Background and aims Protein deficiency is a critical global challenge for public health and food security. The trade-off between maize grain yield (GY) and grain protein content (GPC) remains a major barrier to enhancing both food quantity and nutritional quality. While nitrogen (N) management is crucial for crop productivity, its potential to synergistically improve both GY and GPC across diverse global environments is not fully quantified. This study aims to identify optimal N practices and the environmental drivers that govern the synergy between maize yield and quality. Methods A global meta-analysis was conducted using 699 paired observations from peer-reviewed studies. Mixed-effects models were used to evaluate the effect of N management practices, soil properties and climatic factors on GY, GPC and grain protein yield (GPY). Results The controlled-release fertilizer boosted GY by 11.53% and GPC by 11.93%. Notably, increasing N application rate predominantly enhanced GPC rather than GY, while a modest reduction in N application rate (≤ 20%) did not significantly affect GY, GPC and GPY. Agronomic adjustments further strengthened these effects, with N topdressing increasing GY, GPC and GPY by 7.90%, 11.65% and 20.47%, respectively. Furthermore, these effects were more pronounced under conditions of lower soil bulk density (< 1.4 g/cm 3 ) and warmer climates (≥ 20°C). Conclusion Our findings demonstrate that shifting from conventional N application to synchronized management can achieve a win-win for maize productivity and nutritional security. These results provide a strategic framework for site-specific N management to optimize the soil-plant N balance under a changing climate.

In this study, we examined the influence of dietary PFS powder supplementation on production performance, egg quality, and yolk fatty acid profile in laying hens. A total of 192 Hy-Line® Brown hens, 30 weeks of age, were randomly allocated to four dietary treatments containing 0, 30, 60, and 90 g/kg of PFS powder, administered over a 12-week period. No significant differences were observed in egg weight, feed intake, or feed conversion ratio among the treatment groups (p > 0.05). However, supplementation with 60 and 90 g/kg PFS significantly enhanced egg production and total egg mass (p < 0.05), particularly during weeks 41–44. Egg quality parameters—including albumen height, Haugh unit, yolk color, shell thickness, and shell strength—remained unaffected across treatments (p > 0.05). Serum analyses revealed that PFS supplementation significantly reduced levels of total cholesterol, low-density lipoprotein (LDL), triglycerides, and yolk total cholesterol compared with the control diet (p < 0.05). Moreover, yolk fatty acid composition was notably altered: total PUFAs and n-3 PUFAs increased (p < 0.05), whereas total monounsaturated fatty acids and the n-6/n-3 PUFA ratio decreased (p < 0.05) with rising PFS inclusion. In conclusion, dietary PFS powder improved laying performance and favorably modulated yolk fatty acid composition, without compromising egg quality in laying hens.

Pseudorabies virus (PRV) and Porcine epidemic diarrhea virus (PEDV) are currently co-circulation among swine herd with multiple variant strains, which cause severe economic losses to the global swine industry. In the present study, a recombinant PRV-based vaccine candidate was constructed backbone of a PRV variant via deletion of five genes (gI, gE, US2, US9, and TK) and insertion of the S1 gene derived from a PEDV G2b strain, and the resulting recombinant virus was generated as rPRV-Δ5-S1. The safety of the quintuple-gene-deleted PRV vector (rPRV-Δ5) was firstly evaluated in rabbits and compared with the previously generated triple-gene-deleted vector (rPRV-Δ3). Rabbits inoculated with rPRV-Δ5 exhibited milder clinical manifestations, lower viral loads in tissues, and fewer histopathological lesions, indicating an improved safety profile. Subsequent immunization trials in mice demonstrated that rPRV-Δ5-S1 induced PEDV S1-specific antibodies and PEDV-neutralizing antibodies, which were higher than those observed in the commercial inactivated PEDV vaccine group. Furthermore, rPRV-Δ5-S1 elicited higher neutralizing antibody titers against PRV variants than the Bartha-K61 vaccine and provided complete protection against lethal challenge with the PRV variant. To further validate the immunogenicity and protective efficacy of rPRV-Δ5-S1, challenge experiments were conducted in piglets. The results showed that piglets immunized with rPRV-Δ5-S1 developed detectable antibody responses against both PRV and PEDV. After challenge with the PEDV G2b variant, rPRV-Δ5-S1-immunized piglets exhibited reduced diarrhea severity, decreased viral shedding, alleviated intestinal lesions, and improved weight gain compared with control animals. Collectively, these findings demonstrate that the quintuple-gene-deleted PRV vector exhibits an improved safety profile and can serve as an effective platform for heterologous antigen delivery. The recombinant virus rPRV-Δ5-S1 can induced immune responses against both PRV and PEDV in multiple animal models, which supports its potential as a bivalent vaccine candidate against these two economically critical swine pathogens.

ObjectivesLawsonia intracellularis is an obligately intracellular enteric bacterium that infects intestinal epithelial cells and causes porcine proliferative enteropathy (PPE). This study aimed to investigate the epidemiological prevalence of L. intracellularis in large-scale pig farms in Shaanxi Province, China, and analyze the differences in fecal microbial communities of growing pigs with natural L. intracellularis infection.MethodsA total of 672 fecal samples and 300 serum samples were collected from five intensive pig farms in Shaanxi during 2022–2023. Quantitative PCR (qPCR) was used to detect L. intracellularis in feces and quantify its fecal load, while enzyme-linked immunosorbent assay (ELISA) was employed to detect the seroprevalence of anti-L. intracellularis antibodies in serum. 16S rRNA gene sequencing was performed on fecal samples from L. intracellularis-positive and -negative growing pigs.ResultsThe results showed an overall fecal positive rate of 15.3% (95% CI: 12.7–18.3%) and a serum seroprevalence of 14.7% (95% CI: 11.0–19.3%) for L. intracellularis in the surveyed farms, with growing pigs exhibiting the significantly highest positive rate and fecal pathogen load (p < 0.05). Tongchuan exhibited a significantly lower fecal positive rate than the other surveyed regions (p < 0.05). Fecal microbial diversity analysis revealed that L. intracellularis-positive fecal samples exhibited significantly higher bacterial species richness. LEfSe analysis indicated a significant enrichment of Lactobacillus in L. intracellularis-positive feces relative to negative samples. Network analysis demonstrated a positive correlation between Desulfovibrionaceae and Lachnospiraceae, and random forest analysis identified Erysipelotrichaceae_UCG.003 as the critical microbial biomarker for L. intracellularis infection.ConclusionThis study elucidates the epidemiological characteristics of L. intracellularis in Shaanxi and its interaction with the porcine gut microbiome, thereby providing a theoretical basis for the precise prevention and control of PPE, as well as for further investigations into the interactions between the gut microbiota and L. intracellularis.

Context Selecting low-nitrogen (N)-tolerant maize hybrids is an effective measure for implementing the national ‘nitrogen control and efficiency enhancement’ strategy. However, addressing their combined stress with low light and low N still requires further exploration. Objective To elucidate the effects of combined low light and low N stresses on grain filling, C-N metabolism, pedicel exudation strength (PES) and yield components of different low-N-tolerant hybrids. Methods Field experiments were conducted over two years with ZH311 (low-N-tolerant hybrid) and XY508 (low-N-sensitive hybrid). Treatments included natural sunlight and 35% shading, as well as low N and normal nitrogen rates. Results Low light and low N stresses reduced 100-grain weight 3.7%-10.9%, especially in XY508 and apical grains, causing yield loss. Overall, low N reduced yields by 12.4%-21.0%, low light by 13.7%-18.7%, and combined stress by 25.0%-31.8%. Grain weight (GW) was positively correlated with grain volume, C-N metabolism enzyme activity (CNM-EA), and PES. Among these, PES and CNM-EA contributed the most to GW, and grain volume minimally, especially for apical grains. Low light and low N stress reduced ZT+ZR and IAA content and were accompanied by decreased CNM-EA and PES, thereby slowing grain filling rate and reducing GW. ZH311 improved GW and reduced the difference between apical and basal-middle grains by maintaining hormone balance, sustaining high CNM-EA and PES, and promoting grain filling. Conclusions Low-N-tolerant hybrid ZH311 sustains yield by enhancing assimilate supply, improving hormone balance, and enhancing C-N metabolism under combined low light and low N stresses. Implications These findings further clarify the mechanism of yield difference among different maize hybrids under low light and nitrogen, which could provide theoretical support of stress-tolerant hybrid breeding and N management practice in low light areas.

NADC30-like porcine reproductive and respiratory syndrome virus (PRRSV) has replaced the high-pathogenicity PRRSV (HP-PRRSV) as the predominant circulating strain in China. Unlike HP-PRRSV, NADC30-like PRRSV exhibits increased genetic diversity and enhanced persistence for persistent infection, along with altered host immune responses, which collectively complicate disease control. In this study, we found that infection with the NADC30-like representative strain XM2020 induced a weaker antibody response than infection with the HP-PRRSV strain HuB2, and that SLA-DRA expression was downregulated. In vitro analyses further demonstrated that this suppression of SLA-DR in PBMCs is independently of viral replication. Mechanistically, XM2020-M binds to SLA-DRA and recruits the E3 ubiquitin ligase MARCH6, promoting K33-linked polyubiquitination and proteasomal degradation of SLA-DRA, a function not observed for HuB2-M. In addition, XM2020-M stabilizes MARCH6 by inhibiting its degradation, thereby enhancing its ubiquitin ligase activity. Further mapping identified residue 93 of XM2020-M as a critical determinant of this process. Collectively, these findings reveal a novel immune evasion mechanism by which XM2020-M impairs antigen presentation by targeting the degradation of SLA-DRA, which may contribute to altered adaptive immune responses and viral persistence associated with NADC30-like strains.

Context Low-light-intensity has emerged as the critical constraint on the stalk lodging resistance formation in maize dense planting and climate regional dimming conditions. However, the stage-specific effects of low-light-intensity at basal internode formation in different stages remain poorly understood. Objective and methods The field experiment was conducted in 2021–2023 using white shading nets to simulate low-light-intensity conditions at basal internode formation in different stages. The responses and variations of maize stalk lodging resistance and internode traits to low-light-intensity were systematically investigated from the perspectives of mechanical strength, morphological traits, matter traits and anatomical structures. Results and conclusions Low-light-intensity significantly altered plant morphologies and reduced basal internode mechanical strengths, thereby increasing lodging rate by 42.6 %age points. Low-light-intensity during V6-V12 promoted internode elongation (6.4–11.2%) and reduced internode diameter (11.4–13.6%), accompanied by marked deterioration in anatomical structures, including cross-sectional area, vascular bundle number and area decreased by 23.0–30.4%, 3.5–4.2% and 18.3–28.7%, respectively. In contrast, low-light-intensity after V12 had relatively smaller effects on internode morphologies and anatomical structures, but significantly reduced assimilate accumulation (23.1%), rind thickness (13.7%) and cell wall thickness. Restoration of light-intensity after V12 partially recovered assimilate accumulation, rind thickness and cell wall thickness, leading to improved internode mechanical strengths, particularly rind penetration strength. Low-light-intensity during V12-R1 + 10 caused greater reductions in mechanical strengths than during V6-V12, and bending strength was more sensitive than rind penetration strength. The responses of basal internode mechanical strengths to variations in matter traits were greater than those of morphologies or anatomical structures. Implications These findings demonstrate that the stages of low-light-intensity critically determine its impact on internode development and stalk lodging resistance. This study provides important insights for optimizing lodging-resistant cultivation under dense planting, breeding density-tolerant hybrids, and improving maize production adaptation to climate change (solar radiation reduction) and low solar radiation regions.

With the increasing prevalence of refractory multidrug-resistant Gram-negative bacterial infections, the development of novel antibacterials targeting Gram-negative pathogens has become an urgent priority. The complex cell envelope structure of these bacteria, particularly the lipopolysaccharide (LPS)-containing outer membrane, presents a major barrier to drug penetration and efficacy, driving extensive research on outer membrane-targeting strategies. While progress has been rapid, a focused narrative synthesis of the field remains valuable. This review provides a comprehensive narrative overview of antibacterial strategies directed against Gram-negative bacteria, spanning from fundamental design principles to specific applications. It begins by outlining the structural features of the Gram-negative bacterial envelope that underpin resistance, then surveys and discusses key development approaches—including de novo design and structural modification of existing agents. Within this framework, the mechanisms of action and recent advances of various antibacterial classes are examined and evaluated. In particular, unresolved challenges remain in understanding drug metabolic behavior, mitigating toxicity, and improving the translational efficiency from preclinical design to clinical application. Addressing these issues will likely depend on the coordinated integration of fundamental mechanistic insights, rational drug design strategies, and complete pharmaceutical technologies, ultimately facilitating the development of antibacterial therapies capable of delaying resistance evolution and enabling more precise pathogen eradication.

Since viruses pose a serious threat to human health, rapid and accurate detection methods are necessary to control viral transmission and mitigating harm. Lateral flow immunoassays (LFIAs) have been widely applied for viruses detection with high sensitivity and accuracy owing to its simplicity, rapidity, and cost-effectiveness. Despite significant advancements in LFIA for viruses detection have achieved, a comprehensive and up-to-date review systematically addressing technological optimization, performance enhancement, and clinical applications remains critically needed. To bridge this gap, the review provides an in-depth analysis of LFIA applications in detecting 18 major viruses categories from the novel perspectives of basic sandwich-format LFIA for antigen/antibody detection and nucleic acid amplification-coupled LFIA (NALFIA) for DNA/RNA detection. By evaluating and summarizing the detection capabilities of these two LFIA strategies across diverse virus species, this review aims to guide the development of highly sensitive and multiplexed detection methods while offering evidence-based insights for clinical decision-making, public health policy formulation, and optimized resource allocation.

Since the first ASFV case was reported in China in 2018, troditional control stategies involving whole-herd culling upon detection of ASFV positivity have proved unwieldy because of its high production intensity and complex trade network. To provide an alternative to conventional stamping-out methods, we developed a novel approach termed "Whole-herd-Sampling, qPCR-based-Testing, and Precision-Removal” method. This approach involves whole herd sampling and qPCR testing to determine the status of ASFV in herds, followed by the precision removal of identified infected sows. By applying these techniques, we successfully controlled ASF and eliminated the virus from 4 large swine herds between 2019 and 2020. The time to negative herd (TTNH) was 19, 28, 14, and 1 day from farm 1 to 4, respectively. Retention rates of pigs from farm 1 to farm 4 were 69.7%, 65%, 99.4%, and 99.72%, respectively. We propose that this innovative method would be a good alternative to the conventional stamping-out strategies and greatly facilitate the control and eradication of ASFV both in China and worldwide.

The rapid spread of antibiotic resistance poses a global health crisis. Tigecycline is a last-resort antibiotic, but the recent emergence of the plasmid-borne tet(X3) gene conferring high-level tigecycline resistance is deeply concerning. Here, we report a metabolomics-guided approach to overcome tet(X3)-mediated resistance. Using untargeted metabolomics, we identified adenosine as a key metabolic biomarker associated with tet(X3) expression. Remarkably, supplementation with exogenous adenosine was able to restore tigecycline susceptibility in tet(X3)-positive Escherichia coli both in vitro and in vivo. Our mechanistic investigations reveal that adenosine enhances the bactericidal effects of tigecycline by inducing oxidative stress, DNA/RNA damage, and cell membrane disruption in resistant bacteria. This study establishes a powerful metabolomics-driven strategy to potentiate antibiotic efficacy against drug-resistant pathogens. The adenosine-based adjuvant therapy represents a promising approach to combat the global crisis of antibiotic resistance.IMPORTANCEThe emergence and widespread dissemination of the high-level tigecycline resistance gene tet(X3) have posed a significant challenge to the efficacy of tigecycline, which serves as the "last line of defense" against antimicrobial-resistant bacteria. Although tigecycline has not been approved for veterinary clinical use, constant detection of tet(X3) genes and new subtypes in livestock farming environments poses a substantial threat to public health safety. While developing novel antibiotics is an effective approach to eradicate resistance genes/bacteria, it entails considerable costs and a lengthy timeframe. This study discovered that exogenous adenosine can effectively restore the sensitivity of tet(X3)-positive Escherichia coli to tigecycline through metabolic reprogramming based on a non-targeted metabolomics strategy. The findings are highly significant for exploring comprehensive mechanisms underlying bacterial multidrug resistance, utilizing metabolic reprogramming strategies to curb the spread of novel resistant genes, and treating clinical infections caused by tet(X3)-positive bacteria.

Guanzhong dairy goats, a breed indigenous to the Guanzhong region of China, offer several advantages; however, they are particularly vulnerable to mastitis, a common disease affecting their health and productivity. In this study, we collected milk samples from healthy goats (designated as healthy goat milk samples, n = 13) and goats affected by clinical mastitis (designated as mastitis goat milk samples, n = 24) from two distinct dairy goat farms located in Shaanxi and Gansu (Shaanxi n = 25, Gansu n = 12). We employed 16S rRNA amplicon sequencing to identify the dominant bacterial communities present in both healthy and mastitis-affected goat milk samples. Our findings revealed differences in bacterial populations between the two groups. The alpha diversity of the microbiota in healthy goat milk samples was significantly greater than that in the mastitis-affected samples. Beta diversity analysis demonstrated the presence of pathogen-associated microbiota in healthy goat milk samples, highlighting notable differences in the core microbiota between the two sample groups. Achromobacter was identified as the dominant bacterial genus in healthy goat milk samples, whereas it was either absent or present in minimal quantities in the mastitis samples. In contrast, the mastitis-causing goat milk samples exhibited a more homogeneous microbiota composition, marked by the loss of beneficial probiotic flora. Furthermore, the microbiota structure in goats with severe clinical symptoms, such as gangrenous and streptococcal mastitis, was simplified and comprised exclusively of pathogenic bacteria. Through this study, we identified both probiotic and pathogenic bacteria in healthy and mastitis-affected goat milk samples, providing essential microbiological insights that could inform effective prevention and treatment strategies for mastitis in dairy goats. These findings underscore the importance of understanding the microbial dynamics in dairy goats to enhance herd health and productivity.

Alveolar echinococcosis (AE) is a significant zoonotic disease caused by the larval form of Echinococcus multilocularis (E. multilocularis), posing substantial health risks. However, the roles of circular RNAs (circRNAs), long non-coding RNAs (lncRNAs), and messenger RNAs (mRNAs) in the early stages of E. multilocularis infection, particularly regarding liver involvement, remain unclear. This study aimed to investigate the molecular mechanisms of AE infection in mice during the early stage of secondary infection. The expression profiles of mRNAs, circRNAs and lncRNAs in mouse livers were analyzed at four time points: 2, 4, 8, and 15 days post-infection (dpi). The results identified a substantial number of differentially expressed molecules, including 5004 circRNAs and 8744 lncRNAs. These differentially expressed ncRNAs were mainly involved in immune-related categories, such as antigen processing and presentation and T cell activation. Interactions among circRNAs, lncRNAs, and immune-associated mRNAs formed competing endogenous RNAs (ceRNAs) regulatory networks. Notably, mmu_circ_0008646 showed strong associations with microRNAs such as mmu-miR-466b-3p and mmu-miR-574-5p, while several lncRNAs also interacted with mmu-miR-574-5p. miR-574-5p regulates inflammatory responses and macrophage polarization primarily through the NF-κB pathway. Additionally, different components of E. multilocularis larva distinctively affect macrophage polarization, with the cystic fluid promoting macrophage M2 polarization and protoscoleces stimulating macrophage M1 polarization. This study highlights alterations in circRNA, lncRNA, and mRNA expression patterns in the livers of mice infected with E. multilocularis, enhancing our understanding of the AE transcriptomic profile.

Pseudomonas aeruginosa (P. aeruginosa) is an important zoonotic pathogen. It is also the primary causative agent of systemic infections in the endangered Moschus berezovskii. The emergence of multidrug-resistant strains of P. aeruginosa has made these infections increasingly difficult to control, and bacteriophages are considered important alternatives or adjuncts to antibiotic therapy. This study isolated P. aeruginosa strains that induce suppurative infections in Moschus berezovskii from a farm in Shaanxi Province, China. The bacteriophages vB_PaeP_FMD5 (FMD5) and vB_PaeM_H24-1 (H24-1) were isolated using these bacteria as hosts. The safety and practicality of the two phages were analyzed through methods such as biological characteristic assessment, whole genome sequencing analysis, and animal experiments. FMD5 is classified within the Podoviridae family, whereas H24-1 belongs to Myxoviridae. Biological characterization revealed that both FMD5 and H24-1 exhibit tolerance to temperature, pH, chloroform, and Ultraviolet(UV) exposure. The optimal multiplicity of infection (OMOI) for FMD5 and H24-1 were 0.01 and 0.1, respectively, and the burst sizes from the one-step growth curve were 200 PFU/cell and 150 PFU/cell, respectively. In vitro inhibitory assays demonstrated that FMD5, H24-1, and their cocktail exerted a favorable inhibitory effect for up to 11 hours. Whole genome sequencing confirmed that both phages possess double-stranded DNA genomes, with FMD5 having a length of 72,254 bp and a G+C content of 55.16

Palmatine, a natural isoquinoline alkaloid derived from Fibraureae Caulis, is widely used for its heat-clearing, detoxifying, antibacterial, and anti-inflammatory properties. The emergence of multidrug-resistant Escherichia coli poses a critical challenge to the efficacy of β-lactam antibiotics, particularly cephalosporins such as cefquinome. This study demonstrates that palmatine markedly enhances the antibacterial activity of cefquinome through multi-targeted mechanisms, revealing a new pharmacological potential for this compound. Antimicrobial and synergistic activities were assessed using the microbroth dilution method and checkerboard assay. Bacterial morphology was examined using scanning electron microscopy (SEM), while biofilm inhibition was assessed using confocal laser microscopy. Membrane damage and reactive oxygen species (ROS) levels were detected with fluorescent probe dyes. Transcriptome and RT-qPCR analyses were conducted to identify key mechanistic pathways, and the synergistic effect was further validated in a mouse Escherichia coli infection model. In vitro analyses of 20 tested isolates revealed broad synergistic effects (FICI ≤ 0.5), with cefquinome MICs reduced by 4- and 32-fold. Mechanistic studies revealed that palmatine disrupts membrane integrity, potentiates oxidative stress, and inhibits biofilm formation. Transcriptomic profiling implicated sulfur metabolism as a key pathway, showing that palmatine reversed cefquinome-induced downregulation of sulfur metabolism-related genes. Functional validation confirmed that disruption of taurine uptake in the sulfur metabolic pathway eliminated the synergistic effect. In murine infection models, the combination therapy increased survival by 30%, alleviated diarrhea, and significantly reduced bacterial loads in tissue. This study reveals the novel pharmacological properties of palmatine, identifies metabolic-level reversal regulation as a novel strategy to combat β-lactam resistance, and highlights palmatine as a multi-target adjuvant that enhances cefquinome efficacy against resistant Gram-negative infections.

Porcine circovirus type 2 (PCV2), porcine circovirus type 3 (PCV3), and pseudorabies virus (PRV) are major pathogens posing significant threats to the swine industry. Viral evolution and mutations have limited the efficacy of current commercial vaccines, necessitating the development of more effective prophylactic strategies. In this study, a novel recombinant virus strain, designated as rPRV-ΔTK-PCV3(Cap)/ΔgIgE-PCV2(Cap), was generated using PRV SX-10 variant as the backbone. CRISPR/Cas9-mediated deletion of TK and gE/gI genes was performed, followed by insertion of PCV3 and PCV2 capsid protein genes into the respective loci. The engineered recombinant strain demonstrated stable proliferation in BHK-21 cells, efficiently expressed heterologous PCV3 and PCV2 capsid proteins, while maintaining biological properties comparable to its parental strain. The rPRV-ΔTK-PCV3(Cap)/ΔgIgE-PCV2(Cap) demonstrated favorable safety and immunogenicity profiles in mice and piglets, eliciting robust immune responses characterized by high titers of specific antibodies against PRV, PCV3, and PCV2, along with significantly elevated levels of cytokines (IFN-γ, IL-2, and IL-4). Histopathological analysis and viral load quantification demonstrated that rPRV-ΔTK-PCV3(Cap)/ΔgIgE-PCV2(Cap) immunization significantly attenuated tissue lesions and decreased viral copies of PRV and PCV in mice and piglets. Collectively, these findings suggest that rPRV-ΔTK-PCV3(Cap)/ΔgIgE-PCV2(Cap) serves as a promising candidate vaccine against PRV and PCV infections.

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), a severe hemorrhagic disease with a mortality rate reaching 100%. Despite extensive research on ASFV mechanisms, no safe and effective vaccines or antiviral treatments have been developed. Live attenuated vaccines generated via gene deletion are considered to be highly promising. We developed a novel recombinant ASFV strain by deleting MGF360-10L and MGF505-7R, significantly reducing virulence in pigs. In the inoculation experiment, pigs were infected with 104 50% hemadsorption doses (HAD50) of the mutant strain. All the animals survived the observation period without showing ASF-related clinical signs. Importantly, no significant viral infections were detected in the cohabitating pigs. In the virus challenge experiment, all pigs succumbed after being challenged with the parent strain. RNA-seq analysis showed that the recombinant virus induced slightly higher expression of natural immune factors than the parent ASFV; however, this level was insufficient to provide immune protection. In conclusion, our study demonstrates that deleting MGF360-10L and MGF505-7R from ASFV CN/GS/2018 significantly reduces virulence but fails to provide protection against the parent strain.

The blood-brain barrier (BBB) keeps poisons and infections out of the brain. Some viruses can pass through this barrier and replicate in the central nervous system (CNS). Velogenic Newcastle disease virus (VNDV) is a neurotropic virus that causes avian nonsuppurative encephalitis. VNDV often develops into a chronic infection that seriously affects poultry health in partially immune birds. The routes by which the virus enters the chicken brain are poorly understood. In this study, we discovered that VNDV increased BBB permeability in vivo and in vitro by breaking the tight junction protein zona occludens-1 (ZO-1) continuity of chicken brain microvascular endothelial cells (chBMECs). By investigating the susceptibility of chBMECs to NDV infection, we found that VNDV but not lentogenic NDV was detected in the basolateral compartment in transwell assays after apical infection, suggesting that efficient replication and transcellular transport of the virus across the BBB in vitro. Furthermore, viral replication and BBB permeability were reduced during the early stage of infection by using the dynamin inhibitor dynasore. Our data demonstrate that VNDV invades the chicken brain by infecting and damaging the tight junction of chBMECs directly to increase BBB permeability. VNDV could infect chBMECs via endocytosis. As a result, our findings provide compelling evidence for VNDV entrance into the brain via the BBB, paving the way for the development of medications for NDV prevention and therapy.

The cytokines IFN-γ and CD154 have been well established, and they play pivotal roles in immune protection against Salmonella in mice, but their effects and specific mechanisms in Salmonella-infected chickens are less understood. In this study, we conducted animal experiments to screen the highly immunoprotective chIFN-γ-chCD154 fusion protein compared with single protein chIFN-γ or chCD154 in white Leghorn chickens. The results showed that compared with separate pretreatments with chIFN-γ and chCD154, the fusion protein, chIFN-γ-chCD154, synergistically increased survival of infected chickens, reduced bacterial load in feces and organs, and attenuated pathological damage to the liver and cecum. Pretreatment with chIFN-γ-chCD154 also increased humoral immune responses, expression of the tight junction proteins zo-1, occludin, and claudin-1, and the relative abundance of Enterococcus_cecorum, Lactobacillus_helveticus, and Lactobacillus_agilis, which protect against intestinal inflammation. Compared with single protein pretreatment, chIFN-γ-chCD154 significantly upregulated STAT1, IRF1, and GBP1 in infected chickens while decreasing mRNA expression of TLR4, MyD88, NF-κB, TNF-α, IL-6, and IL-1β. In summary, damage to the cecal epithelial barrier and the inflammation induced by S. typhimurium infection was alleviated by chIFN-γ-chCD154 pretreatment through a mechanism involving the TLR4/MyD88/NF-κB and IFN-γ/STAT/IRF1/GBP1 pathways.

African Swine Fever (ASF) is a highly lethal viral disease in swine. The emergence and rapid spread of African Swine Fever virus (ASFV) in China, since 2018 have caused significant economic losses to the pig farming industry. The complexity of ASFV has impeded the development of effective vaccines, and with no commercial vaccines currently available in China, highlighting the urgent need for safe and efficacious vaccine candidates. In this study, we utilized a highly immunogenic quadruple-gene-deleted recombinant pseudorabies virus (PRV) strain (rPRV SX-10ΔUL24/TK/gI/gE) as a vector to construct two recombinant viral strains expressing ASFV p54, p72, CD2v, and pp62 proteins using the HDR-CRISPR/Cas9 system. These strains, rPRV-p54+p72 and rPRV-CD2v+pp62, demonstrated stable genetic characteristics and efficiently expressed and delivered heterologous proteins while maintaining biological properties similar to their parental strain. Safety evaluation revealed that both recombinant strains exhibited favorable safety profiles in immunized mice and piglets. Furthermore, the strains induced robust humoral and cellular immune responses, as evidenced by specific antibody enzyme-linked immunosorbent assay (ELISA), lymphocyte proliferation assays, and analysis of CD3+, CD4+, and CD8+ T lymphocytes. These findings suggest that rPRV-p54+p72 and rPRV-CD2v+pp62 are promising bivalent vaccine candidates for protecting against both PRV and ASFV infections.