While efforts to eradicate poliovirus are ongoing, non-polio enteroviruses (NPEV) continue to pose a substantial public health threat. NPEVs cause a wide range of diseases, from mild self-limiting infections to severe respiratory, neurological, and cardiac complications, with the highest burden occurring in children and immunocompromised populations. Recent advances in human stem cell-derived cultures, organoids, air-liquid interface systems, and assembloids have transformed the study of NPEV pathogenesis by providing physiologically relevant models that recapitulate human tissue architecture, cellular complexity, and host responses. In this review, we discuss how these advanced in vitro systems have enhanced our understanding of the determinants of viral tropism, including receptor usage, epithelial polarity, tissue-specific susceptibility, and viral genetic variation. We further highlight insights into serotype-dependent differences in innate immune responses, tissue injury, and dissemination mechanisms. Finally, we explore the potential of these models for antiviral discovery and personalized therapeutic development, emphasizing their value for advancing both fundamental and translational NPEV research.

All coronaviruses encode one or two multifunctional papain-like proteases (PLPs) with protease activity that cleaves the viral polyprotein, along with deubiquitinating (DUB) and deISGylase activities that remove ubiquitin and interferon-stimulated gene 15 (ISG15) from viral and host proteins. Studying the role of coronavirus deISGylase activity in viral infection and pathogenesis has been challenging due to the inability to generate a viable deISGylase-deficient coronavirus. To address this issue, we used a recently characterized PLP domain from human coronavirus HKU1 that has intact protease and DUB activity, but minimal deISGylase activity. Switching the HKU1 PLP2 domain with the mouse hepatitis virus strain A59 (MHV-A59) PLP2 domain allowed us to generate a viable chimeric virus, MHV-HP2, that replicated to high titers in cell culture. Analysis of the PLP activities of MHV-HP2 revealed that the virus was impaired in deISGylase activity, intact in its DUB activity, and delayed in proteolytic processing at early time points. Detailed analysis of MHV-HP2 replication kinetics revealed that the delay in proteolytic processing coincided with a transient replication delay in cell culture. This replication delay was not rescued by knockdown of ISG15, suggesting proteolytic delay rather than protein ISGylation drives the delay in replication. Importantly, we found that MHV-HP2 is attenuated for replication and pathogenesis in vivo, with infected mice showing more rapid clearance of MHV-HP2 and reduced evidence of liver damage and inflammation as compared to MHV-A59-infected mice. These results document the generation of a murine coronavirus with diminished deISGylase activity and altered protease activity that highlight the importance of coronavirus PLP in coronavirus pathogenesis.IMPORTANCECoronaviruses are adept at circumventing host antiviral defenses by encoding the multifunctional papain-like protease (PLP). PLP modulates the host cellular environment with its proteolytic, deubiquitinase, and deISGylase activities. Coronavirus deISGylase activity targets the host protein interferon-stimulated gene 15 (ISG15), which can be conjugated to host and viral proteins to promote an antiviral state. To assess the contribution of viral deISGylase activity to pathogenesis, we generated a chimeric mouse coronavirus lacking robust deISGylase activity. Characterization of the chimeric virus revealed that the virus was also inefficient in proteolytic processing. We determined that diminished deISGylase activity and altered PLP protease activity resulted in attenuated pathogenesis in a mouse model system, since mice infected with the chimeric virus exhibited rapid clearance of infection and less severe tissue damage as compared to infection with the parental virus. These results reveal that modulating coronavirus PLP deISGylase and proteolytic activities can reduce disease burden.

Viruses alter host gene expression to create a proviral environment, while the host simultaneously regulates gene expression to restrict viral spread. Owing to RNA viruses' complete reliance on the host translational machinery, it is important to assess translational control during virus infection. Therefore, we used ribosome profiling (Ribo-seq) paired with RNA-seq to observe how red clover necrotic mosaic virus (RCNMV) infection of Arabidopsis plants alters cellular gene expression at the levels of mRNA abundance and translation efficiency. We determined that at 5 days post-inoculation (dpi), the translational response to RCNMV infection is enriched in genes of the innate immune system. Expression of a tumor necrosis factor receptor-associated factor (TRAF)-like protein, a regulator of development and immune response, was translationally but not transcriptionally upregulated early in systemic infection. By 8 dpi, many pathways were regulated/dysregulated, and unfolded protein response (UPR) genes were transcriptionally upregulated but with reduced translation efficiency. Ribosome profiling of RCNMV RNAs revealed (i) -1 programmed ribosomal frameshifting at 7.5%-8.0%, the first direct measurement of frameshift efficiency in infected cells for any plant virus; (ii) that coat protein is translated at extremely high efficiency, while the RNA-dependent RNA polymerase is translated least efficiently; and (iii) an unexpected extremely strong ribosomal pause site in the open reading frame that encodes the movement protein. To our knowledge, this is the first genome-wide study that assesses the translational control of gene expression in plants infected with a virus from the large and diverse Tombusviridae family.IMPORTANCEPositive-strand RNA viruses usurp the host's translation machinery to synthesize viral proteins. Moreover, translation of host mRNAs is altered by virus infection, both as part of the host immune response and by the virus to inhibit host defenses. To assess all these changes globally, we used ribosome profiling of plants infected with a member of the large and ubiquitous Tombusviridae family. We identified key host genes and pathways that were differentially altered in translation efficiency, giving us an understanding of host responses not detectable by conventional RNA sequencing. Moreover, ribosome profiling revealed (i) the most accurate calculation of the efficiency of ribosomal frameshifting during infection for any plant virus, (ii) the extremely high level of translation of viral coat protein, and (iii) an unexpectedly strong ribosomal pause site in the movement protein gene. This work provides an understanding of a new dimension of gene expression control in plant-virus interactions.

DEAD-box RNA helicase 21 (DDX21), a conserved RNA helicase of the DEAD-box family, serves as a key regulator of multiple fundamental cellular processes, including RNA metabolism, ribosome biogenesis, transcriptional regulation, and genomic stability. Our previous work has demonstrated an interaction between DDX21 and the nucleocapsid (N) protein of porcine deltacoronavirus (PDCoV). However, the specific role of DDX21 during PDCoV infection remains largely elusive. In the present study, we show that overexpression of DDX21 significantly restricts PDCoV infection in PK15, IPEC-J2, and HeLa cells, whereas knockout of DDX21 markedly enhances PDCoV replication in HeLa cells. Mechanistically, we identified that the N-terminal domainNTD of PDCoV N binds to the 1-217 amino acid region of DDX21, thereby inducing the cytoplasmic redistribution of DDX21 from the nucleus. Furthermore, DDX21-mediated suppression of PDCoV replication coincides with elevated interferon-β (IFN-β) production and interferon-stimulated geneISG expression. Importantly, PDCoV infection significantly downregulates DDX21 expression to antagonize its antiviral function, and similar antagonistic effects are observed for the coronaviruses from different genera. Collectively, our findings demonstrate that DDX21 interacts with the nucleocapsid protein and correlates with altered interferon signaling during coronavirus infection, which provides novel insights into the development of strategies for the prevention and control of coronavirus infections.

Bat-origin swine acute diarrhea syndrome coronavirus (SADS-CoV) causes diarrhea and death in piglets and also poses a cross-species transmission risk due to its broad host cell tropism. In the absence of commercial vaccines or therapeutics, identifying the host factors exploited by SADS-CoV may provide potential targets for antiviral therapies. Inhibition of glycogen synthase kinase 3 (GSK3) restricts diverse coronavirus replication by targeting the N protein and disrupting its phosphorylation. Here, we identified GSK3 as a crucial host factor for SADS-CoV RNA synthesis. Both GSK3 deficiency and treatment with a classical GSK3 inhibitor, lithium chloride (LiCl), strongly impaired SADS-CoV dsRNA synthesis. LiCl treatment did not alter electrophoretic mobility or expression of SADS-CoV N protein in N-transfected Huh7 cells but significantly reduced NSP12 protein levels. The absence of GSK3 led to the proteasomal degradation of NSP12 to reduce viral RNA synthesis. However, GSK3 interacted with ubiquitin-specific peptidase 27X (USP27X) and promoted the USP27X-mediated deubiquitination of NSP12, thereby enhancing NSP12 stabilization and facilitating viral RNA synthesis. LiCl not only protected neonatal mice from lethal SADS-CoV challenge but also exhibited a notable broad-spectrum antiviral effect against PEDV and TGEV. Our findings demonstrate that GSK3-mediated NSP12 stabilization is essential for SADS-CoV RNA synthesis, underscoring the potential of GSK3 as a broad-spectrum antiviral target for emerging and re-emerging swine coronaviruses.IMPORTANCEZoonotic transmission of coronaviruses continues to pose a significant threat to human populations. Swine acute diarrhea syndrome coronavirus (SADS-CoV) has been identified as a potentially high-risk emerging coronavirus pathogen that could adversely affect the global economy and human health. However, the replication mechanisms of SADS-CoV remain largely unexplored. Our study demonstrates that GSK3 stabilizes NSP12 by interacting with USP27X, thereby enhancing the synthesis of SADS-CoV dsRNA. LiCl, a GSK3 inhibitor, exhibits broad-spectrum antiviral activity against swine enteric alphacoronaviruses. Our work is distinguished for the first time by the GSK3-USP27X regulatory axis that controls the stability of NSP12 and indicates that GSK3 is a promising therapeutic target for the development of broad-spectrum antiviral drugs against emerging or re-emerging swine coronaviruses.

The global outbreak of SARS-CoV-2 has resulted in a renewed focus on coronaviruses with the potential for cross-species transmission and mutation risks. In particular, swine acute diarrhea syndrome coronavirus (SADS-CoV), potentially originating from the intermediate horseshoe bat (Rhinolophus affinis), is a novel coronavirus that causes acute diarrhea, vomiting, and high mortality in suckling piglets. Innate immunity plays a crucial role in defending the host against invading pathogens. Previous studies have identified several host factors that inhibit SADS-CoV replication through innate immunity; however, research on SADS-CoV remains insufficient compared to that on other coronaviruses. This study demonstrates that Moloney leukemia virus 10 protein (MOV10) exerts anti-SADS-CoV activity via its N-terminal domain in a helicase activity-independent manner. Mechanistic analysis reveals that MOV10 induces translocation of tripartite motif-containing 24 (TRIM24) from the nucleus to the cytoplasm. The cytoplasmically localized TRIM24 binds to MOV10 and TNF receptor-associated factor 3 (TRAF3) to form a ternary complex. Functional studies demonstrate that MOV10 strengthens the interaction between TRIM24 and TRAF3 in a dose-dependent manner and facilitates TRIM24-mediated K63-linked ubiquitination of TRAF3, thereby activating interferon immune responses. Furthermore, we identify the strategies by which SADS-CoV evades innate immunity. The viral N protein inhibits the antiviral effect of MOV10 by disrupting the interaction between MOV10 and TRAF3 and by promoting K48-linked polyubiquitination of TRAF3, leading to TRAF3 degradation via the ubiquitin-proteasome pathway. Collectively, our findings reveal the role of MOV10 in antiviral immunity and how SADS-CoV evades host immune defense.

Mammalian hantaviruses are RNA viruses that cause hantavirus cardiopulmonary syndrome in the Americas and hemorrhagic fever with renal syndrome in Eurasia. The cellular entry mechanisms of most hantaviruses remain poorly defined. To examine entry by phylogenetically distinct hantaviruses identified across Africa, Eurasia, and the Americas, we generated replication-competent recombinant vesicular stomatitis viruses (rVSVs) bearing Gn/Gc proteins from Necoclí, Sangassou, Thottapalayam, Kenkeme, Nova, Oxbow, and Tula viruses. All these Gn/Gc proteins except Kenkeme supported infection of primary human endothelial cells, indicating that endothelial cell entry is permissive for a broader range of hantaviruses than previously appreciated. Except for rVSV-Kenkeme, these rVSVs did not acquire additional mutations beyond pre-engineered rescue-enhancing changes during rescue and passaging. Genetic studies in human cells lacking protocadherin-1 (PCDH1) showed that Necoclí, Tula, and Nova viruses use PCDH1 for efficient infection, although the Nova phenotype was weaker. These three Gn/Gc proteins bound soluble PCDH1 with different apparent avidities, and infection by the corresponding rVSVs was inhibited by soluble PCDH1; Necoclí and Tula, but not Nova, were also blocked by a PCDH1-targeting monoclonal antibody. Authentic Tula virus infection was similarly reduced in PCDH1 knockout endothelial cells. Finally, the broadly reactive anti-Gn/Gc human monoclonal antibody ADI-42898 efficiently neutralized Necoclí, Nova, Sangassou, and Kenkeme rVSVs, but showed weak or undetectable activity against Oxbow, Tula, and Thottapalayam rVSVs. Together, these findings expand the range of hantavirus glycoproteins capable of mediating infection of human endothelial cells, broaden the phylogenetic scope of PCDH1-dependent entry, and identify receptor-targeted and viral glycoprotein-targeted strategies with differential activity.IMPORTANCEMany newly discovered hantaviruses are known only from sequence data, leaving their ability to enter human cells and their receptor usage unresolved. Using a BSL2-compatible recombinant vesicular stomatitis virus (rVSV) system, we show that glycoproteins from several divergent hantaviruses can mediate infection of primary human endothelial cells, indicating that endothelial cell entry is permissive for a broader range of hantaviruses than previously appreciated. We also show that protocadherin-1 (PCDH1), previously linked mainly to New World hantaviruses, is used by Necoclí, Tula, and Nova viruses but not universally across the panel, revealing broader but heterogeneous receptor usage. An authentic Tula virus experiment supports this conclusion beyond the surrogate system. Finally, a broadly reactive anti-Gn/Gc antibody neutralizes several, but not all, of these viruses, highlighting both the promise and the limits of broadly protective countermeasures and the utility of these rVSVs for evaluating entry inhibitors.

A recombinant vaccinia virus (vA4-HiBiT) was constructed with a short, split luciferase HiBiT tag at the C-terminus of the A4 core protein open reading frame. When HeLa or A549 cells stably expressing LgBiT were infected with vA4-HiBiT, the interaction of the split luciferase fragments reconstituted NanoLuc enzyme activity. Upon addition of a membrane-permeable luciferase substrate, a strong luminescence signal indicated core entry into the cytoplasm within 5 min after infection and was proportional to virus multiplicity within the range of 0.5-80 PFU/cell. This direct entry assay was dependent on a functional virus entry-fusion complex, was inhibited by a neutralizing antibody, and was unaffected by an inhibitor of protein synthesis. Entry was enhanced by brief pH 5 treatment of cell-bound virus relative to pH 7 and prevented by pH 3 treatment. Bafilomycin A1, an inhibitor of endosomal acidification, exhibited greater inhibition of entry in A549-LgBiT cells compared to HeLa-LgBiT cells, as did treatment with ammonium chloride, suggesting variations in entry pathways or cell metabolism. Inhibition of entry by latrunculin A and cytochalasin D indicated a role for actin polymerization in both cell lines. Despite the discovery of a conserved furin cleavage consensus sequence in the A16 subunit of the entry-fusion complex, a furin inhibitor did not reduce virus entry unlike results obtained with a human papillomavirus control. These data support the use of this NanoBiT assay for studies of vaccinia virus entry, with application to basic research, antivirals, and neutralizing antibodies.

Type I interferon induces multiple protein effectors to inhibit viral replication. Among these, the interferon-induced transmembrane protein (IFITM3) and the cholesterol-25-hydroxylase (CH25h) converge on the inhibition of viral entry by altering the behavior of membranes. Here, we dissect the functional and mechanistic relationship between these two membrane-acting effectors using different viruses. When using HIV-1 and vesicular stomatitis virus (VSV), IFITM3 and CH25h restrict viral entry with similar efficiency but act in a largely redundant manner, a finding that is consistent across infection systems, cell types, and entry assays. However, this is not the case for several orthoflaviviruses against which IFITM3, but not CH25h, provides a strong block to infection. We further uncover that the CH25h-25HC axis remodels IFITM3 trafficking by driving partial relocalization of IFITM3 from endolysosomal compartments to the plasma membrane. This occurs by impairing IFITM3 internalization into early endosomes and leads to an increase in its secretion in exosomal vesicles, overall indicating that CH25h contributes to regulate IFITM3 cellular dynamics. Together, these findings reveal that the apparent redundancy of maintaining two distinct effectors targeting the same step of the viral life cycle is not superfluous but instead allows the cell to compensate and, nonetheless, target viruses that may have developed resistance to one of these effectors. Furthermore, the ability of CH25h to remodel IFITM3 trafficking indicates that these two pathways are connected, adding an additional layer of complexity in the biology of IFITM3.IMPORTANCEIFITM3 and CH25h are two potent, broad-acting inhibitors of viral membrane fusion, but their functional connections have not been explored. Here, we show that although their antiviral activities are largely redundant against HIV-1 and vesicular stomatitis virus (VSV), IFITM3 functionally compensates for a relative resistance of orthoflaviviruses toward CH25h. Furthermore, CH25h redistributes IFITM3 from endosomal compartments to the plasma membrane, enhancing its release in exosomal vesicles. Overall, these findings reveal that despite the fact that IFITM3 and CH25h can act redundantly against certain classes of viruses, IFITM3 can functionally compensate for CH25h resistance against other viruses, highlighting why it is important for the cell to maintain two distinct pathways to block virus-to-cell membrane fusion. In addition, these results also reveal how interferon-driven lipid remodeling can reshape the trafficking behavior of IFITM3, leading it to be more present in exosomes, where it could exert paracrine functions that remain to be explored.

Ubiquitin-specific proteases (USPs) modulate diverse virus-host interaction events through deubiquitination; thus, investigating their functional roles and molecular targets facilitates the interpretation of viral pathogenic mechanisms and informs the design of anti-virus strategies. The deubiquitinase USP8 has been reported to antagonize HIV-1 replication by reversing Vif-mediated ubiquitination of the host restriction factor A3G and shielding it from proteasomal degradation. Here, we identify an additional A3G-independent anti-HIV mechanism of USP8. In A3G-negative cells, USP8 exerts a potent inhibitory effect on HIV-1 replication by significantly suppressing viral transcription. Mechanistically, USP8 interacts with the HIV-1 trans-activator Tat through its C-terminal domain and mediates the removal of Tat's polyubiquitination chains in a manner dependent on USP8 catalytic activity. This disrupts the association between Tat and the HIV-1 LTR transactivation response (TAR) element, thus impairing Tat-mediated viral transactivation. Furthermore, given the essential role of Tat in driving viral gene expression, silencing of USP8 triggers latent HIV-1 reactivation. Collectively, our results, along with previous work, reveal that USP8 acts as a compelling anti-HIV factor that, by regulating both host and viral proteins, ultimately suppresses HIV-1 replication. These results deepen our understanding of USP8-mediated HIV-1 restriction and provide insights for the development of novel anti-HIV therapeutic strategies.IMPORTANCEUbiquitination modification contributes a non-negligible part to the regulation of HIV-1 replication, and the interpretation of key factors involved in this process has significantly benefited our understanding of the virus-host interplay. USP8 is a ubiquitin-specific protease that was recently identified to restrict HIV-1 replication by stabilizing the host restriction factor A3G through reversing the Vif-mediated ubiquitination of A3G. Here, we demonstrate an additional A3G-independent anti-HIV mechanism of USP8. In A3G-negative cells, USP8 directly interacts with and deubiquitinates Tat to cleave its K63-linked ubiquitination, thereby suppressing Tat-mediated HIV-1 trans-activation. The suppression of Tat-driven viral transcription induced by USP8 requires the enzymatic activity of USP8 and is effective in different HIV-1 tropic strains. In addition, the fact that Tat is a novel substrate of USP8 is validated both in cells and by in vitro deubiquitination assays. These findings illustrate the dual regulatory roles of USP8 on both host antiviral factors and viral proteins, expanding our understanding of host-mediated HIV-1 restriction.

Respiratory syncytial virus (RSV) infection causes epithelial injury, barrier disruption, and formation of syncytia in human lung tissue; however, the regulatory host defense mechanisms involved remain poorly defined. ADAM17 is a multifunctional sheddase implicated in inflammatory lung disease and is considered a potential therapeutic target during viral infections. Here, we show that RSV infection induces ADAM17 activity through PKC- and ERK-dependent signaling, increasing surface ADAM17 levels and sheddase activity. Genetic loss or pharmacological inhibition of ADAM17 reduced RSV-induced formation of syncytia, post-entry viral spread, and viral titers. This identifies ADAM17 as a host factor that supports viral dissemination. ADAM17 deficiency simultaneously exacerbated RSV-induced epithelial injury, characterized by loss of intercellular cohesion, impaired barrier function, increased paracellular permeability, and enhanced cytotoxicity. RSV-induced ADAM17 activity promoted the release of multiple epithelial growth factor receptor (EGFR) ligands. Supplementation with recombinant amphiregulin partially restored barrier function in infected ADAM17-deficient cells. Consistent with these functional effects, ADAM17 deficiency was associated with the loss of epithelial differentiation-associated transcriptional programs and increased expression of inflammatory genes. These findings indicate that ADAM17 plays a dual role in RSV infection. It facilitates syncytial viral spread, while preserving epithelial integrity through autocrine EGFR signaling. Thus, ADAM17 activity represents a biological trade-off between viral dissemination and tissue protection, indicating that therapeutic ADAM17 inhibition may reduce viral spread at the cost of aggravating epithelial damage.IMPORTANCEDuring respiratory syncytial virus (RSV) infection, the airway epithelium must maintain barrier integrity while responding to virus-induced syncytium formation and tissue damage. It is poorly understood which host factors balance tissue preservation against viral replication and spread. Here, we demonstrate that the host cellular metalloproteinase ADAM17 is activated upon RSV infection. While RSV-induced ADAM17 activity promotes syncytium formation and viral spread, it concurrently controls the maintenance of epithelial barrier integrity, preserves cell survival, and inhibits hyperinflammatory signaling. By demonstrating that ADAM17 has a dual mode of action controlling both viral propagation and epithelial barrier resilience, this work uncovers a central trade-off: the same host factor exploited by RSV for dissemination is essential for protecting airway tissue. Consequently, targeting ADAM17 for antiviral therapy presents major challenges, as blocking viral spread may compromise vital epithelial defenses.

Coughing, speaking, and even quiet breathing can release virus-laden particles into the air, triggering transmission events that spread through households and communities. Understanding why some respiratory viruses transmit so efficiently, and under which biological or environmental conditions this occurs, is central to anticipating future outbreaks. In this review, we outline the biological and physical factors that shape respiratory virus transmission, highlighting how the site of replication, mechanisms of droplet generation, and environmental conditions influence airborne spread. We examine how viral traits such as receptor usage, tissue tropism, and innate immune evasion intersect with airway physiology to determine the size, composition, and infectiousness of exhaled particles. We lastly draw attention to key gaps in current knowledge, particularly regarding the host and environmental factors that govern both the release and uptake of virions. Filling these gaps will allow us to more accurately assess transmission risk and implement targeted approaches to reduce spread.

Recoviruses (ReCV) serve as valuable surrogates for fastidious human noroviruses (HuNoV). Although HuNoVs can be propagated in small intestinal enteroids, this system has several limitations, including low replication efficiency and an inability to sustain continuous propagation for generating infectious virus stocks beyond certain strains, such as GII.3. In contrast, ReCVs can be efficiently propagated in conventional cell lines (e.g., LLC-MK2), which has significantly advanced understanding of their biological characteristics. As a result, ReCVs are widely used as HuNoV surrogates. However, a physiologically relevant ReCV propagation system that closely mimics HuNoV enteroid cultures has not yet been described. Here, we report the establishment of intestinal stem cell-derived rhesus macaque enteroid lines representing both the small intestine and colon, along with two enteroid-derived cell lines. We further characterize these systems and evaluate their susceptibility to infection with three diverse ReCV isolates. Both small intestine- and colon-derived enteroids, as well as their corresponding cell lines, supported robust ReCV infection. The enteroids contained all major intestinal cell types. The duodenum-derived cell line comprised enteroendocrine cells (chromogranin A) and absorptive enterocytes (villin), whereas the colon-derived cell line included enteroendocrine cells and goblet cells (MUC2). Importantly, both cell lines formed tight barriers (transepithelial electrical resistance [TEER] > 3,000 Ω·cm[2]), making them far superior to LLC-MK2 cells (TEER ~ 100 Ω·cm[2]) for studies of viral entry and pathogenesis. The rhesus enteroids and intestinal cell lines developed in this study will enable comparative studies of HuNoV and ReCV replication in a physiologically relevant system and may have direct applications for culturing HuNoVs.IMPORTANCEHuman intestinal stem cell-derived enteroids have enabled the propagation of human noroviruses; however, this system has several important limitations, including high cost, low replication efficiency, and (except for a recent report for GII.3 human norovirus) the inability to sustain continuous virus propagation for the generation of infectious virus stocks. As a result, research still relies on human norovirus-containing clinical samples as the primary virus source. Here, we developed host-specific rhesus macaque enteroid cultures and two enteroid-derived cell lines that support robust recovirus replication. Studying recovirus replication in rhesus enteroids may help overcome key limitations of the human norovirus enteroid cultures. Moreover, because both natural and experimental human norovirus infections have been described in rhesus macaques, the rhesus enteroids and enteroid-derived cell lines described in this study may be directly applicable to human norovirus cultivation.

Orthoflavivirus virions consist of an envelope enclosing a nucleocapsid core. A critical step in orthoflavivirus assembly is the encapsidation of genomic RNA by the capsid protein to form the nucleocapsid. However, the molecular details of this process remain poorly understood, particularly whether capsid proteins bind genomic RNA through specific interactions. Here, we utilized West Nile virus (WNV) and dengue virus (DENV) as model systems to investigate heterologous genome RNA encapsidation. Employing a chimeric virus strategy, we demonstrated that the DENV-2 capsid protein can package WNV genomic RNA if two critical elements are preserved: cis-acting sequences essential for RNA synthesis and the cleavage site required for capsid maturation. Furthermore, by decoupling the WNV cis-acting elements from the capsid-coding sequence and replacing the WNV capsid protein with orthologous capsid proteins from other orthoflaviviruses, we provide direct virological evidence that orthoflavivirus capsid proteins package genomic RNAs in a non-sequence-specific manner. These findings advance our mechanistic understanding of orthoflavivirus genome packaging.IMPORTANCEThe encapsidation of genomic RNA by capsid proteins as the primary event in orthoflavivirus virion assembly is not well understood in current research; particularly, whether it involves specific packaging signals remains a question. The orthoflavivirus capsid protein plays a key role in both genome replication and viral particle assembly. Our comprehensive virological analyses reveal that orthoflavivirus capsid protein shows considerable interspecies packaging flexibility while critical replication and processing elements are preserved. These insights underscore a key aspect of orthoflavivirus biology: the conserved packaging function of capsid proteins across genus members is mediated by structural interactions, rather than sequence specificity.