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Adenoviruses are the most commonly used viral vectors for clinical applications, mainly for the treatment of cancer and for the prevention of infectious diseases. A large number of adenoviruses—over 100 types have been isolated from humans—have evolved to infect different cells and tissues and to cause a range of diseases. As can be expected, several distinct entry mechanisms have been identified and characterized, which contribute to, or even determine cell and tissue tropism. Knowledge about adenovirus-host factors interactions is important for efficient and specific transduction of adenovirus vectors to cells and tissues of interest. Here we describe the state-of-the-art of human adenovirus cell entry, and discuss with perspectives outstanding questions in the field.

Human adenovirus species D (HAdV-D) contains two-thirds of all known HAdV types (116 in total) and is important as a vector in clinical applications. However, the broad tropism exhibited by several HAdV-D types poses challenges for their use as targeted gene delivery vectors. Since adenoviral tropism is largely governed by receptor usage, we aimed to determine the relative importance of known adenovirus receptors in mediating infection by different HAdV-D types. Here, we generated A549 single-cell CRISPR/Cas9 knockout clones of desmoglein 2 (DSG2), CD46, the coxsackievirus and adenovirus receptor (CAR), and cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS; needed for biosynthesis of sialic acid-containing glycans), and assessed their relative importance for infection by 18 different HAdV-D types. We show that CD46 is the most important receptor for a majority of species D HAdVs. Minor changes in infection levels were noted with A549-ΔCAR and A549-ΔDSG2 cells, whereas A549-ΔCMAS cells displayed an increased susceptibility to infection. We proceed to show that HAdV-D types require CD46 for efficient attachment to A549 cells, and surface plasmon resonance analysis demonstrates that their hexon proteins bind CD46 in an avidity-dependent manner. Strategies to retarget HAdV-D vectors should thus consider hexon-CD46 interactions as a critical determinant of tropism, as CD46 is broadly expressed in vivo. These results increase our understanding of adenovirus-host interactions and will guide the development and targeting of vectors based on HAdV-D types. IMPORTANCE: Several human adenovirus species D (HAdV-D) types are currently used, or under development, as viral vectors for vaccines and gene delivery. However, the unusually broad tropism observed in many HAdV-D types limits their specificity and effectiveness as targeted vectors. Since tropism is largely governed by receptor usage, and previous studies have reported conflicting findings on receptor preferences within this species, clarifying receptor usage is essential. In this study, we systematically investigated receptor usage in 18 different HAdV-D types and identified CD46 as the primary receptor. Since CD46 is widely expressed across human tissues, our findings explain the broad cellular tropism of these viruses and provide valuable insight for the rational design and refinement of HAdV-D-based vectors.

Human adenovirus type 36 (HAdV-D36) has been putatively linked to obesity in animals and has been associated with obesity in humans in some but not all studies. Despite extensive epidemiological research there is limited information about its receptor profile. We investigated the receptor portfolio of HAdV-D36 using a combined structural biology and virology approach. The HAdV-D36 fiber knob domain (FK), which mediates the primary attachment of many HAdVs to host cells, has a significantly elongated DG loop that alters known binding interfaces for established adenovirus receptors such as the coxsackie- and adenovirus receptor (CAR) and CD46. Our data suggest that HAdV-D36 attaches to host cells using a versatile receptor pool comprising sialic acid-containing glycans and CAR. Sialic acids are recognized at the same binding site used by other HAdVs of species D such as HAdV-D37. Using glycan microarrays, we demonstrate that HAdV-D36 displays a binding preference for glycans containing a rare sialic acid variant, 4-O,5-N-diacetylneuraminic acid, over the more common 5-N-acetylneuraminic acid. To date, this sialic acid variant has not been detected in humans, although it can be synthesized by various animal species, including a range of domestic and livestock animals. Taken together, our results indicate that HAdV-D36 has evolved to recognize a specialized set of primary attachment receptors that are different from known HAdV types and coincides with a unique host range and pathogenicity profile.

Although adenoviruses (AdVs) possess advantageous features as vectors, several challenges remain. These include a high prevalence of neutralizing antibodies against certain AdV types and the inability to efficiently transduce CAR-deficient cells and tissues. We showed previously that lactoferricin (Lfcin) enhances CAR-independent HAdV-C5 infection of epithelial and T-cells. Here, we assessed the ability of Lfcin to enable HAdV-C5 infection and transduction of human skeletal muscle cells. Lfcin increases HAdV-C5 infection and transduction of muscle myoblasts and myotubes by 10- to 30-fold. Enhanced infection correlates with increased cell binding, which differs mechanistically from that of coagulation factor X-mediated binding, as it remains unaffected by the removal of heparan sulfate. Additionally, Lfcin reduces the neutralizing effects of serum against HAdV-C5, suggesting it may shield key epitopes. By enabling viral binding to muscle cells and mitigating serum neutralization, Lfcin offers a novel strategy to improve the efficiency and durability of HAdV-C5-based gene delivery systems.

The International Committee on Taxonomy of Viruses (ICTV) holds a ratification vote annually after review of newly proposed taxa by ICTV Study Groups and members of the virology community. In March 2025, the vote outcome of the 11 proposals within the mandate of the Animal DNA Viruses and Retroviruses Subcommittee was made public. Here, we provide a summary of the newly accepted proposals. These include reorganization of taxa in the realm Varidnaviria, classification of the ‘polinton-like’ viruses into a new family (Phypoliviridae) within a new order Archintovirales; establishment of a new phylum (Commensaviricota) in the kingdom Shotokuvirae; the establishment of a new family called Filamentoviridae with two new genera and three new species; the addition of four new genera in the family Anelloviridae with 70 new species; and the addition of 85 new species in the families Adenoviridae (n=16), Baculoviridae (n=5), Circoviridae (n=5), Parvoviridae (n=55) and Polyomaviridae (n=4). Also, in the family Belpaoviridae, 11 species were renamed to comply with the binomial requirement for species names.

Background and Purpose: Human adenovirus (HAdV) causes respiratory or gastrointestinal tract infections depending on the virus subtype. While HAdV infections are generally self-limiting in immunocompetent people, they can result in significant morbidity and mortality in immunocompromised adults and children. Due to the limited availability of effective therapeutic options, there is an urgent need for novel therapeutics to combat HAdV infection and mitigate its severity.

Experimental Approach: Here, we have repurposed the clinically well-used antifungal, itraconazole, to control HAdV infection. We tested the antiviral potential of the itraconazole and the mTOR inhibitor Ku-63794 on the production of infectious HAdV in A549 and Caco-2 cells as well as human intestinal organoids (HIOs). Additionally, we evaluated the benefit of a combination of these host-directed drugs with the direct-acting antiviral brincidofovir.

Key Results: Pharmacological treatment with itraconazole significantly reduced virus titres in different in vitro models, including HIOs. Treatment with itraconazole impairs HAdV entry by entrapping incoming virus particles in endolysosomes and by promoting autophagy in HAdV-infected cells. Moreover, combining itraconazole with brincidofovir, a cidofovir derivative currently under clinical evaluation for anti-HAdV applications, demonstrated a synergistic effect in reducing HAdV titres.

Conclusion and Implications: Given the gastrointestinal toxicity associated with brincidofovir, its combination with the host-directed drug itraconazole allowed lower brincidofovir doses to be used to decrease HAdV titres, thereby minimizing adverse drug effects while maintaining antiviral efficacy.

Mammalian α-defensins are a family of abundant effector peptides of the mucosal innate immune system. Although primarily considered to be antimicrobial, α-defensins can increase rather than block infection by certain prominent bacterial and viral pathogens in cell culture and in vivo. We have shown previously that exposure of mouse and human adenoviruses (HAdVs) to α-defensins is able to overcome competitive inhibitors that block cell binding, leading us to hypothesize a defensin-mediated binding mechanism that is independent of known viral receptors. To test this hypothesis, we used genetic approaches to demonstrate that none of several primary receptors nor integrin co-receptors are needed for human α-defensin-mediated binding of HAdV to cells; however, infection remains integrin dependent. Thus, our studies have revealed a novel pathway for HAdV binding to cells that bypasses viral primary receptors. We speculate that this pathway functions in parallel with receptor-mediated entry and contributes to α-defensin-enhanced infection of susceptible cells. Remarkably, we also found that in the presence of α-defensins, HAdV tropism is expanded to non-susceptible cells, even when viruses are exposed to a mixture of both susceptible and non-susceptible cells. Therefore, we propose that in the presence of sufficient concentrations of α-defensins, such as in the lung or gut, integrin expression rather than primary receptor expression will dictate HAdV tropism in vivo. In summary, α-defensins may contribute to tissue tropism not only through the neutralization of susceptible viruses but also by allowing certain defensin-resistant viruses to bind to cells independently of previously described mechanisms.

Adenovirus (AdV) infection of the respiratory epithelium is common but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackieadenovirus receptor (CAR) for attachment and subsequently integrins for entry. CAR and integrins are however located deep within the tight junctions in the mucosa where they would not be easily accessible. Recently, a model for CAR-independent AdV entry was proposed. In this model, human lactoferrin (hLF), an innate immune protein, aids the viral uptake into epithelial cells by mediating interactions between the major capsid protein, hexon, and yet unknown host cellular receptor(s). However, a detailed understanding of the molecular interactions driving this mechanism is lacking. Here, we present a new cryo-EM structure of HAdV-5C hexon at high resolution alongside a hybrid structure of HAdV-5C hexon complexed with human lactoferrin (hLF). These structures reveal the molecular determinants of the interaction between hLF and HAdV-C5 hexon. hLF engages hexon primarily via its N-terminal lactoferricin (Lfcin) region, interacting with hexon’s hypervariable region 1 (HVR-1). Mutational analyses pinpoint critical Lfcin contacts and also identify additional regions within hLF that critically contribute to hexon binding. Our study sheds more light on the intricate mechanism by which HAdV-C5 utilizes soluble hLF/Lfcin for cellular entry. These findings hold promise for advancing gene therapy applications and inform vaccine development.

Enteric adenovirus types F40 and 41 (EAdVs) are a leading cause of diarrhea and diarrhea-associated death in young children and have recently been proposed to cause acute hepatitis in children. EAdVs have a unique capsid architecture and exhibit — unlike other human adenoviruses — a relatively strict tropism for gastrointestinal tissues with, to date, understudied infection mechanism and unknown target cells. In this study, we turn to potentially limiting host factors by comparing EAdV entry in cell lines with respiratory and intestinal origin by cellular perturbation, virus particle tracking, and transmission electron microscopy. Our analyses highlight kinetic advantages for EAdVs in duodenal HuTu80 cell infection and reveal a larger fraction of mobile particles, faster virus uptake, and infectious particle entry in intestinal cells. Moreover, EAdVs display a dependence on clathrin- and dynamin-dependent pathways in intestinal cells. Detailed knowledge of virus entry routes and host factor requirements is essential to understanding pathogenesis and developing new countermeasures. Hence, this study provides novel insights into the entry mechanisms of a medically important virus with emerging tropism in a cell line originating from a relevant tissue. IMPORTANCE Enteric adenoviruses have historically been difficult to grow in cell culture, which has resulted in lack of knowledge of host factors and pathways required for infection of these medically relevant viruses. Previous studies in non-intestinal cell lines showed slow infection kinetics and generated comparatively low virus yields compared to other adenovirus types. We suggest duodenum-derived HuTu80 cells as a superior cell line for studies to complement efforts using complex intestinal tissue models. We show that viral host cell factors required for virus entry differ between cell lines from distinct origins and demonstrate the importance of clathrin-mediated endocytosis.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by rich deposits of extracellular matrix (ECM), affecting the pathophysiology of the disease. Here, we identified galectin 4 (gal 4) as a cancer cell produced protein deposited into the ECM of PDAC tumors and detected high circulating levels of gal 4 in PDAC patients. In orthotopic transplantation experiments we observed increased infiltration of T-cells and prolonged survival in immunocompetent mice transplanted with cancer cells with reduced expression of gal 4. Increased survival was not observed in immunodeficient RAG1-/- mice, demonstrating that the effect was mediated by the adaptive immune system. Furthermore, by performing single-cell RNA-sequencing we found that the myeloid compartment and cancer-associated fibroblast (CAF) subtypes were altered in the transplanted tumors. Reduced gal 4 expression was associated with higher proportion of myofibroblastic CAFs and reduced numbers of inflammatory CAFs. We also found higher proportions of M1 macrophages, T-cells and antigen presenting dendritic cells in tumors with reduced gal 4 expression. Using a co-culture system, we observed that extracellular gal 4 induced apoptosis in T-cells by binding N-glycosylation residues on CD3 epsilon/delta. Hence, we show that gal 4 is involved in immune evasion and identify gal 4 as a promising drug target for overcoming immunosuppression in PDAC.