Bovine herpesvirus type 1 (BoHV-1) causes severe respiratory and reproductive diseases in cattle, leading to significant economic losses worldwide. Current inactivated vaccines in China fail to induce robust cellular immunity and cannot differentiate infected from vaccinated animals (DIVA). To address these limitations, we constructed two triple-gene-deleted BoHV-1 vaccine candidates, ∆T3 (∆TK-∆gE/gI) and ∆g3 (∆gG-∆gE/gI), using Red/ET two-step recombination technology based on an infectious BAC clone. Their safety and immunogenicity were evaluated in a rabbit model. Both deletion mutants exhibited growth kinetics similar to the wild-type strain but with significantly reduced viral titers and plaque areas. The triple-gene deletion mutants showed superior safety compared with single-gene deletion mutants, with minimal clinical signs and low viral shedding. Immunization with ∆T3 and ∆g3 induced robust gB-specific and neutralizing antibody responses. Following challenge with the virulent BoHV-1 BC01 strain, vaccinated rabbits maintained normal body temperatures, showed significantly reduced viral shedding (approximately 100-fold), and exhibited milder pulmonary lesions. These findings demonstrate that ∆T3 high-dose and ∆g3 low-dose regimens are promising DIVA-compatible vaccine candidates, offering an effective strategy for IBR control and eradication programs.

ABSTRACT Since 2015, duck‐origin novel goose parvovirus (NGPV) has caused severe economic losses to the Chinese duck industry, with no licensed vaccines or treatments available. Our previous study constructed a recombinant duck enteritis virus (rDEV) from an attenuated vaccine strain, which conferred full protection against virulent DEV challenge. NGPV VP2 is the major immunogenic structural antigen. In this study, we generated a recombinant virus rNGPV VP2‐Cre by inserting the NGPV VP2 gene into the rDEV genome. VP2 expression and virus‐like particle assembly were confirmed. Safety and efficacy were evaluated in ducklings. The results showed that rNGPV VP2‐Cre was safe, facilitated clearance of NGPV and provided strong protection against challenge with a highly virulent DEV strain. In summary, rNGPV VP2‐Cre represents a promising bivalent vaccine candidate for concurrent control of DEV and NGPV infections.

Duck plague, caused by a highly contagious α-herpesvirus, poses a major threat to waterfowl farming. Although UL50 homologs have been studied in mammalian α-herpesviruses such as HSV-1 and PRV, their role in avian herpesviruses remains unknown. Here, we investigated the function of the DEV UL50 gene, which encodes a conserved viral dUTPase, using bioinformatics, molecular biology, and virological approaches. Sequence analysis confirmed that DEV UL50 retains conserved catalytic motifs and structural features characteristic of α-herpesvirus homologs. A UL50-deleted mutant (ΔUL50) was constructed using the Red recombination system. In vitro, ΔUL50 exhibited reduced replication efficiency in DEFs, characterized by smaller plaques and lower viral titers, although overall growth kinetics were broadly similar to the WT. Notably, in duck DRG neurons, ΔUL50 replication was nearly abolished. GFP-reporter BAC viruses further confirmed that ΔUL50 failed to spread in DRG neurons but retained propagation in DEFs, indicating a cell-type-dependent replication defect. In vivo, ΔUL50 displayed markedly reduced viral loads and attenuated virulence, with no mortality and milder clinical and histopathological changes. These findings demonstrate that UL50 is dispensable but replication-supportive, particularly in non-dividing neuronal cells, highlighting its role in DEV pathogenicity and extending understanding of α-herpesvirus biology beyond mammalian systems.

Dipeptidyl peptidase-4 (DPP4) and angiotensin-converting enzyme 2 (ACE2) are well-established receptors for merbecoviruses, yet the receptor usage of merbecoviruses of European and Asian hedgehogs (EriCoVs) remains unknown. Here, by testing hedgehog orthologs of known coronavirus receptors, we identify hedgehog aminopeptidase N (APN) as a functional receptor for various EriCoVs. Analysis of APN orthologs from 139 species reveals that EriCoVs exhibit a restricted host range, primarily utilizing APN from hedgehogs and, to a lesser extent, felids and some other species, shaped by specific molecular determinants at the virus–receptor interface. Cryo-EM analysis of EriCoV–APN complex reveals a novel APN-binding mode that is distinct from those used by alpha- and deltacoronaviruses. Functional assays further demonstrate that transmembrane serine protease 2 (TMPRSS2) enhances spike activation and promotes plasma membrane fusion. Neutralizing antibodies targeting the EriCoV RBD and hedgehog APN were generated and effectively blocked EriCoV pseudovirus entry or amplification in hedgehog-APN expressing human cells or primary hedgehog cells. The structural basis underlying the pan-EriCoV neutralization mediated by the RBD-targeting antibody BD23-0177 was further elucidated by cryo-EM analysis. Together, these findings uncover an unexpected convergent evolution of APN utilization among merbecoviruses, establishing a foundation for risk assessment and the development of targeted countermeasures.

Coxsackievirus A2 (CVA2) belongs to the enterovirus group A. In this study, we determined the wholegenome sequences of two strains of CVA2 virus isolated from the faeces of patients with hand, foot and mouth disease in Jinhua, Zhejiang Province, China, in 2024. Their nucleotide and amino acid sequences showed 79.12%-79.50% and 94.47%-94.93% identity with the prototypical Fleetwood strain, respectively, and 97.94% and 98.17% mutual identity, respectively. Recombination analysis showed that the P2 region of the two CVA2 strains recombined with Coxsackievirus A4 (CVA4/JN19838/ CHN/2019). This study obtained the wholegenome sequences of two CVA2 strains, enriching the molecular characterization of CVA2 in China.

Dipeptidyl peptidase-4 (DPP4) is a well-established receptor for several MERS-related coronaviruses (MERSr-CoVs) isolated from humans, camels, pangolins, and bats (1-6). However, the receptor usage of many genetically diverse bat MERSr-CoVs with broad geographical distributions remains poorly understood. Recent studies have identified angiotensin-converting enzyme 2 (ACE2) as an entry receptor for multiple merbecovirus clades. Here, using viral antigen and pseudovirus-based functional assays, we demonstrate that several bat merbecoviruses from the HKU25 clade previously thought to utilize DPP4 (7), employ ACE2 as their functional receptor. Cryo-electron microscopy analysis revealed that HsItaly2011 and VsCoV-a7 recognize ACE2 with a binding mode sharing similarity with that of HKU5 but involving remodeled interfaces and distinct ortholog selectivity, suggesting a common evolutionary origin of ACE2 utilization for these two clades of viruses. EjCoV-3, a strain closely related to the DPP4-using MERSr-CoV BtCoV-422, exhibited relatively broad ACE2 ortholog tropism and could utilize human ACE2 albeit suboptimally. Despite differences in entry mechanisms and spike proteolytic activation compared to MERS-CoV, these viruses remain sensitive to several broadly neutralizing antibodies and entry inhibitors. These findings redefine our understanding of the evolution of receptor usage among MERSr-CoVs and highlight the versatility of ACE2 as a functional receptor for diverse coronaviruses.

Goose astrovirus (GAstV) is an emerging pathogen that is widely distributed throughout China and can cause visceral gout, resulting in serious economic losses for the goose industry. Open reading frame 2 (ORF2) of this virus encodes the precursor capsid protein, which is essential for the assembly and antigenicity of these virions. To construct a bi-valent vaccine for controlling GAstV and duck enteritis virus (DEV) infection, an infectious bacterial artificial chromosome (BAC) clone of the DEV vaccine strain pDEV-EF1 was used to establish a recombinant DEV vector for GAstV ORF2 gene delivery. GAstV ORF2 expression frame was inserted into the US7 and US8 intergenic region of DEV genome by Red E/T two-step recombinant technology, then the recombinant virus rDEV-GAstV ORF2 was rescued by transfecting recombinant clone pDEV-GAstV ORF2 into chicken embryonic fibroblasts (CEFs). The expression of ORF2 in CEFs and formation of virus-like particles (VLPs) were analysed by Western blotting, indirect immunofluorescence assay (IFA) and immunogold electron microscopy (IEM), individually. And protein celluar localization was analysed by IFA. Using this rDEV-GAstV ORF2 vector to infect CEFs was sufficient to elicit GAstV Cap protein expression, as confirmed by Western blotting and IFA. IEM also revealed the formation of VLPs within cells expressing this Cap protein. DEV is a good viral vector for GAstV ORF2 gene delivery and these results provide a basis for the development of a bivalent vaccine for controlling DEV and GAstV infections.

Duck enteritis virus (DEV) is responsible for duck viral enteritis, a contagious and lethal disease in waterfowls. The host proteins targeted by DEV are unknown. In this study, we developed a recombinant DEV rVP26-Flag and identified 17 host proteins that interact with VP26 in infected chicken embryo fibroblast cells using co-immunoprecipitation in conjunction with liquid chromatography-tandem mass spectrometry (Co-IP-MS/MS). The 17 potential targets of VP26 proteins include Xirp1, TMOD3, DCN, ATP5PD, AP3M1, MYO5A, MYH10, MYH9 (non-muscle myosin IIA heavy chain), and GSN. Most of these proteins are microfilament or cytoskeletal proteins with functions such as cytoskeletal protein binding, actin filament interaction, microfilament motor activity, and myosin II interaction. Using the Search Tool for the Retrieval of Interacting Genes analysis, we predicted a functional network of microfilament cytoskeletal proteins interacting with VP26. Interaction between DEV VP26 and the carboxyl-terminus domain of MYH9 (1651-1960 aa) was verified via co-localization and Co-IP assays. We also demonstrated that the inhibition of actin polymerization with cytochalasin D and latrunculin A reduced the DEV titer. Furthermore, siRNA-mediated knockdown of MYH9, which has intrinsic ATPase activity, also resulted in a reduced viral titer. A targeted inhibitor of myosin II ATPase, (-)-Blebbistatin, significantly suppressed DEV infection both in vitro and in vivo. These results suggest that the actin-myosin II network plays a crucial role in DEV proliferation, with MYH9 being an important host factor influencing DEV infection.

Dipeptidyl peptidase-4 (DPP4) is an established receptor for Middle East respiratory syndrome-related coronaviruses (MERSr-CoVs), while recent studies have identified angiotensin-converting enzyme 2 (ACE2) usage in multiple merbecovirus clades. Yet, receptor usage of many genetically diverse bat MERSr-CoVs remains unclear. Here we show that broadly distributed HKU25 clade merbecoviruses use ACE2, rather than DPP4, as their receptor. Cryo-electron microscopy revealed that HsItaly2011 and VsCoV-a7 strains engage ACE2 similarly to HKU5 but with remodelled interfaces and distinct orthologue selectivity, suggesting a shared evolutionary origin of ACE2 recognition. EjCoV-3, a close relative of the DPP4-using BtCoV422, showed broad multi-species ACE2 tropism and preadaptation to human ACE2. Several ACE2 glycans and residues within or near the binding interface were identified as determinants of orthologue selectivity. These viruses remain sensitive to several broadly neutralizing antibodies and entry inhibitors, indicating potential countermeasures for future outbreaks. These findings highlight the versatility of ACE2 as a functional receptor for diverse coronaviruses.

Avipoxvirus (APV) is a prevalent DNA virus in avian species, causing clinical symptoms of fowlpox and leading to reduced egg production, slower broiler growth, and increased mortality. The spread of APV poses a significant threat to the global poultry industry, potentially causing substantial economic losses. Effective control of APV, particularly its major species such as fowlpoxvirus and pigeonpoxvirus, requires the development of rapid and specific diagnostic tools. In this study, a novel multi-enzyme isothermal rapid amplification (MIRA) assay was developed to detect APV. Various primer-probe combinations were screened to identify an optimal pair targeting a conserved region of the viral P4b gene. The MIRA assay operates at a constant temperature and results can be visualized through fluorescence signal detection. The sensitivity, specificity, and applicability of the MIRA assay were evaluated. Additionally, 86 clinical samples were tested to assess the accuracy of the MIRA assay. The MIRA assay provides results within 15 minutes demonstrated high specificity, with no cross-reactivity with other avian pathogens. It achieved a detection limit of 50 copies/μl, which is consistent with the qPCR assay. Further evaluation with 86 clinical samples showed that the accuracy of the MIRA assay was comparable to that of qPCR in detecting fowlpoxvirus and pigeonpoxvirus. The results highlight the convenience, sensitivity, and rapidity of the MIRA assay as a promising tool for diagnosing APV.

Dipeptidyl peptidase-4 (DPP4) and angiotensin-converting enzyme 2 (ACE2) are well-established receptors for merbecoviruses, yet the receptor usage of merbecoviruses of European and Asian hedgehogs (EriCoVs) remains unknown. Here, by testing hedgehog orthologs of known coronavirus receptors, we identify hedgehog aminopeptidase N (APN) as a functional receptor for EriCoVs. Analysis of 94 APN orthologs indicates that EriCoVs have a limited host range, primarily utilizing hedgehog APN and, to a lesser extent, APN from certain felids, shaped by specific determinants at the virus–receptor interface. Cryo-EM reveals an APN-binding mode distinct from those used by alpha- and deltacoronaviruses. Functional assays indicate that hedgehog transmembrane serine protease 2 (TMPRSS2) enhances spike activation and promotes pseudovirus entry. Neutralizing antibodies targeting RBD and APN were developed and could effectively block EriCoV pseudovirus entry and propagation. These findings reveal an unexpected convergent evolution of APN utilization among merbecovirus, establishing a foundation for risk assessment and countermeasure development. Graphic abstract Convergent utilization of hedgehog APN by EriCoVs ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. National Key R&D Program of China, 2024YFC2607300, 2023YFC2605500 the National Natural Science Foundation of China, 82322041, 32270164, 323B2006 Scientific Research Innovation Capability Support Project for Young Faculty, ZYGXONJSKYCXNLZCXM-H16 Natural Science Foundation of Hubei Province, 2023AFA015 Strategic Priority Research Program, XDB1310000 National Science Foundation Grants, 32325004, T2394482, 12034006 Basic Research Program Based on Major Scientific Infrastructures, CAS-JZHKYPT-2021-05, CAS-YSBR-010 Ministry of Science and Technology of China, CPL-1233 Changping Laboratory, 2025D-04-01 [1]: pending:yes

Merbecoviruses comprise four viral species with remarkable genetic diversity: MERS-related coronavirus, Tylonycteris bat coronavirus HKU4, Pipistrellus bat coronavirus HKU5, and Hedgehog coronavirus 1. However, the potential human spillover risk of animal merbecoviruses remains to be investigated. Here, we reported the discovery of HKU5-CoV lineage 2 (HKU5-CoV-2) in bats that efficiently utilize human angiotensin-converting enzyme 2 (ACE2) as a functional receptor and exhibits a broad host tropism. Cryo-EM analysis of HKU5-CoV-2 receptor-binding domain (RBD) and human ACE2 complex revealed an entirely distinct binding mode compared with other ACE2-utilizing merbecoviruses with RBD footprint largely shared with ACE2-using sarbecoviruses and NL63. Structural and functional analyses indicate that HKU5-CoV-2 has a better adaptation to human ACE2 than lineage 1 HKU5-CoV. Authentic HKU5-CoV-2 infected human ACE2-expressing cell lines and human respiratory and enteric organoids. This study reveals a distinct lineage of HKU5-CoVs in bats that efficiently use human ACE2 and underscores their potential zoonotic risk.

The angiotensin-converting enzyme 2 (ACE2) receptor is shared by various coronaviruses with distinct receptor-binding domain (RBD) architectures, yet our understanding of these convergent acquisition events remains elusive. Here, we report that two bat MERS-related coronaviruses (MERSr-CoVs) infecting Pipistrellus nathusii (P.nat)-MOW15-22 and PnNL2018B-use ACE2 as their receptor, with narrow ortholog specificity. Cryoelectron microscopy structures of the MOW15-22/PnNL2018B RBD-ACE2 complexes unveil an unexpected and entirely distinct binding mode, mapping >45 Å away from that of any other known ACE2-using coronaviruses. Functional profiling of ACE2 orthologs from 105 mammalian species led to the identification of host tropism determinants, including an ACE2 N432-glycosylation restricting viral recognition, and the design of a soluble P.nat ACE2 mutant with potent viral neutralizing activity. Our findings reveal convergent acquisition of ACE2 usage for merbecoviruses found in European bats, underscoring the extraordinary diversity of ACE2 recognition modes among coronaviruses and the promiscuity of this receptor.

DPP4 was considered a canonical receptor for merbecoviruses until the recent discovery of African bat-borne MERS-related coronaviruses using ACE2. The extent and diversity with which merbecoviruses engage ACE2 and their receptor species tropism remain unknown. Here, we reveal that HKU5 enters host cells utilizing Pipistrellus abramus (P.abr) and several non-bat mammalian ACE2s through a binding mode distinct from that of any other known ACE2-using coronaviruses. These results show that several merbecovirus clades independently evolved ACE2 utilization, which appears to be a broadly shared property among these pathogens, through an extraordinary diversity of ACE2 recognition modes. We show that MERS-CoV and HKU5 have markedly distinct antigenicity, due to extensive genetic divergence, and identified several HKU5 inhibitors, including two clinical compounds. Our findings profoundly alter our understanding of coronavirus evolution and pave the way for developing countermeasures against viruses poised for human emergence.

Duck reovirus (DRV) is a universal waterfowl virus that causes significant economic losses in the duck industry. However, the role of the host innate immune response of the Bursa of Fabricius to DRV infection is largely unknown. In the present study, we constructed a single-cell resolution transcriptomic atlas of the Bursa of Fabricius of Cairna moschata after infection with HN10 (a novel DRV). Ten cell-type marker genes were used to annotate the cell type, indicating a high degree of cell heterogeneity in the Bursa of Fabricius. Most of the innate and adaptive immune system-related genes were highly expressed in T cells, B cells, neutrophils, macrophages, and DCs. In the Bursa of Fabricius, the proportions of DCs and macrophages were largely increased by HN10 infection at 14 d, suggesting that DCs and macrophages play important roles in the long-term viral response. Notably, a number of innate and adaptive immune system-related genes were highly expressed at 24 h after HN10 infection, indicating that the Bursa of Fabricius has a very strong immune function even in the early developmental stage. In the immune system, the NOD-like receptor signaling pathway and RIG-I-like receptor signaling pathway were significantly activated at the early stage of HN10 infection, while the Toll-like receptor signaling pathway was significantly activated at the late stage. Enrichment analysis suggested that different immune signaling pathways play roles in specific developmental stages. Our data provide an opportunity to reveal the immune response to DRV infection at the single-cell level.

Although coronaviruses use diverse receptors, the characterization of coronaviruses with unknown receptors has been impeded by a lack of infection models1,2. Here we introduce a strategy to engineer functional customized viral receptors (CVRs). The modular design relies on building artificial receptor scaffolds comprising various modules and generating specific virus-binding domains. We identify key factors for CVRs to functionally mimic native receptors by facilitating spike proteolytic cleavage, membrane fusion, pseudovirus entry and propagation for various coronaviruses. We delineate functional SARS-CoV-2 spike receptor-binding sites for CVR design and reveal the mechanism of cell entry promoted by the N-terminal domain-targeting S2L20-CVR. We generated CVR-expressing cells for 12 representative coronaviruses from 6 subgenera, most of which lack known receptors, and show that a pan-sarbecovirus CVR supports propagation of a propagation-competent HKU3 pseudovirus and of authentic RsHuB2019A3. Using an HKU5-specific CVR, we successfully rescued wild-type and ZsGreen-HiBiT-incorporated HKU5-1 (LMH03f) and isolated a HKU5 strain from bat samples. Our study demonstrates the potential of the CVR strategy for establishing native receptor-independent infection models, providing a tool for studying viruses that lack known susceptible target cells. A strategy using engineered functional customized viral receptors enables the development of functional infection models for coronaviruses whose native cellular receptors are unknown.

In 2021, a highly virulent strain of duck enteritis virus (DEV), designated as DEV XJ, was isolated from Zhejiang, China, and its complete genome, spanning 162,234 bp with 78 predicted open reading frames (ORFs), was sequenced. While showing relative homology to the DEV CV strain, DEV XJ exhibited distinctions in 38 ORFs, including various immunogenic and virulence-related genes. Amino acid variation analysis, focusing on UL6 and LORF3, indicated a high degree of homology between DEV XJ and the 2085 strain from Europe, as well as the DEV DP-AS-Km-19 strain from India. Subsequently, a full-length infectious bacterial artificial chromosome clone (BAC) of DEV XJ was successfully constructed to delve into the pathogenic mechanisms of this virulent strain. XJ BAC demonstrated substantial similarity to the parental DEV XJ in both in vitro growth properties and the induction of typical pathogenic symptoms in sheldrakes. Furthermore, the US3, LORF3, UL21, and UL36 genes were individually deleted using a two-step RED recombination approach based on the infectious BAC clone. Our findings revealed that the UL21 and UL36 genes play crucial roles in viral proliferation. Although the US3 and LORF3 genes were dispensable for viral replication and cell-to-cell transmission in vitro, they attenuated the replication and transmission efficiency of DEV compared to the WT. In summary, this study accomplished the whole-genome sequencing of a clinically virulent DEV strain and the successful construction of an infectious DEV XJ clone. Moreover, the functional roles of the above-mentioned mutant genes were preliminarily explored through the analysis of their in vitro biological characteristics.

The recently identified novel duck reovirus (NDRV) is a waterfowl reovirus that can seriously harm or kill various waterfowl species. However, how NDRV interacts with host cells in Muscovy ducklings beyond the typical pathogenesis resulting from a viral infection is unknown. The current study examined the global translation efficiency of the Fabricius bursa of Muscovy ducklings infected with NDRV HN10 using mass spectrometry and ribosome footprint sequencing. Protein-protein interactions were investigated using immunogold labeling, transmission electron microscopy, and immunocytochemistry. An analysis of the relationship between m6A and translation was performed using RNA immunoprecipitation and m6A methylation immunoprecipitation. We found that both in vivo and in vitro, the translation efficiency of RNA modified with m6A could be significantly reduced by σB, a structural protein component of NDRV HN10. Furthermore, σB might simultaneously interact with the stress granule complex CAPRIN1 and G3BP1 and the m6A reader protein YTHDF1/3. Significant overlap was observed between m6A-modified and G3BP1-enriched RNA, indicating that granule stress could capture m6A-methylated RNA. We discovered a new function for NDRV HN10 in translational shutoff by recruiting m6A-modified RNA into stress granules located in the Fabricius bursa of Muscovy ducklings.

IntroductionGoose astrovirus (GAstV) is a newly emerging pathogen that is currently widespread among geese, causing visceral gout and leading to substantial gosling mortalities, posing a severe threat to the waterfowl industry. GAstV II is the predominant epidemic strain, characterized by its high morbidity and mortality rate. Consequently, there is an urgent necessity to develop an effective diagnostic approach to control the dissemination of GAstV II, particularly in clinical farms with limited laboratory resources.MethodsIn this study, a novel multi-enzyme isothermal rapid amplification (MIRA) and lateral flow dipstick (LFD) combined assay was developed. Different primers designed specific targeting a highly conserved region within the viral RdRp gene for the detection of GAstV II. Primers optimized and MIRA-LFD assay analyzed its performance regarding limits of detection, specificity, and efficiency of detection.ResultsThe developed MIRA amplification is conducted at a constant temperature and accomplished within 10 minutes. Subsequent naked-eye observation of the LFD strips merely takes 5 minutes. The established MIRA-LFD method exhibits high specificity, with no cross-reaction with other pathogens and attains a detection sensitivity of 1 copy/μl, which is consistent with the reverse transcription quantitative PCR (RT-qPCR) assay. Further evaluation with clinical samples indicates that the accuracy of this MIRA-LFD method correlates well with RT-qPCR for the detection of GAstV II. ConclusionIn summary, the convenience, sensitivity, and rapidity of this newly developed detection method offer a significant advantage for on-site diagnosis of GAstV II.