Pseudorabies virus (PRV) is one of the most significant pathogens threatening the swine industry, and its infection has caused substantial economic losses to pig farming worldwide. This study utilized two PRV strains isolated by our laboratory: the GXLB-2015 strain, a natural recombinant between a PRV variant strain and the Bartha-K61 vaccine strain, which exhibits stronger virulence, and the GXGG-2016 strain, a classical genotype II PRV strain with a natural 69-amino acid deletion in its TK gene, which demonstrates weak virulence and is non-pathogenic in mice. Focusing on endoplasmic reticulum stress (ERS) and cellular autophagy, this research explored the molecular mechanisms underlying the significant difference in virulence between these two PRV strains. The results revealed that infection with both strains induced noticeable ERS and autophagy, both of which inhibited viral replication. Further investigation showed that upon ERS induction, the GXLB-2015 strain mediated autophagy by activating both the PERK-eIF2α-ATF4-CHOP and IRE1-XBP1 pathways; inhibiting these pathways suppressed viral replication. In contrast, the GXGG-2016 strain infection mediated autophagy primarily through activating only the IRE1-XBP1 pathway, and inhibiting this pathway had no significant impact on viral replication. These findings indicate that while PRV infection can induce cellular autophagy by activating ERS, the specific unfolded protein response (UPR) pathways involved differ significantly between strains of varying virulence. This study provides a foundation for understanding the pathogenesis of different PRV strains and developing novel antiviral drugs.

Mammalian orthoreoviruses (MRVs) infect a wide range of animal hosts, yet epidemiological data from southern China remain limited. Here, we performed regional screening of diarrheic cattle in Guangxi, China, from 2023 to 2025. Using L1-targeted RT-PCR, MRV was detected in 8.4% (15/178) of fecal specimens collected across eight prefectures, with a higher detection rate during the cool (autumn–winter) season (October–March). We obtained two complete S1 segments, assigning one Guangxi strain to MRV1 and the other to MRV3. Phylogenetic analysis of L1 sequences indicated a single local lineage nested within a broader multi-host cluster, which was consistent with segment reassortment. σ1 protein alignment revealed a conserved central/C-terminal scaffold and a variable distal head; notably, the MRV3 σ1 sequence contained an extended ~360–390 aa region compared with MRV1. Overall, these findings provide an epidemiological baseline for Guangxi cattle, integrating detection rate, serotype context, and seasonality to support targeted, seasonally informed surveillance and MRV risk assessment.

Canine influenza virus (CIV), an important lineage of influenza A virus (IAV) adapted to canine hosts, has emerged as a significant concern in zoonotic disease control. In recent years, CIV has exhibited distinct regional epidemic patterns worldwide, with H3N8 and H3N2 being the two predominant subtypes originating from cross-species transmission of equine and avian influenza viruses, respectively. This review systematically outlines the global epidemiological status of CIV and elaborates on the key molecular mechanisms underlying its interspecies barrier crossing and host adaptation. These adaptive mechanisms involve alterations in hemagglutinin receptor-binding specificity (e.g., the convergent W222L mutation), structural variations in the neuraminidase stalk region, adaptive mutations in the polymerase complex (e.g., PB2 I714S, E627K, D701N), immune antagonism by non-structural proteins (e.g., NS1-mediated suppression of the NLRP3 inflammasome), and genetic diversification through reassortment. Collectively, these evolutionary traits enhance viral replication efficiency, transmissibility, and immune evasion in mammalian hosts. A comprehensive understanding of the cross-species transmission patterns and adaptive evolution of CIV is crucial for developing effective prevention strategies and novel vaccines.

Heteroresistance (HR) to commonly used anti-pseudomonal agents in Pseudomonas aeruginosa has been reported in cross-sectional epidemiological surveys; however, longitudinal studies investigating the long-term epidemiological dynamics and clinical correlates of this adaptive phenotype are still lacking. This retrospective time-series study aims to systematically explore the HR phenotypic profiles and temporal dynamic of clinical P. aeruginosa isolates against piperacillin/tazobactam (TZP) and meropenem (MEM), and further clarify their potential clinical correlates. We comprehensively analyzed 380 non-duplicate clinical strains collected from a tertiary hospital between 2011 and 2024. The disk diffusion and Etest gradient diffusion methods were used for the preliminary determination of HR incidence rates in clinical isolates against six anti-pseudomonal antibiotics. Population analysis profiling (PAP) assays was further performed to assess the temporal dynamics and phenotypic characteristics of HR to TZP MEM. HR was defined as the presence of resistant subpopulations with an MIC at least eightfold higher than that of the dominant population at a frequency of ≥ 1 × 10⁻⁷. Subsequently, univariate and multivariate regression analyses were performed to identify clinical factors associated with HR and non-HR strains. Preliminary screening indicated that 55

Getah virus (GETV), an arthropod-borne alphavirus, has emerged as a significant pathogen responsible for reproductive failure and systemic disease in swine, incurring substantial economic losses within the livestock industry and presenting a potential zoonotic threat. Currently, no approved vaccines or antiviral therapies exist. Here, we employed reverse genetics to engineer a recombinant GETV (rGECNLuc) expressing a Nanoluciferase (NLuc) reporter gene. The reporter was inserted at the C-terminus of the viral capsid (Cap) protein via a T2A self-cleavage peptide to facilitate co-translational release. In vitro characterization demonstrated that rGECNLuc replicated efficiently in BHK-21 and Vero cells, with plaque morphology and multi-step growth kinetics comparable to the parental virus, rGETV-GX. The NLuc insertion was genetically stable over six consecutive passages without deletion. In vivo assessment in a 10-day-old suckling mouse model revealed a significant attenuation of virulence for rGECNLuc compared to the parental strain, despite efficient viral replication in joints, lungs, and brain tissues. Using in vivo imaging (IVIS), we established a strong positive correlation between bioluminescent signal intensity and viral load in 3-week-old and 5-week-old mice. Signals disseminated systemically from the inoculation site, with pronounced enrichment in joints and lungs, and exhibited greater intensity in younger animals. Collectively, rGECNLuc enables real-time, non-invasive visualization of GETV infection and provides a tractable platform for quantifying viral dissemination, tissue tropism, and antiviral activity in vivo.

Porcine astrovirus (PAstV) is globally prevalent in swine and is associated with diarrhea and encephalitis in piglets, posing a threat to porcine health. However, its pathogenic mechanisms remain poorly understood. This study used the PAstV1-GX1 strain to infect PK-15 cells, revealing that the virus induces significant apoptosis, with late apoptotic cells reaching 41.2% at 24 hours post-infection. The infection activates caspase-9 and caspase-3, but not caspase-8, and causes mitochondrial damage, indicating apoptosis via the mitochondrial pathway. The apoptosis inhibitor Z-VAD-FMK reduced viral replication, while apoptosis inducer ABT-263 enhanced it at later stages. The nsP1a/3 protein, which interacts with MAVS and localizes to mitochondria, was identified as key in inducing apoptosis. Its 3C-like serine protease domain likely mediates this interaction. Knocking down MAVS reduced apoptosis and increased early-stage replication but decreased it later. Overexpressing MAVS increased apoptosis and decreased replication. Furthermore, we observed that the expression of nsP1a/3 resulted in the cleavage of MAVS and the suppression of the type I interferon (IFN) response. Notably, treatment with Z-VAD-FMK did not influence nsP1a/3-mediated MAVS cleavage or type I IFN inhibition, suggesting that the induction of apoptosis and MAVS cleavage are distinct processes. By employing site-directed mutagenesis to substitute alanine for the catalytic triad residues (His459, Asp487, and Ser549) of the 3C-like serine protease, we significantly reduced the ability of nsP1a/3 to induce apoptosis, cleave MAVS, and suppress the type I IFN response, underscoring the essential role of protease activity in these functions. Furthermore, the use of a serine protease inhibitor markedly decreased PAstV replication. These findings provide significant insights into the pathogenesis of PAstV and establish a foundation for the development of novel antiviral therapies.

The PA gene of the 2009 pandemic H1N1 (pdm09/H1N1) lineage is derived from avian influenza virus (AIV). Together with other polymerase subunits, it enhances the adaptation of avian-origin influenza virus to mammals. However, the functional region of the PA protein remains unclear. Using reverse genetics, we mapped functional domains of pdm09/H1N1 PA in an avian-origin H9N2 canine influenza virus (CIV) background. The N-terminal 169-252 region conferred high intrinsic polymerase activity yet failed to support efficient viral replication alone, revealing a dissociation between catalytic potential and replicative fitness. In contrast, the 85-168 region enabled robust replication despite lower activity. Importantly, synergy between the 169-252 region and its cognate pdm09/H1N1 NP drove high polymerase activity and replication. This pairing also enhanced viral growth in vitro, increased viral titers in mouse lungs and nasal turbinate, and induced pulmonary damage. Mechanistically, the 169-252 region interacts with NP, accelerating nuclear import and cRNA synthesis, thereby optimizing viral RNA replication timing. Thus, PA-NP co-evolution is a key driver of mammalian adaptation, moving beyond single mutations and highlighting internal gene compatibility as a determinant of viral fitness and pandemic potential.

Goose astrovirus (GAstV) is a significant pathogen affecting goslings by inducing visceral gout, yet no commercial vaccine is currently available. This study involved the serial passaging of the GAstV-GXNN strain in LMH cells to investigate alterations in viral replication, genomic stability, and pathogenicity, as well as to assess the potential of a vaccine candidate. The findings indicated that the viral titer increased progressively with each passage, reaching 107.35 TCID50/mL by the 120th passage (GAstV-GXNNP120). Whole-genome sequencing revealed the presence of 6, 19, 26, and 28 nucleotide mutations at the 30th, 60th, 90th, and 120th passages, respectively. Pathogenicity assays demonstrated a reduction in virulence with successive passages, culminating in the complete attenuation of GAstV-GXNNP120, which did not induce clinical signs or lesions in one-day-old goslings. Following five successive passages in goslings, the attenuated strain exhibited stable genetic characteristics without any reversion to virulence. Goslings aged one day, inoculated with GAstV-GXNNP120 at dosages ranging from 102.0 to 105.0 TCID50, developed neutralizing antibodies by the third day post-vaccination. Antibody levels increased in a dose-dependent manner, peaking at day 21 and remaining elevated through day 42. Challenge experiments utilizing the virulent GAstV-GXNN strain revealed that groups vaccinated with doses of 103.0 TCID50 and above achieved complete protection. These groups exhibited no clinical symptoms or pathological damage post-challenge, and both tissue viral loads and virus shedding levels were significantly reduced compared to the control group. Consequently, the minimum effective vaccination dose was established at 103.0 TCID50. These results provide a crucial foundation for the development of a live attenuated GAstV vaccine.

BACKGROUND:Heteroresistance (HR) in Pseudomonas aeruginosa causes misclassification as 'susceptible (S)' on routine antibiotic susceptibility techniques, potentially contributing to subsequent treatment failure. This study aimed to explore clinically relevant risk factors for HR compared with S (S-HR), and resistant (R) compared with HR (HR-R) phenotypes. Additionally, we explored whether integrating medical history and laboratory data can enable rapid and accurate identification of HR and R phenotypes in this pathogen. METHODS:This retrospective study included 420 P. aeruginosa strains collected from 2011 to 2024 in China. The strains were categorized into three groups according to their sensitivity to meropenem: non-heteroresistant susceptible, heteroresistant and non-heteroresistant resistant. Logistic regression, random forest and XGBoost models were constructed using variables identified through LASSO (least absolute shrinkage and selection operator) regression. The models' performance was evaluated via 10-fold cross-validation comparing area under the receiver operating characteristic curve (AUROC), sensitivity and specificity. RESULTS:Multivariate analyses identified central venous catheters as an independent risk factor for S-HR, and malignant solid tumours, pulmonary infections, mechanical ventilation and carbapenem use for HR-R. A discriminating diagnostic model, combining clinical and laboratory data, showed an AUROC of 0.919 for HR-R and 0.856 for S-HR. The calibration plots indicated good alignment between the estimated and observed probabilities. CONCLUSIONS:This study presents a validated two-stage risk assessment model to discriminate the two phases of meropenem heteroresistance in P. aeruginosa. By identifying novel stage-specific risk factors and delivering a practical tool compatible with clinical workflows, this model facilitates the early identification and targeted intervention of HR, offering novel insights into the mechanistic dissection of HR.

Porcine astrovirus (PAstV) is an important and widespread pathogen in swine, linked to diarrheal outbreaks and extraintestinal disease. How PAstV enters host cells has remained unclear, and no cellular factor has been defined for PAstV entry. Here, a genome-wide CRISPR-Cas9 loss-of-function screen in porcine epithelial cells identifies Annexin A1 (ANXA1) as a host factor that facilitates PAstV entry. Genetic ablation or pharmacological/antibody blockade of ANXA1 reduces binding, lowers early viral RNA and capsid signals, and delays the rise of progeny, whereas re-expression restores susceptibility. Biochemical assays and surface plasmon resonance indicate a direct interaction between ANXA1 and the acidic C-terminal domain of the PAstV ORF2 capsid protein, and imaging shows ANXA1 co-localizes with incoming particles at the cell surface and supports attachment and uptake. Loss of ANXA1 does not alter infection by the non-astrovirus panel tested, indicating selectivity for PAstV under our conditions. Notably, infection is reduced but not abolished in ANXA1-deficient cells, consistent with additional entry factors acting alongside ANXA1. These findings position ANXA1 as an entry cofactor for PAstV and provide a mechanistic basis to refine models of astrovirus host-cell recognition.

Porcine astrovirus (PAstV) is widespread in swine, yet the role of its conserved Stem-Loop II motif (s2m) remains unclear. Using a PAstV-GX1 reverse-genetics system, we engineered s2m deletions and point mutants and assessed replication and innate sensing in PK 15 cells. All mutants were viable and genetically stable. Compared with wild type, s2m-edited viruses showed attenuated early replication and delayed ORF2 accumulation, with growth converging by similar to 24 h post-infection. Disruption of s2m also advanced innate responses, increasing IFN-beta mRNA and promoter activity at 8 h. Preinfection with an s2m mutant transiently restricted heterologous virus replication. These findings indicate that s2m is non-essential but optimizes early kinetics and tempers interferon activation, supporting its consideration as a structurally constrained, potentially targetable RNA element.

The N-linked glycosylation of alphavirus envelope proteins plays critical roles in glycoprotein folding, host-receptor interactions, immune evasion, and pathogenicity. Getah virus (GETV) has two putative N-linked glycosylation sites (N-200 and N-262) in the E2 and one (N-141) in E1. We generated seven glycosylation-deficient mutants and evaluated their fitness across mammalian cells, mosquitoes, and mouse models. Loss of glycosylation at E2 N-262 or E1 N-141 enhanced in vitro replication and replication efficiency in mosquitoes, while E2 N-200 glycosylation-deficient mutants retained parental replication capacity in vitro but exhibited accelerated mosquito colonization. Despite these gains, glycan loss reduced viral adsorption/entry in selected settings and decreased measurable virion binding to MXRA8 and LDLR in vitro, while single-site E2 glycan mutants exhibited increased heparin sensitivity/affinity, indicating altered utilization of glycosaminoglycan attachment pathways. In vivo, all mutants remained lethal in 3-day-old mice but showed age-dependent attenuation in 10-day-old mice. Notably, E1 N141-deficient mutant induced no clinical symptoms and exhibited reduced viremia and tissue viral loads. Glycan ablation increased susceptibility to neutralization without impairing induction of neutralizing antibodies. Strikingly, E2 mutants rapidly reacquired glycosylation during in vivo replication, indicating strong evolutionary selection for these sites. Together, our data support an evolutionary trade-off in which GETV envelope glycans-particularly the epidemic-lineage-associated E2-N262 glycan-optimize overall fitness by balancing replication/transmission efficiency with humoral immune evasion.

Eurasian avian-like H1N1 influenza A viruses that have reassorted with the 2009 pandemic H1N1 virus pose a potential public health threat. We previously reported that synergistic mutations in HA and NS genes enhanced the virulence of a mouse-adapted EA H1N1 canine influenza virus (CIV), yet the underlying mechanisms remained unclear. Here, we demonstrate that the adaptive mutations NS1-A53D and NEP-R42K synergistically promote viral replication by enhancing viral ribonucleoprotein (vRNP) nuclear export, while NEP-R42K alone boosts polymerase activity. Critically, we uncover a novel synergy between HA mutations (N198D and A227E) and NS1-A53D, but not NEP-R42K, in facilitating early viral entry, including attachment and internalization. Mechanistically, NS1-A53D significantly enhances HA protein stability without altering receptor binding specificity, thereby compensating for the intrinsic antagonism between the two HA mutations that impairs receptor binding affinity. These findings reveal that HA and NS genes cooperate through distinct but complementary mechanisms: NS1-A53D stabilizes HA to promote entry, while NS1-A53D and NEP-R42K collaboratively enhance vRNP export, collectively driving mammalian adaptation and pathogenicity.

Goose astrovirus (GAstV) is a major pathogen associated with gout in goslings. Currently, no effective therapeutics or vaccines are available against GAstV infection, and disease control primarily relies on strict biosecurity measures. Therefore, the development of rapid and reliable diagnostic methods is critical for clinical management and reducing the impact of the virus. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of GAstV was developed. The N-terminal region (amino acids 130–401) of the GAstV capsid protein was cloned into the prokaryotic expression vector pET-32a(+) and expressed in Escherichia coli BL21 cells. The recombinant protein, designated GAstV-Cap-N, was purified and used as the coating antigen in the ELISA. Following systematic optimization of key parameters, the established indirect ELISA exhibited high specificity, sensitivity, and reproducibility. Comparative analysis with Western blot demonstrated a concordance rate of 87.5%. The assay was subsequently applied to test 228 clinical serum samples from geese, revealing an overall seropositivity rate of 38.19%. These findings indicate that the developed indirect ELISA provides a robust and reliable tool for clinical diagnosis and epidemiological surveillance of GAstV.

Porcine reproductive and respiratory syndrome virus (PRRSV) employs RNA recombination to generate phenotypic variants that evade host immunity, with recombination hotspots frequently observed in the NSP9 gene of field strains. This study investigates the structural determinants of recombination hotspots in the NSP9 gene by integrating bioinformatics analyses and in vitro and in vivo co-infection experiments. We identified three high-frequency recombination hotspots (7492-7624 nt, 7887-7940 nt and 9009-9464 nt) characterized by AU-rich sequences and stem-loop structures. Notably, these hotspot profiles were conserved between field-derived strains and in vitro co-infection models, supporting the use of experimental systems to highlight candidate recombination-prone contexts. To assess the functional role of RNA motifs, synonymous mutations disrupting AU-rich regions or secondary structures were engineered into the 7887-7940 nt hotspot. These mutations significantly reduced recombination rates, underscoring the contribution of local nucleotide composition and structural features to recombination dynamics. Collectively, our findings define sequence/structure contexts that shape PRRSV recombination signatures and provide a mechanistic framework that may inform efforts to improve the genetic stability of live vaccine candidates by reducing recombination propensity while acknowledging that validation in porcine target cells and/or in vivo models is required.

The H3N2 subtype of canine influenza virus (CIV) has emerged as a significant pathogen in canine populations since 2006, causing widespread epidemics globally. The hemagglutinin (HA) protein, particularly the HA1 subunit, plays a critical role in viral entry and is a primary target for vaccine development and antiviral therapies. In this study, we analyzed HA structure and rare codons, immunized BALB/c mice with the truncated HA protein, and generated three HA1-specific mAbs: 2A8, 3C4, and 5A3. mAbs 2A8 and 5A3 targeted the conserved epitope 211QTIIP215, while 3C4 recognized the moderately variable epitope 91SNAFS95. Analysis of H3 CIV HA sequences from GISAID revealed that 211QTIIP215 is highly conserved across H3N2 CIV strains, suggesting its potential as a target for broad-spectrum antibodies development, whereas mutations in 91SNAFS95 exhibits moderate sequence variability. These findings support the design of vaccines and diagnostics for H3N2 CIV, advancing our understanding of CIV biology.

Mycoplasma gallisepticum (MG) is a significant avian pathogen responsible for chronic respiratory disease in chickens and infectious sinusitis in turkeys (Meleagris gallopavo). It infects the respiratory tract, leading to chronic inflammation and, in some cases, conditions such as synovitis and arthritis. MG causes substantial economic losses in the poultry industry due to reduced egg production, hatchability, meat quality, and increased mortality. The primary pathogenic mechanism involves immune dysregulation, enabling the bacterium to persist in the host and establish chronic infection. Key virulence factors include adhesins (e.g., GapA, CrmA, pMGA), variable surface lipoproteins (e.g., VlhA), and recently characterized molecules like the TatD nuclease. Current control measures include antibiotics, management adjustments, and vaccination. However, extensive use of broad-spectrum antibiotics like tetracyclines and macrolides has led to increased drug resistance. Although commercial vaccines (live attenuated, inactivated, and newer epitope-based and recombinant designs) are available, they often provide incomplete or inefficient protection. This review summarizes the current understanding of MG pathogenesis, highlights recent advances in vaccine development, and discusses the limitations and future directions for MG control.

Getah virus (GETV), a mosquito-borne arbovirus of the Alphavirus genus, poses an emerging threat to livestock economies and public health, underscored by its expanding host range and association with recent outbreaks of heightened virulence. While the functional significance of the 3’ untranslated region (3’UTR) in alphavirus biology is recognized, its specific role in GETV remained undefined. Herein, we elucidate the virological functions of the GETV 3’UTR through a reverse genetics approach, generating a panel of viruses with targeted deletions. We demonstrate that the GETV 3’UTR is remarkably plastic, tolerating a consecutive deletion of up to 310 nucleotides while remaining viable. Deletion of conserved repeat sequence elements (RSEs) induced a cell-type-specific replication deficiency in vitro and significantly attenuated virulence in a murine model. A comprehensive deletion mutant (rGETV-KO310) exhibited further impaired replication kinetics in vitro and was profoundly attenuated in vivo, eliciting only transient morbidity with no mortality in both neonatal and weaned mice. Furthermore, this mutant displayed a significant defect in early colonization within mosquito vectors, indicating a role in vector competence. Comparative transcriptomic profiling of knee joints revealed that attenuation correlates with the altered modulation of critical host immune responses, notably the interferon and MAPK signaling pathways. Collectively, these findings establish the GETV 3’UTR as a pivotal regulator of viral fitness, pathogenesis, and transmission. This work provides a foundational rationale for the strategic development of live-attenuated vaccine candidates based on targeted 3’UTR attenuation.

Swine influenza A viruses (swIAVs) undergo frequent reassortment and evolution, posing significant threats to both animal and human health. In this study, we employed reverse genetics to generate five recombinant viruses and evaluated their potential as vaccine candidates. Through pathogenicity evaluation in mice, we identified two promising candidates: an inactivated vaccine (rPR8-JGL16+2) and a live-attenuated vaccine (rPR8-JGL15+3). Both vaccines induced robust hemagglutination inhibition (HI) antibody responses and specific IgG levels against homologous strains. Notably, the attenuated rPR8-JGL15+3 vaccine provided complete protection against both homologous (H1N1) and heterologous (H3N2) viral challenges, with no detectable viral loads in the lungs or nasal turbinate. Although the inactivated rPR8-JGL16+2 vaccine exhibited effective protective efficacy against influenza viruses, it did not fully inhibit the replication of heterologous viruses in mice. Histopathological assessment revealed that rPR8-JGL15+3 immunization prevented significant lung damage, contrasting with the mild pathology observed in rPR8-JGL16+2-vaccinated animals. These findings demonstrate the successful development of novel vaccine candidates against avian-like H1N1 swIAV, with the attenuated vaccine showing promise for broad protection against circulating strains.