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Background: Chikungunya (CHIK) is an underdiagnosed acute febrile illness (AFI) and an important cause of acute encephalitis syndrome (AES). Unavailaibility of rapid and sensitive molecular point-of-care tests (PoCTs) for CHIK at grass-root level, results in increased hospital burden, due to delayed diagnosis or misdiagnosis with other clinically relevant diseases. Since, no therapeutic intervention is readily available, accurate and differential diagnosis of CHIK is the only available option to initiate early supportive treatment. Thus, we aimed to develop a one-pot reverse transcription recombinase polymerase amplification (RT-RPA) mediated CRISPR/Cas12a based detection platform for rapid, specific, and ultrasensitive detection of chikungunya virus (CHIKV) in clinical samples., Results: We have successfully integrated CRISPR/Cas12a technology with reverse transcription recombinase polymerase amplification (RT-RPA) for the detection of Chikungunya virus (CHIKV). The developed assay enabled rapid detection of CHIKV within 35 min, requiring minimal handling process and instrumentation. Next, this assay demonstrated dual mode end-point detection capabilities, employing both fluorescence and lateral flow detection within a reaction. Our one-pot system allows the entire process to be completed without the need to open the reaction tube, thereby eliminating the risk of cross-contamination. Remarkably, the assay exhibits an analytical sensitivity of 412 zg μL -1 (≈1 copy), and 100 % clinical sensitivity and specificity for CHIKV. Furthermore, the developed assay demonstrated limit of detection of 8 gene copies of CHIKV. The assay demonstrates precise detection of CHIKV without any cross-reactivity with other pathogens commonly associated AFI or AES., Significance: The overall findings of this study indicate that the RT-RPA:CRISPR/Cas12a detection assay, with one-pot dual-mode detection approach enables rapid, specific and ultrasensitive molecular detection of CHIKV. This advancement holds significant potential for CHIKV detection in resource-limited settings, providing a robust tool for diagnosis and management of the disease. This developed assay may empower clinicians to initiate prompt supportive therapy for Chikungunya fever, thereby improving patient outcomes and public health responses., Competing Interests: Declaration of competing interest PB and RS are the inventors of the RT-RPA:CRISPR/Cas12a protocol for chikungunya detection described throughout this study and both authors have filed patent related to this technology (202411042360, filed 05 June 2024). The other author declares no conflicts of interest. Indian Council of Medical Research, has a financial interest in disclosure of the sequence and method for detection of chikungunya., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Chikungunya virus (CHIKV) and Mayaro virus (MAYV) are emerging/re-emerging alphaviruses transmitted by Aedes spp. mosquitoes and responsible for recent disease outbreaks in the Americas. The capacity of these viruses to cause epidemics is frequently associated with increased mosquito transmission, which in turn is governed by virus-host-vector interactions. Although many studies have explored virus-vector interactions, significant gaps remain in understanding how vertebrate host factors influence alphavirus transmission by mosquitoes. We previously showed that obesity, a ubiquitous vertebrate host biological factor, reduces alphavirus transmission potential in mosquitoes. We hypothesized that alphavirus-infected obese bloodmeals altered immune genes and/or pathways in mosquitoes, thereby inhibiting virus transmission. To test this, we conducted RNA sequencing (RNA-seq) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) on midgut RNA from mosquitoes fed on alphavirus-infected lean and obese mice. This approach aimed to identify potential antiviral or proviral genes and pathways altered in mosquitoes after consuming infected obese bloodmeals. We found upregulation of the Toll pathway and downregulation of several metabolic and other genes in mosquitoes fed on alphavirus-infected obese bloodmeals. Through gene knockdown studies, we demonstrated the antiviral role of Toll pathway and proviral roles of AAEL009965 and fatty acid synthase (FASN) in the transmission of alphaviruses by mosquitoes. Therefore, this study utilized obesity to identify factors influencing alphavirus transmission by mosquitoes and this research approach may pave the way for designing broadly effective antiviral measures to combat mosquito-borne viruses, such as releasing transgenic mosquitoes deficient in the identified genes., (Journal of Medical Virology© 2024 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.)

Background: Chikungunya cases was reported to be on the rise in Malaysia from 2019 to 2021. Although potential endemicity was described previously, genotype shift during 2008 outbreak originating from the 2004 Indian Ocean Islands outbreak presents the probability of current CHIKV spread from neighboring countries. This is due to the prevalence of the new IOL sub-lineage which consists of E1-226A wildtype or reverted strains that are circulating in the Indian subcontinent before spreading to neighboring Thailand during 2018-2019 outbreak., Method: In this study, samples received mostly from the Tangkak, Johor were analyzed. A total 56 CHIKV positive serum samples received in 2021 by Institute of Medical Research Malaysia (IMR), were collected based on sample selection criteria. Selected samples were subjected to total RNA extraction, whole-genome sequencing as well as bioinformatic analysis such as phylogenetic, variant and mutation analysis., Results: Based on the genomic and phylogenetic analysis, the CHIKV samples from 2021 outbreak were of ECSA-IOL genotype. Genome similarity analysis also revealed that these CHIKVs were highly similar to 2018-2019 outbreak strain from Thailand. In comparison to the 2008 outbreak CHIKV isolate, the current CHIKVs lacked the E1-A226V mutation and harbored the new E1-K211E/E2-V264A sub-linage mutation. Since the E1-K211E/E2-V264A mutation facilitates adaptation to Ae. aegypti as opposed to the E1-A226V mutation which improves adaptation to Ae. albopictus, the emergence 2021 CHIKV outbreak in Malaysia can be postulated due to vector shift. Interestingly, a novel nsP3-T441A/V mutation detected in this study, may also play a role in virus transmission, pathogenicity, fitness and vector adaptation., Conclusion: In summary, the current CHIKV outbreak are strains originated from the Indian subcontinent through Thailand which may have capitalized on vector shifting by adapting to Ae. aegypti. The presence of novel nsP3-T441A/V mutation may also contribute to the spread of this virus across peninsular Malaysia., (© 2024. The Author(s).)

Alphavirus infection induces dramatic remodeling of host cellular membranes, producing filopodia-like and intercellular extensions. The formation of filopodia-like extensions has been primarily assigned to the replication protein nsP1, which binds and reshapes the host plasma membrane when expressed alone. While reported decades ago, the molecular mechanisms behind nsP1 membrane deformation remain unknown. Using mammalian epithelial cells and Chikungunya virus (CHIKV) as models, we characterized nsP1-induced membrane deformations as highly dynamic actin-rich lamellipodia and filopodia-like extensions. Through pharmacological inhibition and genetic invalidation, we identified the critical contribution of the Rac1 GTPase and its downstream effectors PAK1 and the actin nucleator Arp2 in nsP1-induced membrane deformation. An intact Rac1-PAK1-Arp2 signaling axis was also required for optimal CHIKV genome replication. Therefore, our results designate the Rac1-PAK1-Arp2 pathway as an essential signaling node for CHIKV infection and establish a parallel requirement for host factors involved in nsP1-induced plasma membrane reshaping and assembly of a functional replication complex.IMPORTANCEThe alphavirus nsP1 protein dramatically remodels host cellular membranes, resulting in the formation of filopodia-like extensions. Although described decades ago, the molecular mechanisms controlling these membrane deformations and their functional importance remain elusive. Our study provides mechanistic insight, uncovering the critical role of the Rac1 GTPase, along with its downstream effectors PAK1 and the actin nucleator Arp2, in the nsP1-associated phenotype. Furthermore, we demonstrate that the Rac1-PAK1-Arp2 pathway is essential for optimal CHIKV genome replication. Our findings establish a parallel in the cellular mechanisms governing nsP1-induced plasma membrane reshaping and the production of a functional replication complex in infected cells., Competing Interests: The authors declare no conflict of interest.

Arboviruses such as chikungunya virus (CHIKV) and dengue virus (DENV) collectively afflict millions of individuals worldwide particularly in endemic countries like India, leading to substantial morbidity and mortality. With the lack of effective vaccines for both CHIKV and DENV in India, the search for antiviral compounds becomes paramount to control these viral infections. In line with this, our investigation was focused on screening natural compounds for their potential antiviral activity against CHIKV and DENV. Using different assays, including plaque assay, immunofluorescence, and reverse transcription-quantitative real-time PCR (qRT-PCR), out of 109 natural compounds tested, we confirmed lycorine's in vitro antiviral activity against CHIKV and DENV at low micromolar concentrations in different cell types. Time of addition assays indicated that lycorine does not impede viral entry. Additionally, qRT-PCR results along with time of addition assay suggested that lycorine interferes with the synthesis of negative strand viral RNA. Molecular docking analysis was done to understand the mode of inhibition of viral replication. The results revealed that the most likely binding site with the highest binding affinity of lycorine, was at the palm and finger domains, in the vicinity of the catalytic site of CHIKV and DENV RNA-dependent RNA polymerase (RdRp). Collectively, our data underscores the potential of lycorine to be developed as a direct acting inhibitor for DENV and CHIKV, addressing the critical need of requirement of an antiviral in regions where these viruses pose significant public health threats., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Alphaviruses encode an error-prone RNA-dependent RNA polymerase (RdRp), nsP4, required for genome synthesis, yet how the RdRp functions in the complete alphavirus life cycle is not well-defined. Previous work using chikungunya virus has established the importance of the nsP4 residue cysteine 483 in replication. Given the location of residue C483 in the nsP4 palm domain, we hypothesized that other residues within this domain and surrounding subdomains would also contribute to polymerase function. To test this hypothesis, we designed a panel of nsP4 variants via homology modeling based on the coxsackievirus B3 3D polymerase. We rescued each variant in mammalian and mosquito cells and discovered that the palm domain and ring finger subdomain contribute to host-specific replication. In C6/36 cells, we found that while the nsP4 variants had replicase function similar to that of wild-type CHIKV, many variants presented changes in protein accumulation and virion production even when viral nonstructural and structural proteins were produced. Finally, we found that WT CHIKV and nsP4 variant replication and protein production could be enhanced in mammalian cells at 28°C, yet growing virus under these conditions led to changes in virus infectivity. Taken together, these studies highlight that distinct nsP4 subdomains are required for proper RNA transcription and translation, having major effects on virion production., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Martin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Despite their role as innate sentinels, macrophages can serve as cellular reservoirs of chikungunya virus (CHIKV), a highly-pathogenic arthropod-borne alphavirus that has caused large outbreaks among human populations. Here, with the use of viral chimeras and evolutionary selection analysis, we define CHIKV glycoproteins E1 and E2 as critical for virion production in THP-1 derived human macrophages. Through proteomic analysis and functional validation, we further identify signal peptidase complex subunit 3 (SPCS3) and eukaryotic translation initiation factor 3 subunit K (eIF3k) as E1-binding host proteins with anti-CHIKV activities. We find that E1 residue V220, which has undergone positive selection, is indispensable for CHIKV production in macrophages, as its mutation attenuates E1 interaction with the host restriction factors SPCS3 and eIF3k. Finally, we show that the antiviral activity of eIF3k is translation-independent, and that CHIKV infection promotes eIF3k translocation from the nucleus to the cytoplasm, where it associates with SPCS3. These functions of CHIKV glycoproteins late in the viral life cycle provide a new example of an intracellular evolutionary arms race with host restriction factors, as well as potential targets for therapeutic intervention., (© 2024. The Author(s).)

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus causing a debilitating febrile illness with rheumatic disease symptoms of arthralgia and arthritis. Since its spread outside of Africa in 2005, it continues to cause outbreaks and disseminates into new territories. Intervention strategies are urgently required, including vaccination and antiviral approaches. To test efficacy, the use of small animal models is required. Two mouse strains, A129, with a deficiency in their type-I interferon (IFN) receptor, and C57BL/6 are widely used. A direct comparison of these strains alongside the wild-type parental strain of the A129 mice, 129Sv/Ev, was undertaken to assess clinical disease progression, viral loads in key tissues, histological changes and levels of sera biomarkers. Our results confirm the severe disease course in A129 mice which was not observed in the parental 129Sv/Ev strain. Of the two wild-type strains, viral loads were higher in 129Sv/Ev mice compared to C57BL/6 counterparts. Our results have established these models and parameters for the future testing of vaccines and antiviral approaches., Competing Interests: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Chikungunya virus (CHIKV) is a re-emerging, pathogenic alphavirus that is transmitted to humans by Aedesspp. mosquitoes-causing fever and debilitating joint pain, with frequent long-term health implications and high morbidity. The CHIKV replication cycle is poorly understood and specific antiviral therapeutics are lacking. In the current study, we identify host cell Musashi RNA binding protein-2 (MSI-2) as a proviral factor. MSI-2 depletion and small molecule inhibition assays demonstrated that MSI-2 is required for efficient CHIKV genome replication. Depletion of both MSI-2 and MSI-1 homologues was found to synergistically inhibit CHIKV replication, suggesting redundancy in their proviral function. Electromobility shift assay (EMSA) competition studies demonstrated that MSI-2 interacts specifically with an RNA binding motif within the 5' untranslated region (5'UTR) of CHIKV and reverse genetic analysis showed that mutation of the binding motif inhibited genome replication and blocked rescue of mutant virus. For the first time, this study identifies the proviral role of MSI RNA binding proteins in the replication of the CHIKV genome, providing important new insight into mechanisms controlling replication of this significant human pathogen and the potential of a novel therapeutic target., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

RNA interference (RNAi) is a process in which small RNA molecules (such as small interfering RNAs or siRNAs) bind to specific messenger RNAs (mRNAs), leading to its degradation and inhibition of protein synthesis. Our studies have shown that RNAi can effectively silence genes involved in the replication of the Chikungunya virus (CHIKV) in cells. However, these investigations were performed only in laboratory settings and have yet to be tested in human clinical trials. Researchers need to conduct more research to determine the safety and efficacy of RNAi-based therapies as a therapeutic agent to treat viral infections. In this review, the history of evolution of siRNA as an inhibitor of protein synthesis, along with its current developments, is discussed based on our experience. Moreover, this review examines the hurdles and future implications associated with siRNA based therapeutic approaches.

Alphaviruses, such as chikungunya virus (CHIKV), are mosquito-borne viruses that represent a significant threat to human health due to the current context of global warming. Efficient alphavirus infection relies on the activity of the non-structural protein 3 (nsP3), a puzzling multifunctional molecule whose role in infection remains largely unknown. NsP3 is a component of the plasma membrane-bound viral RNA replication complex (vRC) essential for RNA amplification and is also found in large cytoplasmic aggregates of unknown function . Here, we report the cryo-electron microscopy (cryo-EM) structure of the CHIKV nsP3 at 2.35 Å resolution. We show that nsP3 assembles into tubular structures made by a helical arrangement of its alphavirus unique domain (AUD). The nsP3 helical scaffolds are consistent with crown structures found on tomographic reconstructions of the mature viral RCs. In addition, nsP3 helices assemble into cytoplasmic granules organized in a network of tubular structures that contain viral genomic RNA and capsid as well as host factors required for productive infection. Structure-guided mutagenesis identified residues that prevent or disturb nsP3 assemblies, resulting in impaired viral replication or transcription. Altogether, our results reveal an unexpected nsP3-dependent molecular organization essential for different phases of alphavirus infection., (© 2024. The Author(s).)

The current detection method for Chikungunya Virus (CHIKV) involves an invasive and costly molecular biology procedure as the gold standard diagnostic method. Consequently, the search for a non-invasive, more cost-effective, reagent-free, and sustainable method for the detection of CHIKV infection is imperative for public health. The portable Fourier-transform infrared coupled with Attenuated Total Reflection (ATR-FTIR) platform was applied to discriminate systemic diseases using saliva, however, the salivary diagnostic application in viral diseases is less explored. The study aimed to identify unique vibrational modes of salivary infrared profiles to detect CHIKV infection using chemometrics and artificial intelligence algorithms. Thus, we intradermally challenged interferon-gamma gene knockout C57/BL6 mice with CHIKV (20 µl, 1 X 10 5 PFU/ml, n = 6) or vehicle (20 µl, n = 7). Saliva and serum samples were collected on day 3 (due to the peak of viremia). CHIKV infection was confirmed by Real-time PCR in the serum of CHIKV-infected mice. The best pattern classification showed a sensitivity of 83%, specificity of 86%, and accuracy of 85% using support vector machine (SVM) algorithms. Our results suggest that the salivary ATR-FTIR platform can discriminate CHIKV infection with the potential to be applied as a non-invasive, sustainable, and cost-effective detection tool for this emerging disease., (© 2024. The Author(s).)

Chikungunya fever is an acute infectious disease caused by chikungunya virus (CHIKV), which is transmitted by Aedes mosquitoes. Simple, rapid, and sensitive detection of CHIKV is critical for its prevention and spread. To address this issue, we combined one-tube, reverse transcription semi-nested, multi-enzyme isothermal rapid amplification, and lateral flow dipstick strips assay to detect CHIKV RNA. The study used a 318-bp gene fragment of CHIKV NSP4 as the target of the assay. This method of amplification takes 30 min for two-step amplification at 39°C. The dilution of amplification products was added to the LFD strip with results visible to the naked eye after 10 min. The method has a sensitivity of 1 copy/μL for the detection of CHIKV RNA, which is 100-fold higher than the conventional reverse transcription-multi-enzyme isothermal rapid amplification and 10-fold higher than the reverse transcription quantitative PCR (RT-qPCR) method. In addition, the method demonstrated good specificity and a better detection rate (85.7%, 18 of 21) than RT-qPCR (80.9%, 17 of 21) in clinically confirmed patient plasma samples. Thus, the rapid CHIKV RNA assay developed in this study will be an important tool for the rapid and accurate screening of patients for chikungunya fever., Importance: This study presents a new one-tube, reverse transcription semi-nested, multi-enzyme isothermal rapid amplification assay combined with lateral flow dipstick strips for the detection of CHIKV. This technique significantly improves sensitivity and outperforms RT-qPCR for the detection of CHIKV, especially in samples with low viral loads. It is also significantly faster than conventional RT-qPCR and does not require special equipment or a standard PCR laboratory. The combination of the isothermal amplification technology developed in this study with point-of-care molecular testing offers the potential for rapid, on-site, low-cost molecular diagnosis of CHIKV., Competing Interests: The authors declare no conflict of interest.

Chikungunya virus (CHIKV) is a rapidly spreading re-emergent virus transmitted from mosquitoes to humans. The emergence of epidemic variants has been associated with changes in the viral genome, such as the duplication of repeated sequences in the 3' untranslated region (UTR). Indeed, blocks of repeated sequences seemingly favor RNA recombination, providing the virus with a unique ability to continuously change the 3'UTR architecture during host switching. In this work, we provide experimental data on the molecular mechanism of RNA recombination and describe specific sequence and structural elements in the viral 3'UTR that favor template switching of the viral RNA-dependent RNA polymerase on the 3'UTR. Furthermore, we found that a 3'UTR deletion mutant that exhibits markedly delayed replication in mosquito cells and impaired transmission in vivo, recombines in reference laboratory strains of mosquitoes. Altogether, our data provide novel experimental evidence indicating that RNA recombination can act as a nucleic acid repair mechanism to add repeated sequences that are associated to high viral fitness in mosquito during chikungunya virus replication., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Chikungunya virus (CHIKV) is the causative agent of chikungunya fever (CHIKF), and its primary vectors are the mosquitoes Aedes aegypti and Aedes albopictus. CHIKV was initially endemic to Africa but has spread globally in recent years and affected millions of people. According to a risk assessment by the World Health Organization, CHIKV has the potential seriously impact public health. A growing body of research suggests that mutations in the CHIKV gene that enhance viral fitness in the host are contributing to the expansion of the global CHIKF epidemic. In this article, we review the host-adapted gene mutations in CHIKV under natural evolution and laboratory transmission conditions, which can help improve our understanding of the adaptive evolution of CHIKV and provide a basis for monitoring and early warning of future CHIKV outbreaks.

Chikungunya virus (CHIKV) is a mosquito-transmitted, RNA virus that causes an often-severe musculoskeletal illness characterized by fever, joint pain, and a range of debilitating symptoms. The virus has re-emerged as a global health threat in recent decades, spreading from its origin in Africa across Asia and the Americas, leading to widespread outbreaks impacting millions of people. Despite more than 50 years of research into the pathogenesis of CHIKV, there is still no curative treatment available. Current management of CHIKV infections primarily involves providing supportive care to alleviate symptoms and improve the patient's quality of life. Given the ongoing threat of CHIKV, there is an urgent need to better understand its pathogenesis. This understanding is crucial for deciphering the mechanisms underlying the disease and for developing effective strategies for both prevention and management. This review aims to provide a comprehensive overview of CHIKV and its pathogenesis, shedding light on the complex interactions of viral genetics, host factors, immune responses, and vector-related factors. By exploring these intricate connections, the review seeks to contribute to the knowledge base surrounding CHIKV, offering insights that may ultimately lead to more effective prevention and management strategies for this re-emerging global health threat.

Chikungunya virus (CHIKV) has emerged as a significant public health concern due to its rapid spread and potential for causing debilitating epidemics. In Argentina, the virus has garnered attention since its introduction to the Americas in 2013, due to its growing incidence and impact in neighbouring countries. Here we present a comprehensive analysis of the spatiotemporal dynamics of CHIKV in Argentina, focusing on the evolutionary trajectory of its genetic variants. Through a combination of active surveillance, screening of historical and recent samples, and whole-genome sequencing, we traced the evolutionary history of CHIKV lineages circulating within the country. Our results reveal that two distinct genotypes circulated in Argentina: The Asian lineage during the 2016 epidemic and the ECSA lineage in 2023. This distribution reflects the dominance of particular variants across Latin America. Since 2023, the ECSA lineage has led to a surge in cases throughout the Americas, marking a significant shift. The replacement of lineages in the American region constitutes a major epidemiological event, potentially affecting the dynamics of virus transmission and the clinical outcomes in impacted populations. The spatiotemporal analysis highlights CHIKV's distribution across Argentina and underscores the significant role of human mobility, especially when considering recent epidemics in neighbouring countries such as Paraguay and Uruguay, which have facilitated the spread and introduction of the viral strain into different districts. By integrating epidemiological data with genomic insights, we elucidate the patterns of virus dissemination, highlighting key areas of transmission and potential factors contributing to its spread.

Reverse genetic systems are mainly used to rescue recombinant viral strains in cell culture. These tools have also been used to generate, by inoculating infectious clones, viral strains directly in living animals. We previously developed the "Infectious Subgenomic Amplicons" (ISA) method, which enables the rescue of single-stranded positive sense RNA viruses in vitro by transfecting overlapping subgenomic DNA fragments. Here, we provide proof-of-concept for direct in vivo generation of infectious particles following the inoculation of subgenomic amplicons. First, we rescued a strain of tick-borne encephalitis virus in mice to transpose the ISA method in vivo . Subgenomic DNA fragments were amplified using a 3-fragment reverse genetics system and inoculated intramuscularly. Almost all animals were infected when quantities of DNA inoculated were at least 20 µg. We then optimized our procedure in order to increase the animal infection rate. This was achieved by adding an electroporation step and/or using a simplified 2- fragment reverse genetics system. Under optimal conditions, a large majority of animals were infected with doses of 20 ng of DNA. Finally, we demonstrated the versatility of this method by applying it to Japanese encephalitis and Chikungunya viruses. This method provides an efficient strategy for in vivo rescue of arboviruses. Furthermore, in the context of the development of DNA-launched live attenuated vaccines, this new approach may facilitate the generation of attenuated strains in vivo . It also enables to deliver a substance free of any vector DNA, which seems to be an important criterion for the development of human vaccines.

Uruguay experienced its first Chikungunya virus outbreak in 2023, resulting in a significant burden to its healthcare system. We conducted analysis based on real-time genomic surveillance (30 novel whole genomes) to offer timely insights into recent local transmission dynamics and eco-epidemiological factors behind its emergence and spread in the country.

ABSTRACT Alphaviruses are arthropod-borne, single-stranded positive-sense RNA viruses that are recognized as rapidly emerging pathogens. Despite being exquisitely sensitive to the effects of the innate immune response alphaviruses can readily replicate, disseminate, and induce pathogenesis in immunologically competent hosts. Nonetheless, how alphaviruses evade the induction of an innate immune response prior to viral gene expression, or in non-permissive infections, is unknown. Previously we reported the identification of a novel host/pathogen interaction between the viral Capsid (CP) protein and the host IRAK1 protein. The CP/IRAK1 interaction was determined to negatively impact IRAK1-dependent PAMP detection in vitro , however, the precise importance of the CP/IRAK1 interaction to alphaviral infection remained unknown. Here we detail the identification of the CP/IRAK1 interaction determinants of the Sindbis virus (SINV) CP protein and examine the importance of the interaction to alphaviral infection and pathogenesis in vivo using an interaction deficient mutant of the model neurotropic strain of SINV. Importantly, these interaction determinants are highly conserved across multiple Old-World alphaviruses, including Ross River virus (RRV), Mayaro virus (MAYV), Chikungunya virus (CHIKV), and Semliki Forest virus (SFV). In the absence of a functional CP/IRAK1 interaction, SINV replication is significantly restricted and fails to disseminate from the primary site of inoculation due to the induction of a robust type-I Interferon response. Altogether these data indicate that the evasion of IRAK1-dependent signalling is critical to overcoming the host innate immune response and the in vivo data presented here demonstrate the importance of the CP/IRAK1 interaction to neurovirulence and pathogenesis.

Chikungunya virus has caused millions of cases worldwide over the past 20 years, with recent outbreaks in Kedougou region in the southeastern Senegal, West Africa. Genomic characterization highlights that an ongoing epidemic in Kedougou in 2023 is not due to an introduction event but caused by the re-emergence of an endemic strain evolving linearly in a sylvatic context.

Background: Dengue virus (DENV) and Chikungunya virus (CHIKV) are major arboviruses that are transmitted to humans by Aedes aegypti (A. aegypti) and Aedes Albopictus (A. Albopictus) mosquitoes. In absence of specific antivirals and vaccine against these two viruses, prompt diagnosis of acute infections and robust surveillance for outbreak identification remain crucial. Therefore, rapid, robust, high-throughput, accessible, and low-cost assays are essential for endemic countries. This study evaluated our recently developed multiplex RT-PCR and RT-qPCR assays to screen for DENV1-4 and CHIKV circulation in Burkina Faso., Methods and Results: This study, conducted between June to August 2023, enrolled patients with suspected arbovirus infection presenting at healthcare facilities in three Burkina Faso cities (Bobo-Dioulasso, Houndé, and Ouagadougou). Serum samples were collected and screened for DENV serotypes and CHIKV using our newly multiplex RT-PCR and RT-q PCR techniques recently developed. A total of 408 patients (age median = 33, range from 3 to 84 years) participated in this study. Of these, 13.7% (56/408) had DENV infection; DENV-1 was 32.1% (18/56) and DENV-3 was 67.9% (38/56). DENV-2, DENV-4 and CHIKV were not detected., Conclusions: This study demonstrates the effectiveness of our molecular methods for DENV detection and serotyping in Burkina Faso. The affordability of our methods makes them valuable for implementing widespread routine clinical diagnostics or arbovirus surveillance in resource-limited settings., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Chikungunya virus (CHIKV) is a single-stranded RNA virus belonging to the genus Alphavirus and is responsible for causing Chikungunya fever, a type of arboviral fever. Despite extensive research, the pathogenic mechanism of CHIKV within host cells remains unclear. In this study, an in-silico approach was used to predict that CHIKV produces micro-RNAs that target host-specific genes associated with host cellular regulatory pathways. Putative micro-RNAs of CHIKV were predicted using the miRNAFold and Vmir RNA structure web servers, and secondary structure prediction was performed using RNAfold. Host-specific target genes were then predicted, and hub genes were identified using CytoHubba and module selection through MCODE. Functional annotations of hub genes revealed their association with various pathways, including osteoclast differentiation, neuroactive ligand-receptor interaction, and mRNA surveillance. We used the freely available dataset GSE49985 to determine the level of expression of host-specific target genes and found that two genes, F-box and leucine-rich repeat protein 16 (FBXL16) and retinoic acid receptor alpha (RARA), were down-regulated, while four genes, RNA binding protein with serine-rich domain 1 (RNPS1), RNA helicase and ATPase (UPF1), neuropeptide S receptor 1 (NPSR1), and vasoactive intestinal peptide receptor 1 (VIPR1), were up-regulated. These findings provide insight into novel miRNAs and hub genes associated with CHIKV infection and suggest potential targets for therapeutic intervention. Further experimental validation of these targets could lead to the development of effective treatments for CHIKV-mediated diseases., (© 2024. The Author(s).)

Objectives: Encephalitis is a severe neurological syndrome for which herpesvirus and enteroviruses are the most common etiological agents. Arboviruses, a wildly diverse group of pathogens, are also critical epidemiological agents associated with encephalitis. In Brazil, little is known about the causative agents of encephalitis., Methods: We conducted a hospital surveillance for encephalitis between 2020 and 2022. Molecular (RT-PCR and qPCR) and serological (virus-specific IgM and viral antigens) techniques were performed in cerebrospinal fluid and serum samples obtained from study participants., Results: In the 43 participants evaluated, the etiologic agent or the presence of IgM was detected in 16 (37.2%). Nine (20.9%) cases were positive for chikungunya virus (CHIKV), three (7.0%) for dengue virus, two (4.7%) for human adenovirus, one (2.3%) for varicella-zoster virus, and one (2.3%) for enterovirus. Whole-genome sequencing revealed that the CHIKV identified belongs to the East/Central/South African lineage., Conclusion: Herein, CHIKV is a common pathogen identified in encephalitis cases. Our results reinforce previous evidence that chikungunya represents a significant cause of encephalitis during CHIKV outbreaks and epidemics and add to existing information on the epidemiology of encephalitis in Brazil., Competing Interests: Declarations of competing interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Ltd.)

In 2018-2019, Thailand experienced a nationwide spread of chikungunya virus (CHIKV), with approximately 15,000 confirmed cases of disease reported. Here, we investigated the evolutionary and molecular history of the East/Central/South African (ECSA) genotype to determine the origins of the 2018-2019 CHIKV outbreak in Thailand. This was done using newly sequenced clinical samples from travellers returning to Sweden from Thailand in late 2018 and early 2019 and previously published genome sequences. Our phylogeographic analysis showed that before the outbreak in Thailand, the Indian Ocean lineage (IOL) found within the ESCA, had evolved and circulated in East Africa, South Asia, and Southeast Asia for about 15 years. In the first half of 2017, an introduction occurred into Thailand from another South Asian country, most likely Bangladesh, which subsequently developed into a large outbreak in Thailand with export to neighbouring countries. Based on comparative phylogenetic analyses of the complete CHIKV genome and protein modelling, we identified several mutations in the E1/E2 spike complex, such as E1 K211E and E2 V264A, which are highly relevant as they may lead to changes in vector competence, transmission efficiency and pathogenicity of the virus. A number of mutations (E2 G205S, Nsp3 D372E, Nsp2 V793A), that emerged shortly before the outbreak of the virus in Thailand in 2018 may have altered antibody binding and recognition due to their position. This study not only improves our understanding of the factors contributing to the epidemic in Southeast Asia, but also has implications for the development of effective response strategies and the potential development of new vaccines., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Krambrich et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In a previous study to understand how the chikungunya virus (CHIKV) E1 glycoprotein β-strand c functions, we identified several attenuating variants at E1 residue V80 and the emergence of second-site mutations in the fusion loop (E1-M88L) and hinge region (E1-N20Y) with the V80 variants in vivo . The emergence of these mutations led us to question how changes in E1 may contribute to CHIKV infection at the molecular level. Here, we use molecular dynamics to understand how changes in the E1 glycoprotein may influence the CHIKV glycoprotein E1-E2 complex. We found that E1 domain II variants lead to E2 conformational changes, allowing us to hypothesize that emerging variants E1-M88L and E1-N20Y could also change E2 conformation and function. We characterized CHIKV E1-M88L and E1-N20Y in vitro and in vivo to understand how these regions of the E1 glycoprotein contribute to host-specific infection. We found that CHIKV E1-N20Y enhanced infectivity in mosquito cells, while the CHIKV E1-M88L variant enhanced infectivity in both BHK-21 and C6/36 cells and led to changes in viral cholesterol-dependence. Moreover, we found that E1-M88L and E1-N20Y changed E2 conformation, heparin binding, and interactions with the receptor Mxra8. Interestingly, the CHIKV E1-M88L variant increased replication in Mxra8-deficient mice compared to WT CHIKV, yet was attenuated in mouse fibroblasts, suggesting that residue E1-M88 may function in a cell-type-dependent entry. Taken together, these studies show that key residues in the CHIKV E1 domain II and hinge region function through changes in E1-E2 dynamics to facilitate cell- and host-dependent entry.IMPORTANCEArboviruses are significant global public health threats, and their continued emergence around the world highlights the need to understand how these viruses replicate at the molecular level. The alphavirus glycoproteins are critical for virus entry in mosquitoes and mammals, yet how these proteins function is not completely understood. Therefore, it is critical to dissect how distinct glycoprotein domains function in vitro and in vivo to address these gaps in our knowledge. Here, we show that changes in the CHIKV E1 domain II and hinge alter E2 conformations leading to changes in virus-receptor and -glycosaminoglycan interactions and cell-specific infection. These results highlight that adaptive changes in E1 can have a major effect on virus attachment and entry, furthering our knowledge of how alphaviruses infect mammals and insects., Competing Interests: The authors declare no conflict of interest.

Arboviruses such as dengue, Zika, and chikungunya present similar symptoms in the early stages, which complicates their differential and timely diagnosis. In 2022, the PAHO published a guide to address this challenge. This study proposes a methodological framework that transforms qualitative information into quantitative information, establishing differential weights in relation to symptoms according to the medical evidence and the GRADE scale based on recommendation 1 of the said guide. To achieve this, common variables from the dataset were identified using the PAHO guide, and quality rules were established. A linear interpolation function was then parameterised to assign weights to the symptoms according to the evidence. Machine learning was used to compare the different models, achieving 99% accuracy compared with 79% without the methodology. This proposal represents a significant advancement, allowing the direct application of the PAHO recommendations to the dataset and improving the differential classification of arboviruses.

Molecular surveillance is vital for monitoring arboviruses, often employing genus-specific quantitative reverse-transcription polymerase chain reaction (RT-qPCR). Despite this, an overlooked chikungunya fever outbreak occurred in Yunnan province, China, in 2019 and false negatives are commonly encountered during alphaviruses screening practice, highlighting the need for improved detection methods. In this study, we developed an improved alphaviruses-specific RT-qPCR capable of detecting chikungunya virus, eastern equine encephalitis virus, western equine encephalitis virus, Venezuelan equine encephalitis virus, Sindbis virus, Mayaro virus, and Ross River virus with high sensitivity and specificity. The assay identified three chikungunya virus-positive cases out of 188 sera retrospectively. Later genetic characterization suggested that imported cases from neighboring countries may be responsible for the neglected chikungunya fever outbreak of 2019 in Yunnan. Our findings underscore the value of improved alphaviruses-specific RT-qPCR in bolstering alphaviruses surveillance and informing preventive strategies., (© 2024 Wiley Periodicals LLC.)

In Brazil, Dengue, Zika and Chikungunya viruses constitute a major threat to the public health system. Simultaneous circulation of these arboviruses occurs in many regions of the world due to the expansion of transmission vectors. The infection by these arboviruses triggers similar symptoms during their acute phase. However, in some cases, severe symptoms may occur, leading to different types of disabilities and even death. In this context, considering the similarity of the symptoms, the problems caused by the infection of these arboviruses, and the increasing risk of coinfection in humans, the differential diagnosis of these infections is essential for clinical management and epidemiological investigation. Thus, this study aimed to identify, through diagnosis via Quantitative Polymerase Chain Reaction with Reverse Transcription, arbovirus coinfection in patients from the Tocantins state (Northern Brazil). A total of 495 samples were analyzed, three from which were determined to be a coinfection of Dengue and Chikungunya viruses. The data obtained here indicate the co-circulation and coinfection by Dengue and Chikungunya viruses in the Tocantins state. These results highlight the importance of monitoring the circulation of these arboviruses for the development of health actions that aim their prevention and combat, as well as their clinical and therapeutic management., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (Copyright © 2024 Sociedade Brasileira de Infectologia. Published by Elsevier España, S.L.U. All rights reserved.)

We aimed to describe the landscape, including molecular, epidemiological, and clinical aspects of CHIKV infections in the Ribeirao Preto region, an area endemic to dengue. We randomly screened 3744 plasma samples that had undergone DENV diagnosis to evaluate CHIKV-RNA using an in-house RT-PCR assay. Positive samples were followed clinically, and RNA samples were submitted to whole genome sequencing. Seventeen cases (0.5 %) were positive for CHIKV-RNA despite being negative for DENV-RNA. Notably, half of the patients experienced prolonged arthralgia lasting more than 90 days. Compared with the healthy control group, leukopenia and thrombocytopenia were observed in all CHIKV-positive individuals with statistically significant P values (P < 0.0001 and P = 0.0003, respectively). The genomic analysis revealed that the CHIKV strains being studied are classified within the East-Central-South-African (ECSA) genotype. This analysis identified new mutations, E1: K211E and E2: V264A, while the previously known mutation E1: A226V was not detected among these strains. This study highlights the need for epidemiological surveillance and preparedness for potential CHIKV epidemics in Brazil, particularly where other arboviruses co-circulate., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

A recombinant Ross River virus (RRV) that contains the fluorescent protein mCherry fused to the non-structural protein 3 (nsP3) was constructed, which allowed real-time imaging of viral replication. RRV-mCherry contained either the natural opal stop codon after the nsP3 gene or was constructed without a stop codon. The mCherry fusion protein did not interfere with the viral life cycle and deletion of the stop codon did not change the replication capacity of RRV-mCherry. Comparison of RRV-mCherry and chikungunya virus-mCherry infections, however, showed a cell type-dependent delay in RRV-mCherry replication in HEK 293T cells. This delay was not caused by differences in cell entry, but rather by an impeded nsP expression caused by the RRV inhibitor ZAP (zinc finger CCCH-Type, antiviral 1). The data indicate that viral replication of alphaviruses is cell-type dependent, and might be unique for each alphavirus.

Background: Despite dengue virus (DENV) outbreak in Gabon a decade ago, less is known on the potential circulation of DENV serotypes in the country. Previous studies conducted in some areas of the country, are limited to hospital-based surveys which reported the presence of some cases of serotype 2 and 3 seven years ago and more recently the serotype 1. As further investigation, we extend the survey to the community of Moyen Ogooué region with the aim to assess the presence of the dengue virus serotypes, additionally to characterize chikungunya (CHIKV) infection and describe the symptomatology associated with infections., Method: A cross-sectional survey was conducted from April 2020 to March 2021. The study included participants of both sexes and any age one year and above, with fever or history of fever in the past seven days until blood collection. Eligible volunteers were clinically examined, and blood sample was collected for the detection of DENV and CHIKV using RT-qPCR. Positive samples were selected for the target sequencing., Results: A total of 579 volunteers were included. Their mean age (SD) was 20 (20) years with 55% of them being female. Four cases of DENV infection were diagnosed giving a prevalence of 0.7% (95%CI: 0.2-1.8) in our cohort while no case of CHIKV was detected. The common symptoms and signs presented by the DENV cases included fatigue, arthralgia myalgia, cough, and loss of appetite. DENV-1was the only virus detected by RT-qPCR., Conclusion: Our results confirm the presence of active dengue infection in the region, particularly DENV-1, and could suggest the decline of DENV-2 and DENV-3. Continuous surveillance remains paramount to comprehensively describe the extent of dengue serotypes distribution in the Moyen-Ogooué region of Gabon., (© 2024. The Author(s).)

Chikungunya virus ( Togaviridae , Alphavirus ; CHIKV) is a mosquito-borne global health threat. The main urban vector of CHIKV is the Aedes aegypti mosquito, which is found throughout Brazil. Therefore, it is important to carry out laboratory tests to assist in the virus's diagnosis and surveillance. Most molecular biology methodologies use nucleic acid extraction as the first step and require quality RNA for their execution. In this context, four RNA extraction protocols were evaluated in Ae. aegypti experimentally infected with CHIKV. Six pools were tested in triplicates (n = 18), each containing 1, 5, 10, 20, 30, or 40 mosquitoes per pool (72 tests). Four commercial kits were compared: QIAamp ® , Maxwell ® , PureLink ® , and PureLink ® with TRIzol ® . The QIAamp ® and PureLink ® with TRIzol ® kits had greater sensitivity. Two negative correlations were observed: as the number of mosquitoes per pool increases, the Ct value decreases, with a higher viral load. Significant differences were found when comparing the purity and concentration of RNA. The QIAamp ® protocol performed better when it came to lower Ct values and higher RNA purity and concentration. These results may provide help in CHIKV entomovirological surveillance planning.

Background: Chikungunya virus (CHIKV) and O'nyong nyong virus (ONNV) are phylogenetically related alphaviruses in the Semliki Forest Virus (SFV) antigenic complex of the Togaviridae family. There are limited data on the circulation of these two viruses in Burkina Faso. The aim of our study was to assess their circulation in the country by determining seroprevalence to each of the viruses in blood donor samples and by retrospective molecular and serological testing of samples collected as part of national measles and rubella surveillance., Methodology/principal Findings: All blood donor samples were analyzed on the Luminex platform using CHIKV and ONNV E2 antigens. Patient samples collected during national measles-rubella surveillance were screened by an initial ELISA for CHIKV IgM (CHIKjj Detect IgM ELISA) at the national laboratory. The positive samples were then analyzed by a second ELISA test for CHIKV IgM (CDC MAC-ELISA) at the reference laboratory. Finally, samples that had IgM positive results for both ELISA tests and had sufficient residual volume were tested by plaque reduction neutralization testing (PRNT) for CHIKV and ONNV. These same patient samples were also analyzed by rRT-PCR for CHIKV. Among the blood donor specimens, 55.49% of the samples were positive for alphaviruses including both CHIKV and ONNV positive samples. Among patient samples collected as part of national measles and rubella surveillance, 3.09% were IgM positive for CHIKV, including 2.5% confirmed by PRNT. PRNT failed to demonstrate any ONNV infections in these samples. No samples tested by RT-qPCR. had detectable CHIKV RNA., Conclusions/significance: Our results suggest that CHIKV and ONNV have been circulating in the population of Burkina Faso and may have been confused with malaria, dengue fever or other febrile diseases such as measles or rubella. Our study underscores the necessity to enhance arbovirus surveillance systems in Burkina Faso., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Tinto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The genomes of positive-sense (+) single-stranded RNA (ssRNA) viruses are believed to be subjected to a wide range of RNA modifications. In this study, we focused on the chikungunya virus (CHIKV) as a model (+) ssRNA virus to study the landscape of viral RNA modification in infected human cells. Among the 32 distinct RNA modifications analysed by mass spectrometry, inosine was found enriched in the genomic CHIKV RNA. However, orthogonal validation by Illumina RNA-seq analyses did not identify any inosine modification along the CHIKV RNA genome. Moreover, CHIKV infection did not alter the expression of ADAR1 isoforms, the enzymes that catalyse the adenosine to inosine conversion. Together, this study highlights the importance of a multidisciplinary approach to assess the presence of RNA modifications in viral RNA genomes.

Chikungunya virus (CHIKV) is transmitted by mosquito bites and causes chikungunya fever (CHIKF). CHIKV has a single-stranded RNA genome and belongs to a single serotype with three genotypes. The Asian lineage has recently emerged in the Western Hemisphere, likely due to travel-associated introduction. Genetic variation accumulates in the CHIKV genome as the virus replicates, creating new lineages. Whole genome sequencing is ideal for studying virus evolution and spread but is expensive and complex. This study investigated whether specific, highly variable regions of the CHIKV genome could recapitulate the phylogeny obtained with a complete coding sequence (CDS). Our results revealed that concatenated highly variable regions accurately reconstructed CHIKV phylogeny, exhibiting statistically indistinguishable branch lengths and tree confidence compared to CDS. In addition, these regions adequately inferred the evolutionary relationships among CHIKV isolates from the American outbreak with similar results to the CDS. This finding suggests that highly variable regions can effectively capture the evolutionary relationships among CHIKV isolates, offering a simpler approach for future studies. This approach could be particularly valuable for large-scale surveillance efforts.

Acute encephalitis syndrome (AES) outbreaks in children of Eastern Uttar Pradesh (E-UP) region of India have been a longstanding public health issue, with a significant case fatality rate of 20-25%. Since past decade, a rise in chikungunya (CHIK) cases has been occurring, which is a reported etiology of AES. However, the burden of chikungunya virus (CHIKV) among pediatric AES (pAES) is unknown from E-UP. We included 238 hospitalized pAES cases. The presence of IgM antibodies for CHIKV, and Dengue virus (DENV) was tested, and RT-PCR was performed for CHIKV and DENV in serologically confirmed CHIKV and DENV pAES cases. Positive samples were sequenced using Sangers sequencing. Further, to check for co-infection, IgM antibodies for other AES etiologies including Japanese encephalitis virus (JEV), Leptospira and Orientia tsutsugamushi (OT) in serum were also investigated. IgM ELISA demonstrated 5.04% (12) positivity for CHIKV. Among CHIKV IgM positive, 3 (25%, 3/12) pAES patients died. CHIKV genome was detected in 3 pAES specimens. Among which, 2 CHIKV cases were also positive for OT DNA. Partially sequenced CHIKV were genotyped as ECSA. The overall finding indicates evidence of CHIKV infection with high case fatality among pAES patients from E-UP. This study advocates constant serological and molecular surveillance of CHIKV in AES endemic regions of India., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Chikungunya virus (CHIKV) is an alphavirus (genus Alphavirus, family Togaviridae) that is primarily transmitted to humans by Aedes mosquitoes, and can be transmitted from mother to child. Little is known about CHIKV transmission in Vietnam, where dengue is endemic and Aedes mosquitoes are abundant. This study aimed to determine the prevalence and characteristics of vertical CHIKV infection in a birth cohort, and seroprevalence of anti-CHIKV antibodies with or without confirmation by neutralization tests among women bearing children in Vietnam., Methods: We collected umbilical cord blood plasma samples from each newly delivered baby in Nha Trang, Central Vietnam, between July 2017 and September 2018. Samples were subjected to molecular assay (quantitative real-time RT-PCR) and serological tests (anti-CHIKV IgM capture and IgG indirect enzyme-linked immunosorbent assay, and neutralization tests)., Results: Of the 2012 tested cord blood samples from newly delivered babies, the CHIKV viral genome was detected in 6 (0.3%) samples by RT-PCR, whereas, 15 samples (0.7%) were anti-CHIKV-IgM positive. Overall, 18 (0.9%, 95% CI: 0.6-1.5) samples, including three positives for both CHIKV IgM and viral genome on RT-PCR, were regarded as vertical transmission of CHIKV infection. Of the 2012 cord blood samples, 10 (0.5%, 95% CI: 0.2-0.9) were positive for both anti-CHIKV IgM and IgG. Twenty-nine (1.4%, 95% CI: 1.0-2.1) were seropositive for anti-CHIKV IgG while 26 (1.3%, 95% CI: 0.8-1.9) of them were also positive for neutralizing antibodies, and regarded as seropositive with neutralization against CHIKV infection., Conclusion: This is the first report of a possible CHIKV maternal-neonatal infection in a birth cohort in Vietnam. The findings indicate that follow-up and a differential diagnosis of CHIKV infection in pregnant women are needed to clarify the potential for CHIKV vertical transmission and its impact in the newborn., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Chikungunya (CHIKV), Zika (ZIKV), and dengue viruses (DENV) are vector-borne pathogens that cause emerging and re-emerging epidemics throughout tropical and subtropical countries. The symptomatology is similar among these viruses and frequently co-circulates in the same areas, making the diagnosis arduous. Although there are different methods for detecting and quantifying pathogens, real-time reverse transcription-polymerase chain reaction (real-time RT-qPCR) has become a leading technique for detecting viruses. However, the currently developed assays frequently involve probes and high-cost reagents, limiting access in low-income countries. Therefore, this study aims to design and evaluate a quantitative one-step RT-qPCR assay to detect CHIKV, ZIKV, and DENV with high specificity, reproducibility, and low cost in multiple cell substrates. We established a DNA intercalating green dye-based RT-qPCR test that targets nsP1 of CHIKV, and NS5 gene of ZIKV, and DENV for the amplification reaction. The assay exhibited a high specificity confirmed by the melting curve analysis. No cross-reactivity was observed between the three viruses or unspecific amplification of host RNA. The sensitivity of the reaction was evaluated for each virus assay, getting a limit of detection of one RNA copy per virus. Standard curves were constructed, obtaining a reaction efficiency of ~ 100%, a correlation coefficient (R 2 ) of ~ 0.97, and a slope of -3.3. The coefficient of variation (CV) ranged from 0.02 to 1.43. In addition, the method was optimized for viral quantification and tested in Vero, BHK-21, C6/36, LULO, and the Aedes cell lines. Thus, the DNA intercalating green dye-based RT-qPCR assay was a highly specific, sensitive, reproducible, and effective method for detecting and quantifying CHIKV, ZIKV, and DENV in different cell substrates that could also be applied in clinical samples., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Discovering an alternative therapy with a long-lasting effect on symptoms caused by chikungunya virus (CHIKV) infection is prompted by the lack of a vaccine and the absence of safe, effective and non-toxic medications. One potential strategy is synthesizing or identifying small compounds that can specifically target the active site of an essential enzyme and prevent virus replication. Previous site-directed mutagenesis studies have demonstrated the crucial role of the macrodomain, which is a part of non-structural protein 3 (nsP3), in virus replication. Exploiting this fact, the macrodomain can be targeted to discover a natural substance that can inhibit its function and thereby impede virus replication. With this aim, the present study focused on potential CHIKV nsP3 macrodomain (nsP3 MD ) inhibitors through in silico, in vitro and cell-based methods. Through virtual screening of the natural compound library, nine nsP3 MD inhibitors were initially identified. Molecular dynamics (MD) simulations were employed to evaluate these nine compounds based on the stability of their ligand-receptor complexes and energy parameters. Target analysis and ADMET (i.e. absorption, distribution, metabolism, excretion and toxicity) prediction of the selected compounds revealed their drug-like characteristics. Subsequent in vitro investigation allowed us to narrow the selection down to one compound, N-[2-(5-methoxy-1H-indol-3-yl) ethyl]-2-oxo-1,2-dihydroquinoline-4-carboxamide, which exhibited potent inhibition of CHIKV growth. This molecule effectively inhibited CHIKV replication in the stable embryonal rhabdomyosarcoma cell line capable of producing CHIKV. Our findings demonstrate that the selected compound possesses substantial anti-CHIKV nsP3 MD activity both in vitro and in vivo. This work provides a promising molecule for further preclinical studies to develop a potential drug against the CHIKV., (© 2024 Federation of European Biochemical Societies.)

Objectives: We aimed to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) platform for the rapid detection of chikungunya virus (CHIKV) in both patient and mosquito samples from Brazil., Methods: We optimized an RT-LAMP assay and then evaluated the specificity and sensitivity using visual detection. In comparison with the RT-qPCR reference method, we validated the utility of this assay as a molecular diagnostic test in a reference laboratory for arbovirus diagnostics using 100 serum samples collected from suspected CHIKV cases., Results: Our RT-LAMP assay specifically detected CHIKV without cross-reactivity against other arboviruses. The limit of detection of our RT-LAMP was estimated in -1.18 PFU (confidence interval [CI] ranging from -2.08 to 0.45), resulting in a similar analytical sensitivity when directly compared with the reference standard RT-qPCR assay. Then, we demonstrate the ability of our RT-LAMP assay to detect the virus in different human specimens (serum, urine, and saliva), and crude lysate of Aedes aegypti mosquitoes in as little as 20-30 minutes and without a separate RNA isolation step. Lastly, we showed that our RT-LAMP assay could be lyophilized and reactivated by adding water, indicating potential for room-temperature storage. Our RT-LAMP had a clinical sensitivity of 100% (95% CI, 90.97-100.00%), clinical specificity of 96.72% (95% CI, 88.65-99.60%), and overall accuracy of 98.00% (95% CI, 92.96-99.76%)., Discussion: Taken together, these findings indicate that the RT-LAMP assay reported here solves important practical drawbacks to the deployment of molecular diagnostics in the field and can be used to improve testing capacity, particularly in low- and middle-income countries., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Chikungunya virus (CHIKV) is a neglected arthropod-borne and anthropogenic alphavirus. Over the past two decades, the CHIKV distribution has undergone significant changes worldwide, from the original tropics and subtropics regions to temperate regions, which has attracted global attention. However, the interactions between CHIKV and its host remain insufficiently understood, which dampens the need for the development of an anti-CHIKV strategy. In this study, on the basis of the optimal overexpression of non-structural protein 4 (nsP4), we explore host interactions of CHIKV nsP4 using mass spectrometry-based protein-protein interaction approaches. The results reveal that some cellular proteins that interact with nsP4 are enriched in the ubiquitin-proteasome pathway. Specifically, the scaffold protein receptor for activated C kinase 1 (RACK1) is identified as a novel host interactor and regulator of CHIKV nsP4. The inhibition of the interaction between RACK1 and nsP4 by harringtonolide results in the reduction of nsP4, which is caused by the promotion of degradation but not the inhibition of nsP4 translation. Furthermore, the decrease in nsP4 triggered by the RACK1 inhibitor can be reversed by the proteasome inhibitor MG132, suggesting that RACK1 can protect nsP4 from degradation through the ubiquitin-proteasome pathway. This study reveals a novel mechanism by which the host factor RACK1 regulates CHIKV nsP4, which could be a potential target for developing drugs against CHIKV.

We previously reported that deletion of a 44-nucleotide element in the 3' untranslated region (UTR) of the Chikungunya virus (CHIKV) genome enhances the virulence of CHIKV infection in mice. Here, we find that while this 44-nucleotide deletion enhances CHIKV fitness in murine embryonic fibroblasts in a manner independent of the type I interferon response, the same mutation decreases viral fitness in C6/36 mosquito cells. Further, the fitness advantage conferred by the UTR deletion in mammalian cells is maintained in vivo in a mouse model of CHIKV dissemination. Finally, SHAPE-MaP analysis of the CHIKV 3' UTR revealed this 44-nucleotide element forms a distinctive two-stem-loop structure that is ablated in the mutant 3' UTR without altering additional 3' UTR RNA secondary structures.

Chikungunya virus (CHIKV) poses a significant global health threat, re-emerging as a mosquito-transmitted pathogen that caused high fever, rash, and severe arthralgia. In Thailand, a notable CHIKV outbreak in 2019-2020 affected approximately 20,000 cases across 60 provinces, underscoring the need for effective mosquito control protocols. Previous studies have highlighted the role of midgut bacteria in the interaction between mosquito vectors and pathogen infections, demonstrating their ability to protect the insect from invading pathogens. However, research on the midgut bacteria of Aedes (Ae.) aegypti, the primary vector for CHIKV in Thailand remains limited. This study aims to characterize the bacterial communities in laboratory strains of Ae. aegypti, both infected and non-infected with CHIKV. Female mosquitoes from a laboratory strain of Ae. aegypti were exposed to a CHIKV-infected blood meal through membrane feeding, while the control group received a non-infected blood meal. At 7 days post-infection (dpi), mosquito midguts were dissected for 16S rRNA gene sequencing to identify midgut bacteria, and CHIKV presence was confirmed by E1-nested RT-PCR using mosquito carcasses. The study aimed to compare the bacterial communities between CHIKV-infected and non-infected groups. The analysis included 12 midgut bacterial samples, divided into three groups: CHIKV-infected (exposed and infected), non-infected (exposed but not infected), and non-exposed (negative control). Alpha diversity indices and Bray-Curtis dissimilarity matrix revealed significant differences in bacterial profiles among the three groups. The infected group exhibited an increased abundance of bacteria genus Gluconobacter, while Asaia was prevalent in both non-infected and negative control groups. Chryseobacterium was prominent in the negative control group. These findings highlight potential alterations in the distribution and abundance of gut microbiomes in response to CHIKV infection status. This study provides valuable insights into the dynamic relationship between midgut bacteria and CHIKV, underscoring the potential for alterations in bacterial composition depending on infection status. Understanding the relationships between mosquitoes and their microbiota holds promise for developing new methods and tools to enhance existing strategies for disease prevention and control. This research advances our understanding of the circulating bacterial composition, opening possibilities for new approaches in combating mosquito-borne diseases., (© 2024. The Author(s).)

Zika (ZIKV) and Chikungunya (CHIKV) viruses are mosquito-transmitted infections, or vector-borne pathogens, that emerged a few years ago. Reliable diagnostic tools for ZIKV and CHIKV-inexpensive, multiplexed, rapid, highly sensitive, and specific point-of-care (POC) systems-are vital for appropriate risk management and therapy. We recently studied a detection system with great success in Mexico (Villahermosa, state of Tabasco), working with human sera from patients infected with those viruses. The research conducted in Mexico validated the efficacy of a novel two-step rapid isothermal amplification technique (RAMP). This approach, which encompasses recombinase polymerase amplification (RPA) followed by loop-mediated isothermal amplification (LAMP), had been previously established in the lab using lab-derived Zika (ZIKV) and Chikungunya (CHIKV) viruses. Crucially, our findings confirmed that this technique is also effective when applied to human sera samples collected from locally infected individuals in Mexico.

This study aims to determine the frequency and clinical manifestations of dengue and chikungunya viral infections in the district hospital of Mfou, Centre region of Cameroon where malaria is endemic. Blood samples were collected from suspected cases and tested for Plasmodium parasites and for the molecular detection of viral RNAs (dengue, zika and chikungunya viruses) using TRIOPLEX qPCR. A total of 108 patients were clinically suspected among which 25 % were male and 50 % were less than 15.5 years old. Of these 14.8 % (16/108) and 2.8 % (3/108) had acute dengue and chikungunya fevers respectively. Co-infection with malaria was reported in 56.3 % (9/16) of Dengue cases and 33.3 % (1/3) of chikungunya cases. Clinical profiling further revealed that nausea and vomiting show a significant difference in dengue infected individuals to those of non-infected individuals (P = 0.027). The presence of dengue fever and chikungunya fever and the absence of specific clinical manifestations highlight the need to strengthen surveillance of acute febrile infections for a better estimation of the burden of arboviruses., Competing Interests: Declaration of competing interest The authors report no conflicts of interest relevant to this article., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Genetic studies preceded by the observation of an unknown mosquito species in Mikołów (Poland) confirmed that it belongs to a new invasive species in Polish fauna, Aedes japonicus (Theobald, 1901), a known vector for numerous infectious diseases. Ae. japonicus is expanding its geographical presence, raising concerns about potential disease transmission given its vector competence for chikungunya virus, dengue virus, West Nile virus, and Zika virus. This first genetically confirmed identification of Ae. japonicus in Poland initiates a comprehensive review of the literature on Ae. japonicus , its biology and ecology, and the viral infections transmitted by this species. This paper also presents the circumstances of the observation of Ae. japonicus in Poland and a methodology for identifying this species.

Chikungunya fever virus (CHIKV) is a mosquito-borne alphavirus that causes wide-spread human infections and epidemics in Asia, Africa and recently, in the Americas. CHIKV is considered a priority pathogen by CEPI and WHO. Despite recent approval of a live-attenuated CHIKV vaccine, development of additional vaccines is warranted due to the worldwide outbreaks of CHIKV. Previously, we developed immunization DNA (iDNA) plasmid capable of launching live-attenuated CHIKV vaccine in vivo. Here we report the use of CHIKV iDNA plasmid to prepare a novel, live-attenuated CHIKV vaccine V5040 with rearranged RNA genome. In V5040, genomic RNA was rearranged to encode capsid gene downstream from the glycoprotein genes. Attenuated mutations derived from experimental CHIKV 181/25 vaccine were also engineered into E2 gene of V5040. The DNA copy of rearranged CHIKV genomic RNA with attenuated mutations was cloned into iDNA plasmid pMG5040 downstream from the CMV promoter. After transfection in vitro, pMG5040 launched replication of V5040 virus with rearranged genome and attenuating E2 mutations. Furthermore, V5040 virus was evaluated in experimental murine models for general safety and immunogenicity. Vaccination with V5040 virus subcutaneously resulted in elicitation of CHIKV-specific, virus-neutralizing antibodies. The results warrant further evaluation of V5040 virus with rearranged genome as a novel live-attenuated vaccine for CHIKV., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests:P.P. and I.T. are employed by Medigen, Inc. (Frederick, MD, USA). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright: © 2024 Tretyakova et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

RNA viruses quickly evolve subtle genotypic changes that can have major impacts on viral fitness and host range, with potential consequences for human health. It is therefore important to understand the evolutionary fitness of novel viral variants relative to well-studied genotypes of epidemic viruses. Competition assays are an effective and rigorous system with which to assess the relative fitness of viral genotypes. However, it is challenging to quickly and cheaply distinguish and quantify fitness differences between very similar viral genotypes. Here, we describe a protocol for using reverse transcription PCR in combination with commercial nanopore sequencing services to perform competition assays on untagged RNA viruses. Our assay, called the Universal Competition Assay by Nanopore Sequencing (U-CAN-seq), is relatively cheap and highly sensitive. We used a well-studied N24A mutation in the chikungunya virus (CHIKV) nsp3 gene to confirm that we could detect a competitive disadvantage using U-CAN-seq. We also used this approach to show that mutations to the CHIKV 5' conserved sequence element that disrupt sequence but not structure did not affect the fitness of CHIKV. However, similar mutations to an adjacent CHIKV stem loop (SL3) did cause a fitness disadvantage compared to wild-type CHIKV, suggesting that structure-independent, primary sequence determinants in this loop play an important role in CHIKV biology. Our novel findings illustrate the utility of the U-CAN-seq competition assay.