scholarly journals Imd pathway-specific immune assays reveal NF-κB stimulation by viral RNA PAMPs in Aedes aegypti Aag2 cells

2021 ◽  
Vol 15 (2) ◽  
pp. e0008524
Author(s):  
Tiffany A. Russell ◽  
Andalus Ayaz ◽  
Andrew D. Davidson ◽  
Ana Fernandez-Sesma ◽  
Kevin Maringer
Keyword(s):  
Aedes Aegypti ◽  
Viral Rna ◽  
Poly I:C ◽  
Luciferase Reporter ◽  
Toll Pathway ◽  
Experimental Conditions ◽  
Major Barrier ◽  
Imd Pathway ◽  
Arboviral Diseases ◽  
Transgenic Insects

Background The mosquito Aedes aegypti is a major vector for the arthropod-borne viruses (arboviruses) chikungunya, dengue, yellow fever and Zika viruses. Vector immune responses pose a major barrier to arboviral transmission, and transgenic insects with altered immunity have been proposed as tools for reducing the global public health impact of arboviral diseases. However, a better understanding of virus-immune interactions is needed to progress the development of such transgenic insects. Although the NF-κB-regulated Toll and ‘immunodeficiency’ (Imd) pathways are increasingly thought to be antiviral, relevant pattern recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs) remain poorly characterised in A. aegypti. Methodology/Principle findings We developed novel RT-qPCR and luciferase reporter assays to measure induction of the Toll and Imd pathways in the commonly used A. aegypti-derived Aag2 cell line. We thus determined that the Toll pathway is not inducible by exogenous stimulation with bacterial, viral or fungal stimuli in Aag2 cells under our experimental conditions. We used our Imd pathway-specific assays to demonstrate that the viral dsRNA mimic poly(I:C) is sensed by the Imd pathway, likely through intracellular and extracellular PRRs. The Imd pathway was also induced during infection with the model insect-specific virus cricket paralysis virus (CrPV). Conclusions/Significance Our demonstration that a general PAMP shared by many arboviruses is sensed by the Imd pathway paves the way for future studies to determine how viral RNA is sensed by mosquito PRRs at a molecular level. Our data also suggest that studies measuring inducible immune pathway activation through antimicrobial peptide (AMP) expression in Aag2 cells should be interpreted cautiously given that the Toll pathway is not responsive under all experimental conditions. With no antiviral therapies and few effective vaccines available to treat arboviral diseases, our findings provide new insights relevant to the development of transgenic mosquitoes as a means of reducing arbovirus transmission.

scholarly journals Imd pathway-specific immune assays reveal NF-κB stimulation by viral RNA PAMPs in Aedes aegypti Aag2 cells

2020 ◽  
Author(s):  
Tiffany A. Russell ◽  
Andalus Ayaz ◽  
Andrew D. Davidson ◽  
Ana Fernandez-Sesma ◽  
Kevin Maringer
Keyword(s):  
Immune System ◽  
Aedes Aegypti ◽  
Immune Responses ◽  
Health Impact ◽  
Global Public Health ◽  
Toll Pathway ◽  
Experimental Conditions ◽  
Imd Pathway ◽  
Arboviral Diseases ◽  
Transgenic Insects

ABSTRACTBackgroundThe mosquito Aedes aegypti is a major vector for the arthropod-borne viruses (arboviruses) chikungunya, dengue, yellow fever and Zika viruses. Vector immune responses pose a major barrier to arboviral transmission, and transgenic insects with altered immunity have been proposed as tools for reducing the global public health impact of arboviral diseases. However, a better understanding of virus-immune interactions is needed to progress the development of such transgenic insects. Although the NF-κB-regulated Toll and ‘immunodeficiency’ (Imd) pathways are increasingly thought to be antiviral, relevant pattern recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs) remain poorly characterised in A. aegypti.Methodology/Principle FindingsWe developed novel RT-qPCR and luciferase reporter assays to measure induction of the Toll and Imd pathways in the commonly used A. aegypti-derived Aag2 cell line. We thus determined that the Toll pathway is not inducible by exogenous stimulation with bacterial, viral or fungal stimuli in Aag2 cells under our experimental conditions. We used our Imd pathway-specific assays to demonstrate that the viral dsRNA mimic poly(I:C) is sensed by the Imd pathway, likely through intracellular and extracellular PRRs. The Imd pathway was also induced during infection with the model insect-specific virus cricket paralysis virus (CrPV).Conclusions/SignificanceOur demonstration that a general PAMP shared by many arboviruses is sensed by the Imd pathway paves the way for future studies to determine how viral RNA is sensed by mosquito PRRs at a molecular level. Our data also suggest that studies measuring inducible immune pathway activation through antimicrobial peptide (AMP) expression in Aag2 cells should be interpreted cautiously given that the Toll pathway is not responsive under all experimental conditions. With no antiviral therapies and few effective vaccines available to treat arboviral diseases, our findings provide new insights relevant to the development of transgenic mosquitoes as a means of reducing arbovirus transmission.AUTHOR SUMMARYThe mosquito Aedes aegypti, found globally across the tropics and subtropics, transmits viral diseases with a significant global public health impact, including chikungunya, dengue, yellow fever and Zika viruses. There are no antiviral drugs to treat these diseases and few effective vaccines. One way of reducing the global burden of mosquito-borne diseases would be to develop genetically modified mosquitoes unable to transmit viruses. One approach would be to alter the mosquitoes’ immune system to allow them to better fight viral infections. To do so, we first need to understand how viruses are detected by the mosquito immune system. We developed new methods of measuring immune responses in laboratory-cultured mosquito cells and used them to show that one specific arm of the immune system, called the ‘Imd pathway’, can detect the RNA that constitutes the genome of mosquito-borne viruses. These findings pave the way for future immune studies that could inform the development of transmission-incompetent mosquitoes. We also found that another arm of the immune system, called the ‘Toll pathway’, is not functional under any experimental conditions used in this study. This finding has implications for how different laboratories interpret data from these particular cultured cells.

scholarly journals Aedes aegypti (Diptera: Culicidae) Immune Responses with Different Feeding Regimes Following Infection by the Entomopathogenic Fungus Metarhizium anisopliae

Insects ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 95 ◽  
Author(s):  
Sara Cabral ◽  
Adriano de Paula ◽  
Richard Samuels ◽  
Rodrigo da Fonseca ◽  
Simone Gomes ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Immune Responses ◽  
Antimicrobial Peptides ◽  
Fungal Infection ◽  
Fat Body ◽  
Negative Regulator ◽  
Toll Pathway ◽  
Promising Alternative ◽  
Imd Pathway ◽  
Feeding Regimes

The mosquito Aedes aegypti is the most notorious vector of illness-causing viruses. The use of entomopathogenic fungi as bioinsecticides is a promising alternative for the development of novel mosquito control strategies. We investigate whether differences in immune responses could be responsible for modifications in survival rates of insects following different feeding regimes. Sucrose and blood-fed adult A. aegypti females were sprayed with M. anisopliae 1 × 106 conidia mL−1, and after 48 h, the midgut and fat body were dissected. We used RT-qPCR to monitor the expression of Cactus and REL1 (Toll pathway), IMD, REL2, and Caspar (IMD pathway), STAT and PIAS (JAK-STAT pathway), as well as the expression of antimicrobial peptides (Defensin A, Attacin and Cecropin G). REL1 and REL2 expression in both the midgut and fat body were higher in blood-fed fungus-challenged A. aegypti than in sucrose-fed counterparts. Interestingly, infection of sucrose-fed insects induced Cactus expression in the fat body, a negative regulator of the Toll pathway. The IMD gene was upregulated in the fat body in response to fungal infection after a blood meal. Additionally, we observed the induction of antimicrobial peptides in the blood-fed fungus-challenged insects. This study suggests that blood-fed A. aegypti are less susceptible to fungal infection due to the rapid induction of Toll and IMD immune pathways.

A Simple PCR Assay for Discrimination of Dengue Vectors from Nsukka LGA, Nigeria

2020 ◽  
Vol 10 (1) ◽  
pp. 67-77
Author(s):  
Amos Watentena ◽  
Ikem Chris Okoye ◽  
Ikechukwu Eugene Onah ◽  
Onwude Cosmas Ogbonnaya ◽  
Emmanuel Ogudu
Keyword(s):  
Aedes Aegypti ◽  
Aedes Albopictus ◽  
Mosquito Species ◽  
Public Health Problem ◽  
Major Public Health Problem ◽  
Mosquito Vector ◽  
Aedes Mosquitoes ◽  
Allele Specific ◽  
Arboviral Diseases ◽  
Dengue Vectors

Mosquitoes of Aedes species are vectors of several arboviral diseases which continue to be a major public health problem in Nigeria. This study among other things, morphologically identified Aedes mosquitoes collected from Nsukka LGA and used an allele specific PCR amplification for discrimination of dengue vectors. Larval sampling, BG-sentinel traps and modified human landing catches were used for mosquito sampling in two selected autonomous communities of Nsukka LGA (Nsukka and Obimo). A total of 124 Aedes mosquitoes consisting of five (5) different species were collected from April to June, 2019 in a cross-sectional study that covered 126 households, under 76 distinct geographical coordinates. Larvae was mainly collected from plastic containers 73% (n=224), metallic containers 14% (n=43), earthen pots 9% (n=29) and used car tyres 3% (n=9), reared to adult stage 69.35% (n=86), and all mosquitoes were identified using standard morphological keys. Five (5) Aedes mosquito species were captured; Aedes aegypti 83(66.94%), Aedes albopictus 33(26.61%), Aedes simpsoni (4.48%), Aedes luteocephalus (≤1%) and Aedes vittatus (≤1%). Nsukka autonomous community had higher species diversity than Obimo. Allele specific amplification confirmed dengue vectors, Aedes aegypti and Aedes albopictus species on a 2% agarose gel. Since the most recent re-emergence of arboviral diseases is closely associated with Aedes species, findings of this study, therefore, give further evidence about the presence of potential arboviral vectors in Nigeria and describe the role of a simple PCR in discriminating some. Further entomological studies should integrate PCR assays in mosquito vector surveillance.

scholarly journals The Lethal(2)-Essential-for-Life [L(2)EFL] Gene Family Modulates Dengue Virus Infection in Aedes aegypti

2020 ◽  
Vol 21 (20) ◽  
pp. 7520
Author(s):  
Lucky R. Runtuwene ◽  
Shuichi Kawashima ◽  
Victor D. Pijoh ◽  
Josef S. B. Tuda ◽  
Kyoko Hayashida ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Dengue Virus ◽  
Cell Line ◽  
Dengue Infection ◽  
Fruit Fly ◽  
Initiation Factor ◽  
Poly I:C ◽  
Gene Products ◽  
Vector Mosquito

Efforts to determine the mosquito genes that affect dengue virus replication have identified a number of candidates that positively or negatively modify amplification in the invertebrate host. We used deep sequencing to compare the differential transcript abundances in Aedes aegypti 14 days post dengue infection to those of uninfected A. aegypti. The gene lethal(2)-essential-for-life [l(2)efl], which encodes a member of the heat shock 20 protein (HSP20) family, was upregulated following dengue virus type 2 (DENV-2) infection in vivo. The transcripts of this gene did not exhibit differential accumulation in mosquitoes exposed to insecticides or pollutants. The induction and overexpression of l(2)efl gene products using poly(I:C) resulted in decreased DENV-2 replication in the cell line. In contrast, the RNAi-mediated suppression of l(2)efl gene products resulted in enhanced DENV-2 replication, but this enhancement occurred only if multiple l(2)efl genes were suppressed. l(2)efl homologs induce the phosphorylation of eukaryotic initiation factor 2α (eIF2α) in the fruit fly Drosophila melanogaster, and we confirmed this finding in the cell line. However, the mechanism by which l(2)efl phosphorylates eIF2α remains unclear. We conclude that l(2)efl encodes a potential anti-dengue protein in the vector mosquito.

scholarly journals Analysis of Zika virus capsid-Aedes aegypti mosquito interactome reveals pro-viral host factors critical for establishing infection

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Rommel J. Gestuveo ◽  
Jamie Royle ◽  
Claire L. Donald ◽  
Douglas J. Lamont ◽  
Edward C. Hutchinson ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Protein Interactions ◽  
Zika Virus ◽  
Label Free ◽  
Protein Protein Interactions ◽  
Zikv Infection ◽  
Ubiquitin Protein Ligase ◽  
Mosquito Cells ◽  
Endoplasmic Reticulum Protein ◽  
Arboviral Diseases

AbstractThe escalating global prevalence of arboviral diseases emphasizes the need to improve our understanding of their biology. Research in this area has been hindered by the lack of molecular tools for studying virus-mosquito interactions. Here, we develop an Aedes aegypti cell line which stably expresses Zika virus (ZIKV) capsid proteins in order to study virus-vector protein-protein interactions through quantitative label-free proteomics. We identify 157 interactors and show that eight have potentially pro-viral activity during ZIKV infection in mosquito cells. Notably, silencing of transitional endoplasmic reticulum protein TER94 prevents ZIKV capsid degradation and significantly reduces viral replication. Similar results are observed if the TER94 ortholog (VCP) functioning is blocked with inhibitors in human cells. In addition, we show that an E3 ubiquitin-protein ligase, UBR5, mediates the interaction between TER94 and ZIKV capsid. Our study demonstrates a pro-viral function for TER94/VCP during ZIKV infection that is conserved between human and mosquito cells.

Aedes aegypti Toll pathway is induced through dsRNA sensing in endosomes

2021 ◽  
pp. 104138
Author(s):  
Yesseinia I. Angleró-Rodríguez ◽  
Chinmay V. Tikhe ◽  
Seokyoung Kang ◽  
George Dimopoulos
Keyword(s):  
Aedes Aegypti ◽  
Toll Pathway

scholarly journals Duck RIG-I CARD Domain Induces the Chicken IFN-βby Activating NF-κB

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Yang Chen ◽  
Zhengyang Huang ◽  
Bin Wang ◽  
Qinming Yu ◽  
Ran Liu ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Eukaryotic Expression ◽  
Poly I:C ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Regulatory Domains ◽  
Polycytidylic Acid ◽  
Card Domain ◽  
Inducible Gene

Retinoic acid-inducible gene I- (RIG-I-) like receptors (RLRs) have recently been identified as cytoplasmic sensors for viral RNA. RIG-I, a member of RLRs family, plays an important role in innate immunity. Although previous investigations have proved that RIG-I is absent in chickens, it remains largely unknown whether the chicken can respond to RIG-I ligand. In this study, the eukaryotic expression vectors encoding duRIG-I full length (duck RIG-I, containing all domains), duRIG-I N-terminal (containing the two caspase activation and recruitment domain, CARDs), and duRIG-I C-terminal (containing helicase and regulatory domains) labeled with 6*His tags were constructed successfully and detected by western blotting. Luciferase reporter assay and enzyme-linked immunosorbent assay (ELISA) detected the duRIG-I significantly activated NF-κB and induced the expression of IFN-βwhen polyinosinic-polycytidylic acid (poly[I:C], synthetic double-stranded RNA) challenges chicken embryonic fibroblasts cells (DF1 cells), while the duRIG-I was inactive in the absence of poly[I:C]. Further analysis revealed that the CARDs (duRIG-I-N) induced IFN-βproduction regardless of the presence of poly[I:C], while the CARD-lacking duRIG-I (duRIG-I-C) was not capable of activating downstream signals. These results indicate that duRIG-I CARD domain plays an important role in the induction of IFN-βand provide a basis for further studying the function of RIG-I in avian innate immunity.

scholarly journals Characterization of a Viral Phosphoprotein Binding Site on the Surface of the Respiratory Syncytial Nucleoprotein

2012 ◽  
Vol 86 (16) ◽  
pp. 8375-8387 ◽  
Author(s):  
Marie Galloux ◽  
Bogdan Tarus ◽  
Ilfad Blazevic ◽  
Jenna Fix ◽  
Stéphane Duquerroy ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Specific Interaction ◽  
Viral Rna ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Potential Candidate ◽  
Luciferase Reporter Gene ◽  
Interaction Domain ◽  
Rna Complex

The human respiratory syncytial virus (HRSV) genome is composed of a negative-sense single-stranded RNA that is tightly associated with the nucleoprotein (N). This ribonucleoprotein (RNP) complex is the template for replication and transcription by the viral RNA-dependent RNA polymerase. RNP recognition by the viral polymerase involves a specific interaction between the C-terminal domain of the phosphoprotein (P) (PCTD) and N. However, the P binding region on N remains to be identified. In this study, glutathioneS-transferase (GST) pulldown assays were used to identify the N-terminal core domain of HRSV N (NNTD) as a P binding domain. A biochemical characterization of the PCTDand molecular modeling of the NNTDallowed us to define four potential candidate pockets on N (pocket I [PI] to PIV) as hydrophobic sites surrounded by positively charged regions, which could constitute sites complementary to the PCTDinteraction domain. The role of selected amino acids in the recognition of the N-RNA complex by P was first screened for by site-directed mutagenesis using a polymerase activity assay, based on an HRSV minigenome containing a luciferase reporter gene. When changed to Ala, most of the residues of PI were found to be critical for viral RNA synthesis, with the R132A mutant having the strongest effect. These mutations also reduced or abolishedin vitroandin vivoP-N interactions, as determined by GST pulldown and immunoprecipitation experiments. The pocket formed by these residues is critical for P binding to the N-RNA complex, is specific for pneumovirus N proteins, and is clearly distinct from the P binding sites identified so far for other nonsegmented negative-strand viruses.

The Aedes aegypti IMD pathway is a critical component of the mosquito antifungal immune response

2019 ◽  
Vol 95 ◽  
pp. 1-9 ◽  
Author(s):  
José L. Ramirez ◽  
Ephantus J. Muturi ◽  
Ana B.F. Barletta ◽  
Alejandro P. Rooney
Keyword(s):  
Immune Response ◽  
Aedes Aegypti ◽  
Critical Component ◽  
Imd Pathway

scholarly journals Experimental Assessment of Zika Virus Mechanical Transmission by Aedes aegypti

Viruses ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 695 ◽  
Author(s):  
Antoine Boullis ◽  
Nadège Cordel ◽  
Cécile Herrmann-Storck ◽  
Anubis Vega-Rúa
Keyword(s):  
Experimental Design ◽  
Aedes Aegypti ◽  
Zika Virus ◽  
Infectious Virus ◽  
Viral Rna ◽  
Mechanical Transmission ◽  
Experimental Assessment ◽  
Infected Blood ◽  
Virus Titration ◽  
Blood Meals

The pandemic emergence of several mosquito-borne viruses highlights the need to understand the different ways in which they can be transmitted by vectors to human hosts. In this study, we evaluated the propensity of Aedes aegypti to transmit mechanically Zika virus (ZIKV) using an experimental design. Mosquitoes were allowed to feed on ZIKV-infected blood and were then rapidly transferred to feed on ZIKV-free blood until they finished their meal. The uninfected blood meals, the mosquito abdomens, as well as the mouthparts dissected from fully and partially engorged mosquitoes were analyzed using RT-qPCR and/or virus titration. All the fully engorged mosquito abdomens were ZIKV-infected, whereas their mouthparts were all ZIKV-negative. Nonetheless, one of the partially engorged mosquitoes carried infectious particles on mouthparts. No infectious virus was found in the receiver blood meals, while viral RNA was detected in 9% of the samples (2/22). Thus, mechanical transmission of ZIKV may sporadically occur via Ae. aegypti bite. However, as the number of virions detected on mouthparts (2 particles) is not sufficient to induce infection in a naïve host, our results indicate that mechanical transmission does not impact ZIKV epidemiology.

Sign in / Sign up

Export Citation Format

Share Document