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# Chikungunya virus and other emerging arthritogenic alphaviruses

William M de Souza, Marc Lecuit, Scott C Weaver

► To cite this version:

William M de Souza, Marc Lecuit, Scott C Weaver. Chikungunya virus and other emerging arthritogenic alphaviruses. Nature Reviews Microbiology, 2025, 23 (9), pp.585-601. 10.1038/s41579-025-01177-8. pasteur-05320171

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https://pasteur.hal.science/pasteur-05320171v1

Submitted on 17 Oct 2025

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# Chikungunya and other emerging arthritogenic alphaviruses

William M. de Souza<fnref n="1" />, Marc Lecuit<fnref n="2" />,<fnref n="3" />, and Scott C. Weaver<fnref n="4" />*

## Abstract

Arthritogenic alphaviruses are arboviruses (arthropod-borne viruses) transmitted by mosquitoes that cause epidemics on a global scale. These positive-strand RNA viruses are genetically and serologically related, classified into the genus *Alphavirus* (*Togaviridae* family), and cause similar diseases in humans, mainly characterized by fever and often debilitating, sometimes chronic polyarthralgia. At present, most arthritogenic alphaviruses lack approved treatments or vaccines, and the recently licensed vaccines against chikungunya is awaiting implementation in endemic areas. Most arthritogenic alphaviruses are currently limited to specific geographic areas due to vector distributions and amplifying host availability, but they pose a significant risk of emergence. The exception is chikungunya virus, which has emerged repeatedly from Africa, established sustained and efficient transmission in urban areas, including in temperate climates, and caused major epidemics nearly worldwide. In this review, we highlight recent advances about the transmission cycles of the arthritogenic alphaviruses, and their vectors, epidemiology, transmission dynamics, evolution, pathophysiology, and immune responses. We also outline strategies and countermeasures to anticipate and mitigate the impact of arthritogenic alphaviruses on human health.

## INTRODUCTION

Alphaviruses are single-stranded, positive-sense RNA viruses that belong to the *Togaviridae* family<fnref n="1" />. Most of these viruses are transmitted by mosquitoes and categorized into two groups based on the diseases they cause: arthritogenic and encephalitic. Encephalitic alphaviruses (e.g., eastern-, western- and Venezuelan equine encephalitis viruses) are endemic in the Americas and cause outbreaks of central nervous system infections like meningitis and encephalitis, affecting both humans and equids<fnref n="2" />⁻<fnref n="4" />. Arthritogenic alphaviruses are globally distributed and cause predominantly acute and chronic polyarthralgia in humans, resulting in significant public health and economic burdens. Currently, chikungunya virus (CHIKV; species *Alphavirus chikungunya*) causes the highest disease burden, with estimate 16.9 million cases annually, and nearly 2.8 billion people live in areas at risk for infection⁵. Other emerging arthritogenic alphaviruses remain mostly restricted to geographic areas where they were discovered, such as Mayaro virus (MAYV; *Alphavirus mayaro*) in Latin America and the Caribbean, Ross River (RRV; *Alphavirus rossriver*) and Barmah Forest viruses (BFV; *Alphavirus barmah*) in Oceania, and o'nyong-nyong virus (ONNV; *Alphavirus onyong*) in Africa (Fig. 1). These viruses share genetic and antigenic similarities, as well as similar clinical signs and symptoms in humans including fever, rash, myalgia, and debilitating musculoskeletal manifestations. Other severe manifestations reported include central nervous system (CNS)

<footnote n="1">¹ Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, College of Medicine, Lexington, Kentucky, USA.</footnote>

<footnote n="2">² Institut Pasteur, Université Paris Cité, Inserm U1117, Biology of Infection Unit, Paris, France.</footnote>

<footnote n="3">³ Department of Infectious Diseases and Tropical Medicine, APHP, Institut Imagine, Necker-Enfants Malades University Hospital, Paris, France.</footnote>

<footnote n="4">⁴ World Reference Center for Emerging Viruses and Arboviruses, Institute for Human Infections and Immunity, and Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, USA.</footnote>

<footnote>*Email: sweaver@utmb.edu</footnote>
disease, pregnancy and neonatal complications, and deaths⁶⁻⁹. There are no specific treatments or countermeasures available for most arthritogenic alphavirus infections.

<table><thead><tr><th></th><th>CHIKV</th><th>MAYV</th><th>ONNV</th><th>RRV</th><th>BFV</th></tr></thead><tbody><tr><td>Disease</td><td colspan="5">Musculoskeletal diseases often cause debilitating joint pain, ranging from acute to chronic, and affecting one or multiple joints</td></tr><tr><td>Severe outcomes</td><td>CNS infection, pregnancy complications and deaths</td><td>CNS infection</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Cases</td><td>16.9 million per year</td><td>Undetermined (neglected disease)</td><td>2 millions (1959-1962)</td><td>4.000 per year</td><td>400 per year</td></tr><tr><td>Most recent outbreaks</td><td>Ongoing</td><td>Ongoing</td><td>2015-2016</td><td>Ongoing</td><td>Ongoing</td></tr><tr><td>Countries and regions reporting circulation</td><td>111</td><td>11</td><td>17</td><td>8</td><td>2</td></tr><tr><td>Transmission cycles</td><td>Urban and enzootic</td><td>Enzootic</td><td>Enzootic</td><td>Enzootic</td><td>Enzootic</td></tr><tr><td>Main vectors</td><td>Ae. aegypti and Ae. albopictus</td><td>Haemagogus spp. and Sabethes spp.</td><td>An. funestus and An. gambiae</td><td>Ae. vigilax and Ae. camptorhynchus</td><td>Cx. annulirostris and Ae. camptorhynchus</td></tr><tr><td>Aedes mosquitoes transmission</td><td>Yes</td><td>Experimentally</td><td>Experimentally</td><td>Yes</td><td>No</td></tr><tr><td>Vaccines licensed</td><td>Ixchig and Vimkunya (USA, Canada and Europe)</td><td>Unavailable</td><td>Unavailable</td><td>Unavailable</td><td>Unavailable</td></tr></tbody></table>

Geographical distribution

<figure><img src="image_3.png" /><figcaption>**Figure 1.** Overview of key features of the arthritogenic alphaviruses. These include virus, diseases, severe outcomes, number of cases, most recent outbreaks, countries and regions affected, transmission cycles, main vector, *Aedes* mosquito transmission (specifically *Ae. aegypti* or *Ae. albopictus*), licensed vaccines, and geographical distribution about CHIKV⁵,⁹⁻¹¹, MAYV¹²⁻¹⁴, ONNV¹⁵⁻¹⁷, RRV¹⁸,¹⁹, and BFV¹⁸,²⁰,²¹. CNS, central nervous system; CHIKV, chikungunya virus; MAYV, Mayaro virus; RRV, Ross River virus; ONNV, o'nyong-nyong virus; BFV, Barmah Forest virus.</figcaption></figure>

Arthritogenic alphaviruses are primarily spread through enzootic transmission cycles involving sylvatic mosquitoes and wild vertebrate hosts and can spillover to humans. The spillover events are a critical step to emergence that is characterized by newly appearing in a population or having existed but are rapidly increasing in incidence or geographic range. For example, CHIKV has established a sustained human-amplified cycle through two major invasive *Aedes* mosquito species, *Aedes* (Stegomyia) *aegypti* and *Ae. (Stegomyia) albopictus*²²,²³. Human-amplified transmission has been key to the major epidemics reported since 2004. In addition, climate change and other anthropogenic factors, including deforestation, land-use change, and human mobility, also contribute to the geographic expansion of CHIKV and other emerging
arthritogenic alphaviruses. Mechanistically, deforestation and land-use changes may increase the opportunity for spillover from enzootic cycles to initiate human-amplified transmission²⁴. Human mobility and international commerce also contribute to introducing arthritogenic alphaviruses into urban vectors and immunologically naive populations, respectively, while climate change renders regions permissive for transmission in previously temperate regions²⁴.

In this review, we discuss key aspects of arthritogenic alphaviruses within the Semliki Forest serocomplex in the context of recent advances in their study. First, we review viral ecology and transmission dynamics, providing an overview of the transmission cycles, epidemiology, evolution, and factors contributing to viral emergence. This includes advances in the understanding of the dynamics of transmission, the effects of climate change, viral genetic factors, and geographical expansion. Second, we discuss their infection and replication in vertebrates, focusing on viral receptors and host factors, as well as recent advances in the transmission cycle. Third, we summarize recent advances in understanding the pathophysiology and immune response, highlighting similarities and differences across acute, subacute, and chronic manifestations, as well as severe outcomes. Finally, we provide an overview of recent advances in developing vaccines and therapeutics, including the licensed vaccines to prevent chikungunya. We do not explicitly discuss all closely related (e.g., Bebaru, Getah, Uná, and Semliki Forest) viruses due to limited evidence linking them to musculoskeletal diseases. We point out that Sindbis virus is also an important emerging mosquito-borne alphavirus that can cause human musculoskeletal diseases in Africa, Europe, Asia, and Oceania²⁵⁻²⁸. However, this virus belongs to the western equine encephalitis serogroup complex and has been extensively developed as a model for studying central nervous system infections rather than musculoskeletal disease²⁹⁻³¹.

# ECOLOGY, EPIDEMIOLOGY, EVOLUTION, AND VIRAL TRANSMISSION DYNAMICS

## Transmission Cycle and Vectors

Arthritogenic alphaviruses are maintained in enzootic cycles between mosquito vectors and vertebrate hosts, including humans, non-human primates (NHPs), marsupials, birds, and possibly bats. These cycles are often restricted to specific geographic areas where the primary enzootic vector and amplifying host(s) are native. For instance, CHIKV circulates in a sylvatic cycle involving *Aedes* mosquitoes (e.g., *Ae. africanus* and *Ae. furcifer*) and many NHP species in sub-Saharan Africa³². Other wild vertebrates, such as rodents, bats, and birds, may also be involved in this enzootic cycle, but the evidence is inconclusive³³,³⁴ (Fig. 2). Consequently, CHIKV transmission primarily affects rural areas where humans are incidental hosts exposed to spillover transmission from NHPs by sylvatic or bridge vectors, sometimes leading to sporadic outbreaks³³, as well documented in eastern Senegal³⁵.

Between 2004 and 2005, a major epidemic occurred in coastal Kenya and the Comoros Islands, attributed to human-amplified, *Ae. aegypti* transmission³⁶ (Fig. 2). While this CHIKV lineage likely originated from an enzootic cycle in East Africa, the mechanisms and exact location enabling the shift from enzootic to human-amplified transmission remain unclear, with no evidence of initial human- or vector-adaptive viral mutations. This lineage subsequently spread through the Indian Ocean basin, South and Southeast Asia, Europe, the Pacific Islands, and the Americas via *Ae. aegypti*, and, in some locations, *Ae. albopictus* transmission³⁶. Previous CHIKV introductions from Africa to Asia and Oceania probably occurred centuries earlier, following the spread of domesticated *Ae. aegypti* from Africa throughout the tropics and establishment this species as the primary vector responsible for outbreaks, which were first identified definitively in the late 1950s.

Another arthritogenic alphavirus endemic to Africa is ONNV, primarily transmitted between humans by *Anopheles funestus* and *An. gambiae* mosquitoes, which are also malaria parasite vectors³⁷. The enzootic reservoir and amplification hosts for ONNV remain enigmatic, but neutralizing antibodies have been detected in antelopes, forest buffaloes, and mandrills in sub-Saharan Africa³⁸ (Fig. 2).
In Latin America and the Caribbean, MAYV is maintained in an enzootic cycle involving sylvatic *Haemagogus janthinomys* mosquitoes and NHPs, including marmosets (*Calithrix argentina*), as evidenced by virus isolations and serosurveys³⁹. MAYV has also been detected in or isolated from *Mansonia*, *Culex*, *Psorophora*, *Coquillettidia*, and *Sabethes* mosquitoes, and serologic evidence suggests that it can infect many wild mammals, including rodents, marsupials, pilosans, and avians¹²,¹³ (Fig. 2). Although emergence into a human-amplified, *Ae. aegypti* transmission cycle has not been documented, experimental studies indicate that MAYV can be transmitted by *Ae. aegypti* and *Ae. albopictus*⁴⁰,⁴¹. Importantly, human viremia titers in MAYV infections nearly reach those of CHIKV infections, and are sufficient for experimental *Ae. aegypti* and *Ae. albopictus* transmission⁴⁰,⁴¹.

In Oceania, RRV is transmitted in an enzootic cycle between salt-marsh mosquitoes and macropods, such as kangaroos and wallabies. Based on ecological and experimental studies, the main vectors are *Ae. vigilax* and *Ae. camptorhynchus*. In addition, *Coquillettidia linealis* and *Ae. notoscriptus* may transmit in peri-urban areas¹⁹ (Fig. 2). Similarly, BFV is endemic in Australia, and *Cx. annulirostris* is the major vector in inland areas, while *Ae. camptorhynchus* transmits in southern parts of Victoria and Tasmania and *Ae. (Ochlerotatus) vigilax* in New South Wales. The enzootic hosts remain undefined, but the detection of BFV-specific antibodies in kangaroos suggests that macropods are involved.

<figure><img src="image_4.png" /></figure>

**Figure 2.** Transmission cycles of arthritogenic alphaviruses. CHIKV is transmitted by forest *Aedes* mosquitoes (e.g., *Ae. africanus*, *Ae. furcifer*, and *Ae. dalzieli*) among various non-human primates and potentially fruit bats in tropical and subtropical regions of Africa. Humans are usually infected in forest and rural environments by spillover. In urban and peri-urban areas worldwide, CHIKV is transmitted by anthropophilic *Aedes* (*Stegomyia*) mosquitoes (*Ae. aegypti* and *Ae. albopictus*) among humans. RRV is transmitted by *Ae. vigilax* among macropods (kangaroos) and horses in rural and peri-urban areas of Australia, with incidental transmission to humans. MAYV is transmitted by forest mosquitoes (mainly
*Haemagogus janthinomys* and *Sabethes spp.* ) among non-human primates and birds in tropical and
subtropical forests of Central and South America. ONNV is transmitted by *Anopheles* mosquitoes
*(Anopheles funestus* and *An. gambiae*) in rural and peri-urban areas of Africa, with incidental transmission
to humans. The enzootic amplifying host(s) of ONNV remains unknown, but candidates include antelopes,
forest buffaloes, and mandrils. *Potential amplifying host based on serological evidence alone.*

**Epidemiology and Viral Transmission Dynamics**

CHIKV is the most important arthritogenic alphavirus, with over 10 million cases reported in over 110 countries in Africa, Asia, the Americas, Europe, and Oceania since 2004<sup>42</sup> (Fig. 1). The Americas had the highest number, with approximately 3 million cases since 2013<sup>10</sup>. CHIKV was first isolated in 1952 in present-day Tanzania, and outbreaks and epidemics are characterized by their explosive and unpredictable nature, with attack rates ranging from 35% to 86% in an affected area<sup>41</sup>. Herd immunity appears to limit the efficiency of CHIKV transmission because previous infections presumably induce lifelong immunity, protecting against re-infection<sup>7</sup>. However, some countries have experienced recurrent chikungunya outbreaks after years with low incidence and population turnover<sup>43,44,45</sup>. In West Africa, recurrences are attributed to enzootic CHIKV periodically causing spillover infections in people who contact forests, causing limited outbreaks<sup>33</sup>. The periodicity of these spillovers appears to be driven by the cycling of herd immunity among NHPs<sup>32</sup>. In urban environments, chikungunya recurrence is patchy and linked to local transmission pockets with high attack rates followed by spread to another location with remaining naïve populations<sup>43</sup>. The highly incapacitating nature of CHIKV infections may also limit spread, when incapacitated, viremic persons remain in their homes or in hospitals, while the lifetime flight range of *Ae. aegypti* is restricted to approximately 100 meters, limiting CHIKV spread compared to human spread by those infected by other arboviruses that are less often incapacitating, such as dengue and Zika viruses<sup>46</sup>.

MAYV has been endemic yet neglected in Latin America and the Caribbean, at least since
the 1950s (Fig. 1). The burden of Mayaro fever remains unknown, and reports of active circulation
affecting human populations remain scarce. MAYV, first isolated in 1954 in Trinidad and Tobago<sup>47</sup>,
has subsequently been identified in Brazil, Peru, Colombia, Ecuador, Venezuela, French Guiana,
and Haiti<sup>48</sup>. The sylvatic transmission cycle resembles that of yellow fever virus in South America,
which also involves *Haemagogus and Sabethes* mosquitoes and NHPs. From 1977-1978,
outbreaks of both Mayaro and yellow fever occurred in Belterra, Pará State, Brazil<sup>49</sup>.
Approximately 20% of residents were infected by MAYV, while five fatal cases of yellow fever
were reported. The greatest number of Mayaro cases was observed among persons residing
close to forests<sup>49</sup>. Similarly, a serosurvey in French Guiana found that most MAYV infections
occurred in rural areas, particularly among people with frequent forest exposure, such as adult
males<sup>50</sup>. Thus, occupational exposure in forest environments, such as mining, logging, and
fishing, can increase the risk of MAYV infection and may facilitate its introduction in urban settings
with the risk of future urbanization<sup>14</sup>.

ONNV has caused sporadic epidemics, followed by interepidemic disappearance for years or decades. A major epidemic occurred between 1959 and 1962, affecting an estimated 2 million patients in Kenya, Tanzania, Uganda, Malawi, the Central African Republic, the Democratic Republic of Congo, Mozambique, and Senegal<sup>15</sup> (Fig. 1). Later, sporadic outbreaks and cases were reported in Chad, Kenya, Liberia, Ivory Coast, and Uganda<sup>51-54</sup>. Serosurveys in Uganda and Kenya showed IgG positivity ranging from 3% to 45%, with estimated attack rates of 3% to 56%<sup>55,56</sup>. Since the last known outbreak in coastal Kenya, adults have exhibited higher seroprevalence than children, indicating that they are more likely to be exposed to ONNV infection<sup>56</sup>. Although serological cross-reactivity between ONNV and CHIKV complicates serosurveys, some studies suggest that ONNV occurs predominantly in East Africa and is rarely detected in West Africa. Corroborating this serodiagnostic challenge, CHIKV infection in Mali induced an ONNV-reactive immune response in 80% of cases, but ONNV infection led to a cross-
reactive CHIKV response in only 22% of cases⁵⁷. Other studies demonstrated that even neutralization tests cannot reliably distinguish between ONNV and CHIKV infections⁵⁸.

RRV and BFV were discovered in 1959 and 1974 in mosquitoes collected in Queensland and Victoria, Australia, respectively. RRV is endemic in multiple Oceanic countries, including Australia, Papua New Guinea, Fiji, the Cook Islands, American Samoa, French Polynesia, Wallis and Futuna, and New Caledonia, while BFV appears to be limited to Australia⁵⁹⁻⁶¹. A major RRV epidemic occurred in multiple Pacific Islands between 1979 and 1980, affecting approximately 500,000 individuals⁶⁰. In Fiji, the epidemic of polyarthritis was explosive, with nearly 92% of the population in affected villages testing seropositive⁶². This illustrates the potential for human-amplified transmission cycles in the absence of macropod hosts (e.g., kangaroos and wallabies).

Recently, most RRV and BFV cases have been reported in northern Australia during the rainy season, typically between December and February. RRV causes approximately 4,000 annual cases, while BFV has been implicated in over 400 cases¹⁸ (Fig. 1). Increased rainfall is associated with higher RRV notification rates, probably by increasing the abundance and survival of adult mosquitoes⁶³. A mechanistic model suggests that climate warming will likely increase transmission in temperate regions of Australia, where most of the population resides. Conversely, transmission in tropical areas where mean temperatures are already near the ideal (peaking at 26.4°C and dropping to zero at 17.0 and 31.5°C) is projected to decrease⁶⁴. Climate change and other anthropogenic factors may contribute to the redistribution of other arthritogenic alphaviruses worldwide, primarily through the impact of these factors on their vectors. This is anticipated because mosquitoes are ectothermic, with climate and weather patterns significantly affecting vector reproduction, survival, geographic distribution, and their ability to transmit pathogens, as discussed in recent reviews²⁴,⁶⁵,⁶⁶.

## Evolution

CHIKV has been the subject of larger evolutionary studies than other arthritogenic alphaviruses due to its major public health importance; it is classified into three major lineages: East/Central/South African (ECSA) and West African enzootic, and the Asian endemic/epidemic³³. The ECSA is highly diverse, with repeated emergence into human-amplified cycles in Africa, Asia, and the Americas. It gave rise to the Indian Ocean sub-lineage (IOL), which has caused epidemics in the Indian Ocean islands, South and Southeast Asia, and Europe since 2005, as well as the ECSA-American sub-lineage in South America since 2014¹⁰,³³. This Asian lineage was derived from an ECSA ancestor about a century ago and has been associated with human-amplified transmission in South and Southeast Asia since the 1950s¹⁰,³³. It was introduced into the Americas in late 2013 to form the Asian-American sub-lineage. The West African lineage remains in enzootic cycles detected in Senegal, Côte d'Ivoire, and Nigeria³³.

No amino acid substitutions have been associated with improving CHIKV fitness in humans, such as generating higher viremia, more severe disease, or tropism shifts to particular tissues or organs. However, some amino acid substitutions have affected fitness for vector transmission. For example, A226V (alanine change to valine at position 226) in the E1 protein of the CHIKV IOL can increase 40-fold the fitness for infection of Ae. albopictus⁶⁷⁻⁶⁹. Epistasis between amino acids in the envelope glycoproteins can affect the E1-226 phenotype; E1-98A identified in all ECSA African and IOL strains permits the adaptive phenotype in Ae. albopictus, while E1-98T, found in all Asian lineage strains but in no other CHIKV lineages, probably due to a founder effect, prevents penetrance of A226V⁷⁰. Similarly, threonine at E2 residue 211 of ECSA strains allows A226V to enhance infection of Ae. albopictus, but isoleucine at this position renders this substitution without a phenotype⁷⁰,⁷¹. Currently, the Asian-American and ECSA-American sub-lineages circulating in the Americas have E2-211I and lack E1-226V, consistent with their current inability to adapt easily for transmission by Ae. albopictus, a finding also supported by entomological surveillance and sequencing studies¹⁰. Other amino acid substitutions in some IOL strains that impact fitness for vector infection, include E2-K252Q, -K233E, and -L210Q, which
result in a 5-8-fold fitness increase for *Ae. albopictus* transmission⁷². Additionally, the R198Q/S18F substitutions in the E2/E3 proteins lead to a 16-fold increase in this fitness⁷². In contrast, the K211E/V264A substitutions in the E1/E2 proteins, identified in India in 2010, have slightly increased fitness in *Ae. aegypti*³³.

Another major determinant of CHIKV fitness and probably virulence is the 3' untranslated genome region (3'UTR). Asian strains, including those introduced into the Americas, have a low-fitness 3'UTR, believed to have originated as a founder effect when this strain was introduced into Asia from Africa a century ago⁷³. This fitness effect may be manifested in the apparently lower virulence due to a reduced replication capacity of Asian lineage CHIKV strains compared to ECSA strains⁷⁴⁻⁷⁷, including those introduced into Brazil and the Southern Cone of South America.

The evolution of other arthritogenic alphaviruses has been less studied due to the smaller number of cases and countries affected. No amino acid substitutions have been described that are associated with adaptations of MAYV, RRV, BFV, and ONNV to vectors or human amplification. MAYV comprises three genotypes: genotype L detected only in Brazil, genotype D documented in Brazil, Colombia, Peru, Venezuela, French Guiana, Guyana, and Trinidad and Tobago, and genotype N as the latest identified genotype in Peru in 2010⁷⁸. In contrast, the evolutionary relationships among ONNV strains seem not to be influenced by the location and/or year of viral collection, but the limited number of available genomes impedes a broad conclusion⁵¹. RRV comprises four genotypes, mostly reported in Australia: G1 in Queensland State; G2 in Western Australia State; G3 in Western Australia, Tasmania, New South Wales, and Victoria States, as well as Pacific Island countries and territories; and G4 is predominantly in Queensland and Western States⁷⁹. BFV comprises three distinct genotypes (G1–3) that circulate in Australia and Papua New Guinea. G1 is limited to Papua New Guinea, while G2 and G3 co-circulate in Australia, with G3 representing most contemporary strains⁸⁰. The BFV population appears more stable, with infrequent emergences of novel lineages compared to RRV.

# ENTRY AND REPLICATION

## Viral Receptor and Host Factors

Arthritogenic alphaviruses possess approximately a 12 kilobase, single-stranded, positive or messenger sense RNA genome that encodes four nonstructural proteins (nsP1, nsP2, nsP3, and nsP4) encoded by the genomic RNA and five structural proteins (capsid, E3, E2, 6K/TF, and E1) that are encoded by a subgenomic RNA. These proteins contribute to viral transcription and replication, and immune evasion. Mature virions are icosahedral particles composed of 80 trimeric E2-E1 heterodimer spikes arranged in T=4 symmetry with a diameter of approximately 70 nm⁸¹⁻⁸³. Alphavirus entry into cells requires the engagement of attachment factors and cellular receptors, which mediate cellular, tissue, and host tropism. Viral receptors reside on the host cell surface and bind to incoming virions in a specific manner, leading to internalization and productive infection. Matrix remodeling associated 8 (MXRA8) has been identified as the main receptor for arthritogenic alphaviruses, including CHIKV, MAYV, RRV, ONNV, and SFV⁸⁴,⁸⁵. Blocking or deleting MXRA8 *in vitro* and *in vivo* reduces infection, and MXRA8 appears to play a key role in pathogenesis⁸⁴,⁸⁶,⁸⁷. This occurs because of the unique binding mode with both domains of MXRA8 engaging the E2 and E1 proteins of the virion⁸¹,⁸⁸. Other candidate receptors are CD147 and prohibitin-1 (PHB1)⁸⁹,⁹⁰, but further experiments are required to determine their *in vivo* contribution to entry, infection, and pathogenesis. Additionally, heparan sulfate and phosphatidylserine receptor proteins (TIM1 and TIM4, T-cell immunoglobulin and mucin domain, and AXL, Axl receptor tyrosine kinase) can enhance attachment and infection of CHIKV and RRV⁹¹⁻⁹⁵. While adaptation for heparan sulfate binding, typical of cell culture passages, reduces virulence⁹¹⁻⁹³, the role of phosphatidylserine receptor proteins in pathogenesis remains unclear. In addition, some host factors, which can contribute to entry or replication but are insufficient for productive infection, have been identified, although cells lacking such factors likely are not permissive. For instance, four-and-a-half LIM domain protein 1 variant A (FHL1A) is a host factor
required for CHIKV and ONNV replication and pathogenesis (i.e., downstream of viral entry), but does not affect RRV and MAYV⁹⁶,⁹⁷. FHL1A is primarily expressed in the cytoplasm of striated muscles and fibroblasts and interacts with part of the hypervariable domain (HVD) of CHIKV nonstructural protein 3 (nsP3) during replication⁹⁶,⁹⁷. Moreover, several other cellular factors are required during arthritogenic alphavirus replication. For instance, G3BP1 and 2 (Ras-GTPase-activating SH3 domain-binding-proteins) interact with the HVD-nsP3 during CHIKV and MAYV infection, facilitating viral replication and inhibiting stress granule assembly⁹⁸⁻¹⁰². DExH-box RNA helicase 9 (DHX9) reduces CHIKV RNA synthesis and is required for optimal mature nonstructural protein translation¹⁰³. Furthermore, components of the endosomal sorting complex required for transport (ESCRT) are involved in CHIKV genome replication and protein processing¹⁰⁴. Finally, efficient replication of the CHIKV genome depends on chloride channels in both human and mosquito cells¹⁰⁵.

## Infection Cycle

Arthritogenic alphavirus replication begins with the binding of the E2/E1 spikes to the viral receptor (MXRA8) and host attachment factors (heparan sulfate, and phosphatidylserine receptors), facilitating entry (Fig. 3). After attachment, the virion undergoes clathrin-mediated endocytosis and membrane fusion in the endosome, where low pH triggers conformational changes in the viral envelope proteins to expose the E1 fusion peptide. The virion then fuses with the endosomal membrane, and the cytoplasm is extruded into the cytoplasm, which is disassembled to release the viral RNA. At early stages of infection, the nonstructural polyprotein open reading frame (ORF) is translated from the genomic RNA in two precursors of non-structural proteins (nsPs), which are cleaved and generate several forms of nsP1–nsP4. nsPs are critical to viral replication and evasion of host cell defenses. nsP1 is involved in negative strand RNA synthesis and has RNA capping properties. nsP2 possesses RNA helicase, RNA triphosphatase, and proteinase activities and inhibits host cell transcription. nsP3 is part of the replicase, and forms tubular structures that sequester host factors like FHL1, while nsP4 acts as viral RNA-dependent RNA polymerase¹⁰⁶. Together, these proteins form the viral replication complex that synthesizes a negative-strand RNA intermediate, which is used as a template for subgenomic and genomic RNAs¹⁰⁷. The subgenomic mRNA structural polyprotein ORF is translated into the polyprotein C-pE2-6K/TF-E1, with the autoproteolytic serine protease in the capsid protein releasing it into the cytoplasm. The precursor E2 (pE2) and E1 glycoproteins are produced through further processing in the secretory pathway, where host signal peptidases cleave pE2, 6K/TF, and E1. pE2 and E1 combine in the Golgi and then transit to the plasma membrane, where pE2 is cleaved into E2, which is responsible for binding to receptors, and E3, which mediates pE2 folding and its subsequent association with E1. The viral nucleocapsid, formed by the cytoplasmic binding of the capsid protein with genomic RNA, binds to a cytoplasmic domain of the E2 protein found in E2/E1 dimers embedded in the plasma membrane, triggering budding to form mature virions.

In addition to receptor-mediated endocytosis, arthritogenic alphaviruses can spread by cell-to-cell transmission (Fig. 3). For instance, CHIKV remodels the host cell architecture, inducing the formation of long intercellular extensions that form membrane bridges with neighboring cells, mediating efficient intercellular transmission¹⁰⁸ as described for other viruses¹⁰⁹,¹¹⁰. Overall, understanding the molecular interactions between arthritogenic alphaviruses and their host receptors, as well as the viral replication process, could inform the identification of antiviral targets, facilitating the development of therapeutics. However, species-specific differences between replication cycles of arthritogenic alphaviruses remain to be determined because most of the current knowledge is limited to CHIKV and encephalitic alphaviruses.
<figure><img src="image_5.png" /><figcaption>**Figure 3.** Arthritogenic alphavirus replication cycle. Arthritogenic alphavirus initiates infection by binding to specific receptors or host factors (e.g., MXRA8, heparan sulfate, and phosphatidylserine receptors) on susceptible cells. The virus enters the cell through endocytosis and is exposed to an acidic environment within the endosome, triggering conformational changes in the viral envelope. This exposes the E1 fusion peptide, which mediates fusion between the viral and host cell membranes, allowing the release of the viral genome into the cytoplasm. The viral genome is translated into the nsP polyproteins, which are subsequently processed into nsP1-nsP4. A subgenomic RNA is then produced, leading to the expression of the C-pE2-6K-E1 polyprotein precursor. This precursor is autocatalytically processed to release the capsid protein and generate the pE2 and E1 glycoproteins. pE2 and E1 proteins associate with the Golgi apparatus and are transported to the plasma membrane. pE2 is further cleaved into E2, which is involved in receptor binding, and E3, which plays a role in proper pE2 folding and association with E1. Viral assembly involves the binding of the nucleocapsid to the viral genome, followed by recruitment of the envelope glycoproteins from the inner cell membrane, followed by budding. In addition to cell-to-cell transmission, CHIKV can also infect new cells through a unique mechanism. The virus remodels the host cell architecture, inducing the formation of long intercellular extensions that project to form membrane bridges with neighboring cells, facilitating efficient intercellular CHIKV transmission.</figcaption></figure>

## PATHOPHYSIOLOGY AND CLINICAL MANIFESTATIONS

Arthritogenic alphaviruses can cause multisystemic human infections with a broad range of clinical presentations, from asymptomatic or self-limiting febrile episodes to severe and life-threatening diseases (Fig. 4). The clinical manifestations can be classified as acute, subacute, or chronic. Most cases present in the acute phase are characterized by fever (≥39°C), myalgia, headache, rash, and often incapacitating polyarthralgia, primarily involving peripheral joints<sup>42,111</sup>. These manifestations can last up to three weeks. Subacute cases usually lead to fatigue with articular signs and symptoms between three weeks and three months. Chronic cases are defined as patients with debilitating polyarthralgia and polyarthritis, which can persist for over three months or even years<sup>112,113</sup>. The development of long-term arthralgia can vary from 20% to 70%
of infected individuals and appears to be age-dependent, occurring more often in adults than in children¹¹⁴. The mechanisms of chronic disease caused by arthritogenic alphaviruses are not fully understood, in part due to the lack of studies of human tissue samples and experimental models that do not fully recapitulate the diseases observed in humans (Box 1). The chronic disease results in significant hyperplasia and angiogenesis, resembling rheumatoid arthritis, and appears to be exacerbated in patients with underlying arthritis¹¹⁵. The effect of most arthritogenic alphavirus infections on bone health remains poorly understood, particularly their impact on fracture risk, viral persistence in bone during disease progression, and the role of immune cells in bone loss. Likewise, epidemiological studies suggest that CHIKV disproportionately affects adult females compared to males in some locations, potentially due to sex-dependent behaviors⁴³,¹¹⁶. Given that menopause is characterized by significant declines in female hormone levels that can also affect bone health, further studies investigating the potential impact of hormonal fluctuations in females infected with CHIKV or other arthritogenic alphaviruses may provide valuable insights into sex- and age-dependent pathogenesis and disease incidence. The pathophysiology and pathogenesis caused by CHIKV infection and other arthritogenic alphaviruses have been also discussed in previous reviews¹⁹,¹¹⁷⁻¹¹⁹.
<figure><img src="image_6.png" /><figcaption>**Figure 4. Pathophysiology and clinical manifestations of arthritogenic alphaviruses.** Arthritogenic alphavirus infections can lead to various clinical manifestations. The acute phase may include fever, myalgia, polyarthralgia, rash, cutaneous manifestations, and pharmacological and neurological complications. The chronic phase is predominantly characterized by myalgia and polyarthralgia. The virus initially replicates in dermal-resident cells near the inoculation site. Arthritogenic alphaviruses can cause systemic and multi-organ infections, leading to inflammation and dysregulation of proteins related to coagulation factors and complement. Fatal CHIKV infections appear to increase vascular permeability, leading to endothelial dysfunction. Arthritogenic alphaviruses infect macrophages and fibroblasts in the synovial joints, leading to tissue damage, increased production of pro-inflammatory cytokines (e.g., IFN-α, IFN-β, IL-8, IL-6, CCL2), and the recruitment of immune cells including monocytes, macrophages, T cells (CD4+ and CD8+), B cells, natural killer cells, and natural killer T cells. During pregnancy, CHIKV may infect the fetus through placental breaches around term and cause encephalopathy, encephalitis, and meningoencephalitis. CHIKV and MAYV can cause CNS infection, but the mechanisms of neuroinvasion remain unclear. One proposed mechanism for CHIKV neuroinvasion involves cytokine storms, including higher levels of CCL-2, which can contribute to the migration of infected CD14+ and CD16+ monocytes from</figcaption></figure>
blood vessels into the brain parenchyma through a "Trojan horse" strategy. Additionally, CHIKV appears to increase the expression of the zonula occludens 1 (ZO-1) protein and cleaves platelet endothelial cell adhesion molecule 1 (PECAM-1) in the cells of the blood-brain barrier, and CHIKV-cleaved PECAM-1 may facilitate access to the brain parenchyma through a paracellular strategy.

Immune Response

Arthritogenic alphaviruses are transmitted by infected mosquitoes through their saliva during a blood meal. Experimental mouse studies show that initial replication occurs in dermis-resident cells near the inoculation site, such as Langerhans cells, dermal fibroblasts, keratinocytes, and/or macrophages¹²⁰,¹²¹. Then, Ly6C+ monocytes infected in the skin promote local virus replication and facilitate transport to the draining lymph node, which may overcome the macrophage receptor with collagenous structure - lymphatic endothelial cells (MARCO-LEC) barrier¹²²,¹²³. This dissemination into the bloodstream enables viral spread to distal sites¹²⁴ (Fig. 4). Mosquito saliva components may also influence the local immune response, attracting susceptible cells to the local edema¹²². CHIKV and MAYV can cause multi-organ infections in humans, and experimental *in vitro* and *in vivo* studies suggest that endothelial cells and monocytes in the peripheral blood may contribute to the typically high viremia⁸,¹²⁰,¹²⁵,¹²⁶.

During the acute phase, monocytes, macrophages, and type I interferons (e.g., IFN-α and IFN-β) exhibit highly effective protection against arthritogenic alphavirus infection. In experimental mouse studies, both cells contribute to antiviral activity through efferocytosis and resolution of inflammation¹²⁷⁻¹²⁹, and these antiviral cytokines limit the rapid increase in viral replication during the early stages of infection¹³⁰. In addition, clinical and animal studies demonstrate that CHIKV infects macrophages and fibroblasts in the synovial joints, leading to tissue damage, increased production of pro-inflammatory cytokines (e.g., IFN-α, IFN-β, IL-8, IL-6, and CCL2), and the recruitment of immune cells including monocytes, macrophages, T cells, B cells, and natural killer cells, and natural killer T cells¹²¹,¹²⁶,¹³¹⁻¹³³ (Fig. 4). In the adaptive immune response, acute CHIKV infection triggers a robust CD8+ T cell response and killing of virus-infected cells, as described in patients¹³⁴,¹³⁵. However, high initial viral loads appear to lead to CD8+ T cell exhaustion and decreased efficiency in clearance¹³⁴,¹³⁶. Subsequently, CD4+ T cells become predominant during the chronic phase, contributing to inflammation and joint swelling. The chronic phase may be characterized by a decline in CD4+ effector T cell populations as the viral antigens are cleared and the need for T regulatory activation decreases in humans¹³⁷.

The mechanism of arthritogenic alphavirus chronic diseases remains poorly elucidated. Long-term, residual viral RNA and antigens in macrophages, fibroblasts, and myofibers in joints after systemic clearance may contribute to the chronic phase, as suggested by clinical and experimental mouse studies¹²¹,¹³⁸,¹³⁹. Moreover, CHIKV and RRV can directly infect human osteoblasts, stimulating the production of IL-6, which activates RANKL (receptor activator of nuclear factor kappa-B ligand), a protein that plays a key role in stimulating the fusion, attachment, activation, and survival of osteoclasts. Then, RANKL inhibits osteoprotegerin, leading to bone resorption in patients most affected by RRV and CHIKV infection¹⁴⁰⁻¹⁴³. IL-6 is also associated with the severity and chronic phase of chikungunya in humans¹⁴⁴,¹⁴⁵. Additionally, levels of IL-17, another cytokine associated with bone breakdown and resorption, have been reported at higher levels among patients with persistent arthralgia caused by CHIKV¹⁴². Overall, the inflammatory response has been linked to the characteristic joint pain in diseases caused by arthritogenic alphaviruses in patients and mouse models. However, many underlying mechanisms during the acute and chronic phases of arthritogenic alphavirus infection remain unclear.

Central Nervous System Infection

CHIKV can cause various neurological complications, including encephalopathy, including in neonates, encephalitis, meningoencephalitis, and neuropathy⁷,¹¹,¹⁴⁶⁻¹⁴⁸. Chikungunya neurological cases usually occur during the acute phase and can be fatal¹¹,¹⁴⁶,¹⁴⁷,¹⁴⁹,¹⁵⁰. For
instance, a cohort studied in Kenya showed that 9.2% (367 of 3,980) of pediatric patients (≤16 years old) diagnosed as chikungunya had neurological disease⁷. The infants (>12 months) represented 50.1% (184 of 367) of cases⁷. The overall case-fatality rate was 1.4% among CHIKV-positive cases⁷. The mechanism of CNS infection by arthritogenic alphaviruses remains poorly understood. CHIKV infection elicits a robust type I interferon response mediated by various host sensors with direct action on nonhematopoietic cells¹⁵¹, which may contribute to CNS damage due to the neurotoxic effects of type I interferons. Additionally, CHIKV appears to invade the CNS by infecting CD14⁺CD16⁺ monocytes in the presence of high levels of CCL-2 (an inflammatory chemokine), then migrates through the blood-brain barrier (BBB)⁸. This infected monocyte subpopulation may serve as a Trojan horse for CHIKV entry into the CNS. Additionally, CHIKV infection increases the expression of zonula occludens 1 (ZO-1) protein and cleaves platelet endothelial cell adhesion molecule 1 (PECAM-1) in endothelial cells of the BBB, which may contribute to enhanced transmigration of CHIKV-infected leukocytes into the CNS⁸ (Fig. 4). Similarly, MAYV has also been associated with neurological complications, and experimental studies using NHPs demonstrate viral spread to peripheral and central nervous system tissues, as well as multiple organs¹²⁵,¹⁵². Further studies are needed to determine the frequency and severity of sequelae (e.g., cognitive impairment) following CNS infections among chikungunya patients, as well as whether CNS infection occurs after infection by other arthritogenic alphaviruses (e.g., RRV, ONNV, and BFV) and its potential neuroinvasion mechanisms.

## Vertical Transmission and Neonatal Infection

Mother-to-child CHIKV transmission often leads to severe neonatal infection and birth abnormalities, such as encephalopathy, meningoencephalitis, and intravascular coagulation¹¹,¹⁵³-¹⁵⁵ (Fig. 4). Severe neonatal infection occurs predominantly in the third trimester, while miscarriage has been reported in the first and second trimesters, but evidence in the first two trimesters remains inconclusive¹⁵⁶⁻¹⁵⁹. During the 2006-2008 La Réunion epidemic, 9.8% (738 of 7,504) of pregnant women were diagnosed with chikungunya¹¹, and ten neonates developed encephalopathy, with four experiencing persistent disabilities and three resulting in early fetal death¹¹. CHIKV may infect the fetus by perinatal contamination or vertically transmitted through the placenta via micro transfusions or disruptions to the syncytiotrophoblast barrier caused by uterine contractions¹¹. This may lead to histopathological changes due to an increase in pro-inflammatory cells (e.g., CD8⁺ and CD163⁺) and overproduction of cytokines (e.g., IFN-γ, TNF-α, TGF-β, and IL-10) compared to placentas not infected by CHIKV¹⁶⁰. Therefore, CHIKV represents a substantial risk for pregnant women and neonates that should be considered by healthcare workers during epidemics, and MAYV, RRV, and ONNV should be evaluated for similar outcomes.

## Fatal infections

Chikungunya fatal cases have been reported across different subpopulations in several countries affected by different CHIKV lineages⁶,⁷,⁹,¹⁶¹,¹⁶². Also, the incidence of death has been estimated at 8.4 times that of individuals unexposed to CHIKV infection¹⁶³. Deaths usually occur during the acute phase and are often associated with an irreversible, multiple-organ dysfunction syndrome due to hemodynamic disorders and immune system dysregulation⁸,¹⁶². Hyperinflammation in patients who die of CHIKV infection can lead to endothelial injury, vascular permeability, overexpression of tissue factors, and dysregulation of the coagulation cascade, resulting in hemodynamic disturbances⁸. Additionally, mononuclear cell infiltrations in multiple organs are likely a consequence of an inflammatory reaction that triggers the migration of immune cells through the endothelium into these organs⁸,⁹,¹⁶². Although chikungunya deaths do not appear to be exclusively linked to virological or human population characteristics, the impact across different CHIKV lineages and human genetic variants (i.e., single-gene mutations and inborn errors of immunity) remains understudied. Moreover, several factors may affect the risk of death from CHIKV infection, such as healthcare system capacity, underlying diseases (e.g.,
hypertension, diabetes, or obesity), and population demographics. For example, chikungunya deaths have been documented in individuals with or without pre-existing conditions, but elderly individuals (≥ 65 years old) appear to be at higher risk compared to younger age groups¹⁶³. Additionally, the diagnosis of fatal chikungunya cases is often challenging due to resource limitations in countries experiencing epidemics or endemic circulations (Box 2), as well as the co-occurrence of other fatal arboviral diseases with similar clinical manifestations, particularly yellow fever and dengue¹⁰. Consequently, the absence of laboratory confirmation of fatal chikungunya can lead to misdiagnosis and underestimation of the burden of fatal cases.

# PREVENTION AND TREATMENT

## Chikungunya Vaccine

Currently, Ixchiq® (formerly VLA1553) and Vimkunya® (formerly PXVX0317) are the two most advanced vaccines to prevent chikungunya. Ixchiq® was approved in the USA and European Union in 2023 and in Canada in 2024, and Vimkunya® recently received a European Medicines Agency recommendation for a marketing authorization. Ixchiq® is a live-attenuated vaccine with a deletion of 60 amino acids in the hypervariable domain of nsP3⁹⁶,⁹⁷; this is part of the virus replication complex and thus Ixchiq® replicates less efficiently than wild-type CHIKV¹⁶⁴,¹⁶⁵. Clinical trials demonstrated that a single dose exhibits relatively good safety, comparable to other live-attenuated vaccines, and immunogenicity, with stable neutralizing antibody levels persisting for at least 12 months¹⁶⁶⁻¹⁶⁸. The main adverse reactions were headache, fatigue, myalgia, and arthralgia rates of 18%. Although phase 3 efficacy trials are considered the gold standard, this can be challenging for vaccines against sporadically and unpredictably emerging viruses like CHIKV¹⁶⁶,¹⁶⁹,¹⁷⁰. This led regulatory agencies in the USA, Canada, and Europe to accept surrogate endpoints to estimate protection. These were based on previous epidemiologic studies in the Philippines, where neutralizing antibody titers of ≥10 from previous infections were associated with 100% protection from symptomatic infection⁷⁵. Later, an experimental study was conducted where pooled serum from vaccinated humans was transferred to cynomolgus macaques (*Macaca fascicularis*). Then, these NHPs were challenged with wild-type CHIKV, where none receiving passive transfer developed viremia or clinical manifestations¹⁷⁰. Based on these findings, an anti-CHIKV neutralizing antibody titer of >150 (plaque reduction neutralization test 50%, PRNT<sub>50</sub>) at 28 days post-vaccination was established as a correlate of protection for phase 3 endpoints. Protection by Ixchiq® relies on antibodies that neutralize or inactivate virions for destruction and clearance by the immune system¹⁷¹, which also have been hypothesized to at least partially protect against other arthritogenic alphaviruses. For example, antibodies from the serum of individuals vaccinated with Ixchiq® (29- and 365-days post-immunization) or infected with CHIKV (7 to 10 years earlier) neutralize multiple CHIKV strains as well as ONNV and MAYV, but not RRV, suggesting homologous and partial heterologous protection¹⁷².

Vimkunya® is an adjuvanted recombinant vaccine containing CHIKV virus-like particles (VLPs) adsorbed on aluminum hydroxide¹⁷³. The VLPs consist of capsid, E1 and E2 proteins derived from West African CHIKV strain 37997, which generates the authentic viral structure but is replication-incompetent because genomic viral RNA is lacking. Clinical trials demonstrated that a single dose of Vimkunya® exhibits good tolerability and induces a robust antibody response for up to 2 years¹⁷³⁻¹⁷⁶. Like Ixchiq®, the clinical efficacy of Vimkunya® was also inferred from a post-vaccination CHIKV-specific neutralizing antibody titer threshold selected as a surrogate marker and referred to as seroresponse¹⁷⁷. Most adverse events reported during Vimkunya® clinical trials were mild-to-moderate, including fatigue, headache, and myalgia. Vimkunya was approved for use in individuals aged 12 years and older, while Ixchiq® is approved for individuals with 18 years or older. Currently, Ixchiq® is available for travelers in non-endemic countries, and strategies for immunization against chikungunya in endemic countries remain under debate⁵. Importantly, the
potential antibody-dependent enhancement (ADE) caused by immunization with Ixchiq® or Vimkunya® in areas endemic for other arthritogenic alphaviruses has not yet been studied, so the use of Ixchiq is still limited to regions that are non-endemic for related arthritogenic alphaviruses. Also, the exact mechanisms of protection elicited by both vaccines (Ixchiq® and Vimkunya®) have not been determined.

<table><thead><tr><th>Type of vaccine</th><th>Virus</th><th>Vaccine</th><th>Advantages</th><th>Limitations</th><th>Status</th></tr></thead><tbody><tr><td>Live-attenuated virus</td><td>CHIKV</td><td>Ixchiq</td><td><ul><li>Safety and well-tolerated</li><li>Rapid immune response (&gt;14 days)</li><li>Single dose</li><li>Sustained neutralizing antibody levels for at least 12 months</li></ul></td><td><ul><li>Significant severe chikungunya-like adverse (e.g., arthralgia rates of 18%)</li><li>Limited clinical trial data for various age groups and ethnicities</li><li>Uncertainty on protection against chronic diseases</li><li>Currently higher cost</li></ul></td><td>License in USA, Canada, and European Union*</td></tr><tr><td>Virus-like particles</td><td>CHIKV</td><td>PXVX0317</td><td><ul><li>Safety and well-tolerated</li><li>Highly immunogenic</li><li>Single dose</li><li>Sustained neutralizing antibody levels for at least 2 years</li><li>Likely to reduce the adverse effects due to absence of replicating viral</li></ul></td><td><ul><li>Supply chain challenges (stored at -80°C)</li><li>Limited clinical trial data for various age groups and ethnicities</li><li>Uncertainty on protection against chronic diseases</li></ul></td><td>Licensed in European Union* and waiting US FDA approval, expected in 2025</td></tr><tr><td>Recombinant measles vaccine</td><td>CHIKV</td><td>MV-CHIKV/ V184</td><td><ul><li>Safety and well-tolerated</li><li>Likely to reduce the adverse effects due to absence of nsPs proteins</li><li>Enhance measles immunity</li></ul></td><td><ul><li>Two-dose for higher seroconversion rates</li><li>Long-term durability unclear</li><li>Effectiveness against different CHIKV strains uncertain</li><li>Limited clinical trial data for various age groups and ethnicities</li><li>Uncertainty on protection against chronic diseases</li></ul></td><td>Phase 3 concluded Discontinued in Feb 2023 due to a routine pipeline prioritization of manufacturer</td></tr><tr><td>Recombinant simian adenovirus</td><td>CHIKV</td><td>ChAdOx1 Chik</td><td><ul><li>Safety and well-tolerated</li><li>Rapid immune response (&gt;14 days)</li><li>Single dose</li><li>Induced IgG and T-cell response</li><li>Broadly neutralizing antibodies against the four CHIKV lineage</li></ul></td><td><ul><li>Long-term durability unclear</li><li>Limited clinical trial data for various age groups and ethnicities</li><li>Uncertainty on protection against chronic diseases</li></ul></td><td>Phase 2 ongoing</td></tr><tr><td>mRNA vaccine encoding structural proteins</td><td>CHIKV</td><td>mRNA-1388</td><td><ul><li>Safety and well-tolerated</li><li>Sustained neutralizing antibody levels for at least 12 months</li></ul></td><td><ul><li>Multiple doses for higher seroconversion rates</li><li>Effectiveness against different CHIKV strains uncertain</li><li>Limited clinical trial data for various age groups and ethnicities</li><li>Uncertainty on protection against chronic diseases</li></ul></td><td>Phase 1 concluded</td></tr><tr><td>Inactive virus</td><td>CHIKV</td><td>BBV87</td><td><ul><li>Safety and well-tolerated</li><li>Likely to reduce the adverse effects due to absence of replicating viral</li><li>Supply chain challenges due to thermostability (shipping and storage)</li><li>Likely lower cost</li></ul></td><td><ul><li>Two-dose for higher seroconversion rates</li><li>Effectiveness against different CHIKV strains uncertain</li><li>Limited clinical trial data for various age groups and ethnicities</li></ul></td><td>Phase 2/3 ongoing</td></tr><tr><td>Inactive virus</td><td>RRV</td><td>Baxter</td><td><ul><li>Safety and well-tolerated</li><li>Likely to reduce the adverse effects due to absence of replicating viral</li><li>Supply chain challenges due to thermostability (shipping and storage)</li><li>Likely lower cost</li></ul></td><td><ul><li>Three-dose for higher seroconversion rates</li><li>Limited clinical trial data for various age groups and ethnicities</li><li>Uncertainty on protection against chronic diseases</li></ul></td><td>Phase 3 concluded</td></tr></tbody></table>

*Post-approval field efficacy studies were required

<caption>**Figure 5. Vaccine and vaccine candidates for arthritogenic alphaviruses under clinical trials.**
Overview of various vaccines under development for CHIKV, and one for RRV. The stage of development for each vaccine, as well as its advantages, limitations, and status in clinical trials.</caption>

## Other Vaccine Candidates

Candidate vaccines using several technologies have been evaluated in preclinical and clinical studies for chikungunya and other diseases caused by arthritogenic alphaviruses. Many vaccine candidates under development include vectored and mRNA vaccines that also lead to the formation and release of VLPs in transduced cells. These candidates have demonstrated safety and immunogenicity in clinical trials with single or multiple doses¹⁷⁸⁻¹⁸⁰. Similarly to VLP technology, the mRNA technology offers greater safety for immunization of pregnant and immunocompromised individuals compared to live-attenuated vaccines. Additionally, inactivated virus vaccine approach has been used to prevent chikungunya and Ross River disease. For example, BBV87 is a CHIKV vaccine candidate under clinical trial phases 2 and 3 (ClinicalTrials.gov, NCT04603131). Also, an inactivated whole-virus RRV vaccine was well tolerated in an adult population and induced antibody titers correlated with protection against disease during a phase 3 trial (ClinicalTrials.gov, NCT00717834), and no cases of arthritis resembling RRV disease were reported¹⁸¹. Inactivated virus vaccines have lower reactogenicity and greater safety for pregnant and immunocompromised individuals compared to live-attenuated
versions, and their thermostability enables feasible distribution in low- and middle-income countries. However, inactivated vaccines may require secure manufacturing facilities to cultivate live viruses before inactivation and are generally less immunogenic than live-attenuated versions. Other platforms for chikungunya vaccines in preclinical development are chimeric alphaviruses (i.e., Eilat virus, an insect-specific alphavirus)¹⁸²,¹⁸³, internal ribosome entry site (IRES) attenuated¹⁸⁴, viral vector vaccines based on vesicular stomatitis, and modified vaccinia Ankara virus vectors¹⁸⁵,¹⁸⁶. Additionally, preclinical MAYV vaccines include live-attenuated¹⁸⁷, DNA¹⁸⁸, adenoviral-vectored¹⁸⁹,¹⁹⁰, and VLP approaches¹⁹¹. Importantly, a preclinical study demonstrated *in vitro* ADE for MAYV and Uná virus when exposed to serum of mice immunized with adenovirus-vectored vaccine Mayaro and chikungunya vaccines¹⁹². Therefore, the potential for ADE to occur *in vivo* following vaccination against chikungunya or other candidate vaccines against arthritogenic alphaviruses should be further investigated.

## Therapeutics

No licensed antiviral or other specific therapeutics are available for arthritogenic alphavirus infections. Treatment primarily focuses on managing symptoms using analgesics, antipyretics, and nonsteroidal anti-inflammatory drugs. Several compounds have demonstrated antiviral activity against CHIKV, MAYV, and RRV by inhibiting their nsPs or interfering with viral replication *in vitro* and *in vivo*¹⁹³⁻¹⁹⁸. For example, a genome-wide siRNA screen identified antiviral host factors affecting CHIKV replication, such as the calmodulin inhibitor pimozide and the fatty acid synthesis inhibitor TOFA (5-tetradecyloxy-2-furoic acid), which have a therapeutic effect *in vivo*¹⁹⁸. However, further testing in NHPs and humans is required for the clinical advancement of these antivirals (Box 2). Additionally, some studies using NHP and mouse models have shown that monoclonal antibodies (mAbs) are promising prophylactic and therapeutic molecules against arthritogenic alphaviruses¹⁹⁹⁻²⁰⁴. mAbs primarily act by neutralizing viruses during the attachment, entry, and fusion stages or by inhibiting the binding of the virus to its receptor, MXRA8¹⁹⁹,²⁰⁰,²⁰²,²⁰⁴,²⁰⁵. The neutralizing epitopes for mAbs are predominantly located within the A and B domains of the E1 and E2 glycoproteins²⁰³,²⁰⁴,²⁰⁶,²⁰⁷. Moreover, mAbs can offer protection beyond direct neutralization. For instance, pan-protective human mAbs, despite their limited neutralizing ability, protect mice against infection by both arthritogenic (CHIKV and MAYV) and encephalitic (Venezuelan, eastern, and western equine encephalitis) alphaviruses. These mAbs mediate protection through multiple mechanisms, including inhibition of viral egress and monocyte-dependent Fc effector functions²⁰⁸. Further clinical trials are needed to fully evaluate the therapeutic potential of mAbs for arthritogenic alphaviruses. mAbs may be effective in preventing and treating diseases caused by arthritogenic alphaviruses, but further studies should also evaluate these potential therapeutics to prevent severe manifestations, such as chronic disease, CNS infections, and pregnancy and neonatal complications. Meanwhile, the high cost and specialized storage requirements of mAbs may significantly limit their clinical application in lower- and middle-income countries, which have been disproportionately affected by these viruses. Other therapeutic approaches for arthritogenic alphaviruses are reviewed elsewhere¹¹⁷,²⁰⁹⁻²¹².

## Vector control

Currently, the primary method for controlling the transmission of arthritogenic alphaviruses involves controlling mosquito vectors or avoiding human-vector contact. Vector control typically involves a combination of disease and entomological surveillance along with interventions targeting mosquito populations at both immature and adult stages in and around human dwellings. These strategies have traditionally used chemical insecticides (e.g., indoor residual sprays, space sprays, fogging, treated bednets, larvicides) and biological control methods (larvivorous fishes and environmental modification or source reduction)²¹³. However, these methods have become less effective, more costly, and environmentally problematic due to insecticide resistance,
changing vector behaviors (e.g., outdoor biting, altered resting habits), human population movement, and the spread of vectors into new areas, sometimes mediated by climate change<sup>213</sup>. To address these challenges, novel vector control tools have been developed including a precision-guided sterile insect technique using a CRISPR approach<sup>214</sup>, traditional sterile insect techniques using radiation, and mechanical control methods such as the adult oviposition trap<sup>215,216</sup>. Maternally inherited endosymbiotic *Wolbachia* bacteria (wMel strain), which renders mosquitoes incompetent for transmission<sup>217</sup>, have been deployed in several countries (e.g., Singapore, Brazil, and Colombia) with a reduction in dengue and chikungunya cases<sup>218-220</sup>. Effective vector control measures combined with robust surveillance systems are key for controlling arthritogenic alphaviruses, as well as other arboviral diseases.

## CONCLUSIONS

Arthritogenic alphaviruses such as chikungunya pose major public health burdens and global concerns, while MAYV, RRV, and ONNV still remain more local threats. Human activities and climate change may continue to enhance the spread and transmission of these viruses beyond their current endemic regions. Therefore, several actions are needed to reduce the burden of chikungunya and anticipate and prepare for the emergence of other arthritogenic alphaviruses (and reemergence of CHIKV). First, enhanced molecular surveillance, rapid diagnosis, and timely treatment are essential to reduce transmission, improve patient outcomes, and inform effective public health policies. For example, neglected diseases like Mayaro and o'nyong-nyong fever should be considered in differential diagnoses in endemic regions. Second, affordable vaccines for at-risk populations are crucial to mitigating the transmission and disease burden of CHIKV and for preventing or preparing for the emergence of other arthritogenic alphaviruses. A broad-spectrum vaccine that protects against multiple arthritogenic alphaviruses should be prioritized. Considering the long-lasting immunity conferred by some vaccines, chikungunya elimination from urban cycles may be feasible. Third, developing efficient, safe, and accessible antivirals and mAb therapeutics is essential for treating patients, especially those with severe manifestations and chronic disease. This goal also requires refinement or novel experimental models that accurately recapitulate the diseases observed in humans. Fourth, identifying biomarkers associated with severe outcomes and chronic disease will improve clinical management and reduce disease burden. Fifth, a better understanding of the spatiotemporal transmission dynamics of arthritogenic alphaviruses in urban and forest settings, as well as the impact of climate change on vectors and hosts, including humans, are crucial for effective mitigation strategies.

## Acknowledgments

WMdS is supported by a Wellcome Trust - Digital Technology Development Award in Climate Sensitive Infectious Disease Modelling (#226075/Z/22/Z). ML is supported by grants of Institut Pasteur, Institut National de la Santé et de la Recherche Médicale, French National Research Agency and the European Research Council. SCW is supported by National Institutes of Health grants R24AI12094, U01AI151801, and AI121452.

## Author contributions

The authors contributed equally to all aspects of the article.

## Competing interests

The authors declare no competing interests.

## References
1 Chen, R. et al. ICTV Virus Taxonomy Profile: Togaviridae. *J Gen Virol* **99**, 761-762, doi:10.1099/jgv.0.001072 (2018).

2 Lindsey, N. P., Martin, S. W., Staples, J. E. & Fischer, M. Notes from the Field: Multistate Outbreak of Eastern Equine Encephalitis Virus - United States, 2019. *MMWR Morb Mortal Wkly Rep* **69**, 50-51, doi:10.15585/mmwr.mm6902a4 (2020).

3 Campos, A. S. et al. Molecular Epidemiology of Western Equine Encephalitis Virus, South America, 2023-2024. *Emerg Infect Dis* **30**, 1834-1840, doi:10.3201/eid3009.240530 (2024).

4 Rivera, L. F. et al. Characteristics of Madariaga and Venezuelan Equine Encephalitis Virus Infections, Panama. *Emerg Infect Dis* **30**, 94-104, doi:10.3201/eid3014.240182 (2024).

5 Ribeiro dos Santos, G. et al. The global burden of chikungunya virus and the potential benefit of vaccines. *medRxiv*, 2024.2010.2024.24315872, doi:10.1101/2024.10.24.24315872 (2024).

6 Gérardin, P. et al. Chikungunya virus-associated encephalitis: A cohort study on La Réunion Island, 2005-2009. *Neurology* **86**, 94-102, doi:10.1212/WNL.0000000000002234 (2016).

7 Nyamwaya, D. K. et al. Incidence of chikungunya virus infections among Kenyan children with neurological disease, 2014-2018: A cohort study. *PLoS Med* **19**, e1003994, doi:10.1371/journal.pmed.1003994 (2022).

8 de Souza, W. M. et al. Pathophysiology of chikungunya virus infection associated with fatal outcomes. *Cell Host Microbe* **32**, 606-622.e608, doi:10.1016/j.chom.2024.02.011 (2024).

9 de Lima, S. T. S. et al. Fatal Outcome of Chikungunya Virus Infection in Brazil. *Clin Infect Dis* **73**, e2436-e2443, doi:10.1093/cid/ciaa1038 (2021).

10 de Souza, W. M. et al. Chikungunya: a decade of burden in the Americas. *The Lancet Regional Health – Americas* **30**, doi:10.1016/j.lana.2023.100673 (2024).

11 Gerardin, P. et al. Multidisciplinary prospective study of mother-to-child chikungunya virus infections on the island of La Reunion. *PLoS Med* **5**, e60, doi:10.1371/journal.pmed.0050060 (2008).

12 De Sousa, F. B. et al. Report of natural Mayaro virus infection in Mansonia humeralis (Dyar & Knab, Diptera: Culicidae). *Parasit Vectors* **16**, 140, doi:10.1186/s13071-023-05707-2 (2023).

13 Celone, M. et al. A systematic review and meta-analysis of the potential non-human animal reservoirs and arthropod vectors of the Mayaro virus. *PLoS Negl Trop Dis* **15**, e0010016, doi:10.1371/journal.pntd.0010016 (2021).

14 Forato, J. et al. Molecular Epidemiology of Mayaro Virus among Febrile Patients, Roraima State, Brazil, 2018-2021. *Emerg Infect Dis* **30**, 1013-1016, doi:10.3201/eid3005.231406 (2024).

15 Williams, M. C., Woodall, J. P. & Gillett, J. D. O'nyong-Nyong Fever: An Epidemic Virus Diesease in East Africa. Vii. Virus Isolations from Man and Serological Studies up to July 1961. *Trans. R. Soc. Trop. Med. Hyg.* **59**, 186-197 (1965).

16 Mutsaers, M. et al. Vector competence of Anopheles stephensi for O'nyong-nyong virus: a risk for global virus spread. *Parasit Vectors* **16**, 133, doi:10.1186/s13071-023-05725-0 (2023).

17 Vanlandingham, D. L. et al. Differential infectivities of o'nyong-nyong and chikungunya virus isolates in Anopheles gambiae and Aedes aegypti mosquitoes. *Am. J. Trop. Med. Hyg.* **72**, 616-621 (2005).

18 Quinn, H. E., Gatton, M. L., Hall, G., Young, M. & Ryan, P. A. Analysis of Barmah Forest virus disease activity in Queensland, Australia, 1993-2003: identification of a large,
isolated outbreak of disease. *J Med Entomol* **42**, 882-890, doi:10.1093/jmedent/42.5.882 (2005).

19 Harley, D., Sleigh, A. & Ritchie, S. Ross River virus transmission, infection, and disease: a cross-disciplinary review. *Clin. Microbiol. Rev.* **14**, 909-932, table of contents (2001).

20 Boyd, A. M. & Kay, B. H. Experimental infection and transmission of Barmah Forest virus by Aedes vigilax (Diptera: Culicidae). *J Med Entomol* **36**, 186-189, doi:10.1093/jmedent/36.2.186 (1999).

21 Lindsay, M., Johansen, C., Broom, A. K., Smith, D. W. & Mackenzie, J. S. Emergence of Barmah Forest virus in Western Australia. *Emerg Infect Dis* **1**, 22-26 (1995).

22 Iwamura, T., Guzman-Holst, A. & Murray, K. A. Accelerating invasion potential of disease vector Aedes aegypti under climate change. *Nat Commun* **11**, 2130, doi:10.1038/s41467-020-16010-4 (2020).

23 Kraemer, M. U. G. et al. Past and future spread of the arbovirus vectors Aedes aegypti and Aedes albopictus. *Nat Microbiol* **4**, 854-863, doi:10.1038/s41564-019-0376-y (2019).

24 de Souza, W. M. & Weaver, S. C. Effects of climate change and human activities on vector-borne diseases. *Nat Rev Microbiol*, doi:10.1038/s41579-024-01026-0 (2024).

25 Adouchief, S., Smura, T., Sane, J., Vapalahti, O. & Kurkela, S. Sindbis virus as a human pathogen-epidemiology, clinical picture and pathogenesis. *Rev. Med. Virol.* **26**, 221-241, doi:10.1002/rmv.1876 (2016).

26 Ling, J. et al. Introduction and Dispersal of Sindbis Virus from Central Africa to Europe. *J Virol* **93**, doi:10.1128/JVI.00620-19 (2019).

27 Gylfe, Å. et al. Mosquitoborne Sindbis Virus Infection and Long-Term Illness. *Emerg Infect Dis* **24**, 1141-1142, doi:10.3201/eid2406.170892 (2018).

28 Kurkela, S., Helve, T., Vaheri, A. & Vapalahti, O. Arthritis and arthralgia three years after Sindbis virus infection: clinical follow-up of a cohort of 49 patients. *Scand. J. Infect. Dis.* **40**, 167-173, doi:10.1080/00365540701586996 (2008).

29 Calisher, C. H., Karabatsos, N., Lazuick, J. S., Monath, T. P. & Wolff, K. L. Reevaluation of the western equine encephalitis antigenic complex of alphaviruses (family Togaviridae) as determined by neutralization tests. *Am. J. Trop. Med. Hyg.* **38**, 447-452 (1988).

30 Nguyen, B. H., Bartlett, M. L., Troisi, E. M., Stanley, E. & Griffin, D. E. Phenotypic and transcriptional changes in peripheral blood mononuclear cells during alphavirus encephalitis in mice. *mBio* **15**, e0073624, doi:10.1128/mbio.00736-24 (2024).

31 Wang, X. et al. Sindbis Virus Can Exploit a Host Antiviral Protein To Evade Immune Surveillance. *J Virol* **90**, 10247-10258, doi:10.1128/JVI.01487-16 (2016).

32 Althouse, B. M. et al. Role of monkeys in the sylvatic cycle of chikungunya virus in Senegal. *Nat Commun* **9**, 1046, doi:10.1038/s41467-018-03332-7 (2018).

33 Weaver, S. C., Chen, R. & Diallo, M. Chikungunya Virus: Role of Vectors in Emergence from Enzootic Cycles. *Annu Rev Entomol* **65**, 313-332, doi:10.1146/annurev-ento-011019-025207 (2020).

34 de Souza, W. M. et al. Serosurvey of Chikungunya Virus in Old World Fruit Bats, Senegal, 2020-2022. *Emerg Infect Dis* **30**, 1490-1492, doi:10.3201/eid3007.240055 (2024).

35 Dieng, I. et al. Genomic characterization of a reemerging Chikungunya outbreak in Kedougou, Southeastern Senegal, 2023. *Emerg Microbes Infect* **13**, 2373308, doi:10.1080/22221751.2024.2373308 (2024).

36 Sang, R. C. et al. Entomologic investigations of a chikungunya virus epidemic in the Union of the Comoros, 2005. *Am. J. Trop. Med. Hyg.* **78**, 77-82 (2008).

37 Johnson, B. K. et al. Recovery of o'nyong-nyong virus from Anopheles funestus in Western Kenya. *Trans R Soc Trop Med Hyg* **75**, 239-241, doi:10.1016/0035-9203(81)90325-4 (1981).
38 Kading, R. C., Borland, E. M., Cranfield, M. & Powers, A. M. Prevalence of antibodies to alphaviruses and flaviviruses in free-ranging game animals and nonhuman primates in the greater Congo basin. *J. Wildl. Dis.* **49**, 587-599, doi:10.7589/2012-08-212 (2013).

39 Hoch, A. L., Peterson, N. E., LeDuc, J. W. & Pinheiro, F. P. An outbreak of Mayaro virus disease in Belterra, Brazil. III. Entomological and ecological studies. *Am J Trop Med Hyg* **30**, 689-698, doi:10.4269/ajtmh.1981.30.689 (1981).

40 Pereira, T. N., Carvalho, F. D., De Mendonça, S. F., Rocha, M. N. & Moreira, L. A. Vector competence of Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus mosquitoes for Mayaro virus. *PLoS Negl Trop Dis* **14**, e0007518, doi:10.1371/journal.pntd.0007518 (2020).

41 Fernández, D. et al. Differential Susceptibility of Aedes aegypti and Aedes albopictus Mosquitoes to Infection by Mayaro Virus Strains. *Am J Trop Med Hyg* **109**, 115-122, doi:10.4269/ajtmh.22-0777 (2023).

42 Suhrbier, A. Rheumatic manifestations of chikungunya: emerging concepts and interventions. *Nat Rev Rheumatol* **15**, 597-611, doi:10.1038/s41584-019-0276-9 (2019).

43 de Souza, W. M. et al. Spatiotemporal dynamics and recurrence of chikungunya virus in Brazil: an epidemiological study. *Lancet Microbe* **4**, e319-e329, doi:10.1016/S2666-5247(23)00033-2 (2023).

44 Galatas, B. et al. Long-Lasting Immune Protection and Other Epidemiological Findings after Chikungunya Emergence in a Cambodian Rural Community, April 2012. *PLoS Negl Trop Dis* **10**, e0004281, doi:10.1371/journal.pntd.0004281 (2016).

45 Nitatpattana, N. et al. Long-term persistence of Chikungunya virus neutralizing antibodies in human populations of North Eastern Thailand. *Virol J* **11**, 183, doi:10.1186/1743-422X-11-183 (2014).

46 Moore, T. C. & Brown, H. E. Estimating Aedes aegypti (Diptera: Culicidae) Flight Distance: Meta-Data Analysis. *J. Med. Entomol.* **59**, 1164-1170, doi:10.1093/jme/tjac070 (2022).

47 Anderson, C. R., Downs, W. G., Wattley, G. H., Ahin, N. W. & Reese, A. A. Mayaro virus: a new human disease agent. II. Isolation from blood of patients in Trinidad, B.W.I. *Am. J. Trop. Med. Hyg.* **6**, 1012-1016 (1957).

48 Caicedo, E. Y. et al. The epidemiology of Mayaro virus in the Americas: A systematic review and key parameter estimates for outbreak modelling. *PLoS Negl Trop Dis* **15**, e0009418, doi:10.1371/journal.pntd.0009418 (2021).

49 LeDuc, J. W., Pinheiro, F. P. & Travassos da Rosa, A. P. An outbreak of Mayaro virus disease in Belterra, Brazil. II. Epidemiology. *Am. J. Trop. Med. Hyg.* **30**, 682-688 (1981).

50 Hozé, N. et al. Reconstructing Mayaro virus circulation in French Guiana shows frequent spillovers. *Nat Commun* **11**, 2842, doi:10.1038/s41467-020-16516-x (2020).

51 Bessaud, M. et al. O'nyong-nyong Virus, Chad. *Emerg Infect Dis* **12**, 1248-1250, doi:10.3201/eid1208.060199 (2006).

52 Tappe, D. et al. O'nyong-nyong virus infection imported to Europe from Kenya by a traveler. *Emerg Infect Dis* **20**, 1766-1767, doi:10.3201/eid2010.140823 (2014).

53 Posey, D. L. et al. O'Nyong-nyong fever in West Africa. *Am. J. Trop. Med. Hyg.* **73**, 32 (2005).

54 Kiwanuka, N. et al. O'nyong-nyong fever in south-central Uganda, 1996-1997: clinical features and validation of a clinical case definition for surveillance purposes. *Clin. Infect. Dis.* **29**, 1243-1250, doi:10.1086/313462 (1999).

55 Sanders, E. J. et al. O'nyong-nyong fever in south-central Uganda, 1996-1997: description of the epidemic and results of a household-based seroprevalence survey. *J. Infect. Dis.* **180**, 1436-1443, doi:10.1086/315073 (1999).
56 LaBeaud, A. D. et al. High rates of o'nyong nyong and Chikungunya virus transmission in coastal Kenya. *PLoS Negl Trop Dis* **9**, e0003436, doi:10.1371/journal.pntd.0003436 (2015).

57 Hozé, N. et al. Model-based assessment of Chikungunya and O'nyong-nyong virus circulation in Mali in a serological cross-reactivity context. *Nat Commun* **12**, 6735, doi:10.1038/s41467-021-26707-9 (2021).

58 Powers, A. M., Brault, A. C., Tesh, R. B. & Weaver, S. C. Re-emergence of Chikungunya and O'nyong-nyong viruses: evidence for distinct geographical lineages and distant evolutionary relationships. *J. Gen. Virol.* **81**, 471-479 (2000).

59 Aubry, M. et al. Ross River Virus Antibody Prevalence, Fiji Islands, 2013-2015. *Emerg Infect Dis* **25**, 827-830, doi:10.3201/eid2504.180694 (2019).

60 Lau, C. et al. New evidence for endemic circulation of Ross River virus in the Pacific Islands and the potential for emergence. *Int J Infect Dis* **57**, 73-76, doi:10.1016/j.ijid.2017.01.041 (2017).

61 Aubry, M. et al. Silent Circulation of Ross River Virus in French Polynesia. *Int J Infect Dis* **37**, 19-24, doi:10.1016/j.ijid.2015.06.005 (2015).

62 Aaskov, J. G. et al. An epidemic of Ross River virus infection in Fiji, 1979. *Am. J. Trop. Med. Hyg.* **30**, 1053-1059 (1981).

63 Jansen, C. C. et al. Epidemiologic, Entomologic, and Virologic Factors of the 2014-15 Ross River Virus Outbreak, Queensland, Australia. *Emerg Infect Dis* **25**, 2243-2252, doi:10.3201/eid2512.181810 (2019).

64 Shocket, M. S., Ryan, S. J. & Mordecai, E. A. Temperature explains broad patterns of Ross River virus transmission. *Elife* **7**, doi:10.7554/eLife.37762 (2018).

65 Franklinos, L. H. V., Jones, K. E., Redding, D. W. & Abubakar, I. The effect of global change on mosquito-borne disease. *Lancet Infect Dis* **19**, e302-e312, doi:10.1016/S1473-3099(19)30161-6 (2019).

66 Mora, C. et al. Over half of known human pathogenic diseases can be aggravated by climate change. *Nat Clim Chang* **12**, 869-875, doi:10.1038/s41558-022-01426-1 (2022).

67 Tsetsarkin, K. A., Vanlandingham, D. L., McGee, C. E. & Higgs, S. A single mutation in chikungunya virus affects vector specificity and epidemic potential. *PLoS Pathog* **3**, e201, doi:07-PLPA-RA-0664 [pii] 10.1371/journal.ppat.0030201 (2007).

68 Schuffenecker, I. et al. Genome microevolution of chikungunya viruses causing the Indian Ocean outbreak. *PLoS Med* **3**, e263 (2006).

69 Vazeille, M. et al. Two Chikungunya isolates from the outbreak of La Reunion (Indian Ocean) exhibit different patterns of infection in the mosquito, Aedes albopictus. *PLoS One* **2**, e1168, doi:10.1371/journal.pone.0001168 (2007).

70 Tsetsarkin, K. A. et al. Chikungunya virus emergence is constrained in Asia by lineage-specific adaptive landscapes. *Proc Natl Acad Sci U S A* **108**, 7872-7877, doi:10.1073/pnas.1018344108 (2011).

71 Tsetsarkin, K. A. et al. Epistatic roles of E2 glycoprotein mutations in adaption of chikungunya virus to Aedes albopictus and Ae. aegypti mosquitoes. *PLoS One* **4**, e6835, doi:10.1371/journal.pone.0006835 (2009).

72 Tsetsarkin, K. A. et al. Multi-peaked adaptive landscape for chikungunya virus evolution predicts continued fitness optimization in Aedes albopictus mosquitoes. *Nat Commun* **5**, 4084, doi:10.1038/ncomms5084 (2014).

73 Chen, R., Wang, E., Tsetsarkin, K. A. & Weaver, S. C. Chikungunya virus 3' untranslated region: adaptation to mosquitoes and a population bottleneck as major evolutionary forces. *PLoS Pathog* **9**, e1003591, doi:10.1371/journal.ppat.1003591 (2013).
74 Langsjoen, R. M. et al. Chikungunya Virus Strains Show Lineage-Specific Variations in Virulence and Cross-Protective Ability in Murine and Nonhuman Primate Models. *mBio* **9**, doi:10.1128/mBio.02449-17 (2018).

75 Yoon, I. K. et al. High rate of subclinical chikungunya virus infection and association of neutralizing antibody with protection in a prospective cohort in the Philippines. *PLoS Negl Trop Dis* **9**, e0003764, doi:10.1371/journal.pntd.0003764 (2015).

76 Gay, N. et al. Seroprevalence of Asian Lineage Chikungunya Virus Infection on Saint Martin Island, 7 Months After the 2013 Emergence. *Am J Trop Med Hyg* **94**, 393-396, doi:10.4269/ajtmh.15-0308 (2016).

77 Tan, Y. et al. Differing epidemiological dynamics of Chikungunya virus in the Americas during the 2014-2015 epidemic. *PLoS Negl Trop Dis* **12**, e0006670, doi:10.1371/journal.pntd.0006670 (2018).

78 Auguste, A. J. et al. Evolutionary and Ecological Characterization of Mayaro Virus Strains Isolated during an Outbreak, Venezuela, 2010. *Emerg Infect Dis* **21**, 1742-1750, doi:10.3201/eid2110.141660 (2015).

79 Michie, A. et al. Genome-Scale Phylogeny and Evolutionary Analysis of Ross River Virus Reveals Periodic Sweeps of Lineage Dominance in Western Australia, 1977-2014. *J. Virol.* **94**, doi:10.1128/JVI.01234-19 (2020).

80 Michie, A. et al. Phylogenetic and Timescale Analysis of Barmah Forest Virus as Inferred from Genome Sequence Analysis. *Viruses* **12**, doi:10.3390/v12070732 (2020).

81 Basore, K. et al. Cryo-EM Structure of Chikungunya Virus in Complex with the Mxra8 Receptor. *Cell* **177**, 1725-1737.e1716, doi:10.1016/j.cell.2019.04.006 (2019).

82 Voss, J. E. et al. Glycoprotein organization of Chikungunya virus particles revealed by X-ray crystallography. *Nature* **468**, 709-712, doi:nature09555 [pii] 10.1038/nature09555 (2010).

83 Ribeiro-Filho, H. V. et al. Cryo-EM structure of the mature and infective Mayaro virus at 4.4 Å resolution reveals features of arthritogenic alphaviruses. *Nat Commun* **12**, 3038, doi:10.1038/s41467-021-23400-9 (2021).

84 Zhang, R. et al. Mxra8 is a receptor for multiple arthritogenic alphaviruses. *Nature* **557**, 570-574, doi:10.1038/s41586-018-0121-3 (2018).

85 Ng, W. H. et al. Role of MXRA8 in Ross River Virus Disease Pathogenesis. *mBio* **14**, e0058823, doi:10.1128/mbio.00588-23 (2023).

86 Kim, A. S. et al. An Evolutionary Insertion in the Mxra8 Receptor-Binding Site Confers Resistance to Alphavirus Infection and Pathogenesis. *Cell Host Microbe* **27**, 428-440.e429, doi:10.1016/j.chom.2020.01.008 (2020).

87 Zhang, R. et al. Expression of the Mxra8 Receptor Promotes Alphavirus Infection and Pathogenesis in Mice and Drosophila. *Cell Rep* **28**, 2647-2658.e2645, doi:10.1016/j.celrep.2019.07.105 (2019).

88 Song, H. et al. Molecular Basis of Arthritogenic Alphavirus Receptor MXRA8 Binding to Chikungunya Virus Envelope Protein. *Cell* **177**, 1714-1724.e1712, doi:10.1016/j.cell.2019.04.008 (2019).

89 Wintachai, P. et al. Identification of prohibitin as a Chikungunya virus receptor protein. *J Med Virol* **84**, 1757-1770, doi:10.1002/jmv.23403 (2012).

90 De Caluwé, L. et al. The CD147 Protein Complex Is Involved in Entry of Chikungunya Virus and Related Alphaviruses in Human Cells. *Front Microbiol* **12**, 615165, doi:10.3389/fmicb.2021.615165 (2021).

91 Silva, L. A. et al. A single-amino-acid polymorphism in Chikungunya virus E2 glycoprotein influences glycosaminoglycan utilization. *J Virol* **88**, 2385-2397, doi:10.1128/JVI.03116-13 (2014).
92 Tanaka, A. et al. Genome-Wide Screening Uncovers the Significance of N-Sulfation of Heparan Sulfate as a Host Cell Factor for Chikungunya Virus Infection. *J Virol* **91**, doi:10.1128/JVI.00432-17 (2017).

93 Zhang, W., Heil, M., Kuhn, R. J. & Baker, T. S. Heparin binding sites on Ross River virus revealed by electron cryo-microscopy. *Virology* **332**, 511-518 (2005).

94 Kirui, J. et al. The Phosphatidylserine Receptor TIM-1 Enhances Authentic Chikungunya Virus Cell Entry. *Cells* **10**, doi:10.3390/cells10071828 (2021).

95 Jemielity, S. et al. TIM-family proteins promote infection of multiple enveloped viruses through virion-associated phosphatidylserine. *PLoS Pathog* **9**, e1003232, doi:10.1371/journal.ppat.1003232 (2013).

96 Meertens, L. et al. FHL1 is a major host factor for chikungunya virus infection. *Nature* **574**, 259-263, doi:10.1038/s41586-019-1578-4 (2019).

97 Ng, W. H. et al. FHL1 promotes chikungunya and o'nyong-nyong virus infection and pathogenesis with implications for alphavirus vaccine design. *Nat Commun* **14**, 6605, doi:10.1038/s41467-023-42330-2 (2023).

98 Neyret, A. et al. Identification of a non-canonical G3BP-binding sequence in a Mayaro virus nsP3 hypervariable domain. *Front Cell Infect Microbiol* **12**, 958176, doi:10.3389/fcimb.2022.958176 (2022).

99 Götte, B., Utt, A., Fragkoudis, R., Merits, A. & McInerney, G. M. Sensitivity of Alphaviruses to G3BP Deletion Correlates with Efficiency of Replicase Polyprotein Processing. *J Virol* **94**, doi:10.1128/JVI.01681-19 (2020).

100 Scholte, F. E. et al. Stress granule components G3BP1 and G3BP2 play a proviral role early in Chikungunya virus replication. *J Virol* **89**, 4457-4469, doi:10.1128/JVI.03612-14 (2015).

101 Fros, J. J. et al. Chikungunya virus nsP3 blocks stress granule assembly by recruitment of G3BP into cytoplasmic foci. *J Virol* **86**, 10873-10879, doi:10.1128/JVI.01506-12 (2012).

102 Götte, B. et al. Separate domains of G3BP promote efficient clustering of alphavirus replication complexes and recruitment of the translation initiation machinery. *PLoS Pathog* **15**, e1007842, doi:10.1371/journal.ppat.1007842 (2019).

103 Matkovic, R. et al. The Host DHX9 DExH-Box Helicase Is Recruited to Chikungunya Virus Replication Complexes for Optimal Genomic RNA Translation. *J Virol* **93**, doi:10.1128/JVI.01764-18 (2019).

104 Torii, S. et al. Host ESCRT factors are recruited during chikungunya virus infection and are required for the intracellular viral replication cycle. *J Biol Chem* **295**, 7941-7957, doi:10.1074/jbc.RA119.012303 (2020).

105 Müller, M. et al. Chikungunya virus requires cellular chloride channels for efficient genome replication. *PLoS Negl Trop Dis* **13**, e0007703, doi:10.1371/journal.pntd.0007703 (2019).

106 Kril, V. et al. Alphavirus nsP3 organizes into tubular scaffolds essential for infection and the cytoplasmic granule architecture. *Nat Commun* **15**, 8106, doi:10.1038/s41467-024-51952-z (2024).

107 Glanville, N., Ranki, M., Morser, J., Kääriäinen, L. & Smith, A. E. Initiation of translation directed by 42S and 26S RNAs from Semliki Forest virus in vitro. *Proc Natl Acad Sci U S A* **73**, 3059-3063, doi:10.1073/pnas.73.9.3059 (1976).

108 Yin, P. et al. Chikungunya virus cell-to-cell transmission is mediated by intercellular extensions in vitro and in vivo. *Nat Microbiol* **8**, 1653-1667, doi:10.1038/s41564-023-01449-0 (2023).

109 Sattentau, Q. Avoiding the void: cell-to-cell spread of human viruses. *Nat Rev Microbiol* **6**, 815-826, doi:10.1038/nrmicro1972 (2008).
110 Zeng, C. et al. SARS-CoV-2 spreads through cell-to-cell transmission. Proc Natl Acad Sci U S A **119**, doi:10.1073/pnas.2111400119 (2022).

111 Halsey, E. S. et al. Mayaro virus infection, Amazon Basin region, Peru, 2010-2013. *Emerg Infect Dis* **19**, 1839-1842, doi:10.3201/eid1911.130777 (2013).

112 Rama, K. et al. Clinical outcomes of chikungunya: A systematic literature review and meta-analysis. *PLoS Negl Trop Dis* **18**, e0012254, doi:10.1371/journal.pntd.0012254 (2024).

113 Schilte, C. et al. Chikungunya virus-associated long-term arthralgia: a 36-month prospective longitudinal study. *PLoS Negl Trop Dis* **7**, e2137, doi:10.1371/journal.pntd.0002137 (2013).

114 Warnes, C. M. et al. Longitudinal analysis of post-acute chikungunya-associated arthralgia in children and adults: A prospective cohort study in Managua, Nicaragua (2014-2018). *PLoS Negl Trop Dis* **18**, e0011948, doi:10.1371/journal.pntd.0011948 (2024).

115 van Aalst, M., Nelen, C. M., Goorhuis, A., Stijnis, C. & Grobusch, M. P. Long-term sequelae of chikungunya virus disease: A systematic review. *Travel Med Infect Dis* **15**, 8-22, doi:10.1016/j.tmaid.2017.01.004 (2017).

116 Salje, H. et al. How social structures, space, and behaviors shape the spread of infectious diseases using chikungunya as a case study. Proc Natl Acad Sci U S A **113**, 13420-13425, doi:10.1073/pnas.1611391113 (2016).

117 Bartholomeeusen, K. et al. Chikungunya fever. Nat Rev Dis Primers **9**, 17, doi:10.1038/s41572-023-00429-2 (2023).

118 Zaid, A. et al. Arthritogenic alphaviruses: epidemiological and clinical perspective on emerging arboviruses. Lancet Infect. Dis. **21**, e123-e133, doi:10.1016/S1473-3099(20)30491-6 (2021).

119 Liu, X., Tharmarajah, K. & Taylor, A. Ross River virus disease clinical presentation, pathogenesis and current therapeutic strategies. Microbes Infect **19**, 496-504, doi:10.1016/j.micinf.2017.07.001 (2017).

120 Lentscher, A. J. et al. Chikungunya virus replication in skeletal muscle cells is required for disease development. *J Clin Invest* **130**, 1466-1478, doi:10.1172/JCI129893 (2020).

121 Young, A. R. et al. Dermal and muscle fibroblasts and skeletal myofibers survive chikungunya virus infection and harbor persistent RNA. *PLoS Pathog* **15**, e1007993, doi:10.1371/journal.ppat.1007993 (2019).

122 Pingen, M. et al. Host Inflammatory Response to Mosquito Bites Enhances the Severity of Arbovirus Infection. Immunity **44**, 1455-1469, doi:10.1016/j.immuni.2016.06.002 (2016).

123 Li, F. S. et al. Species-specific MARCO-alphavirus interactions dictate chikungunya virus viremia. Cell Rep **42**, 112418, doi:10.1016/j.celrep.2023.112418 (2023).

124 Holmes, A. C. et al. Ly6C. Cell Rep **43**, 113876, doi:10.1016/j.celrep.2024.113876 (2024).

125 Weber, W. C. et al. Mayaro virus pathogenesis and immunity in rhesus macaques. *PLoS Negl Trop Dis* **17**, e0011742, doi:10.1371/journal.pntd.0011742 (2023).

126 Labadie, K. et al. Chikungunya disease in nonhuman primates involves long-term viral persistence in macrophages. *J. Clin. Invest.* **120**, 894-906, doi:10.1172/JCI40104 (2010).

127 Haist, K. C., Burrack, K. S., Davenport, B. J. & Morrison, T. E. Inflammatory monocytes mediate control of acute alphavirus infection in mice. *PLoS Pathog* **13**, e1006748, doi:10.1371/journal.ppat.1006748 (2017).

128 Fox, J. M. et al. Optimal therapeutic activity of monoclonal antibodies against chikungunya virus requires Fc-FcγR interaction on monocytes. *Sci Immunol* **4**, doi:10.1126/sciimmunol.aav5062 (2019).
129 Poo, Y. S. et al. CCR2 deficiency promotes exacerbated chronic erosive neutrophil-dominated chikungunya virus arthritis. *J Virol* **88**, 6862-6872, doi:10.1128/JVI.03364-13 (2014).

130 Rudd, P. A. et al. Interferon response factors 3 and 7 protect against Chikungunya virus hemorrhagic fever and shock. *J Virol* **86**, 9888-9898, doi:10.1128/JVI.00956-12 (2012).

131 Wilson, J. A. et al. RNA-Seq analysis of chikungunya virus infection and identification of granzyme A as a major promoter of arthritic inflammation. *PLoS Pathog* **13**, e1006155, doi:10.1371/journal.ppat.1006155 (2017).

132 Hoarau, J. J. et al. Persistent chronic inflammation and infection by Chikungunya arthritogenic alphavirus in spite of a robust host immune response. *J. Immunol.* **184**, 5914-5927, doi:10.4049/jimmunol.0900255 (2010).

133 Michlmayr, D. et al. Comprehensive innate immune profiling of chikungunya virus infection in pediatric cases. *Mol Syst Biol* **14**, e7862, doi:10.15252/msb.20177862 (2018).

134 Dias, C. N. S. et al. Human CD8 T-cell activation in acute and chronic chikungunya infection. *Immunology* **155**, 499-504, doi:10.1111/imm.12992 (2018).

135 Wauquier, N. et al. The acute phase of Chikungunya virus infection in humans is associated with strong innate immunity and T CD8 cell activation. *J. Infect. Dis.* **204**, 115-123, doi:10.1093/infdis/jiq006 (2011).

136 Mueller, S. N. & Ahmed, R. High antigen levels are the cause of T cell exhaustion during chronic viral infection. *Proc Natl Acad Sci U S A* **106**, 8623-8628, doi:10.1073/pnas.0809818106 (2009).

137 Chang, A. Y. et al. The Natural History of Post-Chikungunya Viral Arthritis Disease Activity and T-cell Immunology: A Cohort Study. *J Cell Immunol* **6**, 64-75, doi:10.33696/immunology.6.191 (2024).

138 Chang, A. Y. et al. Chikungunya Arthritis Mechanisms in the Americas: A Cross-Sectional Analysis of Chikungunya Arthritis Patients Twenty-Two Months After Infection Demonstrating No Detectable Viral Persistence in Synovial Fluid. *Arthritis Rheumatol* **70**, 585-593, doi:10.1002/art.40383 (2018).

139 Kam, Y. W. et al. Early Appearance of Neutralizing Immunoglobulin G3 Antibodies Is Associated With Chikungunya Virus Clearance and Long-term Clinical Protection. *J. Infect. Dis.* **205**, 1147-1154, doi:10.1093/infdis/jis033 (2012).

140 Roy, E., Shi, W., Duan, B. & Reid, S. P. Chikungunya Virus Infection Impairs the Function of Osteogenic Cells. *mSphere* **5**, doi:10.1128/mSphere.00347-20 (2020).

141 Chen, W. et al. Arthritogenic alphaviral infection perturbs osteoblast function and triggers pathologic bone loss. *Proc Natl Acad Sci U S A* **111**, 6040-6045, doi:10.1073/pnas.1318859111 (2014).

142 Chow, A. et al. Persistent arthralgia induced by Chikungunya virus infection is associated with interleukin-6 and granulocyte macrophage colony-stimulating factor. *J. Infect. Dis.* **203**, 149-157, doi:10.1093/infdis/jiq042 (2011).

143 Noret, M. et al. Interleukin 6, RANKL, and osteoprotegerin expression by chikungunya virus-infected human osteoblasts. *J. Infect. Dis.* **206**, 455-457: 457-459, doi:10.1093/infdis/jis368 (2012).

144 Ng, L. F. et al. IL-1beta, IL-6, and RANTES as biomarkers of Chikungunya severity. *PLoS ONE* **4**, e4261, doi:10.1371/journal.pone.0004261 (2009).

145 Manimunda, S. P. et al. Clinical progression of chikungunya fever during acute and chronic arthritic stages and the changes in joint morphology as revealed by imaging. *Trans. R. Soc. Trop. Med. Hyg.* **104**, 392-399, doi:10.1016/j.trstmh.2010.01.011 (2010).

146 Rampal, Sharda, M. & Meena, H. Neurological complications in Chikungunya fever. *J Assoc Physicians India* **55**, 765-769 (2007).
147 Lemant, J. et al. Serious acute chikungunya virus infection requiring intensive care during the Reunion Island outbreak in 2005-2006. *Crit Care Med* **36**, 2536-2541, doi:10.1097/CCM.0b013e318183f2d2 (2008).

148 Brito Ferreira, M. L. et al. Neurological disease in adults with Zika and chikungunya virus infection in Northeast Brazil: a prospective observational study. *Lancet Neurol* **19**, 826-839, doi:10.1016/S1474-4422(20)30232-5 (2020).

149 Robin, S. et al. Neurologic manifestations of pediatric chikungunya infection. *J Child Neurol* **23**, 1028-1035, doi:10.1177/0883073808314151 (2008).

150 Lewthwaite, P. et al. Chikungunya virus and central nervous system infections in children, India. *Emerg Infect Dis* **15**, 329-331, doi:10.3201/eid1502.080902 (2009).

151 Schilte, C. et al. Type I IFN controls chikungunya virus via its action on nonhematopoietic cells. *J. Exp. Med.* **207**, 429-442, doi:10.1084/jem.20090851 (2010).

152 Mutricy, R. et al. Mayaro virus infection in French Guiana, a cross sectional study 2003-2019. *Infect Genet Evol* **99**, 105243, doi:10.1016/j.meegid.2022.105243 (2022).

153 Robillard, P. Y. et al. [Vertical maternal fetal transmission of the chikungunya virus. Ten cases among 84 pregnant women]. *Presse Med* **35**, 785-788, doi:10.1016/s0755-4982(06)74690-5 (2006).

154 Sagay, A. S. et al. Chikungunya virus antepartum transmission and abnormal infant outcomes in a cohort of pregnant women in Nigeria. *Int J Infect Dis* **139**, 92-100, doi:10.1016/j.ijid.2023.11.036 (2024).

155 Ramful, D. et al. Mother-to-child transmission of Chikungunya virus infection. *Pediatr Infect Dis J* **26**, 811-815, doi:10.1097/INF.0b013e3180616d4f (2007).

156 Lenglet, Y. et al. [Chikungunya infection in pregnancy: Evidence for intrauterine infection in pregnant women and vertical transmission in the parturient. Survey of the Reunion Island outbreak]. *J Gynecol Obstet Biol Reprod (Paris)* **35**, 578-583, doi:10.1016/s0368-2315(06)76447-x (2006).

157 Foeller, M. E. et al. Chikungunya infection in pregnancy - reassuring maternal and perinatal outcomes: a retrospective observational study. *BJOG* **128**, 1077-1086, doi:10.1111/1471-0528.16562 (2021).

158 Gupta, S. & Gupta, N. Short-term pregnancy outcomes in patients chikungunya infection: An observational study. *J Family Med Prim Care* **8**, 985-987, doi:10.4103/jfmpc.jfmpc_274_18 (2019).

159 Ali, A. A., Abdallah, T. M., Alshareef, S. A., Al-Nafeesah, A. & Adam, I. Maternal and perinatal outcomes during a Chikungunya outbreak in Kassala, eastern Sudan. *Arch Gynecol Obstet* **305**, 855-858, doi:10.1007/s00404-021-06204-6 (2022).

160 Salomão, N. et al. Histopathological and immunological characteristics of placentas infected with chikungunya virus. *Front Microbiol* **13**, 1055536, doi:10.3389/fmicb.2022.1055536 (2022).

161 Kang, H. et al. Chikungunya seroprevalence, force of infection, and prevalence of chronic disability after infection in endemic and epidemic settings: a systematic review, meta-analysis, and modelling study. *Lancet Infect Dis* **24**, 488-503, doi:10.1016/S1473-3099(23)00810-1 (2024).

162 Sharp, T. M. et al. Clinical Characteristics, Histopathology, and Tissue Immunolocalization of Chikungunya Virus Antigen in Fatal Cases. *Clin. Infect. Dis.* **73**, e345-e354, doi:10.1093/cid/ciaa837 (2021).

163 Cerqueira-Silva, T. et al. Risk of death following chikungunya virus disease in the 100 Million Brazilian Cohort, 2015-18: a matched cohort study and self-controlled case series. *Lancet Infect. Dis.* **24**, 504-513, doi:10.1016/S1473-3099(23)00739-9 (2024).

164 Hallengard, D. et al. Novel attenuated Chikungunya vaccine candidates elicit protective immunity in C57BL/6 mice. *J. Virol.* **88**, 2858-2866, doi:10.1128/JVI.03453-13 (2014).
165 Roques, P. et al. Attenuated and vectored vaccines protect nonhuman primates against Chikungunya virus. *JCI Insight* 2, e83527, doi:10.1172/jci.insight.83527 (2017).

166 Schneider, M. et al. Safety and immunogenicity of a single-shot live-attenuated chikungunya vaccine: a double-blind, multicentre, randomised, placebo-controlled, phase 3 trial. *Lancet*, doi:10.1016/S0140-6736(23)00641-4 (2023).

167 Wressnigg, N. et al. Single-shot live-attenuated chikungunya vaccine in healthy adults: a phase 1, randomised controlled trial. *Lancet Infect. Dis.* **20**, 1193-1203, doi:10.1016/S1473-3099(20)30238-3 (2020).

168 Buerger, V. et al. Safety and immunogenicity of a live-attenuated chikungunya virus vaccine in endemic areas of Brazil: interim results of a double-blind, randomised, placebo-controlled phase 3 trial in adolescents. *Lancet Infect Dis*, doi:10.1016/S1473-3099(24)00458-4 (2024).

169 Rezza, G. & Weaver, S. C. Chikungunya as a paradigm for emerging viral diseases: Evaluating disease impact and hurdles to vaccine development. *PLoS Negl Trop Dis* **13**, e0006919, doi:10.1371/journal.pntd.0006919 (2019).

170 Roques, P. et al. Effectiveness of CHIKV vaccine VLA1553 demonstrated by passive transfer of human sera. *JCI Insight* 7, doi:10.1172/jci.insight.160173 (2022).

171 Ng, L. F. P. & Rénia, L. Live-attenuated chikungunya virus vaccine. *Cell* **187**, 813-813.e811, doi:10.1016/j.cell.2024.01.033 (2024).

172 Weber, W. C. et al. The Approved Live-Attenuated Chikungunya Virus Vaccine (IXCHIQ. Vaccines (Basel) **12**, doi:10.3390/vaccines12080893 (2024).

173 Bennett, S. R. et al. Safety and immunogenicity of PXVX0317, an aluminium hydroxide-adjuvanted chikungunya virus-like particle vaccine: a randomised, double-blind, parallel-group, phase 2 trial. *Lancet Infect Dis* **22**, 1343-1355, doi:10.1016/S1473-3099(22)00226-2 (2022).

174 Chen, G. L. et al. Effect of a Chikungunya Virus-Like Particle Vaccine on Safety and Tolerability Outcomes: A Randomized Clinical Trial. *JAMA* **323**, 1369-1377, doi:10.1001/jama.2020.2477 (2020).

175 Goo, L. et al. A Virus-Like Particle Vaccine Elicits Broad Neutralizing Antibody Responses in Humans to All Chikungunya Virus Genotypes. *J. Infect. Dis.* **214**, 1487-1491, doi:10.1093/infdis/jiw431 (2016).

176 Chang, L. J. et al. Safety and tolerability of chikungunya virus-like particle vaccine in healthy adults: a phase 1 dose-escalation trial. *Lancet* **384**, 2046-2052, doi:10.1016/S0140-6736(14)61185-5 (2014).

177 Akahata, W. et al. A virus-like particle vaccine for epidemic Chikungunya virus protects nonhuman primates against infection. *Nat. Med.* **16**, 334-338, doi:10.1038/nm.2105 (2010).

178 Folegatti, P. M. et al. A single dose of ChAdOx1 Chik vaccine induces neutralizing antibodies against four chikungunya virus lineages in a phase 1 clinical trial. *Nat Commun* **12**, 4636, doi:10.1038/s41467-021-24906-y (2021).

179 Reisinger, E. C. et al. Immunogenicity, safety, and tolerability of the measles-vectored chikungunya virus vaccine MV-CHIK: a double-blind, randomised, placebo-controlled and active-controlled phase 2 trial. *Lancet* **392**, 2718-2727, doi:10.1016/S0140-6736(18)32488-7 (2019).

180 Shaw, C. A. et al. A phase 1, randomized, placebo-controlled, dose-ranging study to evaluate the safety and immunogenicity of an mRNA-based chikungunya virus vaccine in healthy adults. *Vaccine* **41**, 3898-3906, doi:10.1016/j.vaccine.2023.04.064 (2023).

181 Wressnigg, N. et al. An inactivated Ross River virus vaccine is well tolerated and immunogenic in an adult population in a randomized phase 3 trial. *Clin Vaccine Immunol* **22**, 267-273, doi:10.1128/CVI.00546-14 (2015).
182 Wang, E., Kim, D. Y., Weaver, S. C. & Frolov, I. Chimeric Chikungunya viruses are nonpathogenic in highly sensitive mouse models but efficiently induce a protective immune response. *J. Virol.* **85**, 9249-9252, doi:10.1128/JVI.00844-11 (2011).

183 Adam, A. et al. A safe insect-based Chikungunya fever vaccine affords rapid and durable protection in cynomolgus macaques. *bioRxiv*, doi:10.1101/2024.05.21.595029 (2024).

184 Partidos, C. D. et al. Cross-protective immunity against o'nyong-nyong virus afforded by a novel recombinant chikungunya vaccine. *Vaccine* **30**, 4638-4643, doi:10.1016/j.vaccine.2012.04.099 (2012).

185 Chattopadhyay, A., Wang, E., Seymour, R., Weaver, S. C. & Rose, J. K. A chimeric vesiculo/alphavirus is an effective alphavirus vaccine. *J. Virol.* **87**, 395-402, doi:10.1128/JVI.01860-12 (2013).

186 García-Arriaza, J. et al. A novel poxvirus-based vaccine, MVA-CHIKV, is highly immunogenic and protects mice against chikungunya infection. *J Virol* **88**, 3527-3547, doi:10.1128/JVI.03418-13 (2014).

187 Weise, W. J. et al. A novel live-attenuated vaccine candidate for mayaro Fever. *PLoS Negl Trop Dis* **8**, e2969, doi:10.1371/journal.pntd.0002969 (2014).

188 Choi, H. et al. Protective immunity by an engineered DNA vaccine for Mayaro virus. *PLoS Negl Trop Dis* **13**, e0007042, doi:10.1371/journal.pntd.0007042 (2019).

189 Powers, J. M. et al. Non-replicating adenovirus based Mayaro virus vaccine elicits protective immune responses and cross protects against other alphaviruses. *PLoS Negl Trop Dis* **15**, e0009308, doi:10.1371/journal.pntd.0009308 (2021).

190 Kim, Y. C., Lücke, A. C., López-Camacho, C., Kümmerer, B. M. & Reyes-Sandoval, A. Development of Viral-Vectored Vaccines and Virus Replicon Particle-Based Neutralisation Assay against Mayaro Virus. *Int J Mol Sci* **23**, doi:10.3390/ijms23084105 (2022).

191 Abbo, S. R. et al. Comparative Efficacy of Mayaro Virus-Like Particle Vaccines Produced in Insect or Mammalian Cells. *J. Virol.* **97**, e0160122, doi:10.1128/jvi.01601-22 (2023).

192 Weber, W. C. et al. Nonreciprocity in CHIKV and MAYV Vaccine-Elicited Protection. *Vaccines (Basel)* **12**, doi:10.3390/vaccines12090970 (2024).

193 Ashbrook, A. W. et al. Antagonism of the Sodium-Potassium ATPase Impairs Chikungunya Virus Infection. *MBio* **7**, doi:10.1128/mBio.00693-16 (2016).

194 Bassetto, M. et al. Computer-aided identification, design and synthesis of a novel series of compounds with selective antiviral activity against chikungunya virus. *Antiviral Res* **98**, 12-18, doi:10.1016/j.antiviral.2013.01.002 (2013).

195 Cruz, D. J. et al. Identification of novel compounds inhibiting chikungunya virus-induced cell death by high throughput screening of a kinase inhibitor library. *PLoS Negl Trop Dis* **7**, e2471, doi:10.1371/journal.pntd.0002471 (2013).

196 Yin, P. et al. 4'-Fluorouridine inhibits alphavirus replication and infection. *mBio* **15**, e0042024, doi:10.1128/mbio.00420-24 (2024).

197 Langendries, L., Abdelnabi, R., Neyts, J. & Delang, L. Repurposing Drugs for Mayaro Virus: Identification of EIDD-1931, Favipiravir and Suramin as Mayaro Virus Inhibitors. *Microorganisms* **9**, doi:10.3390/microorganisms9040734 (2021).

198 Karlas, A. et al. A human genome-wide loss-of-function screen identifies effective chikungunya antiviral drugs. *Nat Commun* **7**, 11320, doi:10.1038/ncomms11320 (2016).

199 Pal, P. et al. Development of a highly protective combination monoclonal antibody therapy against Chikungunya virus. *PLoS Pathog* **9**, e1003312, doi:10.1371/journal.ppat.1003312 (2013).

200 Smith, S. A. et al. Isolation and Characterization of Broad and Ultrapotent Human Monoclonal Antibodies with Therapeutic Activity against Chikungunya Virus. *Cell Host Microbe* **18**, 86-95, doi:10.1016/j.chom.2015.06.009 (2015).
201 Pal, P. et al. Chikungunya viruses that escape monoclonal antibody therapy are clinically attenuated, stable, and not purified in mosquitoes. *J. Virol.* **88**, 8213-8226, doi:10.1128/JVI.01032-14 (2014).

202 Zhou, Q. F. et al. Structural basis of Chikungunya virus inhibition by monoclonal antibodies. *Proc Natl Acad Sci U S A* **117**, 27637-27645, doi:10.1073/pnas.2008051117 (2020).

203 Earnest, J. T. et al. The mechanistic basis of protection by non-neutralizing anti-alphavirus antibodies. *Cell Rep* **35**, 108962, doi:10.1016/j.celrep.2021.108962 (2021).

204 Powell, L. A. et al. Human monoclonal antibodies against Ross River virus target epitopes within the E2 protein and protect against disease. *PLoS Pathog* **16**, e1008517, doi:10.1371/journal.ppat.1008517 (2020).

205 Long, F. et al. Cryo-EM structures elucidate neutralizing mechanisms of anti-chikungunya human monoclonal antibodies with therapeutic activity. *Proc Natl Acad Sci U S A* **112**, 13898-13903, doi:10.1073/pnas.1515558112 (2015).

206 Earnest, J. T. et al. Neutralizing antibodies against Mayaro virus require Fc effector functions for protective activity. *J. Exp. Med.* **216**, 2282-2301, doi:10.1084/jem.20190736 (2019).

207 Malonis, R. J. et al. Near-germline human monoclonal antibodies neutralize and protect against multiple arthritogenic alphaviruses. *Proc Natl Acad Sci U S A* **118**, doi:10.1073/pnas.2100104118 (2021).

208 Kim, A. S. et al. Pan-protective anti-alphavirus human antibodies target a conserved E1 protein epitope. *Cell* **184**, 4414-4429.e4419, doi:10.1016/j.cell.2021.07.006 (2021).

209 Hucke, F. I. L. & Bugert, J. J. Current and Promising Antivirals Against Chikungunya Virus. *Front Public Health* **8**, 618624, doi:10.3389/fpubh.2020.618624 (2020).

210 Kovacikova, K. & van Hemert, M. J. Small-Molecule Inhibitors of Chikungunya Virus: Mechanisms of Action and Antiviral Drug Resistance. *Antimicrob Agents Chemother* **64**, doi:10.1128/AAC.01788-20 (2020).

211 Powers, A. M. Vaccine and Therapeutic Options To Control Chikungunya Virus. *Clin. Microbiol. Rev.* **31**, doi:10.1128/CMR.00104-16 (2018).

212 Abdelnabi, R., Jacobs, S., Delang, L. & Neyts, J. Antiviral drug discovery against arthritogenic alphaviruses: Tools and molecular targets. *Biochem Pharmacol* **174**, 113777, doi:10.1016/j.bcp.2019.113777 (2020).

213 Montenegro-Quiñonez, C. A. et al. Interventions against Aedes/dengue at the household level: a systematic review and meta-analysis. *EBioMedicine* **93**, 104660, doi:10.1016/j.ebiom.2023.104660 (2023).

214 Li, M. et al. Suppressing mosquito populations with precision guided sterile males. *Nat Commun* **12**, 5374, doi:10.1038/s41467-021-25421-w (2021).

215 Lee, C. et al. Gravitraps for management of dengue clusters in Singapore. *Am J Trop Med Hyg* **88**, 888-892, doi:10.4269/ajtmh.12-0329 (2013).

216 Ho, S. H. et al. Singapore's 5 decades of dengue prevention and control-Implications for global dengue control. *PLoS Negl Trop Dis* **17**, e0011400, doi:10.1371/journal.pntd.0011400 (2023).

217 Zheng, X. et al. Incompatible and sterile insect techniques combined eliminate mosquitoes. *Nature* **572**, 56-61, doi:10.1038/s41586-019-1407-9 (2019).

218 Ribeiro Dos Santos, G. et al. Estimating the effect of the wMel release programme on the incidence of dengue and chikungunya in Rio de Janeiro, Brazil: a spatiotemporal modelling study. *Lancet Infect Dis* **22**, 1587-1595, doi:10.1016/S1473-3099(22)00436-4 (2022).

219 Aliota, M. T. et al. The wMel Strain of Wolbachia Reduces Transmission of Chikungunya Virus in Aedes aegypti. *PLoS Negl Trop Dis* **10**, e0004677, doi:10.1371/journal.pntd.0004677 (2016).
220 Tan, C. H. et al. wMel limits zika and chikungunya virus infection in a Singapore Wolbachia-introgressed Ae. aegypti strain, wMel-Sg. *PLoS Negl Trop Dis* 11, e0005496, doi:10.1371/journal.pntd.0005496 (2017).
## Glossary

**Spillover**: pathogen transmission from zoonotic wildlife hosts to humans.

**Enzootic**: Ancestral, often continuous cycles of zoonotic arboviruses involving wild animals serving as amplification and/or reservoir hosts.

**Epistasis**: genetic phenomenon when the expression of a gene is modified by the expression of one or more other genes.

**Viral clearance**: a process by which the immune system eliminates a virus from the body.

**Efferocytosis**: a process by which professional and non-professional phagocytes clear apoptotic cells.

**Transduced cells**: cells that have been genetically modified by introducing foreign DNA into their genome.

**Antibody-dependent enhancement**: a phenomenon where pre-existing antibodies generated from prior infections with the related viruses or vaccination, instead of neutralizing the virus, facilitate its entry into host cells, leading to increased viral replication and potentially more severe disease.

## Box 1. Animal models

Animal models are an essential component of research on arthritogenic alphaviruses. They have provided valuable knowledge regarding viral pathogenesis, transmission dynamics, and the development of potential therapeutic interventions. Each animal model has its strengths and limitations, which should be selected based on specific research questions. Currently, wild-type, knockout, and humanized mice are the most widely used due to their ease of handling and genetic modification, as well as their relatively low cost. Despite being essential to pathogenesis studies, mouse models have limitations because they only partially mimic diseases observed in humans, and thus are a limited platform for the study of vaccine testing. Hamsters (*Mesocricetus auratus*) are susceptible to arthritogenic alphavirus infection (CHIKV) and develop myositis and tenosynovitis like human patients. However, the viral clearance usually takes only four days, and there are limited biological resources available compared to mice. NHPs (e.g., *Macaca fascicularis* and *Macaca mulatta*) have been valuable for studying arthritogenic alphaviruses because they share many physiological and immunological similarities with humans, and pathogenesis resembles that observed in humans (CHIKV and MAYV). The use of an NHP model was pivotal to the approval of a vaccine against chikungunya in the USA and Canada in the absence of a clinical efficacy trial. However, the utilization of NHPs has high costs and significant logistical challenges. Alternative animal models have been explored, such as Zebrafish (*Danio rerio*), which offers a unique advantage for visualizing viral spread and host responses at a cellular level, providing valuable insights into the early stages of infection, particularly for neurological infection. The use of animal models should be limited on ethical grounds when alternative *in vitro* or *in silico* methods are not available. All animal experimentation must comply with local regulations, and the principles of Reduction, Refinement, Replacement, and Responsibility should be considered.
<table><thead><tr><th>Animal model</th><th>Advantages</th><th>Disadvantages</th></tr></thead><tbody><tr><td>Mice</td><td><ul><li>Ease of handling</li><li>Genetic modification</li><li>Relatively low cost</li><li>Several biological resources available</li></ul></td><td><ul><li>Partially mimic diseases observed in humans</li><li>Limited for vaccine testing</li></ul></td></tr><tr><td>Hamster</td><td><ul><li>Ease of handling</li><li>Myositis and tenosynovitis like human patients</li></ul></td><td><ul><li>Short viral clearance (~ 4 days)</li><li>Limited biological resources available</li><li>Limited for vaccine testing</li></ul></td></tr><tr><td>Non-human primate</td><td><ul><li>Physiologically and immunologically similar with humans</li><li>Pathology resembled the diseases observed in humans</li><li>Suitable for antiviral and vaccine testing</li></ul></td><td><ul><li>High cost</li><li>Significant logistical issues</li></ul></td></tr><tr><td>Zebra fish</td><td><ul><li>Ease of handling</li><li>Genetic modification</li><li>Relatively low cost</li><li>Visualizing viral spread and host response at cellular level</li></ul></td><td><ul><li>Partially mimic diseases observed in humans</li><li>Limited for viral and vaccine testing</li><li>Limited biological resources available</li></ul></td></tr></tbody></table>

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**Figure. Arthritogenic alphaviruses and animal models.** Examples of animal models used in experimental infection with arthritogenic alphaviruses: advancements and limitations.

## Box 2. Laboratory diagnosis

Following an infected mosquito bite, arthritogenic alphaviruses replicate in skin and fibroblast cells before entering the bloodstream and disseminating to multiple organs, including the liver, muscle, joints, and lymphoid tissues. The incubation period usually is between 2 and 7 days; then, symptoms typically appear abruptly, including high fever, muscle pain, skin rash, weakness, and headache, which typically last for 4 to 8 days. Clinical presentations of arthritogenic alphavirus infections can be similar to other arboviruses (e.g., dengue, Zika, and yellow fever viruses), making laboratory testing essential for diagnosis. Acute infection diagnosis can be performed by detecting viral RNA in serum or plasma using directed methods up to 8 days from the onset of symptoms and, in some patients, up to 14 days. The methods can include reverse transcription polymerase chain reactions (RT-PCRs) and reverse transcription loop-mediated isothermal amplification (RT-LAMP). During the viremic period, positive samples can also be used for genomic sequencing to determine the viral lineage or genotype. Acute infection can be detected by a serological test for IgM, which appears around 4 days after symptom onset and can remain detectable for up to 3 months. IgM testing can be useful in patients with signs and symptoms after a negative nucleic acid test result. For IgM testing, there are enzyme-linked immunosorbent assay (ELISA) and immunochromatographic tests available for CHIKV, MAYV, and RRV. Additionally, a retrospective diagnosis of arthritogenic alphavirus infections can be performed by the detection of IgG antibodies in serum samples, which usually appear 7 to 10 days after the onset of symptoms and persist for years. The plaque reduction neutralization test (PRNT) is the most specific serological test for confirming infection, but it is technically demanding and not widely available in clinical settings.
<figure><img src="image_11.png" /><figcaption>**Figure. Arthritogenic alphaviruses and laboratory diagnosis.** Diagnostic methods employed during the acute and convalescent phases of arthritogenic alphavirus infection.</figcaption></figure>