Nipah virus (NiV) is an emerging zoonotic pathogen with a high mortality rate, posing a serious threat to public health. Its clinical manifestations are often indistinguishable from those of Japanese encephalitis virus (JEV), making rapid and accurate differential diagnosis essential for effective prevention and control. In this study, we developed a one-pot duplex detection assay that integrates loop-mediated isothermal amplification (LAMP) with the Pyrococcus furiosus Argonaute (PfAgo) cleavage, guided by 3 '-mismatched gDNA to enhance detection efficiency. The assay exhibited high sensitivity in duplex detection, detecting as few as 1.5 copies per reaction for both NiV and JEV without cross-reactivity to major porcine pathogens. Simulated clinical validation further confirmed its on-site applicability, achieving a sensitivity of 3 copies/mu L for NiV using a portable 3D-printed device. This work highlights the utility of an Ago-based detection platform for rapid, sensitive, and fielddeployable diagnosis of emerging animal diseases, and offers a promising tool for the detection of other pathogens in resource-limited settings.

Porcine epidemic diarrhea virus (PEDV) is the most dominant virus causing diarrhea in swine, and suckling piglets exhibit extremely high mortality. Continuous surveillance is essential for understanding the evolutionary trajectory of PEDV and for disease control. In this study, we analyzed the temporal evolutionary dynamics of PEDV in China from 2013 to 2023 by integrating newly sequenced genomic data with sequences available in public databases. Phylogenetic reconstruction, evolutionary rate estimation, selective pressure analysis, population dynamics modeling, and prediction of N-glycosylation sites in the Spike gene were performed. Our results revealed dynamic genotype turnover over the past decade, with the G2c subtype in China gradually became the dominant strain. The evolution rate of the S gene of the G2 genotype in China showed variation between time-defined periods. The dN/dS ratio for the Spike gene was consistently below 1 and exhibited a general declining trend. The dN/dS ratio exhibited a transient increase of 25.8% from its lowest point in late 2018 to a peak in 2021. Analysis of N-glycosylation sites showed that, during 2013-2023, the pattern characterized by reduced site diversity but increased site number underwent a shift. However, these observations are based on computational predictions and represent temporal trends. This study highlights the ongoing diversification of PEDV under changing epidemiological, offering a scientific basis for assessing its ongoing threat to the swine sector.

African swine fever (ASF) is an acute, hemorrhagic, and highly contagious disease caused by African swine fever virus (ASFV), which causes severe economic losses in the swine industry. ASFV has evolved multiple strategies to evade host antiviral immune responses. Here, we report that ASFV pMGF360-3 L promotes host mitophagy by manipulating chaperone-mediated autophagy (CMA), thereby inhibiting the production of type I interferon (IFNB/IFN-β). Mechanistically, pMGF360-3 L targets the SKP1 protein via its N-terminal ankyrin (ANK) repeat domain, promoting the degradation of SKP1 through the CMA pathway, which inhibits the proteasomal degradation of BNIP3 to increase its expression level in mitochondria. Subsequently, BNIP3 binds to MAP1LC3B/LC3B to induce mitophagy, a process that leads to the degradation of mitochondria. Notably, the CMA-mediated degradation of SKP1 depends on its K94 site, and the SKP1-BNIP3 axis is critical for pMGF360-3 L-mediated IFNB inhibition. In summary, our study reveals a mechanism through which ASFV pMGF360-3 L facilities CMA-dependent degradation of the E3 complex component SKP1. This stabilizes mitochondrial BNIP3 to initiate mitophagy and block IFNB production. This deepens our understanding of the immune evasion strategies of ASFV and provides potential drug targets for controlling viral infection.Abbreviations: 3-MA: 3-methyladenine; ASFV: African swine fever virus; BafA1: bafilomycin A1; BNIP3: BCL2 interacting protein 3; CMA: chaperone-mediated autophagy; co-IP: co-immunoprecipitation; CQ: chloroquine; CHX: cycloheximide; CUL1: cullin 1; DAPI: 4', 6-diamidino-2'-phenylindole; EV: emptor vector; FBXL4: F-box and leucine rich repeat protein 4; hpi: hours post-infection; IFNB: interferon beta; ISGs: IFN-stimulated genes; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MG132: cbz-leu-leu-leucinal; MAVS: mitochondrial antiviral signaling protein; MOI: multiplicity of infection; PAMs: porcine alveolar macrophages; PPTC7: protein phosphatase targeting COQ7; RBX1: ring-box 1; RT-PCR: real-time polymerase chain reaction; siRNA: small interfering RNA; SKP1: S-phase kinase associated protein 1; TCID50: 50% tissue culture infectious doses; Ub: ubiquitin; WCL: whole-cell lysate; WT: wild-type.

African swine fever (ASFV) leads a highly contagious and lethal hemorrhagic disease, causing a huge economic loss to the global pig industry. Recently, lethal genotype I/II recombinants were isolated and characterized in China and Vietnam. To differentiate the three genotypes (I, II and I/II recombinants) of ASFV in China, a triplex quantitative PCR (qPCR) assay was developed by targeting B646L, A151R and MGF360-14L genes. The detection limitation for the A151R, MGF360-14L, and B646L genes were 10 copies/μl, demonstrating a high sensitivity. Meanwhile, no cross-reactivity was observed with nucleic acids from ASFV genotype I and II or other swine viruses, confirming a high specificity for this assay. The coefficient of assay variation was below 2

African swine fever (ASF), a pig disease caused by ASFV, is highly contagious and often lethal. The recent emergence of novel ASFV with I/II genomic recombination has posed significant challenges to global ASF prevention and control. In this study, we used the Chinese I/II genomic recombinant virulent strain ASFV-HN10005 (ASFV-HN) to construct two gene-deleted viruses. ASFV-HNΔMGF lacks the MGF505-1R-MGF360-14L genes cluster (including MGF505-1R, -2R, -3R, and MGF360-12L, -13L, -14L), and ASFV-HNΔCD2vΔMGF lacks both EP402R and the MGF505-1R-MGF360-14L genes cluster. ASFV-HNΔMGF still showed pathogenicity in domestic pigs, while ASFV-HNΔCD2vΔMGF showed markedly attenuated virulence, with all inoculated pigs surviving. However, these pigs did not gain complete protection against subsequent lethal challenge from the parental ASFV-HN strain or the virulent II-type strain ASFV-GZ. This implies that I/II genomic recombinant ASFV may have unique biological traits and immune evasion mechanisms. Our findings indicate the need to reassess current ASFV control strategies and provide a basis for future vaccine target selection.

African swine fever (ASF), caused by the African swine fever virus (ASFV), is a highly contagious and often fatal disease that poses a serious threat to the global pig industry. The emergence of genotype I/II recombinant ASFV strains has further complicated vaccine development and disease control. This study reveals that ASFV-GZΔI177LΔCD2vΔMGF, an effective candidate vaccine strain against genotype II ASFV, fails to provide protection against lethal challenge from the genotype I/II recombinant strain ASFV-HN. Although a five-gene-deleted strain based on ASFV-HN with I177L deletion (ASFV-HNΔI177LΔCD2vΔMGF) exhibits significantly reduced pathogenicity in pigs and was observed to delay mortality in pigs upon lethal challenge, it did not effectively protect against lethal challenge from either the parental strain or genotype II virulent strains. These findings reveal the unique virulence characteristics of genotype I/II recombinant ASFV strains and highlight the limitations of current vaccine strategies. They also emphasize the need to develop new approaches to address the risks posed by recombinant ASFV strains and to account for the importance of viral genotype and recombination events.

ABSTRACT African swine fever virus (ASFV) causes a severe hemorrhagic disease, posing a significant threat to the global pig industry. Although the ASFV encodes nearly 200 proteins, the functions of many remain unknown. Here, we identify the inner envelope protein pE146L as essential for ASFV replication. pE146L, localized in the endoplasmic reticulum lumen, induces endoplasmic reticulum (ER) perinuclear aggregation, and its absence disrupts viral factory formation. Mechanistic studies revealed the first high-resolution crystal structure of pE146L-ΔTM. Structural and biochemical analyses revealed that disrupting the intermolecular disulfide bonds of the dimers (C103A) abrogates ER aggregation and impedes viral replication. Furthermore, pE146L binds lipids via a positively charged surface patch, a function critical for replication, suggesting a role in lipid regulation. These findings highlight the multifunctionality of pE146L in ASFV morphogenesis and provide a promising target for anti-ASFV drug development. IMPORTANCE African swine fever virus (ASFV) causes a highly lethal infectious disease in swine; however, our understanding of its replication and assembly mechanisms remains limited, which hinders the development of vaccines and drugs. In this study, we identified the uncharacterized pE146L, a protein of the inner envelope that is required for the viral life cycle. Notably, we found that pE146L showed distinct colocalization and the ability to induce noticeable ER aggregation. Moreover, we solved the first high-resolution crystal structure of the extracellular soluble region of pE146L and discovered that it is a lipid-binding protein. Interestingly, structural and biochemical analyses suggest the potentially significant impact of intermolecular disulfide bonds on ER aggregation and viral replication. These results highlight the multifunctionality of ASFV pE146L, providing new insights for the development of specific antiviral drugs.

Nipah virus (NiV) is a zoonotic pathogen that causes severe encephalitis and respiratory disease in humans and multiple mammalian species. However, no licensed vaccines or therapeutics are currently available against NiV infection. In this study, we developed three mRNA vaccine candidates using a lipid nanoparticle (LNP) delivery platform: mRNA-F-LNP, comprising mRNA encoding the fusion protein (F); mRNA-G-LNP, containing mRNA encoding the attachment glycoprotein (G); and mRNA-GF-LNP, in which mRNAs encoding both F and G proteins were co-encapsulated at a 1:1 molar ratio. All three mRNA-LNPs induced robust and sustained immune responses in both mice and Syrian hamsters. Sera from immunized Syrian hamster showed high levels of cross-neutralizing antibodies against both NiV-Malaysia (NiV-M) and NiV-Bangladesh (NiV-B) strains. Notably, all three mRNA-LNPs conferred complete protection against a lethal challenge with NiV-M in Syrian hamsters. These findings demonstrate that these mRNA-based vaccines are highly immunogenic and efficacious, highlighting their potential as promising candidates for NiV vaccine development.

The African swine fever virus (ASFV) is a significant threat to the global swine industry, with no effective vaccine available. ASFV subverts cellular processes, including programmed cell death mechanisms, to enhance its transmission and pathogenicity. Here, we show that the ASFV p54 manipulates the secretory carrier membrane protein 3 (SCAMP3)-dependent apoptotic bodies (ApoBDs) pathway to facilitate cell-cell transmission, thereby enhancing viral replication. We demonstrate that p54 interacts with SCAMP3 via its carboxyl-terminal 150-184 amino acids, which is critical for the formation of ApoBDs induced by ASFV. Deletion of this motif in ASFV significantly impairs the release of virions, leading to intracellular viral accumulation and triggering ferroptosis instead of apoptosis. This shift in cell death mechanism significantly reduces virulence and provides protection against lethal challenges with wild-type ASFV. Our findings reveal a mechanism of ASFV-induced cell death and intercellular transmission, providing a theoretical foundation for developing future vaccine strategies against ASFV.

Interferon regulatory factor 7 (IRF7)-mediated type I interferon antiviral response is crucial for regulating the host following viral infection in chickens. Infectious bursal disease virus (IBDV) is a double-stranded RNA virus that induces immune suppression and high mortality rates in chickens aged 3-6 weeks. Previous studies have shown that IBDV infection antagonizes the type I interferon production to facilitate viral replication in the cell, and IRF7 signaling might play an important role. However, the underlying mechanisms that enable IBDV to block the IRF7 pathway remain unclear. In this study, we found that IRF7 and IFN-β expression were suppressed in DF-1 cells during infection with very virulent IBDV (vvIBDV), but not with attenuated IBDV, while the virus continued to replicate. Overexpression of IRF7 inhibits IBDV replication while knocking down IRF7 promotes IBDV replication. Overexpression of IRF7 couldn’t compensate the IRF7 protein level in vvIBDV-infected cells, which suggested that IRF7 protein was degraded by IBDV infection. By using inhibitors, the degradation of IRF7 was found to be related to the proteasome pathway. Further study revealed that IRF7 was observed to interact and colocalize with the IBDV VP3 protein. Consistent with IBDV infection results, IBDV VP3 protein was observed to inhibit the IRF7-IFN-β expression, affect the degradation of IRF7 protein via proteasome pathway. All these results suggest that the IBDV exploits IRF7 by affecting its expression and proteasome degradation via the viral VP3 protein to facilitate viral replication in the cells. These findings revealed a novel mechanism that IBDV uses to evade host antiviral defense.

Influenza A viruses (IAVs) constitute a major threat to human and animal health. Currently, M2 ion-channel inhibitors, neuraminidase (NA) inhibitors, RNA polymerase inhibitors, and cap-dependent endonuclease inhibitors have been applied clinically as therapeutics against IAVs. However, IAVs possess adaptive mutations to these inhibitors, especially M2 ion channel and NA inhibitors. Thus, novel antiviral agents should be developed. In the present study, we screen approximately 5500 compounds and identify an IAV inhibitor, nanchangmycin, which possesses a robust antiviral activity both in vitro and in vivo. In addition, it exhibits broad-spectrum antiviral activity for additional virus infections, including pseudorabies virus, herpes virus, porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus. Most importantly, it has antiviral activity against oseltamivir-resistant strains in sub-μM ranges and promotes the survival of MDCK cells infected with the oseltamivir-resistant influenza A virus strain. Further studies reveal that it blocks the nuclei migration of viral nuclear proteins (NPs), resulting in NP accumulation in the cytoplasm, particularly within perinuclear endosomes. Also, it inhibits IAVs by blocking endosomal acidification. Overall, nanchangmycin has the potential to be developed as an anti-influenza agent.

African swine fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious swine disease that has spread globally. Effective control strategies are not yet available. In this study, we prepared K205R mRNA, which was then formulated using Lipid Nanoparticle (LNP). The resulting K205R mRNA-LNP showed a particle size of approximately 86.27 nm and an mRNA encapsulation efficiency of 96.24%. Efficient expression of the K205R protein was confirmed in both HEK293T and PK15 cells. We further evaluated the immunogenicity of K205R mRNA-LNP in mice and pigs. All immunized animals developed significantly higher levels of IgG antibodies against K205R compared to the control group in the first week after the second immunization, with antibody titers reaching up to 105. Challenge experiments showed that K205R mRNA delayed the time of death. Our results suggested the successful implementation of the mRNA platform in the preparation and application of ASFV mRNA.

Enveloped viruses such as coronaviruses are highly transmissible, necessitating effective prevention and inactivation strategies. In this study, we engineered uniform, small-sized (∼57 nm) iron-phenolic nano-networks (NA-Fe) that exhibit high lipoxidase-like activity for the inactivation of enveloped virus, encompassing both DNA virus (Pseudorabies, PRV) and RNA virus (Transmissible Gastroenteritis, TGEV; and Porcine epidemic diarrhea virus, PEDV). Theoretical studies indicate that NA-Fe facilitates the adsorption of pentadiene moieties within the viral envelope through interactions between the d-orbital electrons of iron and the π-electrons of pentadiene. This interaction leads to activation of the C=C bonds, potentially resulting in disruption of the viral envelope. Besides its extracellular antiviral effects, NA-Fe also suppresses intracellular viral replication by stimulating antiviral innate immune responses, and upregulate the expression of interferon-stimulating genes (ISGs). In vivo studies demonstrate that NA-Fe exhibits favorable biosafety, and intranasal administration significantly reduces viral titers while providing effective antiviral protection in PRV-infected mice. Notably, surfaces coated with NA-Fe, such as fences and air purifiers in farm facilities, provide effective antiviral protection. Collectively, these findings demonstrate that NA-Fe exhibits potent broad-spectrum antiviral activity against both extracellular and intracellular viruses, highlighting its potential for application in comprehensive biosecurity and infectious disease control.

Infectious bursal disease(IBD)is an acute,highly contagious disease that affects chicks(Müller et al.2003).IBD mainly damages the immune organs of chicks,especially the central immune organ,causing immune suppression in diseased chicks(Muller et al.2012).The pathogenic infectious bursal disease virus(IBDV)is a member of the Avira virus genus in the Birnaviridae family.(Harkness et al.1975;Dobos et al.1979;Müller et al.1979).IBDV is prevalent worldwide,causing serious economic losses to the global poultry industry.Currently,vaccination remains the most cost-effective way to prevent IBDV.

Classical swine fever (CSF), caused by the classical swine fever virus (CSFV), remains a significant threat to the global pig industry. Recent advances in mRNA vaccines offered a promising platform for combating CSFV. In this study, we designed and evaluated three lipid nanoparticle (LNP)-encapsulated mRNA vaccine candidates encoding the ectodomain of E2 glycoprotein (E2_EX), E2_EX fused with the transmembrane (TM) region of the PEDV S protein (E2tm), and E2_EX fused with the TM region of the influenza virus HA protein (E2tm-HA). Among these, the E2tm mRNA vaccine induced the most robust antibody responses in pigs. Immunization of piglets with the E2tm mRNA vaccine showed that its immunogenicity was not impaired by maternal antibodies. Comparative analysis of pseudouridine (Ψ)-modified (ΨE2tm) and unmodified (E2tm) mRNA vaccines revealed that E2tm induced significantly higher antibodies titers than ΨE2tm. All vaccinated pigs survived the CSFV challenge, with the 150 µg E2tm dose providing optimal protection, effectively suppressing viremia and preventing viral dissemination to tissues while also resulting in undetectable viral RNA in swab samples. Our findings provide a promising novel mRNA vaccine that could be used as an alternative vaccination strategy against CSFV infection. IMPORTANCE:Classical swine fever virus (CSFV) remains a significant threat to the global pig industry. While live attenuated and subunit vaccines are currently in use, there is an urgent need for more effective and safer vaccination strategies. Here, we present a novel mRNA vaccine encoding the CSFV E2 glycoprotein, which provides protective immunity against the CSFV challenge in pigs. Our findings underscore the promising efficacy of this mRNA-based vaccine platform and offer an alternative strategy for CSFV prevention and control.

ABSTRACT Since the first human infection reported in 2013, H7N9 avian influenza virus (AIV) has been regarded as a serious threat to human health. In this study, we sought to identify the virulence determinant of the H7N9 virus in mammalian hosts. By comparing the virulence of the SH/4664 H7N9 virus, a non-virulent H9N2 virus, and various H7N9-H9N2 hybrid viruses in infected mice, we first pinpointed PB2 as the primary viral factor accounting for the difference between H7N9 and H9N2 in mammalian virulence. We further analyzed the in vivo effects of individually mutating H7N9 PB2 residues different from the closely related H9N2 virus and consequently found residue 473, alongside the well-known residue 627, to be critical for the virulence of the H7N9 virus in mice and the activity of its reconstituted viral polymerase in mammalian cells. The importance of PB2-473 was further strengthened by studying reverse H7N9 substitutions in the H9N2 background. Finally, we surprisingly found that species-specific usage of ANP32A, a family member of host factors connecting with the PB2-627 polymorphism, mediates the contribution of PB2 473 residue to the mammalian adaption of AIV polymerase, as the attenuating effect of PB2 M473T on the viral polymerase activity and viral growth of the H7N9 virus could be efficiently complemented by co-expression of chicken ANP32A but not mouse ANP32A and ANP32B. Together, our studies uncovered the PB2 473 residue as a novel viral host range determinant of AIVs via species-specific co-opting of the ANP32 host factor to support viral polymerase activity. IMPORTANCE The H7N9 avian influenza virus has been considered to have the potential to cause the next pandemic since the first case of human infection reported in 2013. In this study, we identified PB2 residue 473 as a new determinant of mouse virulence and mammalian adaptation of the viral polymerase of the H7N9 virus and its non-pathogenic H9N2 counterparts. We further demonstrated that the variation in PB2-473 is functionally linked to differential co-opting of the host ANP32A protein in supporting viral polymerase activity, which is analogous to the well-known PB2-627 polymorphism, albeit the two PB2 positions are spatially distant. By providing new mechanistic insight into the PB2-mediated host range determination of influenza A viruses, our study implicated the potential existence of multiple PB2-ANP32 interfaces that could be targets for developing new antivirals against the H7N9 virus as well as other mammalian-adapted influenza viruses.

African swine fever has caused substantial economic losses to China`s pig industry in recent years. Currently, the highly pathogenic African swine fever virus strain of genotype II is predominantly circulating in China, accompanied by a series of emerging isolates displaying unique genetic variations. The pathogenicity of these emerging strains is still unclear. Recently, a novel ASFV strain with a distinguishable three-large-fragment gene deletion was obtained from the field specimens, and its in vivo pathogenicity and transmission were evaluated in this study. The animal experiment involved inoculating a high dose of YNFN202103 and comparing its effects with those of the highly pathogenic strain GZ201801_2. Results showed that pigs infected by YNFN202103 exhibited significantly prolonged onset and survival time, lower viremia levels, and less severe histopathological lesions compared to GZ201801_2. These findings contributed valuable insights into the pathogenicity and transmission of ASFV and its prevention and eradication strategies in practical settings.

Antiviral vaccine is essential for preventing and controlling virus spreading, along with declining morbidity and mortality. A major challenge in effective vaccination lies in the ability to enhance both the humoral and cellular immune responses by adjuvants. Herein, self-assembled nanoparticles based on graphene oxide quantum dots with components of carnosine, resiquimod and Zn2+ ions, namely ZnGC-R, are designed as a new adjuvant for influenza vaccine. With its high capability for antigen-loading, ZnGC-R enhances antigen utilization, improves DC recruitment, and activates antigen-presenting cells. Single cell analysis of lymphocytes after intramuscular vaccination revealed that ZnGC-R generated multifaceted immune responses. ZnGC-R stimulated robust CD4+CCR7loPD-1hi Tfh and durable CD8+CD44hiCD62L- TEM immune responses, and simultaneously promoted the proliferation of CD26+ germinal center B cells. Besides, ZnGC-R elicited 2.53-fold higher hemagglutination-inhibiting antibody than commercial-licensed aluminum salt adjuvant. ZnGC-R based vaccine induced 342% stronger IgG antibody responses compared with vaccines with inactivated virus alone, leading to 100% in vivo protection efficacy against the H1N1 influenza virus challenge.

Virus replication relies on complex interactions between proteins. A comprehensive understanding of the protein interactions may provide clues for developing new antiviral strategies. In the case of African swine fever virus (ASFV), only a few protein interactions have been identified so far. In this study, we demonstrate that ASFV protein p72 interacts with p11.5 using co-immunoprecipitation and liquid chromatography-mass spectrometry (LC-MS). It was found that protein p72 interacts specifically with p11.5 at sites amino acids (aa) 1–216 of p72 and aa 1–68 of p11.5. To assess the importance of p11.5 in ASFV infection, we developed a recombinant virus (ASFVGZΔA137R) by deleting the A137R gene from the ASFVGZ genome. Compared with ASFVGZ, the infectious progeny virus titers of ASFVGZΔA137R were reduced by approximately 1.0 logs. In addition, we demonstrated that the growth defect was partially attributable to a higher genome copies-to-infectious virus titer ratios produced in ASFVGZΔA137R-infected MA104 cells than in those infected with ASFVGZ. This finding suggests that MA104 cells infected with ASFVGZΔA137R may generate larger quantities of noninfectious particles. Importantly, we found that p11.5 did not affect virus-cell binding or endocytosis. Collectively, we show for the first time that the interaction between ASFV p72 and p11.5. Our results effectively provide the relevant information of the p11.5 protein. These results extend our understanding of complex interactions between viral proteins, paving the way for further studies of the potential mechanism and pathogenesis of ASFV infection.

African swine fever (ASF), caused by the African swine fever virus (ASFV), has resulted in significant losses in the global pig industry. Considering the absence of effective vaccines, developing drugs against ASFV may be a crucial strategy for its prevention and control in the future. In this study, punicalagin, a polyphenolic substance extracted from pomegranate peel, was found to significantly inhibit ASFV replication in MA-104, PK-15, WSL, and 3D4/21 cells by screening an antiviral compound library containing 536 compounds. Time-of-addition studies demonstrated that punicalagin acted on early viral replication stages, impinging on viral attachment and internalization. Meanwhile, punicalagin could directly inactivate the virus according to virucidal assay. RT-qPCR and Western blot results indicated that punicalagin modulated the NF-κB/STAT3/NLRP3 inflammasome signaling pathway and reduced the levels of inflammatory mediators induced by ASFV. In conclusion, this study reveals the anti-ASFV activity of punicalagin and the mechanism of action, which may have great potential for developing effective drugs against ASFV.