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BackgroundGlass membrane feeders are used in malaria research for artificial blood feeding. This study investigates the use of Hemotek membrane feeders as a standardized alternative feeding system.MethodsHemotek feeders were compared with glass feeders by assessing mosquito feeding rate, imbibed blood meal volume and Plasmodium falciparum infection intensity on mosquito guts.ResultsWhile mosquito feeding rate and blood meal volume were comparable between Hemotek and glass feeders, a loss in transmission was observed using the Hemotek feeder with a conventional collagen membrane. There was no difference in transmission between both feeders when Parafilm was used as the membrane.ConclusionsHemotek feeders with a Parafilm membrane can be used as an alternative feeding system for malaria transmission research.

We describe a protocol for single-cell RNA sequencing of SARS-CoV-2-infected human induced pluripotent stem cell (iPSC)-derived kidney organoids. After inoculation of kidney organoids with virus, we use mechanical and enzymatic disruption to obtain single cell suspensions. Next, we process the organoid-derived cells into sequencing-ready SARS-CoV-2-targeted libraries. Subsequent sequencing analysis reveals changes in kidney cells after virus infection. The protocol was designed for kidney organoids cultured in a 6-well transwell format but can be adapted to organoids with different organ backgrounds. For complete details on the use and execution of this protocol, please refer to Jansen et al. (2022).

Kidney failure is frequently observed during and after COVID-19, but it remains elusive whether this is a direct effect of the virus. Here, we report that SARS-CoV-2 directly infects kidney cells and is associated with increased tubule-interstitial kidney fibrosis in patient autopsy samples. To study direct effects of the virus on the kidney independent of systemic effects of COVID-19, we infected human-induced pluripotent stem-cell-derived kidney organoids with SARS-CoV-2. Single-cell RNA sequencing indicated injury and dedifferentiation of infected cells with activation of profibrotic signaling pathways. Importantly, SARS-CoV-2 infection also led to increased collagen 1 protein expression in organoids. A SARS-CoV-2 protease inhibitor was able to ameliorate the infection of kidney cells by SARS-CoV-2. Our results suggest that SARS-CoV-2 can directly infect kidney cells and induce cell injury with subsequent fibrosis. These data could explain both acute kidney injury in COVID-19 patients and the development of chronic kidney disease in long COVID.

Aedes aegypti mosquitoes are responsible for the transmission of arthropod-borne (arbo) viruses including dengue and chikungunya virus (CHIKV) but in contrast to human hosts, arbovirus-infected mosquitoes are able to efficiently control virus replication to sub-pathological levels. Yet, our knowledge of the molecular interactions of arboviruses with their mosquito hosts is incomplete. Here, we aimed to identify and characterize novel host genes that control arbovirus replication in Aedes mosquitoes. RNA binding proteins (RBPs) are well-known to regulate immune signaling pathways in all kingdoms of life. We therefore performed a knockdown screen targeting 461 genes encoding predicted RBPs in Aedes aegypti Aag2 cells and identified 15 genes with antiviral activity against Sindbis virus. Amongst these, the three DEAD-box RNA helicases AAEL004419/Dhx15, AAEL008728, and AAEL004859 also acted as antiviral factors in dengue and CHIKV infections. Here, we explored the mechanism of Dhx15 in regulating an antiviral transcriptional response in mosquitoes by silencing Dhx15 in Aag2 cells followed by deep-sequencing of poly-A enriched RNAs. Dhx15 knockdown in uninfected and CHIKV infected cells resulted in differential expression of 856 and 372 genes, respectively. Interestingly, amongst the consistently downregulated genes, glycolytic process was the most enriched gene ontology (GO) term as the expression of all core enzymes of the glycolytic pathway was reduced, suggesting that Dhx15 regulates glycolytic function. A decrease in lactate production indicated that Dhx15 silencing indeed functionally impaired glycolysis. Modified rates of glycolytic metabolism have been implicated in controlling the replication of several classes of viruses and strikingly, infection of Aag2 cells with CHIKV by itself also resulted in the decrease of several glycolytic genes. Our data suggests that Dhx15 regulates replication of CHIKV, and possibly other arboviruses, by controlling glycolysis in mo

Small RNA mediated responses are essential for antiviral defence in mosquitoes, however, they appear to differ per virus-vector combination. To further investigate the diversity of small RNA responses against viruses in mosquitoes, we applied a small RNA deep sequencing approach on five mosquito cell lines: Culex tarsalis CT cells, Aedes albopictus U4.4 and C6/36 cells, Ae. aegypti Aag2 cells (cleared from cell fusing agent virus and Culex Y virus (CYV) by repetitive dsRNA transfections) and Ae. pseudoscutellaris AP-61 cells. De novo assembly of small RNAs revealed the presence of Phasi Charoen-like virus (PCLV), Calbertado virus, Flock House virus and a novel narnavirus in CT cells, CYV in U4.4 cells, and PCLV in Aag2 cells, whereas no insect-specific viruses (ISVs) were detected in C6/36 and AP-61 cells. Next, we investigated the small RNA responses to the identified ISVs and to acute infection with the arthropod-borne West Nile virus (WNV). We demonstrate that AP-61 and C6/36 cells do not produce siRNAs to WNV infection, suggesting that AP-61, like C6/36, are Dicer-2 deficient. CT cells produced a strong siRNA response to the persistent ISVs and acute WNV infection. Interestingly, CT cells also produced viral PIWI-interacting (pi)RNAs to PCLV, but not to WNV or any of the other ISVs. In contrast, in U4.4 and Aag2 cells, WNV siRNAs, and pi-like RNAs without typical ping-pong piRNA signature were observed, while this signature was present in PCLV piRNAs in Aag2 cells. Together, our results demonstrate that mosquito small RNA responses are strongly dependent on both the mosquito cell type and/or the mosquito species and family of the infecting virus.

Deep sequenced small RNAs from Culex tarsalis CT, Aedes albopictus U4.4 and C6/36, Aedes aegypti Aag2 and Aedes pseudoscutellaris AP-61 cell lines to detect persistent insect-specific viruses. Also infected these cell lines with West Nile virus to investigate the small RNA responses to acute West Nile virus infection, and to the detected persistent insect-specific viruses., Deep sequenced small RNAs from Culex tarsalis CT, Aedes albopictus U4.4 and C6/36, Aedes aegypti Aag2 and Aedes pseudoscutellaris AP-61 cell lines to detect persistent insect-specific viruses. Also infected these cell lines with West Nile virus to investigate the small RNA responses to acute West Nile virus infection, and to the detected persistent insect-specific viruses.