Chikungunya Virus Research Articles

Unveiling Actin Cytoskeleton Role in Mediating Chikungunya-Associated Arthritis: An Integrative Proteome-Metabolome Study.

Background: Chikungunya is a zoonotic disease caused by the Chikungunya virus (CHIKV), primarily transmitted to humans through infected Aedes mosquitoes. The infection is characterized by symptoms such as high fever, musculoskeletal pain, polyarthritis, and a rash, which can lead to severe complications such as encephalitis, meningitis, and even fatalities. While many disease manifestations resemble those of other viral infections, chronic arthritis caused by CHIKV is unique, and its molecular mechanisms remain ill-defined. Materials and Methods: Proteomics data from both cellular and patient levels of CHIKV infection were curated from PubMed and screened using inclusion and exclusion criteria. Patient serum proteomics data obtained from P RIDE underwent reanalysis using Proteome Discoverer 2.2. Enrichment and protein-protein interaction network analysis were conducted on differentially expressed proteins from both serum and cellular datasets. Metabolite data from CHIKV-infected patients were further retrieved, and their protein binding partners were identified using BindingDB. The protein-metabolite interaction pathway was further developed using MetaboAnalyst. Results: The proteomics data analysis revealed differential expression of proteins involved in critical host mechanisms, such as cholesterol metabolism and mRNA splicing, during CHIKV infection. Consistent upregulation of two actin cytoskeleton proteins, TAGLN2 and PFN1, was noted in both serum and cellular datasets, and their upregulations are associated with arthritis. Furthermore, alterations in purine metabolism were observed in the integrative proteome-metabolome analysis, correlating with cytoskeletal remodelling. Conclusion: Collectively, this integrative view sheds light on the involvement of actin cytoskeleton remodeling proteins and purine metabolic pathways in the development of arthritis during CHIKV infection.

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.

High prevalence of dengue, Zika, and chikungunya viruses in blood donors during a dengue outbreak and an endemic period in Colombia.

Arboviruses pose a challenge in ensuring the supply of pathogen-free blood components because they are not routinely screened in blood banks, and blood components from infected asymptomatic donors could be transfused. This study aimed to detect and characterize arboviral infections in Colombian blood donors. In a cross-sectional study, the prevalence of dengue (DENV), Zika (ZIKV), and chikungunya (CHIKV) viruses and co-infections of blood donors were compared between an epidemic period (November 2019-February 2020, n = 462) and an endemic period (November 2021-August 2022, n = 1,119). Viral RNA from each donor serum was purified, and the viruses were detected using a previously standardized multiplex hemi-nested RT-PCR protocol. Subsequently, donors who tested positive were surveyed 15 days after the detection of the virus to identify clinical characteristics related to the arboviral infection. The prevalences of each virus were presented as percentages and compared between epidemic and endemic periods. Significantly higher prevalences were found in the epidemic period compared with the endemic period for DENV (14.5 vs. 1.9%), ZIKV (7.8 vs. 0.3%), CHIKV (8 vs. 3.3%), and co-infections (4.3 vs. 0.2%). The survey response rate of positive donors in the two periods was 83/175 (47%). In total, 57% of the donors surveyed were asymptomatic. Symptomatic donors most frequently reported headache (31%), malaise (13%), arthralgia (10%), and fever/chills (8%). The prevalence observed in epidemic and endemic periods was higher than that reported in other studies in the Americas. The high proportion of asymptomatic cases found, in addition to the mild and nonspecific manifestations among the symptomatic, may limit the effectiveness of the donor selection criteria used to mitigate the risk of transfusion-transmitted arboviruses.

Identification of RACK1 as a novel regulator of non-structural protein 4 of chikungunya virus.

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.

Chikungunya-virus-specific CD4+ T cells are associated with chronic chikungunya viral arthritic disease in humans

Abstract Chikungunya virus (CHIKV) is a mosquito-borne Alphavirus that causes recurrent outbreaks in tropical and subtropical regions of the world. In the first 2-7 days of infection, most individuals experience high fever, skin rash and joint pain and swelling. However, about 30-60% of the infected individuals, primarily middle-age females, develop debilitating chronic arthritis-like symptoms such as joint pain, muscle aches and edema. Despite ongoing treatment and vaccine development efforts, the immune responses to CHIKV in humans remain largely understudied. In this study, we tested peripheral blood mononuclear cells (PBMCs) collected from a cohort of 39 individuals diagnosed with CHIKV disease during the 2014-2015 epidemic in Colombia against pools of overlapping sequentially spanning the entire CHIKV genome. Using high-resolution spectral flow cytometry, we detected robust CHIKV-specific CD4+ T cell responses, but not CD8+ T cell responses in the periphery. Patients experiencing chronic symptoms displayed a significantly higher abundance of CHIKV-specific memory CD4+ T cells, compared to their recovered counterparts. Furthermore, these CD4+ T cells displayed a significantly lower Th1 phenotype but were enriched in the Th17 phenotype, with the predominant production of TNFɑ. Overall, our work demonstrates that CD4+ T cells play a pivotal role in the pathogenesis of CHIKV and provides insight into possible therapeutic options.

Investigating the Antiviral Properties of Nyctanthes arbor-tristis Linn against the Ebola, SARS-CoV-2, Nipah, and Chikungunya Viruses: A Computational Simulation Study.

Background: The hunt for naturally occurring antiviral compounds to combat viral infection was expedited when COVID-19 and Ebola spread rapidly. Phytochemicals from Nyctanthes arbor-tristis Linn were evaluated as significant inhibitors of these viruses. Methods: Computational tools and techniques were used to assess the binding pattern of phytochemicals from Nyctanthes arbor-tristis Linn to Ebola virus VP35, SARS-CoV-2 protease, Nipah virus glycoprotein, and chikungunya virus. Results: Virtual screening and AutoDock analysis revealed that arborside-C, beta amyrin, and beta-sitosterol exhibited a substantial binding affinity for specific viral targets. The arborside-C and beta-sitosterol molecules were shown to have binding energies of -8.65 and -9.11 kcal/mol, respectively, when interacting with the major protease. Simultaneously, the medication remdesivir exhibited a control value of -6.18 kcal/mol. The measured affinity of phytochemicals for the other investigated targets was -7.52 for beta-amyrin against Ebola and -6.33 kcal/mol for nicotiflorin against Nipah virus targets. Additional molecular dynamics simulation (MDS) conducted on the molecules with significant antiviral potential, specifically the beta-amyrin-VP35 complex showing a stable RMSD pattern, yielded encouraging outcomes. Conclusions: Arborside-C, beta-sitosterol, beta-amyrin, and nicotiflorin could be established as excellent natural antiviral compounds derived from Nyctanthes arbor-tristis Linn. The virus-suppressing phytochemicals in this plant make it a compelling target for both in vitro and in vivo research in the future.

Características de la infección aguda por el virus chikungunya en niños: un estudio epidemiológico en el Departamento de Caaguazú, Paraguay

Introduction: The rapid transmission and severe symptoms associated with acute chikungunya virus (CHIKV) infection in children make it a highly concerning health issue. Objective: This study aimed to describe the characteristics of acute chikungunya virus infection in children from the Department of Caaguazú, Paraguay. Material and Methods: A retrospective observational study was conducted in the Department of Caaguazú, Paraguay, in 2023, with all patients who came to the Regional Hospital within 5 days of developing characteristic symptoms of acute CHIKV infection and tested positive for the virus by RT-PCR. Patients with Dengue or Zika infections were excluded. We collected data on clinical characteristics using a standardized case record form and created an electronic dataset for analysis. Results: A total of 461 children were included in the study. 51.6% were women. Cases were divided into groups based on pediatric age: infants (0-23 months, n=88, 19.1%), preschoolers (2-5 years, n=115, 24.9%), schoolchildren (6-11 years, n=163, 35.4%), and adolescents (12-17 years and 11 months, n=95, 20.6%). Schoolchildren and adolescents experienced a higher prevalence of myalgia (64.6%) and arthralgia (63.7%). Vomiting (89%), headache (89.4%), and retro-orbital pain (95%) were more common in preschoolers, schoolchildren, and adolescents, while rash (39.5%) and petechiae (18.5%) were more prevalent in infants. Four children died during the study period. Conclusion: The 2023 CHIKV virus epidemic in Paraguay had different clinical presentations depending on the age of the affected children, with fatal outcomes occurring in a small percentage of cases.

Acquiring insights through a sequence-based approach to the critical Zika virus MTase domain

Background: ZIKV is one of the re-emerging arboviruses (viruses carried by arthropods), which is spread through the Aedes mosquito. It is an RNA virus with only one strand that is appropriate to the family Flaviviridae's Flavivirus (genus) & has been linked to other Flaviviruses such as the West Nile virus, chikungunya virus, & dengue (DENV) virus. The envelope, precursor membrane, and capsid are three structural proteins, and seven nonstructural proteins are also encoded by the Zika virus genome.Methods: We conducted an in-silico analysis of the Zika virus' MTase domain protein for this publication. We predicted that methylation would play a significant role in the available Prosite, Pfam, and InterProScan tools to aid in locating the MTase domain. Along with alignment, amino acid composition, charged amino acids, atomic level studies, & molecular weight, we also make predictions for these variables, including theoretical Pi.Results: We also examine the MTase domain's simulated structure (alpha helix, beta sheet, turn) and its specifics, including secondary structure. We also pinpoint the locations where proteins, DNA, and RNA bind. Potential phosphorylation sites can be found on the Ser, Thr, and Tyr residues in the MTase domain.Conclusion: These outcomes imply a complicated interaction between different phosphorylation modifications that modulates the activity of the MTase domain. To fully appreciate the auxiliary and practical perspectives and to clarify the varied roles of PTM in the MTase domain will be a primary goal of future study.Keywords: I-TASSER; Secondary structure; Prosite; α-helix; Pfam; InterProScan; Binding sites; Posttranslational modification; SOPMA; Phyre2

Winged Threat on the Offensive: A Literature Review Due to the First Identification of Aedes japonicus in Poland.

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.

In silico identification of antiviral compounds for the treatment of chikungunya virus infection: qsar modelling and md simulation analysis

Chikungunya virus (CHIKV), transmitted by arthropods, has gained global recognition for its impact on public health. It has expanded globally, including Africa, Asia, and the Indian subcontinent, and has a helicase protein in its genome that is crucial for its replication. Thus, the study targeted the helicase protein of CHIKV with 745 antiviral compounds using an ML-based QSAR model and molecular docking. Top binders (5279172, 78161839, 6474310, and 5330286) were selected for MD simulation based on the control (Silvestrol). All compounds had the highest binding scores, with 78161839 showing the most consistent RMSD and the least conformational variation, indicating high stability. It also showed the lowest binding free energy (ΔG ​= ​−31.31 ​kcal/mol), indicating energetically favourable binding. PCA and FEL also depicted the stable complex confirmation of the protein and 78161839 complex during the 100 ns simulation. Overall, this study aimed to identify helicase function antiviral binders that could be experimentally tested for treating CHIKV.

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.

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. 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. 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. 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.

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.

Possible vertical transmission of Chikungunya virus infection detected in the cord blood samples from a birth cohort in Vietnam

BackgroundChikungunya 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. MethodsWe 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). ResultsOf 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. ConclusionThis 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.

Awareness and Knowledge of Chikungunya Virus Infection Among Healthcare Workers in Hulu Langat District, Selangor, Malaysia.

As Chikungunya virus (CHIKV) infection continues to rise globally, including in Malaysia, it is essential for healthcare workers (HCWs) to have adequate knowledge about the disease for diagnostic accuracy and to improve public health surveillance systems. This study aimed to assess awareness and measure the level of knowledge of CHIKV infection among HCWs in the Hulu Langat district and explore associated sociodemographic and skill-related factors. This was a cross-sectional study in which the questionnaire was physically distributed to participants using the universal sampling method. All participants (100%) were aware of CHIKV infection, and most (80.1%) had knowledge of the disease. Furthermore, networks such as professional members, family, and friends (27.8%), followed by professional development programs (23.1%), were identified as the common platforms utilized by HCWs to access information regarding CHIKV infection. Ordinal logistic regression analysis further demonstrated that the level of education (odds ratio [OR] = 2.23,) and HCWs who attended Continuing Medical Education (CME)/courses on CHIKV infection (OR = 1.73,) and had experience in handling the case (OR = 3.23,) were significantly associated with awareness and knowledge of the disease. Implementing continuous education and training can enhance HCWs' understanding of CHIKV infection.

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.

Design, development, and validation of multi-epitope proteins for serological diagnosis of Zika virus infections and discrimination from dengue virus seropositivity.

Zika virus (ZIKV), an arbovirus from the Flaviviridae family, is the causative agent of Zika fever, a mild and frequent oligosymptomatic disease in humans. Nonetheless, on rare occasions, ZIKV infection can be associated with Guillain-Barré Syndrome (GBS), and severe congenital complications, such as microcephaly. The oligosymptomatic disease, however, presents symptoms that are quite similar to those observed in infections caused by other frequent co-circulating arboviruses, including dengue virus (DENV). Moreover, the antigenic similarity between ZIKV and DENV, and even with other members of the Flaviviridae family, complicates serological testing due to the high cross-reactivity of antibodies. Here, we designed, produced in a prokaryotic expression system, and purified three multiepitope proteins (ZIKV-1, ZIKV-2, and ZIKV-3) for differential diagnosis of Zika. The proteins were evaluated as antigens in ELISA tests for the detection of anti-ZIKV IgG using ZIKV- and DENV-positive human sera. The recombinant proteins were able to bind and detect anti-ZIKV antibodies without cross-reactivity with DENV-positive sera and showed no reactivity with Chikungunya virus (CHIKV)- positive sera. ZIKV-1, ZIKV-2, and ZIKV-3 proteins presented 81.6%, 95%, and 66% sensitivity and 97%, 96%, and 84% specificity, respectively. Our results demonstrate the potential of the designed and expressed antigens in the development of specific diagnostic tests for the detection of IgG antibodies against ZIKV, especially in regions with the circulation of multiple arboviruses.

Detectable Viremia at Presentation Is a Predictor of Disease Severity in Chikungunya.

Background Chikungunya is a mosquito-borne re-emerging disease that has caused a significant number of outbreaks recently in diverse geographic settings across the globe. It leads to severe debilitating illness in a significant proportion of persons who are infected. Measures to limit the impact produced by recurrent outbreaks of the disease are limited and there is an urgent clinical need for early identification of those predisposed to develop severe disease. A comprehensive understandingregarding the proportion of individuals predisposed to developing severe disease is lacking as its correlation with detectable viremia is hinted at by some studies. In this context, we hypothesized that detectable viremia reflected in the diagnostic RT-PCR assay could be significantly associated with the development of severe disease in Chikungunya among those diagnosed on the basis of seroconversion. Our study aims to confirm the same in relation to disease severity among the suspected patients of Chikungunya in the setting of a tertiary care center. Methods In a prospective observational study at a tertiary care center, a total number of 1021 Chikungunya suspects presenting within seven days of illness were screened with Chikungunya Virus IgM ELISA from 2021 to 2023. Those having positive IgM results were further tested with RT-PCR in a blinded manner. According to the information entered into the predesigned form and the hospital follow-up/discharge data, the cases where symptoms like fever and joint pain persisted beyond two weeks were classified as severe versus those resolving within two weeks as mild. The patients in each group were compared for their clinical symptoms and association with the disease severity with detectable viremia (RT-PCR positivity). Results We identified a total of 178 (17.4%) lab-confirmed Chikungunya IgM-positive cases amongst the recruited patients. Here a total of 31 (18.9%) cases could be classified as severe and 133 (74.7%) as mild illness, the remaining 14 patients were excluded from analysis due to insufficientclinical data. Severe illness was significantly higher in elderly individuals belonging to more than 60 years (p = 0.01). Viremia was detected in 16 (9%), those with detectable viremia had higher odds (OR = 4.1) of manifesting as severe disease. Among the severe cases, the proportion of cases with RT-PCR positivity (8, 25.8%) at presentation was significantly higher (P = 0.01) versus those who presented with mild disease (7, 5.5%). Conclusion Our study reveals a correlation betweendetectable viremia in Chikungunya virus (CHIKV) patients and an increased risk of manifesting into a severe disease, where severe cases exhibited a significantly higher proportion of viremia, indicated by RT-PCR positivity. This study hints at the presence of viremia, joint symptoms, and elderly age as potentially useful clinical predictors of disease outcomes, these may serve as indicators for closer monitoring among individuals seeking medical attention due to Chikungunya infection.However, we need to validate these findings in future longitudinal studies incorporating multiple, time-bound follow-up data on clinical outcomes, viral titers, and itslong-term complications.

Influence of diimine bidentate ligand in the nitrosyl and nitro terpyridine ruthenium complex on the HSA/DNA interaction and antiviral activity

Nitric oxide (NO) acts in different physiological processes, such as blood pressure control, antiparasitic activities, neurotransmission, and antitumor action. Among the exogenous NO donors, ruthenium nitrosyl/nitro complexes are potential candidates for prodrugs, due to their physicochemical properties, such as thermal and physiological pH stability. In this work, we proposed the synthesis and physical characterization of the new nitro terpyridine ruthenium (II) complexes of the type [RuII(L)(NO2)(tpy)]PF6 where tpy = 2,2':6′,2″-terpyridine; L = 3,4-diaminobenzoic acid (bdq) or o-phenylenediamine (bd) and evaluation of influence of diimine bidentate ligand NH.NHq-R (R = H or COOH) in the HSA/DNA interaction as well as antiviral activity. The interactions between HSA and new nitro complexes [RuII(L)(NO2)(tpy)]+ were evaluated. The Ka values for the HSA–[RuII(bdq)(NO2)(tpy)]+ is 10 times bigger than HSA–[RuII(bd)(NO2)(tpy)]+. The sites of interaction between HSA and the complexes via synchronous fluorescence suppression indicate that the [RuII(bdq)(NO2)(tpy)]+ is found close to the Trp-241 residue, while the [RuII(bd)(NO2)(tpy)]+ complex is close to Tyr residues. The interaction with fish sperm fs-DNA using direct spectrophotometric titration (Kb) and ethidium bromide replacement (KSV and Kapp) showed weak interaction in the system fs-DNA-[RuII(bdq)(NO)(tpy)]+. Furthermore, fs-DNA–[RuII(bd)(NO2)(tpy)]+ and fs-DNA–[RuII(bd)(NO)(tpy)]3+ system showed higher intercalation constant. Circular dichroism spectra for fs-DNA–[RuII(bd)(NO2)(tpy)]+ and fs-DNA–[RuII(bd)(NO)(tpy)]3+, suggest semi-intercalative accompanied by major groove binding interaction modes. The [RuII(bd)(NO2)(tpy)]+ and [RuII(bd)(NO)(tpy)]3+ inhibit replication of Zika and Chikungunya viruses based in the nitric oxide release under S-nitrosylation reaction with cysteine viral.

Metagenomic Analysis for Diagnosis of Hemorrhagic Fever in Minas Gerais, Brazil.

Viral hemorrhagic fever poses a significant public health challenge due to its severe clinical presentation and high mortality rate. The diagnostic process is hindered by similarity of symptoms across different diseases and the broad spectrum of pathogens that can cause hemorrhagic fever. In this study, we applied viral metagenomic analysis to 43 serum samples collected by the Public Health Laboratory (Fundação Ezequiel Dias, FUNED) in Minas Gerais State, Brazil, from patients diagnosed with hemorrhagic fever who had tested negative for the standard local hemorrhagic disease testing panel. This panel includes tests for Dengue virus (DENV) IgM, Zika virus IgM, Chikungunya virus IgM, yellow fever IgM, Hantavirus IgM, Rickettsia rickettsii IgM/IgG, and Leptospira interrogans IgM, in addition to respective molecular tests for these infectious agents. The samples were grouped into 18 pools according to geographic origin and analyzed through next-generation sequencing on the NextSeq 2000 platform. Bioinformatic analysis revealed a prevalent occurrence of commensal viruses across all pools, but, notably, a significant number of reads corresponding to the DENV serotype 2 were identified in one specific pool. Further verification via real-time PCR confirmed the presence of DENV-2 RNA in an index case involving an oncology patient with hemorrhagic fever who had initially tested negative for anti-DENV IgM antibodies, thereby excluding this sample from initial molecular testing. The complete DENV-2 genome isolated from this patient was taxonomically classified within the cosmopolitan genotype that was recently introduced into Brazil. These findings highlight the critical role of considering the patient's clinical condition when deciding upon the most appropriate testing procedures. Additionally, this study showcases the potential of viral metagenomics in pinpointing the viral agents behind hemorrhagic diseases. Future research is needed to assess the practicality of incorporating metagenomics into standard viral diagnostic protocols.