Tripartite interactions between viruses, parasites, and mosquitoes.

Mosquito-borne diseases have a major impact on global human health. Biological agents that colonize the mosquito vector are increasingly explored as an intervention strategy to prevent vector-borne disease transmission. For instance, the release of mosquitoes carrying the endosymbiotic bacterium Wolbachia effectively reduced dengue virus incidence and disease. Insect-specific viruses are likewise considered as biocontrol agents against vector-borne diseases. While most studies focused on insect-specific viruses as an intervention against arthropod-borne viruses, we here consider whether mosquito-specific viruses may affect the transmission of the malaria-causing Plasmodium parasite by Anopheles mosquitoes. Although there is no direct experimental evidence addressing this question, we found that viral infections in dipteran insects activate some of the immune pathways that are antiparasitic in Anopheles. These findings suggest that indirect virus-parasite interactions could occur and that insect-specific viruses may modulate malaria transmission. Tripartite interactions between viruses, parasites, and Anopheles mosquitoes thus merit further investigation.

The impact of BNT162b2 mRNA vaccine on adaptive and innate immune responses.

The mRNA-based BNT162b2 protects against severe disease and mortality caused by SARS-CoV-2 via induction of specific antibody and T-cell responses. Much less is known about its broad effects on immune responses against other pathogens. Here, we investigated the adaptive immune responses induced by BNT162b2 vaccination against various SARS-CoV-2 variants and its effects on the responsiveness of immune cells upon stimulation with heterologous stimuli. BNT162b2 vaccination induced effective humoral and cellular immunity against SARS-CoV-2 that started to wane after six months. We also observed long-term transcriptional changes in immune cells after vaccination. Additionally, vaccination with BNT162b2 modulated innate immune responses as measured by inflammatory cytokine production after stimulation - higher IL-1/IL-6 release and decreased IFN-α production. Altogether, these data expand our knowledge regarding the overall immunological effects of this new class of vaccines and underline the need for additional studies to elucidate their effects on both innate and adaptive immune responses.

Chromatin profiling identifies transcriptional readthrough as a conserved mechanism for piRNA biogenesis in mosquitoes.

The piRNA pathway in mosquitoes differs substantially from other model organisms, with an expanded PIWI gene family and functions in antiviral defense. Here, we define core piRNA clusters as genomic loci that show ubiquitous piRNA expression in both somatic and germline tissues. These core piRNA clusters are enriched for non-retroviral endogenous viral elements (nrEVEs) in antisense orientation and depend on key biogenesis factors, Veneno, Tejas, Yb, and Shutdown. Combined transcriptome and chromatin state analyses identify transcriptional readthrough as a conserved mechanism for cluster-derived piRNA biogenesis in the vector mosquitoes Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Anopheles gambiae. Comparative analyses between the two Aedes species suggest that piRNA clusters function as traps for nrEVEs, allowing adaptation to environmental challenges such as virus infection. Our systematic transcriptome and chromatin state analyses lay the foundation for studies of gene regulation, genome evolution, and piRNA function in these important vector species.

Composition and global distribution of the mosquito virome - A comprehensive database of insect-specific viruses.

Mosquitoes are vectors for emerging and re-emerging infectious viral diseases of humans, livestock and other animals. In addition to these arthropod-borne (arbo)viruses, mosquitoes are host to an array of insect-specific viruses, collectively referred to as the mosquito virome. Mapping the mosquito virome and understanding if and how its composition modulates arbovirus transmission is critical to understand arboviral disease emergence and outbreak dynamics. In recent years, next-generation sequencing as well as PCR and culture-based methods have been extensively used to identify mosquito-associated viruses, providing insights into virus ecology and evolution. Until now, the large amount of mosquito virome data, specifically those acquired by metagenomic sequencing, has not been comprehensively integrated. We have constructed a searchable database of insect-specific viruses associated with vector mosquitoes from 175 studies, published between October 2000 and February 2022. We identify the most frequently detected and widespread viruses of the Culex, Aedes and Anopheles mosquito genera and report their global distribution. In addition, we highlight the challenges of extracting and integrating published virome data and we propose that a standardized reporting format will facilitate data interpretation and re-use by other scientists. We expect our comprehensive database, summarizing mosquito virome data collected over 20 years, to be a useful resource for future studies.

Longitudinal proteomic profiling of the inflammatory response in dengue patients.

BACKGROUND: The immunopathogenesis of dengue virus (DENV) infection remains incompletely understood. To increase our understanding of inflammatory response in non-severe dengue, we assessed longitudinal changes in the inflammatory proteome in patients with an acute DENV infection. METHODS: Using a multiplex proximity extension assay (PEA), we measured relative levels of 368 inflammatory markers in plasma samples from hospitalized patients with non-severe DENV infection in the acute (n = 43) and convalescence (n = 35) phase of the infection and samples of healthy controls (n = 10). RESULTS: We identified 203 upregulated and 39 downregulated proteins in acute versus convalescent plasma samples. The upregulated proteins had a strong representation of interferon (IFN) and IFN-inducible effector proteins, cytokines (e.g. IL-10, IL-33) and cytokine receptors, chemokines, pro-apoptotic proteins (e.g. granzymes) and endothelial markers. A number of differentially expressed proteins (DEPs) have not been reported in previous studies. Functional network analysis highlighted a central role for IFNγ, IL-10, IL-33 and chemokines. We identified different novel associations between inflammatory proteins and circulating concentrations of the endothelial glycocalyx disruption surrogate marker syndecan-1. Conclusion: This unbiased proteome analysis provides a comprehensive insight in the inflammatory response in DENV infection and its association with glycocalyx disruption.

Parallel evolution and enhanced virulence upon in vivo passage of an RNA virus in Drosophila melanogaster.

Virus evolution is strongly affected by antagonistic co-evolution of virus and host. Host immunity positively selects for viruses that evade the immune response, which in turn may drive counter-adaptations in host immune genes. We investigated how host immune pressure shapes virus populations, using the fruit fly Drosophila melanogaster and its natural pathogen Drosophila C virus (DCV), as a model. We performed an experimental evolution study in which DCV was serially passaged for ten generations in three fly genotypes differing in their antiviral RNAi response: wild-type flies and flies in which the endonuclease gene Dicer-2 was either overexpressed or inactivated. All evolved virus populations replicated more efficiently in vivo and were more virulent than the parental stock. The number of polymorphisms increased in all three host genotypes with passage number, which was most pronounced in Dicer-2 knockout flies. Mutational analysis showed strong parallel evolution, as mutations accumulated in a specific region of the VP3 capsid protein in every lineage in a host genotype-independent manner. The parental tyrosine at position ninety-five of VP3 was substituted with either one of five different amino acids in fourteen out of fifteen lineages. However, no consistent amino acid changes were observed in the viral RNAi suppressor gene 1A, nor elsewhere in the genome in any of the host backgrounds. Our study indicates that the RNAi response restricts the sequence space that can be explored by viral populations. Moreover, our study illustrates how evolution towards higher virulence can be a highly reproducible, yet unpredictable process.

Arbovirus-vector protein interactomics identifies Loquacious as a co-factor for dengue virus replication in Aedes mosquitoes.

Efficient virus replication in Aedes vector mosquitoes is essential for the transmission of arboviral diseases such as dengue virus (DENV) in human populations. Like in vertebrates, virus-host protein-protein interactions are essential for viral replication and immune evasion in the mosquito vector. Here, 79 mosquito host proteins interacting with DENV non-structural proteins NS1 and NS5 were identified by label-free mass spectrometry, followed by a functional screening. We confirmed interactions with host factors previously observed in mammals, such as the oligosaccharyltransferase complex, and we identified protein-protein interactions that seem to be specific for mosquitoes. Among the interactors, the double-stranded RNA (dsRNA) binding protein Loquacious (Loqs), an RNA interference (RNAi) cofactor, was found to be essential for efficient replication of DENV and Zika virus (ZIKV) in mosquito cells. Loqs did not affect viral RNA stability or translation of a DENV replicon and its proviral activity was independent of its RNAi regulatory activity. Interestingly, Loqs colocalized with DENV dsRNA replication intermediates in infected cells and directly interacted with high affinity with DENV RNA in the 3' untranslated region in vitro (KD = 48-62 nM). Our study provides an interactome for DENV NS1 and NS5 and identifies Loqs as a key proviral host factor in mosquitoes. We propose that DENV hijacks a factor of the RNAi mechanism for replication of its own RNA.

Using human iPSC-derived kidney organoids to decipher SARS-CoV-2 pathology on single cell level.

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).

Cationic Geminoid Peptide Amphiphiles Inhibit DENV2 Protease, Furin, and Viral Replication.

Dengue is an important arboviral infectious disease for which there is currently no specific cure. We report gemini-like (geminoid) alkylated amphiphilic peptides containing lysines in combination with glycines or alanines (C15H31C(O)-Lys-(Gly or Ala)nLys-NHC16H33, shorthand notation C16-KXnK-C16 with X = A or G, and n = 0-2). The representatives with 1 or 2 Ala inhibit dengue protease and human furin, two serine proteases involved in dengue virus infection that have peptides with cationic amino acids as their preferred substrates, with IC50 values in the lower µM range. The geminoid C16-KAK-C16 combined inhibition of DENV2 protease (IC50 2.3 µM) with efficacy against replication of wildtype DENV2 in LLC-MK2 cells (EC50 4.1 µM) and an absence of toxicity. We conclude that the lysine-based geminoids have activity against dengue virus infection, which is based on their inhibition of the proteases involved in viral replication and are therefore promising leads to further developing antiviral therapeutics, not limited to dengue.