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Newcastle disease (ND) is a highly contagious avian disease, causing considerable economic losses to the poultry industry. To obtain a safe, inexpensive, and effective ND vaccine to meet the international trade requirements of differentiating infected from vaccinated animals (DIVA), here we report the production of Oryza sativa recombinant fusion (F) protein in stably transformed transgenic rice seeds via agroinfiltration. The F protein expression level was enhanced 3.6-fold with a genetic background in low glutelin. Inoculation of plant-produced F antigen into Specific Pathogen Free (SPF) chickens markedly elicited neutralizing antibody responses against homologous and heterologous ND virus strains. Two doses of 4.5 μg fully protected chickens from a lethal ND challenge without any clinical symptoms. The mean weight gain of F protein-immunized chickens within 15 days after challenge was significantly higher than that of traditional whole virus vaccine-immunized chickens, thereby obtaining higher economic benefits. Moreover, the sera from the chickens vaccinated with the plant-produced F vaccine did not show reactivity in an immunochromatographic strip targeting the haemagglutinin-neuraminidase protein (HN) protein, and DIVA could be achieved within 10 minutes. Our results demonstrate that the plant-derived F vaccine along with immunochromatographic strips could be useful in the implementation of an NDV eradication program.

Antibody-dependent enhancement (ADE) contributes to the pathogenesis of porcine reproductive and respiratory syndrome virus (PRRSV)-persistent infection. However, the mechanisms of PRRSV-ADE infection are still confusing. A clear understanding of the event upon virus infection by the ADE pathway has become crucial for developing efficient intervention of the PRRSV infection. In this study, an ADE assay showed that PRRSV-ADE infection in porcine alveolar macrophages (AMs) significantly decreased the production of interferon-α (IFN-α) and tumor necrosis factor-α (TNF-α), and significantly increased the production of interleukine-10 (IL-10). A gene knockdown assay based on small interfering RNA (siRNA) showed that both Fc gamma receptor I (FcγRI) and FcγRIII in porcine AMs were involved in PRRSV-ADE infection. An activation assay showed that specific activation of FcγRI or FcγRIII in porcine AMs during PRRSV infection not only significantly decreased the production of IFN-α and TNF-α, but also significantly increased the production of IL-10 and significantly facilitated PRRSV replication. In conclusion, our studies suggested that ADE downregulated the production of IFN-α and TNF-α in porcine AMs maybe via FcγRI and FcγRIII, thereby leading to enhanced PRRSV infection.

MYH9 has been identified as an indispensable cellular protein for porcine reproductive and respiratory syndrome virus (PRRSV) entry into permissive cells using the monoclonal anti-idiotypic antibody (Mab2-5G2) recognizing an antibody that specifically interacts with PRRSV glycoprotein 5 (GP5). More recently, we found that Mab2-5G2 interacted with the MYH9 C-terminal domain, designated PRA, which is required for PRRSV internalization. In this study, we demonstrate that blocking of MYH9 with Mab2-5G2 significantly diminished PRRSV internalization by porcine alveolar macrophage (PAM) via interruption of direct interaction between GP5 and MYH9, and thus remarkably inhibited subsequent infection of PAMs by PRRSV-2 isolates. Moreover, the three-dimensional structure of the Mab2-5G2 Fab-PRA complex determined via homology modeling predicted potential docking sites required for PRRSV internalization. Further analysis of Mab2-5G2-binding sites within PRA highlighted that the amino acids E1670, K1673, E1679, and I1683 in PRA are the key Mab2-5G2-binding residues. Notably, recombinant PRA protein blocked the interaction between PRRSV GP5 and cellular MYH9 by preventing translocation of MYH9 from the cytoplasm to the cell membrane, an essential step for PRRSV virion internalization. Meanwhile, porcine cell line permissive for PRRSV bearing point mutation of E1670A in MYH9 demonstrated reduced susceptibility for PRRSV infection. In conclusion, this work increases understanding of both PRRSV pathogenesis and the mechanistic role played by MYH9 in PRRSV infection.

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals that has caused tremendous economic losses worldwide. In this study, we designed a chimeric nanoparticles (CNPs) vaccine that displays the predominant epitope of the serotype O foot-and-mouth disease virus (FMDV) VP1 131-160 on the surface of MS2 phage. The recombinant protein was expressed in Escherichia Coli and can self-assemble into CNPs with diameter at 25–30 nm in vitro. A tandem repeat peptide epitopes (TRE) was prepared as control. Mice were immunized with CNPs, TRE and commercialized synthetic peptide vaccines (PepVac), respectively. The ELISA results showed that CNPs stimulated a little higher specific antibody levels to PepVac, but was significantly higher than the TRE groups. Moreover, the results from specific IFN-γ responses and lymphocyte proliferation test indicated that CNP immunized mice exhibited significantly enhanced cellular immune response compared to TRE. These results suggested that the CNPs constructed in current study could be a potential alternative vaccine in future FMDV control.

Macrophages are crucial members of the mononuclear phagocyte system essential to protect the host from invading pathogens and are central to the inflammatory response with their ability to acquire specialized phenotypes of inflammatory (M1) and anti-inflammatory (M2) and to produce a pool of inflammatory mediators. Equipped with a broad range of receptors, such as Toll-like receptor 4 (TLR4), CD14, and Fc gamma receptors (FcγRs), macrophages can efficiently recognize and phagocytize invading pathogens and secrete cytokines by triggering various secondary signaling pathways. Phospholipase C (PLC) is a family of enzymes that hydrolyze phospholipids, the most significant of which is phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Cleavage at the internal phosphate ester generates two second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), both of which mediate in diverse cellular functions including the inflammatory response. Recent studies have shown that some PLC isoforms are involved in multiple stages in TLR4-, CD14-, and FcγRs-mediated activation of nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and interferon regulatory factors (IRFs), all of which are associated with the regulation of the inflammatory response. Therefore, secondary signaling by PLC is implicated in the pathogenesis of numerous inflammatory diseases. This review provides an overview of our current knowledge on how PLC signaling regulates the macrophage-mediated inflammatory response.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection strongly modulates the host’s immune response. The RNA silencing pathway is an intracellular innate response to viral infections. However, it is unknown whether PRRSV interacts with cellular RNA silencing to facilitate the viral infection. Here, we report for the first time the interaction between PRRSV and RNA silencing in both the porcine macrophages and African green monkey kidney cell line (MARC-145) cell line, which were derived from African green monkey kidney cells and highly permissive for PRRSV infection. Our data demonstrated that PRRSV suppressed RNA silencing induced by short-hairpin (sh) RNA, double-strand (ds) RNA and microRNA (miRNA) and downregulated the expression of argonaute protein-2 (Ago-2), which is a key protein of the RNA silencing pathway in animal cells. Further, exogenous introduction of siRNA and shRNA downregulated Dicer or Ago-2 proteins of the cellular RNA silencing apparatus in MARC-145 cells and porcine macrophages, which, in turn, increased the viral replication and titers. The viral non-structure protein 1α (nsp-1α) and nsp11 of PRRSV were identified as the suppressors for cellular RNA silencing (RSSs) to downregulate the Ago-2 protein. Our results identify that PRRSV, through its nsp proteins, suppresses the cellular RNA silencing apparatus in favor of viral infection and supports a co-evolutionary process of the virus and the cellular RNA silencing process.

The antibiotic resistance of Salmonella has become increasingly serious due to the increased use of antibiotics, and antimicrobial peptides have been considered as an ideal antibiotic alternative. Salmonella can induce macrophage apoptosis and thus further damage the immune system. The antimicrobial peptide JH-3 has been shown to have a satisfactory anti-Salmonella effect in previous research, but its mechanism of action remains unknown. In this study, the effects of JH-3 on macrophages infected with Salmonella Typhimurium CVCC541 were evaluated at the cellular level. The results showed that JH-3 significantly alleviated the damage to macrophages caused by S. Typhi infection, reduced the release of lactic dehydrogenase (LDH), and killed the bacteria in macrophages. In addition, JH-3 decreased the phosphorylation level of p65 and the expression and secretion of interleukin 2 (IL-2), IL-6, and tumor necrosis factor-α (TNF-α) by inhibiting the activation of the mitogen-activated protein kinase (MAPK) (p38) signaling pathway and alleviating the cellular inflammatory response. From confocal laser scanning microscopy and flow cytometry assays, JH-3 was observed to inhibit the release of cytochrome c in the cytoplasm; the expression of TNF-αR2, caspase-9, and caspase-8; to further weaken caspase-3 activation; and to reduce the S.-Typhi-induced apoptosis of macrophages. In summary, the mechanism by which JH-3 inhibits Salmonella infection was systematically explored at the cellular level, laying the foundation for the development and utilization of JH-3 as a therapeutic alternative to antibiotics.

Hepatitis E virus (HEV) infects both humans and animals, with an overall human mortality rate generally less than 1%, but as high as 20% among pregnant women. HEV strains fall into 4 major genotypes. Zoonotic genotypes 3 and 4 associate with sporadic human and animal HEV cases in many industrialized countries. To date, collective evidence implicates pigs as the main HEV reservoir, justifying the importance of monitoring HEV infection rates in pig herds to prevent human illness. Due to the lack of a robust in vitro cell culture system for viral propagation, no "gold standard" assay has yet been developed to detect HEV infection in domestic pigs. 1E4, a monoclonal antibody (mAb) specific for the C-terminal 268 amino acids of HEV genotype 4 ORF2 capsid protein (sORF2-C), was generated and conjugated to horseradish peroxidase (HRP) for use in a blocking ELISA (bELISA). Optimal sORF2-C coating antigen concentration (8 μg/ml), HRP-1E4 dilution (1:1000), and test pig serum dilution (1:20) were determined using a checkerboard titration test. A cut-off value of 16.9% was chosen to differentiate between positive vs. negative sera after mean percent inhibition (PI) testing of 230 negative pig sera. Compared with the indirect ELISA (iELISA), western blot, and a commercial ELISA kit for detecting anti-HEV antibodies in human sera, the bELISA showed no statistical differences and statistically high coincidence of 93.23%, 92%, and 95% with the other tests, respectively. A blocking ELISA (bELISA) for detecting anti-HEV antibodies in pig serum samples was developed with high sensitivity and high specificity comparable to that of the indirect ELISA. The bELISA results exhibited high agreement with iELISA, western blot, and a commercial ELISA kit designed to detect human anti-HEV antibodies. Therefore, bELISA should serve as an ideal method for large-scale serological investigation of anti-HEV antibodies in domestic pigs.

Bovine herpesvirus 1 (BoHV-1) is an important pathogen of cattle that causes lesions in mucosal surfaces, genital tracts and nervous systems. As a novel oncolytic virus, BoHV-1 infects and kills numerous human tumor cells. However, the mechanisms underlying the virus-induced cell damages are not fully understood. In this study, we demonstrated that virus infection of MDBK cells induced high levels of DNA damage, because the percentage of comet tail DNA (tailDNA%) determined by comet assay, a direct indicator of DNA damage, and the levels of 8-hydroxyguanine (8-oxoG) production, an oxidative DNA damage marker, consistently increased following the virus infection. The expression of 8-oxoguanine DNA glycosylase (OGG-1), an enzyme responsible for the excision of 8-oxoG, was significantly decreased due to the virus infection, which corroborated with the finding that BoHV-1 infection stimulated 8-oxoG production. Furthermore, the virus replication in human tumor cells such as in A549 cells and U2OS cells also induced DNA damage. Chemical inhibition of reactive oxidative species (ROS) production by either ROS scavenger N-Acetyl-l-cysteine or NOX inhibitor diphenylene iodonium (DPI) significantly decreased the levels of tailDNA%, suggesting the involvement of ROS in the virus induced DNA lesions. Collectively, these results indicated that BoHV-1 infection of these cells elicits oxidative DNA damages, providing a perspective in understanding the mechanisms by which the virus induces cell death in both native host cells and human tumor cells.

Porcine reproductive and respiratory syndrome (PRRS) is identified as one of the most important etiological agents in multifactorial respiratory disease of swine and can predispose pigs to secondary infections by other pathogens, usually bacteria. To understand the mechanism for an increased susceptibility to secondary bacterial infections, we investigated the antibody-dependent phagocytosis behaviour and killing ability of PMNs after infection by PRRSV strains BJ-4 or HN07-1. PMN's antibody-dependent phagocytosis and their ability to kill E.coli were both noticeably decreased following PRRSV infection, in particular with the highly pathogenic strain HN07-1. As the change in this function of the PMNs may reflect a variation in the expression of FcγRs, the expression profiles of the activating and the inhibitory FcγRs were examined. We found that RNA expression of the inhibitory receptor FcγRIIB was up-regulated post-infection, and this was greater after infection with the more virulent PRRSV strain HN07-1. The activating receptor FcγRIIIA RNA expression was on the other hand inhibited to the same extent by both PRRSV strains. Neutralizing antibody titers post-infection by PRRSV strains BJ-4 or HN07-1 were also detected. All of the pigs in infection groups showed viraemia by the end of the study (56 DPI). These observations may help to understand the mechanism of increased susceptibility to secondary bacterial infections following PRRSV infection.

OBJECTIVE: Organophosphorus (OP) pesticides are considered hazardous substances because of their high toxicity to nontarget species and their persistence in the environment and agricultural products. Therefore, it is important to develop a rapid, sensitive, and economical method for detecting OP pesticides and their residues in food and the environment. METHODS: A broad, selective monoclonal antibody (MAb) for organophosphorus pesticides was produced. Based on the MAb, an enzyme linked immunosorbent assay (ELISA) and an immunochromatography assay (ICA) for detecting OP pesticides in different agricultural products were developed using a binding inhibition format on microtiter plates and a membrane strip, respectively. RESULTS: Under the optimized conditions, the IC(50) values of the ELISA ranged from 3.7 to 162.2 ng mL(-1) for the 8 OP pesticides. The matrix interferences of Apple, Chinese cabbage, and greengrocery were removed by 40-fold dilution, the recoveries from spiked samples ranged from 79.1% to 118.1%. The IC(50) values of ICA for the 8 OP pesticides ranged from 11.8 to 470.4 ng mL(-1). The matrix interference was removed from the Chinese cabbage and Apple samples with 5-fold dilution, and the interference was removed from the greengrocery samples with 20-fold dilution. The recoveries from the spiked samples ranged between 70.6 and 131.9%. The established ELISA and ICA were specific selectivity for the 8 OP pesticides. CONCLUSIONS: The established ELISA is a sensitive screening method for the detection of OP pesticides, but the ELISA detection method depends on a laboratory platform and requires a relative long assay time and several steps operation. The established ICA is very useful as a screening method for the quantitative, semi-quantitative or qualitative detection of OP pesticides in agricultural products, and it has advantages over ELISA methods with regard to factors such as the testing procedure, testing time, and matrix interferences, among others.

Antibody-dependent enhancement (ADE) of virus infection caused by the uptake of virus-antibody complexes by FcγRs is a significant obstacle to the development of effective vaccines to control certain human and animal viral diseases. The activation FcγRs, including FcγRI and FcγRIIa have been shown to mediate ADE infection of virus. In the present paper, we showed that pocine FcγRIIb, an inhibitory FcγR, mediates ADE of PRRSV infection. Stable Marc-145 cell lines expressing poFcγRIIb (Marc-poFcγRII) were established. The relative yield of progeny virus was significantly increased in the presence of sub-neutralization anti-PRRSV antibody. The Fab fragment and normal porcine sera had no effect. Anti-poFcγRII antibody inhibited the enhancement of infection when cells were infected in the presence of anti-PRRSV antibody, but not when cells were infected in the absence of antibody. These results indicate that enhancement of infection in these cells by anti-PRRSV virus antibody is FcγRII-mediated. Identification of the inhibitory FcγR mediating ADE infection should expand our understanding of the mechanisms of pathogenesis for a broad range of infectious diseases and may open many approaches for improvements to the treatment and prevention of such diseases.

Lomefloxacin (LMF), a third-generation fluoroquinolone antibacterial agent, is often used to treat bacterial and mycoplasma infections. However, due to its prolonged half-life and slow metabolism, it is prone to residues in animal-derived foods, posing a potential food safety risk. Therefore, it is particularly urgent and important to establish a method for detecting lomefloxacin. In this study, direct and indirect competitive fluorescence-linked immunosorbent assay (dc-FLISA and ic-FLISA) based on quantum dots (QDs) was established for the detection of LMF. As for dc-FLISA, the half-maximal inhibitory concentration (IC 50 ) and limit of detection (LOD) were 0.84 ng/mL, 0.04 ng/mL, respectively, the detection ranges from 0.08 to 9.11 ng/mL. The IC 50 and LOD of ic-FLISA were 0.43 ng/mL and 0.03 ng/mL, respectively, meanwhile the detection ranges from 0.05 to 3.49 ng/mL. The recoveries of dc-FLISA and ic-FLISA in animal-derived foods (milk, fish, chicken, and honey), ranged from 95.8% to 105.2% and from 96.3% to 103.4%, respectively, with the coefficients of variation less than 8%. These results suggest that the dc-FLISA and ic-FLISA methods, which are based on QD labelling, are highly sensitive and cost-effective, and can be effectively used to detect LMF in animal-derived foods.

An ultrasensitive and broad-specific monoclonal antibody recognising cyproheptadine hydrochloride and six phenothiazines was produced. The 50% inhibition concentration against cyproheptadine hydrochloride was 0.036 ng/mL, and the cross-reactivities for six phenothiazines were from 6.33% to 63.16%. Based on the developed monoclonal antibody, an immunochromatographic strip was established, with the visual detection limits (cut-off values) of seven drugs ranging from 5 to 100 ng/g in feedstuffs. With the strip reader, the 50% inhibition concentration of the developed immunochromatographic strip for seven drugs ranged from 0.570 to 7.750 ng/g. The intra-assay recoveries were from 79.8% to 103.4% with the highest coefficient of variation of 11.3%. The inter-assay recoveries were from 79.0% to 96.6% with the highest coefficient of variation of 12.7%. In summary, the proposed immunochromatographic strip was considered suitable for simultaneously monitoring cyproheptadine hydrochloride and phenothiazines in feedstuffs.