UBE2J1 promotes ALV-A proviral DNA synthesis through the STAT3/IRF1 signaling pathway.

The ubiquitin-binding enzyme E2J1 is located on the endoplasmic reticulum membrane. It plays a role in transport throughout the process of ubiquitination. In mammals, UBE2J1 can promote RNA virus replication. However, the biological function of chicken UBE2J1 is unclear. In this study, chicken UBE2J1 was cloned for the first time, and UBE2J1 overexpression and shRNA knockdown plasmids were constructed. In chicken embryo fibroblasts, overexpression of UBE2J1 promoted the replication of subtype A avian leukosis virus, while knockdown of UBE2J1 inhibited the replication of ALV-A virus. In addition, we divided virus replication into virus adsorption and invasion into DF-1 cells, synthesis of proviral DNA, and release of viral particles. UBE2J1 promoted the replication of ALV-A virus by promoting the synthesis of proviral DNA. This result was caused by UBE2J1 inhibiting the production of interferon by inhibiting the STAT3/IRF1 pathway. We mutated ser at position 184 of UBE2J1 to Gly and found that this site plays a role as the phosphorylation site of UBE2J1. We confirmed that UBE2J1 promotes ALV-A replication in chicken embryo fibroblasts by inhibiting the STAT3/IRF1 pathway. This study provides new ideas and insights into ubiquitin-related proteins and antiviral immunity.

Two-component system LiaSR negatively regulated the acid resistance and pathogenicity of Listeria monocytogenes 10403S.

The glutamate decarboxylase (GAD) system is one of the acid-resistant systems of Listeria monocytogenes (L. monocytogenes), while the regulatory mechanism of GadT2/GadD2, which plays the major role in the GAD system for acid resistance, is not clear. The two-component system (TCS) is a signal transduction system that is also involved in regulating acid resistance in bacteria. By screening the TCSs of L. monocytogenes 10403S, we found that knocking out the TCS LisSR (encoded by lmo1021/lmo1022) led to a significant increase in the transcription and expression of the gadT2/gadD2 cluster. Subsequently, we constructed a complemental strain CΔliaSR. and a complemental strain with LiaS His157 to Ala, which was designated as CΔliaSRH157A. Survival assay, transcriptional and expression analysis and pathogenicity assay revealed that liaSR deletion significantly enhanced the acid resistance and pathogenicity of 10403S and significantly increased the gadT2/gadD2 transcription and expression. Mutating LiaS His157 to Ala significantly enhanced the acid resistance and pathogenicity of CΔliaSR and significantly increased the gadT2/gadD2 transcription and expression. The results suggest that the two-component system LiaSR mediates the acid resistance and pathogenicity in 10403S by inhibiting the gadT2/gadD2 cluster, and the key activation site of LiaS is His157. This study provides novel knowledge on the regulation of GAD system and the control of this foodborne pathogen.

Destroying glutathione peroxidase improves the oxidative stress resistance and pathogenicity of Listeria monocytogenes.

Introduction: Glutathione peroxidase is abundant in eukaryotes as an important antioxidant enzyme. However, prokaryotic glutathione peroxidase has not been thoroughly studied. Listeria monocytogenes is a facultative intracellular pathogen that is capable of causing listeriosis in animals as well as humans. Despite the fact that L. monocytogenes encodes a putative glutathione peroxidase, GSH-Px (encoded by lmo0983)), the functions of the enzyme are still unknown. Here we revealed the unusual roles of L. monocytogenes GSH-Px in bacterial antioxidants and pathogenicity. Methods: L. monocytogenes Lm850658 was taken as the parental strain to construct the gsh-px deletion strain and related complement strain. The effect of the gsh-px gene on the resistance of L. monocytogenes to oxidative stress was determined by measuring the concentrations of glutathione and assaying the stress survival rates under different oxidative conditions. In addition, the pathogenicity of L. monocytogenes was determined by cellular adhesion and invasion assays and mice virulence tests, and the expression of virulence factors was determined by Western blot. Results: Deficiency of GSH-Px not only increased glutathione concentrations in L. monocytogenes but also enhanced its resistance to oxidative stress when exposed to copper and iron ions. In addition, the absence of gsh-px significantly improved the adhesion and invasion efficiency of L. monocytogenes to Caco-2 cells. More importantly, L. monocytogenes lacking GSH-Px could colonize and proliferate more efficiently in mice livers and spleens, enhancing the pathogenicity of L. monocytogenes by increasing the expression of virulence factors like InlA, InlB, and LLO. Discussion: Taken together, we confirmed that GSH-Px of L. monocytogenes has a counter-intuitive effect on the antioxidant capacity and pathogenicity.

GadR4 mediates the acid resistance and pathogenicity of Listeria monocytogenes 10403S by negatively regulating the gadT2/gadD2 cluster.

Listeria monocytogenes is an important foodborne pathogen that can survive under acidic conditions. The glutamate decarboxylase (GAD) system is one of the acid resistance systems of L. monocytogenes. It usually comprises two glutamate transporters (GadT1/T2) and three glutamate decarboxylases (GadD1/D2/D3). Among them, gadT2/gadD2 contributes most significantly to the acid resistance of L. monocytogenes. However, the regulation mechanisms of gadT2/gadD2 still remain unclear. The results of this study indicated that gadT2/gadD2 deletion significantly decreases the survival rate of L. monocytogenes under different acidic conditions, including brain and heart infusion (BHI) broth, with a pH of 2.5, 2% citric acid, 2% acetic acid and 2% lactic acid. Further, gadT2/gadD2 cluster was expressed in the representative strains in response to alkaline stress rather than acid stress. To explore the regulation of gadT2/gadD2, we knocked out the five transcriptional factors belonging to the Rgg family in L. monocytogenes 10403S. We found that the deletion of gadR4, which exhibits the highest homology with the gadR of Lactococcus lactis, resulted in a significant increase in the survival rate of L. monocytogenes upon acid stress. Western blot analysis showed that gadR4 deletion significantly increased the gadD2 expression of L. monocytogenes under alkaline and neutral conditions. Furthermore, the gfp reporter gene showed that gadR4 deletion significantly increased the expression of the gadT2/gadD2 cluster. Adhesion and invasion assays indicated that gadR4 deletion significantly increased the rates of adhesion and invasion of L. monocytogenes to epithelial Caco-2 cells. Virulence assays showed that gadR4 knockout significantly improved the colonization ability of L. monocytogenes in the livers and spleens of the infected mice. Taken together, our results showed that GadR4, a transcription factor belonging to the Rgg family, negatively regulates the gadT2/gadD2 cluster, thus, reducing the acid stress tolerance and pathogenicity of L. monocytogens 10403S. Our results provide a better understanding of the regulation of the GAD system of L. monocytogenes and a novel approach to potentially prevent and control listeriosis.

Enhanced pathogenicity by up-regulation of A20 after avian leukemia subgroup a virus infection.

Avian leukemia virus subgroup A (ALV-A) infection slows chicken growth, immunosuppression, and tumor occurrence, causing economic loss to the poultry industry. According to previous findings, A20 has a dual role in promoting and inhibiting tumor formation but has rarely been studied in avians. In this study, A20 overexpression and shRNA interference recombinant adenoviruses were constructed and inoculated into chicken embryos, and ALV-A (rHB2015012) was inoculated into 1-day-old chicks. Analysis of body weight, organ index, detoxification, antibody production, organ toxin load, and Pathological observation revealed that A20 overexpression could enhance ALV-A pathogenicity. This study lays the foundation for subsequent exploration of the A20-mediated tumorigenic mechanism of ALV-A.

Isolation and characterization of avian leukosis virus subgroup J associated with hemangioma and myelocytoma in layer chickens in China.

A strain of avian leukosis virus (ALV) belonging to a new envelope subgroup J (ALV-J) emerged in 1988 as a new subgroup of ALV and spread rapidly throughout the world. Due to the infection and spread of ALV-J, the global poultry industry experienced a significant loss. Although the disease had been prevented and controlled effectively by culling domestic chickens in the infected zone, a few field cases of ALV-J infection were reported in China in recent years. This study was conducted to characterize the genome and analyze the lesions and histopathology of the ALV-J strain named HB2020, which was isolated from layer chickens in Hubei Province, China. The full-length proviral genome sequence analysis of ALV-J HB2020 revealed that it was a recombinant strain of ev-1 and HPRS-103 in the gag gene in comparison to ALV-J prototype HPRS-103. In the 3'-untranslated region (3'UTR) of the nucleotide sequence, there were found 205-base pairs (bp) deletion, of which 175 were detected in the redundant transmembrane (rTM) region. Besides, the surface glycoprotein gene gp85 had five mutations in a conservative site, whereas the transmembrane protein gene gp37 was relatively conserved. The animal experiments conducted later on this strain have shown that HB2020 can cause various neoplastic lesions in chickens, including enlarged livers with hemangiomas and spleens with white nodules. Additionally, as the exposure time increased, the number of tumor cells that resembled myelocytes in the blood smears of infected chickens gradually increased. These results indicated that HB2020 on recombination with ALV subgroup E (ALV-E) and ALV-J could induce severe hemangiomas and myelocytomas. This inference might provide a molecular basis for further research about the pathogenicity of ALV and emphasize the need for control and prevention of avian leukosis.

A20 Enhances the Expression of the Proto-Oncogene C-Myc by Downregulating TRAF6 Ubiquitination after ALV-A Infection.

Hens infected with avian leukosis virus subgroup A (ALV-A) experience stunted growth, immunosuppression, and potentially, lymphoma development. According to past research, A20 can both promote and inhibit tumor growth. In this study, DF-1 cells were infected with ALV-A rHB2015012, and Gp85 expression was measured at various time points. A recombinant plasmid encoding the chicken A20 gene and short hairpin RNA targeting chicken A20 (A20-shRNA) was constructed and transfected into DF-1 cells to determine the effect on ALV-A replication. The potential signaling pathways of A20 were explored using bioinformatics prediction, co-immunoprecipitation, and other techniques. The results demonstrate that A20 and ALV-A promoted each other after ALV-A infection of DF-1 cells, upregulated A20, inhibited TRAF6 ubiquitination, and promoted STAT3 phosphorylation. The phosphorylated-STAT3 (p-STAT3) promoted the expression of proto-oncogene c-myc, which may lead to tumorigenesis. This study will help to further understand the tumorigenic process of ALV-A and provide a reference for preventing and controlling ALV.

Recombinant invasive Lactobacillus plantarum expressing the J subgroup avian leukosis virus Gp85 protein induces protection against avian leukosis in chickens.

Avian leukosis, caused by avian leukosis virus (ALV), is an infectious tumor disease and severely hinders the development of the poultry industry. The use of Lactobacillus plantarum (L. plantarum) could effectively alleviate viremia in the early period of J subgroup ALV (ALV-J) infection. In this study, an invasive L. plantarum NC8 expressing Gp85 protein of ALV-J was constructed. After chickens were orally administered the recombinant invasive NC8, the levels of expression of CD4+ and CD8+ T lymphocytes in peripheral blood and spleen by flow cytometry and the proliferation ability of splenocytes by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were examined, and the contents of cytokines, the anti-ALV-J antibody in serum, and mucosal antibody sIgA in intestinal lavage fluid were detected by enzyme-linked immunosorbent assay (ELISA). The immunoprotective efficiency was evaluated by monitoring the infection rate, the percent of cloacal swabs and survival, body weight gain, the organ indexes, and relative virus loads after challenge with ALV-J. The results showed that the recombinant invasive strain (FnBPA-gp85) could promote the expression levels of the CD8+T cells in peripheral blood and spleen, the proliferation of splenocytes, the secretions of cytokines interleukin 2 (IL-2) and γ-interferon (IFN-γ), and the production of IgG and sIgA compared with the PBS and FnBPA control groups in chickens. The FnBPA-gp85 group was exhibited the highest immune protection against ALV-J infection. The above results indicated that the recombinant invasive NC8 could promote the cellular immunity, humoral immunity, and mucosal immunity responses in chicken and provide a new method for exploring the live vaccine against ALV-J.Key points• The FnBPA-gp85 strain could enhance cellular immunity response.• The FnBPA-gp85 strain could improve the immune protection against ALV-J infection.

Preparation of a novel monoclonal antibody against Avian leukosis virus subgroup J Gp85 protein and identification of its epitope.

Avian leukosis virus subgroup J (ALV-J) is an avian oncogenic retrovirus that has caused huge economic losses in the poultry industry due to its great pathogenicity and transmission ability. However, the continuous emergence of new strains would bring challenges to diagnosis and control of ALV-J. .This study focuses on preparing the monoclonal antibody (MAb) against ALV-J Gp85 and identifying its epitope. The truncated ALV-J gp85 gene fragment was amplified and then cloned into expression vectors. Purified GST-Gp85 was used to immune mice and His-Gp85 was used to screen MAb. Finally, a hybridoma cell line named J16 that produced specific MAb against the ALV-J. Immunofluorescence assay showed that MAb J16 specifically recognized ALV-J rather than ALV-A or ALV-K infected DF-1 cells. To identify the epitope recognized by MAb J16, fourteen partially overlapping ALV-J Gp85 fragments were prepared and tested by Western blot. The results indicated that peptide 150-LIRPYVNQ-157 was the minimal epitope of ALV-J Gp85 recognized by MAb J16. Alignment analysis of Gp85 from different ALV subgroups showed that the epitope keep high conservation among 36 ALV-J strains, but significant different from that of ALV subgroup A, B, C, D, E and K. Overall, we prepared a MAb specific against ALV-J and identified peptide 150-LIRPYVNQ-157 as a novel specific epitope of ALV-J Gp85, which may assist in laying the foundation for specific ALV-J detection methods.

Rapid Quantum Dot Nanobead-mAb Probe-Based Immunochromatographic Assay for Antibody Monitoring of Trichinella spiralis Infection.

PURPOSE: Sensitive and selective point-of-care biosensor is an urgent pursuit of serological antibody detection to control parasite pathogen. For specific, quantitative and on-site screening of Trichinella spiralis infection in livestock, a quantum dot nanobead-monoclonal antibody (QB-mAb) probe-based immunochromatographic assay (ICA) was developed by introducing a competitive sandwich strategy (QB-CICA). METHODS: In the QB-CICA, QB-mAb probes competed with serum antibody for a particular epitope, followed by immunocomplexes binding to capture antibody on the test line. With the accumulation of target antibody, captured probes served as signal elements for fluorescent readout in a "turn off" mode, along with the fluorescence gradually weakened. The sensitivity and standard calibration curve of the QB-CICA were quantified using swine sera as negative control (n = 200) and artificial infected swine sera (n = 80) compared with a commercial ELISA kit. Besides, Trichinella spiralis-antibody targeting test ability of the QB-CICA, instead of other parasites or viruses antibodies (n = 10), was evaluated. RESULTS: The QB-CICA exhibited a good linear range, a low detection limit of 189.92 ng mL-1 and 100% selectivity that was higher than commercial ELISA kit (90%), as well as the same serological positive rate (100%) with commercial ELISA kit in different infection dose models. CONCLUSION: Taking advantage of its simplicity, short response time (25 min), sensitivity and specificity, the proposed QB-CICA has potential applications for parasite-related antibody monitoring in food safety and clinical diagnosis fields.

Identification of a novel epitope specific for Gp85 protein of avian leukosis virus subgroup K.

During the past two decades, avian leukosis virus (ALV) caused tremendous economic losses to poultry industry in China. ALV-K as a newly found subgroup in recent years, which made the control and eradication of ALV more difficult as they were originated from the recombination of different subgroups. To date, specific rapid detection methods refer to ALV-K are still missing. Gp85 is the main structural protein of the virus, which mediates the invasion of host cells by the virus and determinates the classification of subgroups. In this study, we prepared a monoclonal antibody (Mab) named Km3 against Gp85 of ALV-K. Immunofluorescence assay showed that Km3 specifically recognized the strains of ALV-K rather than the strains of ALV-A or ALV-J. To explain the subgroups specificity of Km3, the epitope cognized by the Mab was identified by Western blotting using 15 overlapping fragments spanning the Gp85. Finally, the peptide 129AFGPRSIDTLSDWSRPQ145 was identified as the minimal linear epitope recognized by Km3. Alignment of Gp85 from different subgroups showed that the epitope was highly conserved among ALV-K strains, which was quite different from that of the strains from ALV -A, -B and -J. In conclusion, the Mab Km3 may serve as a useful reagent for ALV-K detection and diagnosis in the future.

Gold nanorods-based multicolor immunosensor for visual detection of enterovirus 71 infection.

Based on the etching of gold nanorods (GNRs) and enzyme-linked immunosorbent assay (ELISA), a multicolor immunosensor for visual detection of enterovirus 71 infection is proposed. Once the immunocomplex is formed, the horseradish peroxidase bound to the ELISA plate oxidizes 3,3',5,5'-tetramethylbenzidine (TMB) into TMB2+ in the presence of hydrogen peroxide. Subsequently, TMB2+ quantitatively etches GNRs to the short GNRs, leading to a blue shift of longitudinal localized surface plasmon resonance and corresponding color responses. This change is used to develop two types of cut-off standards, which respond to the human anti-enterovirus at a concentration of 71 IgM antibody. The method has been validated with clinical serum samples and showed high sensitivity and specificity . This visual immunosensor has an important application value for point-of-care detection of EV71, especially in areas lacking detection equipment. Graphical abstract.

Cysteamine triggered "turn-on" fluorescence sensor for total detection of fumonisin B1, B2 and B3.

A "turn-on" fluorescence sensor was developed for total detection of fumonisin B1, B2 and B3 (FB1, FB2 and FB3) in maize samples. Rhodamine B isothiocyanate (RBITC) and gold nanoparticles (AuNPs) were employed as the energy donor-acceptor pairs. The intensity of restored fluorescence was inversely proportional to the concentration of the FBs. The limit of detection (LOD) of the sensor for FB1 was 23.80 pg/mL. The coefficient of variation (CV) was both less than 7% for intra- and inter-assay. The linear range was from 51.39 to 2125.92 pg/mL. The average recoveries of FBs from maize samples were ranged from 88.7% to 107.2%. The correlation coefficient of the results between the developed sensor and LC-MS/MS was 0.9970. To fulfill the procedure of the assay, only 46 min was needed. These results suggest that the fluorescence "turn-on" sensor has great potential applications in the analysis of FBs in maize samples.

Carbon nanotubes in electrochemical, colorimetric, and fluorimetric immunosensors and immunoassays: a review.

This review (with 126 references) summarizes the progress that has been made in the use of carbon nanotubes (CNTs) in immunoassays and immunosensors. Following an introduction into the field, a first large section covers functional group-modified CNTs as carriers for antibodies (with subsections on CNTs modified with amino, carboxy and by various other materials). The next section treats CNTs coupled with inorganic or organic materials as active components (with the use of nanoparticles such as Fe3O4, ZnO, gold) or with indium tin oxide (ITO), NiCoBP, ionic liquids, TiO2 nanosheets, metal clusters; chitosan, polystyrene, polydopamine, graphene, or enzymes. Another section discusses the synergistic effects of CNTs with metals, metal ions, and organic/inorganic materials. Several Tables are presented that give an overview on the wealth of methods and materials. A concluding section summarizes the current status, addresses current challenges, and gives an outlook on potential future trends. Graphical abstract Schematic presentation of the roles of carbon nanotubes (CNTs) in immunosensors and immunoassays. (I) Functional group-modified CNTs as carriers for antibody; (II) CNTs coupled with metal or organic materials as active component; and (III) Synergistic effects of CNTs with other materials to improve the parameters of immunosensors.

Identification and characterization of a novel natural recombinant avian leucosis virus from Chinese indigenous chicken flock.

Avian leukosis virus (ALV) caused tremendous economic losses to poultry industry all over the world, especially in China. One natural recombinant ALV strain, designated as HB2015032, was isolated from indigenous chickens with neoplastic diseases in Hubei, China. The complete proviral genome of HB2015032 is 7703 bp in length. Sequence analysis showed that the Env of HB2015032 exhibited 99.3% similarity with that of a ALV subgroup K (ALV-K) isolate JS11C1 at amino acid level. Phylogenetic analysis revealed that both gp85 and gp37 of HB2015032 were clustered in the same branch with JS11C1 and other ALV-K strains isolated from Chinese indigenous chickens in recent years. However, the pol gene, the 3' untranslated region (3' UTR), and the 3' long terminal repeat (3' LTR) of HB2015032 were more closely related to ALV-J prototype HPRS-103, and clustered in the same branch with ALV-J strains. Furthermore, the pol gene of HB2015032 contained a premature stop codon that resulted in a truncated Pol protein with 22 amino acid residues missing, which was a unique feature of the pol gene of ALV-J. 3'UTR of HB2015032 containing entire DR1, E element and U3. E element of HB2015032 contained one base deletion, which resulted in a c-Ets-1 binding site. In addition, U3 region of HB2015032 contains most of the transcription regulatory elements of ALV-J, including two CAAT boxes, Y boxes, CArG boxes, PRE boxes, NFAP-1 boxes, and one TATA box. These results suggest that isolate HB2015032 was a novel recombinant ALV-K containing the ALV-K env gene and the ALV-J backbone and exhibiting high pathogenicity.

The regulation roles of miR-125b, miR-221 and miR-27b in porcine Salmonella infection signalling pathway.

miRNAs are non-coding RNA molecules typically 18-22 nucleotides long that can suppress the expression of their target genes. Several laboratories have attempted to identify miRNAs from the pig that are involved in Salmonella infection. These bioinformatics strategies using the newly available genomic sequence are generally successful. Here, we report an in silico identification of miRNAs in pig focusing on the Salmonella infection pathway, and further investigated the differential expression of those miRNAs by quantitative real-time PCR during pre- and post-natal stage of Salmonella inoculation from the peripheral blood of commercially breed pigs. We identified 29 miRNAs that have predicted targets in the Salmonella infection pathway and nine of them were not yet described in pig. In addition, the expression of nine selected miRNAs was validated in the peripheral blood by northern blotting. Through expression analyses, differences were found between pre- and post-natal stages of Salmonella inoculation for miR-221, miR-125b and miR-27b-all of them were suppressed 2 days after Salmonella inoculation. The predicted targets of those three miRNAs were validated by luciferase reporter assays. We show that FOS is a direct target of miR-221, miR-125b can suppress MAPK14, and miR-27b can target IFNG. These findings will be helpful in understanding the function and processing of these miRNAs in Salmonella infection. The miRNA differentially expressed in the peripheral blood of commercial breed pigs suggest that it can be used as genetic markers for salmonella infection resistance in pigs.

Outbreak of Listeria Monocytogenes in Pheasants.

Listeria monocytogenes is capable of infecting almost all animals. However, outbreaks of listeriosis are infrequent in birds. This report describes an outbreak of listeriosis in a small pheasant (Phasianus colchicus) breeder farm with more than 2,000 pheasants from Hubei province of the People's Republic of China. The affected flock consisted of adult and young birds. Approximately 300 young birds and a few adult birds were found dead within a few days of the onset of clinical signs. Twenty-five dead birds were collected for further examination. Histopathological lesions in the visceral organs were characterized by monocyte infiltration and proliferation. Localized encephalitis and meningitis were detected in the brains of dead birds. Gram-positive organisms were observed in heart blood smear, liver, and brain impression smears. The organisms were isolated from fresh liver and were identified as L. monocytogenes serotype 4b based on multiplex polymerase chain reaction (PCR) and hlyA gene sequence analysis. This is the first report describing outbreak of listeriosis in pheasant flock.