Streptococcus suis is a worldwide pathogen that impacts the swine industry, causing severe clinical signs, including meningitis and arthritis, in postweaning piglets. A key virulence mechanism of S. suis is biofilm formation, which improves its persistence and resistance to external factors. Here, we assessed the in vitro biofilm formation of 240 S. suis isolates from Spanish swine farms and evaluated the effects of serovars (SVs) and coinfections with other porcine respiratory disease complex (PRDC) pathogens. Our study revealed significant heterogeneity in biofilm formation among S. suis SVs. Notably, SV2 resulted in the lowest degree of biofilm formation, in contrast with the high biofilm-forming capacities of SV1, SV7, and SV9. Other PRDC pathogens, including Actinobacillus pleuropneumoniae, Glaesserella parasuis, and Pasteurella multocida, formed biofilms, although they were generally less robust than those of S. suis (except for SV2), which contrasts with the high biofilm formation of Staphylococcus hyicus. Coinfections enhanced biofilm formation in mixed cultures of S. suis, particularly with P. multocida. Other coinfections revealed variable results in pathogen interactions, suggesting the potential of biofilms for increased persistence and pathogenicity in coinfections. In conclusion, this study underscores the importance of serovar-specific differences in biofilm formation among S. suis isolates, with significant implications for pathogenicity and persistence. The heterogeneous biofilm formation observed in coinfections with other PRDC pathogens reveals a complex interplay that could exacerbate disease severity. These findings provide a foundation for further research on biofilm mechanisms to mitigate the impact of PRDC in the swine industry.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant diseases affecting the pig industry worldwide and is caused by the PRRS virus (PRRSV), which has complex genetic variation due to frequent mutations, indels, and recombination. The emergence of PRRSV L1C.5 in 2020 in the United States has raised worldwide concerns about PRRSV with the RFLP 1-4-4 pattern and lineage 1C. However, studies on the pathogenic characteristics, epidemiological distribution, and effectiveness of vaccines against PRRSV with L1C and RFLP1-4-4 pattern in China are still insufficient. In this study, a novel recombinant variant of PRRSV with RFLP 1-4-4 and lineage 1C features, different from L1C.5 in the United States, was isolated in China in 2021. In pathogenicity experiments in specific pathogen-free piglets or farm piglets, 60-100% of artificially infected experimental piglets died with high fever and respiratory symptoms. Inflammatory cytokine and chemokine levels were upregulated in infected piglets. A commercially modified live vaccine against highly pathogenic PRRSV did not provide effective protection when the vaccinated piglets were challenged with the novel L1C-1-4-4 variant. Therefore, this strain merits special attention when devising control and vaccine strategies. These findings suggest that extensive joint surveillance is urgently needed and that vaccine strategies should be updated to prevent the disease from spreading further.

The genus Megalocytivirus, belonging to the family Iridoviridae, is one of the most detrimental virus groups to fish aquaculture. Megalocytivirus creates a virus-mock basement membrane (VMBM) on the surface of infected cells. This membrane provides attachment sites for lymphatic endothelial cells (LECs), disrupting fish's endothelial cell-extracellular matrix system. This disruption triggers injury to the vascular system and can result in death. Exploring the VMBM-cell interaction mechanism is crucial for uncovering the pathogenesis of Megalocytivirus and identifying therapeutic targets. Claudins, a class of tetra transmembrane proteins, play a key role in creating tight junctions between endothelial or epithelial cells. In this study, we demonstrated that the expression of Claudin2, a member of the Claudin family in fish, was significantly up-regulated by Megalocytivirus infection. Claudin2 was found in LECs attached to the surface of infected cells. It interacted with the VMBM viral components VP23R, VP08R, and VP33L at multiple binding sites through its two extracellular loops. However, it did not interact with the host basement membrane's nidogen. Therefore, Claudin2 is involved in the interaction of LEC with VMBM and plays a role in the disturbed distribution of extracellular matrix and endothelial cells in Megalocytivirus-infected fish tissues. This study aims to uncover the molecular mechanisms by which Megalocytivirus infection leads to pathological changes in the vascular system.

Bovine parainfluenza virus type 3 (BPIV3) is a viral respiratory pathogen that infects cattle and causes significant economic losses. We generated a recombinant adenovirus called rHAd5-F + HN by expressing the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein of BPIV3 using the human adenovirus serotype 5 (rHAd5). We evaluated its effects on humoral and cellular immune responses in mice (n = 45) and calves (n = 9). Serum antibody responses were assessed by enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition (HI), and neutralising antibodies (NAb). After boosting immunity with rHAd5-F + HN, mice produced significantly higher levels of antibodies against the BPIV3 genotype A and genotype C strains. The production of antibodies exceeded those produced by adenoviruses rHAd5-F and rHAd5-HN, which express the F and HN glycoprotein, respectively. The percentages of splenic CD3/CD8T lymphocytes and IL-4 cytokines in rHAd5-F + HN mice were considerably higher than those in the control group. Mice immunised with rHAd5-F + HN exhibited much lower viral loads in the lungs and tracheas compared to the control group. Additionally, the lungs of mice vaccinated with rHAd5-F + HN showed no notable histopathological changes. On the other hand, rHAd5-F + HN produced a humoral immune response in calves. Following the booster intramuscular injection with the rHAd5-F + HN, the serum antibody levels against BPIV3 genotype C strain were 1:20 452, 1:1024, and 1:426 in calves, as detected by ELISA, HI, and NAb, respectively. The HI and NAb levels against the BPIV3 genotype A strain were 1:213 and 1:85 in calves, respectively. These results indicate that rHAd5-F + HN effectively induced immunity against BPIV3 infection.

Rosavirus is a newly discovered member of the family Picornaviridae that was initially detected in wild rodents and subsequently in children with diarrhoea. Nevertheless, there is a significant gap in our understanding of the geographical distribution, phylogenetic relationships, evolutionary patterns, and transmission of rosaviruses. To address these issues, we analysed 434 rodents and shrews from five different species that were collected in southern China. Using PCR screening of faecal samples, we detected rosaviruses in Norway rats (Rattus norvegicus) and identified two previously undocumented host species: tanezumi rats (Rattus tanezumi) and Asian house shrews (Suncus murinus). Rosaviruses were particularly common in these animals, with an overall prevalence rate of 32.49% (141/434). For genetic and evolutionary analyses, we selected six representative positive samples to amplify the complete genomes of rosaviruses. Bayesian phylogenetic analysis suggested that our sequences clustered within the genus Rosavirus, where genotype B sequences are the closest relatives. The elevated nonsynonymous-to-synonymous ratios observed in rosavirus B may be attributed to relaxed selection pressures driven by virus spillover events. On the basis of the available data, it is hypothesized that the genus Rosavirus may have originated from Norway rats around the year 1339. In summary, these findings provide valuable insights into the complex evolutionary history of rosaviruses and underscore the urgent need for ongoing surveillance of this virus.

Porcine dermatitis and nephropathy syndrome (PDNS) is a severe condition that affects mainly growing pigs and is considered to be caused by a type III hypersensitivity reaction. Although porcine circovirus 2 (PCV-2) is the antigen linked to this condition, porcine circovirus 3 (PCV-3) has also been proposed to be causally associated with PDNS. Moreover, the initial description of porcine circovirus 4 (PCV-4) also related this novel agent to this clinicopathological entity. Therefore, this retrospective study included a large number of PDNS cases (n = 102) fulfilling specific histologic criteria in search of known porcine circoviruses (PCV-1 to PCV-4) by conventional and/or quantitative PCR (qPCR). All the samples were subjected to PCV-2 immunohistochemistry (IHC) or conventional in situ hybridization (C-ISH), and RNAscope (R-ISH) was used to study PCV-2 and PCV-3 localization in a subset of the samples. All PDNS cases were PCV-2 positive by qPCR, while 30 of them (29.4%) yielded PCV-3 qPCR positivity; PCV-2 viral loads were significantly greater than PCV-3 viral loads. All animals were negative for PCV-1 and PCV-4. By C-ISH/IHC, 63 cases (61.8%) were positive for PCV-2, with low to moderate amounts of antigen. R-ISH demonstrated higher sensitivity, as all studied cases were positive; however, neither PCV-2 nor PCV-3 were consistently found within characteristic PDNS lesions. These results indicate that all PDNS-affected pigs were infected with PCV-2, emphasizing the likelihood that this viral antigen is causally linked to this condition. In contrast, no evidence of the association of PCV-1, PCV-3 or PCV-4 with PDNS was found.

Porcine parvovirus (PPV) infection induces germ cell death, leading to reproductive disorders in first-pregnant sows. Porcine placental trophoblast cells (PTCs) are the major target of PPV, and we have previously found that PPV infection leads to the death of PTCs by a non-apoptotic process, which may be related to PPV pathogenicity. The Z-nucleic acid-binding protein 1 (ZBP1) is often activated after virus invasion and mediates subsequent cell death. Here, we found that PPV infection induced ZBP1-mediated necroptosis of porcine PTCs in the presence of the apoptosis inhibitor, AC-DEVD-CHO. ZBP1 expression was upregulated during PPV infection, and ZBP1 knockout or RNA interference significantly reduced its expression along with the PPV-induced necroptosis. We discovered that RIPK3 and MLKL, but not Caspase-8, were involved in downstream signaling of ZBP1 during PPV infection; the phosphorylation levels of RIPK3 and MLKL were enhanced, but Caspase-8 was not significantly cleaved. The knockout of RIPK3 and MLKL significantly reduced the PPV infection-induced necroptosis of porcine PTCs. RIPK3 knockout did not affect the PPV infection-induced upregulation of ZBP1 expression, but blocked the activation of MLKL. MLKL knockout did not affect the upregulation of ZBP1 expression and RIPK3 phosphorylation during PPV infection. UV-inactivated PPV induced significantly less necroptosis of porcine PTCs than non-irradiated PPV. A PPV strain with a mutation in the translation initiation codon was still able to induce necroptosis of PTCs through the ZBP1/RIPK3/MLKL pathway. These results provide new insights into the pathogenic mechanism of PPV infection and suggest that PPV infection of porcine PTCs induces necroptosis through the viral DNA-dependent ZBP1/RIPK3/MLKL pathway.

Elizabethkingia miricola is a multidrug-resistant pathogen that can cause life-threatening infections in immunocompromised humans and outbreaks in amphibians. However, the specific virulence factors of this microorganism have not been described. In this study, we identified the polysaccharide biosynthesis protein-encoding gene capD, which is located in the conserved region of the Wzy-dependent capsule synthesis gene cluster in the E. miricola strain FL160902, and investigated its role in the pathogenesis of E. miricola. Our results revealed that the capD deletion strain (ΔcapD) lost its typical encapsulated structure, with a 45% reduction in cell wall thickness. CapD affects wza expression in the capsule polysaccharide synthesis pathway. Furthermore, the survival rates were significantly reduced in ΔcapD in response to complement-mediated killing, desiccation stress, and macrophage phagocytosis, whereas biofilm formation, surface hydrophobicity, and adherence to both endothelial and epithelial cells were increased. Additionally, the deletion of capD sharply attenuated the virulence of E. miricola in a frog infection model. Complementation of the capD gene restored the biological properties and virulence to wild-type levels. Overall, these findings suggest that CapD contributes to polysaccharide synthesis and plays a crucial role in the pathogenesis of E. miricola.

In 2018, African swine fever virus (ASFV) emerged in China, causing extremely serious economic losses to the domestic pig industry. Infection with ASFV can cause disseminated coagulation, leading to the consumption of platelets and coagulation factors and severe bleeding. However, the mechanism of virus-induced coagulation has yet to be established. In our study, ASFV downregulated the coagulation process, as detected by D-dimer (D2D) and Factor X (F10) expression in pigs challenged with ASFV HLJ/18. In vitro, ASFV infection increased Factor IX (F9) and Factor XII (F12) expression while downregulating F10 expression in porcine alveolar macrophages (PAMs). African swine fever virus induced both intrinsic and extrinsic coagulation cascades. In addition, several encoded proteins affect the expression of the crucial coagulation protein F10, and among the encoded proteins, p72 inhibits the activity and expression of F10. Proteomic analysis also revealed that p72 is involved in the coagulation cascade. p72 can interact with F10, and its inhibitory functional domains include amino acids 423-432 and amino acids 443-452. Finally, we found that F10 and p72 interact with tripartite motif-containing protein 28 (TRIM28). TRIM28 knockdown resulted in a decrease in F10 expression. Importantly, TRIM28 contributes to the reduction in F10 protein expression regulated by p72. Our findings revealed an inhibitory effect of the viral protein p72 on the ASFV infection-induced coagulation cascade and revealed a role of TRIM28 in reducing F10 expression, revealing a molecular mechanism of ASFV-associated coagulation.

Porcine reproductive and respiratory syndrome virus (PRRSV) has led to significant economic losses in the global swine industry. Type I interferon (IFN-I) plays a crucial role in the host's resistance to PRRSV infection. Despite extensive research showing that PRRSV employs multiple strategies to antagonise IFN-I induction, the underlying mechanisms remain to be fully elucidated. In this study, we have discovered that PRRSV inhibits the production of IFN-I by degrading TANK-binding kinase 1 (TBK1) through chaperon-mediated autophagy (CMA). From a mechanistic standpoint, PRRSV nonstructural protein 2 (Nsp2) increases the interaction between the heat shock protein member 8 (HSPA8) and TBK1. This interaction leads to the translocation of TBK1 into lysosomes for degradation, mediated by lysosomal-associated membrane protein 2A (LAMP2A). As a result, the downstream activation of IFN regulatory factor 3 (IRF3) and the production of IFN-I are hindered. Together, these results reveal a new mechanism by which PRRSV suppresses host innate immunity and contribute to the development of new antiviral strategies against the virus.

Mastitis, often caused by bacterial infection, is an inflammatory condition affecting the mammary glands. The condition is particularly prevalent in dairy cattle. Current treatment of bovine mastitis heavily relies on the use of antibiotics. To identify alternative solutions to antibiotic use, we evaluated the antimicrobial activity of 14 cathelicidins reported from 10 animal species. In conjunction, we assessed two bacteriocins against the bovine-mastitis causative bacterial panel, consisting of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus equi. Among the antimicrobial peptides (AMPs), cc-CATH3, ML-CATH, and PD-CATH proved to be highly active (minimum inhibitory concentration of 2-41 μg/mL, 0.2-10.3 μM) against all bacterial strains in the panel and field isolates from milk, with elevated somatic cell counts (≥ 500,000 cells/mL). Of the AMPs tested in this study, ML-CATH presented the highest level of effectiveness in controlling mastitis-associated bacterial strains while also possessing minimal cytotoxicity and functional stability against pH change and a high salt condition. The results of in silico analyses on the biochemical features of 12 helical cathelicidins revealed that the charge of AMPs appears to be a major determinant in killing Gram-negative bacteria. Furthermore, we observed a unique motif, "N-P-N", from the sequences of PMAP-36, cc-CATH3, ML-CATH, and PD-CATH that exhibits potent antimicrobial activity against a broad spectrum of bacteria compared to others. Our findings support the proposition that AMPs could serve as effective antimicrobial alternatives to conventional antibiotics in treating complex animal diseases caused by microbial infection, such as bovine mastitis.

Interferon α (IFNα) and interferon γ (IFNγ) play pivotal roles in mediating crucial biological functions, including antiviral activity and immune regulation. However, the efficacy of monomeric IFN is limited, and its administration relies solely on injection. To address this issue, we successfully expressed and purified a recombinant porcine IFNα and IFNγ fusion protein (rPoIFNα/γ). Furthermore, we developed a pH-triggered humic acid hydrogel delivery system that effectively protects rPoIFNα/γ from gastric acid degradation, enhancing its oral bioavailability. Neither the humic acid hydrogel nor rPoIFNα/γ exhibited cytotoxic effects on porcine kidney-15 (PK-15) cells in vitro. The replication of vesicular stomatitis virus and pseudorabies virus (PRV) was effectively inhibited by rPoIFNα/γ, resulting in an antiviral activity of approximately 10 U/mL. Scanning electron microscopy revealed that the humic acid hydrogel had a loose and porous honeycomb structure. The IFNα/γ@PAM hydrogel effectively adsorbed rPoIFNα/γ, as confirmed by Fourier transform infrared spectroscopy analysis, demonstrating a favourable IFN-loading capacity. In vitro experiments revealed that IFNα/γ@PAM swelled and released IFNα/γ rapidly at pH 7.4 but not at pH 1.2. The oral administration of IFNα/γ@PAM in mice enhanced the proliferation and differentiation of CD4 and CD8 cells. Additionally, mice infected with PRV and treated with IFNα/γ@PAM presented increased transcription levels of interferon-stimulated genes in the serum, reduced mortality rates, lower viral loads in various tissues, and decreased levels of organ damage. In conclusion, this study demonstrates that orally administered IFNα/γ@PAM has antiviral and immunomodulatory effects, highlighting its potential as a therapeutic agent against PRV infection.

Highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b viruses were first detected in St. John's, Canada in late 2021. To investigate the patterns of avian influenza virus (AIV) infection and immune responses subsequent to the arrival of H5N1, we sampled the wild urban duck population in this area for a period of 16 months after the start of the outbreak and compared these findings to those from archived samples. Antibody seroprevalence was relatively stable before the outbreak (2011-2014) at 27.6% and 3.9% for anti-AIV (i.e., NP) and H5-specific antibodies, respectively. During the winter of 2022, AIV-NP and H5-specific antibody seroprevalence both reached 100%, signifying a population-wide infection event, which was observed again in late February 2023 following a second H5N1 incursion from Eurasia. As expected, population-level immunity waned over time, with ducks seropositive for anti-AIV-NP antibodies for approximately twice as long as for H5-specific antibodies, with the population seronegative to the latter after approximately six months. We observed a clear relationship of increasing antibody levels with decreasing viral RNA loads that allowed for interpretation of the course of infection and immune response in infected individuals and applied these findings to two cases of resampled ducks to infer infection history. Our study highlights the value of applying both AIV surveillance and seroprevalence monitoring to provide a better understanding of AIV dynamics in wild populations, which may be crucial following the global dissemination of clade 2.3.4.4b H5Nx subtypes to assess the threats they pose to both wild and domestic animals, and to humans.

The 13th International Veterinary Immunology Symposium (IVIS) was initially due to take place in August 2022, but as many things in our lives, the COVID-19 pandemic that hit the world two years prior, forced the organising committee to postpone the meeting until November 2023. As it is tradition, the veterinary immunology symposium was organised as a satellite meeting of the IUIS International Congress of Immunology, which in 2023 took place in Cape Town, and it is where veterinary immunologists from all over the world get together to discuss advances and challenges in the field of animal health. The 2023 International Veterinary Immunology Symposium (IVIS) was held from November 17th to 21st at Kruger National Park, Skukuza, South Africa. This was the first time the symposium was hosted on the African continent. This event gathered 210 veterinary professionals and scientists from 38 countries to discuss the latest advancements and challenges in veterinary immunology. A highlight of the event was that over 70% of the delegates were first-time attendees, contributing to the symposium's global reach. The symposium featured a series of 83 oral presentations and 104 poster presentations, including topics relating to protective immunity, vaccine strategies, important disease targets, and methodological advancements in veterinary immunology. Workshops provided hands-on experiences and discussions on new technologies such as next-generation sequencing and vaccine development strategies against bacterial infections. The symposium also provided opportunities for networking and engagements with leaders in the field, set against the backdrop of one of Africa's most iconic game reserves, enhancing the experience with a unique blend of professional exchange and natural beauty.

Infectious bronchitis virus (IBV) is the causative agent of infectious bronchitis (IB), a severe disease that primarily affects young chickens and poses a significant challenge to the global poultry industry. Understanding the complex interaction between the virus and its host is vital for developing innovative antiviral strategies. Long non-coding RNA (lncRNA) plays a crucial role in regulating host antiviral immune responses. Our previous studies have shown that IBV infection disrupts the stability of lncRNA in host cells, indicating a potential regulatory role for lncRNA in IBV pathogenesis. It is still not clear how lncRNA precisely modulates IBV replication. In this study, we observed down-regulation ofMSTRG.26120.58 (named lncRNA-DRNR) expression in various chicken cell lines upon IBV infection. We demonstrated that silencing lncRNA-DRNR using siRNA enhances intracellular replication of IBV. Through exploring genes encoding proteins upstream and downstream of lncRNA-DRNR within a 100 kb range, we identified chJMJD6 (chicken JMJD6) as a potential target gene negatively regulated by lncRNA-DRNR expression levels. Furthermore, chJMJD6 inhibits STAT1 methylation, thereby affecting the induction of interferon-stimulated genes (ISGs) through the activation of the IFN-β-mediated JAK-STAT signalling pathway, ultimately promoting the intracellular replication of IBV. In summary, our findings reveal the critical role played by lncRNA-DRNR during IBV infection, providing novel insights into mechanisms underlying coronavirus-induced disruption in lncRNA stability.

The present study aimed to determine the dynamics of influenza A virus (IAV) infection in two endemically infected farms (F1 and F2), where a longitudinal follow-up of piglets was performed from birth to 8-12 weeks of age. During the study, a highly virulent isolate of porcine reproductive and respiratory syndrome virus (PRRSV) was introduced on both farms. This allowed us to examine the impact of such introduction on the patterns of infection, disease, and the antibody response of pigs to IAV infection. The introduction of the new PRRSV strain coincided with a change in the dynamics of IAV infection on both farms. In F1, the cumulative incidence of IAV increased from 20% before the outbreak to 67.5%, together with the existence of animals that tested positive for IAV (RT‒qPCR) in nasal swabs for two or more consecutive samples. In F2, the cumulative incidence of IAV increased from 50% before the PRRSV outbreak to 70%, and the proportion of prolonged IAV shedders increased sharply. Additionally, some animals were infected with the same IAV twice during the observation period. In contrast to previous reports, our study revealed that prolonged shedding was not related to the titres of maternally derived antibodies at the time of infection but was significantly (p < 0.05) related to PRRSV infection status. Notably, both before and after the PRRSV outbreak, a high proportion of IAV-infected piglets did not seroconvert, which was significantly (p < 0.05) related to the hemagglutination inhibition titres against IAV when infected.

Glaesserella parasuis (GPS) is an important bacterial pathogen of swine. Serotype identification has presented a bottleneck in GPS research since it was first identified as the pathogen causing Glässer's disease in pigs in 1910. This paper presents a systematic review of the history of the development and application of gel immunodiffusion (GID), indirect hemagglutination assay (IHA), and polymerase chain reaction (PCR) typing methods for GPS, and the discovery of their shared antigenic basis. It provides a systematic theoretical overview of the immunology and principles underlying the three typing methods and offers new ideas for research into the prevention and control of Glässer's disease. In 1992, GPS was first classified into serotypes 1-15 using GID based on GPS heat-stable antigens, but about 25% of the strains were found to be non-typeable, and the composition of their antigens for serotyping was unclear. In 2003, the IHA method was established based on saline-extracted antigens of GPS, whose sensitivity and typing rate were higher than for GID, although about 15% of strains were still found to be non-typeable. The results of IHA and GID typing are roughly consistent, since they share the same GPS surface polysaccharide serotyping antigens, although whether these are capsular polysaccharides, lipopolysaccharides, or other polysaccharides, remains to be determined. In 2013, the Capsular polysaccharide (CPS) synthetic gene clusters from GPS serotypes 1-15 were successfully analyzed, confirming that CPS is essential for the formation of antigens for serotyping. In 2015, primers were designed based on the specific target genes of GPS capsules to establish a PCR typing method (H-PCR) for GPS, which, however, could not identify serotypes 5 and 12. In 2017, a new PCR typing method (J-PCR) was established based on the specific target genes of GPS capsules, which could identify serotypes 5 and 12. A combination of the two PCR typing methods enables the typing of almost all GPS strains, and the consistency with GID and IHA was verified using molecular biological methods. The antigenic basis of the three typing methods was shown to involve the GPS capsule. PCR typing methods are characterized by simple operation, fast speed, and low cost, and can successfully solve many problems in GID and IHA serotyping, and so have become widely adopted.

Duck plague virus (DPV) causes the highly pathogenic duck plague, and the envelope glycoprotein I (gI), as one of the key virulence genes, has not yet had its critical virulence sites identified through screening. This study used reverse genetics technology to target the gI, specifically within the DPV genome. Four DPV mutants with gI N-glycosylation site mutations were designed and constructed, and these mutant strains were successfully rescued. Our results confirmed that three asparagine residues of gI (N, N, and N) are N-glycosylation sites, and western blot analysis substantiated that glycosylation at each predicted N-glycosylation site was compromised. The deglycosylation of gI leads to the protein misfolding and subsequent retention in the endoplasmic reticulum (ER). The subsequent deglycosylated gI is carried into the Golgi apparatus (GM130) in the interaction of gE. Compared to the parental virus, the mutated virus shows a 66.3% reduction in intercellular transmission capability. In ducks, the deglycosylation of gI significantly reduces DPV replication in vivo, thereby weakening the virulence of DPV. This study represents the first successful creation of a weak DPV virus strain by specific mutation at the N-glycosylation site. The findings provide a foundational understanding of DPV pathogenesis and form the basis for developing live attenuated vaccines against the disease.

The Riemerella anatipestifer bacterium is known to cause infectious serositis in ducklings. Moreover, its adherence to the host's respiratory mucosa is a critical step in pathogenesis. Membrane cofactor protein (MCP; CD46) is a complement regulatory factor on the surface of eukaryotic cell membranes. Bacteria have been found to bind to this protein on host cells. Outer membrane proteins (OMPs) are necessary for adhesion, colonisation, and pathogenicity of Gram-negative bacteria; however, the mechanism by which R. anatipestifer adheres to duck cells remains unclear. In this study, pull-down assays and LC-MS/MS identified eleven OMPs interacting with duck CD46 (dCD46), with OMP71 exhibiting the strongest binding. The ability of an omp71 gene deletion strain to bind dCD46 is weaker than that of the wild-type strain, suggesting that this interaction is important. Further evidence of this interaction was obtained by synthesising OMP71 using an Escherichia coli recombinant protein expression system. Adhesion and invasion assays and protein and antibody blocking assays confirmed that OMP71 promoted the R. anatipestifer YM strain (RA-YM) adhesion to duck embryo fibroblasts (DEFs) by binding to CD46. Tests of the pathogenicity of a Δomp71 mutant strain of RA-YM on ducks compared to the wild-type parent supported the hypothesis that OMP71 was a key virulence factor of RA-YM. In summary, the finding that R. anatipestifer exploits CD46 to bind to host cells via OMP71 increases our understanding of the molecular mechanism of R. anatipestifer invasion. The finding suggests potential targets for preventing and treating diseases related to R. anatipestifer infection.

The nasal mucosa forms a critical barrier against the invasion of respiratory pathogens. Composed of a heterogeneous assortment of cell types, the nasal mucosa relies on the unique characteristics and complex intercellular dynamics of these cells to maintain their structural integrity and functional efficacy. In this study, single-cell RNA sequencing (scRNA-seq) of porcine nasal mucosa was performed, and nineteen distinct nasal cell types, including nine epithelial cell types, five stromal cell types, and five immune cell types, were identified. The distribution patterns of three representative types of epithelial cells (basal cells, goblet cells, and ciliated cells) were subsequently detected by immunofluorescence. We conducted a comparative analysis of these data with published human single-cell data, revealing consistent differentiation trajectories among porcine and human nasal epithelial cells. Specifically, basal cells serve as the initial stage in the differentiation process of nasal epithelial cells, which then epithelial cells. This research not only enhances our understanding of the composition and transcriptional signature of porcine nasal mucosal cells but also offers a theoretical foundation for developing alternative models for human respiratory diseases.