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An indirect enzyme-linked immunosorbent assay (I-ELISA) based on the VP2 protein of duck hepatitis A virus type 3 (DHAV-3) was established in this study. The optimal dilutions of antigen, serum and goat anti-duck IgG conjugate were 1:1600 (2.23 μg/mL), 1:160 and 1:2000, respectively. The optimal blocking buffer was 1% skim milk. The cut-off value for the method was 0.25, and the analytical sensitivity of the method was 1:5120. The results of specific evaluation showed that except for DHAV-1, DHAV-3 antisera did not cross-react with any other common duck-sensitive pathogens, indicating that this method can be used to detect DHAV-3 and DHAV-1 antibodies. The coefficients of variation (CVs) were lower than 10 %. The coincidence rate between the VP2-DHAV-3-ELISA and the neutralization test was 93.3 %. In summary, the I-ELISA method based on VP2 protein has high sensitivity, specificity, and coincidence rate compared with the neutralization test and has advantages in serum monitoring. The I-ELISA method based on VP2 protein provides a simple and rapid method for the detection of anti-DHAV antibodies and the epidemiological monitoring of DHAV.

Duck hepatitis A virus type 1 (DHAV-1) is one of the most harmful pathogens in the duck industry. The infection of adult ducks with DHAV-1 was previously shown to result in transient cytokine storms in their kidneys. To understand how DHAV-1 infection impacts the host liver, we conducted animal experiments with the virulent CH DHAV-1 strain and the attenuated CH60 commercial vaccine strain. Visual observation and standard hematoxylin and eosin staining were performed to detect pathological damage in the liver, and viral copy numbers and cytokine expression in the liver were evaluated by quantitative PCR. The CH strain (10

Background UL13 multifunctional tegument protein of duck enteritis virus (DEV) is predicted as protein kinase (CHPK); however, little is known concerning its subcellular localization signal. Results In this study, by transfection with two predicted nuclear signals of DEV UL13 fused to enhanced green fluorescent protein (EGFP), two bipartite nuclear localization signal (NLS) was identified. We identified the nuclear localization signals (NLSs) that control its nuclear importing using fluorescence assay and proved that nuclear localization of DEV UL13 is a classical importin α/β-dependent process. And we constructed the mutant DEV, with the NLSs of UL13 deleted, to explore whether it will affect the replication of virus particles. Conclusions DEV UL13 protein is directed by amino acids 4 to 7 and 90 to 96, and proved that this nuclear import occurs via the classical importin α/β-dependent process. And Entry nucleus of UL13 protein has no effect on DEV replication in cell culture. Our study enhances the understanding of DEV pUL13. Taken together, these results would provide significant information for the stud y of the biological function of UL13 during DEV infection.

Excessive iron in the bacterial cytoplasm can potentiate the production of harmful reactive oxygen species (ROS). Riemerella anatipestifer (R. anatipestifer, RA), a gram-negative bacterium, encodes an iron uptake system, but its iron detoxification mechanism is unknown. Here, the dps gene of R. anatipestifer CH-1 (RA-CH-1) was deleted using sacB as a counterselection marker. The dps mutant was more sensitive to H2O2 than the wild type in iron-rich conditions but not in iron-limited conditions, suggesting that Dps prevents H2O2-induced damage through iron binding. However, the dps mutant and wild type were identically sensitive to bactericidal antibiotics, and antibiotic treatment did not enhance RA-CH-1 ROS production. Furthermore, Dps prevents DNA damage by binding DNA. The RA-CH-1 dps transcript level was higher in the stationary phase than in the early and exponential phases and was increased by OxyR in the presence of H2O2. Finally, duckling colonization by the dps mutant was similar to that by the wild type at 48 h postinfection but significantly lower at 60 h postinfection, suggesting that RA-CH-1 Dps is not involved in host invasion but increases resistance to host clearance. Dps thus likely plays an important role in R. anatipestifer physiology and pathogenesis through protecting against oxidative stress.

Carbapenems are traditionally recognized to be the last resort drugs to treat infections due to MDR organisms such as E. coli. As such, the emergence of New Delhi metallo-β-lactamase-producing E. coli strains have become a challenging threat to the public health. In this regard, we examined the molecular characteristics of carbapenem-resistant E. coli (CRE) isolated from waterfowls in China's tropical island, Hainan. A total of 311 single E. coli strains were obtained from 20 various farms of healthy ducks and geese in 2 districts of Hainan island. The CRE strains were initially identified via phenotypic resistance and modified Hodge test. PCR assay and subsequent nucleotide sequencing were used to detect different types of carbapenemase encoding genes (blaNDM, blaVIM, blaIMP, blaOXA and blaKPC). In addition, MLST and PFGE analyses were also performed. Among the 311 E. coli strains, 8 strains were detected to produce a single type of carbapenemase i.e. NDM-1 (2.6%). A total of 5 sequence types (STs) were observed, of which ST10 was the most prevalent accounting for 37.5% (3/8). Moreover, these 8 isolates yielded 6 different PFGE clusters but showed approximately related PFGE types, suggesting the propagation of similar clone between the farms. This is the first report on the identification of NDM-1-producing E. coli from waterfowls in Hainan island, China. Our results emphasize the need for better efforts to control the further spread of NDM-1-producing E. coli strains in this tropical island.

Duck Tembusu virus (DTMUV) has recently appeared in ducks in China and the key cellular determiners for DTMUV replication in host cells remain unknown. Autophagy is an evolutionarily conserved cellular process that has been reported to facilitate flavivirus replication. In this study, we utilized primary duck embryo fibroblast (DEF) as the cell model and found that DTMUV infection triggered LC3-II increase and polyubiquitin-binding protein sequestosome 1 (p62) decrease, confirming that complete autophagy occurred in DEF cells. The induction of autophagy by pharmacological treatment increased DTMUV replication in DEF cells, whereas the inhibition of autophagy with pharmacological treatments or RNA interference decreased DTMUV replication. Inhibiting autophagy enhanced the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-&kappa, B) and interferon regulatory factor 7 (IRF7) pathways and increased the p62 protein level in DTMUV-infected cells. We further found that the overexpression of p62 decreased DTMUV replication and inhibited the activation of the NF-&kappa, B and IRF7 pathways, and changes in the NF-&kappa, B and IRF7 pathways were consistent with the level of phosphorylated TANK-binding kinase 1 (p-TBK1). Opposite results were found in p62 knockdown cells. In summary, we found that autophagy-mediated p62 degradation acted as a new strategy for DTMUV to evade host innate immunity.

Additional file 1: Table S1. Sequence and characteristics of RT-qPCR primers. The primers of DEV UL21, UL54, UL13, US2, β-actin were designed with Oligo 7. Figure S1. Complete image of UL21 genotype identification. GCV represents DEV-infected cells adding ganciclovir and CHX is adding cycloheximide. The (−) represents negative control and (+) were positive control.

Duck enteritis virus (DEV) can successfully evade the host innate immune responses and establish a lifelong latent infection in the infected host. However, the study about how DEV escapes host innate immunity is still deficient up to now. In this study, for the first time, we identified a viral protein VP16 by which DEV can obviously downregulate the production of IFN-β in duck embryo fibroblast (DEF). Our results showed that ectopic expression of VP16 decreased duck IFN-β (duIFN-β) promoter activation and significantly inhibited the mRNA transcription of IFN-β. Further study showed that VP16 can also obviously inhibit the mRNA transcription of interferon-stimulated genes (ISGs), such as myxovirus resistance protein (Mx) and interferon-induced oligoadenylate synthetase-like (OASL). Furthermore, we found that this anti-interferon activity of VP16 depended on its N-terminus (aa1-200). Coexpression analysis revealed that VP16 selectively blocked duIFN-β promoter activity at the duIRF7 level rather than duIRF1. Based on the results of coimmunoprecipitation analysis (co-IP) and indirect immunofluorescence assay (IFA), VP16 was able to bind to duck IRF7 (duIRF7) directly, but did not interact with duck IRF1 (duIRF1) in vitro.

Duck tembusu virus (DTMUV) is a single-stranded, positive-polarity RNA flavivirus that has caused considerable economic losses in China in recent years. Innate immunity represents the first line of defense against invading pathogens and serves as an important role in resisting viral infections. In this study, we found that the infection of ducks by DTMUV triggers Toll-like receptors (TLRs) and (RIG-I)-like receptors (RLRs) signaling pathways and inducing abundant of pro-inflammatory factors and type I interferons (IFNs), in which melanoma differentiation-associated gene 5 (MDA5) and Toll-like receptor 3 (TLR3) play important immunity roles, they can inhibit the replication process of DTMUV via inducing type I IFNs. Moreover, we demonstrated that type I IFNs can inhibit the DTMUV replication process in a time- and dose-dependent manner. Exosomes are small membrane vesicles that have important roles in intercellular communication. MicroRNAs (miRNAs) are small non-coding RNAs that can modulate gene expression and are common substances in exosomes. In our experiment, we successfully isolated DEF cells derived exosome for the first time and explored its function. Firstly, we found the expression of miR-148a-5p is significantly decreased following DTMUV infect. Then we found miR-148a-5p can target TLR3 and down-regulate the expression of TLR3, serving as a negative factor in innate immunity. Unfortunately, we cannot find miRNAs with different expression changes that can target MDA5. Lastly, our experimental results showed that TLR3 was one of the causes of miR-148a-5p reduction, suggesting that the high level of TLR3 after DTMUV infect can both trigger innate immunity and suppress miR-148a-5p to resist DTMUV.

Four oxide clusterfullerenes Ho2O@C90 (I–IV) were prepared by a modified arc discharge method and isolated by a multistage high performance liquid chromatography procedure. Based on their vis-NIR spectra, the carbon cage symmetries in Ho2O@C90 (I, III, IV) are confirmed to be C 2(43)-C90, C 2(40)-C90 and C 2(41)-C90, respectively. Electrochemical studies display that the first and second reduction potentials of Ho2O@C90 isomers are close to those of Er2C2@C90 ones, but much lower than those of Sm@C90 ones. Moreover, it is found that the electrochemical band gaps of Ho2O@C90 isomers are also very close to those of Er2C2@C90 isomers. A detailed investigation on the redox potentials of C90 based EMFs reveals that endohedral clusters/atoms have much greater effect than carbon cage symmetries on the electrochemical properties of EMFs.

Long-term excessive exposure to fluoride from environmental sources can cause serious public health problems such as dental fluorosis and skeletal fluorosis. The aberrant activation of osteoblasts in the early stage is one of the critical steps during the pathogenesis of skeletal fluorosis and canonical Wnt signaling pathway participate in the progress. However, the specific mechanism that how canonical Wnt signaling pathway was mediated is not yet clear. In this study, we found that miR-21-5p induced the activation of canonical Wnt signaling pathway via targeting PTEN and DKK2 during fluoride induced osteoblasts activation and firstly demonstrated the forward loop between canonical Wnt signaling and miR-21-5p in the process. These findings suggested an important regulatory role of miR-21-5p on canonical Wnt signaling pathway during skeletal fluorosis and miR-21-5p might be a potential therapeutic target for skeletal fluorosis.

The duck hepatitis A virus 1 (DHAV-1) 2C protein was predicted to be a superfamily III helicase member and includes nucleotide binding (NTB) and putative RNA helicase activity motifs. To study whether DHAV-1 2C protein has NTB activity, we expressed DHAV-1 2C protein with maltose binding protein (MBP) to solve its poor solubility in a prokaryotic expression system. We showed that the DHAV-1 2C protein has nucleoside triphosphatase (NTPase) activity by measuring the released phosphate. The NTPase of the DHAV-1 2C protein is Mg

Duck Tembusu virus (DTMUV) is a newly emerged causative agent of avian disease. The protease-dependent immune evasion of flaviviruses has been reported; however, the molecular details of this process are unclear. In this study, we found that DTMUV nonstructural protein 2B-3, a NS2B3 protease, can inhibit IFN-β production. DTMUV NS2B3 inhibited RIG-I–, MDA5-, MAVS-, and STING-directed IFN-β transcription, but not TBK1- and IRF7-mediated induction of IFN-β. Further analysis showed that DTMUV NS2B3 could cleave duck STING (duSTING); the cleavage was dependent on the protease activity of NS2B3. Moreover, the STING cleavage event occurred in a not-strictly-species-specific manner. The scissile bond of duSTING cleaved by NS2B3 was mapped between the R84 and G85 residues. The ability of NS2B3 to reduce duSTING cleavage-resistant mutant-mediated IFN-β, and ISG production was significantly reduced, demonstrating that duSTING cleavage is essential for NS2B3-induced suppression of type I IFN responses. Remarkably, the binding of NS2B3 to duSTING, which is a prerequisite for cleavage, was found to depend on NS2B, but not NS3, the cofactor of the enzyme. Unexpectedly, we found that the region between aa residues 221–225 of duSTING, distal from the site of the scissile bond, was essential for the binding of NS2B3 to duSTING and/or the cleavage of duSTING by NS2B3. Thus, we identified the molecular mechanism by which DTMUV subverts the host innate immunity using its protease. More importantly, our study provides insight into NS2B3-mediated STING cleavage events in general.

Background Riemerella anatipestifer is one of the most serious infectious disease-causing pathogens in the duck industry. Drug administration is an important method for prevention and treatment of infection in duck production, leading to widespread drug resistance in R. anatipestifer. Methods For a total of 162 isolates of R. anatipestifer, the MICs were determined for a quinolone antimicrobial agent, namely, nalidixic acid, and three fluoroquinolones, namely, ciprofloxacin, enrofloxacin and ofloxacin. The gyrA, parC, and parE gene fragments were amplified by PCR to identify the mutation sites in these strains. Site-directed mutants with mutations that were detected at a high frequency in vivo were constructed (hereafter referred to as site-directed in vivo mutants), and the MICs of these four drugs for these strains were determined. Results In total, 100, 97.8, 99.3 and 97.8% of the 137 R. anatipestifer strains isolated between 2013 and 2018 showed resistance to nalidixic acid, ciprofloxacin, enrofloxacin, and ofloxacin, respectively. The high-frequency mutation sites were detected in a total of 162 R. anatipestifer strains, such as Ser83Ile and Ser83Arg, which are two types of substitution mutations of amino acid 83 in GyrA; Val799Ala and Ile811Val in ParC; and Val357Ile, His358Tyr, and Arg541Lys in ParE. MIC analysis results for the site-directed in vivo mutants showed that the strains with only the Ser83Ile mutation in GyrA exhibited an 8–16-fold increase in MIC values, and all mutants showed resistance to ampicillin and ceftiofur. Conclusions The resistance of R. anatipestifer to quinolone agents is a serious problem. Amino acid 83 in GyrA is the major target mutation site for the fluoroquinolone resistance mechanism of R. anatipestifer.

Riemerella anatipestifer (R. anatipestifer, RA) is an important bacterial pathogen of ducks and other birds; infection with RA causes high poultry mortality and heavy economic losses in the poultry industry. However, the pathogenesis of this bacterium is poorly understood, in part due to the lack of a suitable array of methods for genetic manipulation. In this study, we first examined the efficacy of the mutated pheS gene (pheS*) as a counterselectable marker in R. anatipestifer. A suicide vector carrying pheS*, pOES, was constructed and used for markerless deletion of the gene RA0C_2053 which encode a putative TonB-dependent receptor in RA ATCC11845. The suicide plasmid pOES was also used to introduce a "knock-in" Myc-tag into the C-terminus of RA0C_1912 which encode a putative Fur protein. Using pheS* as a counterselectable marker, markerless mutagenesis and "knock-in" genetic manipulation techniques were also developed based on natural transformation. Furthermore, this marker was used to generate a point mutation in the RA0C_1912 gene of the RA ATCC11845 genome. The genetic methods developed in this study provide new and useful tools required to investigate the physiology and pathogenic mechanisms of this bacterium. These techniques may also have wider application in many other members of the Flavobacteria.

Microbial electrolysis cells (MECs) are an efficient technology for generating hydrogen gas from organic matters, but an additional voltage is needed to overcome the thermodynamic barrier of the reaction. A combined system of MEC and the aluminum-air battery (Al-air battery) was designed for hydrogen generation, coagulant production and operated in an energy self-sufficient mode. The Al-air battery (28 mL) produced a voltage ranged from 0.58 V to 0.80 V, which powered an MEC (28 mL) to produce hydrogen. The hydrogen production rate reached 0.19 ± 0.01 m3 H2/m3/d and 14.5 ± 0.9 mmol H2/g COD. The total COD removal rate was 85 ± 1%, of which MEC obtained 75 ± 1% COD removal and 10 ± 1% COD removal was achieved by in-situ coagulating process. The microorganisms removal of MEC effluent was 97 ± 2% through ex-situ coagulating process. These results showed that the Al-air battery-MEC system can be operated in energy self-sufficient mode and recovered energy from wastewater with high quality effluent.

Nickel-based hydroxide/bamboo fiber-derived carbon (BFC) composite (BFCNi), with an open and interconnected pore structure inherited from BFC, is constructed for the first time by redxo etching reaction based on MnO2 templates. Benefiting from the outstanding porous structure of BFC and high theoretical capacity of nickel-based hydroxide, BFCNi exhibits excellent advantages for electrochemical energy storage. Typically, in the three-electrode system, BFCNi delivers high specific capacitance of 1310 F g−1 at 1 A g−1 and superior rate capability (841 F g−1 at 15 A g−1). The energy storage mechanism of BFCNi is deeply studied, which reveals that the energy storage process of BFCNi combines surface control behavior and diffusion control behavior. Additionally, a coin-type asymmetric supercapacitor on the basic of BFCNi and BFC is assembled (BFCNi//BFC), which achieves an extraordinary energy density of 74.8 Wh kg−1 at a power density of 799.8 W kg−1, good cycle stability (87.5% capacitance retention after 5000 cycles) and satisfactory rate capability, indicating a great promise of BFCNi in electrochemical energy storage.

// Honglin Luo 1, 2, * , Huizan Yang 1, 3, * , Yong Lin 1, * , Yongde Zhang 1 , Chuanyan Pan 1 , Pengfei Feng 1 , Yanling Yu 1 and Xiaohan Chen 1 1 Guangxi Key Laboratory for Aquatic Genetic Breeding and Healthy Aquaculture, Guangxi Institute of Fishery Sciences, Nanning, P.R. China 2 Guangxi Medical University, Nanning, P.R. China 3 College of Animal Science and Technology, Guangxi University, Nanning, P.R. China * These authors have contributed equally to this work Correspondence to: Honglin Luo, email: 541365548@qq.com Xiaohan Chen, email: Chnxhn@163.com Keywords: long non-coding RNA; oreochromis niloticus ; macrophages; HSP70; Streptococcus agalactiae Received: June 22, 2017 Accepted: August 17, 2017 Published: September 30, 2017 ABSTRACT Objectives: To investigate the lncRNA profiling during tilapia peritoneal macrophages (TPMs) activation and discuss the relationship between lncRNA and mRNA. Materials and Methods: RNA sequencing was used to investigate the lncRNA and mRNA profiles of TPMs activation following stimulation with Streptococcus agalactiae (Sa) antigen, heat shock protein 70 (HSP70) and HSP70+Sa. The expressions of lncRNA and mRNA were confirmed by qPCR. 356 lncRNA, 10173 mRNA and 1782 transcripts of uncertain coding potential (TUCP) were differentially expressed by pairwise comparison. These lncRNAs were shorter in length, fewer in exon number and higher in expression levels as compared with mRNAs. 683 lncRNAs and 4320 mRNAs were co-located, while 316 lncRNAs and 9997 mRNAs were in co-expression networks. Seven mRNAs (ANKRD34A, FMODA, GJA3, CNTN5, BMP10, BAI2 and HS3ST6) were involved in both networks of LNC_00035 and LNC_000466. Differentially expressed genes were involved in signaling pathways, such as “phosphorylation”, “cytokine-cytokine receptor interaction”, “endocytosis” and “MHC protein complex”. LNC_000792, LNC_000215, LNC_000035 and LNC_000310, with cis and/or trans relationships with mRNAs, were also involved in ceRNA network. Conclusions: These results might represent the first identified expression profile of lncRNAs and mRNAs in tilapia macrophages activated by HSP70 and Sa.

The biologically pretreated cellulosic ethanol wastewater poses a serious environmental concern because of its refractory and color compounds. The decolorization of electrochemical oxidation using Sb doped Ti/SnO 2 electrode for advanced treatment of cellulosic ethanol wastewater under different current density (5–30 mA·cm −2 ), initial pH (3–8.9) and supporting electrolyte (0–0.25 M NaCl) was investigated in this study. Complete decolorization, 8.5% chemical oxygen demand (COD) and 69.1% dissolved organic carbon removal efficiencies were achieved under the optimal conditions (20 mA·cm −2 , pH 5 and supporting electrolyte of 0.1 M NaCl) after 150 min. The energy consumption required to meet National Discharge Standard (GB 27631-2011) is 93.8 kWh kg COD −1 . Further investigation revealed that hydroxyl radicals played a primary role in the degradation of organic contaminants, while active chlorine formed from chloride oxidation played a less important role. Direct anodic oxidation and indirect reaction via peroxodisulfate generated from sulfate oxidation could be negligible. The formation of chlorination by-products appeared to be low since the final total Trihalomethanes concentration detected was 263 µg L −1 , with the detection of chloroform as the main Trihalomethanes.

A coral-like polyaniline/activated biocarbon (PANI/AB) composites were prepared via in-site polymerization method. First, the activated biocarbon (AB) was derived from sugarcane bagasse, and then, the PANI/AB composites were obtained using AB as the scaffolds for PANI growth and [CMMIm]Cl ionic liquid as dopant. The results indicated that chemical composition and microstructure of PANI/AB were closely related to the mass ratio of aniline monomer (An) and AB, and which further impacted electrochemical performance of PANI/AB profoundly. Especially, the PANI/AB-4 (the mass ratio of An/AB is 40) exhibited a highest specific capacitance of 447 F g − 1 at a current density of 0.5 A g − 1 . Furthermore, the asymmetric two-electrode system based on PANI/AB-4 as positive electrode and AB as negative electrode is successfully assembled with a voltage window of 0–1.6 V, exhibiting high energy density (27.3 Wh kg − 1 ) and power density (800 W kg − 1 ), and excellent cycling stability (87% capacitance retention after 2000 cycles).

In this paper, MnO 2 /allium-giganteum-like biocarbon (KWB) composites (KWBM) have been prepared by a simple method. First, the biocarbon was derived from sugarcane bagasse by one-step carbonization and activation method, and then, MnO 2 nanosheets anchored on the surface of biocarbon and the KWBM was obtained. The analysis results demonstrated that chemical composition and microstructure of KWBM were closely related to the mass ratio of KMnO 4 /KWB, and which further impacted electrochemical performance of KWBM profoundly. Especially, the KWBM-4 (the mass ratio of KMnO 4 /KWB is 2) exhibited a highest specific capacitance of 402 F g − 1 at a current density of 1 A g − 1 in three-electrode system. The asymmetric two-electrode system with outstanding energy density was assembled by employing the KWBM-4 as the positive electrode and the KWB as the negative electrode. The two-electrode system displays a high energy density of 25.9 Wh kg − 1 at a power density of 750 W kg − 1 within a potential rage of 0–1.5 V. Furthermore, the system exhibited high cycle stability with only 5.8% loss of its initial capacitance after 2000 cycles.

Background/Aims: The induction of endothelial injury by hyperglycemia in diabetes has been widely accepted. Naringin is a bio-flavonoid. Some studies showed that naringin alleviates diabetic complications, but the exact mechanisms by which naringin improves diabetic anomalies are not yet fully understood. The aim of this research was to study the protective effect of naringin on high glucose-induced injury of human umbilical vein endothelial cells (HUVECs). Methods: HUVECs were cultured with or without high glucose in the absence or presence of naringin for 5 days. The expression of CX3CL1 was determined by quantitative real-time RT-PCR (qPCR) and western blot. The cellular bioenergetic analysis oxygen consumption rate (OCR) was measured with a Seahorse Bioscience XF analyzer. Results: The production of reactive oxygen species (ROS), the expression of CX3CL1 and the level of AKT phosphorylation were increased in HUVECs cultured with high glucose compared with controls. However, naringin rescued these increases in ROS production, CX3CL1 expression and AKT phosphorylation. Nitric oxide (NO) production and OCR were lower in the high glucose group, and naringin restored the changes induced by high glucose. Molecular docking results suggested that Naringin might interact with the CX3CL1 protein. Conclusion: Naringin protects HUVECs from high-glucose-induced damage through its antioxidant properties by downregulating CX3CL1 and by improving mitochondrial function.

To discuss the physical adsorption mechanism of the adsorption process of cellulase, a commercial enzyme cocktail sourced from Acremonium was immobilized in mesoporous silica with various pore sizes by pure physical adsorption in this study. Mesoporous silica materials with 17.6 nm and 3.8 nm pore sizes (hereafter referred to as MS-17.6 nm and MS-3.8 nm, respectively) were synthesized in the manner of a seeded-growth method. Other available mesoporous silica materials denoted as H-32 and diatomite were also used as sorbents. Then, the sorbents were characterized via small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), scanning electron microscopy (SEM), the Barrett–Emmett–Teller (BET) method and the Barrett–Joyner–Halenda (BJH) method to confirm their mesostructure. Furthermore, the adsorption abilities of different sorbents and enzymatic activities of immobilized cellulase were studied. The adsorption amounts exhibited a clear correlation with the pore size of the sorbents; i.e., the adsorption amount of MS-17.6 nm (410 mg g−1) with the pore size similar to the long axes of cellulase molecules was higher than that of MS-3.8 nm (315 mg g−1) with the pore size approximated to the short axes of cellulase (which was realized at 50 °C). Besides, the adsorption behavior of diatomite (with a pore size of about 200 nm) revealed a periodicity because the pore size was significantly larger than cellulase molecules. Moreover, the pore size was suggested to be a critical factor for the enzymatic activity of cellulase. When the average pore size of MS-3.8 nm just matched the short axes of cellulase molecules, immobilized cellulase preserved the active sites of cellulase intactly and showed the best activity (i.e. 63.3% of free cellulase activity at 50 °C). Consequently, the pore size of the sorbents had a significant influence on cellulase immobilization.

Timely diagnosis of the service condition of concrete dam is required to prevent the potential great loss of life and property in case of dam failure. Scientific and reliable diagnosis indexes are significant for effective dam diagnosis. Traditional dam diagnosis focused on displacement at a single point. Rational combined diagnosis based on multi-point displacement is rare. This paper presents a multi-block combined diagnosis method for concrete dam displacement. This method combines comprehensive block displacement and multidimensional confidence region method. The hybrid model, together with structure analysis, is selected to perform prediction. Comprehensive block displacement, a displacement system made up of single-point displacements with different weights, is introduced to describe block displacement. The weights are determined with improved projection pursuit method, which is adept in analyzing and processing high-dimensional data. Multi-block combined diagnosis indexes are then established in multidimensional space based on the residual sequences, and the obtained ellipse diagram can be a straightforward and visualized tool for dam diagnosis. The multi-block combined diagnosis method is recommended because of its high accuracy and reliability.

Background Goose parvovirus (GPV) is the etiological agent of Derzsy’s disease and is fatal for gosling. Research on the molecular basis of GPV pathogenicity has been hampered by the lack of a reliable reverse genetics system. At present, the GPV infectious clone has been rescued by transfection in the goose embryo, but the growth character of it is unclear in vitro. Methods In this study, we identified the full-length genome of GPV RC16 from the clinical sample, which was cloned into the pACYC177, generating the pIRC16. The recombinant virus (rGPV RC16) was rescued by the transfection of pIRC16 into goose embryo fibroblasts (GEFs). The rescued virus was characterized by whole genome sequencing, indirect immunofluorescence assays (IFA) and western blot (WB) using rabbit anti-GPV Rep polyclonal antibody as the primary antibody. Previously, we found the 164 K, 165 K, and 167 K residues in the 160YPVVKKPKLTEE171 are required for the nuclear import of VP1 (Chen S, Liu P, He Y, et al. Virology 519:17–22). According to that, the GPV infectious clones with mutated K164A, K165A, or K167A in VP1 were constructed, rescued and passaged. Results The rGPV RC16 has been successfully rescued by transfection of pIRC16 into the GEFs and can proliferate in vitro. Furthermore, the progeny virus produced by pIRC16 transfected cells was infectious in GEFs. Moreover, mutagenesis experiments showed that the rGPV RC16 with mutated 164 K, 165 K and 167 K in VP1 could not proliferate in GEFs based on the data of IFA and WB in parental virus and progeny virus. Conclusions The rGPV RC16 containing genetic maker and the progeny virus are infectious in GEFs. The 164 K, 165 K, and 167 K of VP1 are vital for the proliferation of rGPV RC16 in vitro.

Background Duck tembusu virus (DTMUV, genus Flaviviruses, family Flaviviridae) is an emerging flavivirus that can infect a wide range of cells and cell lines in vitro, though the initial step of virus invasion remains obscure. Methods In this study, drug treatments that including heparin, chondroitin sulfate, heparinase I, chondroitinase ABC and trypsin were applied to detect the influence of DTMUV absorption, subsequently, the copy number of viral genome RNA was analyzed by quantitative real-time PCR. The inhibition process of viral absorption or entry by heparin was determined by western blotting, and the cytotoxicity of drug treated cells was detected by cell counting kit-8. Results We found that the desulfation of glycosaminoglycans (GAGs) with sodium chlorate had a significant effect on the adsorption of DTMUV in both BHK21 and DEF cells. Based on this result, we incubated cells with a mixture of DTMUV and GAGs competition inhibitors or pre-treated cells with inhibitors, after incubation with the virus, the NS5 expression of DTMUV and viral titers were detected. The data suggested that heparin can significantly inhibit the absorption of DTMUV in a dose dependent manner but not at the step of viral entry in BHK21 and DEF cells. Meanwhile, heparinase I can significantly inhibit DTMUV attachment step. Conclusions Our results clearly proved that heparin sulfate plays an important role in the first step of DTMUV entry, viral attachment, in both BHK21 and DEF cells, which sheds light on the entry mechanism of DTMUV.

Background pUL21 is a conserved protein of Alphaherpesvirinae that performs multiple important functions. The C-terminus of pUL21 in other members of this subfamily has RNA-binding ability; this domain contributes to pseudorabies virus (PRV) retrograde axonal transport in vitro and in vivo and participates in newly replicated viral DNA packaging and intracellular virus transport. However, knowledge regarding duck enteritis virus (DEV) pUL21 is limited. Methods In our study, recombinant pUL21 was expressed using an pET-32c (+) vector in Escherichia coli BL21 cells induced with 0.4 mM isopropyl β-D-thiogalactoside for 8 h at 30°C. The antibody used for the indirect immunofluorescence (IFA) and western blotting (WB) analysis were prepared. Pharmacological inhibition, WB and quantitative reverse transcription PCR (RT-qPCR) were performed. A coimmunoprecipitation (CO-IP) assay was conducted to test the interaction between pUL21 and pUL16. Results We verified that DEV UL21 is a γ2 gene that encodes a structural protein. Moreover, we observed that pUL21 localized to the nucleus and cytoplasm. DEV pUL21 interacted with pUL16 and formed a complex in transfected human embryonic kidney (HEK) 293T cells and DEV-infected duck embryo fibroblasts (DEFs). These results were further confirmed by CO-IP assays. Conclusions The DEV UL21 gene is a late gene, and pUL21 localizes to the nucleus and cytoplasm. DEV UL21 is a virion component. In addition, pUL21 can interact with pUL16. These findings provide insight into the characteristics of UL21 and the interaction between pUL21 and its binding partner pUL16. Our study enhances the understanding of DEV pUL21. Keywords: Duck enteritis virus, UL21, UL16, late gene, interaction

The duck plague virus (DPV) US3 protein, a homolog of the herpes simplex virus-1 (HSV-1) US3 protein that is reported to be critical for viral replication, has been minimally studied. Therefore, to investigate the function of the DPV US3 protein, we used scarless Red recombination technology based on an infectious bacterial artificial chromosome (BAC) containing the DPV Chinese virulent strain (CHv) genome and successfully constructed and rescued a US3-deleted mutant and the corresponding revertant virus (BAC-CHv-ΔUS3 and BAC-CHv-ΔUS3R, respectively). For viral growth characteristics, compared to the parental and revertant viruses, the US3-deleted mutant showed an approximately 100-fold reduction in viral titers but no significant reduction in genome copies, indicating that the US3-deleted mutant exhibited decreased viral replication but not decreased viral DNA generation. In addition, the US3-deleted mutant formed viral plaques that were 33% smaller on average than those formed by the parental and revertant viruses, demonstrating that US3 protein affected the viral cell-to-cell spread of DPV. Finally, the results of electron microscopy showed that the deletion of US3 resulted in a large number of virions accumulating in the nucleus and perinuclear space, thus blocking virion nuclear egress. In this study, we found that the DPV US3 protein played pivotal roles in viral replication by promoting viral cell-to-cell spread and virion nuclear egress, which may provide some references for research on the function of the DPV US3 protein.

Background MicroRNAs (miRNAs) is increasingly recognized as an important element in regulating virus-host interactions. Our previous results showed that cellular miR-30a-5p was significantly downregulated after duck enteritis virus (DEV) infection cell. However, whehter or not the miR-30a-5p is involved in DEV infection has not been known. Methods Quantitative reverse-transcription PCR (qRT-PCR) was used to measure the expression levels of miRNAs(miR-30a-5p) and Beclin-1 mRNA. The miR-30a-5p - Beclin-1 target interactions were determined by Dual luciferase reporter assay (DLRA). Western blotting was utilized to analyze Beclin-1-mediated duck embryo fibroblast (DEF) cells autophagy activity. DEV titers were estimated by the median tissue culture infective dose (TCID50). Results The miR-30a-5p was significantly downregulated and the Beclin-1 mRNA was significantly upregulated in DEV-infected DEF cells. DLRA confirmed that miR-30a-5p directly targeted the 3′- UTR of the Beclin-1 gene. Overexpression of miR-30a-5p significantly reduced the expression level of Beclin-1protein (p P P Conclusions This study shows that miR-30a-5p can inhibit DEV replication through reducing autophagy by targeting Beclin-1. These findings suggest a new insight into virus-host interaction during DEV infection and provide a potential new antiviral therapeutic strategy against DEV infection.

Duck Tembusu virus (DTMUV), which is similar to other mosquito-borne flaviviruses that replicate well in most mammalian cells, is an emerging pathogenic flavivirus that has caused epidemics in egg-laying and breeding waterfowl. Immune organ defects and neurological dysfunction are the main clinical symptoms of DTMUV infection. Preinfection with DTMUV makes the virus impervious to later interferon (IFN) treatment, revealing that DTMUV has evolved some strategies to defend against host IFN-dependent antiviral responses. Immune inhibition was further confirmed by screening for DTMUV-encoded proteins, which suggested that NS2A significantly inhibited IFN-β and IFN-stimulated response element (ISRE) promoter activity in a dose-dependent manner and facilitated reinfection with duck plague virus (DPV). DTMUV NS2A was able to inhibit duck retinoic acid-inducible gene-I (RIG-I)-, and melanoma differentiation-associated gene 5 (MDA5)-, mitochondrial-localized adaptor molecules (MAVS)-, stimulator of interferon genes (STING)-, and TANK-binding kinase 1 (TBK1)-induced IFN-β transcription, but not duck TBK1- and interferon regulatory factor 7 (IRF7)-mediated effective phases of IFN response. Furthermore, we found that NS2A competed with duTBK1 in binding to duck STING (duSTING), impaired duSTING-duSTING binding, and reduced duTBK1 phosphorylation, leading to the subsequent inhibition of IFN production. Importantly, we first identified that the W164A, Y167A, and S361A mutations in duSTING significantly impaired the NS2A-duSTING interaction, which is important for NS2A-induced IFN-β inhibition. Hence, our data demonstrated that DTMUV NS2A disrupts duSTING-dependent antiviral cellular defenses by binding with duSTING, which provides a novel mechanism by which DTMUV subverts host innate immune responses. The potential interaction sites between NS2A and duSTING may be the targets of future novel antiviral therapies and vaccine development. IMPORTANCE Flavivirus infections are transmitted through mosquitos or ticks and lead to significant morbidity and mortality worldwide with a spectrum of manifestations. Infection with an emerging flavivirus, DTMUV, manifests with clinical symptoms that include lesions of the immune organs and neurological dysfunction, leading to heavy egg drop and causing serious harm to the duck industry in China, Thailand, Malaysia, and other Southeast Asian countries. Mosquito cells, bird cells, and mammalian cell lines are all susceptible to DTMUV infection. An in vivo study revealed that BALB/c mice and Kunming mice were susceptible to DTMUV after intracerebral inoculation. Moreover, there are no reports about DTMUV-related human disease, but antibodies against DTMUV and viral RNA were detected in serum samples of duck industry workers. This information implies that DTMUV has expanded its host range and may pose a threat to mammalian health. However, the pathogenesis of DTMUV is largely unclear. Our results show that NS2A strongly blocks the STING-induced signal transduction cascade by binding with STING, which subsequently blocks STING-STING binding and TBK1 phosphorylation. More importantly, the W164, Y167, or S361 residues in duSTING were identified as important interaction sites between STING and NS2A that are vital for NS2A-induced IFN production and effective phases of IFN response. Uncovering the mechanism by which DTMUV NS2A inhibits IFN in the cells of its natural hosts, ducks, will help us understand the role of NS2A in DTMUV pathogenicity.

Duck enteritis virus (DEV) can successfully evade the host innate immune responses and establish a lifelong latent infection in the infected host. However, the study about how DEV escapes host innate immunity is still deficient up to now. In this study, for the first time, we identified a viral protein VP16 by which DEV can obviously downregulate the production of IFN-β in duck embryo fibroblast (DEF). Our results showed that ectopic expression of VP16 decreased duck IFN-β (duIFN-β) promoter activation and significantly inhibited the mRNA transcription of IFN-β. Further study showed that VP16 can also obviously inhibit the mRNA transcription of interferon-stimulated genes (ISGs), such as myxovirus resistance protein (Mx) and interferon-induced oligoadenylate synthetase-like (OASL). Furthermore, we found that this anti-interferon activity of VP16 depended on its N-terminus (aa1-200). Coexpression analysis revealed that VP16 selectively blocked duIFN-β promoter activity at the duIRF7 level rather than duIRF1. Based on the results of coimmunoprecipitation analysis (co-IP) and indirect immunofluorescence assay (IFA), VP16 was able to bind to duck IRF7 (duIRF7) directly, but did not interact with duck IRF1 (duIRF1) in vitro.

Duck hepatitis A virus (DHAV) is prevalent worldwide and has caused significant economic losses. As the predominant serotype in China, DHAV-3 has become a major challenge to the local duck industry. Here the genetics and pathogenesis of a virulent DHAV-3 strain and its embryo-passaged strain were assessed. There were only two amino acid substitutions (Y164N in VP0 protein and L71I in 2C protein) introduced during the adaptation process. The pathogenicity of these strains was further evaluated in vivo. Clinical signs, gross pathology, and histopathological analysis showed that the embryo-passaged strain was attenuated. Meanwhile, the viral RNA loads were significantly lower in the liver tissues of the ducklings infected with the attenuated strain. As expected, infection with the virulent and attenuated strains led to the activation of different innate immune genes. We suspected that the loss of replication efficiency in ducklings was responsible for the attenuation phenotype of the embryo-passaged strain. In addition, different innate immune responses in the liver of ducklings were at least partly responsible for the differential infectivity phenotype. These findings provide new insights into the genetics and pathogenesis of DHAV-3, which may aid the development of new vaccines and the implementation of immunization strategies.

A colloidal gold strip (CGS) for detecting antibodies to duck plague virus (DPV) was developed. Colloidal gold-labeled DPV gI protein and goat anti-rabbit IgG were dispensed on a conjugate pad as tracers. The recombinant DPV gI protein and rabbit IgG were used as capture reagents at the test line and control line, respectively. The detection limit of this assay was 1:256. Additionally, the CGS did not react with antisera from other common duck diseases, only reacting with anti-DPV serum and yielding a specific and strong red signal. 123 serum samples were tested by CGS and enzyme-linked immunosorbent assay (ELISA), and the results showed good agreement. The CGS test results can be observed in 15 min with the naked eye, should be suitable for clinical testing and large-scale detection.

The access of wind turbines to the power grid will bring new challenges to the safe and stable operation of power systems. When the wind turbine adopts the virtual synchronous control strategy, it can match the characteristics of the traditional synchronous generator and solve the unstable operation problem caused by the lack of inertia. In this paper, the influence mechanism of virtual synchronous machine (VSM) model on voltage stability of power system is analyzed with bifurcation theory. The increase of the damping coefficient of the VSM and the reduction of the inertia time constant will be beneficial to the voltage stability, which can provide a basis for the wind power transmission limit.

Galleria mellonella larvae have been used as a host model to study interactions between pathogens and hosts for several years. However, whether the model is useful to interrogate Riemerella anatipestifer infection biology remained unknown. This study aimed to exploit the potential of G. mellonella larvae and reveal their limitations as a host model for R. anatipestifer infection. G. mellonella larvae were shown to be effective for virulence evaluations of different R. anatipestifer strains. Furthermore, the virulent strain R. anatipestifer CH-1 had a stronger ability to proliferate than the attenuated strain R. anatipestifer ATCC 11845 in both G. mellonella larvae and ducklings. Unconventionally it was shown that G. mellonella larvae cannot be used to evaluate the efficacy of antimicrobials and their combinations. Additionally, it was shown that certain virulence factors, such as OmpA (B739_0861), B739_1208, B739_1343, and Wza (B739_1124), were specific only for ducklings, suggesting that G. mellonella larvae must be cautiously used to identify virulence factors of R. anatipestifer. Evaluation of heme uptake-related virulence genes, such as tonB1 and tonB2, required preincubating the strains with hemoglobin before infection of G. mellonella larvae since R. anatipestifer cannot obtain a heme source from G. mellonella larvae. In conclusion, this study revealed the applicability and limitations of G. mellonella as a model with which to study the pathogen-host interaction, particularly in the context of R. anatipestifer infection.

The reduction of nitrogen oxide (DeNOx) from flue gas by microalgae is a promising technology that has attracted increasing attention. Because the water source is a major limitation of microalgae application in the DeNOx from flue gas, we investigated the feasibility of using domestic wastewater (WW) as a water source. As a result, a biomass accumulation rate of 0.27 ± 0.01 mg L−1 d−1 was achieved by Tetradesmus obliquus PF3 cultivated in WW for 8 d, and 30 mg L−1 of nitrate nitrogen was added to the WW to fulfill the nutrient requirements of the microalgae cells. The ammonium (NH4+) nitrogen present in WW exerted inhibitory effects on the removal of nitric oxide (NO), thereby leading to 8% decrease removal efficiency in comparison with that using clean water and nutrients (BG11 medium). However, these inhibitory effects disappeared following the exhaustion of NH4+ by T. obliquus PF3 after 1 d. To overcome the inhibition of NH4+ and to achieve a high NO removal efficiency, a strategy of connecting two reactors in series was presented. The removal efficiency of NO by the two series reactors reached up to 71.2 ± 2.9%, which was significantly higher than that obtained by a single reactor (43.1 ± 3.6%). In addition, 70.9 ± 4.8% of the supplied NO was fixed into microalgae cells in the two reactors, which was 1.75 times higher than that in the single reactor (40.6 ± 5.1%), thereby suggesting that connecting two reactors in series rendered effective recovery of NO from flue gas using WW as a water source. In this study, we provided an economically viable water source for the application of microalgae in the biological DeNOx from flue gases.

Background The picornaviral 3C protease mediates viral polyprotein maturation and multiple cleavages of host proteins to modulate viral translation and transcription. The 3C protease has been regarded as a valid target due to its structural similarity among different picornaviruses and minimal sequence similarity with host proteins; therefore, the development of potent inhibitors against the 3C protease as an antiviral drug is ongoing. Duck hepatitis A virus (DHAV) belongs to the Picornavidea family and is a major threat to the poultry industry. To date, little is known about the roles of the DHAV 3C protease plays during infection. Methods In this study, we compared the full-length DHAV 3C protein sequence with other 3C sequences to obtain an alignment for the construction of a phylogenetic tree. Then, we expressed and purified recombinant DHAV 3C protease in the BL21 expression system using nickel-NTA affinity chromatography. The optimization of the cleavage assay conditions and the kinetic analysis for DHAV 3C protease were done by in vitro cleavage assays with a fluorogenic peptide respectively. The inhibitory activity of rupintrivir against the DHAV 3C protease was further evaluated. The localization of the 3C protease in infected and transfected cells was determined using immunofluorescence and confocal microscopy. Results Under different expression conditions, the 3C protease was found to be highly expressed after induction with 1 mM IPTG at 16 °C for 10 h. We synthesized a fluorogenic peptide derived from the cleavage site of the DHAV polyprotein and evaluated the protease activity of the DHAV 3C protease for the first time. We used fluorimetric based kinetic analysis to determine kinetic parameters, and V max and K m values were determined to be 16.52 nmol/min and 50.78 μM, respectively. Rupintrivir was found to exhibit inhibitory activity against the DHAV 3C protease. Using polyclonal antibody and an indirect immunofluorescence microscopy assay (IFA), it was determined that the DHAV 3C protease was found in the nucleus during infection. In addition, the DHAV 3C protease can enter into the nucleus without the cooperation of viral proteins. Conclusions This is the first study to examine the activity of the DHAV 3C protease, and the activity of the DHAV 3C protease is temperature-, pH- and NaCl concentration- dependent. The DHAV 3C protease localizes throughout DHAV-infected cells and can enter into the nucleus in the absence of other viral proteins. The kinetic analysis was calculated, and the V max and K m values were 16.52 nmol/min and 50.78 μM, respectively, using the Lineweaver–Burk plot.

Tembusu virus (TMUV), a member of the genus flavivirus, primarily causes egg-drop syndrome in ducks and is associated with low disease mortality but high morbidity. The commercially available live vaccines for treating TMUV currently include the main WF100, HB, and FX2010-180P strains, and efficient treatment and/or preventative measures are still urgently needed. Capsid-targeted viral inactivation (CTVI) is a conceptually powerful new antiviral strategy that is based on two proteins from the capsid protein of a virus and a crucial effector molecule. The effector molecule can destroy the viral DNA/RNA or interfere with the proper folding of key viral proteins, while the capsid protein mainly plays a role in viral integration and assembly; the fusion proteins are incorporated into virions during packaging. This study aimed to explore the potential use of this strategy in duck TMUV. Our results revealed that these fusion proteins can be expressed in susceptible BHK21 cells without cytotoxicity and possess excellent Ca2+-dependent nuclease activity, and their expression is also detectable in DF-1 cells. Compared to those in the negative controls (BHK21 and BHK21/pcDNA3.1(+) cells), the numbers of viral RNA copies in TMUV-infected BHK21/Cap-SNase and BHK21/Cap-Linker-SNase cells were reduced by 48 h, and the effect of Cap-Linker-SNase was superior to that of Cap-SNase. As anticipated, these results suggest that these fusion proteins contribute to viral resistance to treatment. Thus, CTVI might be applicable for TMUV inhibition as a novel antiviral therapeutic candidate during viral infection.

Background: Duck plague virus (DPV) can induce apoptosis in duck embryo fibroblasts (DEFs) and in infected ducks, but the molecular mechanism of DPV-induced apoptosis remains unknown. Methods: We first used qRT-PCR and a Caspase-Glo assay to determine whether the caspase protein family plays an important role in DPV-induced apoptosis. Then, we used an intracellular ROS detection kit and the mitochondrial probe JC-1 to respectively detect ROS levels and mitochondrial membrane potential (MMP). Finally, flow cytometry was used to detect apoptosis and cell cycle progression. Results: In this study, the mRNA levels and enzymatic activities of caspase-3, caspase-7, caspase-8, and caspase-9 were significantly increased during DPV-induced apoptosis. The caspase inhibitors Z-DEVD-FMK, Z-LEHD-FMK, and Q-VD-OphA could inhibit DPV-induced apoptosis and promote viral replication. Subsequently, a significant decrease in MMP and an increase in the intracellular ROS levels were observed. Further study showed that pretreating infected cells with NAC (a ROS scavenger) decreased the intracellular ROS levels, increased the MMP, inhibited apoptosis, and promoted viral replication. Finally, we showed that DPV infection can cause cell cycle S-phase arrest. Conclusions: This study shows that DPV causes cell cycle S-phase arrest and leads to apoptosis through caspase activation and increased intracellular ROS levels. These findings may be useful for gaining an understanding of the pathogenesis of DPV and the apoptotic pathways induced by &alpha, herpesviruses.

Additional file 7: Table S7. Primers and plasmids used in this study

Riemerella anatipestifer is a gram-negative bacterium that mainly infects ducks, turkeys and other birds. In a previous study, we established a markerless mutation system based on the pheS mutant as a counterselectable marker. However, the toxic effect of p-Cl-Phe on the R. anatipestifer strain expressing the pheS mutant was weak on blood agar plates. In this study, we successfully obtained streptomycin-resistant derivative of R. anatipestifer ATCC11845 using 100 μg/mL streptomycin as a selection pressure. Then, we demonstrate that rpsL can be used as a counterselectable marker in the R. anatipestifer ATCC11845 rpsL mutant strain, namely, R. anatipestifer ATCCs. A suicide vector carrying wild-type rpsL, namely, pORS, was constructed and used for markerless deletion of the gene RA0C_1534, which encodes a putative sigma-70 family RNA polymerase sigma factor. Using rpsL as a counterselectable marker, markerless mutagenesis of RA0C_1534 was also performed based on natural transformation. R. anatipestifer ATCCsΔRA0C_1534 was more sensitive to H2O2-generated oxidative stress than R. anatipestifer ATCCs. Moreover, transcription of RA0C_1534 was upregulated under 10 mM H2O2 treatment and upon mutation of fur. These results suggest that RA0C_1534 is involved in oxidative stress response in R. anatipestifer. The markerless gene mutation method developed in this study provides new tools for investigation of the physiology and pathogenic mechanisms of this bacterium.

We report for the first time the occurrence of integrative conjugative elements (ICEs) in Riemerella anatipestifer (R.anatipestifer) isolated from diseased ducks in China. For this purpose, a total of 48 genome sequences were investigated, which comprised 30 publicly available R. anatipestifer genome sequences, and 18 clinical isolates genomes sequences. Two ICEs, named ICERanRCAD0133-1 and ICERanRCAD0179-1 following the classic nomenclature system, were identified in R. anatipestifer through the use of bioinformatics tools. Comparative analysis revealed that three ICEs in Ornithobacterium rhinotracheale showed a high degree of conservation with the core genes of ICERanRCAD0133-1, while 13 ICEs with high similarity to ICERanRCAD0179-1 were found in Bacteroidetes. Based on the definition of ICE family, ICERanRCAD0179-1 was grouped in CTnDOT/ERL family; however, ICERanRCAD0133-1, which had no significant similarity with known ICEs, might be classified into a novel ICE family. The sequences of ICERanRCAD0133-1 and ICERanRCAD0179-1 were 70890 bp and 49166 bp in length, had 33.14 and 50.34% GC content, and contained 77 CDSs and 51 CDSs, respectively. Cargo genes carried by these two ICEs were predicted to encode: R-M systems, IS elements, a putative TonB-dependent receptor, a bacteriocin/lantibiotic efflux ABC transporter, a tetracycline resistance gene and more. In addition, phylogenetic analyses revealed that ICERanRCAD0179-1 and related ICEs were derived from a common ancestor, which may have undergone divergence prior to integartation into the host bacterial chromosome, and that the core genes co-evolved via a related evolutionary process or experienced only a low degree of recombination events during spread from a common CTnDOT/ERL family ancestor. Collectively, this study is the first identification and characterization of ICEs in R. anatipestifer; and provides new insights into the genetic diversity, evolution, adaptation, antimicrobial resistance, and virulence of R. anatipestifer.

Riemerella anatipestifer is a gram-negative bacterium that leads to severe contagious septicemia in ducks, turkeys, chickens, and wild waterfowl. Here, a pan-genome with 32 R. anatipestifer genomes is re-established, and the mathematical model is calculated to evaluate the expansion of R. anatipestifer genomes, which were determined to be open. Average nucleotide identity (ANI) and phylogenetic analysis preliminarily clarify intraspecies variation and distance. Comparative genomic analysis of R. anatipestifer found that horizontal gene transfer events, which provide an expressway for the recruitment of novel functionalities and facilitate genetic diversity in microbial genomes, play a key role in the process of acquiring and transmitting antibiotic-resistance genes in R. anatipestifer. Furthermore, a new antibiotic-resistance gene cluster was identified in the same loci in 14 genomes. The uneven distribution of virulence factors was also confirmed by our results. Our study suggests that the ability to acquire foreign genes (such as antibiotic-resistance genes) increases the adaptability of R. anatipestifer, and the virulence genes with little mobility are highly conserved in R. anatipestifer.

Biocathode microbial electrochemical system (MES) is considered as a sustainable and eco-friendly technology for wastewater treatment. With domestic wastewater, the anode/cathode matching ratios of biocathode MES are optimized to match the anodic respiration and cathodic oxygen reduction rates. One anode module matched with two identical cathode modules obtains the highest power density. The long-term stability of MES is examined for one year with optimized architecture. Stable power generation and organic removal performance are achieved. A low effluent chemical oxygen demand (COD) is obtained at 36 ± 14 mg L−1 with a COD removal of 89 ± 4%. To meet the discharge standard in China (COD

The low-cost biocathode microbial electrochemical system (MES) with microbial separator (MS) is promising for the practical application in wastewater treatment. To evaluate the potential realizable advantages for future full-size MESs, four 2-Liters MES modules with MS are constructed and their energy conversion, biomass metabolism and pollution degradation are systematically investigated. The mass and energy flow for MES with raw domestic wastewater is built under maximum power point (Pmax), maximum current point (Imax), and open circuit (OC) modes. The chemical oxygen demand (COD) removal rates are 0.831 and 0.846 kg m−3 day−1 in Pmax and Imax modes, while that of OC is 0.609 kg m−3 day−1. The effluent COD concentrations at Pmax and Imax are stable at 41 ± 2 and 37 ± 2 mg L−1, while that of OC is up to 101 ± 4 mg L−1. The observed sludge yield is only 23.4% (Pmax) or 19.5% (Imax) of the OC mode with much higher sludge calorific values. For the raw wastewater treatment, the direct electricity harvest and reuse from MES units are unrealistic and uneconomical due to its low energy efficiency. But the high COD removal efficiency and low observed sludge yield are the realizable advantages for practical applicated MESs.