Project description:This study seeks to understand the mechanism of midgut escape and identify candidate genes and potential biochemical pathways involved in the midgut infection of chikungunya virus (CHIKV) in vector mosquito Aedes aegypti. We conducted a comparative transcriptomic analysis of midgut samples of female mosquitoes,after feeding a saline meal (SM) or a protein meal (PM) containing CHIKV. Our results allow the conclusion that midgut-expressed genes not involved in blood or protein digestion can be identified by substituting either a bloodmeal or PM for a SM. In presence of orally acquired CHIKV in midguts of SM fed mosquitoes, the majority of the upregulated DE genes belonged to the categories immune system and catalytic activity. These genes included several serine-type endopeptidases, trypsins, collagenases, and M1 zinc metalloproteases, which potentially could be involved in the midgut escape mechanism of CHIKV. One of the serine metalloproteinase genes, AeLT, was further analyzed showing strong (MMP) collagenase activity in vitro. Our results present a set of candidate genes potentially responsible for overcoming the arbovirus midgut escape barrier (MEB) in Ae. aegypti.
Project description:The midgut of hematophagous insects is the initial site of infection by arthropod-borne viruses (arboviruses) and plays a crucial role in vector competence. To further understand processes that occur in the midgut in response to infection by an arbovirus, DNA microarrays were used to analyze gene expression changes following infection by the alphavirus, Sindbis (MRE16 Malaysian strain). Midgut transcription profiles from mosquitoes fed blood containing 108 pfu/ml of virus were compared with those from mosquitoes ingesting blood meals having no virus. Transcription profiles from both experimental groups were analyzed at 1, 4, and 8 days post feeding. Among the many transcription changes observed by microarray analysis, the most dramatic involved three genes that had twenty-five to forty-fold increases in transcript levels in virus infected mosquitoes at 4 days post infection . These genes were synaptic vesicle protein-2 (SV2), potassium-dependent sodium/calcium exchanger (NCKX), and a homologue of C. elegans Unc-93, a putative component of a two-pore potassium channel. We speculate that these changes represent changes in vesicle transport processes. In addition to these observations, transcript changes were observed in infected mosquitoes that suggested involvement of Toll and JNK immune cascades as a response to viral infection
Project description:Aedes mosquitoes transmit pathogenic arthropod-borne (arbo) viruses, putting nearly half the world’s population at risk. Blocking virus replication in mosquitoes rather than in humans serves as a promising approach to prevent arbovirus transmission, which requires in-depth knowledge of mosquito immunity. By integrating multi-omics data, we identified that heat shock factor 1 (Hsf1) regulates eight small heat shock protein (sHsp) genes within one topological associated domain. This Hsf1-sHsp cascade acts as an early response against chikungunya virus (CHIKV) infection and shows pan-antiviral activity in three vector mosquitoes, Aedes aegypti, Aedes albopictus, and Anopheles gambiae. We then assessed the baseline expression of sHsp genes in different tissues of female Ae. aegypti using RNA-seq, and we observed a highly dynamic expression pattern of sHsp genes that varied dramatically across different tissues. Interestingly, sHsp genes were expressed at low levels in two main barrier tissues, the midgut and salivary glands, compared to other tissues such as the crop. Importantly, activation of Hsf1 led to a reduced CHIKV infection rate in adult Ae. aegypti mosquitoes, demonstrating Hsf1 as a promising target for the development of novel intervention strategies to limit arbovirus transmission by mosquitoes.
Project description:High-throughput sequencing was performed to identify miRNAs profiles of Anopheles anthropophagus midguts after blood feeding and Plasmodium infection. The expression patterns of miRNA in different midgut libraries were compared basing on transcripts per million reads (TPM). This study provides novel regulated miRNAs information of An. anthropophagus during blood feeding and parasite infection
Project description:The emergence of mosquito-borne diseases because of climate change emphasizes the need to study arbovirus-vector protein-protein interactions (PPI) to better understand viral replication and transmission. One such human pathogenic arbovirus is Zika virus (ZIKV; Flaviviridae), transmitted by Aedes aegypti mosquitoes. With the lack of molecular tools to study mosquito cells, we developed an Ae. aegypti AF5 cell line stably expressing ZIKV capsid to investigate PPI through label-free quantification proteomics. We identified 157 interactors with 8 potentially pro-viral during ZIKV infection and showed that the transitional endoplasmic reticulum 94 (TER94) protein of the ubiquitin-proteasome pathway (UPP) was important during ZIKV infection in mosquito cells. Silencing TER94 in AF5 cells prevented ZIKV capsid degradation and significantly reduced the establishment of replication at the early stages of infection. Human TER94 ortholog, valosin containing protein (VCP), identified through ortholog mapping, was found to have a similar function during ZIKV infection in A549 cells. ZIKV had reduced ability to replicate when ubiquitination and VCP function were blocked by chemical inhibitors. Furthermore, ubiquitin protein ligase E3 component n-recognin 5 (UBR5) was identified as a TER94/VCP co-factor for capsid interaction. Our study demonstrates a conserved function for TER94/VCP-UPP during early ZIKV infection in mosquito and human cells.
Project description:We report the application of whole transcriptome sequencing technology for high-throughput profiling of coding and non-coding RNAs associated with RBSDV infection in Laodelphax striatellus (Fallén) midgut. Over 20 and 21 million clean reads from virus free (VF) and RBSDV infected (RB) libraries were obtained, respectively. Q30 base percentages of each sequencing sample were no less than 93.89%. More than 50.37% of the clean reads were perfectly mapped to the L. striatellus genome. The uniquely mapping ratio was ranged from 48.03% to 52.08%. A total of 35,316 mRNAs and 13,927 novel lncRNAs were identified from VF and RB libraries. 169 mRNAs and 176 lncRNAs were differentially expressed during RBSDV infection. RNA-seq data had a linear relationship with qRT–PCR results. Our study represents the first detailed transcriptomes analysis of L. striatellus midgut during RBSDV infection, with biologic replicates, generated by RNA-seq technology. Our data contribute to the understanding of the function of coding and non-coding RNAs in the regulation of viral infection.
Project description:The impact of global climate change on the transmission dynamics of infectious diseases is the subject of extensive debate. The transmission of mosquito-borne viral diseases is particularly complex, with climatic variables directly affecting many parameters associated with the prevalence of disease vectors. While evidence shows that warmer temperatures often decrease the extrinsic incubation period of an arthropod-borne virus (arbovirus), exposure to cooler temperatures often predisposes disease vector mosquitoes to higher infection rates. RNA interference pathways are essential to antiviral immunity in the mosquito; however, few experiments have explored the effects of temperature on the RNAi machinery.