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Human T-cell immunoglobulin and mucin domain 1 (TIM-1) facilitates infection of re-emerging viruses, including alphaviruses, through its phosphatidylserine (PS) binding domain. Although alphaviruses are transmitted to humans via mosquito bite, it is unclear whether mosquito-derived viruses also use TIM-1 to infect human cells. Since viruses acquire their PS-containing envelope from the host cell and insect cell membranes differ in lipid composition from mammalian membranes, we here investigate the role of TIM-1 at the mosquito-mammalian interface. We show that TIM-1 promotes infection with mosquito cell-derived, replication-competent alphaviruses, including Chikungunya virus, O’nyong’nyong virus (ONNV), and Sindbis virus. The TIM-1 PS-binding domain is essential for enhancing mosquito cell-derived ONNV infection as shown by TIM-1 mutagenesis. According to untargeted lipidomics, mosquito cell-derived ONNV virions contain higher levels of phosphatidylethanolamine (PE) and PS compared to mammalian cell-derived ONNV. Notably, TIM-1 engages PE as well as PS, as demonstrated by liposome competition. PS decarboxylase experiments suggest that PS is an important mediator of mosquito-cell derived ONNV entry into mammalian cells. Taken together, our data show that TIM-1 promotes mosquito cell-derived alphavirus infection of mammalian cells. This work provides implications for the understanding of the TIM-1 ligand repertoire and advances our understanding of arbovirus transmission at the mosquito-mammalian interface.

Raccoon dogs (Nyctereutes procyonoides) originated from East Asia and are listed as invasive alien species of Union concern in the European Union. Apart from their potential negative impact on native fauna as predators and competitors, they are discussed as reservoirs and vectors for various infectious diseases. Between 2021 and 2022, comprehensive post-mortem examinations of 110 raccoon dogs were performed as part of a health and risk assessment study of selected invasive alien species in Schleswig-Holstein, Germany. Parasitic infections, often accompanied by eosinophilic and/or granulomatous inflammation, were one of the most common and significant findings. In total, 64% animals had an ectoparasitosis and up to 76% an endoparasitosis. Alaria alata and Trichinella spp were the most relevant endoparasite species found. Eggs of A. alata were detected in 30% of faecal samples, while the prevalence of Trichinella spp in muscle samples was 0.9%. Influenza A virus, canine distemper virus, rustrela virus, Borna disease virus 1 and severe acute respiratory syndrome corona virus 2 were not detected. Carnivore protoparvovirus 1 was detected with a prevalence of 3%, but was not associated with pathognomonic lesions. In general, most of the pathological findings were of minor significance regardless of whether pathogens were detected or not, and therefore did not appear to affect the overall health status of the examined animals. Our study shows that raccoon dogs may contribute to the spread and persistence of certain pathogens by acting as carriers of infectious diseases. This underpins the need to further investigate the risk they pose to endemic European wildlife, livestock and human health.

Tetraspanins (Tspans) are transmembrane proteins that coordinate life cycle steps of viruses from distinct families. Here, we identify the human Tspan10 and Tspan15, both members of the TspanC8 subfamily, as replication factors for alphavirus Venezuelan equine encephalitis virus (VEEV) in astrocytoma cells. Pharmacological inhibition and small interfering RNA (siRNA)-mediated silencing of TspanC8 interactor a disintegrin and metalloproteinase 10 (ADAM10) reduced VEEV infection. Silencing of Tspan10, Tspan15, and ADAM10 did not affect VEEV entry but diminished viral genome replication. We report that Tspan10 is important for VEEV infection of several cell lines, while silencing of Tspan15 diminishes infection with several alphaviruses, but not flaviviruses, in astrocytoma cells. Conversely, we demonstrate that siRNA-mediated silencing of Tspan14, another member of the TspanC8 family, enhances infection with lentiviral pseudoparticles harbouring the envelope proteins of VEEV, identifying it as a restriction factor for VEEV entry. Silencing of ADAM10/Tspan15 substrate neuronal (N)-cadherin reduced VEEV infectivity, suggesting potential roles of ADAM10 substrates in VEEV infection. In sum, our study identifies three TspanC8s and ADAM10 as important modulators of VEEV infectivity.

Transmission of Zika virus (ZIKV) has been reported in 92 countries and the geographical spread of invasive virus-borne vectors has increased in recent years. Arboviruses naturally survive between vertebrate hosts and arthropod vectors. Transmission success requires the mosquito to feed on viraemic hosts. There is little specific understanding of factors that may promote ZIKV transmission-success. Here we show that mosquito host-seeking behaviour is impacted by viral infection of the vertebrae host and may be essential for the effective transmission of arboviruses like ZIKV. Human skin fibroblasts produce a variety of metabolites, and we show that ZIKV immediately alters gene/protein expression patterns in infected-dermal fibroblasts, altering their metabolism to increase the release of mosquito-attractive volatile organic compounds (VOCs), which improves its transmission success. We demonstrate that at the invasion stage, ZIKV differentially altered the emission of VOCs by significantly increasing or decreasing their amounts, while at the transmission stage of the virus, all VOCs are significantly increased. The findings are complemented by an extensive meta-proteome analysis. Overall, we demonstrate a multifaceted role of virus-host interaction and shed light on how arboviruses may influence the behaviour of their vectors as an evolved means of improving transmission-success.

Three-dimensional culture models of the brain enable the study of neuroinfection in the context of a complex interconnected cell matrix. Depending on the differentiation status of the neural cells, two models exist: 3D spheroids also called neurospheres and cerebral organoids. Here, we describe the preparation of 3D spheroids and cerebral organoids and give an outlook on their usage to study Rift Valley fever virus and other neurotropic viruses.

The synthesis of a library of halogenated rocaglate derivatives belonging to the flavagline class of natural products, of which silvestrol is the most prominent example, is reported. Their antiviral activity and cytotoxicity profile against a wide range of pathogenic viruses, including hepatitis E, Chikungunya, Rift Valley Fever virus and SARS-CoV-2, were determined. The incorporation of halogen substituents at positions 4′, 6 and 8 was shown to have a significant effect on the antiviral activity of rocaglates, some of which even showed enhanced activity compared to CR-31-B and silvestrol.

Human CD81 and CD9 are members of the tetraspanin family of proteins characterized by a canonical structure of four transmembrane domains and two extracellular loop domains. Tetraspanins are known as molecular facilitators, which assemble and organize cell surface receptors and partner molecules forming clusters known as tetraspanin-enriched microdomains. They have been implicated to play various biological roles including an involvement in infections with microbial pathogens. Here, we demonstrate an important role of CD81 for the invasion of epithelial cells by Salmonella enterica. We show that the overexpression of CD81 in HepG2 cells enhances invasion of various typhoidal and non-typhoidal Salmonella serovars. Deletion of CD81 by CRISPR/Cas9 in intestinal epithelial cells (C2BBe1 and HT29-MTX-E12) reduces S. Typhimurium invasion. In addition, the effect of human CD81 is species-specific as only human but not rat CD81 facilitates Salmonella invasion. Finally, immunofluorescence microscopy and proximity ligation assay revealed that both human tetraspanins CD81 and CD9 are recruited to the entry site of S. Typhimurium during invasion but not during adhesion to the host cell surface. Overall, we demonstrate that the human tetraspanin CD81 facilitates Salmonella invasion into epithelial host cells.

Affinity enrichment coupled with liquid chromatography–tandem mass spectrometry (AE–LC–MS/MS) enables a comprehensive study of virus–host protein–protein interactions in cells and tissues infected with Rift Valley fever virus (RVFV) or ectopically expressing RVFV proteins. Depending on the research question, different experimental setups with carefully chosen controls are needed. Here, we describe the detailed workflow of sample preparation, processing, and cleanup, while also outlining critical points to consider when designing and performing AE–LC–MS/MS experiments.

Severe acute respiratory coronavirus 2 (SARS-CoV-2) causes neurological disease in the peripheral and central nervous system (PNS and CNS, respectively) of some patients. It is not clear whether SARS-CoV-2 infection or the subsequent immune response are the key factors that cause neurological disease. Here, we addressed this question by infecting human induced pluripotent stem cell-derived CNS and PNS neurons with SARS-CoV-2. SARS-CoV-2 infected a low number of CNS neurons and did not elicit a robust innate immune response. On the contrary, SARS-CoV-2 infected a higher number of PNS neurons. This resulted in expression of interferon (IFN) λ1, several IFN-stimulated genes and proinflammatory cytokines. The PNS neurons also displayed alterations characteristic of neuronal damage, as increased levels of sterile alpha and Toll/interleukin receptor motif-containing protein 1, amyloid precursor protein and α-synuclein, and lower levels of cytoskeletal proteins. Interestingly, blockade of the Janus kinase and signal transducer and activator of transcription pathway by Ruxolitinib did not increase SARS-CoV-2 infection, but reduced neuronal damage, suggesting that an exacerbated neuronal innate immune response contributes to pathogenesis in the PNS. Our results provide a basis to study coronavirus disease 2019 (COVID-19) related neuronal pathology and to test future preventive or therapeutic strategies.

Assembly of infectious hepatitis C virus (HCV) particles requires multiple cellular proteins including for instance apolipoprotein E (ApoE). To describe these protein-protein interactions, we performed an affinity purification mass spectrometry screen of HCV-infected cells. We used functional viral constructs with epitope-tagged envelope protein 2 (E2), protein (p) 7, or nonstructural protein 4B (NS4B) as well as cells expressing a tagged variant of ApoE. We also evaluated assembly stage-dependent remodeling of protein complexes by using viral mutants carrying point mutations abrogating particle production at distinct steps of the HCV particle production cascade. Five ApoE binding proteins, 12 p7 binders, 7 primary E2 interactors, and 24 proteins interacting with NS4B were detected. Cell-derived PREB, STT3B, and SPCS2 as well as viral NS2 interacted with both p7 and E2. Only GTF3C3 interacted with E2 and NS4B, highlighting that HCV assembly and replication complexes exhibit largely distinct interactomes. An HCV core protein mutation, preventing core protein decoration of lipid droplets, profoundly altered the E2 interactome. In cells replicating this mutant, E2 interactions with HSPA5, STT3A/B, RAD23A/B, and ZNF860 were significantly enhanced, suggesting that E2 protein interactions partly depend on core protein functions. Bioinformatic and functional studies including STRING network analyses, RNA interference, and ectopic expression support a role of Rad23A and Rad23B in facilitating HCV infectious virus production. Both Rad23A and Rad23B are involved in the endoplasmic reticulum (ER)-associated protein degradation (ERAD). Collectively, our results provide a map of host proteins interacting with HCV assembly proteins, and they give evidence for the involvement of ER protein folding machineries and the ERAD pathway in the late stages of the HCV replication cycle.IMPORTANCEHepatitis C virus (HCV) establishes chronic infections in the majority of exposed individuals. This capacity likely depends on viral immune evasion strategies. One feature likely contributing to persistence is the formation of so-called lipo-viro particles. These peculiar virions consist of viral structural proteins and cellular lipids and lipoproteins, the latter of which aid in viral attachment and cell entry and likely antibody escape. To learn about how lipo-viro particles are coined, here, we provide a comprehensive overview of protein-protein interactions in virus-producing cells. We identify numerous novel and specific HCV E2, p7, and cellular apolipoprotein E-interacting proteins. Pathway analyses of these interactors show that proteins participating in processes such as endoplasmic reticulum (ER) protein folding, ER-associated protein degradation, and glycosylation are heavily engaged in virus production. Moreover, we find that the proteome of HCV replication sites is distinct from the assembly proteome, suggesting that transport process likely shuttles viral RNA to assembly sites.