Releasing Intracellular NS1 from Mosquito Cells for the Detection of Dengue Virus-Infected Mosquitoes

Dengue virus (DENV), the pathogen that causes dengue fever, is mainly transmitted by Aedes aegypti. Surveillance of infected mosquitoes is a major component of integrated mosquito control methods for reducing the risk of vector-born disease outbreaks. However, a specialized rapid test for DENV detection in mosquitoes is not currently available. Utilizing immunoblotting, we found that the secretion of NS1 from both a DENV-infected mosquito cell line and mosquito bodies was below the detection threshold. However, when Triton X-100 was used to lyse infected mosquitoes, intracellular NS1 was released, and could then be effectively detected by the NS1 rapid test. The distribution of DENV NS1 in intrathoracically infected mosquitoes was different from that of orally infected mosquitoes. Next, we performed sensitivity tests by bisecting mosquitoes longitudinally; one half of each mosquito was subjected to the NS1 rapid test while the other half was used for qPCR confirmation. This modified test had a sensitivity of nearly 90% from five days post-infection onwards, while DENV had escaped from the midgut barrier. This adapted test offers a valuable, easy-to-use tool for mosquito surveillance, which is a crucial component of DENV disease control.

Download Full-text

Ultrastructural Characterization and Three-Dimensional Architecture of Replication Sites in Dengue Virus-Infected Mosquito Cells

Journal of Virology ◽

10.1128/jvi.00118-14 ◽

2014 ◽

Vol 88 (9) ◽

pp. 4687-4697 ◽

Cited By ~ 113

Author(s):

J. Junjhon ◽

J. G. Pennington ◽

T. J. Edwards ◽

R. Perera ◽

J. Lanman ◽

...

Keyword(s):

Dengue Virus ◽

Three Dimensional ◽

Infected Mosquito ◽

Mosquito Cells ◽

Ultrastructural Characterization ◽

Replication Sites

Download Full-text

Secretion of Nonstructural Protein 1 of Dengue Virus from Infected Mosquito Cells: Facts and Speculations

Journal of Virology ◽

10.1128/jvi.00275-18 ◽

2018 ◽

Vol 92 (14) ◽

Cited By ~ 4

Author(s):

Ana C. Alcalá ◽

Laura A. Palomares ◽

Juan E. Ludert

Keyword(s):

Dengue Virus ◽

Nonstructural Protein ◽

Infected Mosquito ◽

Nonstructural Protein 1 ◽

Mosquito Cells

ABSTRACTDengue virus nonstructural protein 1 (NS1) is a multifunctional glycoprotein. For decades, the notion in the field was that NS1 is secreted exclusively from vertebrate cells and not from mosquito cells. However, recent evidence shows that mosquito cells also secrete NS1 efficiently. In this review, we discuss the evidence for secretion of NS1 of dengue virus, and of other flaviviruses, from mosquito cells, differences between NS1 secreted from mosquito and NS1 secreted from vertebrate cells, and possible roles of soluble NS1 in the insect flavivirus vector.

Download Full-text

The dengue virus non-structural protein 1 (NS1) is secreted from infected mosquito cells via a non-classical caveolin-1-dependent pathway

Journal of General Virology ◽

10.1099/jgv.0.000881 ◽

2017 ◽

Vol 98 (8) ◽

pp. 2088-2099 ◽

Cited By ~ 12

Author(s):

Ana C. Alcalá ◽

Raiza Hernández-Bravo ◽

Fernando Medina ◽

David S. Coll ◽

Jose L. Zambrano ◽

...

Keyword(s):

Dengue Virus ◽

Infected Mosquito ◽

Structural Protein ◽

Caveolin 1 ◽

Mosquito Cells ◽

Dependent Pathway

Download Full-text

The dengue virus non-structural protein 1 (NS1) is secreted efficiently from infected mosquito cells

Virology ◽

10.1016/j.virol.2015.11.020 ◽

2016 ◽

Vol 488 ◽

pp. 278-287 ◽

Cited By ~ 18

Author(s):

Ana C. Alcalá ◽

Fernando Medina ◽

Arturo González-Robles ◽

Lizbeth Salazar-Villatoro ◽

Rogelio J. Fragoso-Soriano ◽

...

Keyword(s):

Dengue Virus ◽

Infected Mosquito ◽

Structural Protein ◽

Mosquito Cells

Download Full-text

Life as a Vector of Dengue Virus: The Antioxidant Strategy of Mosquito Cells to Survive Viral Infection

Antioxidants ◽

10.3390/antiox10030395 ◽

2021 ◽

Vol 10 (3) ◽

pp. 395

Author(s):

Chih-Chieh Cheng ◽

Eny Sofiyatun ◽

Wei-June Chen ◽

Lian-Chen Wang

Keyword(s):

Dengue Virus ◽

Viral Infection ◽

Denv Infection ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Infected Mosquito ◽

Mosquito Vector ◽

Evolutionary Pathway ◽

Antioxidant Genes ◽

Mosquito Cells

Dengue fever is a mosquito-borne viral disease of increasing global importance. The disease has caused heavy burdens due to frequent outbreaks in tropical and subtropical areas of the world. The dengue virus (DENV) is generally transmitted between human hosts via the bite of a mosquito vector, primarily Aedes aegypti and Ae. albopictus as a minor species. It is known that the virus needs to alternately infect mosquito and human cells. DENV-induced cell death is relevant to the pathogenesis in humans as infected cells undergo apoptosis. In contrast, mosquito cells mostly survive the infection; this allows infected mosquitoes to remain healthy enough to serve as an efficient vector in nature. Overexpression of antioxidant genes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), glutaredoxin (Grx), thioredoxin (Trx), and protein disulfide isomerase (PDI) have been detected in DENV2-infected mosquito cells. Additional antioxidants, including GST, eukaryotic translation initiation factor 5A (eIF5a), and p53 isoform 2 (p53-2), and perhaps some others, are also involved in creating an intracellular environment suitable for cell replication and viral infection. Antiapoptotic effects involving inhibitor of apoptosis (IAP) upregulation and subsequent elevation of caspase-9 and caspase-3 activities also play crucial roles in the ability of mosquito cells to survive DENV infection. This article focused on the effects of intracellular responses in mosquito cells to infection primarily by DENVs. It may provide more information to better understand virus/cell interactions that can possibly elucidate the evolutionary pathway that led to the mosquito becoming a vector.

Download Full-text

Direct nucleotide sequencing using total cellular RNA from dengue-virus-infected mosquito cells

Research in Virology ◽

10.1016/s0923-2516(06)80103-2 ◽

1992 ◽

Vol 143 ◽

pp. 193-197 ◽

Cited By ~ 2

Author(s):

I.M. Kautner ◽

S.-K. Lam

Keyword(s):

Dengue Virus ◽

Infected Mosquito ◽

Nucleotide Sequencing ◽

Mosquito Cells

Download Full-text

Monitoring Mitochondrial Function in Aedes albopictus C6/36 Cell Line during Dengue Virus Infection

Insects ◽

10.3390/insects12100934 ◽

2021 ◽

Vol 12 (10) ◽

pp. 934

Author(s):

María E. Santana-Román ◽

Paola Maycotte ◽

Salvador Uribe-Carvajal ◽

Cristina Uribe-Alvarez ◽

Nayeli Alvarado-Medina ◽

...

Keyword(s):

Dengue Virus ◽

Cell Line ◽

Aedes Albopictus ◽

Mitochondrial Function ◽

Mitochondrial Fission ◽

Hemorrhagic Fever ◽

Denv Infection ◽

Infected Mosquito ◽

Mosquito Cells ◽

Induced Changes

Aedes aegypti and Aedes albopictus mosquitoes are responsible for dengue virus (DENV) transmission in tropical and subtropical areas worldwide, where an estimated 3 billion people live at risk of DENV exposure. DENV-infected individuals show symptoms ranging from sub-clinical or mild to hemorrhagic fever. Infected mosquitoes do not show detectable signs of disease, even though the virus maintains a lifelong persistent infection. The interactions between viruses and host mitochondria are crucial for virus replication and pathogenicity. DENV infection in vertebrate cells modulates mitochondrial function and dynamics to facilitate viral proliferation. Here, we describe that DENV also regulates mitochondrial function and morphology in infected C6/36 mosquito cells (derived from Aedes albopictus). Our results showed that DENV infection increased ROS (reactive oxygen species) production, modulated mitochondrial transmembrane potential and induced changes in mitochondrial respiration. Furthermore, we offer the first evidence that DENV causes translocation of mitofusins to mitochondria in the C6/36 mosquito cell line. Another protein Drp-1 (Dynamin-related protein 1) did not localize to mitochondria in DENV-infected cells. This observation therefore ruled out the possibility that the abovementioned alterations in mitochondrial function are associated with mitochondrial fission. In summary, this report provides some key insights into the virus–mitochondria crosstalk in DENV infected mosquito cells.

Download Full-text

A Role for the Insulin Receptor in the Suppression of Dengue Virus and Zika Virus in Wolbachia-Infected Mosquito Cells

Cell Reports ◽

10.1016/j.celrep.2018.12.068 ◽

2019 ◽

Vol 26 (3) ◽

pp. 529-535.e3 ◽

Cited By ~ 9

Author(s):

Gholamreza Haqshenas ◽

Gerard Terradas ◽

Prasad N. Paradkar ◽

Jean-Bernard Duchemin ◽

Elizabeth A. McGraw ◽

...

Keyword(s):

Dengue Virus ◽

Insulin Receptor ◽

Zika Virus ◽

Infected Mosquito ◽

Mosquito Cells

Download Full-text

Laboratory Evaluation of the Rapid Analyte Measurement Platform Assay to Detect Dengue Virus in Mosquito Pools

Journal of the American Mosquito Control Association ◽

10.2987/21-7013.1 ◽

2021 ◽

Vol 37 (3) ◽

pp. 152-156

Author(s):

Kristen L. Burkhalter ◽

Harry M. Savage

Keyword(s):

Dengue Virus ◽

Negative Control ◽

Infected Mosquito ◽

Laboratory Evaluation ◽

Denv Serotypes ◽

Complete Inactivation ◽

Detection Assay ◽

Triton X ◽

Discrete Sensitivity ◽

Biosafety Level

ABSTRACT We report the results of a laboratory sensitivity and specificity evaluation of the Rapid Analyte Measurement Platform (RAMP®) Dengue Virus (DENV) antigen detection assay, which is designed to detect all serotypes of DENV in mosquito pools. The RAMP DENV assay was able to detect geographically distinct strains of all 4 DENV serotypes in virus-spiked mosquito pools that contained at least 4.3 log10 plaque forming units/ml, although discrete sensitivity limits varied slightly for each serotype. The RAMP DENV assay also detected DENV 1–4 in mosquito pools containing a single infected mosquito and 24 laboratory-reared uninfected mosquitoes. No false positives were detected in negative control mosquito pools or in samples containing high titers of nontarget arboviruses. We found that while the kit-supplied RAMP buffer reduced the infectious titer of DENV, it did not completely inactivate all serotypes. We recommend adding a detergent, Triton X-100, to the buffer to ensure complete inactivation of DENV if the assay is to be conducted at a lower biosafety level than required for DENV handling.

Download Full-text