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Fluorescent and electrochemical dual-mode detection of Chikungunya virus E1 protein using fluorophore-embedded and redox probe-encapsulated liposomes.

ABSTRACT: The critical goal of sensitive virus detection should apply in the early stage of infection, which may increase the probable survival rate. To achieve the low detection limit for the early stage where a small number of viruses are present in the sample, proper amplified signals from a sensor can make readable and reliable detection. In this work, a new model of fluorescent and electrochemical dual-mode detection system has been developed to detect virus, taking recombinant Chikungunya virus E1 protein (CHIK-VP) as an example. The hydrophobic quantum dots (QDs) embedded in the lipid bilayer of liposome and methylene blue (MB) encapsulated in the inner core of liposomes played a role of dual-signaling modulator. After CHIK-VP addition, the nanocomposites and APTES-coated Fe3O4 nanoparticles (Fe3O4 NPs) were conjugated with antibodies to form a sandwich structure and separated from the medium magnetically. The nanoconjugates have been burst out by chloroform as surfactant, and both the QDs and MB are released from the liposome and were then monitored through changes in the fluorescence and electrochemical signals, respectively. These two fluorometric and electrochemical signals alteration quantified the CHIK-VP in the range of femtogram to nanogram per milliliter level with a LOD of 32 fg mL-1, making this liposomal system a potential matrix in a virus detection platform. Graphical abstract.

SUBMITTER: Nasrin F

PROVIDER: S-EPMC7688297 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Fluorescent and electrochemical dual-mode detection of Chikungunya virus E1 protein using fluorophore-embedded and redox probe-encapsulated liposomes.

Nasrin Fahmida F Chowdhury Ankan Dutta AD Ganganboina Akhilesh Babu AB Achadu Ojodomo J OJ Hossain Farzana F Yamazaki Masahito M Park Enoch Y EY

Mikrochimica acta 20201125 12

The critical goal of sensitive virus detection should apply in the early stage of infection, which may increase the probable survival rate. To achieve the low detection limit for the early stage where a small number of viruses are present in the sample, proper amplified signals from a sensor can make readable and reliable detection. In this work, a new model of fluorescent and electrochemical dual-mode detection system has been developed to detect virus, taking recombinant Chikungunya virus E1 p ...[more]

PMID: 33241435

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Project description:Chikungunya virus (CHIKV) is a reemerging arthropod-borne alphavirus and a serious threat to human health. Therefore, efforts toward elucidating how this virus causes disease and the molecular mechanisms underlying steps of the viral replication cycle are crucial. Using an in vivo transmission system that allows intrahost evolution, we identified an emerging CHIKV variant carrying a mutation in the E1 glycoprotein (V156A) in the serum of mice and saliva of mosquitoes. E1 V156A has since emerged in humans during an outbreak in Brazil, cooccurring with a second mutation, E1 K211T, suggesting an important role for these residues in CHIKV biology. Given the emergence of these variants, we hypothesized that they function to promote CHIKV infectivity and subsequent disease. Here, we show that E1 V156A and E1 K211T modulate virus attachment and fusion and impact binding to heparin, a homolog of heparan sulfate, a key entry factor on host cells. These variants also exhibit differential neutralization by antiglycoprotein monoclonal antibodies, suggesting structural impacts on the particle that may be responsible for altered interactions at the host membrane. Finally, E1 V156A and E1 K211T exhibit increased titers in an adult arthritic mouse model and induce increased foot-swelling at the site of injection. Taken together, this work has revealed new roles for E1 where discrete regions of the glycoprotein are able to modulate cell attachment and swelling within the host. IMPORTANCE Alphaviruses represent a growing threat to human health worldwide. The reemerging alphavirus chikungunya virus (CHIKV) has rapidly spread to new geographic regions in the last several decades, causing overwhelming outbreaks of disease, yet there are no approved vaccines or therapeutics. The CHIKV glycoproteins are key determinants of CHIKV adaptation and virulence. In this study, we identify and characterize the emerging E1 glycoprotein variants, V156A and K211T, that have since emerged in nature. We demonstrate that E1 V156A and K211T function in virus attachment to cells, a role that until now has only been attributed to specific residues of the CHIKV E2 glycoprotein. We also demonstrate E1 V156A and K211T increase foot-swelling of the ipsilateral foot in mice infected with these variants. Observing that these variants and other pathogenic variants occur at the E1-E1 interspike interface, we highlight this structurally important region as critical for multiple steps during CHIKV infection. Together, these studies further define the function of E1 in CHIKV infection and can inform the development of therapeutic or preventative strategies.

Project description:BackgroundChikungunya fever is a pandemic disease caused by the mosquito-borne Chikungunya virus (CHIKV). E1 glycoprotein mediation of viral membrane fusion during CHIKV infection is a crucial step in the release of viral genome into the host cytoplasm for replication. How the E1 structure determines membrane fusion and whether other CHIKV structural proteins participate in E1 fusion activity remain largely unexplored.MethodsA bicistronic baculovirus expression system to produce recombinant baculoviruses for cell-based assay was used. Sf21 insect cells infected by recombinant baculoviruses bearing wild type or single-amino-acid substitution of CHIKV E1 and EGFP (enhanced green fluorescence protein) were employed to investigate the roles of four E1 amino acid residues (G91, V178, A226, and H230) in membrane fusion activity.ResultsWestern blot analysis revealed that the E1 expression level and surface features in wild type and mutant substituted cells were similar. However, cell fusion assay found that those cells infected by CHIKV E1-H230A mutant baculovirus showed little fusion activity, and those bearing CHIKV E1-G91D mutant completely lost the ability to induce cell-cell fusion. Cells infected by recombinant baculoviruses of CHIKV E1-A226V and E1-V178A mutants exhibited the same membrane fusion capability as wild type. Although the E1 expression level of cells bearing monomeric-E1-based constructs (expressing E1 only) was greater than that of cells bearing 26S-based constructs (expressing all structural proteins), the sizes of syncytial cells induced by infection of baculoviruses containing 26S-based constructs were larger than those from infections having monomeric-E1 constructs, suggesting that other viral structure proteins participate or regulate E1 fusion activity. Furthermore, membrane fusion in cells infected by baculovirus bearing the A226V mutation constructs exhibited increased cholesterol-dependences and lower pH thresholds. Cells bearing the V178A mutation exhibited a slight decrease in cholesterol-dependence and a higher-pH threshold for fusion.ConclusionsCells expressing amino acid substitutions of conserved protein E1 residues of E1-G91 and E1-H230 lost most of the CHIKV E1-mediated membrane fusion activity. Cells expressing mutations of less-conserved amino acids, E1-V178A and E1-A226V, retained membrane fusion activity to levels similar to those expressing wild type E1, but their fusion properties of pH threshold and cholesterol dependence were slightly altered.

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Fluorescent and electrochemical dual-mode detection of Chikungunya virus E1 protein using fluorophore-embedded and redox probe-encapsulated liposomes.

Publications

Fluorescent and electrochemical dual-mode detection of Chikungunya virus E1 protein using fluorophore-embedded and redox probe-encapsulated liposomes.

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