Japanese encephalitis virus (JEV) preferentially targets brain regions rich in dopaminergic neurons, including the thalamus, midbrain, and striatum, leading to severe neuroinflammation. However, the underlying pathological mechanisms remain unclear. This study hypothesizes that JEV-induced pyroptosis exacerbates neuroinflammation by activating the NLRP3/caspase-1/GSDMD signaling pathway. In vivo experiments using JEV-infected mice revealed virus-specific targeting of dopaminergic neurons, concurrent activation of the NLRP3 inflammasome, and induction of inflammatory responses. In vitro studies with mouse midbrain dopaminergic cells showed significant viral replication, progressive cell membrane damage, and colocalization of JEV with pyroptotic markers. The pharmacological inhibition of NLRP3, while suppressing viral load and NLRP3 inflammasome activation, fails to delay the onset of neuroinflammation, potentially due to the activation of glial cells. This suggests that glial cell-mediated inflammatory pathways independent of NLRP3 may drive neuroinflammation despite NLRP3 inhibition. The unimpeded activation of glia could sustain pro-inflammatory responses, highlighting the need to target glial activation alongside NLRP3 to effectively mitigate neuroinflammation.

Newcastle disease virus (NDV) has been well established as a versatile vector platform for heterologous antigen delivery in poultry vaccine development. However, the prospects for combined application of two recombinant NDV (rNDV) vaccines remain poorly understood. In previous studies, we generated two rNDVs based on the thermostable TS09-C strain, expressing glycoprotein B (gB) of infectious laryngotracheitis virus (ILTV) and hemagglutinin (HA) of the H9N2 subtype avian influenza virus (H9N2 AIV). In the present study, we evaluated the potential interference between the two rNDVs and using both in vitro and in vivo models. When two rNDVs co-infected BHK-21 and CEK cells, their viral replication kinetics and expression levels of heterologous antigens (gB and HA) were comparable to those in the respective single-virus infection groups. This finding indicates that the two rNDVs do not exhibit significant interference in vitro. SPF chickens co-immunized with the two rNDVs via intranasal and intraocular routes elicited effective H9N2-, ILTV-, and NDV-specific antibody responses comparable to those in the single-virus immunization group. Compared with control chickens, co-immunized chickens displayed milder clinical signs and reduced viral shedding following challenge with H9N2 AIV, ILTV, and NDV. Based on these findings, spray co-immunization was further tested in commercial chickens and proven to confer effective protection against challenges with NDV, H9N2 AIV, and ILTV. In conclusion, this study confirms the feasibility of co-immunization using the two rNDVs, thereby providing a highly viable strategy for the simultaneous control of multiple avian pathogens.

We evaluated the effects of different levels of DON (LD 441 and HD 1223 μg DON/kg in diet) on the growth performance, immunity, reproductive hormones, and intestinal health of immature gilts. No significant differences were observed in average daily gain, average daily feed intake, or feed to gain ratio between the LD group and the HD group (p > 0.05). The red blood cell count and hematocrit were higher in the LD group compared with the HD group on d 21 (p < 0.05). The gamma-glutamyl transferase activity in the LD group on d 1, 21, 28, 35, and 42 was higher (p < 0.05) compared with the HD group. The aspartate aminotransferase, total antioxidant capacity, and lactic dehydrogenase levels on d 35 were higher in the LD group than those in the HD group (p < 0.05). On d 35, the levels of interleukin 1β, interleukin-4, interleukin-10, tumor necrosis factor-α, and interferon-γ in LD were higher than those in the HD group (p < 0.05). The levels of immunoglobulin A, immunoglobulin M, immunoglobulin G, and complement 4 on d 35 were higher in the LD group compared with those in the HD group (p < 0.05). The gonadotrophin-releasing hormone, luteotrophic hormone, follicle-stimulating hormone, or estradiol did not differ between LD and HD groups throughout the experiment (p > 0.05). For fecal microbiota, Streptococcus in the HD group was reduced compared with the LD group (p < 0.05). In summary, feeding diets contaminated with 1223 μg DON/kg exerted adverse effects on serum profiles of gilts but did not affect their growth performance or reproductive hormones in the present study.

Vaccination remains the core strategy for the prevention and control of Newcastle disease (ND). The inherent thermosensitivity of traditional Newcastle disease virus (NDV) vaccines imposes major limitations on their transportation, storage, and field application. To address these challenges, a novel liquid, thermostable, live ND vaccine was developed in the present study. Firstly, Tris/HCl buffer at near-neutral pH was identified as the optimal basic buffer system. On this basis, further screening and formulation optimization of vaccine stabilizers were conducted, and NDV strains with excellent thermal stability were used to verify the stability-conferring properties of the developed stabilizer. The results showed that the formulation composed of 0.5% gelatin, 4% trehalose, 0.1% L-glutamic acid, and 0.5% thiourea was confirmed as the optimal stabilizer for ND liquid vaccines. This formulation maintained the stable storage of the tested NDV for 12 months at 4 °C and exhibited promising stability for 30 days at 25 °C, marking a significant advancement toward development thermostable NDV vaccines that are independent of a continuous cold chain. More importantly, the liquid vaccine stored at 4 °C for 12 months still induced high levels of NDV-specific antibodies in specific pathogen-free chicks and provided 100% protective efficacy against challenge with virulent NDV. In conclusion, the liquid vaccine stabilizer developed in this study not only significantly enhanced the thermostability of the vaccine but also effectively maintained its immunogenicity, thereby providing an important theoretical basis for the research and development of liquid ND vaccines.

Importance: The Japanese encephalitis virus (JEV) is a massive threat to the pig-raising industry and attenuated-live vaccines are used widely to prevent JEV infections. Primary hamster kidney cells and Vero cells are mainly used to propagate JEV-attenuated vaccines. Objective: This study aimed to construct porcine mesangial cells (PMCs) for JEV cultivation and study its characteristics. Methods: An immunofluorescence assay was used to analyze the homogeneity ofPMCs. Karyotypic analysis and flow cytometry were used to detect the cell chromosome numbers and cell cycle, respectively. TCID50 was used to measure the viral titers. Results: The prepared PMCs were uniform in shape and without variation in chromosome numbers. The PMCs exhibit strong division and proliferation ability, and the JEV could replicate efficiently, having a similar cytopathic effect to that in Vero cells. The viral titer results showed that the JEV grows faster in PMCs than in Vero cells. Conclusions and Relevance: This study generated an immortalized PMC cell line suitable for cultivating the JEV, which has significance for producing pig JEV-attenuated vaccines.

Respiratory illnesses present a significant threat to porcine health, with co-infections involving Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Streptococcus suis (SS), Porcine Circovirus Type 2 (PCV2), and Porcine Circovirus Type 3 (PCV3) acting as the primary causative agents. As a result, the precise diagnosis of PRRSV, PCV2, PCV3 and SS is of paramount importance in the prevention and control of respiratory diseases in swine. Therefore, we conducted a molecular bioinformatical analysis to concurrently detect and differentiate PRRSV, PCV2, PCV3 and SS. We selected the ORF6 gene of PRRSV, the ORF2 gene of PCV2 and PCV3, and the glutamate dehydrogenase (GDH) gene of SS as targets. Specific primers and probes were designed for each pathogen, and following meticulous optimization of reaction conditions, we established a multiple TaqMan fluorescence quantitative PCR detection method. Subsequently, we subjected this method to a comprehensive assessment, evaluating its specificity, sensitivity, and repeatability. The research results demonstrated that the established multiple TaqMan fluorescence quantitative PCR detection method displays displayed exemplary specificity, with no instances of cross-reactivity with other pathogens. The method’s minimum detection concentrations for PRRSV, PCV2, PCV3, and SS were 2.80 × 101 copies/µL, 1.96 × 102 copies/µL, 2.30 × 102 copies/µL, and 1.75 × 103 copies/µL, respectively. When applied to the analysis of 30 clinical samples, the results closely mirrored those obtained through Chinese standard uniplex real-time qPCR detection method for PRRSV, as well as the general PCR methods for SS, PCV2, and PCV3. This study underscores the robust specificity, high sensitivity, and consistent stability of the multiple TaqMan fluorescence quantitative PCR detection method that we have developed. It is ideally suited to the clinical monitoring of PRRSV, PCV2, PCV3, and SS, and it carries significant importance in ongoing efforts to prevent and manage respiratory diseases in porcine populations.

Ulcerative colitis (UC) is one of the primary inflammatory bowel diseases (IBDs) and causes a serious threat to human public health around the world. Currently, there are no proven safe and effective treatment options to treat UC. Fraxetin (Fxt) is a widely recognized antioxidant and anti-inflammatory legume derived from ash bark. In the present study, we investigated the protective effect and mechanism of Fxt on UC. Our results showed that Fxt significantly attenuated the body weight, colon length reduction, tissue damage, and disease activity index induced by dextran sodium sulphate (DSS). Moreover, the DSS-induced activation of the NF-κB pathway and NLRP3 inflammasomes was inhibited, and the inflammatory response was reduced. Fxt restored gut barrier function by increasing the number of goblet cells and the levels of tight junction proteins (ZO-1 and occludin). In addition, Fxt can alter the intestinal microbiota by enhancing the diversity of the microbiota, increasing the relative abundance of beneficial bacteria and inhibiting the growth of harmful bacteria. These results revealed that Fxt alleviates DSS-induced colitis by modulating the inflammatory response, enhancing epithelial barrier integrity and regulating the gut microbiota. This study may provide a scientific basis for the potential therapeutic effect of Fxt in the prevention of colitis and other related diseases.

The primary cause of viral encephalitis (VE) is invasion of the central nervous system (CNS) by the virus, which leads to neuroinflammation and poses a significant threat to global public health. Microglia, as CNS-resident macrophages, play a crucial role in neuroinflammation and are often identified as the preferred target for the prevention or treatment of VE. In this study, we used pseudorabies virus (PRV)-induced VE in mice and pigs as a model to investigate the regulation of microglial responses during viral encephalitis and explored the mechanism of microglial activation. Cellular experiments revealed that microglial activation was accompanied by cell migration, characteristic morphological changes, phagocytosis, inflammatory cytokine production, and antigen presentation. Transcriptome analysis revealed that genes related to inflammation in PRV-infected BV2 cells were significantly enriched. The expression of the NOD1 gene in BV2 cells was significantly increased during PRV infection, after which NOD1 in BV2 cells was silenced by siRNA and overexpressed via a plasmid. NOD1 was found to be involved in the secretion of cytokines in BV2 cells by regulating the MAPK/NF-κB signalling pathway. Mouse and pig experiments have shown that NOD1 is involved in the secretion of cytokines by microglia by regulating the MAPK/NF-κB signalling pathway during PRV infection.

Recent epidemiological studies have discovered that a lot of cases of porcine epidemic diarrhea virus (PEDV) infection are frequently accompanied by porcine kobuvirus (PKV) infection, suggesting a potential relationship between the two viruses in the development of diarrhea. To investigate the impact of PKV on PEDV pathogenicity and the number of intestinal lymphocytes, piglets were infected with PKV or PEDV or co-infected with both viruses. Our findings demonstrate that co-infected piglets exhibit more severe symptoms, acute gastroenteritis, and higher PEDV replication compared to those infected with PEDV alone. Notably, PKV alone does not cause significant intestinal damage but enhances PEDV's pathogenicity and alters the number of intestinal lymphocytes. These results underscore the complexity of viral interactions in swine diseases and highlight the need for comprehensive diagnostic and treatment strategies addressing co-infections.

The pathogens responsible for porcine viral diarrhea are diverse, causing significant economic losses to the pig industry. PEDV and TGEV are well-known pathogens causing diarrheal diseases in pigs, leading to significant economic losses in the breeding industry. In contrast, the newly identified diarrhea virus, PKV, has not garnered as much attention. However, co-infection of PKV with PEDV results in more severe symptoms in piglets, such as acute gastroenteritis, and promotes increased replication of PEDV. Rapid and accurate diagnosis of viral diarrhea is essential for farms to identify pathogens early and mitigate economic losses. This study describes the development of a triplex real-time fluorescent quantitative RT-qPCR technique that can simultaneously detect three RNA viruses associated with porcine viral diarrhea: PEDV, TGEV, and PKV. To establish the triplex RT-qPCR method for the simultaneous detection and identification of the above three diarrhea viruses, conserved regions of the M gene of TGEV, the N gene of PEDV, and the 3D gene of PKV were selected to design specific primers and probes. After optimizing the reaction conditions, the method’s specificity, sensitivity, and reproducibility were evaluated. The triplex RT-qPCR method did not show a significant difference in PCR efficiency compared to the single RT-qPCR method. The method is specific to TGEV, PKV, and PEDV, exhibits no cross-reactivity with other pathogens, and demonstrates satisfactory sensitivity and reproducibility; the limit of detection (LOD) of PEDV, TGEV, and PKV is 11.42 copies/μL. Furthermore, the performance of the triplex RT-qPCR assay was compared with the Chinese standard single-assay method for detecting TGEV, PKV, and PEDV, showing complete consistency between the two methods (100% compliant). Subsequently, 1502 clinical diarrhea samples were collected from the Guangxi Zhuang Autonomous Region to investigate the local prevalence of TGEV, PKV, and PEDV and the positive rates were 16.38% (246/1502), 1.46% (22/1502), and 45.14% (678/1502), respectively. Co-infection of PEDV and PKV were most common, with a rate of 12.12% (182/1502). This study presents a valuable method for the rapid and simultaneous identification of PEDV, TGEV, and PKV in clinical animal farming practices, and provides a reassessment of the epidemiology of these diarrhea-causing viral pathogens in the Guangxi Zhuang Autonomous Region.

Porcine rotavirus is a significant pathogen that causes rotavirus sickness in both humans and animals. G9 rotavirus, in particular, is thought to be a new rotavirus that has lately spread over the world between humans and pigs. However, the understanding of the pathogenicity of G9 rotavirus is limited to date. In the current study, a G9 porcine rotavirus strain was isolated in the fecal samples of diarrheal piglets from a large-scale pig farm located in Guangxi, China. Then, the virus was identified by western blotting, and the production dynamics of the virus were confirmed by an indirect immunofluorescence test with a viral titer of 107.46 TCID50/mL. Based on the VP7, VP4 and VP6 genes, phylogenetic analysis and sequencing data indicated that the GX9579 strain was of genotype G9P [23]I5. In addition, to explore the pathogenicity of the isolated GX9579 strain, animal tests were performed herein. Fecal viral shedding was detected, and fecal excretion was the highest at 105 copies/mu L 24h after the challenge and then gradually decreased. Histopathological test results suggested the intestinal villous arrangement of infected piglets became shortened, partially shed, and broken, intestinal villous epithelial cells shed into the intestinal lumen, and the villi propria lamina became hyperemic with severe bleeding and submucosal edema. Positive signals were seen throughout the intestinal villi's epithelial cells and the small intestine glands, according to immunohistochemical studies. These results provide reference data for further studies on the epidemiology and pathogenesis of G9 swine rotavirus.

Porcine viral diarrhea is a common ailment in clinical settings, causing significant economic losses to the swine industry. Notable culprits behind porcine viral diarrhea encompass transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA). Co-infections involving the viruses are a common occurrence in clinical settings, thereby amplifying the complexities associated with differential diagnosis. As a consequence, it is therefore necessary to develop a method that can detect and differentiate all four porcine diarrhea viruses (TGEV, PEDV, PDCoV, and PoRVA) with a high sensitivity and specificity. Presently, polymerase chain reaction (PCR) is the go-to method for pathogen detection. In comparison to conventional PCR, TaqMan real-time PCR offers heightened sensitivity, superior specificity, and enhanced accuracy. This study aimed to develop a quadruplex real-time RT-qPCR assay, utilizing TaqMan probes, for the distinctive detection of TGEV, PEDV, PDCoV, and PoRVA. The quadruplex real-time RT-qPCR assay, as devised in this study, exhibited the capacity to avoid the detection of unrelated pathogens and demonstrated commendable specificity, sensitivity, repeatability, and reproducibility, boasting a limit of detection (LOD) of 27 copies/μL. In a comparative analysis involving 5483 clinical samples, the results from the commercial RT-qPCR kit and the quadruplex RT-qPCR for TGEV, PEDV, PDCoV, and PoRVA detection were entirely consistent. Following sample collection from October to March in Guangxi Zhuang Autonomous Region, we assessed the prevalence of TGEV, PEDV, PDCoV, and PoRVA in piglet diarrhea samples, revealing positive detection rates of 0.2% (11/5483), 8.82% (485/5483), 1.22% (67/5483), and 4.94% (271/5483), respectively. The co-infection rates of PEDV/PoRVA, PEDV/PDCoV, TGEV/PED/PoRVA, and PDCoV/PoRVA were 0.39%, 0.11%, 0.01%, and 0.03%, respectively, with no detection of other co-infections, as determined by the quadruplex real-time RT-qPCR. This research not only established a valuable tool for the simultaneous differentiation of TGEV, PEDV, PDCoV, and PoRVA in practical applications but also provided crucial insights into the prevalence of these viral pathogens causing diarrhea in Guangxi.

Salmonella, a prevalent foodborne pathogen, poses a significant social and economic strain on both food safety and public health. The application of phages in the control of foodborne pathogens represents an emerging research area. In this study, Salmonella pullorum phage vB_SpuM_X5 (phage X5) was isolated from chicken farm sewage samples. The results revealed that phage X5 is a novel Myoviridae phage. Phage X5 has adequate temperature tolerance (28 °C–60 °C), pH stability (4–12), and a broad host range of Salmonella bacteria (87.50% of tested strains). The addition of phage X5 (MOI of 100 and 1000) to milk inoculated with Salmonella reduced the number of Salmonella by 0.72 to 0.93 log10 CFU/mL and 0.66 to 1.06 log10 CFU/mL at 4 °C and 25 °C, respectively. The addition of phage X5 (MOI of 100 and 1000) to chicken breast inoculated with Salmonella reduced bacterial numbers by 1.13 to 2.42 log10 CFU/mL and 0.81 to 1.25 log10 CFU/mL at 4 °C and 25 °C, respectively. Phage X5 has bactericidal activity against Salmonella and can be used as a potential biological bacteriostatic agent to remove mature biofilms of Salmonella or for the prevention and control of Salmonella.

Background: Japanese encephalitis virus (JEV) is a highly neurotropic virus that can enter the central nervous system after high levels of peripheral replication, causing severe neuroinflammation. JEV targets dopaminergic neuron-rich areas such as the thalamus, midbrain, and striatum, causing pyroptosis. Pyroptosis, which is a novel mode of programmed cell death, plays an important role in infection by other flaviviruses, but whether it plays a role in JEV-infected neurons has not been confirmed. Methods: In this study, the damaging effect of JEV infection on dopaminergic neurons was examined in vivo and in vitro through HE staining, immunohistochemical staining, double immunofluorescence labeling, determination of cytotoxicity, qPCR and western blotting for testing pyroptosis. Results: Mice infected with JEV showed glial cell activation and proliferation, neuronal degeneration and necrosis, perivascular cuffing, and neuronophilia in brain tissue, and a large number of microglia were significantly activated around the virus-positive signal, as shown by immunofluorescence staining. JEV specifically targeted dopaminergic neurons, activated the nucleotide oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3)/cysteinyl aspartate specific proteinase-1 (caspase-1)/gasdermin D (GSDMD) signaling pathway and induced an inflammatory response. In vitro infection of mouse midbrain dopaminergic neuron cells revealed that JEV significantly replicated in MN9D cells. Infected cells were significantly lesioned at 24 hpi, and the number of lesioned cells increased significantly with time. JEV infection resulted in a gradual increase in LDH release, indicating damage to the cytosol. JEV clearly colocalized with MN9D cells, and apoptosis occurred via an NLRP3/caspase-1/GSDMD-dependent pathway. Conclusions: These results show that inhibiting pyroptosis can delay motor impairment caused by JEV to some extent and that protecting dopaminergic neurons from viral infection may be an effective strategy for the treatment of JEV with late effects.

Japanese encephalitis virus (JEV) infection can cause brain tissue lesions characterized by neuronal death, and apoptosis is involved in JEV-induced neuronopathy. In the present study, mouse microglia were infected with JEV, and pyknosis with dark-staining nuclei of infected cells was detected using Hoechst 33342 staining. TUNEL staining showed that JEV infection promoted the apoptosis of BV2 cells, and the apoptosis rate was significantly increased at 24-60 hours postinfection (hpi) ( P < 0.01) and was the highest at 36 h ( P < 0.0001). Western blot results showed that the expression of the Bcl-2 protein in JEV-infected cells was downregulated significantly at 60 hpi ( P < 0.001), whereas that of the Bax protein was observably upregulated at 60 hpi ( P < 0.001). At the same time, the level of cytochrome c (Cyt c) was significantly increased ( P < 0.001), and the expression levels of two apoptosis-related proteins, namely, cleaved caspase-3 ( P < 0.01) and caspase-9 ( P < 0.001), were elevated significantly. Immunofluorescence staining showed that the amount of Cyt c increased with time after infection. After BV2 cells were infected with JEV, the expression of RIG-1 increased significantly from 24 hpi to 60 h ( P < 0.001). The expression of MAVS increased significantly at 24 h ( P < 0.001) and decreased gradually from 24 h to 60 hpi. The expression of TBK1 and NF-κB (p65) was not significantly changed. The expression of p-TBK1 and p-NF-κB (p-p65) increased significantly within 24 h ( P < 0.001) and decreased from 24 to 60 hpi. The expression levels of IRF3 and p-IRF3 peaked at 24 hpi ( P < 0.001) and decreased gradually from 24 to 60 hpi. However, the expression levels of JEV proteins showed no significant change at 24 and 36 hpi but were markedly elevated at 48 and 60 hpi. Interference with the expression of the RIG-1 protein in BV2 cells resulted in a dramatic increase in the expression of the anti-apoptotic protein Bcl-2 ( P < 0.05), whereas the pro-apoptotic protein Bax, cleaved caspase-9, and especially cleaved caspase-3 were downregulated ( P < 0.05), and viral protein expression was notably reduced ( P < 0.05). These results indicate that JEV induces apoptosis through mitochondrial-dependent apoptosis pathways, interfering with the expression of RIG-1 in BV2 cells can inhibit viral replication and inhibit apoptosis.

Abstract Pseudorabies virus (PRV) can infect multiple hosts and lead to fatal encephalitis. There is a significant increase in the number of microglia in the brain of animals infected with PRV. However, whether and how microglia contribute to central nervous system damage in PRV infection remain unknown. In the present study, we elucidated that PRV infection can cause more severe inflammatory cell infiltration, thicker and more numerous vessel sleeve walls, and more severe inflammatory responses in the brains of natural hosts (pigs) than in those of nonnatural hosts (mice). In a mice infection model, activated microglia restricted viral replication in the early stage of infection. Acute neuroinflammation caused by microglia hyperactivation at late-stage of infection. Furthermore, in vitro experiments revealed that microglia restricted viral replication and decreased viral infectivity. This may be associated with the phagocytic ability of microglia because we observed a significant increase in the expression of the membrane receptor TREM2 in microglia, which is closely related to phagocytosis, we observed that depletion of microglia exacerbated neurological symptoms, blood–brain barrier breakdown, and peripheral lymphocyte infiltration. Taken together, we revealed the dual role of microglia in protecting the host and neurons from PRV infection.

为探究猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)感染仔猪导致的腹股沟淋巴结的组织病理学变化及致病机制,对PRRSV感染仔猪腹股沟淋巴结进行组织病理学观察及转录组分析.结果显示,PRRSV感染仔猪腹股沟淋巴结肿胀出血,镜检观察可见淋巴组织结构模糊,淋巴细胞减少,出血;淋巴小结数量减少,界限不清,生发中心大量淋巴细胞坏死.基于Illumina测序平台,运用RNA-seq技术进行建库测序和生物信息分析,发现TLR8、CCL5、CCL8、IFNG、IRF5、TNFSF13B等炎性因子差异性表达,Toll样受体通路、NF-κB信号通路、TNF信号通路等通路被激活,提示这些细胞因子及信号通路参与PRRSV感染引起的淋巴结炎的病理变化过程.

Enterococcus faecalis is a potential animal and human pathogen. Improper use of antibiotics encourages resistance. Bacteriophages and their derivatives are promising for treating drug-resistant bacterial infections. In this study, phylogenetic and electron microscopy analyses of phage vB_EfaS_WH1 (WH1) isolated from chicken feces revealed it to be a novel phage in the family Siphoviridae. WH1 showed good pH stability (4–11), temperature tolerance (4–60 °C), and broad E. faecalis host range (60% of isolates). Genome sequencing revealed a 56,357 bp double-stranded DNA genome with a G+C content of 39.21%. WH1 effectively destroyed E. faecalis EF01 biofilms, even at low concentrations. When WH1 was applied at 1 × 105 to 1 × 109 PFU/g to chicken breast samples stored at 4 °C, surface growing E. faecalis were appreciably eradicated after 24 h. The phage WH1 showed good antibacterial activity, which could be used as a potential biocontrol agent to reduce the formation of E. faecalis biofilm, and could also be used as an alternative for the control of E. faecalis in chicken products.

In ovo vaccination is an attractive immunization approach for chickens. However, most live Newcastle disease virus (NDV) vaccine strains used safely after hatching are unsafe as in ovo vaccines due to their high pathogenicity for chicken embryos. The mechanism for viral pathogenicity in chicken embryos is poorly understood. Our previous studies reported that NDV strain TS09-C was a safe in ovo vaccine, and the F protein cleavage site (FCS) containing three basic amino acids (3B-FCS) was the crucial determinant of the attenuation of TS09-C in chicken embryos. Here, five trypsin-like proteases that activated NDV in chicken embryos were identified. The F protein with 3B-FCS was sensitive to the proteases Tmprss4, Tmprss9, and F7, was present in fewer tissue cells of chicken embryos, which limited the viral tropism, and was responsible for the attenuation of NDV with 3B-FCS, while the F protein with FCS containing two basic amino acids could be cleaved not only by Tmprss4, Tmprss9, and F7 but also by Prss23 and Cfd, was present in most tissue cells, and thereby was responsible for broad tissue tropism and high pathogenicity of virus in chicken embryos. Furthermore, when mixed with the protease inhibitors aprotinin and camostat, NDV with 2B-FCS exhibited greatly weakened pathogenicity in chicken embryos. Thus, our results extend the understanding of the molecular mechanism of NDV pathogenicity in chicken embryos and provide a novel molecular target for the rational design of in ovo vaccines, ensuring uniform and effective vaccine delivery and earlier induction of immune protection by the time of hatching. IMPORTANCE As an attractive immunization approach for chickens, in ovo vaccination can induce a considerable degree of protection by the time of hatching, provide support in closing the window in which birds are susceptible to infection, facilitate fast and uniform vaccine delivery, and reduce labor costs by the use of mechanized injectors. The commercial live Newcastle disease virus (NDV) vaccine strains are not safe for in ovo vaccination and cause the death of chicken embryos. The mechanism for viral pathogenicity in chicken embryos is poorly understood. In the present study, we identified five trypsin-like proteases that activate NDV in chicken embryos and elucidated their roles in the tissue tropism and pathogenicity of NDV used as in ovo vaccine. Finally, we revealed the molecular basis for the pathogenicity of NDV in chicken embryos and provided a novel strategy for the rational design of in ovo ND vaccines.

The thymus, the central immune organ in mammals, plays an important role in immune defense. Porcine reproductive and respiratory syndrome virus (PRRSV) infection in piglets can cause thymus injury and immunosuppression. However, the mechanisms of thymus injury remain unknown. This study was aimed at investigating the specific manifestations of thymus injury through the construction of a PRRSV-infected piglet model and histopathological observation. In this study, fourteen 40-day-old PRRSV-free piglets were randomly divided into two groups, eleven of which were intramuscularly injected with 3 mL of PRRSV WUH3 virus suspension (10(6) PFU /mL) in the infection group, and three of which were sham-inoculated with 3 mL of RPMI-1640 medium in the control group. Clinical necropsy and samples collection were performed on day 8 after artificial infection. With the Illumina platform, the transcriptomes of piglet thymus tissues from infected and control piglets were sequenced to explore the relationships of differentially expressed genes (DEGs) and signaling pathways with thymus injury. The immune organs of PRRSV-infected piglets were severely damaged. The histopathological findings in the thymus indicated that PRRSV infection was associated with a large decrease in lymphocytes, cell necrosis and cell apoptosis; an increase in blood vessels and macrophages; thymic corpuscle hyperplasia; and interstitial widening of the thymic lobules. The transcriptomic analysis results revealed that the Gene Ontology functions of DEGs were enriched primarily in biological processes such as angiogenesis, regulation of angiogenesis and positive regulation of cell migration. Moreover, greater numbers of blood vessels and macrophages were observed in the thymus in PRRSV-infected than control piglets. KEGG pathway enrichment analysis revealed that the DEGs were significantly enriched in the Toll-like receptor signaling pathway, chemokine signaling pathway, IL-17 signaling pathway and TNF signaling pathway. The expression of TLR8, IRF5, the chemokines CCL2, CCL3L1 and CCL5; and their receptors CCR1, CCR2 and CCR5 was significantly up-regulated in PRRSV infection, thus suggesting that these cytokines were associated with the pathological processes of thymus injury.