Expression and immunological characteristics of the surface-localized pyruvate kinase in Mycoplasma gallisepticum.

The widespread avian pathogen Mycoplasma gallisepticum is a causative agent of respiratory disease. The wall-less prokaryotes lack some tricarboxylic acid cycle enzymes, therefore, the glycolysis metabolic pathway is of great importance to these organisms. Pyruvate kinase (PK) is one of the key enzymes of the glycolytic pathway, and its immunological characteristics in Mycoplasma are not well known. In this study, the M. gallisepticum pyruvate kinase fusion protein (PykF) was expressed in a pET system. The full-length of the gene was subcloned into the expression vector pET28a(+) to construct the pET28a-rMGPykF plasmid, which was then transformed into Escherichia coli strain BL21 (DE3) cells. The expression of the 62 kDa recombinant protein of rMGPykF in E. coli strain BL21 (DE3) was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie blue staining. Purified rMGPykF exhibited PK catalytic activity, which could reflect the conversion of NADH to NAD(+). Mouse anti-PykF antibodies were generated by immunization of mice with rMGPykF. Immunoblot and immunoelectron microscopy assays identified PykF as an immunogenic protein expressed on the surface of M. gallisepticum cells. Bactericidal assay showed that anti-rMGPykF antiserum killed 70.55% of M. gallisepticum cells, suggesting the protective potential of PykF. Adherence inhibition assay on immortalized chicken fibroblasts (DF-1) cells revealed more than 39.31% inhibition of adhesion in the presence of anti-rMGPykF antiserum, suggesting that PykF of M. gallisepticum participates in bacterial adhesion to DF-1 cells.

Virulent and attenuated strains of duck hepatitis A virus elicit discordant innate immune responses in vivo.

Previous studies of duck hepatitis A virus infection have focused only on the pathogenicity and host response of one strain. Here, we show that the virulent SH strain and the attenuated FC64 strain induced varied pathogenicity, apoptosis and immune responses in the livers of 1-day-old ducklings. SH infection caused apoptosis and visible lesions in the liver; serum aspartate aminotransferase, alanine transaminase, alkaline phosphatase, γ-glutamyltransferase and total bilirubin activities were markedly upregulated; and ducklings died at 36 h post-infection (p.i.). However, FC64 infection did not induce significant symptoms or impair liver function, and all of the infected ducklings remained healthy. In addition, both virus strains replicated well in the liver, spleen and intestine, whilst the SH strain replicated more efficiently than FC64. IFN-γ, IL-2, inducible nitric oxide synthase and nitric oxide were strongly induced by SH infection, and may be associated with the pathogenicity of the SH strain. IFN-α, IFN-ß, IFN-stimulated transmembrane protein 1, IFN-stimulated gene 12, 2',5'-oligoadenylate synthetase-like and IL-6 were moderately induced by SH infection at 24 h p.i., and dramatically induced by FC64 infection at 36 h p.i. The intensive induction of cytokines by FC64 may be involved in restriction of virus replication and stimulation of adaptive immune responses. Ducklings inoculated with FC64 produced high levels of antiviral antibodies within 45 days p.i. The low virulence and strong immune response of FC64 rendered this strain a good vaccine candidate, as confirmed by a protective assay in this study.

Effect of age on the pathogenesis of DHV-1 in Pekin ducks and on the innate immune responses of ducks to infection.

Duck hepatitis virus (DHV) affects 1-week-old but not 3-week-old ducks, and it causes a more severe disease in the younger ducks. These differences may be partially due to the host response to DHV infection. In order to understand this difference, we characterized the pathobiology of and innate immune response to DHV infection in 1-day-old (1D) and 3-week-old (3 W) ducks. Viral RNA was detected in duck livers at 24, 36 and 72 h after inoculation with DHV at a dose of 10(3) LD50. Virus-induced pathology ranged from no clinical signs to severe disease and death, and it was more severe in the 1D ducks. Infection with DHV induced up-regulation of gene expression of Toll-like receptor (TLR)-7, TLR3, retinoic-acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA-5), interleukin (IL)-6, interferon (IFN)-α, interferon-induced transmembrane protein 1 (IFITM1), interferon-stimulated gene 12 (ISG12), and 2'-5' oligoadenylate synthetase-like gene (OASL) in the livers of 3 W ducks. Of these, IL-6, OASL and ISG12 mRNA levels were more than 100-fold higher in infected 3 W ducks than in mock-infected ducks of the same age. These genes were induced much less in infected 1D ducklings. We present evidence that a lower level of viral replication in the hepatocytes of 3 W ducks, whose basal level of cytokines is higher than that in 1D ducklings, may be related to the strong innate immunity induced. From our data, we conclude that duck age plays an important role in the pathogenicity of and innate immune responses to DHV.

Whole genome sequencing and biological characterization of Duck/JS/10, a new lentogenic class I Newcastle disease virus.

A lentogenic Newcastle disease virus (NDV), Duck/JS/10 (JS10), was isolated from an unvaccinated duck in China. The complete genome of the virus contained 15,198 nucleotides. Based on length of the genome and a partial sequence of the F gene, the virus was classified as a class I genotype 4 NDV. The antigenicity of the virus was compared with that of NDV strain La Sota via hemagglutination inhibition (HI), virus neutralization (VN) assay and animal experiments. Our results show that JS10 generates higher HI and VN titers than La Sota against both class I and II virulent NDV strains. Experiments on animals demonstrate that virus shedding from chickens vaccinated with JS10 is significantly reduced when compared to those vaccinated with La Sota. Overall, this study strongly suggests that JS10 may qualify as a new vaccine candidate against Newcastle disease.

The Mycoplasma gallisepticum α-enolase is cell surface-exposed and mediates adherence by binding to chicken plasminogen.

The α-enolase protein is reported to be an adhesin in several pathogenic bacterial species, but its role in Mycoplasma gallisepticum is unknown. In this study, the M. gallisepticum α-enolase gene was adapted to heterologous expression in Escherichia coli by performing overlapping polymerase chain reaction with site-directed mutagenesis to introduce A960G and A1158G mutations in the nucleotide sequence. The full-length mutated gene was cloned into a pGEM-T Easy vector and subcloned into the expression vector pET32a(+) to construct the pET-rMGEno plasmid. The expression of rMGEno in E. coli strain DE3 was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis with Coomassie blue staining. Purified rMGEno exhibited α-enolase catalytic activity that it could reflect the conversion of NADH to NAD(+). Mouse antiserum to α-enolase was generated by immunization with rMGEno. Immunoblotting and immunofluorescence assay with the antiserum identified α-enolase on the surface of M. gallisepticum cells. Enzyme-linked immunosorbent assay characterized rMGEno as a chicken plasminogen binding protein. An adherence inhibition assay on immortalized chicken fibroblasts (DF-1) demonstrated more than 77% inhibition of adhesion in the presence of mouse antiserum, suggesting that α-enolase of M. gallisepticum participates in bacterial adhesion to DF-1 cells.

Safety and Efficacy Evaluation of Recombinant Marek's Disease Virus with REV-LTR.

Recently, chickens vaccinated with the CVI988/Rispens vaccine showed increased tumor incidence. Moreover, many strains of Marek's disease virus (MDV) that were naturally integrated with the long terminal repeat (LTR) of the avian reticuloendotheliosis virus (REV) have been isolated, which means it is necessary to develop a new vaccine. In this study, two LTR sequences were inserted into Rispens to construct a recombinant MDV (rMDV). Then, the safety and efficacy of rMDV were evaluated separately in chickens. The growth rate curves showed that the insertion of REV-LTR into MDV enabled a faster replication in vitro than Rispens. Chickens immunized with high or repeated dose rMDV had no MD clinical signs. Further, no tumor, tissue lesions, or evident pathological changes were observed in the chicken organs. Polymerase chain reaction (PCR) and virus isolation revealed that rMDV had the ability to spread horizontally to non-immunized chickens and had no impact on the environment. After five passages in chickens, there were no obvious lesions, and the LTR insertion was stable. There were also no deletions or mutations, which indicates that rMDV is safe in chickens. In addition, rMDV has an advantage over Rispens against vvMDV Md5 at low doses. All results demonstrate that the transgenic strain of rMDV with REV-LTR can be used as a live attenuated vaccine candidate.

Newcastle disease virus NP and P proteins induce autophagy via the endoplasmic reticulum stress-related unfolded protein response.

Newcastle disease virus (NDV) can replicate and trigger autophagy in human tumor cells. Our previous study confirmed the critical role of autophagy in NDV infection. Here we studied the role of NDV structural proteins in the induction of autophagy through endoplasmic reticulum (ER) stress-related unfolded protein response (UPR) pathways. Ectopic expression of the NDV nucleocapsid protein (NP) or phosphoprotein (P) was sufficient to induce autophagy. NP or P expression also altered ER homeostasis. The PERK and ATF6 pathways, but not the XBP1 pathway, all of which are components of the UPR, were activated in both NDV-infected and NP or P-transfected cells. Knockdown of PERK or ATF6 inhibited NDV-induced autophagy and reduced the extent of NDV replication. Collectively, these data suggest not only roles for the NDV NP and P proteins in autophagy, but also offer new insights into the mechanisms of NDV-induced autophagy through activation of the ER stress-related UPR pathway.

Development of a loop-mediated isothermal amplification targeting a gene within the pyruvate dehydrogenase complex, the pdhA gene, for rapid detection of Mycoplasma gallisepticum.

Mycoplasma gallisepticum infections impose a significant economic burden on the poultry industry. In the current study, a loop-mediated isothermal amplification (LAMP) assay was developed and optimized to detect M. gallisepticum based on a gene within the pyruvate dehydrogenase complex, the pdhA gene, which codes for the major subunit (E1α) in the complex. The reaction conditions were optimized, and the specificity was confirmed by successful amplification of several M. gallisepticum strains, while no amplification was detected with 20 other major bacterial and viral pathogens of poultry. Additionally, the LAMP assay achieved 10-fold higher sensitivity than an existing polymerase chain reaction (PCR) method. The LAMP assay was applied to swab samples collected from poultry farms and compared with PCR. The positive detection rate was 20.2% (37/183) by LAMP and 13.1% (24/183) by PCR. The LAMP assay could provide a cost-effective, quick, and sensitive method for the detection of M. gallisepticum.

[Full-length genome analysis of four genotype 3 letogenic Newcastle disease viruses isolated from different hosts].

The purpose of this study is trying to analysis the homology between four lentogenic Class I genotype 3 Newcastle disease virus isolates from different hosts with NDV strain NDV 08-004, which was the first obtained complete genome sequence virus of class I genotype 3. The full-length genome of NDV isolates, JS/3/09/Ch, ZJ/3/10/Ch, AH/2/10/Du and JS/9/08/Go,were determined by RT-PCR and then an alyzed. All the genomes are 15 198 nucleotides (nt) in length. Compared with the full genome sequences of Class II NDV stains (genotype IV-IX),four isolates has a 6-nt deletion in the non-coding region of nuclear phosphoprotein gene between nucleotides 1 640-1 641 and 12-nt insertion in the coding region of phospho protein gene between nucleotides 2 381-2 382. All the isolates have the motifs 112EQ/RQE/GRL117 at the cleavage site of the fusion protein, which is typical of lenogenic NDV strains, and it is in agreement with the result of pathogenic tests. The full-length genome of 4 genotype 3 NDV isolates shared 93% nucleotide identity with NDV08-004. The results of alignment of 6 viral genes showed that NP gene shared the highest identity (98.3%-96.4%) and P gene shared the lowest identity (96.1%-91.9%). The results show the following two points. First, it is concluded that the isolates from different hosts share the same genotype has the insignificant divergence in the genetic information. Second, it is proposed that the mutation rates of NP/F/L genes are lower than P/M/HN genes.