Interleukin 27, like interferons, activates JAK-STAT signaling and promotes pro-inflammatory and antiviral states that interfere with dengue and chikungunya viruses replication in human macrophages.

Interferons (IFNs) are a family of cytokines that activate the JAK-STAT signaling pathway to induce an antiviral state in cells. Interleukin 27 (IL-27) is a member of the IL-6 and/or IL-12 family that elicits both pro- and anti-inflammatory responses. Recent studies have reported that IL-27 also induces a robust antiviral response against diverse viruses, both in vitro and in vivo, suggesting that IFNs and IL-27 share many similarities at the functional level. However, it is still unknown how similar or different IFN- and IL-27-dependent signaling pathways are. To address this question, we conducted a comparative analysis of the transcriptomic profiles of human monocyte-derived macrophages (MDMs) exposed to IL-27 and those exposed to recombinant human IFN-α, IFN-γ, and IFN-λ. We utilized bioinformatics approaches to identify common differentially expressed genes between the different transcriptomes. To verify the accuracy of this approach, we used RT-qPCR, ELISA, flow cytometry, and microarrays data. We found that IFNs and IL-27 induce transcriptional changes in several genes, including those involved in JAK-STAT signaling, and induce shared pro-inflammatory and antiviral pathways in MDMs, leading to the common and unique expression of inflammatory factors and IFN-stimulated genes (ISGs)Importantly, the ability of IL-27 to induce those responses is independent of IFN induction and cellular lineage. Additionally, functional analysis demonstrated that like IFNs, IL-27-mediated response reduced chikungunya and dengue viruses replication in MDMs. In summary, IL-27 exhibits properties similar to those of all three types of human IFN, including the ability to stimulate a protective antiviral response. Given this similarity, we propose that IL-27 could be classified as a distinct type of IFN, possibly categorized as IFN-pi (IFN-π), the type V IFN (IFN-V).

Chikungunya virus infection in human microglial C20 cells induces mitochondria-mediated apoptosis.

Introduction: Chikungunya virus (CHIKV) infection is associated with acute clinical manifestations and chronic joint inflammation. CHIKV has emerged as a significant causative agent of central nervous system (CNS) complications, including encephalitis and related sequelae. Microglial cells, crucial for immune responses and tissue repair in the CNS, play a vital role in the host response to viral infections, with their activation potentially leading to either protection or pathology. In this study, the infection biology of CHIKV in the C20 human microglial cell line was investigated. Methods: The permissiveness of C20 cells to CHIKV infection was assessed, and viral replication kinetics were compared to Vero E6 cells. Cytopathic effects of CHIKV infection on C20 cells were examined, along with ultrastructural changes using transmission electron microscopy. Additionally, apoptosis induction, mitochondrial membrane potential, and alterations in cell surface marker expression were evaluated by flow cytometry. Results: CHIKV infection demonstrated permissiveness in C20 cells, similar to Vero cells, resulting in robust viral replication and cytopathic effects. Ultrastructural analysis revealed viral replication, mature virion formation, and distinctive cytoplasmic and nuclear changes in infected C20 cells. CHIKV infection induced significant apoptosis in C20 cells, accompanied by mitochondrial membrane depolarization and altered expression of cell surface markers such as CD11c, CD14, and HLA-DR. Notably, decreased CD14 expression was observed in CHIKV-infected C20 cells. Discussion: The study findings suggest that CHIKV infection induces apoptosis in C20 microglial cells via the mitochondrial pathway, with significant alterations in cell surface marker expression, particularly CD14 that is linked with apoptosis induction. These observations provide valuable insights into the role of human microglial cells in the host response to CHIKV infection and contribute to the knowledge on the neuropathogenesis of this virus.

Comparative analysis of midgut bacterial communities in Chikungunya virus-infected and non-infected Aedes aegypti Thai laboratory strain mosquitoes.

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.

Aedes albopictus is a competent vector of five arboviruses affecting human health, greater Paris, France, 2023.

Aedes albopictus collected in 2023 in the greater Paris area (Île-de-France) were experimentally able to transmit five arboviruses: West Nile virus from 3 days post-infection (dpi), chikungunya virus and Usutu virus from 7 dpi, dengue virus and Zika virus from 21 dpi. Given the growing number of imported dengue cases reported in early 2024 in France, surveillance of Ae. albopictus should be reinforced during the Paris Olympic Games in July, when many international visitors including from endemic countries are expected.

Exposure to Zika and chikungunya viruses impacts aspects of the vectorial capacity of Aedes aegypti and Culex quinquefasciatus.

Zika (ZIKV) and chikungunya (CHIKV) are arboviruses that cause infections in humans and can cause clinical complications, representing a worldwide public health problem. Aedes aegypti is the primary vector of these pathogens and Culex quinquefasciatus may be a potential ZIKV vector. This study aimed to evaluate fecundity, fertility, survival, longevity, and blood feeding activity in Ae. aegypti after exposure to ZIKV and CHIKV and, in Cx. quinquefasciatus exposed to ZIKV. Three colonies were evaluated: AeCamp (Ae. aegypti-field), RecL (Ae. aegypti-laboratory) and CqSLab (Cx. quinquefasciatus-laboratory). Seven to 10 days-old females from these colonies were exposed to artificial blood feeding with CHIKV or ZIKV. CHIKV caused reduction in fecundity and fertility in AeCamp and reduction in survival and fertility in RecL. ZIKV impacted survival in RecL, fertility in AeCamp and, fecundity and fertility in CqSLab. Both viruses had no effect on blood feeding activity. These results show that CHIKV produces a higher biological cost in Ae. aegypti, compared to ZIKV, and ZIKV differently alters the biological performance in colonies of Ae. aegypti and Cx. quinquefasciatus. These results provide a better understanding over the processes of virus-vector interaction and can shed light on the complexity of arbovirus transmission.

A Highly Sensitive Molecular Technique for RNA Virus Detection.

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.

Molecular identification and phylogenetic analysis of chikungunya virus among dengue-negative patients in Kolkata, India.

Dengue and chikungunya are co-circulating vector-borne diseases that share a significant number of clinical symptoms. To identify variables to aid physicians in making rapid and effective diagnostic decisions, we performed molecular diagnosis of the chikungunya virus and examined the clinical manifestations of chikungunya cases to identify the prevalence among dengue-negative individuals in Kolkata. Dengue suspected patients' samples were collected during January 2020-December 2021 and Enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) methods have been performed to confirm the prevalence of chikungunya infection among dengue-negative patients. By performing phylogenetic analysis, comparing clinical classifications, identifying disease aetiology using clinical and laboratory factors, and evaluating the time course of several clinical variables, we have evaluated the clinical manifestations linked to dengue and chikungunya virus infections. Chikungunya infection was found in 15.1% and 6.3% of the 635 dengue-negative patients, as determined by ELISA and RT-PCR, respectively. Arthritis and myalgia were more common in chikungunya-infected patients at the time of hospital admission while conjunctivitis, photosensitivity, arthralgia, Anorexia, fatigue, retro-orbital pain, vomiting, dermatitis, or swollen glands were significantly presented as an overlapping symptom. Although dengue and chikungunya infections have significant clinical overlap, basic clinical and laboratory criteria can predict these diseases at presentation for proper management. Effective management enables doctors to treat and care for patients properly and contributes to the development of control measures for these infections in a medical setting.

A case series of post-infectious chikungunya myeloradiculoneuropathy.

Chikungunya fever is an arboviral illness caused by chikungunya virus (CHIKV) and transmitted by the bite of Aedes aegypti and Aedes albopictus. It is an RNA virus belonging to the genus Alphavirus and family Togaviridae. We present a case series of three patients with chikungunya illness developing para/post-infectious myeloradiculoneuropathy.These patients developed neurological symptoms in the form of bilateral lower limb weakness with sensory and bowel involvement after the recovery from the initial acute episode of chikungunya fever. Clinical examination findings suggested myeloradiculoneuropathy with normal Magnetic Resonance Imaging of the Spine, with the nerve conduction study showing sensorimotor axonal polyneuropathy. All the patients were treated with 1 g of methylprednisolone once a day for five days, and case 2 was given intravenous immunoglobulin also. In the follow-up, cases 1 and 2 showed complete recovery without recurrence, and case 3 did not show improvement at one month.

Bone erosions and joint damage caused by chikungunya virus: a systematic review.

BACKGROUND: Chikungunya fever is an emerging global infection transmitted by Aedes mosquitoes that manifests as an acute febrile illness with joint pain and can lead to chronic arthritis. The mechanism underlying chronic joint damage remains unclear; however, chronic chikungunya arthritis shares similarities with rheumatoid arthritis. Disease-modifying antirheumatic drugs have revolutionized rheumatoid arthritis treatment by preventing joint damage. However, the role of these therapies in chronic chikungunya arthritis has not been determined. We conducted a systematic review to evaluate the burden of joint structural damage in chronic chikungunya arthritis to help to define the role of disease-modifying therapy in this disease. METHODS: This systematic review included retrospective and prospective studies, trials, and case reports evaluating joint damage caused by chikungunya virus. Various databases were searched without any date or language restrictions. Study selection was conducted independently by two researchers, and data were extracted from the articles selected. RESULTS: A total of 108 studies were initially evaluated, with 8 meeting the inclusion criteria. Longitudinal studies have reported persistent joint pain from chikungunya infection and the progression of radiographic joint damage up to 13 years post-infection. Joint imaging revealed synovial inflammation, bone erosion, and cartilage destruction in patients with chronic chikungunya arthritis. CONCLUSIONS: Few studies have addressed chikungunya-induced joint damage, limiting our understanding of chronic chikungunya arthritis. Nevertheless, chronic chikungunya arthritis has similarities to rheumatoid arthritis. The success of early disease-modifying antirheumatic drug therapy in rheumatoid arthritis underscores the need for comprehensive research on its role in chikungunya arthritis.

The first confirmed outbreak of chikungunya reported in Timor-Leste, 2024.

Abstract: Timor-Leste is a mountainous, half-island nation with a population of 1.3 million, which shares a land border with Indonesia and is 550 km from Darwin, Australia. Since independence in 2002, Timor-Leste has achieved significant development; however, high levels of poverty remain. Chikungunya virus (CHIKV) is endemic in over 100 countries in Africa, Asia, Europe and in the Americas. It is transmitted by the bite of infected Aedes aegypti or Ae. albopictus mosquitoes, which are present in Timor-Leste and which contribute to annual rainy-season dengue virus (DENV) outbreaks. Symptomatic people typically suffer from acute onset of fever, usually accompanied by severe arthritis or arthralgia. Joint pain can be debilitating for several days, and may sometimes last for weeks, months or years. Unlike DENV infection which has significant mortality, most people recover completely. Between 2002 and 2023, there were 26 cases of CHIKV notified in Australia who acquired their infection in Timor-Leste; however, laboratory testing capability for CHIKV in Timor-Leste only became available in 2021 using polymerase chain reaction (PCR). The first locally diagnosed case was notified in November 2023. In January 2024, an outbreak of CHIKV was recognised in Timor-Leste for the first time, with 195 outbreak cases reported during 1-31 January 2024; all were PCR positive. There were no cases hospitalised, and no deaths. The median age of cases was 17 years (range 1-76 years); 51% were males. Cases were reported across the country; most (88/195) were from Dili, although the highest incidence was seen in the neighbouring municipality of Ermera (monthly incidence rate of 58.8 cases per 100,000 population). This first reported outbreak of CHIKV in Timor-Leste highlights the need for improved mosquito-borne illness control and response strategies, including minimising breeding sites and promoting early presentation for treatment and differential diagnosis from DENV, and consideration of the deployment of Wolbachia-infected mosquitoes, particularly as they have shown to reduce the transmission of CHIKV, DENV and Zika virus, all of which pose threats in Timor-Leste.

GS-441524-Diphosphate-Ribose Derivatives as Nanomolar Binders and Fluorescence Polarization Tracers for SARS-CoV-2 and Other Viral Macrodomains.

Viral macrodomains that can bind to or hydrolyze protein adenosine diphosphate ribosylation (ADP-ribosylation) have emerged as promising targets for antiviral drug development. Many inhibitor development efforts have been directed against the severe acute respiratory syndrome coronavirus 2 macrodomain 1 (SARS-CoV-2 Mac1). However, potent inhibitors for viral macrodomains are still lacking, with the best inhibitors still in the micromolar range. Based on GS-441524, a remdesivir precursor, and our previous studies, we have designed and synthesized potent binders of SARS-CoV-2 Mac1 and other viral macrodomains including those of Middle East respiratory syndrome coronavirus (MERS-CoV), Venezuelan equine encephalitis virus (VEEV), and Chikungunya virus (CHIKV). We show that the 1'-CN group of GS-441524 promotes binding to all four viral macrodomains tested while capping the 1″-OH of GS-441524-diphosphate-ribose with a simple phenyl ring further contributes to binding. Incorporating these two structural features, the best binders show 20- to 6000-fold increases in binding affinity over ADP-ribose for SARS-CoV-2, MERS-CoV, VEEV, and CHIKV macrodomains. Moreover, building on these potent binders, we have developed two highly sensitive fluorescence polarization tracers that only require nanomolar proteins and can effectively resolve the binding affinities of nanomolar inhibitors. Our findings and probes described here will facilitate future development of more potent viral macrodomain inhibitors.

Live-attenuated CHIKV vaccine with rearranged genome replicates in vitro and induces immune response in mice.

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.

The High Capacity of Brazilian Aedes aegypti Populations to Transmit a Locally Circulating Lineage of Chikungunya Virus.

The incidence of chikungunya has dramatically surged worldwide in recent decades, imposing an expanding burden on public health. In recent years, South America, particularly Brazil, has experienced outbreaks that have ravaged populations following the rapid dissemination of the chikungunya virus (CHIKV), which was first detected in 2014. The primary vector for CHIKV transmission is the urban mosquito species Aedes aegypti, which is highly prevalent throughout Brazil. However, the impact of the locally circulating CHIKV genotypes and specific combinations of local mosquito populations on vector competence remains unexplored. Here, we experimentally analyzed and compared the infectivity and transmissibility of the CHIKV-ECSA lineage recently isolated in Brazil among four Ae. aegypti populations collected from different regions of the country. When exposed to CHIKV-infected AG129 mice for blood feeding, all the mosquito populations displayed high infection rates and dissemination efficiency. Furthermore, we observed that all the populations were highly efficient in transmitting CHIKV to a vertebrate host (naïve AG129 mice) as early as eight days post-infection. These results demonstrate the high capacity of Brazilian Ae. aegypti populations to transmit the locally circulating CHIKV-ECSA lineage. This observation could help to explain the high prevalence of the CHIKV-ECSA lineage over the Asian lineage, which was also detected in Brazil in 2014. However, further studies comparing both lineages are necessary to gain a better understanding of the vector's importance in the epidemiology of CHIKV in the Americas.

U-CAN-seq: A Universal Competition Assay by Nanopore Sequencing.

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.

Isolation and molecular detection of dengue and chikungunya virus from field-collected adult mosquitoes in Kelantan, Malaysia.

BACKGROUND OBJECTIVES: Dengue and chikungunya infections are one of the major health problems that have plagued the human population globally. All dengue virus (DENV) serotypes circulate within Malaysia with particular serotypes dominating in different years/outbreaks. In the state of Kelantan, an increasing number of DENV and chikungunya virus (CHIKV) new cases have been reported, including several deaths. This study aimed to isolate and detect these arboviruses from adult mosquitoes in Kelantan. METHODS: Adult mo squito samples were collected from January to August 2019 and were identified according to gender, species and locality. The isolation of the virus was done in C6/36 cells. Dengue NS1 antigen was carried out using direct mosquito lysate and mosquito culture supernatant. Detection and serotyping of the DENV was performed using multiplex RT-PCR and CHIKV detection using a one-step RT-PCR assay. RESULTS: Of 91 mosquito pools, four were positive for NS1 antigen comprising two pools (2.2%) of male Ae. albopictus (Pulau Melaka and Kubang Siput) and two pools (2.2%) of Ae. aegypti (Kampung Demit Sungai). DENV 1 was detected in one pool (0.9%) of female Ae. albopictus among 114 tested Aedes pools. Two pools of 114 pools (1.7%) from both male Aedes species were positive with double serotypes, DENV 1 and DENV 2 (Pulau Melaka). However, no pool was positive for CHIKV. INTERPRETATION CONCLUSION: The presence of DENV and the main vectors of arboviruses in Kelantan are pertinent indicators of the need to improve vector controls to reduce arbovirus infections among people in the localities.

Antiviral and Virucidal Activities of Uncaria tomentosa (Cat's Claw) against the Chikungunya Virus.

Uncaria tomentosa (UT) is a medicinal plant popularly known as cat's claw belonging to the Rubiaceae family that has been reported to display antiviral and anti-inflammatory activities. The chikungunya virus (CHIKV) outbreaks constitute a Brazilian public health concern. CHIKV infection develops an abrupt onset of fever, usually accompanied by a skin rash, besides incapacitating polyarthralgia. There is no vaccine available or treatment for CHIKV infection. The present study evaluates the hydroalcoholic extract of UT bark as a potential antiviral against CHIKV. The in vitro antiviral activity of the UT extract against the Brazilian CHIKV strain was assessed using quantitative reverse transcription polymerase chain reaction, flow cytometry, and plaque assay. Results obtained demonstrated that UT inhibits CHIKV infection in a dose-dependent manner. At the non-cytotoxic concentration of 100 µg/mL, UT exhibited antiviral activity above 90% as determined by plaque reduction assay, and it reduced the viral cytopathic effect. Similarly, a significant virucidal effect of 100 µg/mL UT was observed after 24 and 48 h post-infection. This is the first report on the antiviral activity of UT against CHIKV infection, and the data presented here suggests UT as a potential antiviral to treat CHIKV infection.

Potential Serological Misdiagnosis of Barmah Forest Virus and Ross River Virus Diseases as Chikungunya Virus Infections in Australia: Comparison of ELISA with Neutralization Assay Results.

To evaluate the frequency of errors in the diagnosis of medical laboratory-diagnosed Chikungunya virus (CHIKV) infections in Australia, we studied 42 laboratory-diagnosed CHIKV serum samples from one Queensland medical laboratory by ELISA IgG/IgM and measured the specific neutralization antibodies (Nab) against Barmah Forest virus (BFV), CHIKV and Ross River virus (RRV). The sero-positivity rates for the sera were as follows: anti-BFV IgG+ 19% (8/42), IgM+ 2.4% (1/42) and Nab+ 16.7% (7/42); anti-CHIKV IgG+ 90.5% (38/42), IgM+ 21.4% (9/42) and Nab+ 90.5% (38/42); anti-RRV IgG+ 88.1% (37/42), IgM+ 28.6% (12/42) and Nab+ 83.2% (35/42), respectively. Among the samples with multiple antibody positivity, 2.4% (1/42) showed triple ELISA IgM+, and 14.3% (6/42) exhibited double IgM RRV+CHIKV+; 9.5% (4/42) showed triple IgG+, 76.2% (32/42) displayed double IgG RRV+CHIKV+, 4.8% (2/42) showed IgG BFV+RRV+ and 4.8% (2/42) showed IgG BFV++CHIKV+; and 9.5% (4/42) showed triple Nab+ and 69% (29/42) exhibited double Nab RRV+CHIKV+, respectively. Our analysis of the single-virus infection control Nab results suggested no cross-neutralization between RRV and BFV, and only mild cross-neutralization between CHIKV and RRV, BFV and CHIKV, all with a ≥4-fold Nab titre ratio difference between the true virus infection and cross-reactivity counterpart virus. Subsequently, we re-diagnosed these 42 patients as 1 BFV+, 8 CHIKV+ and 23 RRV+ single-virus infections, along with five RRV+/BFV+ and four RRV+/CHIKV+ double infections, and one possible RRV+/BFV+ or RRV+CHIKV+, respectively. These findings suggests that a substantial proportion of medically attended RRV and BFV infections were misdiagnosed as CHIKV infections, highlighting the imperative need for diagnostic laboratory tests capable of distinguishing between CHIKV infections and actively co-circulating RRV and BFV. For a correct diagnosis, it is crucial to consider reliable diagnostic methods such as the neutralization assay to exclude RRV and BFV.

ß-D-N4-hydroxycytidine (NHC, EIDD-1931) inhibits chikungunya virus replication in mosquito cells and ex vivo Aedes aegypti guts, but not when ingested during blood-feeding.

Chikungunya virus (CHIKV) is a mosquito-borne virus transmitted by Aedes mosquitoes. While there are no antiviral therapies currently available to treat CHIKV infections, several licensed oral drugs have shown significant anti-CHIKV activity in cells and in mouse models. However, the efficacy in mosquitoes has not yet been assessed. Such cross-species antiviral activity could be favorable, since virus inhibition in the mosquito vector might prevent further transmission to vertebrate hosts. Here, we explored the antiviral effect of ß-d-N4-hydroxycytidine (NHC, EIDD-1931), the active metabolite of molnupiravir, on CHIKV replication in Aedes aegypti mosquitoes. Antiviral assays in mosquito cells and in ex vivo cultured mosquito guts showed that NHC had significant antiviral activity against CHIKV. Exposure to a clinically relevant concentration of NHC did not affect Ae. aegypti lifespan when delivered via a bloodmeal, but it slightly reduced the number of eggs developed in the ovaries. When mosquitoes were exposed to a blood meal containing both CHIKV and NHC, the compound did not significantly reduce virus infection and dissemination in the mosquitoes. This was confirmed by modelling and could be explained by pharmacokinetic analysis, which revealed that by 6 h post-blood-feeding, 90% of NHC had been cleared from the mosquito bodies. Our data show that NHC inhibited CHIKV replication in mosquito cells and gut tissue, but not in vivo when mosquitoes were provided with a CHIKV-infectious bloodmeal spiked with NHC. The pipeline presented in this study offers a suitable approach to identify anti-arboviral drugs that may impede replication in mosquitoes.