to a reduction in IL-6 levels and a primary outcome of death or the need for intubation in cases with severe COVID-19. Researchers indicated that among patients with greater levels of inflammation at randomization, PCSK9 inhibition increased survival when compared to placebo, indicating that the therapeutic benefits may depend on the severity of inflammation [107]. Researchers investigated if the results above could be repeated in primary human monocytes and monocyte-derived dendritic cells (MoDCs), as myeloid cells are the main targets of DENV. Using the 250-gene human inflammatory panel on the NanoString nCounter platform, gene expression analysis of primary monocytes treated with PCSK9 and infected with DENV showed 23 genes that were considerably downregulated and 7 that were slightly elevated. The NF-κB and IFN pathways were shown to be significantly downregulated by Gene Ontology (GO) biological pathway analysis. The observed increase by researchers in DENV replication may be explained by these data, which imply that PCSK9 supplementation reduces the antiviral response against DENV. The addition of alirocumab reversed these alterations in DENV replication and antiviral responses such as IFN-β and C-X-C motif chemokine 10 (CXCL10), suggesting that these effects were unique to elevations in PCSK9 concentrations. All of these results point to the possibility that elevated PCSK9 expression enhances DENV infection in human myeloid-derived cells by diminishing antiviral responses in low-oxygen settings that are typical of lymph node microenvironments. Despite DENV infection, LDLR-C absorption would have dispersed cholesterol throughout the cell. In contrast, researchers demonstrated that de novo cholesterol synthesis increased ER cholesterol levels, which inhibited the phosphorylation of tank-binding kinase (TBK) and the stimulator of the IFN gene (STING). The production of IFN-I and the downstream antiviral IFN-stimulated genes (ISGs) was therefore decreased by decreased STING and TBK activation. Clinical studies that demonstrated a clear connection between plasma levels of PCSK9 and increased viremia levels and illness severity in dengue patients corroborated researchers’ in vitro results. Additionally, researchers suggested that PCSK9 serves as a host factor for DENV in target cells found in hypoxic conditions and that blocking PCSK9 [108, 109].

Adverse results during COVID-19 are correlated with the degree of inflammation. LDLR homeostasis is mediated by PCSK9, which may also have an impact on the inflammatory response to COVID-19 and vascular inflammation. In individuals with severe COVID-19, researchers looked at the effects of PCSK9 inhibition vs a placebo on clinical and laboratory results. Sixty patients with ground-glass opacity pneumonia, arterial partial oxygen pressure to a fraction of inspired oxygen ratio ≤ 300 mm Hg, and hospitalized for severe COVID-19 were randomly assigned 1:1 to receive a single 140-mg subcutaneous injection of either evolocumab or placebo in this double-blind, placebo-controlled, multicenter pilot study. Death or the requirement for intubation at 30 days was the primary outcome. Changes in circulating IL-6 at 7 and 30 days from baseline were the primary secondary endpoints. Researchers demonstrated that PCSK9 inhibition decreased IL-6 levels and the primary outcome of mortality or intubation in patients with severe COVID-19 when compared to a placebo. PCSK9 inhibition improved survival for patients with more severe inflammation at randomization compared to placebo, suggesting that therapeutic effects may be driven by inflammatory severity. IMPACT-SIRIO 5: The Effect of PCSK9 Inhibition on Clinical Outcome in Patients During the Inflammatory Stage of COVID-19 [107].

Further evidence suggested that PCSK9 blocks HCV entrance and replication. PCSK9 impacted neither HCV translation nor assembly/secretion. Also, researchers showed that both in HCV genomic replicon cells and after infection with HCV generated from cell culture (HCVcc), overexpression of PCSK9 suppressed HCV replication in a dose-dependent fashion. Replication of HCV was enhanced by knocking down PCSK9. The fact that the HCV replication suppression by PCSK9 was not caused by LDLR degradation is supported by the fact that the gain-of-function (D374Y) or loss-of-function (Δaa. 31-52) PCSK9 mutants for LDLR degradation did not impact HCV replication. The down-regulation of HCV replication by uncleaved ProPCSK9, but not by cleaved PCSK9, indicates that auto-cleavage of PCSK9 impacted HCV replication. In addition, researchers discovered that PCSK9 interacted with NS5A via NS5A aa. 95-215; this area was critical for HCV replication, NS5A-RNA binding, and dimerization of NS5A. Importantly, PCSK9 interaction inhibited NS5A dimerization and NS5A-RNA binding. Based on these findings, researchers suggested that PCSK9 interacted with NS5A to prevent HCV replication. Patients with aberrant lipid profiles could benefit from researchers’s findings in optimizing their anti-HCV medication regimen [110].

Inhibition with PCSK9 antibodies (PCSK9i) reduces cardiovascular events in people with coronary artery disease, while elevated levels are linked to an increased risk of cardiovascular events. People with dyslipidemia and HIV also tend to have higher PCSK9 levels. Researchers assessed the hypothesis that PCSK9i improves impaired coronary endothelial function in dyslipidemia without coronary artery disease and in PLWH with nearly optimal/above-goal LDL-C levels. This is because impaired coronary endothelial function is associated with increased PCSK9 in PLWH, which is an indicator of coronary vascular health. The percentage increases in cross-sectional area from rest to isometric handgrip exercise were + 5.6 ± 5.5% in the PLWH group and + 4.5 ± 3.1% in the dyslipidemia group after 6 weeks of evolocumab, with both groups showing a significant improvement compared to baseline. In all groups, researchers showed that there was a notable increase in coronary blood flow that occurred with an improved cross-sectional area. Researchers showed that PLWH and dyslipidemia patients benefit from PCSK9 inhibition on coronary artery health [111]. Hepatocyte LDLR, ApoER2, and VLDLR are just a few of the membrane-bound receptors that human PCSK9 is known to facilitate the degradation of. Scientists looked at the possibility that PCSK9 might influence CD81 levels, an essential HCV receptor, due to the LDLR’s role in HCV entrance. Scientists found that CD81 and LDLR expression were significantly reduced when PCSK9 or its active membrane-bound version (PCSK9-ACE2) was expressed consistently. So, they used the JFH1 HCV genotype virus to test PCSK9’s antiviral effects in vitro. According to the findings, cells expressing PCSK9 or PCSK9-ACE2, but not the control protein ACE2, were shown to be resistant to HCV infection. Purified soluble PCSK9, when added to cell culture supernatant, inhibited HCV infection in a dose-dependent manner. The researchers anticipated the resistance of HuH7 cells expressing PCSK9-ACE2 to infection by HCV pseudoparticles. Furthermore, researchers demonstrated that PCSK9 modulates CD81 cell surface expression independently of LDLR. The quantities of LDLR and CD81 proteins were considerably decreased in the livers of Pcsk9 and Ldlr knockout mice, but there was no change in the amounts of transferrin or scavenger receptor class B type 1 proteins. Based on their findings, the researchers concluded that circulating liver PCSK9 inhibits the amount of mouse liver CD81 expression in vivo and has an antiviral impact on HCV in cells. Consequently, scientists have postulated that PCSK9 activity and plasma levels may regulate HCV infection in humans [112].

Research by Esther Shuyi Gan et al. [113] has shown that DENV infection triggers the production of PCSK9, a protein that acts as a negative regulator of the LDLR. A recent study found that hypoxic cells with elevated PCSK9 levels had lower LDLR and LDL-C uptake, leading to an increase in de novo CHO production. Despite DENV infection, de novo CHO production increased levels of ER CHO, which reduced the phosphorylation of STING and TBK instead of LDLR absorption, which would have disseminated CHO throughout the cell. Downstream antiviral ISGs and IFN-I expression were both decreased as a result of reduced STING and TBK activation. Clinical studies supporting a direct connection between plasma levels of PCSK9 and increased viremia levels and illness severity in dengue patients corroborated researchers’ in vitro results. In addition to HMGCoA reductase, researchers found that PCSK9 is a host factor for DENV in target cells in hypoxic microenvironments. Therefore, blocking PCSK9 instead might be a good way to treat dengue. Elevated plasma PCSK9 levels in dengue patients with high viremia and greater severity of plasma leakage further corroborated researchers’ in vitro results. Dengue patients may benefit from a host-directed medication that targets PCSK9, according to researchers, who also found that PCSK9 had an unknown function in dengue pathogenesis. A drawback of the in vitro trials is the doses of recombinant PCSK9 (rPCSK9) that were supplied, even though researchers have pointed out the critical function that PCSK9 plays in DENV infection. The levels of PCSK9 in the researchers’ clinical trial cohort were lower than the concentrations of 400 ng/mL PCSK9 that were supplemented in vitro. This discrepancy is because PCSK9 activity in vivo is higher than rPCSK9 effectiveness. This impact may be explained in several ways. One of the many posttranslational alterations that PCSK9 experiences in vivo is Ser phosphorylation by FAM20C. The binding affinity of PCSK9 to the LDLR is enhanced as a result. The capacity of rPCSK9 to degrade LDLR is diminished in vitro due to the lack of phosphorylation by FAM20C. Secondly, in contrast to in vitro settings, the liver exhibits elevated levels of CAP-1. Endocytosis and lysosomal breakdown of LDLR are facilitated by CAP-1 binding to PCSK9. This means that PCSK9 activity is reduced in vitro when CAP-1 levels are decreased. This precludes any comparison between PCSK9 supplementation concentrations in vitro and plasma PCSK9 levels [90, 113, 114]. PCSK9 functions in DENV infection are illustrated in Fig. 5.

Fig. 5

PCSK9 expression is induced by DENV infection. Unlike LDLR CHO absorption, which would have distributed CHO throughout the cell, de novo CHO generation enriched ER CHO levels that prevented the phosphorylation of STING and TBK despite DENV infection. Reduced STING and TBK activation led to a reduction in the production of IFN-I and the downstream antiviral ISGs. Further evidence that PCSK9 serves as a host factor for DENV in target cells inhabiting hypoxic microenvironments has been obtained by researchers, indicating that targeting PCSK9 rather than only HMG-CoA reductase might be a useful tactic to bridge the therapeutic gap for dengue treatment [113]

Another interesting method for modifying PCSK9 activity is vaccination. Compared to other treatment techniques, vaccines offer several potential benefits. For example, vaccines are generally cheap to make, which might lower patient expenses, and they usually need fewer doses, which could improve patient compliance. Immunological processes of self-tolerance usually restrict the capacity to induce antibody responses against self-antigens such as PCSK9. On the other hand, large concentrations of self-antigens on the surface of virus-like particles (VLPs), a kind of nanoparticle-based vaccination platform, may efficiently evade these processes. Targeting self-antigens, VLP-based vaccinations have been evaluated in human clinical trials; they are safe and may induce high titer antibody responses. To test their hypothesis that these unique linear peptides from human PCSK9 might interact with LDLR, researchers developed VLP vaccines. Researchers found many vaccine candidates that induced high titer anti-PCSK9 antibody responses and reduced overall CHO levels in injected mice. Researchers have created and combined vaccines that display two species-specific linear peptides from PCSK9 to assess the efficacy of VLP-based immunizations in reducing CHO levels and triggering anti-PCSK9 antibody responses in a range of animal models. Researchers supported the effective reduction of LDL-C levels without the need for statin co-administration by using a bivalent VLP-based immunization that targets two PCSK9 epitopes [115].

Additionally, studies revealed that upregulating CHO synthesis via PCSK9-dependent reductions in LDLR-mediated CHO uptake may have lessened the anti-DENV efficacy of statins, at least in part. Thus, rather than total cellular CHO levels, researchers suggested that the proviral determinant of DENV infection is the subcellular location of CHO. While researchers have used alirocumab to demonstrate the role of PCSK9 in DENV infection, their findings suggest that anti-PCSK9 inhibitors may have anti-dengue benefits as well [113]. Severe dengue cases have been linked to decreased circulating LDL-C levels [62]. Additionally, the PCSK9 result implies that elevated LDL-C uptake was not the cause of the reduced plasma LDL-C levels seen in individuals with severe dengue. Instead, the reduction in CHO production might have been caused by poor hepatic synthesis since people with severe dengue are known to have inflammation in their livers. On the other hand, the correlated elevation in endothelial permeability could have also led to the loss of CHO molecules into the extravascular area, thereby reducing the levels of LDL-C in plasma. When cells in low-oxygen organs are infected with DENV, their metabolism of CHO is changed, which promotes pathogenesis. Significantly, researchers postulated that RNA interference or inhibitory mAb-based PCSK9 activity reduction would be a secure therapy alternative for dengue patients [113]. In Table 3, we created a list of lipid-lowering drugs in DENV infection and explained their function.

Table 3 The role of the lipid-lowering drug in DENV infection

Future and landscape

Long-standing public health concerns about DENV have been compounded by the COVID-19 pandemic, which will place further strain on many Asian nations, particularly in terms of the effects on the health and economic sectors. The problem of cross-reaction caused by infection with distinct DENV serotypes and flaviviruses remains unresolved as of yet. Furthermore, there is also a greater chance that an ADE problem would develop and exacerbate the illness. In addition to these issues, one of the factors to be taken into account in order to manage the DENV infection properly is the virus’s tendency to evade immune response [116]. A significant focus of research for dengue therapy has been developing DENV-specific antivirals since a higher virus load may promote severe dengue sickness. Clinical trials using repurposed medications that have been shown to have antiviral action in pre-clinical investigations, such as celgosivir, balapiravir, chloroquine, and LOV, have not yet shown any effectiveness in lowering viremia or improving clinical outcomes. In vitro, JNJ-A07 demonstrated antiviral efficacy against 21 clinical isolates and all four dengue serotypes. The viremia and viral load in organs of immunocompromised mouse models infected with both fatal and sub-lethal DENV-2 dosages and in models of ADE exhibited a rapid reduction, regardless of whether JNJ-A07 therapy was initiated at the commencement of infection or delayed. In addition, the survival rate of the drug-treated infected rodents increased, and pro-inflammatory cytokines, including IL-18, IFN-γ, TNF, and IL-6, were reduced. The drug-treated group’s immunological response metrics, such as neutralizing antibody titers after infection, hemoconcentration, or fluid buildup in organs, were not measured. This is important because a subsequent dengue infection might result in severe dengue due to the medication’s rapid decrease of viremia, which could hinder the generation of antibodies. An analog of JNJ-A07, JNJ-64281802, is presently being studied in two phase 2 randomized, double-blind, placebo-controlled clinical trials to see if it works for prevention against the disease in healthy individuals (NCT05201794) and for treating dengue in patients with confirmed DF (NCT04906980) [117].

Intravenous Immunoglobulin (IVIG) infused intravenously is often used to treat idiopathic thrombocytopenia purpura (ITP). Platelet-associated IgG (PAIgG) autoantibodies are present in conjunction with ITP-associated thrombocytopenia. Researchers noticed that FcγR expressed on mononuclear phagocytic cells causes enhanced clearance of platelets coated with PAIgG. The competitive inhibition of FcγR or the ligation of inhibitory receptors is the mechanism of action of IVIGs. Researchers conducted a 4-day clinical experiment with 36 dengue patients to evaluate the efficacy of IVIG therapy. Regretfully, studies could not show that IVIG therapy was beneficial in facilitating platelet recovery [118]. Phase 2 trial results from separate research showed that the IVM group, administered for 3 days, tended to have a shorter plasma NS1 clearance time than the placebo group. Combining phase 2 and phase 3 trials (100 and 103 patients receiving IVM and placebo, respectively) resulted in 203 patients being included in the intention-to-treat analysis. Of the patients on IVM therapy, 24 developed DHF, whereas 32 were given a placebo. A single 400 µg/kg oral IVM dose each day for 3 days was safe and accelerated the clearance of NS1 antigenemia in dengue patients. However, at this dosage level, IVM’s therapeutic efficacy was not seen by researchers [119].

Currently, there is no strongly approved vaccine or therapeutic agent against dengue, and antiviral medications have a limited function in the treatment of DF. There is a continuing need for novel medicines since virus strains resistant to antiviral drugs are emerging. Since most presently available antiviral medicines are non-specific for certain viruses, medical research is now focused on developing targeted and cost-effective antiviral regimens that incorporate herbal therapy. Numerous potent phytoconstituents, including hirsutin, α-mangostin, quercetin, castanospermine, and schisandrin-A, have shown potential in inhibiting all four DENV serotypes [120]. In certain studies employing combinational drug testing for DENV, ribavirin has been combined with the α-glucosidase inhibitor CM-10–18, the nucleoside analog INX-08189, or the E protein inhibitor BP34610. The studies show that combination therapies increase antiviral efficacy in vitro (INX-08189, BP34610, or ribavirin with CM-10–18) as well as in vivo (ribavirin with CM-10–18). Moreover, ribavirin and CM-10–18 combined demonstrated a synergistic antiviral impact even when CM-10–18 was administered at a subeffective dose. A greater focus on evaluating the antiviral efficacy of different drug combinations is necessary, given the knowledge gained from studying other viruses and the encouraging early results for DENV [121, 122].

It is challenging to produce a safe and effective antiviral medication for DENV. Finding a molecule with low toxicity, pan-protective antiviral capabilities, reduced risk of viral resistance, and appropriate stability to guarantee absorption and distribution is a barrier to creating antiviral medications [122].

At almost every stage of their life cycle, viruses rely on lipid rafts for efficient infection. It’s now evident how many viruses utilize various lipid raft modification techniques to attach, internalize, fuse membranes, replicate their genomes, assemble, and release [123]. CHO in viral infection is possible to see from the perspectives of the virus and the host cell. The capacity of a virus particle to absorb the necessary quantity of sterol from the infected cell to maintain the form of a single macromolecular assembly—its envelope membrane bilayer—is all that counts since virus particles lack internal membrane structures. Therefore, broad-spectrum antivirals may help patients achieve better health outcomes and control viral infections more quickly [124].

To summarize, this extensive cohort research using propensity scores matching showed that patients with hyperlipidemia who were on statin medication may have a decreased chance of contracting a virus. It is important to remember that the study’s design was observational. The mechanisms behind the antiviral effects of statins were not investigated and provided conflicting results. It is necessary to gather more data from masked randomized controlled trials to support pertinent changes in clinical practice [125]. To determine the phases of the flaviviral replication cycle CHO is engaged in, several research has been conducted. For instance, sequestering CHO from the plasma membrane in WNV led to decreased viral titers and unsuccessful virus internalization. Still, elevated CHO levels facilitated the fusing of a target membrane with a liposome model. Investigators suggested that CHO affects the first phases of infection in some flaviviruses. It is recommended that the entry-stage CHO contribution results from the viruses’ need to bind to receptors before infection. Lipid rafts, CHO-rich clusters on the plasma membrane, are where receptors are concentrated. Receptor assembly will be impacted by CHO depletion, which will lessen the likelihood of viral adherence to the host. It is also thought that CHO contributes to the flaviviral replication stage. To maximize the surface area accessible for viral replication and to concentrate the replication components inside the vesicle packets, lipid rafts are thought to be active at this step. It has been shown that lipid rafts or CHO microdomains inside cells are the replication sites for both JEV and DENV. However, given that the application of an RCT inhibitor impacted the trafficking of DENV within infected cells rather than preventing binding, CHO may play a more significant role in the early phases of flaviviral biogenesis than just binding. It’s interesting to note that a different DENV research showed that impairing CHO biosynthesis reduced virus generation rather than inhibiting replication, suggesting that CHO may play a function in the latter phases of viral biogenesis [126].

LOV has been extensively researched as a statin to suppress DENV. Although researchers showed no clinical or virological advantage, LOV treatment is safe for adult dengue patients. The results reassure doctors about the safety of maintaining statin treatment in patients who get dengue, even if the researchers’ findings do not support supplementary statin therapy for dengue. Good supportive care, early identification, and detection of severe characteristics continue to be essential components of clinical treatment [127].

However, clinical studies and research have linked the usage of statins to several possible adverse effects. These include hemorrhagic stroke, muscular damage, liver problems, increased risk of diabetes, and cognitive decline. Myopathy, which is characterized by unexplained muscular weakness or discomfort, is one dangerous side effect. Rhabdomyolysis is one of the rarest forms of myopathy; even at the highest dosages, fewer than 0.1% of people experience it. It is pretty uncommon for statins to cause severe liver damage. For individuals with a history of cerebrovascular illness, statins may raise the risk of hemorrhagic stroke. There is insufficient data to connect the use of statins with several additional adverse effects, including peripheral neuropathy, cognitive problems, tendon rupture, cataracts, and AKI [128]. Consequently, it is essential to examine the dose utilized in DF patients with various conditions and at different ages. Additionally, research should be done on the adverse effects of these medications and ways to lessen them.

Expression of PCSK9 was triggered by DENV infection. As a result, there is less LDLR recycling, which promotes CHO translocation into the ER and prevents IFN-I activation and STING residing there. PCSK9 inhibitors greatly enhanced IFN-I production to counteract this. Additionally, statins treat individuals who have recovered from influenza and Ebola illnesses. Overall, it is evident that statins have antiviral effects by preventing or changing the trafficking of CHO, its redistribution, the attachment of viruses, the formation of virions, and their maturation, release, and stability [129]. In DF, PCSK9 inhibitors must be further investigated as, when administered appropriately, they might lessen the disease’s symptoms.

Conclusion

The endemic expansion of DF harms the health of people living in tropical and subtropical climates. It is a significant global public health concern. Climate change, human lifestyles, poor sanitary systems, viral genetic development, socioeconomic factors, devasting urbanization, unchecked population growth, and international travel and trade processes are the primary causes of DENV transmission. Scientists have not been able to create a safe dengue vaccine or treatment agent. Thus, repurposing medications that decrease CHO may be a valuable strategy for DENV. Lowering CHO seems to be a successful antiviral strategy, yet it may also weaken the host’s defenses. It’s important to remember that because viruses depend on lipid flow, it may be more beneficial to use statins to lower CHO levels necessary for viral maintenance rather than to decrease CHO overall. Patients with high CHO who are infected with DENV should keep taking medications that decrease CHO. Adding PCSK9 inhibitors, statins, and fenofibrates to the usual medications used to treat DENV may be regarded as an adjuvant therapy. PCSK9 inhibitors may be an appropriate host-directed therapy for dengue patients since PCSK9 has a hitherto unknown involvement in the pathophysiology of the disease. To determine if there may be a connection between PCSK9 inhibitor usage and infection, further research is necessary.