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.

SARS-CoV-2 RNA in exhaled air of hospitalized COVID-19 patients.

Knowledge about contagiousness is key to accurate management of hospitalized COVID-19 patients. Epidemiological studies suggest that in addition to transmission through droplets, aerogenic SARS-CoV-2 transmission contributes to the spread of infection. However, the presence of virus in exhaled air has not yet been sufficiently demonstrated. In pandemic situations low tech disposable and user-friendly bedside devices are required, while commercially available samplers are unsuitable for application in patients with respiratory distress. We included 49 hospitalized COVID-19 patients and used a disposable modular breath sampler to measure SARS-CoV-2 RNA load in exhaled air samples and compared these to SARS-CoV-2 RNA load of combined nasopharyngeal throat swabs and saliva. Exhaled air sampling using the modular breath sampler has proven feasible in a clinical COVID-19 setting and demonstrated viral detection in 25% of the patients.

The calcium channel inhibitor lacidipine inhibits Zika virus replication in neural progenitor cells.

After decades of being considered non-pathogenic, Zika virus (ZIKV) emerged as an important threat to human health during the epidemic of 2015-2016. ZIKV infections are usually asymptomatic, but can cause Guillain-Barré syndrome in adults and microcephaly in newborns. As there are currently no approved antiviral drugs against ZIKV, we tested anti-ZIKV activity of compounds from the NIH Clinical Collection for which we previously showed antiviral activity against the related dengue virus. One of the top hits from the screen was lacidipine, a 1,4-dihydropyridine calcium antagonist that is approved as an antihypertensive drug. Our data show that lacidipine is antiviral against ZIKV (strain H/PF/2013) in both Vero cells and induced pluripotent stem cell (iPSC)-derived human neural progenitor cells with IC50 values of 3.0 µM and <50 nM, respectively. The antiviral effect was also observed against four other ZIKV strains from the African and Asian lineages. Time-of-addition and replicon assays indicated that lacidipine acts at the post-entry stage of the viral replication cycle, inhibiting viral genome replication. Lacidipine altered the subcellular distribution of free cholesterol and neutral lipids, suggesting that the antiviral effect of lacidipine is mediated by altered trafficking of lipids. Together, these results identify lacidipine as a novel inhibitor of ZIKV replication that likely disturbs trafficking of lipids needed for replication organelle formation.

SARS-CoV-2 infects the human kidney and drives fibrosis in kidney organoids.

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.

Posaconazole inhibits multiple steps of the alphavirus replication cycle.

Repurposing drugs is a promising strategy to identify therapeutic interventions against novel and re-emerging viruses. Posaconazole is an antifungal drug used to treat invasive aspergillosis and candidiasis. Recently, posaconazole and its structural analog, itraconazole were shown to inhibit replication of multiple viruses by modifying intracellular cholesterol homeostasis. Here, we show that posaconazole inhibits replication of the alphaviruses Semliki Forest virus (SFV), Sindbis virus and chikungunya virus with EC50 values ranging from 1.4 µM to 9.5 µM. Posaconazole treatment led to a significant reduction of virus entry in an assay using a temperature-sensitive SFV mutant, but time-of-addition and RNA transfection assays indicated that posaconazole also inhibits post-entry stages of the viral replication cycle. Virus replication in the presence of posaconazole was partially rescued by the addition of exogenous cholesterol. A transferrin uptake assay revealed that posaconazole considerably slowed down cellular endocytosis. A single point mutation in the SFV E2 glycoprotein, H255R, provided partial resistance to posaconazole as well as to methyl-ß-cyclodextrin, corroborating the effect of posaconazole on cholesterol and viral entry. Our results indicate that posaconazole inhibits multiple steps of the alphavirus replication cycle and broaden the spectrum of viruses that can be targeted in vitro by posaconazole, which could be further explored as a therapeutic agent against emerging viruses.

Interferon gamma immunotherapy in five critically ill COVID-19 patients with impaired cellular immunity: A case series.

BACKGROUND: Prolonged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) shedding has been described in immunocompromised coronavirus disease 2019 (COVID-19) patients, resulting in protracted disease and poor outcome. Specific therapy to improve viral clearance and outcome for this group of patients is currently unavailable. METHODS: Five critically ill COVID-19 patients with severe defects in cellular immune responses, high SARS-CoV-2 viral RNA loads, and no respiratory improvement were treated with interferon gamma, 100 µg subcutaneously, thrice weekly. Bronchial secretion was collected every 48 h for routine diagnostic SARS-CoV-2 RT-PCR and viral culture. FINDINGS: Interferon gamma administration was followed by a rapid decline in SARS-CoV-2 load and a positive-to-negative viral culture conversion. Four patients recovered, and no signs of hyperinflammation were observed. CONCLUSIONS: Interferon gamma may be considered as adjuvant immunotherapy in a subset of immunocompromised COVID-19 patients. FUNDING: A.v.L. and R.v.C. are supported by National Institutes of Health (R01AI145781). G.J.O. and R.P.v.R. are supported by a VICI grant (016.VICI.170.090) from the Dutch Research Council (NWO). W.F.A. is supported by a clinical fellowship grant (9071561) of Netherlands Organization for Health Research and Development. M.G.N. is supported by an ERC advanced grant (833247) and a Spinoza grant of the Netherlands Organization for Scientific Research.

A piRNA-lncRNA regulatory network initiates responder and trailer piRNA formation during mosquito embryonic development.

PIWI-interacting (pi)RNAs are small silencing RNAs that are crucial for the defense against transposable elements in germline tissues of animals. In Aedes aegypti mosquitoes, the piRNA pathway also contributes to gene regulation in somatic tissues, illustrating additional roles for piRNAs and PIWI proteins besides transposon repression. Here, we identify a highly abundant endogenous piRNA (propiR1) that associates with both Piwi4 and Piwi5. PropiR1-mediated target silencing requires base-pairing in the seed region with supplemental base-pairing at the piRNA 3' end. Yet, propiR1 represses a limited set of targets, among which is the lncRNA AAEL027353 (lnc027353). Slicing of lnc027353 initiates production of responder and trailer piRNAs from the cleavage fragment. Expression of propiR1 commences early during embryonic development and mediates degradation of maternally provided lnc027353 Both propiR1 and its lncRNA target are conserved in the closely related Aedes albopictus mosquito, underscoring the importance of this regulatory network for mosquito development.

Endogenous piRNA-guided slicing triggers responder and trailer piRNA production from viral RNA in Aedes aegypti mosquitoes.

In the germline of animals, PIWI interacting (pi)RNAs protect the genome against the detrimental effects of transposon mobilization. In Drosophila, piRNA-mediated cleavage of transposon RNA triggers the production of responder piRNAs via ping-pong amplification. Responder piRNA 3' end formation by the nuclease Zucchini is coupled to the production of downstream trailer piRNAs, expanding the repertoire of transposon piRNA sequences. In Aedes aegypti mosquitoes, piRNAs are generated from viral RNA, yet, it is unknown how viral piRNA 3' ends are formed and whether viral RNA cleavage gives rise to trailer piRNA production. Here we report that in Ae. aegypti, virus- and transposon-derived piRNAs have sharp 3' ends, and are biased for downstream uridine residues, features reminiscent of Zucchini cleavage of precursor piRNAs in Drosophila. We designed a reporter system to study viral piRNA 3' end formation and found that targeting viral RNA by abundant endogenous piRNAs triggers the production of responder and trailer piRNAs. Using this reporter, we identified the Ae. aegypti orthologs of Zucchini and Nibbler, two nucleases involved in piRNA 3' end formation. Our results furthermore suggest that autonomous piRNA production from viral RNA can be triggered and expanded by an initial cleavage event guided by genome-encoded piRNAs.

Zooming in on targets of mosquito small RNAs.

Small RNAs are crucial for the regulation of basic cellular processes and protection against viruses and transposons in mosquitoes. Rozen-Gagnon et al. established CLIP (cross-linking and immunoprecipitation) for Argonaute proteins in Aedes aegypti. Their study sheds light on small RNA-target interactions in mosquitoes and provides an important resource for further study.

Neutrophil Extracellular Traps in Dengue Are Mainly Generated NOX-Independently.

Neutrophil extracellular traps (NETs) are increasingly recognized to play a role in the pathogenesis of viral infections, including dengue. NETs can be formed NADPH oxidase (NOX)-dependently or NOX-independently. NOX-independent NETs can be induced by activated platelets and are very potent in activating the endothelium. Platelet activation with thrombocytopenia and endothelial dysfunction are prominent features of dengue virus infection. We postulated that dengue infection is associated with NOX-independent NET formation, which is related to platelet activation, endothelial perturbation and increased vascular permeability. Using our specific NET assays, we investigated the time course of NET formation in a cohort of Indonesian dengue patients. We found that plasma levels of NETs were profoundly elevated and that these NETs were predominantly NOX-independent NETs. During early recovery phase (7-13 days from fever onset), total NETs correlated negatively with platelet number and positively with platelet P-selectin expression, the binding of von Willebrand factor to platelets and levels of Syndecan-1. Patients with gall bladder wall thickening, an early marker of plasma leakage, had a higher median level of total NETs. Ex vivo, platelets induced NOX-independent NET formation in a dengue virus non-structural protein 1 (NS1)-dependent manner. We conclude that NOX-independent NET formation is enhanced in dengue, which is most likely mediated by NS1 and activated platelets.

PIWI proteomics identifies Atari and Pasilla as piRNA biogenesis factors in Aedes mosquitoes.

As in most arthropods, the PIWI-interacting RNA (piRNA) pathway in the vector mosquito Aedes aegypti is active in diverse biological processes in both soma and germline. To gain insights into piRNA biogenesis and effector complexes, we mapped the interactomes of the somatic PIWI proteins Ago3, Piwi4, Piwi5, and Piwi6 and identify numerous specific interactors as well as cofactors associated with multiple PIWI proteins. We describe the Piwi5 interactor AAEL014965, the direct ortholog of the Drosophila splicing factor pasilla. We find that Ae. aegypti Pasilla encodes a nuclear isoform and a cytoplasmic isoform, the latter of which is required for efficient piRNA production. In addition, we characterize a splice variant of the Tudor protein AAEL008101/Atari that associates with Ago3 and forms a scaffold for PIWI proteins and target RNAs to promote ping-pong amplification of piRNAs. Our study provides a useful resource for follow-up studies of somatic piRNA biogenesis, mechanism, and function in Aedes mosquitoes.