CeO2 Nanoparticle-Loaded MnO2 Nanoflowers for Selective Catalytic Reduction of NOx with NH3 at Low Temperatures.

CeO2 nanoparticle-loaded MnO2 nanoflowers, prepared by a hydrothermal method followed by an adsorption-calcination technique, were utilized for selective catalytic reduction (SCR) of NOx with NH3 at low temperatures. The effects of Ce/Mn ratio and thermal calcination temperature on the NH3-SCR activity of the CeO2-MnO2 nanocomposites were studied comprehensively. The as-prepared CeO2-MnO2 catalysts show high NOx reduction efficiency in the temperature range of 150-300 °C, with a complete NOx conversion at 200 °C for the optimal sample. The excellent NH3-SCR performance could be ascribed to high surface area, intimate contact, and strong synergistic interaction between CeO2 nanoparticles and MnO2 nanoflowers of the well-designed composite catalyst. The in situ diffuse reflectance infrared Fourier transform spectroscopy (DRIFTs) characterizations evidence that the SCR reaction on the surface of the CeO2-MnO2 nanocomposites mainly follows the Langmuir-Hinshelwood (L-H) mechanism. Our work provides useful guidance for the development of composite oxide-based low temperature NH3-SCR catalysts.

LAMP real-time turbidity detection for fowl adenovirus.

BACKGROUND: Fowl adenovirus (FAdV) is an infectious agent that can cause jaundice, severe anaemia, dyspnoea and reproductive disorders in fowls. Since 2015, FAdV disease has been rapidly spreading among broiler chickens in China, where it has caused significant economic losses. In this study, a loop-mediated isothermal amplification (LAMP) real-time turbidity method with strong specificity to FAdV was established. RESULTS: The established assay was specific to FAdV-4, and the lower limit of detection was 75 copies/µL of extracted DNA. The positive detection rate for the suspected tissue samples was 33.3% (14/42), which was consistent with that of the real-time PCR. CONCLUSION: The proposed LAMP method can quickly and accurately detect prevalent FAdV via real-time turbidity assay, thereby providing a diagnostic platform for the prevention and control of the FAdV disease.

Isolation and genetic characterization of avian influenza virus H4N6 from ducks in China.

An avian influenza virus (AIV) strain belonging to the H4 subtype and provisionally designated as A/duck/China/J1/2012(H4N6) was isolated from diseased ducks with respiratory disease at a commercial poultry farm in Shandong, China, in 2012. The genomic coding sequences of all eight segments of this J1 isolate were determined and used for subsequent analysis. Phylogenetic analysis of all eight segments showed that this duck H4N6 virus was of Eurasian lineage and not American lineage. The results show that the virus probably emerged because of a reassortment event involving other avian H4N6 and H6N1 viruses. Interestingly, this H4N6 virus had all the conserved features common to low-pathogenic AIVs, including the HA cleavage sequence, receptor-binding sequences for the 2,3-linked sialic acid receptor in avian species, and the PB2 627E motif. These results suggest that the duck H4N6 isolate could not cross the species barrier to infect and replicate in mammals, including humans. In addition, screening of the duck serum samples showed that only 0.57 % (2/352) of the individuals had weak but measurable hemagglutination inhibition (HI) antibody titers. The low antibody prevalence data were also supported by the failure to detect H4N6 virus (0/56) in clinical nasal swabs of the ducks. These data indicate an alternate reservoir for the H4N6 virus.

[Evolution of non-coding nucleotide sequences in Newcastle disease virus genomes ].

[OBJECTIVE] Although much is done in the coding genes of Newcastle disease virus (NDV) , limited papers can be found with non-coding sequences. In this paper, the evolution tendency of non-coding sequences was studied. [METHODS] NDV strain LC12 isolated from duck with egg drop syndrome in 2012, and others 35 strains genome cDNA of different NDV genotype were sought and obtained from GenBank. Analytical approaches including nucleotide homology, nucleotide alignment and phylogenetic tree were associated with the leading sequences, trailer sequences, intergenic sequences (IGS), and coding gene between 5 'and 3' UTR nucleotide, respectively. [RESULTS] The location and the length of the non-coding sequences highly conserve, and the variation trend of non-coding sequences is synchronous with the entire genomes and coding genes. [ CONCLUSION] The molecular variation of the coding gene was indistinguishable with the non-coding gene in view of the NDV genome.

Surveillance and molecular characterization of Newcastle disease virus in seafowl from coastal areas of China in 2011.

Four Newcastle disease virus (NDV) isolates were obtained from 997 fecal and tissue samples were collected in 2011 from seafowl that included seagull, sea duck, and swan from the coastal areas of Guangdong, Jiangsu, and Shandong in China. These isolates (SD1, SD2, GD1, and JS1) were characterized for their pathogenicity according to their mean death time, intracerebral pathogenicity index and intravenous pathogenicity index. Full-length fusion protein genes containing the cleavage site were sequenced, and amino-acid sequences around the cleavage site were deduced. One isolate (SD2) was virulent to poultry as indicated by its mean death time, intracerebral pathogenicity index, and fusion gene cleavage site sequence, which was specific for virulent NDV ((112)R-R-Q-K-R-F(117)). The phylogenic analysis indicated that three of the isolates (SD1, GD1, and JS1) belonged to genotype II and the virulent isolate (SD2) belonged to genotype VIId. These findings suggest that some seafowl NDVs in the coastal areas of China have different virulences and molecular characterizations, and these NDVs have some similarity with vaccine- or poultry-adapted isolates.

Genetic and biological characterizations of a Newcastle disease virus from swine in China.

BACKGROUND: Newcastle Disease Virus (NDV) has been considered to only infect avian species. However, one paramyxovirus named as Xiny10 was isolated from swine. The differences of Xiny10, another previous swine NDV (JL01) and vaccine strain La Sota were compared on the basis of sequences of the whole-lengthen Fusion (F) gene and biological characteristics. FINDINGS: Through serologic tests and sequence alignment, Xiny10 was proved as NDV. It has great differences with JL01 in virulence, biological characteristics, genotype and amino acid homology of F gene. The sequence alignment showed Xiny10 and La Sota both belonged to genotype II. It shared 97.3% to 98.7% identities with genotype II NDVs, which was higher than these strains from the other genotypes. CONCLUSIONS: These above data suggested that the swine virus was NDV and it might be generated from La Sota.

Rapid detection of newly isolated Tembusu-related Flavivirus by reverse-transcription loop-mediated isothermal amplification assay.

BACKGROUND: From April 2010 to January 2011, a severe new viral disease had devastated most duck-farming regions in China. This disease affected not only laying ducks but also meat ducks, causing huge economic losses for the poultry industry. The objective of this study is to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of the new virus related to Tembusu-related Flavivirus. RESULTS: The RT-LAMP assay is very simple and rapid, and the amplification can be completed within 50 min under isothermal conditions at 63°C by a set of 6 primers targeting the E gene based on the sequences analysis of the newly isolated viruses and other closely related Flavivirus.The monitoring of gene amplification can also be visualized by using SYBR green I fluorescent dye. In addition, the RT-LAMP assay for newly isolated Tembusu-related Flavivirus showed higher sensitivity with an RNA detection-limit of 2 copies/µL compared with 190 copies/µL of the conventional RT-PCR method. The specificity was identified without cross reaction to other common avian pathogens. By screening a panel of clinical samples this method was more feasible in clinical settings and there was higher positive coincidence rate than conventional RT-PCR and virus isolation. CONCLUSION: The RT-LAMP assay for newly isolated Tembusu-related Flavivirus is a valuable tool for the rapid and real-time detection not only in well-equipped laboratories but also in general conditions.

The Novel Tumor Suppressor Gene ZNF24 Induces THCA Cells Senescence by Regulating Wnt Signaling Pathway, Resulting in Inhibition of THCA Tumorigenesis and Invasion.

OBJECT: Clinically, the effective treatment options available to thyroid cancer (THCA) patients are very limited. Elucidating the features of tumor suppressor genes (TSGs) and the corresponding signal transduction cascade may provide clues for the development of new strategies for targeted therapy of THCA. Therefore, this paper aims to explore the mechanism of ZNF24 underlying promoting THCA cell senescence at molecular level. METHODS: We performed RT-PCR and Western Blotting for evaluating associated RNA and protein expression. CCK8, colony forming, wound healing and Transwell chamber assays were conducted to examine THCA cell proliferation, invasion and migration. ß-galactosidase staining assay was performed to detect THCA cells senescence. The size and volume of xenotransplanted tumors in nude mice are calculated to asses ZNF24 effect in vivo. RESULTS: Ectopic expression of ZNF24 significantly inhibited the cell viability, colony forming, migration and invasion abilities of THCA cell lines (K1/GLAG-66i and BCPAPi) (P < 0.05). ZNF24 induced BCPAPi cells senescence through regulating Wnt signaling pathway. ZNF24 inhibited Wnt signaling pathway activition by competitively binding ß-catenin from LEF1/TCF1-ß-catenin complex. In nude mice, both Ectopic expression of ZNF24 and 2,4-Da (the strong ß-catenin/Tcf-4 inhibitor) treatment significantly decreased both the size and weight of xenotransplanted tumors when compared with control mice (P < 0.05). CONCLUSION: Results obtained in vivo and in vitro reveal the role of ZNF24 in significantly suppressing THCA tumorigenesis and invasion by regulating Wnt signaling pathway.

Identification, Pathogenicity of Novel Fowl Adenovirus Serotype 4 SDJN0105 in Shandong, China and Immunoprotective Evaluation of the Newly Developed Inactivated Oil-emulsion FAdV-4 Vaccine.

Since mid-2015, numerous outbreaks of hydropericardium-hepatitis syndrome (HHS), which is caused by a novel fowl adenovirus serotype 4 (FAdV-4), have been reported in chickens in parts of China, thereby causing huge economic losses to the poultry industry. Thus, an effective vaccine to control the further spread of infections with this hyper-virulent FAdV-4 is imperative. In this study, we isolated a novel FAdV-4 strain SDJN0105 from a broiler farm with HHS disease in Shandong Province. Pathogenicity was evaluated by the observation of clinical symptoms, necropsy changes, and pathological tissue sections after oral and intramuscular (IM) infection of Specific pathogen free (SPF) chickens. The chickens infected by IM injection all died within three days, and chickens infected via the oculonasal route died within five days post-infection (dpi). Histopathological examination revealed that the pathology was confined to heart, liver, spleen, lung, kidney, and particularly the liver. Irrespective of the inoculation route, the highest viral DNA copy numbers were detected in the livers of infected chickens. The mRNA expression levels of IL-1ß, IL-6, IL-8, IFNs, TNF-α, Mx, and OASL were significantly upregulated during the viral infection. In addition, an inactivated oil-emulsion FAdV-4 vaccine was developed. The vaccine could provide full protection for SPF chickens against a lethal dose of the FAdV-4 strain SDJN0105 and a high level of antibodies. These results improve our understanding of the innate immune responses in chickens infected with FAdV-4 and the pathogenesis of FAdV-4 caused by host factors, and the developed FAdV-4 vaccine is promising as a drug candidate for the prevention and reduction of the spread of HHS in poultry in China.

Genome analysis of newly emerging goose-origin nephrotic astrovirus in China reveals it belongs to a novel genetically distinct astrovirus.

Since 2017, a new type of goose-origin astrovirus (GoAstV) disease occurred in China. This disease can cause joint swelling of sick geese, and the anatomy shows a clear urate precipitation in the viscera. The rate of death or amputation can reach more than 30%, revealing its severe pathogenicity. One novel goose-origin astrovirus strain, designated as CXZ18, was isolated from diseased geese with a fatal infection characterized by visceral urate deposition. Similar clinical anatomy symptoms were partially reproduced by attacking infection of healthy geese. The CXZ18 has no hemagglutination with chicken erythrocyte, only reproduced in goose embryos, not in SPF chicken or duck embryos. The complete genome-encoded three open reading frames (ORFs) of CXZ18 were 7252 nt in length. BLAST-based homology analysis of viral complete genome showed that CXZ18 has only 53.0%-61.8% with other classic avian astrovirus from various hosts. Further analysis of ORF 1a, ORF 1b, and ORF 2 genes revealed that the isolate was genetically distinct from known astroviruses and belonged to a distinctive branch of avian astroviruses. To conclude, a naturally occurring novel nephrotic astrovirus, distinguished with all previously reported avian astroviruses, was derived from goose.

Genetic characterization of one duck-origin paramyxovirus type 4 strain in China.

Avian paramyxovirus type 4 (APMV-4) has been frequently reported from wildfowl and waterfowl in recent year. However, few studies have reported on the molecular characteristics and regional transmission of APMV-4, knowledge of which is important for understanding the genetic diversity and epidemiology of avian paramyxovirus. Herein, we report the isolation of one APMV-4 strain, designated as QY17, from the duck in eastern China. The determined complete genome of the isolate with six gene segments 3'-N-P-M-F-HN-L-5' was 15,054 nt in length. Genetic analysis of the whole-fusion gene of this isolate showed that QY17 was derived from a Eurasian lineage. Further phylogenetic analysis showed that the duck-origin strain QY17 had a highly genetic relationship with representative APMV-4 strains from wildfowl in neighbouring regions. These genetic results suggested that APMV-4 viral exchange may occur in wildfowl and poultry via wild bird migration.

Pathotypical characterization and molecular epidemiology of Newcastle disease virus isolates from different hosts in China from 1996 to 2005.

Thirty Newcastle disease virus (NDV) strains isolated from outbreaks in China during 1996 to 2005 were characterized pathotypically and genotypically. All strains except one were velogenic. An analysis of the variable region (nucleotides 47 to 420) of the F gene indicated that 6 isolates belonged to genotype II, 3 to genotype III, 1 (isolated from a pigeon) to genotype VI, and 20 to genotype VII. Isolates belonging to genotype VII were further divided into five subtypes, VIIa, VIIb, VIIc, VIId, and VIIe, and subtype VIId was made up of VIId1 to VIId5. These results showed that genotype VII isolates might have been the most prevalent in China during the past two decades. Genotype VII isolates shared high homology, but the homology was less than that between genotype VII viruses and the vaccine virus LaSota. Among these NDV isolates, 25 isolates had the velogenic motif (112)R/K-R-Q-K/R-R-F(117) that is consistent with results of the biological tests. However, four of five LaSota-type isolates that contained the lentogenic motif (112)G-R-Q-G-R-L(117) were velogenic, except SY/03, in the view of the biological test. The majority of genotype VII isolates had lost one or two N-glycosylation sites. Finally, a cross-protection experiment in which specific-pathogen-free chickens vaccinated with LaSota were challenged by six NDV isolates showed that more than three isolates were antigenic variants that could be responsible for recent outbreaks of Newcastle disease.

Molecular characterization of three new virulent Newcastle disease virus variants isolated in China.

Three cases of Newcastle disease virus (NDV) found in nature had the lentogenic motif (112)G-R-Q-G-R-L(117) in their fusion protein cleavage sites. However, both intracerebral pathogenicity and intravenous pathogenicity indexes showed that these NDV isolates were virulent. In comparison with the LaSota live virus vaccine, these viruses had significant genetic variations in the hemagglutinin-neuraminidase gene.

[Correlation of the neutralization index in chicken embryo with the homologies of F and HN gene of different Newcastle-disease isolates].

Thirteen prevailed Newcastle-disease viruses (NDV) isolated in China during 2001-2004 were purified by chick embryo fibroblast (CEF) plaque assay and characterized pathotypically and genotypically. The biological tests showed that these viruses were highly virulent. Sequence analysis based on the variable region (nucleotide 47-420) of the F gene indicated that of the 13 NDV isolates 2 belonged to genotype II, 2 to genotype IX and 9 to genotype VII. Isolates with genotype VII shared 94.6%-99.3% nucleotide (nt) homology with the F gene, whereas for genotype VII and La Sota was only 82.7%-84.1%. In addition, these NDV isolates all shared 95.2%-100% nt homology with the hemagglutinin-neuraminidase (HN) gene, whereas only 79.1%-84.3% compared these viruses with La Sota. The cross neutralization assays were done using positive serums in specific pathogen free (SPF) chicken embryos respectively. Correlation of the neutralization index in chicken embryo with the homologies of F and HN gene of different NDV isolates were analyzed by SPSS8.0 software. The result showed that the neutralization index was closely correlated with nt sequence (P < 0.01, r = 0.35) or deduced amino acid sequence (P < 0.01, r = 0.34) of the HN gene, whereas weekly correlated (P < 0.05, r = 0.20 or 0.19) with the F gene, and non-correlated with 374 nt segment. This implied that the genetic mutations of HN resulted in antigenic variations of these viruses and the search for new vaccines would be necessary.

Establishment and Application of Rapid Diagnosis for Reverse Transcription-Quantitative PCR of Newly Emerging Goose-Origin Nephrotic Astrovirus in China.

In 2017, a new type of goose-origin astrovirus (GoAstV) that is completely different from previously identified avian astroviruses (which have only 30.0% to 50.5% homology with GoAstV) has been isolated from diseased geese in China. This disease can cause joint swelling in sick geese, and the anatomy shows a clear precipitation of urate in the kidney. The rate of death and culling can reach more than 30%, revealing the disease's severe pathogenicity. To quickly and accurately diagnose the newly emerging disease, we established a highly specific reverse transcription-quantitative PCR (RT-qPCR) method of detecting GoAstV. Sensitivity testing showed that the minimum amount of test sample for this method is 52.5 copies/µl. Clinical application confirmed that this method can quickly and effectively detect GoAstV, providing a diagnostic platform for the prevention and control of goose disease.IMPORTANCE Goose-origin astrovirus (GoAstV), as a newly emerging virus in 2017, is different from previously known astroviruses in the genus Avastrovirus So far, few studies have focused on the novel virus. Considering the infectious development of astrovirus (AstV), we established a reverse transcription-quantitative PCR (RT-qPCR) assay with a strong specificity to quickly and accurately diagnose GoAstV. Confirmed by clinical application, this method can quickly and accurately detect prevalent GoAstV. The assay is thus convenient for clinical operation and is applicable to the monitoring of GoAstV disease.

[Study on immune efficacy of Newcastle disease chitosan microsphere vaccine].

Newcastle disease is an acute and highly contagious disease caused by Newcastle disease virus (NDV), one of which does great harms to the poultry industry. The most basic measure of controlling New Castle disease is to alid vaccine, now we usually use La Sota live vaccine and inactivated NDV vaccine, but these two vaccines both have more or less limitation. It can produce higher mucosal immunity titers by taking vaccine orally, meanwhile it can induce humoral and cell-mediated immune response and mucosal immunity strongly. Therefore, it becomes the focus of the research, which prepare new pattern vaccines taking orally. NDV chitosan microsphere vaccine was prepared using chitosan as capsule wall material, NDV as core material, glutaraldehyde as cross-linking material, and its even particle diameter was 5.83um, and its surface was smooth and glossy, no obviously pore space, yellow brown pykno-ball, and its safety and potency were evaluated. The SPF chickens were immunized with NDV chitosan microsphere vaccine, La Sota live vaccine and inactivated NDV vaccine respectively. To evaluate vaccine's immune efficacy, using MTT to measure lymphocytes proliferation in vitro, using HI to measure serum special IgG and using ELISA tests to detect mucosal sIgA titers. The results show that NDV chitosan microsphere vaccine was safe, could induce humoral and cell-mediated immune response and mucosal immunity strongly. The results of the potency tests conformed that the vaccine could produce good protective effect.

[Newcastle disease virus heredity mutation and correlation of HN and F gene].

Prevailed Newcastle disease virus isolates were collected during 1999-2005 in China. These isolates were purified by CEF plaque assay and replicated in SPF embryos. The fusion protein (F) gene and hemagglutinin-neuraminidase (HN) gene of these isolated viruses were cloned and sequenced. Some of the F gene and HN gene sequences from GenBank were also used in this study. The homologies of nucleotide and amino acids were compared and correlations were analyzed by SPSS8.0 software among different length sequences of the F gene or HN gene. The nucleotide homologies and correlation among the F gene and HN gene were also analyzed. The results indicated there are good correlation among different length sequences of the F gene or HN gene and the F genome or HN genome (r > or = 0.973). There was also good correlations among different length amino acids of NDV F protein or HN protein (0.911< or = r < or = 0.968). But, there was only a less correlation between the whole F gene and HN gene (r = 0.312). The heredity mutation of HN genes had the character of geographical areas. The sequences of HN gene in Chinese isolates had an identity of more than 97%. But there was only 79.2% - 80.7% in HN nucleotide homology among the Chinese isolates and La Sota (vaccine).

H9N2 avian influenza virus-derived natural reassortant H5N2 virus in swan containing the hemagglutinin segment from Eurasian H5 avian influenza virus with an in-frame deletion of four basic residues in the polybasic hemagglutinin cleavage site.

We isolated a novel H5N2 avian influenza virus from swans in China. The virus was derived from a widespread H9N2 avian influenza virus but acquired the hemagglutinin gene from Eurasian H5 subtype with a naturally occurring in-frame deletion of four basic residues in the polybasic hemagglutinin cleavage site.

The pathogenicity of novel duck reovirus in Cherry Valley ducks.

The novel duck reovirus (NDRV) is an emerging, contagious infection. To better realize the pathogenic mechanism of NDRV in ducks, an infection experiment was conducted. The resulting data demonstrated that typical gross lesions were observed in the infected ducks. NDRV was able to replicate in various tissues, leading to these pathological lesions, especially on the liver and spleen. Real-time quantitative PCR showed that the expression of most innate immune-related genes was up-regulated and the antiviral innate immune response could be established in both the liver and spleen. This study indicates that NDRV is a pantropic virus. To resist viral infection, several pathogen recognition receptors can cooperatively recognize NDRV and initiate innate immunity, but the responses are different between different tissues. As far as we know, this is the first systematic investigation of the pathogenicity of NDRV in Cherry Valley ducks based on the host's innate immunity, and these data will provide new insights into the further study of the disease.

Genomic and phylogenetic characterization of novel, recombinant H5N2 avian influenza virus strains isolated from vaccinated chickens with clinical symptoms in China.

Infection of poultry with diverse lineages of H5N2 avian influenza viruses has been documented for over three decades in different parts of the world, with limited outbreaks caused by this highly pathogenic avian influenza virus. In the present study, three avian H5N2 influenza viruses, A/chicken/Shijiazhuang/1209/2013, A/chicken/Chiping/0321/2014, and A/chicken/Laiwu/0313/2014, were isolated from chickens with clinical symptoms of avian influenza. Complete genomic and phylogenetic analyses demonstrated that all three isolates are novel recombinant viruses with hemagglutinin (HA) and matrix (M) genes derived from H5N1, and remaining genes derived from H9N2-like viruses. The HA cleavage motif in all three strains (PQIEGRRRKR/GL) is characteristic of a highly pathogenic avian influenza virus strain. These results indicate the occurrence of H5N2 recombination and highlight the importance of continued surveillance of the H5N2 subtype virus and reformulation of vaccine strains.