The Red Queen Hypothesis (RQH) is an evolutionary concept that describes the coevolution between RNA viruses and their hosts. This theory is based on an excerpt from the book “Through the Looking Glass”, by Lewis Carroll, in which the Red Queen states: “Here it is necessary to run as fast as possible to stay in the same place”[1]. This idea of constant racing and competitiveness applies to the world of RNA viruses and their hosts[2]. RNA viruses are infectious agents that have ribonucleic acid as their genetic material. They have a relatively high mutation rate and, as a result, can evolve quickly. The Red Queen theory suggests that because of this high mutation rate, RNA viruses are constantly adapting and evolving to evade their host's defense mechanisms[3]. Hosts, in turn, are also constantly evolving to develop defense strategies against viruses, such as methylation, an epigenetic modification, that plays a pivotal role in regulating gene expression and cellular functions in host organisms. The addition of methyl groups to DNA or proteins can alter their activity, impacting various biological processes. In the context of viral infections, methylation patterns in host cells can act as a regulatory mechanism that influences the host's susceptibility to viral attacks[4]. Changes in methylation patterns can affect the expression of genes involved in antiviral defense mechanisms, thereby influencing the host's ability to recognize and combat invading viruses[5]. Differential methylation profiles in host cells can determine the outcome of viral interactions, ranging from successful immune clearance to persistent infection[6]. DNA methylation alterations can regulate the expression of antimicrobial peptides, cytokines, and other immune-related factors, thereby enhancing the host's ability to mount an effective immune response, plus DNA methylation patterns induced by pathogens can act as signals that alert the host immune system to the presence of foreign invaders[7]. These epigenetic modifications can help in distinguishing self from non-self, enabling the host to initiate targeted immune responses against pathogens while maintaining tolerance to self-antigens. On the other hand and alongside with mutations the methylation of viral genomes has also been implicated in this arms race. The N6-methyl-adenosine (m6A) modification of viral RNA is involved in all phases of RNA metabolism, influencing viral gene expression, replication, and interactions with host cells[8]. This modification plays a crucial role in regulating viral RNA functions and dynamics during infection., such modifications can either support (proviral) or obstruct (antiviral) viral infections, indicating a complex pattern of epitranscriptomic regulation of viral gene expression and adds a new layer of regulation to viral RNA functions, influencing processes such as RNA stability, splicing, and translation[9]. The dynamic nature of m6A modification suggests that it occurs co-transcriptionally as viral RNAs are being transcribed. Methylation of specific regions in viral RNA contribute to the evasion of the host innate immune response, potentially enhancing viral replication and spread thus the modifications of viral RNA structures through methylation can impact interactions with host factors involved in immune recognition and response8, 10.

This leads to an evolutionary arms race, in which hosts are constantly trying to keep up with viral changes, while viruses are adapting to overcome host defenses[11]. This dynamic interaction between RNA viruses and their hosts has important implications for human and animal health. For example, RNA viruses such as the Influenza virus and human immunodeficiency virus (HIV) are known for their ability to evolve rapidly and evade immune responses12, 13. This makes the development of effective vaccines and long-lasting treatments against these infections challenging and a continuous non-stop process. The RQH in the context of RNA viruses highlights the evolutionary race between viruses and their hosts, where both are constantly adapting to survive and reproduce. Understanding this evolutionary dynamic is essential for the development of effective strategies to control and mitigate viral disease burden[14].

Dengue virus (DENV) is a mosquito-borne pathogen that causes a febrile illness called dengue fever, which affects millions of people around the world annually[15]. This virus belongs to the Flaviviridae family and has a positive single-stranded RNA genome. The genomic nature of the dengue virus and its replication rate give it a remarkable ability to evolve as evidenced in in vitro studies[16]. The DENV mutation rate of viral RNA is about 10,000 times greater than that of eukaryotic organisms. These mutations can lead to changes in viral proteins, allowing the virus to escape the host's immune system or acquire new replication properties. In addition, genetic recombination between different strains of the Dengue virus can generate new viral variants with unique characteristics. On the other hand, there are several constraints in Dengue virus evolution, mutations that lead to fitness enhancement are normally counterbalanced by long distance mutations with replication effects[16]. Natural selection also plays an important role in the evolution of the Dengue virus. Favorable genetic variations can increase the virus's ability to replicate its transmissibility. These well-adapted variants are more likely to spread in the host population, resulting in changes in the prevalence of different virus strains over time[17].

The ongoing evolution of the Dengue virus presents significant challenges for disease control and prevention[18]. Vaccination will begin shortly but in the meantime vector control and molecular diagnostics are key strategies to mitigate the spread of the Dengue virus. However, the constant genetic changes of the virus can lead to immune evasion and reduced vaccine effectiveness[19]. In addition, the evolution of the virus can lead to the emergence of more virulent variants or those with greater dissemination capacity, aggravating the impact of the disease, as it has been suggested for the DENV2 cosmopolitan variant20, 21. Therefore, understanding the evolution of the Dengue virus, including analysis of its genome is essential for the development of effective control and prevention strategies. Continuous genomic surveillance of the Dengue virus, combined with functional and epidemiological studies, makes it possible to monitor changes in the virus and adapt public health strategies to address the evolutionary and epidemiological challenges presented by this pathogen22, 23.

One of the first lines of antiviral host immune response against viral pathogens are through dendritic cells (DCs), these sentinel cells recognize pathogens and activate adaptive responses24, 25. The C-type lectin adhesion molecule, called DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin, CD209), is expressed on DCs and has been identified as an important receptor for DENV[26]. The interactions between DENV envelope protein (DENV_E) domain 2 (EDII) and DC-SIGN on DCs is known to be critical for virus entry into host cells. Mutation on the EDII can lead to increased viral replication and virus shedding26, 27. Several studies have investigated the molecular mechanisms involved in the EDII-DC-SIGN interaction and its impact on DENV pathogenesis24, 25, 26, 28. Binding of EDII to DC-SIGN was found to activate specific intracellular signaling pathways in DCs, resulting in modulation of the immune response[25]. Furthermore, this interaction promotes the transmission of DENV to other cell types, such as T cells, thus enhancing viral spread[29]. Detailed understanding of these interactions is essential for the development of effective therapeutic strategies against DENV infection. Therapeutic targets based on the EDII-DC-SIGN interaction can be exploited to prevent virus entry into DCs and modulate the immune response, potentially reducing the severity of the infection[28]. Interactions between EDII and DC-SIGN play a crucial role in the pathogenesis of DENV infection and specific binding regions are expected to be under intense antagonistic evolutionary pressure following the RQH[30].

The DENV_E is a transmembrane glycoprotein that plays a key role in virus entry into the host cell and in inducing the host's immune response[31]. The protein is composed of three main domains, domain I (EDI), domain II (EDII) and domain III (EDIII). EDI is a small cytoplasmic domain that binds to cellular proteins during virion assembly. EDII is an extracellular hydrophobic domain that penetrates the lipid bilayer of the cell membrane, allowing the virus to enter the cell[32]. EDII is also responsible for binding the virus to cell receptors. From a structural point of view, the DENV_E is composed of four domains, EDI (residues 1-52, 132-193, 280-296), EDII (residues 53-131, 194-279), EDIII (residues 297-394), which form the globular shape of the protein that lies on the lipidic surface, while a stem domain (residues 395-486) connects the EDI-EDIII domains to the transmembrane region[32] (Figure 1). The glycoprotein is formed by the cleavage of the precursor protein prM, which is cleaved during the maturation of the virus, forming the M protein and the DENV_E[32]. The M protein is necessary for the correct folding and stability of the DENV_E. The envelope protein is highly glycosylated and contains multiple glycosylation sites that may affect virus-host cell interaction and host immune response[32]. The DENV_E also contains several conserved regions that are targets for the production of neutralizing antibodies[31].

The EDI is an important region of the Dengue virus envelope protein. EDI is composed of a beta-hairpin loop, which is considered a highly conserved region of the Dengue envelope, and is important for the host immune response against the virus[32]. EDI is a critical region for the development of vaccines and therapies against the Dengue virus. Many studies have investigated the EDI-induced immune response in humans and animals, and some vaccination approaches based on EDI antigens have been tested in clinical trials[31]. In addition, some studies have explored the possibility of using EDI-specific neutralizing antibodies as a therapy for Dengue infection[33].

The main receptor for the DENV_E is a protein complex known as the flavivirus entry receptor (FVCR), which is composed of four different cellular proteins, CLEC5A, DC-SIGN, L-SIGN and TIM/TAM. DC-SIGN is a type II transmembrane lectin expressed in DCs and macrophages, which recognizes and binds to high mannose carbohydrates in viral proteins, including the DENV_E DII27, 34. The interaction between EDII and the DC-SIGN protein is a crucial process in the pathogenesis of DENV infection. Binding of EDII to DC-SIGN facilitates virus entry into host cells, allowing DENV to evade the innate immune response and replicate within host cells27, 35. Furthermore, the interaction between ED2 and DC-SIGN promotes cell-to-cell transmission of the virus, increasing viral shedding outside the host[27]. Understanding the interaction between the DENV EDII and the DC-SIGN has important implications for the development of Dengue prevention and treatment strategies. Inhibitors of EDII-DC-SIGN binding, for example, have the potential to block virus entry into host cells and therefore reduce the severity of infection27, 35.

The EDIII of the DENV_E also participates in the binding of the virus to cell receptors. This domain is highly conserved among different DENV serotypes and is considered an important target for vaccine development31, 32. The structure of EDIII consists of an antiparallel beta sheet with two loops that extend outwards. These loops contain amino acid residues that interact with cell receptors27, 32, 35. These interactions promote virus entry into the host cell and the spread of infection. Furthermore, EDIII is also an important target for the production of neutralizing antibodies[31]. Specific antibodies for EDIII can inhibit virus binding to cell receptors and thus prevent virus entry into the host cell. These antibodies can also activate the host immune system to destroy the virus[31].

In addition to DC-SIGN, the Dengue virus envelope can also bind to other receptors on the cell surface, such as cell adhesion protein 1 (DCAMKL1), integrin adhesion protein αvβ3 and the TIM protein (T-cell immunoglobulin and mucin domain)[36]. This discovery could have important implications for the development of new therapies and vaccines against the dengue virus. Several other cell surface receptors have been identified as playing crucial roles in Dengue virus infection in humans. Heparan Sulfate Proteoglycans (HSPGs) act as attachment factors, mediating the initial binding of Dengue virus particles to host cells. The laminin receptor has also been implicated as a Dengue virus receptor, aiding in virus entry into target cells. Furthermore, members of the Tyro3/Axl/Mer (TAM) family of receptor tyrosine kinases and certain integrin receptors are suggested to play roles in viral entry and infection. T cell Immunoglobulin and Mucin-domain containing proteins (TIMs) and Tyro3/Axl/Mer (TAM) family proteins may contribute to viral pathogenesis and immune evasion[36].

The DC-SIGN protein has the ability to recognize and bind to a wide variety of pathogens, including the Dengue virus[34]. The interaction between the Dengue virus envelope and DC-SIGN is mediated by specific amino acid residues in both proteins. In particular, DC-SIGN phenylalanine at position 313 (Phe 313) is a critical residue that is responsible for Dengue virus binding to DC-SIGN34, 35. Initial studies have shown that the mutation from phenylalanine to alanine (Phe 313 Ala) results in a significant reduction in the ability of the Dengue virus to bind to DC-SIGN and infect dendritic cells32, 34. The DC-SIGN structure is composed of a head domain and a neck domain. The head domain contains a CRD domain (carbohydrate recognition domain), which is responsible for recognizing and binding carbohydrates on the surface of pathogens. The neck domain contains a calcium-binding domain, which is essential for protein stability. The interaction between DC-SIGN and the Dengue virus envelope is mediated by the CRD domain of DC-SIGN and the viral envelope fusion region27, 28, 32. The CRD domain of DC-SIGN recognizes and binds to carbohydrates on the surface of the viral envelope, while the fusion region of the viral envelope is responsible for the fusion of the virus with the cell membrane26, 27, 35. Phe 313 in DC-SIGN is a critical residue that directly interacts with several residues in the DENV_E. Structural studies have shown that Phe 313 fits into a hydrophobic pocket present in the EDII, allowing Dengue virus binding to the protein27, 32, 36. The interaction between EDII residues and Phe 313 of the DC-SIGN receptor is a critical step in Dengue virus infection and is reinforced by other amino acid interactions in the viral envelope fusion region. In addition, Phe 313 is also considered a potential target for the development of new antiviral drugs that can interrupt the entry of the virus into the host cell, preventing the progression of the infection28, 36.

The history of dengue in Brazil begins with reference to the colonial period, where the first cases were registered in the city of Recife in 1685, followed by historical records in Salvador, Rio de Janeiro and São Paulo starting in the year 1846[37]. In the early 20th century, the pioneer Brazilian Doctor Oswaldo Cruz, implemented a mosquito control program that lasted for many years. The great challenge at the time was the yellow fever epidemic and Aedes aegypti was eradicated in Brazil in the 1950s by the continued efforts of the Oswaldo Cruz program, but was unfortunately reintroduced in the 1980s37, 38.

With the reemergence of dengue, the first major occurrence of dengue infection in Brazil was registered in the State of Roraima in 1981, with the registration, isolation and identification of serotypes DENV1 and DENV4[39]. Some time later, in 1986, the DENV1 serotype was again isolated and identified in the state of Rio de Janeiro and in some urban areas in the Northeast[37]. In 1990, the DENV2 serotype was isolated and identified in Rio de Janeiro and other cities in the Southeast of the country[37]. It was only in 2000 that the first records of isolation and identification of the DENV3 serotype occurred in Rio de Janeiro and later in the states of southern Brazil. From these initial records, DENV spread to other states across the country40, 41, 42, 43. All four DENV serotypes have circulated in Brazil, including the introduction of novel genotypes such as the DENV-2 Cosmopolitan genotype (DENV-2-GII). Among these, DENV-2-GII is one of the most prevalent genotypes, circulating across Asia-Pacific, the Middle East, Africa, and Oceania, significantly contributing to the global dengue burden[21].

Here, we combine phylogenetic inferences, structural modeling, and molecular dynamics (MD) analyses to describe the epidemiological characteristics of major Brazilian DENV strains that circulated from 1990 to 2022 from a combined perspective, thus providing us with a more detailed picture on the dynamics of such interactions over time. The goal of this study was to assess the influence of the mutations on circulating strains of DENV versus its interactions with a known receptor to all four serotypes via phylogenomics and structural biology methods such as modeling, guided docking, MD and MM/PBSA. Several authors have investigated similar hypotheses for other viruses but to our knowledge this is the first systematic report combining these methods to evaluate binding interactions with MD over time considering phylogenetic history as a basis[44].