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.

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.

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.

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.

Unlocking the antiviral potential of rosmarinic acid against chikungunya virus via IL-17 signaling pathway.

Background: The Chikungunya virus is an Alphavirus that belongs to the Togaviridae family and is primarily transmitted by mosquitoes. It causes acute infection characterized by fever, headache, and arthralgia. Some patients also experience persistent chronic osteoarthritis-like symptoms. Dedicated antiviral treatments are currently unavailable for CHIKV. This study aims to explore the potential anti-CHIKV effect of rosmarinic acid using network pharmacology. Methods: This study employed network pharmacology to predict and verify the molecular targets and pathways associated with ROSA in the context of CHIKV. The analysis outcomes were further validated using molecular docking and in vitro experiments. Results: The analysis of CHIKV targets using the Kyoto Encyclopedia of Genes and Genomes and MCODE identified IL-17 as an important pathogenic pathway in CHIKV infection. Among the 30 targets of ROSA against CHIKV, nearly half were found to be involved in the IL-17 signaling pathway. This suggests that ROSA may help the host in resisting CHIKV invasion by modulating this pathway. Molecular docking validation results showed that ROSA can stably bind to 10 core targets out of the 30 identified targets. In an in vitro CHIKV infection model developed using 293T cells, treatment with 60 µM ROSA significantly improved the survival rate of infected cells, inhibited 50% CHIKV proliferation after CHIKV infection, and reduced the expression of TNF-α in the IL-17 signaling pathway. Conclusion: This study provides the first confirmation of the efficacy of ROSA in suppressing CHIKV infection through the IL-17 signaling pathway. The findings warrant further investigation to facilitate the development of ROSA as a potential treatment for CHIKV infection.

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).

In vivo rescue of arboviruses directly from subgenomic DNA fragments.

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.

4'-Fluorouridine inhibits alphavirus replication and infection in vitro and in vivo.

Chikungunya virus (CHIKV) is an enveloped, positive-sense RNA virus that has re-emerged to cause millions of human infections worldwide. In humans, acute CHIKV infection causes fever and severe muscle and joint pain. Chronic and debilitating arthritis and joint pain can persist for months to years. To date, there are no approved antivirals against CHIKV. Recently, the ribonucleoside analog 4'-fluorouridine (4'-FlU) was reported as a highly potent orally available inhibitor of SARS-CoV-2, respiratory syncytial virus, and influenza virus replication. In this study, we assessed 4'-FlU's potency and breadth of inhibition against a panel of alphaviruses including CHIKV, and found that it broadly suppressed alphavirus production in cell culture. 4'-FlU acted on the viral RNA replication step, and the first 4 hours post-infection were the critical time for its antiviral effect. In vitro replication assays identified nsP4 as the target of inhibition. In vivo, treatment with 4'-FlU reduced disease signs, inflammatory responses, and viral tissue burden in mouse models of CHIKV and Mayaro virus infection. Treatment initiated at 2 hours post-infection was most effective; however, treatment initiated as late as 24-48 hours post-infection produced measurable antiviral effects in the CHIKV mouse model. 4'-FlU showed effective oral delivery in our mouse model and resulted in the accumulation of both 4'-FlU and its bioactive triphosphate form in tissues relevant to arthritogenic alphavirus pathogenesis. Together, our data indicate that 4'-FlU inhibits CHIKV infection in vitro and in vivo and is a promising oral therapeutic candidate against CHIKV infection.IMPORTANCEAlphaviruses including chikungunya virus (CHIKV) are mosquito-borne positive-strand RNA viruses that can cause various diseases in humans. Although compounds that inhibit CHIKV and other alphaviruses have been identified in vitro, there are no licensed antivirals against CHIKV. Here, we investigated a ribonucleoside analog, 4'-fluorouridine (4'-FlU), and demonstrated that it inhibited infectious virus production by several alphaviruses in vitro and reduced virus burden in mouse models of CHIKV and Mayaro virus infection. Our studies also indicated that 4'-FlU treatment reduced CHIKV-induced footpad swelling and reduced the production of pro-inflammatory cytokines. Inhibition in the mouse model correlated with effective oral delivery of 4'-FlU and accumulation of both 4'-FlU and its bioactive form in relevant tissues. In summary, 4'-FlU exhibits potential as a novel anti-alphavirus agent targeting the replication of viral RNA.

Tracing the evolution of the chikungunya virus in Argentina, 2016-2023: independent introductions and prominence of Latin American lineages.

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.

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.

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.

Development and field validation of a reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) for the rapid detection of chikungunya virus in patient and mosquito samples.

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.

Quantitative detection of chikungunya, Zika, and dengue viruses by one-step real-time PCR in different cell substrates.

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 (R2) 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.

Chikungunya virus perturbs the Wnt/ß-catenin signaling pathway for efficient viral infection.

IMPORTANCE: Being obligate parasites, viruses use various host cell machineries in effectively replicating their genome, along with virus-encoded enzymes. In order to carry out infection and pathogenesis, viruses are known to manipulate fundamental cellular processes in cells and interfere with host gene expression. Several viruses interact with the cellular proteins involved in the Wnt/ß-catenin pathway; however, reports regarding the involvement of protein components of the Wnt/ß-catenin pathway in Chikungunya virus (CHIKV) infection are scarce. Additionally, there are currently no remedies or vaccines available for CHIKV. This is the first study to report that modulation of the Wnt/ß-catenin pathway is crucial for effective CHIKV infection. These investigations deepen the understanding of the underlying mechanisms of CHIKV infection and offer new avenue for developing effective countermeasures to efficiently manage CHIKV infection.

Development of disposable electrode for the detection of mosquito-borne viruses.

Development of disposable, rapid, and convenient biosensor with high sensitivity and reliability is the most desired method of viral disease prevention. To achieve this goal, in this work, a practical impedimetric biosensor has been implemented into a disposable electrode on a screen-printed carbon electrode (SPCE) for the detection of two mosquito-borne viruses. The biosensor fabrication has step-wisely carried out on the disposable electrode surface at room temperature: starting from conductive film formation, physical binding of the gold nanoparticles (AuNPs)-polyaniline (PAni) into the conductive film, and biofunctionalization. To get the maximum efficiency of the antibody, biotinylated antibody has been conjugated on the surface of AuNP-PAni/PAni-SPCE via the streptavidin-biotin conjugation method which is a critical factor for the high sensitivity. Using the antibody-antigen interaction, this disposable electrode has designed to detect mosquito-borne infectious viruses, Chikungunya virus (CHIKV), and Zika virus (ZIKV) separately in a wide linear range of 100 fg mL-1 to 1 ng mL-1 with a low detection limit of 1.33 and 12.31 fg mL-1 , respectively.

Vitamin D modulates the expression of Toll-like receptors and pro-inflammatory cytokines without affecting Chikungunya virus replication, in monocytes and macrophages.

Chikungunya virus (CHIKV) is a zoonotic arthropod-borne virus that causes Chikungunya fever (CHIKF), a self-limiting disease characterized by myalgia and acute or chronic arthralgia. CHIKF pathogenesis has an important immunological component since higher levels of pro-inflammatory factors, including cytokines and chemokines, are detected in CHIKV-infected patients. In vitro studies, using monocytes and macrophages have shown that CHIKV infection promotes elevated production of pro-inflammatory cytokines and antiviral response factors. Vitamin D3 (VD3) has been described as an important modulator of immune response and as an antiviral factor for several viruses. Here, we aimed to study the effects of VD3 treatment on viral replication and pro-inflammatory response in CHIKV-infected human monocytes (VD3-Mon) and monocyte-derived macrophages differentiated in the absence (MDMs) or the presence of VD3 (VD3-MDMs). We found that VD3 treatment did not suppress CHIKV replication in either VD3-Mon or VD3-MDMs. However, the expression of VDR, CAMP and CYP24A1 mRNAs was altered by CHIKV infection. Furthermore, VD3 treatment alters TLRs mRNA expression and production of pro-inflammatory cytokines, including TNFα and CXCL8/IL8, but not IL1ß and IL6, in response to CHIKV infection in both VD3-Mon and VD3-MDMs. While a significant decrease in CXCL8/IL8 production was observed in CHIKV-infected VD3-Mon, significantly higher production of CXCL8/IL8 was observed in CHIKV-infected VD3-MDM at 24 hpi. Altogether, our results suggest that vitamin D3 may play an important role in ameliorating pro-inflammatory response during CHIKV infection in human Mon, but not in MDMs. Although further studies are needed to evaluate the efficacy of VD3; nevertheless, this study provides novel insights into its benefits in modulating the inflammatory response elicited by CHIKV infection in humans.

Two Conserved Phenylalanine Residues in the E1 Fusion Loop of Alphaviruses Are Essential for Viral Infectivity.

Alphaviruses infect cells by a low pH-dependent fusion reaction between viral and host cell membranes that is mediated by the viral E1 glycoprotein. Most reported alphavirus E1 sequences include two phenylalanines (F87 and F95) in the fusion loop, yet the role of these residues in viral infectivity remains to be defined. Following introduction of wild type (WT), E1-F87A, and E1-F95A chikungunya virus (CHIKV) RNA genomes into cells, viral particle production was similar in magnitude. However, CHIKV E1-F87A and E1-F95A virions displayed impaired infectivity compared with WT CHIKV particles. Although WT, E1-F87A, and E1-F95A particles bound cells with similar efficiencies, E1-F87A and E1-F95A particles were unable to undergo fusion and entry into cells. Introduction of an F95A mutation in the E1 fusion loop of Mayaro virus or Venezuelan equine encephalitis virus also resulted in poorly infectious virions. We further tested whether an E1-F87A or E1-F95A mutation could be incorporated into a live-attenuated vaccine strain, CHIKV 181/25, to enhance vaccine safety. Infection of immunocompromised Ifnar1-/- and Irf3-/-Irf5-/-Irf7-/- mice with 181/25E1-F87A or 181/25E1-F95A resulted in 0% mortality, compared with 100% mortality following 181/25 infection. Despite this enhanced attenuation, surviving Ifnar1-/- and Irf3-/-Irf5-/-Irf7-/- mice were protected against virulent virus re-challenge. Moreover, single-dose immunization of WT mice with either 181/25, 181/25E1-F87A, or 181/25E1-F95A elicited CHIKV-specific antibody responses and protected against pathogenic CHIKV challenge. These studies define a critical function for residues E1-F87 and E1-F95 in alphavirus fusion and entry into target cells and suggest that incorporation of these mutations could enhance the safety of live-attenuated alphavirus vaccine candidates. IMPORTANCE Alphaviruses are human pathogens that cause both debilitating acute and chronic musculoskeletal disease and potentially fatal encephalitis. In this study, we determined that two highly conserved phenylalanine residues in the alphavirus E1 glycoprotein are required for fusion of viral and host cell membranes and viral entry into target cells. We further demonstrated that mutation of these phenylalanines results in a substantial loss of viral virulence but not immunogenicity. These data enhance an understanding of the viral determinants of alphavirus entry into host cells and could contribute to the development of new antivirals targeting these conserved phenylalanines or new live-attenuated alphavirus vaccines.

Seroprevalence of dengue, Zika, chikungunya and Ross River viruses across the Solomon Islands.

Across the Pacific, and including in the Solomon Islands, outbreaks of arboviruses such as dengue, chikungunya, and Zika are increasing in frequency, scale and impact. Outbreaks of mosquito-borne disease have the potential to overwhelm the health systems of small island nations. This study mapped the seroprevalence of dengue, Zika, chikungunya and Ross River viruses in 5 study sites in the Solomon Islands. Serum samples from 1,021 participants were analysed by ELISA. Overall, 56% of participants were flavivirus-seropositive for dengue (28%), Zika (1%) or both flaviviruses (27%); and 53% of participants were alphavirus-seropositive for chikungunya (3%), Ross River virus (31%) or both alphaviruses (18%). Seroprevalence for both flaviviruses and alphaviruses varied by village and age of the participant. The most prevalent arboviruses in the Solomon Islands were dengue and Ross River virus. The high seroprevalence of dengue suggests that herd immunity may be a driver of dengue outbreak dynamics in the Solomon Islands. Despite being undetected prior to this survey, serology results suggest that Ross River virus transmission is endemic. There is a real need to increase the diagnostic capacities for each of the arboviruses to support effective case management and to provide timely information to inform vector control efforts and other outbreak mitigation interventions.