Development and application of a colloidal-gold immunochromatographic strip for detecting Getah virus antibodies.

Getah virus (GETV) is a re-emerging mosquito-borne alphavirus that is highly pathogenic, mainly to pigs and horses. There are no vaccines or treatments available for GETV in swine in China. Therefore, the development of a simple, rapid, specific, and sensitive serological assay for GETV antibodies is essential for the prevention and control of GETV. Current antibody monitoring methods are time-consuming, expensive, and dependent on specialized instrumentation, and these features are not conducive to rapid detection in clinical samples. To address these problem, we developed immunochromatographic test strips (ICTS) using eukaryotically expressed soluble recombinant p62-E1 protein of GETV as a labelled antigen, which has good detection sensitivity and no cross-reactivity with other common porcine virus-positive sera. The ICTS is highly compatible with IFA and ELISA and can be stored for 1 month at 37 °C and for at least 3 months at room temperature. Hence, p62-E1-based ICTS is a rapid, accurate, and convenient method for rapid on-site detection of GETV antibodies. KEY POINTS: • We established a rapid antibody detection method that can monitor GETV infection • We developed colloidal gold test strips with high sensitivity and specificity • The development of colloidal gold test strips will aid in the field serologic detection of GETV.

Virus-specific antibody secreting cells reside in the peritoneal cavity and systemic immune sites of Atlantic salmon (Salmo salar) challenged intraperitoneally with salmonid alphavirus.

The development and persistence of antibody secreting cells (ASC) after antigenic challenge remain inadequately understood in teleosts. In this study, intraperitoneal (ip) injection of Atlantic salmon (Salmo salar) with salmonid alphavirus (WtSAV3) increased the total ASC response, peaking 3-6 weeks post injection (wpi) locally in the peritoneal cavity (PerC) and in systemic lymphoid tissues, while at 13 wpi the response was only elevated in PerC. At the same time point a specific ASC response was induced by WtSAV3 in PerC and systemic tissues, with the highest frequency in PerC, suggesting a local role. Inactivated SAV (InSAV1) induced comparatively lower ASC responses in all sites, and specific serum antibodies were only induced by WtSAV3 and not by InSAV1. An InSAV1 boost did not increase these responses. Expression of immune marker genes implies a role for PerC adipose tissue in the PerC immune response. Overall, the study suggests the Atlantic salmon PerC as a secondary immune site and an ASC survival niche.

Evolutionary Profile of Mayaro Virus in the Americas: An Update into Genome Variability.

The Mayaro virus (MAYV) is an arbovirus with emerging potential, though with a limited understanding of its epidemiology and evolution due to the lack of studies and surveillance. Here, we investigated 71 MAYV genome sequences from the Americas available at GenBank and characterized the phylogenetic relationship among virus strains. A phylogenetic analysis showed that sequences were grouped according to the genotypes L, D, and N. Genotype D sequences were closely related to sequences collected in adjacent years and from their respective countries, suggesting that isolates may have originated from circulating lineages. The coalescent analysis demonstrated similar results, indicating the continuous circulation of the virus between countries as well. An unidentified sequence from the USA was grouped with genotype D, suggesting the insertion of this genotype in the country. Furthermore, the recombination analysis detected homologous and three heterologous hybrids which presented an insertion into the nsP3 protein. Amino acid substitutions among sequences indicated selective pressure sites, suggesting viral adaptability. This also impacted the binding affinity between the E1-E2 protein complex and the Mxra8 receptor, associated with MAYV entry into human cells. These results provide information for a better understanding of genotypes circulating in the Americas.

Pathogenicity and virulence of O'nyong-nyong virus: A less studied Togaviridae with pandemic potential.

O'nyong-nyong virus (ONNV) is a neglected mosquito-borne alphavirus belonging to the Togaviridae family. ONNV is known to be responsible for sporadic outbreaks of acute febrile disease and polyarthralgia in Africa. As climate change increases the geographical range of known and potential new vectors, recent data indicate a possibility for ONNV to spread outside of the African continent and grow into a greater public health concern. In this review, we summarise the current knowledge on ONNV epidemiology, host-pathogen interactions, vector-virus responses, and insights into possible avenues to control risk of further epidemics. In this review, the limited ONNV literature is compared and correlated to other findings on mainly Old World alphaviruses. We highlight and discuss studies that investigate viral and host factors that determine viral-vector specificity, along with important mechanisms that determine severity and disease outcome of ONNV infection.

Mayaro virus, a potential threat for Europe: vector competence of autochthonous vector species.

BACKGROUND: Mayaro virus (MAYV) is an emerging alphavirus, primarily transmitted by the mosquito Haemagogus janthinomys in Central and South America. However, recent studies have shown that Aedes aegypti, Aedes albopictus and various Anopheles mosquitoes can also transmit the virus under laboratory conditions. MAYV causes sporadic outbreaks across the South American region, particularly in areas near forests. Recently, cases have been reported in European and North American travelers returning from endemic areas, raising concerns about potential introductions into new regions. This study aims to assess the vector competence of three potential vectors for MAYV present in Europe. METHODS: Aedes albopictus from Italy, Anopheles atroparvus from Spain and Culex pipiens biotype molestus from Belgium were exposed to MAYV and maintained under controlled environmental conditions. Saliva was collected through a salivation assay at 7 and 14 days post-infection (dpi), followed by vector dissection. Viral titers were determined using focus forming assays, and infection rates, dissemination rates, and transmission efficiency were calculated. RESULTS: Results indicate that Ae. albopictus and An. atroparvus from Italy and Spain, respectively, are competent vectors for MAYV, with transmission possible starting from 7 dpi under laboratory conditions. In contrast, Cx. pipiens bioform molestus was unable to support MAYV infection, indicating its inability to contribute to the transmission cycle. CONCLUSIONS: In the event of accidental MAYV introduction in European territories, autochthonous outbreaks could potentially be sustained by two European species: Ae. albopictus and An. atroparvus. Entomological surveillance should also consider certain Anopheles species when monitoring MAYV transmission.

Molecular Epidemiology of Mayaro Virus among Febrile Patients, Roraima State, Brazil, 2018-2021.

We detected Mayaro virus (MAYV) in 3.4% (28/822) of febrile patients tested during 2018-2021 from Roraima State, Brazil. We also isolated MAYV strains and confirmed that these cases were caused by genotype D. Improved surveillance is needed to better determine the burden of MAYV in the Amazon Region.

Exposure dynamics of Ross River virus in horses - Horses as potential sentinels (a One Health approach).

Ross River virus (RRV), the most medically and economically important arbovirus in Australia, has been the most prevalent arbovirus infections in humans for many years. Infected humans and horses often suffer similar clinical symptoms. We conducted a prospective longitudinal study over a 3.5-year period to investigate the exposure dynamics of RRV in three foal cohorts (n = 32) born in a subtropical region of South East Queensland, Australia, between 2020 and 2022. RRV-specific seroconversion was detected in 56% (n = 18) of foals with a median time to seroconversion, after waning of maternal antibodies, of 429 days (95% CI: 294-582). The median age at seroconversion was 69 weeks (95% CI: 53-57). Seroconversion events were only detected between December and March (Southern Hemisphere summer) over the entire study period. Cox proportion hazards regression analyses revealed that seroconversions were significantly (p < 0.05) associated with air temperature in the month of seroconversion. Time-lags in meteorological variables were not significantly (p > 0.05) associated with seroconversion, except for relative humidity (p = 0.036 at 2-month time-lag). This is in contrast to research results of RRV infection in humans, which peaked between March and May (Autumn) and with a 0-3 month time-lag for various meteorological risk factors. Therefore, horses may be suitable sentinels for monitoring active arbovirus circulation and could be used for early arbovirus outbreak detection in human populations.

Trans-Amplifying RNA: A Journey from Alphavirus Research to Future Vaccines.

Replicating RNA, including self-amplifying RNA (saRNA) and trans-amplifying RNA (taRNA), holds great potential for advancing the next generation of RNA-based vaccines. Unlike in vitro transcribed mRNA found in most current RNA vaccines, saRNA or taRNA can be massively replicated within cells in the presence of RNA-amplifying enzymes known as replicases. We recently demonstrated that this property could enhance immune responses with minimal injected RNA amounts. In saRNA-based vaccines, replicase and antigens are encoded on the same mRNA molecule, resulting in very long RNA sequences, which poses significant challenges in production, delivery, and stability. In taRNA-based vaccines, these challenges can be overcome by splitting the replication system into two parts: one that encodes replicase and the other that encodes a short antigen-encoding RNA called transreplicon. Here, we review the identification and use of transreplicon RNA in alphavirus research, with a focus on the development of novel taRNA technology as a state-of-the art vaccine platform. Additionally, we discuss remaining challenges essential to the clinical application and highlight the potential benefits related to the unique properties of this future vaccine platform.

DEAD box RNA helicase 5 is a new pro-viral host factor for Sindbis virus infection.

BACKGROUND: RNA helicases are emerging as key factors regulating host-virus interactions. The DEAD-box ATP-dependent RNA helicase DDX5, which plays an important role in many aspects of cellular RNA biology, was also found to either promote or inhibit viral replication upon infection with several RNA viruses. Here, our aim is to examine the impact of DDX5 on Sindbis virus (SINV) infection. METHODS: We analysed the interaction between DDX5 and the viral RNA using imaging and RNA-immunoprecipitation approaches. The interactome of DDX5 in mock- and SINV-infected cells was determined by mass spectrometry. We validated the interaction between DDX17 and the viral capsid by co- immunoprecipitation in the presence or absence of an RNase treatment. We determined the subcellular localization of DDX5, its cofactor DDX17 and the viral capsid protein by co-immunofluorescence. Finally, we investigated the impact of DDX5 depletion and overexpression on SINV infection at the viral protein, RNA and infectious particle accumulation level. The contribution of DDX17 was also tested by knockdown experiments. RESULTS: In this study we demonstrate that DDX5 interacts with the SINV RNA during infection. Furthermore, the proteomic analysis of the DDX5 interactome in mock and SINV-infected HCT116 cells identified new cellular and viral partners and confirmed the interaction between DDX5 and DDX17. Both DDX5 and DDX17 re-localize from the nucleus to the cytoplasm upon SINV infection and interact with the viral capsid protein. We also show that DDX5 depletion negatively impacts the viral replication cycle, while its overexpression has a pro-viral effect. Finally, we observed that DDX17 depletion reduces SINV infection, an effect which is even more pronounced in a DDX5-depleted background, suggesting a synergistic pro-viral effect of the DDX5 and DDX17 proteins on SINV. CONCLUSIONS: These results not only shed light on DDX5 as a novel and important host factor to the SINV life cycle, but also expand our understanding of the roles played by DDX5 and DDX17 as regulators of viral infections.

Assessment of Mayaro virus vector competence of the mosquito Aedes aegypti (Linnaeus, 1762) populations in Argentine using dose-response assays.

Mayaro virus (MAYV; Alphavirus: Togaviridae) is an emerging pathogen in Latin America, causing fever and polyarthritis. Sporadic outbreaks of MAYV have occurred in the region, with reported human cases being imported to Europe and North America. Although primarily a risk for those residing in the Amazon basin's tropical forests, recent reports highlight that urbanization would increase the risk of MAYV transmission in Latin America. Urban emergence depends on human susceptibility and the ability of mosquitos like Aedes aegypti  (Linnaeus, 1762) (Diptera: Culicidae) to transmit MAYV. Despite the absence of active MAYV transmission in Argentine, the risk of introduction is substantial due to human movement and the presence of Ae. aegypti in the region. This study aimed to evaluate the susceptibility of different Argentine Ae. aegypti populations to MAYV genotype L (MAYV-L) using dose-response assays and determine barriers to virus infection, dissemination and transmission. Immature mosquito stages were collected in Buenos Aires, Córdoba and Rosario cities. Female Ae. aegypti (F2) were orally infected by feeding on five concentrations of MAYV-L, ranging from 1.0 to 6.0 log10 PFU/mL. Abdomens, legs and saliva were analysed using viral plaque assays. Results revealed that MAYV-L between infection and dissemination were associated with viral doses rather than the population origin. Infection rates varied between 3% and 65%, with a 50% infectious dose >5.5 log10 PFU/mL. Dissemination occurred at 39%, with a 50% dissemination dose of ~6.0 log10 PFU/mL. Dissemination among infected mosquitoes ranged from 60% to 86%, and transmission from disseminated mosquitoes ranged from 11% to 20%. Argentine Ae. aegypti populations exhibited a need for higher viral doses of MAYV-L than those typically found in humans to become infected. In addition, only a small proportion of infected mosquitoes were capable of transmitting the virus. Understanding MAYV transmission in urban areas is crucial for public health interventions.

Extreme infectious titer variability in individual Aedes aegypti mosquitoes infected with Sindbis virus is associated with both differences in virus population structure and dramatic disparities in specific infectivity.

Variability in how individuals respond to pathogens is a hallmark of infectious disease, yet the basis for individual variation in host response is often poorly understood. The titer of infectious virus among individual mosquitoes infected with arboviruses is frequently observed to vary by several orders of magnitude in a single experiment, even when the mosquitoes are highly inbred. To better understand the basis for this titer variation, we sequenced populations of Sindbis virus (SINV) obtained from individual infected Aedes aegypti mosquitoes that, despite being from a highly inbred laboratory colony, differed in their titers of infectious virus by approximately 10,000-fold. We observed genetic differences between these virus populations that indicated the virus present in the midguts of low titer mosquitoes was less fit than that of high titer mosquitoes, possibly due to founder effects that occurred during midgut infection. Furthermore, we found dramatic differences in the specific infectivity or SI (the ratio of infectious units/viral genome equivalents) between these virus populations, with the SI of low titer mosquitoes being up to 10,000-fold lower than that of high titer mosquitoes. Despite having similar amounts of viral genomes, low titer mosquitoes appeared to contain less viral particles, suggesting that viral genomes were packaged into virions less efficiently than in high titer mosquitoes. Finally, antibiotic treatment, which has been shown to suppress mosquito antiviral immunity, caused an increase in SI. Our results indicate that the extreme variation that is observed in SINV infectious titer between individual Ae. aegypti mosquitoes is due to both genetic differences between virus populations and to differences in the proportion of genomes that are packaged into infectious particles.

Testing the intrinsic mechanisms driving the dynamics of Ross River Virus across Australia.

The mechanisms driving dynamics of many epidemiologically important mosquito-borne pathogens are complex, involving combinations of vector and host factors (e.g., species composition and life-history traits), and factors associated with transmission and reporting. Understanding which intrinsic mechanisms contribute most to observed disease dynamics is important, yet often poorly understood. Ross River virus (RRV) is Australia's most important mosquito-borne disease, with variable transmission dynamics across geographic regions. We used deterministic ordinary differential equation models to test mechanisms driving RRV dynamics across major epidemic centers in Brisbane, Darwin, Mandurah, Mildura, Gippsland, Renmark, Murray Bridge, and Coorong. We considered models with up to two vector species (Aedes vigilax, Culex annulirostris, Aedes camptorhynchus, Culex globocoxitus), two reservoir hosts (macropods, possums), seasonal transmission effects, and transmission parameters. We fit models against long-term RRV surveillance data (1991-2017) and used Akaike Information Criterion to select important mechanisms. The combination of two vector species, two reservoir hosts, and seasonal transmission effects explained RRV dynamics best across sites. Estimated vector-human transmission rate (average ß = 8.04x10-4per vector per day) was similar despite different dynamics. Models estimate 43% underreporting of RRV infections. Findings enhance understanding of RRV transmission mechanisms, provide disease parameter estimates which can be used to guide future research into public health improvements and offer a basis to evaluate mitigation practices.

The impact of arthritogenic viruses in oral tissues.

Arthritis and periodontitis are inflammatory diseases that share several immunopathogenic features. The expansion in the study of virus-induced arthritis has shed light on how this condition could impact other parts of the human body, including the mouth. Viral arthritis is an inflammatory joint disease caused by several viruses, most notably the alphaviruses Chikungunya virus (CHIKV), Sindbis virus (SINV), Ross River virus (RRV), Mayaro virus (MAYV), and O'nyong'nyong virus (ONNV). These viruses can induce an upsurge of matrix metalloproteinases and immune-inflammatory mediators such as Interleukin-6 (IL6), IL-1ß, tumor necrosis factor, chemokine ligand 2, and receptor activator of nuclear factor kappa-B ligand in the joint and serum of infected individuals. This can lead to the influx of inflammatory cells to the joints and associated muscles as well as osteoclast activation and differentiation, culminating in clinical signs of swelling, pain, and bone resorption. Moreover, several data indicate that these viral infections can affect other sites of the body, including the mouth. The human oral cavity is a rich and diverse microbial ecosystem, and viral infection can disrupt the balance of microbial species, causing local dysbiosis. Such events can result in oral mucosal damage and gingival bleeding, which are indicative of periodontitis. Additionally, infection by RRV, CHIKV, SINV, MAYV, or ONNV can trigger the formation of osteoclasts and upregulate pro-osteoclastogenic inflammatory mediators, interfering with osteoclast activation. As a result, these viruses may be linked to systemic conditions, including oral manifestations. Therefore, this review focuses on the involvement of alphavirus infections in joint and oral health, acting as potential agents associated with oral mucosal inflammation and alveolar bone loss. The findings of this review demonstrate how alphavirus infections could be linked to the comorbidity between arthritis and periodontitis and may provide a better understanding of potential therapeutic management for both conditions.

Mayaro virus infection elicits a robust pro-inflammatory and antiviral response in human macrophages.

Mayaro virus (MAYV), the etiological agent of Mayaro fever (MAYF), is an emergent arbovirus pathogen belonging to Togaviridae family. MAYF is characterized by high inflammatory component that can cause long-lasting arthralgia that persists for months. Macrophages are viral targets and reservoirs, key components of innate immunity and host response. Given the importance of this pathogen, our aim was to determine the inflammatory and antiviral response of human monocyte-derived macrophages (MDMs) infected with MAYV. First, we established the replication kinetics of the virus. Thereafter, we determined the expression of pattern recognition receptors, NF-ĸB complex, interferons (IFNs), two interleukin 27 (IL27) subunits, IFN-stimulated genes (ISGs), and the production of cytokines/chemokines. We found that human MDMs are susceptible to MAYV infection in vitro, with a peak of viral particles released between 24- and 48-hours post-infection (h.p.i) at MOI 0.5, and between 12 and 24 h.p.i at MOI 1. Interestingly, we observed a significant decline in the production of infectious viral particles at 72 h.p.i that was associated with the induction of antiviral response and high cytotoxic effect of MAYV infection in MDMs. We observed modulation of several genes after MAYV infection, as well, we noted the activation of antiviral detection and response pathways (Toll-like receptors, RIG-I/MDA5, and PKR) at 48 h.p.i but not at 6 h.p.i. Furthermore, MAYV-infected macrophages express high levels of the three types of IFNs and the two IL27 subunits at 48 h.p.i. Moreover, we found higher production of IL6, IL1ß, CXCL8/IL8, CCL2, and CCL5 at 48 h.p.i as compared to 6 h.p.i. A robust antiviral response (ISG15, APOBEC3A, IFITM1, and MX2) was observed at 48 but not at 6 h.p.i. The innate and antiviral responses of MAYV-infected MDMs differ at 6 and 48 h.p.i. We conclude that MAYV infection induces robust pro-inflammatory and antiviral responses in human primary macrophages.

The low-density lipoprotein receptor promotes infection of multiple encephalitic alphaviruses.

Members of the low-density lipoprotein receptor (LDLR) family, including LDLRAD3, VLDLR, and ApoER2, were recently described as entry factors for different alphaviruses. However, based on studies with gene edited cells and knockout mice, blockade or abrogation of these receptors does not fully inhibit alphavirus infection, indicating the existence of additional uncharacterized entry factors. Here, we perform a CRISPR-Cas9 genome-wide loss-of-function screen in mouse neuronal cells with a chimeric alphavirus expressing the Eastern equine encephalitis virus (EEEV) structural proteins and identify LDLR as a candidate receptor. Expression of LDLR on the surface of neuronal or non-neuronal cells facilitates binding and infection of EEEV, Western equine encephalitis virus, and Semliki Forest virus. Domain mapping and binding studies reveal a low-affinity interaction with LA domain 3 (LA3) that can be enhanced by concatenation of LA3 repeats. Soluble decoy proteins with multiple LA3 repeats inhibit EEEV infection in cell culture and in mice. Our results establish LDLR as a low-affinity receptor for multiple alphaviruses and highlight a possible path for developing inhibitors that could mitigate infection and disease.

Essential role of the CCL2-CCR2 axis in Mayaro virus-induced disease.

Mayaro virus (MAYV) is an emerging arbovirus member of the Togaviridae family and Alphavirus genus. MAYV infection causes an acute febrile illness accompanied by persistent polyarthralgia and myalgia. Understanding the mechanisms involved in arthritis caused by alphaviruses is necessary to develop specific therapies. In this work, we investigated the role of the CCL2/CCR2 axis in the pathogenesis of MAYV-induced disease. For this, wild-type (WT) C57BL/6J and CCR2-/- mice were infected with MAYV subcutaneously and evaluated for disease development. MAYV infection induced an acute inflammatory disease in WT mice. The immune response profile was characterized by an increase in the production of inflammatory mediators, such as IL-6, TNF, and CCL2. Higher levels of CCL2 at the local and systemic levels were followed by the significant recruitment of CCR2+ macrophages and a cellular response orchestrated by these cells. CCR2-/- mice showed an increase in CXCL-1 levels, followed by a replacement of the macrophage inflammatory infiltrate by neutrophils. Additionally, the absence of the CCR2 receptor protected mice from bone loss induced by MAYV. Accordingly, the silencing of CCL2 chemokine expression in vivo and the pharmacological blockade of CCR2 promoted a partial improvement in disease. Cell culture data support the mechanism underlying the bone pathology of MAYV, in which MAYV infection promotes a pro-osteoclastogenic microenvironment mediated by CCL2, IL-6, and TNF, which induces the migration and differentiation of osteoclast precursor cells. Overall, these data contribute to the understanding of the pathophysiology of MAYV infection and the identification future of specific therapeutic targets in MAYV-induced disease.IMPORTANCEThis work demonstrates the role of the CCL2/CCR2 axis in MAYV-induced disease. The infection of wild-type (WT) C57BL/6J and CCR2-/- mice was associated with high levels of CCL2, an important chemoattractant involved in the recruitment of macrophages, the main precursor of osteoclasts. In the absence of the CCR2 receptor, there is a mitigation of macrophage migration to the target organs of infection and protection of these mice against bone loss induced by MAYV infection. Much evidence has shown that host immune response factors contribute significantly to the tissue damage associated with alphavirus infections. Thus, this work highlights molecular and cellular targets involved in the pathogenesis of arthritis triggered by MAYV and identifies novel therapeutic possibilities directed to the host inflammatory response unleashed by MAYV.

LDLR is used as a cell entry receptor by multiple alphaviruses.

Alphaviruses are arboviruses transmitted by mosquitoes and are pathogenic to humans and livestock, causing a substantial public health burden. So far, several receptors have been identified for alphavirus entry; however, they cannot explain the broad host range and tissue tropism of certain alphaviruses, such as Getah virus (GETV), indicating the existence of additional receptors. Here we identify the evolutionarily conserved low-density lipoprotein receptor (LDLR) as a new cell entry factor for GETV, Semliki Forest virus (SFV), Ross River virus (RRV) and Bebaru virus (BEBV). Ectopic expression of LDLR facilitates cellular binding and internalization of GETV, which is mediated by the interaction between the E2-E1 spike of GETV and the ligand-binding domain (LBD) of LDLR. Antibodies against LBD block GETV infection in cultured cells. In addition, the GST-LBD fusion protein inhibits GETV infection both in vitro and in vivo. Notably, we identify the key amino acids in LDLR-LBD that played a crucial role in viral entry; specific mutations in the CR4 and CR5 domain of LDLR-LBD reduce viral entry to cells by more than 20-fold. These findings suggest that targeting the LDLR-LBD could be a potential strategy for the development of antivirals against multiple alphaviruses.

Establishment and application of an indirect ELISA for Getah virus E2 antibody detection.

Getah virus (GETV) is a mosquito-transmitted disease that affects animals, causing fever, aseptic meningitis, and abortion. Its prevalence in China poses risks to both animal health and public well-being. Currently, there is a scarcity of seroepidemiological data on GETV due to the absence of commercial antibody detection kits for pigs. The aim of this study is to develop a rapid, accurate, and sensitive ELISA, providing a reliable tool for GETV seroepidemiology and laying the foundation for future commercial assay development. In this study, we removed specific hydrophobic domains and intracellular structures from E2 proteins and constructed the recombinant plasmid pCold-TF-E2. The recombinant protein was expressed using a prokaryotic expression system, and efficient purification of the rE2 protein was achieved using a nickel affinity column. The purified rE2 protein is suitable for the development of an indirect ELISA (rE2 ELISA). Following the optimization of reaction conditions for the rE2-ELISA, the cut-off value was 0.356. Additionally, the rE2-ELISA method showed a positive rate of 37.1% for IgG antibodies against GETV when testing 986 pig clinical serum samples collected from pigs in Sichuan between May 2022 and September 2022. The rE2-ELISA method displayed a 95.1% overall agreement with VNT, boasting a sensitivity of 98.2% and a specificity of 92.6%. These results indicate that IgG ELISA based on rE2 protein is an efficient and economical method for the detection of GETV antibodies in pigs, facilitating the diagnosis and prevention of GETV.