In the Citespace analysis of alphavirus receptors from 2013 to 2024, a total of 167 publications were retrieved. The annual number of articles is shown in Figure 2 . The number of publications can be divided into three phases. The first phase is from 2013 to 2017 and presents a general downward trend, except for a slight increase in 2016. Notably, the number in 2013 reached a high point (n = 21), partly due to the 2012 outbreak of the East/Central/South African strain of CHIKV in Papua New Guinea and the spread of CHIKV to the Western Hemisphere (Zimmerman et al., 2023a). The number of studies reached its lowest point in 2017 (N = 8). The second phase is from 2017 to 2021 and shows a trend of continuous growth. The number in 2021 also reaches a high point (n = 20). The third phase is from 2022 to 2024, with a high level maintained in 2021 and 2023 and an additional article in the year 2024. Each trend may represent an outbreak of a specific alphavirus or new research on the receptor structure of the viruses and the host range of alphaviruses.

The publication of authors and their co-authorship networks can help identify outstanding scholars leading research in the field and provide information on collaboration among authors, which is crucial for future research development. We analyzed publications using a time-slicing of 1 year, resulting in a total of 270 authors involved in alphavirus receptor research. Each node in Figure 3A generated using Citespace (6.2. R7) represents an author, with the circle’s size indicating the number of articles published by the author. Diamond published the most papers (n = 26), followed by Kim (n = 8), Fremont (n = 8), Basore (n = 7), and Fox (n = 7). Figure 3A also illustrates the authors’ collaboration and contribution through the node size and link color depth. Diamond had the highest link strength, and their research collaborations appear to be steady and close. We then analyzed cited authors using a time-slicing of 2 years, selecting the top 50 levels per slice. Co-citation networks often have a vast number of links, and displaying links indiscriminately can cause clustering. According to citation analysis in Figure 3B generated using Citespace (6.2. R7), the most cited author was Weaver (n = 55), followed by Zhang (n = 45), Voss (n = 42), and Coudert (n = 37). It is apparent that many authors tend to collaborate with a relatively stable group of collaborators, resulting in several major author clusters typically consisting of two or more authors.

The institutional analysis runs over a time-slicing of 2 years. The collaboration map in Figure 4 illustrates the level of collaboration among these institutions. Washington University School of Medicine (n = 19), Johns Hopkins Bloomberg School of Public Health (n = 6), Griffith University (n = 5), and the University of North Carolina at Chapel Hill (n = 5) are the most prominent institutions in the collaboration map, indicating their significant role in this field.

An analysis of national publication counts reveals the countries/regions that have made the most contributions in this field. Figure 5A generated using Citespace (6.2. R7), Figure 5B analyzed by VOS viewer, and Figure 5C conducted by bibliometrix display the distribution of publications across 33 countries, primarily located in America, South and East Asia, Europe, and Australia. The USA leads with the highest number of publications (n = 100), followed by the UK (n = 15) and India (n = 12). Further analysis of publication numbers was used to construct a collaborative network based on the relationships between countries. Notably, there is extensive cooperation between different countries. The USA, China, Germany, and Australia demonstrate strong collaboration, as indicated by the thickness of the links. The USA, being the largest country, holds a central position in the alphavirus receptor field with a centrality value of 0.84. The UK ranks second with a centrality value of 0.32.

Each line segment in Figure 6 represents a period of time, and the red bar represents strong citation burstiness. The reference with the first citation burst is “Glycoprotein organization of CHIKV particles revealed by X-ray crystallography” (Voss et al., 2010), which gained prominence from 2013 to 2015. This study unveiled the molecular mechanism underlying the CHIKV invasion of susceptible cells, which is facilitated by two viral glycoproteins, E1 and E2 (Voss et al., 2010). The reference with the second citation burst is “Structural changes of envelope proteins during alphavirus fusion” (Li et al., 2010), cited from 2010 to 2014. In this article, the author reported the structure of SINV and the trimeric spike structure at low pH, representing an intermediate stage in the fusion process and elucidating the maturation process (Li et al., 2010). The reference with the third citation burst is “Mxra8 is a receptor for multiple arthritogenic alphaviruses” (Zhang et al., 2018), which has been widely referenced since 2019 and continues to be a topic of interest. With the application of a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-based screen, they identified the cell adhesion molecule Mxra8 as an entry mediator for multiple arthritogenic alphaviruses, including CHIKV, RRV, and ONNV (Zhang et al., 2018).

A word cloud was created by Bibliometrix to visualize the count of author keywords and present a visualization of the words that appear most frequently in research papers ( Figure 7A ). The most common word was “CHIKV”, the second most common word was “nonhuman”, and the third most common word was “alphavirus”. It is evident that receptors in different animal hosts are a key research topic in the field, with a significant portion of research focusing on humans as hosts. The word cloud provides more importance to author keywords that appear more frequently and serves as a visualization method to reveal the known research focus and trends in alphavirus receptor research.

To further investigate the hotspots in this field, keyword analysis was performed using Citespace (6.2.R7). Through cluster calculation, a total of eight clusters were obtained based on the co-occurrence of the keywords ( Figure 7B ). The keyword analysis showed the most occurring keywords related to alphavirus receptor were divided into the following eight clusters: sensing viral invasion, cross-protective ability, receptor ldlrad3, infected mice, Venezuelan equine, alphavirus encephalomyelitis, capsid protein, and authentic CHIKV cell entry. These eight clusters demonstrate the main themes of exploration in this field. The keywords of eight clusters were described along the landscape view ( Figure 7C ) according to the time order, which shows the research progress in the alphavirus receptor field from 2013 to 2024.

The infected mice were considered an important mammal model in the research process on virus receptors. The research process of NRAMP/NRAMP2 involved the mouse model for SINV. As for LDLRAD3, gene editing of mouse ldlrad3 can result in a low viral load in mouse neuronal cells. Next, it was discovered that another receptor, LDLR, was found in mouse neuronal cells. The multifunctional arthritogenic alphavirus capsid protein is crucial for viral infection and has roles in genome encapsulation, budding, and virion assembly. The arthritogenic alphaviral capsid protein can play an important role in vaccine design, can be regarded as a therapeutic target, and can play an important role in the development of diagnosis (Rao and Taylor, 2021). CHIKV can be genetically divided into three lineages: West African; East, Central, and South African; Indian Ocean; and Asian. Data suggested that IOL-based vaccine strains can offer robust cross-protection against strains from other lineages (Langsjoen et al., 2018). Other experiments suggest that antibodies against one of the arthritogenic alphaviruses can cross-react with other group members. However, effective cross-protection would require a higher dose and more vaccinations (Nguyen et al., 2020). The New World alphaviruses, which consist of VEEV, EEEV, and WEEV, induce encephalomyelitis. Cellular infection with alphaviruses can ultimately result in several outcomes. During infection with VEEV, the severity of the disease ranges from asymptomatic to rapid death, with clinically affected equids demonstrating fever and lethargy (Baxter and Heise, 2020).

Figure 7D displays the top 25 keywords with the strongest citation bursts. Keywords with citation bursts refer to keywords that are frequently cited by scholars in a specific field over time. Figure 7D illustrates each line segment representing a period of time, and the red bar represents strong citation burstiness. Citation bursts for keywords appeared as early as 2013 and as late as 2024. The keyword with the first citation burst is titled “Chikungunya alphavirus”. CHIKV is transmitted by arboreal Aedes mosquitoes in an enzootic cycle with nonhuman primates as the main reservoir (Vu et al., 2017). Research on the receptors of CHIKV includes PHB1 and MXRA8 (with bovine receptors not belonging to the host range). The keyword with the second strongest citation burst is titled “Nucleotide sequence”, which ended in 2014. The newly strongest citation bursts include virus flow cytometry, enzyme-linked immunosorbent assay, Vero cell line, tumor necrosis factor, glycosylation, and immune response.

Experimentally establishing the role of a surface protein as a virus receptor should meet four criteria: direct interaction between the virus and receptors; mediation of virus internalization by the receptor; inhibition of virus infection by neutralizing antibodies against the receptor, soluble receptor decoy molecules, or mutagenesis of the virus receptor binding domain; and correlation between susceptibility of a permissive cell type and receptor expression level (Holmes et al., 2020). Research on alphavirus receptors can be traced back to 1992, when the high-affinity laminin receptor was identified as the first receptor for SINV in mammalian cells ( Figure 8 ) (Wang et al., 1992). This receptor, a 67-kDa protein present on the cell surface, binds to basement membrane laminin and plays a role in tumor invasion. Blocking SINV binding to mammalian and mosquito cells was achieved using a monoclonal antibody against the C-terminal domain of the high-affinity laminin receptor ( Figure 9 ) (Wang et al., 1992).

In 2011, the second alphavirus receptor, NRAMP/NRAMP2, was discovered as a cellular receptor for SFV in both insect and mammalian hosts ( Figure 8 ) (Rose et al., 2011). The NRAMP is crucial for the binding and entry of SINV into Drosophila cells. Through genome-wide RNAi screening in Drosophila, it was found that dNRAMP, the single Drosophila homolog of mammalian NRAMPs, is important for virus binding and entry ( Figure 9 ). Overexpression of NRAMP2 increased the percentage of infected cells, while NRAMP2 deficiency rendered mammalian cells nonpermissive to SINV infection (Rose et al., 2011).

Another receptor, PHB1, was identified as a cellular receptor for CHIKV in human cells in 2012 ( Figure 8 ) (Wintachai et al., 2012). It was observed to interact with the E2 protein of CHIKV under confocal microscopy. Using a two-dimensional virus overlay protein-binding assay, PHB1 was identified as one of the CHIKV-binding proteins. Experimental results showed a significant reduction in the number of infected cells in the presence of anti-PHB1 antibodies. Co-localization, co-immunoprecipitation, and antibody- and siRNA-mediated infection inhibition studies confirmed the interaction between PHB1 and the CHIKV E2 protein in human cells ( Figure 9 ) (Wintachai et al., 2012). Interestingly, PHB1 also serves as a putative receptor for DENV-3 in SH-SY5Y and CHME-3 cells (Sharma et al., 2020).

In 2018, a significant breakthrough in the field of alphavirus receptor research coincided with the widespread use of gene editing in scientific research. CRISPR, a natural element of the bacterial immune system, has been widely harnessed by prokaryotes as a crucial survival mechanism for self-defense, enabling the degradation of invading plasmid DNA or bacteriophage DNA (Loureiro and da Silva, 2019). The introduction of the CRISPR/Cas9 system enabled the targeted modification, insertion, or replacement of specific nucleic acid sequences (Cong et al., 2013; Mali et al., 2013; Sternberg et al., 2014). Published in 2018 by Zhang et al., an article discovered that MXRA8 could play a crucial role in the entry of several alphaviruses in both humans and mice ( Figure 8 ). Gene editing of mouse MXRA8 or human MXRA8 resulted in reduced viral infection levels in fibroblasts and skeletal muscle cells ( Figure 10 ) (Zhang et al., 2018). Over the following years, as science and technology continued to advance, researchers gained a deeper understanding of the protein structure of MXRA8 from multiple perspectives. Several articles attempted to explain the molecular connection between the virus and its receptor.

MXRA8, a cell adhesion molecule composed of two Ig-like domains, binds to a surface-exposed region across the A and B domains of the CHIKV E2 protein ( Figure 10 ) (Zhang et al., 2018; Basore et al., 2019; Song et al., 2019). Domain 1 is formed by the hinge region and domain 2 (Zhang et al., 2018). MXRA8 directly binds to CHIKV particles, enhancing virus attachment and internalization in humans. The determinants of the MXRA8-binding site in CHIKV are generally conserved among arthritogenic alphaviruses (44% conserved) but more divergent in encephalitic alphaviruses (14% conserved) (Basore et al., 2019).

Another publication in 2020 by Kim et al. (2020) expanded the range of species to include mice, rats, chimpanzees, dogs, horses, goats, sheep, and humans. Within the cattle MXRA8, a 15-amino acid insertion was identified in the C′–C″ loop of domain D1. This insertion prevented MXRA8 from binding to CHIKV, while other species did not contain this amino acid sequence insertion ( Figure 10 ). Experimental results presented in the article showed divergent outcomes when the amino acids were inserted or removed from bovine animals or when the 15-amino acids were removed from other animals.

In addition, MXRA8 can act as a receptor for SINV, WEEV, and related alphaviruses in avian reservoirs ( Figure 8 ). Structural analysis suggests that duck MXRA8 D1 contacts E1 of WEEV, while mammalian MXRA8 predominantly contacts CHIKV E2 (Zimmerman et al., 2023b). Interactions occur with the wrapped, intraspike, and interspike heterodimers. Duck MXRA8 D1 makes primary contact with WEEV E1 in four sites: the C–C′ loop, C″–D loop, D–E loop, and B–C connector ( Figure 10 ) (Zimmerman et al., 2023b). When D1 of the duck is replaced with D1 of the mouse MXRA8, MXRA8 fails to bind to WEEV. Further research suggests that reptiles may also serve as potential future hosts (Zimmerman et al., 2023b).

Accompanied by the application of CRISPR-Cas9 in the field of alphavirus receptors, several receptors were subsequently discovered. In 2021, LDLRAD3, VLDLR, and ApoER2, members of the LDLR family, were identified as receptors for alphaviruses ( Figure 8 ). The LDLRAD3 contains three LDLa repeats, which in other LDL receptor family members function to recognize ligands. LDLRAD3 also contains two conserved PPxY motifs, which can function as interaction motifs, and a conserved dileucine motif, which has been shown to mediate endocytosis in other proteins (Noyes et al., 2016). In 2020, Ma et al. (2020) found that gene editing of mouse Ldlrad3 or human LDLRAD3 resulted in highly reduced VEEV infection of neuronal cells. LDLRAD3 involves three LDL-receptor class-A domains involving D1, D2, and D3, with D1 predicted to interact with the VEEV as it is the farthest end of the plasma membrane. Injection of LDLRAD3(D1)-Fc can help mice against the infection of VEEV, further suggesting that D1 can interact with the virus (Ma et al., 2020). Both the antibodies for the LDLRAD3 and LDLRAD3-Fc fusion proteins block VEEV infection, which suggests a strategy for the prevention of severe VEEV infection (Ma et al., 2020). Two studies solved the structure of VEEV and LDLRAD3 in 2021, and domain 1 of LDLRAD3 is necessary and sufficient to support infection by VEEV ( Figure 8 ) (Basore et al., 2021; Ma et al., 2021). The soluble decoy proteins with multiple LA3 repeats inhibit EEEV infection in mouse cell culture. LDLRAD3 was also identified as an ACE2-independent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor through genome-wide CRISPR activation screening (Zhu et al., 2022).

In 2021, Clark et al. (2022) found the E2 and E1 glycoproteins of SFV, EEEV, and SINV can interact with the ligand-binding domains of VLDLR and ApoER2. Under the use of CRISPR-Cas9, VLDLR and ApoER2 were identified as the entry receptors for EEEV/SFV/SINV. VLDLR contains an N-terminal ligand-binding domain with cysteine-rich repeats, a cluster of ECG modules containing a β-propeller domain, and a membrane-proximal O-linked sugar domain. The study found that the E2 and E1 glycoproteins of SFV, EEEV, and SINV interact with the ligand-binding domains of VLDLR and ApoER2 (Clark et al., 2022). The administration of a VLDLR LBD-Fc fusion protein can help against a rapidly fatal Semliki Forest virus infection in mice.

In 2024, Ma et al. (2024) performed a CRISPR-Cas9 genome-wide loss-of-function screen with the expression of the chimeric alphavirus expressing the EEEV structural proteins in mouse neuronal cells and identified LDLR as a low-affinity receptor for EEEV, WEEV, and SFV entry ( Figure 8 ). Expression of LDLR on the surface of neuronal or non-neuronal cells can help facilitate the binding and infection of EEEV, WEEV, and SFV ( Figure 11 ). LDLR is a membrane protein composed of seven LA repeats, two ECG-like domains, a beta-propeller domain, another ECG-like domain, and a stretch of residues with multiple O-linked glycosylation sites. LA domain proteins with three or five tandem repeats were designed and expressed in the context of an Fc-fusion protein, which helped to counteract the activity of EEEV and WEEV in cell culture (Ma et al., 2024).

CD147 acts as an alternative receptor for SARS-CoV-2 entry into cells with low or undetectable ACE2 expression (Lim et al., 2022). It is also an entry factor for various viruses, including human immunodeficiency virus (HIV) (Pushkarsky et al., 2001) and SARS-CoV (Chen et al., 2005). Additionally, it was discovered as a receptor for alphavirus in 2021 by De Caluwé et al. (2021) ( Figure 8 ). It was found that affinity purification mass spectrometry can allow the identification of factors that facilitate entry of CHIKV in human cells, and it was further validated using CRISPR-Cas9 (De Caluwé et al., 2021). CD147 contains similar protein domains as another alphavirus receptor, MXRA8. The CD147 protein complex is involved in a step prior to viral RNA synthesis and can play an important role in the replication cycle of alphaviruses ( Figure 11 ) (De Caluwé et al., 2021). In summary, many recent studies focused on the hosts of various alphavirus receptors, the structure of receptors, and their binding with alphaviruses. They employed a variety of methods to identify potential receptors and gain a more detailed understanding of them. They not only provide guidance and support for further exploration of alphavirus receptors in the future but also lay the groundwork for investigating receptors of other viruses.