Erratum: Detection of two alphaviruses: Middelburg virus and Sindbis virus from enzootic amplification cycles in southwestern Uganda.

[This corrects the article DOI: 10.3389/fmicb.2024.1394661.].

Interaction of chikungunya virus glycoproteins with macrophage factors controls virion production.

Despite their role as innate sentinels, macrophages can serve as cellular reservoirs of chikungunya virus (CHIKV), a highly-pathogenic arthropod-borne alphavirus that has caused large outbreaks among human populations. Here, with the use of viral chimeras and evolutionary selection analysis, we define CHIKV glycoproteins E1 and E2 as critical for virion production in THP-1 derived human macrophages. Through proteomic analysis and functional validation, we further identify signal peptidase complex subunit 3 (SPCS3) and eukaryotic translation initiation factor 3 subunit K (eIF3k) as E1-binding host proteins with anti-CHIKV activities. We find that E1 residue V220, which has undergone positive selection, is indispensable for CHIKV production in macrophages, as its mutation attenuates E1 interaction with the host restriction factors SPCS3 and eIF3k. Finally, we show that the antiviral activity of eIF3k is translation-independent, and that CHIKV infection promotes eIF3k translocation from the nucleus to the cytoplasm, where it associates with SPCS3. These functions of CHIKV glycoproteins late in the viral life cycle provide a new example of an intracellular evolutionary arms race with host restriction factors, as well as potential targets for therapeutic intervention.

Nanopore sequencing provides snapshots of the genetic variation within salmonid alphavirus-3 (SAV3) during an ongoing infection in Atlantic salmon (Salmo salar) and brown trout (Salmo trutta).

Frequent RNA virus mutations raise concerns about evolving virulent variants. The purpose of this study was to investigate genetic variation in salmonid alphavirus-3 (SAV3) over the course of an experimental infection in Atlantic salmon and brown trout. Atlantic salmon and brown trout parr were infected using a cohabitation challenge, and heart samples were collected for analysis of the SAV3 genome at 2-, 4- and 8-weeks post-challenge. PCR was used to amplify eight overlapping amplicons covering 98.8% of the SAV3 genome. The amplicons were subsequently sequenced using the Nanopore platform. Nanopore sequencing identified a multitude of single nucleotide variants (SNVs) and deletions. The variation was widespread across the SAV3 genome in samples from both species. Mostly, specific SNVs were observed in single fish at some sampling time points, but two relatively frequent (i.e., major) SNVs were observed in two out of four fish within the same experimental group. Two other, less frequent (i.e., minor) SNVs only showed an increase in frequency in brown trout. Nanopore reads were de novo clustered using a 99% sequence identity threshold. For each amplicon, a number of variant clusters were observed that were defined by relatively large deletions. Nonmetric multidimensional scaling analysis integrating the cluster data for eight amplicons indicated that late in infection, SAV3 genomes isolated from brown trout had greater variation than those from Atlantic salmon. The sequencing methods and bioinformatics pipeline presented in this study provide an approach to investigate the composition of genetic diversity during viral infections.

Prevalence of Flavivirus and A lphavirus in bats captured in the state of Yucatan, southeastern Mexico.

Flavivirus (family Flaviviridae) and Alphavirus (family Togaviridae) are mosquito-borne viruses that poses a significant risk to public health worldwide. Examples of these viruses include Dengue virus (DENV) and Zika virus (ZIKV) in the Flavivirus genus, and Chikungunya virus (CHIKV) in the Alphavirus genus. The potential contribution of bats in the mosquito-to-human transmission cycle of these viral genera in the tropics has not been studied. Here, a total of 144 bats belonging to three families (Emballonuridae, Phyllostomidae, and Molossidae) and six species were captured for one year using mist nets in sites with different landscapes (forest and grassland) in the state of Yucatan, southeastern Mexico. Blood samples and rectal and oral swabs were collected to detect Flavivirus and Alphavirus RNA genomes through RT-PCR. Flavivirus RNA was detected in 53 individuals (36.8%; 95% CI: 29.4%-44.9%), and Alphavirus RNA was detected in 59 individuals (40.1%; 95% CI: 33.2%-49.2%). The sequences obtained were consistent with ZIKV and DENV, into the Flavivirus, and CHIKV into the Alphavirus positive samples. The prevalence of both Flavivirus and Alphavirus was higher during the dry season compared with the rainy season. This high positivity rate, highlighted in both Flavivirus and Alphavirus, suggests a potential contribution of bats in the circulation of these viral genera in sylvatic environments. Seasonal variation in viral genera prevalence, with higher prevalence during dry seasons than rainy seasons, may suggest specific viral activity patterns in response to climatic conditions.

Unraveling the complex interplay: immunopathology and immune evasion strategies of alphaviruses with emphasis on neurological implications.

Arthritogenic alphaviruses pose a significant public health concern due to their ability to cause joint inflammation, with emerging evidence of potential neurological consequences. In this review, we examine the immunopathology and immune evasion strategies employed by these viruses, highlighting their complex mechanisms of pathogenesis and neurological implications. We delve into how these viruses manipulate host immune responses, modulate inflammatory pathways, and potentially establish persistent infections. Further, we explore their ability to breach the blood-brain barrier, triggering neurological complications, and how co-infections exacerbate neurological outcomes. This review synthesizes current research to provide a comprehensive overview of the immunopathological mechanisms driving arthritogenic alphavirus infections and their impact on neurological health. By highlighting knowledge gaps, it underscores the need for research to unravel the complexities of virus-host interactions. This deeper understanding is crucial for developing targeted therapies to address both joint and neurological manifestations of these infections.

Mayaro Virus: An Emerging Alphavirus in the Americas.

Mayaro virus (MAYV) is an arbovirus first isolated in Trinidad and Tobago in 1954. MAYV is the causative agent of Mayaro fever, which is characterised by high fever, maculopapular rash, myalgia and arthralgia. The potential for chronic arthralgia is of particular clinical concern. Currently, MAYV outbreaks are restricted to South and Central America, with some cases reported in Africa as well as several imported cases in Europe. However, in recent years, MAYV has become a growing global concern due to its potential to emerge into urban transmission cycles. Challenges faced with diagnostics, as well as a lack of specific antivirals or licensed vaccines further exacerbate the potential global health threat posed by MAYV. In this review, we discuss this emerging arboviral threat with a particular focus on the current treatment and vaccine development efforts. Overall, MAYV remains a neglected arbovirus due to its limited area of transmission. However, with the potential of its urbanisation and expanding circulation, the threat MAYV poses to global health cannot be overlooked. Further research into the improvement of current diagnostics, as well as the development of efficacious antivirals and vaccines will be crucial to help prevent and manage potential MAYV outbreaks.

Chikungunya and Mayaro Viruses Induce Chronic Skeletal Muscle Atrophy Triggered by Pro-Inflammatory and Oxidative Response.

Chikungunya (CHIKV) and Mayaro (MAYV) viruses are arthritogenic alphaviruses that promote an incapacitating and long-lasting inflammatory muscle-articular disease. Despite studies pointing out the importance of skeletal muscle (SkM) in viral pathogenesis, the long-term consequences on its physiology and the mechanism of persistence of symptoms are still poorly understood. Combining molecular, morphological, nuclear magnetic resonance imaging, and histological analysis, we conduct a temporal investigation of CHIKV and MAYV replication in a wild-type mice model, focusing on the impact on SkM composition, structure, and repair in the acute and late phases of infection. We found that viral replication and induced inflammation promote a rapid loss of muscle mass and reduction in fiber cross-sectional area by upregulation of muscle-specific E3 ubiquitin ligases MuRF1 and Atrogin-1 expression, both key regulators of SkM fibers atrophy. Despite a reduction in inflammation and clearance of infectious viral particles, SkM atrophy persists until 30 days post-infection. The genomic CHIKV and MAYV RNAs were still detected in SkM in the late phase, along with the upregulation of chemokines and anti-inflammatory cytokine expression. In agreement with the involvement of inflammatory mediators on induced atrophy, the neutralization of TNF and a reduction in oxidative stress using monomethyl fumarate, an agonist of Nrf2, decreases atrogen expression and atrophic fibers while increasing weight gain in treated mice. These data indicate that arthritogenic alphavirus infection could chronically impact body SkM composition and also harm repair machinery, contributing to a better understanding of mechanisms of arthritogenic alphavirus pathogenesis and with a description of potentially new targets of therapeutic intervention.

Eilat virus (EILV) causes superinfection exclusion against West Nile virus (WNV) in a strain-specific manner in Culex tarsalis mosquitoes.

West Nile virus (WNV) is the leading cause of mosquito-borne illness in the USA. There are currently no human vaccines or therapies available for WNV, and vector control is the primary strategy used to control WNV transmission. The WNV vector Culex tarsalis is also a competent host for the insect-specific virus (ISV) Eilat virus (EILV). ISVs such as EILV can interact with and cause superinfection exclusion (SIE) against human pathogenic viruses in their shared mosquito host, altering vector competence for these pathogenic viruses. The ability to cause SIE and their host restriction make ISVs a potentially safe tool to target mosquito-borne pathogenic viruses. In the present study, we tested whether EILV causes SIE against WNV in mosquito C6/36 cells and C. tarsalis mosquitoes. The titres of both WNV strains - WN02-1956 and NY99 - were suppressed by EILV in C6/36 cells as early as 48-72 h post-superinfection at both m.o.i. values tested in our study. The titres of WN02-1956 at both m.o.i. values remained suppressed in C6/36 cells, whereas those of NY99 showed some recovery towards the final timepoint. The mechanism of SIE remains unknown, but EILV was found to interfere with NY99 attachment in C6/36 cells, potentially contributing to the suppression of NY99 titres. However, EILV had no effect on the attachment of WN02-1956 or internalization of either WNV strain under superinfection conditions. In C. tarsalis, EILV did not affect the infection rate of either WNV strain at either timepoint. However, in mosquitoes, EILV enhanced NY99 infection titres at 3 days post-superinfection, but this effect disappeared at 7 days post-superinfection. In contrast, WN02-1956 infection titres were suppressed by EILV at 7 days post-superinfection. The dissemination and transmission of both WNV strains were not affected by superinfection with EILV at either timepoint. Overall, EILV caused SIE against both WNV strains in C6/36 cells; however, in C. tarsalis, SIE caused by EILV was strain specific potentially owing to differences in the rate of depletion of shared resources by the individual WNV strains.

Molecular Epidemiology of Western Equine Encephalitis Virus, South America, 2023-2024.

Western equine encephalitis virus (WEEV) is a mosquitoborne virus that reemerged in December 2023 in Argentina and Uruguay, causing a major outbreak. We investigated the outbreak using epidemiologic, entomological, and genomic analyses, focusing on WEEV circulation near the Argentina‒Uruguay border in Rio Grande do Sul state, Brazil. During November 2023‒April 2024, the outbreak in Argentina and Uruguay resulted in 217 human cases, 12 of which were fatal, and 2,548 equine cases. We determined cases on the basis of laboratory and clinical epidemiologic criteria. We characterized 3 fatal equine cases caused by a novel WEEV lineage identified through a nearly complete coding sequence analysis, which we propose as lineage C. Our findings highlight the importance of continued surveillance and equine vaccination to control future WEEV outbreaks in South America.

Mosquitoes as Vectors of Mycobacterium ulcerans Based on Analysis of Notifications of Alphavirus Infection and Buruli Ulcer, Victoria, Australia.

Alphavirus infections are transmitted by mosquitoes, but the mode of transmission for Mycobacterium ulcerans, which causes Buruli ulcer, is contested. Using notification data for Victoria, Australia, during 2017-2022, adjusted for incubation period, we show close alignment between alphavirus and Buruli ulcer seasons, supporting the hypothesis of mosquito transmission of M. ulcerans.

Exploring Barmah Forest virus pathogenesis: molecular tools to investigate non-structural protein 3 nuclear localization and viral genomic determinants of replication.

Barmah Forest virus (BFV) is a mosquito-borne virus that causes arthralgia with accompanying rash, fever, and myalgia in humans. The virus is mainly found in Australia and has caused outbreaks associated with significant health concerns. As the sole representative of the Barmah Forest complex within the genus Alphavirus, BFV is not closely related genetically to other alphaviruses. Notably, basic knowledge of BFV molecular virology has not been well studied due to a lack of critical investigative tools such as an infectious clone. Here we describe the construction of an infectious BFV cDNA clone based on Genbank sequence and demonstrate that the clone-derived virus has in vitro and in vivo properties similar to naturally occurring virus, BFV field isolate 2193 (BFV2193-FI). A substitution in nsP4, V1911D, which was identified in the Genbank reference sequence, was found to inhibit virus rescue and replication. T1325P substitution in nsP2 selected during virus passaging was shown to be an adaptive mutation, compensating for the inhibitory effect of nsP4-V1911D. The two mutations were associated with changes in viral non-structural polyprotein processing and type I interferon (IFN) induction. Interestingly, a nuclear localization signal, active in mammalian but not mosquito cells, was identified in nsP3. A point mutation abolishing nsP3 nuclear localization attenuated BFV replication. This effect was more prominent in the presence of type I interferon signaling, suggesting nsP3 nuclear localization might be associated with IFN antagonism. Furthermore, abolishing nsP3 nuclear localization reduced virus replication in mice but did not significantly affect disease.IMPORTANCEBarmah Forest virus (BFV) is Australia's second most prevalent arbovirus, with approximately 1,000 cases reported annually. The clinical symptoms of BFV infection include rash, polyarthritis, arthralgia, and myalgia. As BFV is not closely related to other pathogenic alphaviruses or well-studied model viruses, our understanding of its molecular virology and mechanisms of pathogenesis is limited. There is also a lack of molecular tools essential for corresponding studies. Here we describe the construction of an infectious clone of BFV, variants harboring point mutations, and sequences encoding marker protein. In infected mammalian cells, nsP3 of BFV was located in the nuclei. This finding extends our understanding of the diverse mechanisms used by alphavirus replicase proteins to interact with host cells. Our novel observations highlight the complex synergy through which the viral replication machinery evolves to correct mutation errors within the viral genome.

Cross-Neutralizing Anti-Chikungunya and Anti-Dengue 2 IgG Antibodies from Patients and BALB/c Mice against Dengue and Chikungunya Viruses.

Dengue (DENV) and Chikungunya (CHIKV) viruses can be transmitted simultaneously by Aedes mosquitoes, and there may be co-infections in humans. However, how the adaptive immune response is modified in the host has yet to be known entirely. In this study, we analyzed the cross-reactivity and neutralizing activity of IgG antibodies against DENV and CHIKV in sera of patients from the Mexican Institute of Social Security in Veracruz, Mexico, collected in 2013 and 2015 and using IgG antibodies of BALB/c mice inoculated with DENV and/or CHIKV. Mice first inoculated with DENV and then with CHIKV produced IgG antibodies that neutralized both viruses. Mice were inoculated with CHIKV, and then with DENV; they had IgG antibodies with more significant anti-CHIKV IgG antibody neutralizing activity. However, the inoculation only with CHIKV resulted in better neutralization of DENV2. In sera obtained from patients in 2013, significant cross-reactivity and low anti-CHIKV IgG antibody neutralizing activity were observed. In CHIKV-positive 2015 sera, the anti-DENV IgG antibody neutralizing activity was high. These results suggest that CHIKV stimulates DENV2-induced memory responses and vice versa. Furthermore, cross-reactivity between the two viruses generated neutralizing antibodies, but exchanging CHIKV for DENV2 generated a better anti-CHIKV neutralizing response.

Semliki Forest Virus (SFV) Self-Amplifying RNA Delivered to J774A.1 Macrophage Lineage by Its Association with a Purified Recombinant SFV Capsid Protein.

The Semliki Forest virus capsid protein (C) is an RNA binding protein which exhibits both specific and unspecific affinities to single-strand nucleic acids. The putative use of the self-amplifying RNAs (saRNAs) of alphaviruses for biotechnological purpose is one of the main studied strategies concerning RNA-based therapies or immunization. In this work, a recombinant C protein from SFV was expressed and purified from bacteria and used to associate in vitro with a saRNA derived from SFV. Results showed that the purified form of C protein can associate with the saRNA even after high temperature treatment. The C protein was associated with a modified saRNA coding for the green fluorescent protein (GFP) and delivered to murine macrophage cells which expressed the GFP, showing that the saRNA was functional after being associated with the recombinant purified C protein.

Chikungunya virus release is reduced by TIM-1 receptors through binding of envelope phosphatidylserine.

T-cell immunoglobin and mucin domain protein-1 (TIM-1) mediates entry of chikungunya virus (CHIKV) into some mammalian cells through the interaction with envelope phospholipids. While this interaction enhances entry, TIM-1 has been shown to tether newly formed HIV and Ebola virus particles, limiting their efficient release. In this study, we investigate the ability of surface receptors such as TIM-1 to sequester newly budded virions on the surface of infected cells. We established a luminescence reporter system to produce chikungunya viral particles that integrate nano-luciferase and easily quantify viral particles. We found that TIM-1 on the surface of host cells significantly reduced CHIKV release efficiency in comparison to other entry factors. Removal of cell surface TIM-1 through direct cellular knock-out or altering the cellular lipid distribution enhanced CHIKV release. Over the course of infection, CHIKV was able to counteract the tethering effect by gradually decreasing the surface levels of TIM-1 in a process mediated by the nonstructural protein 2. This study highlights the importance of phosphatidylserine receptors in mediating not only the entry of CHIKV but also its release and could aid in developing cell lines capable of enhanced vaccine production. IMPORTANCE: Chikungunya virus (CHIKV) is an enveloped alphavirus transmitted by the bites of infectious mosquitoes. Infection with CHIKV results in the development of fever, joint pain, and arthralgia that can become chronic and last for months after infection. Prevention of this disease is still highly focused on vector control strategies. In December 2023, a new live attenuated vaccine against CHIKV was approved by the FDA. We aimed to study the cellular factors involved in CHIKV release, to better understand CHIKV's ability to efficiently infect and spread among a wide variety of cell lines. We found that TIM-1 receptors can significantly abrogate CHIKV's ability to efficiently exit infected cells. This information can be beneficial for maximizing viral particle production in laboratory settings and during vaccine manufacturing.

Displaying alphavirus physicochemical consensus antigens on immunogenic liposomes enhances antibody elicitation in mice.

Cobalt-porphyrin phospholipid displays recombinant protein antigens on liposome surfaces via antigen polyhistidine-tag (His-tag), and when combined with monophosphorylated lipid A and QS-21 yields the "CPQ" vaccine adjuvant system. In this proof of principle study, CPQ was used to generate vaccine prototypes that elicited antibodies for two different alphaviruses (AV). Mice were immunized with computationally designed, His-tagged, physicochemical property consensus (PCPcon) protein antigens representing the variable B-domain of the envelope protein 2 (E2) from the serotype specific Venezuelan Equine Encephalitis Virus (VEEVcon) or a broad-spectrum AV-antigen termed EVCcon. The CPQ adjuvant enhanced the antigenicity of both proteins without eliciting detectable anti-His-tag antibodies. Antibodies elicited from mice immunized with antigens admixed with CPQ showed orders-of-magnitude higher levels of antigen-specific IgG compared to alternative control adjuvants. The ELISA results correlated with antiviral activity against VEEV strain TC83 and more weakly to Chikungunya virus 118/25. Thus, display of E.coli-produced His-tagged E2 protein segments on the surface of immunogenic liposomes elicits high levels of antigen-specific and AV neutralizing antibodies in mice with vaccination, while facilitating vaccine preparation and providing dose-sparing potential.

Comparative pathogenesis of three Mayaro virus genotypes in the cynomolgus macaque.

Mayaro virus (MAYV), a mosquito-borne alphavirus, is considered an emerging threat to public health with epidemic potential. Phylogenetic studies show the existence of three MAYV genotypes. In this study, we provide a preliminary analysis of the pathogenesis of all three MAYV genotypes in cynomolgus macaques (Macaca facicularis, Mauritian origin). Significant MAYV-specific RNAemia and viremia were detected during acute infection in animals challenged intravenously with the three MAYV genotypes, and strong neutralizing antibody responses were observed. MAYV RNA was detected at high levels in lymphoid tissues, joint muscle and synovia over 1 month after infection, suggesting that this model could serve as a promising tool in studying MAYV-induced chronic arthralgia, which can persist for years. Significant leucopenia was observed across all MAYV genotypes, peaking with RNAemia. Notable differences in the severity of acute RNAemia and composition of cytokine responses were observed among the three MAYV genotypes. Our model showed no outward signs of clinical disease, but several major endpoints for future MAYV pathology and intervention studies are described. Disruptions to normal blood cell counts and cytokine responses were markedly distinct from those observed in macaque models of CHIKV infection, underlining the importance of developing non-human primate models specific to MAYV infection.

Safety concern of recombination between self-amplifying mRNA vaccines and viruses is mitigated in vivo.

Self-amplifying mRNA (SAM) vaccines can be rapidly deployed in the event of disease outbreaks. A legitimate safety concern is the potential for recombination between alphavirus-based SAM vaccines and circulating viruses. This theoretical risk needs to be assessed in the regulatory process for SAM vaccine approval. Herein, we undertake extensive in vitro and in vivo assessments to explore recombination between SAM vaccine and a wide selection of alphaviruses and a coronavirus. SAM vaccines were found to effectively limit alphavirus co-infection through superinfection exclusion, although some co-replication was still possible. Using sensitive cell-based assays, replication-competent alphavirus chimeras were generated in vitro as a result of rare, but reproducible, RNA recombination events. The chimeras displayed no increased fitness in cell culture. Viable alphavirus chimeras were not detected in vivo in C57BL/6J, Rag1-/- and Ifnar-/- mice, in which high levels of SAM vaccine and alphavirus co-replicated in the same tissue. Furthermore, recombination between a SAM-spike vaccine and a swine coronavirus was not observed. In conclusion we state that although the ability of SAM vaccines to recombine with alphaviruses might be viewed as an environmental safety concern, several key factors substantially mitigate against in vivo emergence of chimeric viruses from SAM vaccine recipients.

In situ fate of Chikungunya virus replication organelles.

Chikungunya virus (CHIKV) is a mosquito-borne pathogen responsible for an acute musculoskeletal disease in humans. Replication of the viral RNA genome occurs in specialized membranous replication organelles (ROs) or spherules, which contain the viral replication complex. Initially generated by RNA synthesis-associated plasma membrane deformation, alphavirus ROs are generally rapidly endocytosed to produce type I cytopathic vacuoles (CPV-I), from which nascent RNAs are extruded for cytoplasmic translation. By contrast, CHIKV ROs are poorly internalized, raising the question of their fate and functionality at the late stage of infection. Here, using in situ cryogenic-electron microscopy approaches, we investigate the outcome of CHIKV ROs and associated replication machinery in infected human cells. We evidence the late persistence of CHIKV ROs at the plasma membrane with a crowned protein complex at the spherule neck similar to the recently resolved replication complex. The unexpectedly heterogeneous and large diameter of these compartments suggests a continuous, dynamic growth of these organelles beyond the replication of a single RNA genome. Ultrastructural analysis of surrounding cytoplasmic regions supports that outgrown CHIKV ROs remain dynamically active in viral RNA synthesis and export to the cell cytosol for protein translation. Interestingly, rare ROs with a homogeneous diameter are also marginally internalized in CPV-I near honeycomb-like arrangements of unknown function, which are absent in uninfected controls, thereby suggesting a temporal regulation of this internalization. Altogether, this study sheds new light on the dynamic pattern of CHIKV ROs and associated viral replication at the interface with cell membranes in infected cells.IMPORTANCEThe Chikungunya virus (CHIKV) is a positive-stranded RNA virus that requires specialized membranous replication organelles (ROs) for its genome replication. Our knowledge of this viral cycle stage is still incomplete, notably regarding the fate and functional dynamics of CHIKV ROs in infected cells. Here, we show that CHIKV ROs are maintained at the plasma membrane beyond the first viral cycle, continuing to grow and be dynamically active both in viral RNA replication and in its export to the cell cytosol, where translation occurs in proximity to ROs. This contrasts with the homogeneous diameter of ROs during internalization in cytoplasmic vacuoles, which are often associated with honeycomb-like arrangements of unknown function, suggesting a regulated mechanism. This study sheds new light on the dynamics and fate of CHIKV ROs in human cells and, consequently, on our understanding of the Chikungunya viral cycle.

Changes in the chikungunya virus E1 glycoprotein domain II and hinge influence E2 conformation, infectivity, and virus-receptor interactions.

In a previous study to understand how the chikungunya virus (CHIKV) E1 glycoprotein ß-strand c functions, we identified several attenuating variants at E1 residue V80 and the emergence of second-site mutations in the fusion loop (E1-M88L) and hinge region (E1-N20Y) with the V80 variants in vivo. The emergence of these mutations led us to question how changes in E1 may contribute to CHIKV infection at the molecular level. Here, we use molecular dynamics to understand how changes in the E1 glycoprotein may influence the CHIKV glycoprotein E1-E2 complex. We found that E1 domain II variants lead to E2 conformational changes, allowing us to hypothesize that emerging variants E1-M88L and E1-N20Y could also change E2 conformation and function. We characterized CHIKV E1-M88L and E1-N20Y in vitro and in vivo to understand how these regions of the E1 glycoprotein contribute to host-specific infection. We found that CHIKV E1-N20Y enhanced infectivity in mosquito cells, while the CHIKV E1-M88L variant enhanced infectivity in both BHK-21 and C6/36 cells and led to changes in viral cholesterol-dependence. Moreover, we found that E1-M88L and E1-N20Y changed E2 conformation, heparin binding, and interactions with the receptor Mxra8. Interestingly, the CHIKV E1-M88L variant increased replication in Mxra8-deficient mice compared to WT CHIKV, yet was attenuated in mouse fibroblasts, suggesting that residue E1-M88 may function in a cell-type-dependent entry. Taken together, these studies show that key residues in the CHIKV E1 domain II and hinge region function through changes in E1-E2 dynamics to facilitate cell- and host-dependent entry.IMPORTANCEArboviruses are significant global public health threats, and their continued emergence around the world highlights the need to understand how these viruses replicate at the molecular level. The alphavirus glycoproteins are critical for virus entry in mosquitoes and mammals, yet how these proteins function is not completely understood. Therefore, it is critical to dissect how distinct glycoprotein domains function in vitro and in vivo to address these gaps in our knowledge. Here, we show that changes in the CHIKV E1 domain II and hinge alter E2 conformations leading to changes in virus-receptor and -glycosaminoglycan interactions and cell-specific infection. These results highlight that adaptive changes in E1 can have a major effect on virus attachment and entry, furthering our knowledge of how alphaviruses infect mammals and insects.