Rapid Genotyping of Campylobacter coli Strains from Poultry Meat by PFGE, Sau-PCR, and fla-DGGE.

The genotyping of Campylobacter coli was done using three methods, pulsed-field gel electrophoresis (PFGE), Sau-polymerase chain reaction (Sau-PCR), and denaturing gradient gel electrophoresis assay of flagellin gene (fla-DGGE) and the characteristics of these assays were compared. The results showed that a total of 53 strains of C. coli were isolated from chicken and duck samples in three markets. All isolates were clustered into 31, 33, and 15 different patterns with Simpson's index of diversity (SID) values of 0.972, 0.974, and 0.919, respectively. Sau-PCR assay was simpler, more rapid, and had higher discriminatory power than PFGE assay. Fla-DGGE assay could detect and illustrate the number of contamination types of C. jejuni and C. coli without cultivation, which saved more time and cost than Sau-PCR and PFGE assays. Therefore, Sau-PCR and fla-DGGE assays are both rapid, economical, and easy to perform, which have the potential to be promising and accessible for primary laboratories in genotyping C. coli strains.

Comparison of pulsed-field gel electrophoresis and a novel amplified intergenic locus polymorphism method for molecular typing of Campylobacter jejuni.

Campylobacter is regarded as the leading cause of zoonotic diseases and Campylobacter jejuni (C. jejuni) is one of the predominant pathogenic species. To track C. jejuni infections, various genotyping methods have been used. In this study, amplified intergenic locus polymorphism (AILP) was used to type C. jejuni for the first time. To confirm its feasibility, pulsed-field gel electrophoresis (PFGE) was performed as a control, and the results obtained by the AILP and PFGE methods were compared. Fifty-one isolates were resolved into 34 and 29 different genotypes with Simpson's indices of 0.976 and 0.967 using the AILP and PFGE methods, respectively. The adjusted Rand coefficient of the two approaches was as high as 0.845. In summary, the data showed that the two genotyping methods were similar for discriminating isolates and were both appropriate methods to distinguish whether two isolates were indistinguishable, but the AILP was faster and less costly than PFGE. Therefore, the AILP is a reliable, rapid, and highly discriminative method to genotype C. jejuni collected from poultry meat, which is helpful to effectively monitor C. jejuni.

Gene order rearrangement of the M gene in the rabies virus leads to slower replication.

The matrix protein (M) is one of only five genes in the RV genome and is an important multifunctional protein. Besides to allow for the release of newly replicated virions pairing with G, the M protein also functions in virus replication, pathogenicity, and host cell apoptosis. The goal of present study is to generate recombinant viruses with M gene rearranged, thus laying the foundation for further exploring what will happen when the gene for M is relocated on the RV single-strand RNA. We used rHEP-Flury, an attenuated virus that remains virulent for less than 3 days in sucking mice, to reshuffle the M gene, using an approach that leaves the other viral nucleotide sequence intact. Two viruses with translocated M genes (N1M2 and N1M4) were recovered from each of the rearranged cDNAs, whose gene order is 3'-N-M-P-G-L-5' and 3'-N-P-G-M-L-5' respectively. The growth dynamics of these viruses showed slower replication than the wild-type virus in multiple-step growth curves, but they can grow to a comparable titer in tests of single-step growth curves. Further experimentation with these rearranged viruses will provide insights into the relationships between genome structure and virus phenotypes.

Molecular phylogenetic and positive selection analysis of Japanese encephalitis virus strains isolated from pigs in China.

Japanese encephalitis virus (JEV) is one of the most important virus which causes encephalitis. This disease is most prevalent in the south, southeast and the east region of Asia. In this study, two JEV strains, named JEV/SW/GD/01/2009 and JEV/SW/GZ/09/2004, were isolated from aborted fetuses and seminal fluid of pigs in China. To determine the characteristic of these virus isolates, the virulence of two newly JEV isolates was investigated, the result evidenced that the JEV/SW/GD/01/2009 did not kill mice, while the JEV/SW/GZ/09/2004 displayed neurovirulence with 0.925log10 p.f.u./LD50. Additionally, the full genome sequences of JEV were determined and compared with other known JEV strains. Results demonstrated that the genome of two JEV isolates was 10,976 nucleotides (nt) in length. As compared to the Chinese vaccine strain SA14-14-2, the JEV/SW/GD/01/2009 and the JEV/SW/GZ/09/2004 showed 99.7% and 97.5% identity at the nucleotide level, 99.6% and 96.7% identity at the amino acid level, respectively. Phylogenetic analysis, based on the full-length genome revealed that two JEV isolates were all clustered into genotype III compared to the reference strains. Furthermore, selection analyses revealed that dominant selective pressure acting on the JEV genome was purifying selection. Four sites under positive selection were identified: codon 521 (amino acid E-227), 2296 (amino acid NS4b-24), 3048 (amino acid NS5-521) and 3055 (amino acid NS5-528). Amino acid E-227 was proved to be related to neurovirulence. Taken together, the molecular epidemiology and functional of positively selected amino acid sites of two newly JEV isolates were fully understood, which might be helpful to predict possible changes in virulence.

Classical swine fever virus triggers RIG-I and MDA5-dependent signaling pathway to IRF-3 and NF-κB activation to promote secretion of interferon and inflammatory cytokines in porcine alveolar macrophages.

BACKGROUND: Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious disease of pigs. The RNA helicases retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5) are differentially involved in the detection of various RNA viruses. In present study, we investigated the roles of RIG-I and MDA-5 in eliciting antiviral and inflammatory responses to CSFV shimen strain in Porcine alveolar macrophages (PAMs). METHODS: CSFV Shimen strain was used as challenge virus in this study and PAMs were cultured in vitro. Interferon regulatory factor (IRF)-3 and nuclear factor-kappa B (NF-κB) translocation was detected using immunofluorescent staining; RIG-I, MDA5, interferon promoter-stimulating factor 1 (IPS-1), IRF-3 and NF-κB expression was measured by Western Blotting; Interferon beta (IFN-ß), IFN-α, interleukin-1beta (IL-1ß), IL-6 and tumor necrosis factor (TNF-α) expression was tested by Enzyme-linked immunosorbent assays (ELISA) and shRNA-mediated knockdown of MDA5 or RIG-I was performed. RESULTS: The findings suggested that the initial response to CSFV infection resulted in the higher expression of RIG-I and MDA5 leading to the activation of IPS-1, IRF-3 and NF-κB in a dose-dependent manner. Evaluation of IFN-α, IFN-ß, IL-1ß, IL-6 or TNF-α expressed by PAMs showed significant differences between infected and uninfected cells. CSFV infected cells induced to express high levels of IFN-α, IFN-ß, IL-1ß, IL-6 and TNF-α in a dose-dependent way within 24 h post-infection (hpi). At the same time, CSFV improved the nuclear translocation of IRF-3 and NF-κB. We also directly compared and assessed the roles of RIG-I and MDA5 in triggering innate immune actions during CSFV infection through shRNA-mediated knockdown of MDA5 or RIG-I. We found that, compared to the control, the production of IFN-α, IFN-ß, IL-1ß, IL-6 and TNF-α in response to CSFV infection was heavily reduced in RIG-I knockdown cells while it was moderately decreased in MDA5 knockdown cells. PAMs derived from knockdown of both RIG-I and MDA5 almost failed to produce IFNs and inflammatory cytokines. CONCLUSIONS: It indicates that CSFV can be recognized by both RIG-I and MDA5 to initiate the RIG-I signaling pathway to trigger innate defenses against infection.

Classical swine fever virus failed to activate nuclear factor-kappa b signaling pathway both in vitro and in vivo.

BACKGROUND: Classical swine fever virus (CSFV) is the cause of CSF which is a severe disease of pigs, leading to heavy economic losses in many regions of the world. Nuclear factor-kappa B (NF-κB) is a critical regulator of innate and adaptive immunity, and commonly activated upon viral infection. In our previous study, we found that CSFV could suppress the maturation and modulate the functions of monocyte-derived dendritic cells (Mo-DCs) without activating NF-κB pathway. To further prove the effects of CSFV on the NF-κB signaling pathway, we investigated the activity of NF-κB after CSFV infection in vivo and in vitro. METHODS: Attenuated Thiverval strain and virulent wild-type GXW-07 strain were used as challenge viruses in this study. Porcine kidney 15 (PK-15) cells were cultured in vitro and peripheral blood mononuclear cells (PBMCs) were isolated from the blood of CSFV-infected pigs. DNA binding of NF-κB was measured by electrophoretic mobility shift assays (EMSA), NF-κB p65 translocation was detected using immunofluorescent staining, and p65/RelA and IκBα expression was measured by Western Blotting. RESULTS: Infection of cells with CSFV in vitro and in vivo showed that compared with tumor necrosis factor alpha (TNF-α) stimulated cells, there was no distinct DNA binding band of NF-κB, and no significant translocation of p65/RelA from the cytoplasm to the nucleus was observed, which might have been due to the apparent lack of IkBa degradation. CONCLUSIONS: CSFV infection had no effect on the NF-κB signaling pathway, indicating that CSFV could evade host activation of NF-κB during infection.

Entry of tiger frog virus (an Iridovirus) into HepG2 cells via a pH-dependent, atypical, caveola-mediated endocytosis pathway.

Tiger frog virus (TFV), in the genus Ranavirus of the family Iridoviridae, causes high mortality of cultured tiger frog tadpoles in China. To explore the cellular entry mechanism of TFV, HepG2 cells were treated with drugs that inhibit the main endocytic pathways. We observed that TFV entry was inhibited by NH(4)Cl, chloroquine, and bafilomycin, which can all elevate the pH of acidic organelles. In contrast, TFV entry was not influenced by chlorpromazine or overexpression of a dominant-negative form of Esp15, which inhibit the assembly of clathrin-coated pits. These results suggested that TFV entry was not associated with clathrin-mediated endocytosis, but was related to the pH of acidic organelles. Subsequently, we found that endocytosis of TFV was dependent on membrane cholesterol and was inhibited by the caveolin-1 scaffolding domain peptide. Dynamin and actin were also required for TFV entry. In addition, TFV virions colocalized with the cholera toxin subunit B, indicating that TFV enters as caveola-internalized cargo into the Golgi complex. Taken together, our results demonstrated that TFV entry occurs by caveola-mediated endocytosis with a pH-dependent step. This atypical caveola-mediated endocytosis is different from the clathrin-mediated endocytosis of frog virus 3 (FV3) by BHK cells, which has been recognized as a model for iridoviruses. Thus, our work may help further the understanding of the initial steps of iridovirus infection in lower vertebrates.

Global landscape of structural proteins of infectious spleen and kidney necrosis virus.

Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus Megalocytivirus in the family Iridoviridae, causes severe damage to mandarin fish cultures in China. Little is known about the proteins of ISKNV virions. In this study, a total of 38 ISKNV virion-associated proteins were identified by four different workflows with systematic and comprehensive proteomic approaches. Among the 38 identified proteins, 21 proteins were identified by the gel-based workflows (one-dimensional [1-D] and two-dimensional [2-D] gel electrophoresis). Fifteen proteins were identified by 1-D gel electrophoresis, and 16 proteins were identified by 2-D gel electrophoresis, with 10 proteins identified by both methods. Another 17 proteins were identified only by liquid chromatography (LC)-based workflows (LC-matrix-assisted laser desorption ionization [MALDI] and linear trap quadrupole [LTQ]-Orbitrap). Among these 17 LC-identified proteins, 5 proteins were identified uniquely by the LC-MALDI workflow, whereas another 6 proteins were identified only by the LTQ-Orbitrap workflow. These results underscore the importance of incorporation of multiple approaches in identification of viral proteins. Based on viral genomic sequence, genes encoding these 38 viral proteins were cloned and expressed in vitro. Antibodies were produced against these 38 proteins to confirm the ISKNV structural proteins by Western blotting. Of the newly identified proteins, ORF 056L and ORF 118L were identified and confirmed as two novel viral envelope proteins by Western blotting and immunoelectron microscopy (IEM). The ISKNV proteome reported here is currently the only characterized megalocytivirus proteome. The systematic and comprehensive identification of ISKNV structural proteins and their localizations in this study will facilitate future studies of the ISKNV assembly process and infection mechanism.

Cloning of IRAK1 and its upregulation in symptomatic mandarin fish infected with ISKNV.

Interleukin-1 receptor activated kinases (IRAKs) play crucial roles in the Toll-like receptor (TLR) mediated signal transduction pathways that control host innate immune responses. Here we report the cloning of an IRAK1 cDNA (named ScIRAK1) from the mandarin fish. The predicted ScIRAK1 peptide contains a death domain and a serine/threonine-specific kinase domain. Quantitative RT-PCR showed that ScIRAK1 mRNA was primarily expressed in blood cells and posterior kidney. Seven days following infection with infectious spleen and kidney necrosis virus (ISKNV), the ScIRAK1 mRNA level was significantly higher in the blood cells of clinically symptomatic fish than in the blood cells of asymptomatic fish or control fish injected with phosphate-buffered saline. Additional experiments showed that overexpression of ScIRAK1 in the 293T cells could induce NF-kappaB activation. These results suggest that ScIRAK1 may play a role in the pathology of ISKNV infection in the mandarin fish.

A novel C-type lectin from the shrimp Litopenaeus vannamei possesses anti-white spot syndrome virus activity.

C-type lectins play key roles in pathogen recognition, innate immunity, and cell-cell interactions. Here, we report a new C-type lectin (C-type lectin 1) from the shrimp Litopenaeus vannamei (LvCTL1), which has activity against the white spot syndrome virus (WSSV). LvCTL1 is a 156-residue polypeptide containing a C-type carbohydrate recognition domain with an EPN (Glu(99)-Pro(100)-Asn(101)) motif that has a predicted ligand binding specificity for mannose. Reverse transcription-PCR analysis revealed that LvCTL1 mRNA was specifically expressed in the hepatopancreas of L. vannamei. Recombinant LvCTL1 (rLvCTL1) had hemagglutinating activity and ligand binding specificity for mannose and glucose. rLvCTL1 also had a strong affinity for WSSV and interacted with several envelope proteins of WSSV. Furthermore, we showed that the binding of rLvCTL1 to WSSV could protect shrimps from viral infection and prolong the survival of shrimps against WSSV infection. Our results suggest that LvCTL1 is a mannose-binding C-type lectin that binds to envelope proteins of WSSV to exert its antiviral activity. To our knowledge, this is the first report of a shrimp C-type lectin that has direct anti-WSSV activity.

N-Phenyl-anthranilic anhydride.

The complete mol-ecule of the title compound, C(26)H(20)N(2)O(3), is generated by crystallographic twofold symmetry, with the central O atom lying on the rotation axis. The conformation is stabilized by an intra-molecular N-H⋯O hydrogen bond. The dihedral angle between the inner and outer aromatic ring planes is 61.12 (5)°.