The recombinant H3N8 avian influenza virus (AIV) continues to circulate among poultry in China, with sporadic human infections reported, posing a significant threat to both the poultry industry and public health. However, no commercial vaccine against H3N8 AIV is currently available. Our previous studies have showed that serotype 4 fowl adenovirus (FAdV-4) with the deletion of fiber-2 gene is highly attenuated and serves as an ideal vaccine vector for expressing foreign genes. In this study, a novel recombinant FAdV-4 carrying HA of H3N8 was generated by targeting fiber-2 through the CRISPR/Cas9 and Cre-LoxP systems, designed as FA4-CMV-H3. In vitro, studies demonstrated that FA4-CMV-H3 not only efficiently expressed the HA protein of H3N8 but also replicated efficiently in LMH cells stably expressing Fiber-2 protein of FAdV-4. Chicken studies showed that FA4-CMV-H3 is highly attenuated in vivo and induces high neutralizing antibodies against FAdV-4 and high HI titers against H3N8. Moreover, FA4-CMV-H3 could provide full protection against both H3N8 and FAdV-4 challenges. All these demonstrate that the generated recombinant virus FA4-CMV-H3 is a promising bivalent efficient vaccine candidate against both FAdV-4 and H3N8.

The pathogenesis of the immune dysregulation in porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) infection is not fully understood yet. In this context, immune checkpoints have been identified as pivotal regulators of inflammatory and immune responses. This study evaluated the expression of CTLA-4 at early and late stages of PRRSV-1 infection in piglets with two strains of different virulence (3249 and Rosalía). Animals infected with the highly virulent Rosalía strain showed more severe clinical signs and a significantly reduced average daily gain. In addition, they presented more severe lung lesions in the early stages of the infection, characterised by interstitial pneumonia, with marked thymus atrophy and enlargement of the tracheobronchial lymph nodes. Although seroconversion was similar in both groups, Rosalía induced earlier and higher viraemia, as well as higher viral loads in the thymus, where the infection persisted for up to 42 days. Gene expression analysis revealed up-regulation of CTLA4 and TIM3 in the lung, tracheobronchial lymph node, and thymus during the early stages of infection, particularly in Rosalía-infected animals, decreasing until the advanced stage in the lung. This was associated with a higher frequency of CD4[+]CTLA-4[+] and CD8β[+]CTLA-4[+] T cells in peripheral blood and lung, which persisted, although to a lesser extent, until 42 dpi in animals infected with Rosalía strain. Our findings support the high virulence of Rosalía strain and point out to a role of CTLA-4, as well as other immune checkpoints, such as TIM-3, in modulating the host's immune response.

Porcine deltacoronavirus (PDCoV) causes a significant threat to the global swine industry. The PDCoV accessory protein NS6 has the capability to antagonize type I interferon (IFN-I) production and influence viral proliferation. In this study, we discovered that tripartite motif-containing 28 (TRIM28) interacts with NS6 via its RING and coiled-coil (CC) domains, facilitating K48-linked ubiquitination of NS6, thereby leading to the degradation of NS6. Furthermore, NS6 attenuates the expression of interferon-stimulated genes (ISGs). The inhibition of the interferon (IFN) response is further intensified when the degradation of NS6 is inhibited. This study further identifies TRIM28 as an antiviral factor that restricts PDCoV replication.

Porcine circovirus type 3 (PCV3), a newly identified pathogen linked to multiple clinical diseases, threatens the global swine industry. Nucleolin, a multifunctional protein involved in various biological events, has been confirmed to suppresses PCV3 replication; however, the underlying molecular mechanism remains unclear. Herein, we demonstrate that nucleolin inhibits PCV3 replication by activating the type I interferon (IFN)/Janus kinase (JAK)-signal transducer and activator of transcription 1 (STAT1) pathway, a critical host antiviral defense axis. Mechanistically, nucleolin enhances STAT1/STAT2 phosphorylation, promotes their nuclear translocation, and interacts with IFN regulatory factor 9 (IRF9), thereby driving the formation of the IFN-stimulated gene factor 3 (ISGF3) complex and the subsequent production of interferon-stimulated genes (ISGs). Furthermore, PCV3 infection antagonizes JAK-STAT1 activation, an effect that is reversed by nucleolin overexpression. Notably, the JAK-STAT1 pathway agonist RO8191 not only inhibits PCV3 replication in cultured cells but also reduces PCV3 viral load and alleviates PCV3-induced pathological lesions in the lungs and spleens of mice. Collectively, these results indicate that PCV3 facilitates its own replication by suppressing this pathway, whereas nucleolin exerts its antiviral activity by counteracting this suppression, specifically by targeting the STAT1-STAT2-IRF9 signaling axis.

Infectious bovine rhinotracheitis (IBR) is a globally recognized infectious disease. The causative agent infects the respiratory and reproductive tracts of cattle, resulting in substantial economic losses to the cattle industry. Endoplasmic reticulum stress (ERS) and mitophagy play critical roles in maintaining cellular homeostasis during viral infections. However, whether Infectious bovine rhinotracheitis virus (IBRV) infection activates ERS and regulates the initiation of mitophagy remains unclear. In this study, IBRV infection robustly activates the ERS unfolded protein response (UPR) pathway and concurrently induces mitophagy and the innate immune pathway mediated by RIG-I-MAVS. Pharmacological inhibition of ERS, 4-PBA significantly abrogated IBRV-induced mitophagy. Further investigations revealed that inhibition of PERK with GSK2606414 and IRE1 with STF-083010 reverse IBRV-induced mitophagy, whereas the ATF6 inhibitor AEBSF had no such effect. Collectively, these findings suggested that IBRV-induced ER stress promotes mitophagy, at least in part, through the PERK and IRE1 pathways. Additionally, the mitophagy induced by IBRV may be related to the weakened transmission of type I interferon pathway mediated by RIG-I-MAVS, thereby establishing a permissive intracellular microenvironment for viral replication. These results implied the mechanism by which IBRV facilitates self-replication by impairing innate immunity through the induction of endoplasmic reticulum stress-mediated activation of mitophagy, likely offering novel insights for the prevention and control of IBRV infection.

The objectives of this study were to determine the virulence genes in E. coli recovered from diarrheic and non-diarrheic pigs, and to assess genetic susceptibility to E. coli-F4 and F18 on a subset of farms in Ontario, Canada. Rectal swabs from 10 diarrheic and 5 non-diarrheic pigs on each farm were cultured and the isolates genotyped. Blood samples were used to test genetic susceptibility. In total, 199 E. coli isolates carrying at least one virulence gene were recovered from 55% diarrheic and 46% non-diarrheic pigs (P = 0.14). The most prevalent fimbriae genes were F4 (9.0%) and F18 (8.3%), and enterotoxin genes were EAST1 (35.7%), STb (24.7%), and LT (24.3%). In total, 52 virotypes were identified with F4:STa:STb:Paa (5.5%), AIDA-I:LT:STb:EAST1 (5.5%), F4:LT:STb:Paa:EAST1 (4.0%), F18:LT:STb:Paa:EAST1 (3.0%), and F18:STa:STb (3.0%) as the most commonly detected. ETEC (carrying at least one fimbriae or AIDA-I gene and one enterotoxin gene) was recovered from 24.5% diarrheic and 17.0% non-diarrheic pigs (P = 0.14). Diarrheic pigs were more likely to shed E. coli-carrying STb (P = 0.03) or LT (P = 0.02) gene. Overall, 42.7% (MUC4) and 56.3% (CHCF1) pigs were susceptible to E. coli- F4, and 93.7% to E. coli-F18. There was no significant agreement between the MUC4 and CHCF1 (P < 0.001). Pigs that were susceptible to E. coli-F4 based on MUC4 (OR=3.3; P = 0.02) or CHCF1 (OR=2.8; P = 0.06) were more likely to shed E. coli-F4. These findings will help to develop an effective E. coli vaccine and can be used to breed E. coli resistant pigs.

Caseous lymphadenitis (CLA), caused by Corynebacterium pseudotuberculosis, is endemic in Korean native goats, but relationships among antimicrobial resistance (AMR), spatial distribution, and population structure remain unclear. We profiled AMR and multidrug resistance (MDR) in 286 pld-positive isolates from CLA lesions of 1177 goats in the Jeolla provinces, South Korea, integrating spatial analysis with five-locus multilocus sequence typing (MLST) and phylogenetics. Susceptibility to 12 antimicrobials was determined by disk diffusion, and MDR was defined as resistance to ≥ 3 antimicrobial classes. Farm-level spatial analyses included 54 farms with valid MDR estimates. Resistance was dominated by aminoglycosides, with streptomycin and gentamicin resistance of 99.7% and 88.1%, respectively. MDR occurred in 96 of 286 isolates (33.6%) and comprised 35 resistance-class patterns; the eight most frequent accounted for 61.5% of MDR isolates. Local indicators of spatial association (LISA) identified three significant farm-level patterns: one high-low outlier, one low-high outlier, and one low-low cluster, whereas no significant local patterns were detected at the gun/si level. Global Moran's I was non-significant at both scales. The CLA-MDR association was non-significant at the farm level but positive at the gun/si level by Pearson correlation. Neither AMR-profile dissimilarity nor differences in MDR prevalence showed significant isolation by distance using Haversine or road-network distances. MLST of 49 representative isolates indicated a cohesive, locally diversified lineage, with MDR distributed across related isolates rather than confined to a dominant typed lineage. These findings support herd-level antimicrobial stewardship and integrated phenotypic, spatial, and molecular surveillance for CLA control.

Feline calicivirus (FCV) shows extensive genetic and antigenic diversity and typically causes oral-respiratory disease (ORD), although some isolates are associated with virulent systemic disease (VSD). The molecular basis of FCV-associated VSD remains unclear and cannot be attributed to a single VP1 residue, phylogenetic lineage, or recombination event. Here, we comparatively characterized three VSD-associated isolates (XA13_VSD, GZ_VSD, and AH1_VSD) and one ORD-associated isolate (SH2022_ORD) by integrating phylogenetic background, modular genomic variation, in vitro replication, experimental pathogenicity, tissue viral RNA distribution, and serological cross-neutralization. All four isolates belonged to genogroup GI, but the VSD-associated isolates were distributed across different GI sublineages. RDP5 analysis of 253 complete genomes identified 90 recombination events, including 27 spanning the Region A/Region B boundary; AH1_VSD showed a representative modular recombination pattern near the ORF1/ORF2 junction. Within this four-isolate panel, the three VSD-associated isolates showed higher in vitro replication levels and caused more severe disease than SH2022_ORD in experimentally infected cats, accompanied by greater viral RNA shedding, whole-blood FCV RNA detection at 3-9 dpi, broader tissue viral RNA distribution, and more severe histopathological lesions. After homologous neutralizing titers were standardized to 2 [1] [0], AH1_VSD antiserum displayed the broadest cross-neutralizing profile within the tested panel. Collectively, these findings support a multifactorial view of FCV-associated systemic disease involving genomic background, replication and pathogenicity traits, viral dissemination, tissue involvement, and capsid antigenic variation. The comparatively broad cross-neutralization profile of AH1_VSD provides a rationale for its further evaluation as a potential parental candidate for attenuation and vaccine development.

Enterococcus species are clinically important opportunistic pathogens capable of acquiring and disseminating antimicrobial resistance (AMR). Although vancomycin-resistant enterococci (VRE) and linezolid-resistant enterococci (LRE) are increasingly reported in human medicine, their prevalence and genomic features in companion animals remain poorly characterized. This study investigated the global distribution and genetic traits of acquired vancomycin and linezolid resistance determinants in enterococci from dogs and cats. Among 32,971 genomes analyzed, 34 carried vanA, optrA, poxtA, and/or cfr. VanA-positive strains were predominantly Enterococcus faecium and were identified in Europe, Oceania, Asia, and South America between 1995 and 2021. All harbored the vanHAX cluster, mainly associated with Tn1546-like transposons. Transferable oxazolidinone resistance genes were identified in 25 strains, mostly Enterococcus faecalis, recovered exclusively from China and the United States between 2015 and 2023. The optrA gene (OptrA variants RDK and KLDK, defined by specific amino acid substitutions) and optrA-fexA genetic context predominated, whereas poxtA and cfr were each identified in a single isolate. One E. faecium strain co-harbored optrA and poxtA. VRE and LRE populations displayed high sequence type diversity, including lineages previously reported in human healthcare settings (ST78 and ST80) and livestock (ST27), as well as novel lineages (ST3090, ST3091, and ST2180). Genomic analyses and literature data indicate that VRE and LRE in dogs and cats remain uncommon but are globally distributed and associated with diverse lineages and mobile resistance platforms shared across human, animal, and environmental reservoirs. Enhanced surveillance is needed to better understand the epidemiology and dissemination of these clinically relevant resistance determinants.

FMDV infection induces rearrangement of host cell membrane structures and recruits various proteins to form a replication complex for viral replication. Vimentin, a major component of type III intermediate filaments, has been implicated in the replication processes of multiple viruses. This study identifies that Vimentin interacts with FMDV 2 C and participates in replication complex formation. Knockout of Vimentin significantly suppressed FMDV replication in BHK-21 cells. FMDV infection did not affect Vimentin expression but instead induced the rearrangement of Vimentin network structure. Treatment with IDPN and acrylamide disrupted Vimentin structural rearrangement and inhibited FMDV replication. Furthermore, we demonstrated that FMDV upregulates CaMKIIγ expression, which in turn promotes Vimentin phosphorylation at Ser39 and Ser83 to induce structural rearrangement. Inhibiting CaMKIIγ activity resulted in a significant reduction in FMDV replication. Overexpression of Vimentin effectively enhanced the replication of several RNA viruses, including Poliovirus type 1, Enterovirus 71, Respiratory enteric orphan virus type 3 strain 176, Coxsackievirus A16, and Human parainfluenza virus type 2. Our results indicate that Vimentin regulates the replication of multiple RNA viruses, providing a theoretical reference for in-depth investigation into the mechanisms of RNA virus infection and the development of antiviral drugs.

Salmonella enterica subsp. enterica serovar Typhimurium (S.Typhimurium) is a critical foodborne zoonotic pathogen that impairs poultry production and threatens public health. The virulence effector SptP encoded by Salmonella Pathogenicity Island 1 (SPI-1) exerts unclear pathogenic functions in indigenous Wenchang chickens, a high-susceptible local broiler breed in China. Here, wild-type (WT) S. Typhimurium and an isogenic ΔSptP knockout mutant were combined with in vitro stress assays, HD11 chicken macrophage infection, in vivo oral challenge of Wenchang chicks, and ileal RNA sequencing (RNA-seq) to systematically dissect the roles of SptP in bacterial environmental fitness, intracellular replication, and intestinal inflammatory injury. In vitro tests revealed that SptP significantly boosted bacterial tolerance to acid, oxidative and hyperosmotic stresses, and accelerated biofilm formation. Within HD11 macrophages, SptP promoted intracellular bacterial proliferation and upregulated pro-inflammatory mediators IL-1β, IL-6 and inducible nitric oxide synthase (INOS). In vivo trials demonstrated that WT-infected chicks exhibited more severe growth retardation, higher diarrhoea incidence, elevated bacterial loads in visceral and intestinal tissues, and aggravated macroscopic lesions compared with the ΔSptP group. SptP markedly suppressed the transcription of intestinal tight junction genes Occludin, Claudin-1 and ZO-1. A total of 126 differentially expressed genes (DEGs) were identified from ileal tissues, which were mainly enriched in lipid metabolism, immune response and the peroxisome proliferator-activated receptor (PPAR) signalling pathway. Collectively, SptP acts as a core virulence determinant that facilitates intestinal colonization, triggers inflammatory cascades and disrupts epithelial barrier integrity. Deletion of sptP drastically attenuates bacterial virulence. This study provides theoretical support for developing live attenuated vaccine candidates and antibiotic-free green control strategies against avian salmonellosis.

African swine fever virus (ASFV) pA137R is a key immune evasion protein known to target TBK1 and suppress the cGAS-STING pathway; however, whether it modulates type I interferon (IFN) signal transduction remains unknown. In this study, we investigated the role of the A137R protein (pA137R) in the JAK-STAT type I IFN signaling cascade. We found that pA137R directly interacts with TYK2 via a 10-amino-acid motif (aa81-90), interferes with the IFNAR1-TYK2 interaction. This disruption inhibited the phosphorylation and nuclear translocation of both STAT1 and STAT2, leading to a pronounced suppression of interferon-stimulated gene (ISG) expression. Consistently, an A137R-deleted ASFV strain displayed markedly enhanced sensitivity to IFN stimulation and produced significantly higher levels of ISGs compared to the parental virus. Collectively, these findings identify pA137R as a potent antagonist of the JAK-STAT pathway that, by targeting TYK2, blocks STAT1/2 activation and ISG induction. This work reveals a previously unrecognized mechanism by which this virulence factor disarms type I IFN-mediated innate antiviral responses and, together with its known activity against cGAS-STING, underscores the pivotal role of A137R in ASFV immune evasion.

The poultry respiratory microbiome is increasingly recognized as a critical determinant of poultry health, mucosal immunity, and host resistance to opportunistic pathogens. Chronic Respiratory Disease (CRD) exerts a severe economic toll on the global broiler industry. While conventionally attributed to specific etiological agents such as Mycoplasma gallisepticum, disease progression is fundamentally tied to broad ecological dysbiosis within the respiratory tract. This comprehensive review synthesizes current knowledge on the functional roles and compositional dynamics of the poultry respiratory microbiome in both healthy and diseased states. Notably, we acknowledge the emerging threat of climate change, elucidating how heat stress physically and immunologically disrupts the respiratory mucosal microenvironment, acting as a primary catalyst for microbiome destabilization and CRD susceptibility. By shifting the paradigm from a single-pathogen model to a comprehensive pathobiome perspective, we detail the microbial shifts that characterize CRD. Furthermore, because investigating this low-biomass environment presents unique analytical challenges, we critically evaluate current technical methodologies. We address critical bottlenecks in sample collection, DNA extraction biases, and the comparative efficacy of 16S rRNA amplicon versus shotgun metagenomic sequencing. Ultimately, this synthesis provides a foundational framework for optimizing diagnostic methodologies and developing microbiome-targeted interventions -such as next-generation probiotics- to mitigate CRD in an era of escalating environmental stressors.

Cryptosporidium parvum is a ubiquitous enteric parasite that infects a diverse range of vertebrate species. The detection of C. parvum can be confounded when oocysts are intermittently shed below an assay's limit of detection, yielding a false-negative result. We therefore investigated the utility of Cryptosporidium parvum virus 1 (CSpV1), a putative symbiont of Cryptosporidium parvum, as a surrogate target for detecting the parasite in bovine calves. Using real-time polymerase chain reaction (qPCR), we tested 422 samples for Cryptosporidium spp., C. parvum-associated targets, and CSpV1. Among the 189 samples positive for at least one target, CSpV1 was detected in 24 (12.70%) samples without concurrent detection of Cryptosporidium. Additionally, we analyzed CSpV1 genomic sequences to ascertain its value as an epidemiological biomarker. Evaluation of dsRNA1 amino acid sequences identified country-associated patterns, suggesting potential utility for geographic distribution analyses. These findings suggest that CSpV1 may serve as a biological signature of C. parvum and support further investigation into its usefulness as an adjunct molecular target.

Coronavirus hijacks a large number of host factors to complete the replication cycle. However, the engagement of a specific host factor in a particulate viral replication stage and the underlying mechanisms are poorly understood. Our previous transcriptomics studies have found that serum and glucocorticoid-induced kinase 1 (SGK1) is significantly up-regulated in cells infected with coronavirus avian infectious bronchitis virus (IBV). In this study, we confirm that SGK1 is upregulated in cells infected with coronaviruses in three genera, i.e., gammacoronavirus IBV, alphacoronaviruses porcine epidemic diarrhoea virus (PEDV) and betacoronavirus human coronavirus-OC43 (HCoV-OC43). Suppression of SGK1 expression by either siRNA-mediated knockdown or use of an SGK1-specific inhibitor showed the accumulation of significantly more viral structural proteins and higher viral titres in the total cell lysates, but comparatively less in the supernatants, compared with that in the control cells. In cells overexpressing SGK1, the accumulation of viral proteins was significantly reduced in the total lysates, but were slightly upregulated in the culture supernatants. These effects were further reinforced by overexpressing a constitutively active mutant SGK1-S422D and a functionally inactive mutant SGK1-K127M. Furthermore, knockdown and/or inhibition of upstream signalling molecules and downstream effectors demonstrated that IBV infection may activate the PI3K-SGK1-NEDD4L signal axis, playing a regulatory role in the IBV replication cycle. In addition, knockdown of SGK1 was shown to suppress the expression of several proinflammatory cytokines/chemokines and enhance the activation of apoptosis in IBV-infected cells. These findings reveal that induction of SGK1 expression by coronavirus infection may enhance the release of mature virions, identifying a potential target for designing novel intervention approaches.