Dataset Information

Dynamic proteomic analysis of Aedes aegypti Aag-2 cells infected with Mayaro virus.

ABSTRACT:

Background

Mayaro virus (MAYV) is responsible for a mosquito-borne tropical disease with clinical symptoms similar to dengue or chikungunya virus fevers. In addition to the recent territorial expansion of MAYV, this virus may be responsible for an increasing number of outbreaks. Currently, no vaccine is available. Aedes aegypti is promiscuous in its viral transmission and thus an interesting model to understand MAYV-vector interactions. While the life-cycle of MAYV is known, the mechanisms by which this arbovirus affects mosquito host cells are not clearly understood.

Methods

After defining the best conditions for cell culture harvesting using the highest virus titer, Ae. aegypti Aag-2 cells were infected with a Brazilian MAYV isolate at a MOI of 1 in order to perform a comparative proteomic analysis of MAYV-infected Aag-2 cells by using a label-free semi-quantitative bottom-up proteomic analysis. Time-course analyses were performed at 12 and 48 h post-infection (hpi). After spectrum alignment between the triplicates of each time point and changes of the relative abundance level calculation, the identified proteins were annotated and using Gene Ontology database and protein pathways were annotated using the Kyoto Encyclopedia of Genes and Genomes.

Results

After three reproducible biological replicates, the total proteome analysis allowed for the identification of 5330 peptides and the mapping of 459, 376 and 251 protein groups, at time 0, 12 hpi and 48 hpi, respectively. A total of 161 mosquito proteins were found to be differentially abundant during the time-course, mostly related to host cell processes, including redox metabolism, translation, energy metabolism, and host cell defense. MAYV infection also increased host protein expression implicated in viral replication.

Conclusions

To our knowledge, this first proteomic time-course analysis of MAYV-infected mosquito cells sheds light on the molecular basis of the viral infection process and host cell response during the first 48 hpi. Our data highlight several mosquito proteins modulated by the virus, revealing that MAYV manipulates mosquito cell metabolism for its propagation.

SUBMITTER: Vasconcellos AF

PROVIDER: S-EPMC7285477 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Publications

Dynamic proteomic analysis of Aedes aegypti Aag-2 cells infected with Mayaro virus.

Vasconcellos Anna Fernanda AF Mandacaru Samuel Coelho SC de Oliveira Athos Silva AS Fontes Wagner W Melo Reynaldo Magalhães RM de Sousa Marcelo Valle MV Resende Renato Oliveira RO Charneau Sébastien S

Parasites & vectors 20200610 1

<h4>Background</h4>Mayaro virus (MAYV) is responsible for a mosquito-borne tropical disease with clinical symptoms similar to dengue or chikungunya virus fevers. In addition to the recent territorial expansion of MAYV, this virus may be responsible for an increasing number of outbreaks. Currently, no vaccine is available. Aedes aegypti is promiscuous in its viral transmission and thus an interesting model to understand MAYV-vector interactions. While the life-cycle of MAYV is known, the mechanis ...[more]

PMID: 32522239

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Project description:Newly emerging or re-emerging arthropod-borne viruses (arboviruses) are important causes of human morbidity and mortality worldwide. Arboviruses such as Dengue (DENV), Zika (ZIKV), Chikungunya (CHIKV), and West Nile virus (WNV) have undergone extensive geographic expansion in the tropical and sub-tropical regions of the world. In the Americas the main vectors of DENV, ZIKV, and CHIKV are mosquito species adapted to urban environments, namely Aedes aegypti and Aedes albopictus, whereas the main vector of WNV is Culex quinquefasciatus. Given the widespread distribution in the Americas and high permissiveness to arbovirus infection, these mosquito species may play a key role in the epidemiology of other arboviruses normally associated with sylvatic vectors. Here, we test this hypothesis by determining the vector competence of Ae. aegypti, Ae. albopictus, and Cx. quinquefasciatus to Mayaro (MAYV) virus, a sylvatic arbovirus transmitted mainly by Haemagogus janthinomys that has been causing an increasing number of outbreaks in South America, namely in Brazil. Using field mosquitoes from Brazil, female mosquitoes were experimentally infected, and their competence for infection and transmission rates of MAYV was evaluated. We found consistent infection rate for MAYV in Ae. aegypti (57.5%) and Ae. albopictus (61.6%), whereas very low rates were obtained for Cx. quinquefasciatus (2.5%). Concordantly, we observed high potential transmission ability in Ae. aegypti and Ae. albopictus (69.5% and 71.1% respectively), in contrast to Cx. quinquefasciatus, which could not transmit the MAYV. Notably, we found that very low quantities of virus present in the saliva (undetectable by RT-qPCR) were sufficiently virulent to guarantee transmission. Although Ae. aegypti and Ae. albopictus mosquitoes are not the main vectors for MAYV, our studies suggest that these mosquitoes could play a significant role in the transmission of this arbovirus, since both species showed significant vector competence for MAYV (Genotype D), under laboratory conditions.

Project description:BackgroundThe primary disease vectors for dengue virus (DENV) transmission between humans are the mosquitoes Aedes aegypti and Aedes albopictus, with Ae. aegypti population size strongly correlated with DENV outbreaks. When a mosquito is infected with DENV, the virus migrates from the midgut to the salivary glands to complete the transmission cycle. How the virus crosses the hemocoel, resulting in systemic infection, is still unclear however. During viral infection and migration, the innate immune system is activated in defense. As part of cellular-mediated immunity, hemocytes are known to defend against bacteria and Plasmodium infection and may also participate in defending against DENV infection. Hemocytes are categorized into three cell types: prohemocytes, granulocytes, and oenocytoids. Here, we investigated which hemocytes can be infected by DENV and compare hemocyte infection between Ae. aegypti and Ae. albopictus.MethodsHemocytes were collected from Ae. aegypti and Ae. albopictus mosquitoes that were intrathoracically infected with DENV2-GFP. The collected hemocytes were then identified via Giemsa staining and examined microscopically for morphological differences and viral infection.ResultsAll three types of hemocytes were infected by DENV, though the predominantly infected cell type was prohemocytes. In Ae. aegypti, the highest and lowest infection rates at 7 days post infection occurred in prohemocytes and granulocytes, respectively. Prohemocytes were also the primary infection target of DENV in Ae. albopictus, with similar infection rates across the other two hemocyte groups. The ratios of hemocyte composition did not differ significantly between non-infected and infected mosquitoes for either species.ConclusionsIn this study, we showed that prohemocytes were the major type of hemocyte infected by DENV in both Ae. aegypti and Ae. albopictus. The infection rate of prohemocytes in Ae. albopictus was lower than that in Ae. aegypti, which may explain why systemic DENV infection in Ae. albopictus is less efficient than in Ae. aegypti and why Ae. albopictus is less correlated to dengue fever outbreaks. Future work in understanding the mechanisms behind these phenomena may help reduce arbovirus infection prevalence.

Project description:BACKGROUND: The understanding of mosquito immune responses can provide valuable tools for development of novel mosquito control strategies. Aiming the study at insect innate immunity, continuous insect cell lines have been established and used as research tools due to the fact that they constitute more homogeneous, sensitive, and reproducible systems than the insects from which they originated. More recently, Aag-2, an Aedes aegypti cell lineage, began to be frequently used as a model for studies of mosquito immunity. Nevertheless, to our knowledge, no study has systematically characterized the responses of Aag-2 cell line against different kinds of pathogens and compared its response to those exhibited by whole mosquitoes. For this reason, in this study we characterized gene expression profiles of the Aag-2 cell line in response to different kinds of immune challenges, such as Gram negative and positive bacteria, fungi and viruses, comparing the obtained results with the ones already described in the literature for whole mosquitoes. METHODS: Aedes aegypti Aag-2 cells were exposed to different immune stimuli (gram-positive and gram negative heat inactivated bacteria, zymosan or Sindbis virus) for 24 hours and the expression of selected marker genes from toll, IMD and Jak/STAT pathways was analyzed by qPCR. Also, cells were incubated with fluorescent latex beads for evaluation of its phagocytosis capacity. RESULTS: Aag-2 cells were stimulated with two concentrations of heat-killed Gram negative (Enterobacter cloacae) or Gram positive (Micrococcus luteus) bacteria, Zymosan or infected with Sindbis virus and the expression of key genes from the main immune related pathways, Toll, IMD and Jak/STAT, were investigated. Our results suggest that Toll and IMD pathways are activated in response to both Gram positive and negative bacteria and Zymosan in Aag-2 cells, displaying an immune profile similar to those described in the literature for whole mosquitoes. The same stimuli were also capable of activating Jak/STAT pathway in Aag-2 cells. Infection with Sindbis virus led to an up-regulation of the transcription factor STAT but was not able to induce the expression of any other gene from any of the pathways assayed. We also showed that this cell line is able to phagocytose latex beads in culture. CONCLUSIONS: Our results characterize the expression profile of Aag-2 cells in response to different immune stimuli and demonstrate that this cell lineage is immune-competent and closely resembles the response described for whole Ae. aegypti mosquitoes. Hence, our findings support the use of Aag-2 as a tool to comprehend Ae. aegypti immune response both at cellular and humoral levels.

Dataset Information

Dynamic proteomic analysis of Aedes aegypti Aag-2 cells infected with Mayaro virus.

Background

Methods

Results

Conclusions

Publications

Dynamic proteomic analysis of Aedes aegypti Aag-2 cells infected with Mayaro virus.

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