Chikungunya virus release is reduced by TIM-1 receptors through binding of envelope phosphatidylserine.

T-cell immunoglobin and mucin domain protein-1 (TIM-1) mediates entry of chikungunya virus (CHIKV) into some mammalian cells through the interaction with envelope phospholipids. While this interaction enhances entry, TIM-1 has been shown to tether newly formed HIV and Ebola virus particles, limiting their efficient release. In this study, we investigate the ability of surface receptors such as TIM-1 to sequester newly budded virions on the surface of infected cells. We established a luminescence reporter system to produce chikungunya viral particles that integrate nano-luciferase and easily quantify viral particles. We found that TIM-1 on the surface of host cells significantly reduced CHIKV release efficiency in comparison to other entry factors. Removal of cell surface TIM-1 through direct cellular knock-out or altering the cellular lipid distribution enhanced CHIKV release. Over the course of infection, CHIKV was able to counteract the tethering effect by gradually decreasing the surface levels of TIM-1 in a process mediated by the nonstructural protein 2. This study highlights the importance of phosphatidylserine receptors in mediating not only the entry of CHIKV but also its release and could aid in developing cell lines capable of enhanced vaccine production. IMPORTANCE: Chikungunya virus (CHIKV) is an enveloped alphavirus transmitted by the bites of infectious mosquitoes. Infection with CHIKV results in the development of fever, joint pain, and arthralgia that can become chronic and last for months after infection. Prevention of this disease is still highly focused on vector control strategies. In December 2023, a new live attenuated vaccine against CHIKV was approved by the FDA. We aimed to study the cellular factors involved in CHIKV release, to better understand CHIKV's ability to efficiently infect and spread among a wide variety of cell lines. We found that TIM-1 receptors can significantly abrogate CHIKV's ability to efficiently exit infected cells. This information can be beneficial for maximizing viral particle production in laboratory settings and during vaccine manufacturing.

In situ fate of Chikungunya virus replication organelles.

Chikungunya virus (CHIKV) is a mosquito-borne pathogen responsible for an acute musculoskeletal disease in humans. Replication of the viral RNA genome occurs in specialized membranous replication organelles (ROs) or spherules, which contain the viral replication complex. Initially generated by RNA synthesis-associated plasma membrane deformation, alphavirus ROs are generally rapidly endocytosed to produce type I cytopathic vacuoles (CPV-I), from which nascent RNAs are extruded for cytoplasmic translation. By contrast, CHIKV ROs are poorly internalized, raising the question of their fate and functionality at the late stage of infection. Here, using in situ cryogenic-electron microscopy approaches, we investigate the outcome of CHIKV ROs and associated replication machinery in infected human cells. We evidence the late persistence of CHIKV ROs at the plasma membrane with a crowned protein complex at the spherule neck similar to the recently resolved replication complex. The unexpectedly heterogeneous and large diameter of these compartments suggests a continuous, dynamic growth of these organelles beyond the replication of a single RNA genome. Ultrastructural analysis of surrounding cytoplasmic regions supports that outgrown CHIKV ROs remain dynamically active in viral RNA synthesis and export to the cell cytosol for protein translation. Interestingly, rare ROs with a homogeneous diameter are also marginally internalized in CPV-I near honeycomb-like arrangements of unknown function, which are absent in uninfected controls, thereby suggesting a temporal regulation of this internalization. Altogether, this study sheds new light on the dynamic pattern of CHIKV ROs and associated viral replication at the interface with cell membranes in infected cells.IMPORTANCEThe Chikungunya virus (CHIKV) is a positive-stranded RNA virus that requires specialized membranous replication organelles (ROs) for its genome replication. Our knowledge of this viral cycle stage is still incomplete, notably regarding the fate and functional dynamics of CHIKV ROs in infected cells. Here, we show that CHIKV ROs are maintained at the plasma membrane beyond the first viral cycle, continuing to grow and be dynamically active both in viral RNA replication and in its export to the cell cytosol, where translation occurs in proximity to ROs. This contrasts with the homogeneous diameter of ROs during internalization in cytoplasmic vacuoles, which are often associated with honeycomb-like arrangements of unknown function, suggesting a regulated mechanism. This study sheds new light on the dynamics and fate of CHIKV ROs in human cells and, consequently, on our understanding of the Chikungunya viral cycle.

Clinical manifestations of dengue, Zika and chikungunya in the Pacific Islands: A systematic review and meta-analysis.

Dengue, Zika and chikungunya outbreaks pose a significant public health risk to Pacific Island communities. Differential diagnosis is challenging due to overlapping clinical features and limited availability of laboratory diagnostic facilities. There is also insufficient information regarding the complications of these arboviruses, particularly for Zika and chikungunya. We conducted a systematic review and meta-analysis to calculate pooled prevalence estimates with 95% confidence intervals (CI) for the clinical manifestations of dengue, Zika and chikungunya in the Pacific Islands. Based on pooled prevalence estimates, clinical features that may help to differentiate between the arboviruses include headache, haemorrhage and hepatomegaly in dengue; rash, conjunctivitis and peripheral oedema in Zika; and the combination of fever and arthralgia in chikungunya infections. We estimated that the hospitalisation and mortality rates in dengue were 9.90% (95% CI 7.67-12.37) and 0.23% (95% CI 0.16-0.31), respectively. Severe forms of dengue occurred in 1.92% (95% CI 0.72-3.63) of reported cases and 23.23% (95% CI 13.58-34.53) of hospitalised patients. Complications associated with Zika virus included Guillain-Barré syndrome (GBS), estimated to occur in 14.08 (95% CI 11.71-16.66) per 10,000 reported cases, and congenital brain malformations such as microcephaly, particularly with first trimester maternal infection. For chikungunya, the hospitalisation rate was 2.57% (95% CI 1.30-4.25) and the risk of GBS was estimated at 1.70 (95% CI 1.06-2.48) per 10,000 reported cases. Whilst ongoing research is required, this systematic review enhances existing knowledge on the clinical manifestations of dengue, Zika and chikungunya infections and will assist Pacific Island clinicians during future arbovirus outbreaks.

Results of a nationally representative seroprevalence survey of chikungunya virus in Bangladesh.

There is an increasing global burden from chikungunya virus (CHIKV). Bangladesh reported a major epidemic in 2017, however, it was unclear if there had been prior widespread transmission. We conducted a nationally representative seroprevalence survey in 70 randomly selected communities immediately prior to the epidemic. We found 69/2,938 (2.4%) of sampled individuals were seropositive to CHIKV. Being seropositive to dengue virus (aOR 3.13 [95% CIs: 1.86-5.27]), male sex (aOR 0.59 [95% CIs: 0.36-0.99]), and community presence of Aedes aegypti mosquitoes (aOR: 1.80, 95% CI: 1.05-3.07) were significantly associated with CHIKV seropositivity. Using a spatial prediction model, we estimated that across the country, 4.99 (95% CI: 4.89 - 5.08) million people had been previously infected. These findings highlight high population susceptibility prior to the major outbreak and that previous outbreaks must have been spatially isolated.

Serosurvey of Chikungunya Virus in Old World Fruit Bats, Senegal, 2020-2022.

We conducted a cross-sectional serosurvey for chikungunya virus (CHIKV) exposure in fruit bats in Senegal during 2020-2023. We found that 13.3% (89/671) of bats had CHIKV IgG; highest prevalence was in Eidolon helvum (18.3%, 15/82) and Epomophorus gambianus (13.7%, 63/461) bats. Our results suggest these bats are naturally exposed to CHIKV.

Development of Multiplex Molecular Assays for Simultaneous Detection of Dengue Serotypes and Chikungunya Virus for Arbovirus Surveillance.

The major arboviruses mainly belong to the Bunyaviridae, Togaviridae, and Flaviviridae families, among which the chikungunya virus and dengue virus have emerged as global public health problems. The main objective of this study was to develop specific, sensitive, and cost-effective molecular multiplex RT-PCR and RT-qPCR assays for the rapid and simultaneous detection of CHIKV and the four serotypes of DENV for arbovirus surveillance. Specific primers for all viruses were designed, and one-step multiplex RT-PCR (mRT-PCR) and RT-qPCR (mRT-qPCR) were developed using reference strains of the CHIKV and DENV serotypes. The specificity of the test for all the viruses was confirmed through sequencing. The standard curves showed a high correlation coefficient, R2 = 0.99, for DENV-2 and DENV-3; R2 = 0.98, for DENV-4; and CHIKV; R2 = 0.93, for DENV-1. The limits of detection were calculated to be 4.1 × 10-1 copies/reaction for DENV-1, DENV-3, and CHIKV and 4.1 × 101 for DENV-2 and DENV-4. The specificity and sensitivity of the newly developed mRT-PCR and mRT-qPCR were validated using positive serum samples collected from India and Burkina Faso. The sensitivity of mRT-PCR and mRT-qPCR are 91%, and 100%, respectively. The specificity of both assays was 100%. mRT-PCR and mRT-qPCR assays are low-cost, and a combination of both will be a useful tool for arbovirus surveillance.

Immunogenicity, safety and duration of protection afforded by chikungunya virus vaccines undergoing human clinical trials.

Background. Chikungunya virus (CHIKV) causes chikungunya fever and has been responsible for major global epidemics of arthritic disease over the past two decades. Multiple CHIKV vaccine candidates are currently undergoing or have undergone human clinical trials, with one vaccine candidate receiving FDA approval. This scoping review was performed to evaluate the 'efficacy', 'safety' and 'duration of protection' provided by CHIKV vaccine candidates in human clinical trials.Methods. This scoping literature review addresses studies involving CHIKV vaccine clinical trials using available literature on the PubMed, Medline Embase, Cochrane Library and Clinicaltrial.gov databases published up to 25 August 2023. Covidence software was used to structure information and review the studies included in this article.Results. A total of 1138 studies were screened and, after removal of duplicate studies, 12 relevant studies were thoroughly reviewed to gather information. This review summarizs that all seven CHIKV vaccine candidates achieved over 90 % seroprotection against CHIKV after one or two doses. All vaccines were able to provide neutralizing antibody protection for at least 28 days.Conclusions. A variety of vaccine technologies have been used to develop CHIKV vaccine candidates. With one vaccine candidate having recently received FDA approval, it is likely that further CHIKV vaccines will be available commercially in the near future.

Oxidative stress governs mosquito innate immune signalling to reduce chikungunya virus infection in Aedes-derived cells.

Arboviruses such as chikungunya, dengue and zika viruses cause debilitating diseases in humans. The principal vector species that transmits these viruses is the Aedes mosquito. Lack of substantial knowledge of the vector species hinders the advancement of strategies for controlling the spread of arboviruses. To supplement our information on mosquitoes' responses to virus infection, we utilized Aedes aegypti-derived Aag2 cells to study changes at the transcriptional level during infection with chikungunya virus (CHIKV). We observed that genes belonging to the redox pathway were significantly differentially regulated. Upon quantifying reactive oxygen species (ROS) in the cells during viral infection, we further discovered that ROS levels are considerably higher during the early hours of infection; however, as the infection progresses, an increase in antioxidant gene expression suppresses the oxidative stress in cells. Our study also suggests that ROS is a critical regulator of viral replication in cells and inhibits intracellular and extracellular viral replication by promoting the Rel2-mediated Imd immune signalling pathway. In conclusion, our study provides evidence for a regulatory role of oxidative stress in infected Aedes-derived cells.

Inhibition of early RNA replication in Chikungunya and Dengue virus by lycorine: In vitro and in silico studies.

Arboviruses such as chikungunya virus (CHIKV) and dengue virus (DENV) collectively afflict millions of individuals worldwide particularly in endemic countries like India, leading to substantial morbidity and mortality. With the lack of effective vaccines for both CHIKV and DENV in India, the search for antiviral compounds becomes paramount to control these viral infections. In line with this, our investigation was focused on screening natural compounds for their potential antiviral activity against CHIKV and DENV. Using different assays, including plaque assay, immunofluorescence, and reverse transcription-quantitative real-time PCR (qRT-PCR), out of 109 natural compounds tested, we confirmed lycorine's in vitro antiviral activity against CHIKV and DENV at low micromolar concentrations in different cell types. Time of addition assays indicated that lycorine does not impede viral entry. Additionally, qRT-PCR results along with time of addition assay suggested that lycorine interferes with the synthesis of negative strand viral RNA. Molecular docking analysis was done to understand the mode of inhibition of viral replication. The results revealed that the most likely binding site with the highest binding affinity of lycorine, was at the palm and finger domains, in the vicinity of the catalytic site of CHIKV and DENV RNA-dependent RNA polymerase (RdRp). Collectively, our data underscores the potential of lycorine to be developed as a direct acting inhibitor for DENV and CHIKV, addressing the critical need of requirement of an antiviral in regions where these viruses pose significant public health threats.

Rapid and sensitive detection of chikungunya virus using one-tube, reverse transcription, semi-nested multi-enzyme isothermal rapid amplification, and lateral flow dipstick assays.

Chikungunya fever is an acute infectious disease caused by chikungunya virus (CHIKV), which is transmitted by Aedes mosquitoes. Simple, rapid, and sensitive detection of CHIKV is critical for its prevention and spread. To address this issue, we combined one-tube, reverse transcription semi-nested, multi-enzyme isothermal rapid amplification, and lateral flow dipstick strips assay to detect CHIKV RNA. The study used a 318-bp gene fragment of CHIKV NSP4 as the target of the assay. This method of amplification takes 30 min for two-step amplification at 39°C. The dilution of amplification products was added to the LFD strip with results visible to the naked eye after 10 min. The method has a sensitivity of 1 copy/µL for the detection of CHIKV RNA, which is 100-fold higher than the conventional reverse transcription-multi-enzyme isothermal rapid amplification and 10-fold higher than the reverse transcription quantitative PCR (RT-qPCR) method. In addition, the method demonstrated good specificity and a better detection rate (85.7%, 18 of 21) than RT-qPCR (80.9%, 17 of 21) in clinically confirmed patient plasma samples. Thus, the rapid CHIKV RNA assay developed in this study will be an important tool for the rapid and accurate screening of patients for chikungunya fever. IMPORTANCE: This study presents a new one-tube, reverse transcription semi-nested, multi-enzyme isothermal rapid amplification assay combined with lateral flow dipstick strips for the detection of CHIKV. This technique significantly improves sensitivity and outperforms RT-qPCR for the detection of CHIKV, especially in samples with low viral loads. It is also significantly faster than conventional RT-qPCR and does not require special equipment or a standard PCR laboratory. The combination of the isothermal amplification technology developed in this study with point-of-care molecular testing offers the potential for rapid, on-site, low-cost molecular diagnosis of CHIKV.

An improved alphaviruses-specific RT-qPCR facilitates monitoring and prevention of alphaviruses.

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

Viral methyltransferase inhibitors: berbamine, venetoclax, and ponatinib as efficacious antivirals against chikungunya virus.

Chikungunya virus (CHIKV), transmitted by mosquitoes, poses a significant global health threat. Presently, no effective treatment options are available to reduce the disease burden. The lack of approved therapeutics against CHIKV and the complex spectrum of chronic musculoskeletal and neurological manifestations raise significant concerns, and repurposing drugs could offer swift avenues in the development of effective treatment strategies. RNA capping is a crucial step meditated by non-structural protein 1 (nsP1) in CHIKV replication. In this study, FDA-approved antivirals targeting CHIKV nsP1 methyltransferase (MTase) have been identified by structure-based virtual screening. Berbamine Hydrochloride (BH), ABT199/Venetoclax (ABT), and Ponatinib (PT) were the top-hits, which exhibited robust binding energies. Tryptophan fluorescence spectroscopy-based assay confirmed binding of BH-, ABT-, and PT to purified nsP1 with KD values ∼5.45 µM, ∼161.3 µM, and ∼3.83 µM, respectively. In a capillary electrophoresis-based assay, a decrease in CHIKV nsP1 MTase activity was observed in a dose-dependent manner. Treatment with BH, ABT, and PT lead to a dose-dependent reduction in the virus titer with IC50 < 100, ∼6.75, and <3.9 nM, respectively, and reduced viral mRNA levels. The nsP1 MTases are highly conserved among alphaviruses; therefore, BH, ABT, and PT, as expected, inhibited replication machinery in Sindbis virus (SINV) replicon assay with IC50 ∼1.94, ∼0.23, and >1.25 µM, respectively. These results highlight the potential of repurposing drugs as rapid and effective antiviral therapeutics against CHIKV.

Attenuation of neurovirulence of chikungunya virus by a single amino acid mutation in viral E2 envelope protein.

BACKGROUND: Chikungunya virus (CHIKV) has reemerged as a major public health concern, causing chikungunya fever with increasing cases and neurological complications. METHODS: In the present study, we investigated a low-passage human isolate of the East/ Central/South African (ECSA) lineage of CHIKV strain LK(EH)CH6708, which exhibited a mix of small and large viral plaques. The small and large plaque variants were isolated and designated as CHIKV-SP and CHIKV-BP, respectively. CHIKV-SP and CHIKV-BP were characterized in vitro and in vivo to compare their virus production and virulence. Additionally, whole viral genome analysis and reverse genetics were employed to identify genomic virulence factors. RESULTS: CHIKV-SP demonstrated lower virus production in mammalian cells and attenuated virulence in a murine model. On the other hand, CHIKV-BP induced higher pro-inflammatory cytokine levels, compromised the integrity of the blood-brain barrier, and led to astrocyte infection in mouse brains. Furthermore, the CHIKV-SP variant had limited transmission potential in Aedes albopictus mosquitoes, likely due to restricted dissemination. Whole viral genome analysis revealed multiple genetic mutations in the CHIKV-SP variant, including a Glycine (G) to Arginine (R) mutation at position 55 in the viral E2 glycoprotein. Reverse genetics experiments confirmed that the E2-G55R mutation alone was sufficient to reduce virus production in vitro and virulence in mice. CONCLUSIONS: These findings highlight the attenuating effects of the E2-G55R mutation on CHIKV pathogenicity and neurovirulence and emphasize the importance of monitoring this mutation in natural infections.

Evaluation of assays for nucleic acid testing for the prevention of chikungunya and dengue virus transmission by blood transfusion.

BACKGROUND: The large dengue (DENV) and chikungunya (CHIKV) outbreaks observed during the last decade across the world, as well as local transmissions in non-endemic areas are a growing concern for blood safety. The aim of this study was to evaluate and compare the sensitivity of nucleic acid tests (NAT) detecting DENV and CHIKV RNA. MATERIALS AND METHODS: Using DENV 1 to 4 International Standards, the limits of detection (LODs) calculated by probit analysis of two NAT assays; the cobas CHIKV/DENV assay (Roche Diagnostics) and the Procleix Dengue Virus Assay (Grifols) were compared. In addition, CHIKV-RNA LOD of the cobas CHIKV/DENV assay was evaluated. RESULTS: For dengue, the 95% LOD of the cobas assay ranged between 4.10 [CI95%: 2.70-8.19] IU/mL (DENV-2) and 7.07 [CI95%: 4.34-14.89] IU/mL (DENV-4), and between 2.19 [CI95%: 1.53-3.83] IU/mL (DENV-3) and 5.84 [CI95%: 3.84-10.77] IU/mL (DENV-1) for Procleix assay. The Procleix assay had a significant lower LOD for DENV-3 (2.19 vs. 5.89 IU/mL) when compared to the cobas assay (p = 0.005). The 95% LOD for CHIKV-RNA detection of the cobas assay was 4.76 [CI95%: 3.08-8.94] IU/mL. DISCUSSION: The two NAT assays developed for blood donor screening evaluated in this study demonstrated high and similar analytical performance. Subject to an appropriate risk-benefit assessment, they can be used to support blood safety during outbreaks in endemic areas or in non-endemic areas as an alternative to deferring blood donors during local transmission likely to affect the blood supply. The development of multiplex assays is expected to optimize laboratory organization.

Detecting the impacts of humidity, rainfall, temperature, and season on chikungunya, dengue and Zika viruses in Aedes albopictus mosquitoes from selected sites in Cebu city, Philippines.

BACKGROUND: Aedes albopictus is the secondary vector for dengue virus (DENV) in the Philippines, and also harbors chikungunya (CHIKV) and Zika (ZIKV) viruses. This study aimed to determine the minimum infection rates (MIRs) of CHIKV, DENV serotypes, and ZIKV in Ae. albopictus collected from selected two-site categories by altitude (highland [H] and lowland [L] sites) in Cebu city, Philippines during the wet (WS) and dry seasons (DS) of 2021-2022, and to explore the relationships between these arboviral MIRs and the local weather. METHODS: The viral RNA extracts in pooled and reared adult Ae. albopictus collected during the DS and WS from two-site categories were subjected to RT-PCR to amplify and detect gene loci specific for CHIKV, DENV-1 to DENV-4, and ZIKV and analyzed with the weather data. RESULTS: The range of CHIKV MIRs was higher in the WS (13.61-107.38 infected individuals per 1,000 mosquitoes) than in the DS (13.22-44.12), but was similar between the two-site categories. Rainfall (RF) influenced the CHIKV MIR. The MIR ranges of both DENV-2 (WS: H = 0, L = 0; DS: H = 0-5.92; L = 0-2.6) and DENV-4 (WS: H = 0, L = 0-2.90; DS: H = 2.96-6.13, L = 0-15.63) differed by season but not between the two-site categories. Relative humidity (RH), RF, and temperature did not influence DENVs' MIRs. The MIR range of ZIKV was similar in both seasons (WS: 11.36-40.27; DS: 0-46.15) and two-site categories (H = 0-90.91, L = 0-55.56). RH and temperature influenced ZIKV MIR. CONCLUSIONS: RF influenced CHIKV MIR in Ae. albopictus, whereas RH and temperature influenced that of ZIKV. Season influenced the MIRs of CHIKV and DENVs but not in ZIKV. Ae. albopictus were co-infected with CHIKV, DENVs, and ZIKV in both highland and lowland sites in Cebu city. Recommendations include all-year-round implementation of the Philippine Department of Health's  4S enhanced strategy and installation of water pipelines in rural highlands for vector and disease control. Our findings are relevant to protect public health in the tropics in this climate change.

Repurposed drugs in combinations exert additive anti-chikungunya virus activity: an in-vitro study.

Chikungunya virus (CHIKV) infection causes chikungunya, a viral disease that currently has no specific antiviral treatment. Several repurposed drug candidates have been investigated for the treatment of the disease. In order to improve the efficacy of the known drugs, combining drugs for treatment is a promising approach. The current study was undertaken to explore the antiviral activity of a combination of repurposed drugs that were reported to have anti-CHIKV activity. We explored the effect of different combinations of six effective drugs (2-fluoroadenine, emetine, lomibuvir, enalaprilat, metyrapone and resveratrol) at their non-toxic concentrations against CHIKV under post infection treatment conditions in Vero cells. Focus-forming unit assay, real time RT-PCR, immunofluorescence assay, and western blot were used to determine the virus titre. The results revealed that the combination of 2-fluoroadenine with either metyrapone or emetine or enalaprilat exerted inhibitory activity against CHIKV under post-infection treatment conditions. The effect of these drug combinations was additive in nature compared to the effect of the individual drugs. The results suggest an additive anti-viral effect of these drug combinations against CHIKV. The findings could serve as an outline for the development of an innovative therapeutic approach in the future to treat CHIKV-infected patients.

Urban arbovirus exposure in blood donations from an endemic area of Brazil.

BACKGROUND AND OBJECTIVES: In Brazil, urban arboviruses, such as dengue virus (DENV), Zika virus (ZIKV) and chikungunya virus (CHIKV), constitute a major public health problem, and due to their endemicity and asymptomatic cases, they pose a potential threat to blood donations. Rio de Janeiro (RJ), Brazil, has been impacted by extensive DENV epidemics over the last 30 years and, after 2015, by CHIKV and ZIKV. MATERIALS AND METHODS: Urban arboviruses DENV, ZIKV and CHIKV were investigated in blood donations (n = 778) at the State Institute of Hematology, HEMORIO (RJ) from 2019 to 2022 by serological and molecular methods. RESULTS: An overall arbovirus exposure was observed in 26.1% of the blood donations. Anti-DENV IgM was detected in 4.0% of samples and two donations were DENV NS1 positive. Positive anti-CHIKV IgM was observed in 4.7% of the donations. Co-detection of anti-CHIKV IgM and anti-DENV IgM was observed in 1.0% of donors, and CHIKV prevalence was 21.3%. All blood donations tested were negative for the DENV, ZIKV and CHIKV RNA. CONCLUSION: IgM seroprevalence to the arboviruses analyzed here is an indicator of recent infection in asymptomatic donors, showing that the population of blood donors can be a vehicle for new infections, especially during epidemic periods.

Molecular and serological evidence of chikungunya virus infection with high case fatality among pediatric population with acute encephalitis syndrome: first report from Eastern Uttar Pradesh, India.

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

Utilization of novel molecular multiplex methods for the detection and, epidemiological surveillance of dengue virus serotypes and chikungunya virus in Burkina Faso, West Africa.

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

The impact of arthritogenic viruses in oral tissues.

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