Hongning Wang

Complete genome sequence and recombination analysis of infectious bronchitis virus attenuated vaccine strain H120

The strain H120 of infectious bronchitis virus (IBV) is one of the earliest and representative attenuated live Infectious Bronchitis vaccine strains. To investigate the genomic feature of H120 and further understand its role in the epidemiology of IBV, complete genome of H120 was sequenced and compared with sequences of other IBV strains by phylogenetic and recombination analysis. The complete genome of H120 is 27631 nucleotides in length and has a similar structure with that of Beaudette strain. We found that strain ZJ971 is probably a virulence revertant of H120. Nine amino acids changes and a three-nucleotide deletion were identified in ZJ971. Besides, potential recombination events associated with H120 were found in five IBV strains including H52, KQ6, SAIBK, Ark DPI 11, and Ark DPI 101. This study suggested that H120 might have contributed to the emergence of new IBV variants through both virulence reversion and recombination.

Improving Phytase Enzyme Activity in a Recombinant phyA Mutant Phytase from Aspergillus niger N25 by Error-Prone PCR

The mutant acid phytase (phyAm) gene was modified by random mutagenesis to improve enzymatic activity by using an error-prone PCR (ep-PCR) strategy. The mutated gene was linearized and inserted into plasmid vector pPIC9K and transformed by electroporation into Pichia pastoris GS115. A single transformant, PP-NPep-6A, showing the strongest phytase activity from among the 5,500 transformants, was selected for detailed analyses. Southern blot analysis of the mutant yeast transformant showed that phyAep gene was integrated into the chromosome genome through single crossover with one copy of phyA. The kinetic parameters indicated that the mutant one showed 61% higher specific activity and 53% lower km value than that of PP-NPm-8 (Pu2009<u20090.05). In addition, the overall catalytic efficiency (kcat/km) of the mutant one was 84% higher (Pu2009<u20090.05) than that of PP-NPm-8. Nine bases were altered in the mutant sequences, which resulted in three amino acid changes, namely, Glu156Gly, Thr236Ala, and Gln396Arg. The structural predictions indicated that the mutations generated by ep-PCR somehow reorganized or remodeled the active site, which could lead to increasing catalytic efficiency.

Phenotypic and genotypic characterisation of antimicrobial resistance in faecal bacteria from 30 Giant pandas

To study the prevalence of antimicrobial resistance in faecal bacteria from Giant pandas in China, 59 isolates were recovered from faecal pats of 30 Giant pandas. Antimicrobial susceptibility testing of the isolates was performed by the standardised disk diffusion method (Kirby-Bauer). Of the 59 study isolates, 32.20% were resistant to at least one antimicrobial and 16.95% showed multidrug-resistant phenotypes. Thirteen drug resistance genes [aph(3)-IIa, aac(6)-Ib, ant(3)-Ia, aac(3)-IIa, sul1, sul2, sul3, tetA, tetC, tetM, cat1, floR and cmlA] were analysed using four primer sets by multiplex polymerase chain reaction (PCR). The detection frequency of the aph(3)-IIa gene was the highest (10.17%), followed by cmlA (8.47%). The genes aac(6)-Ib, sul2 and tetA were not detected. PCR products were confirmed by DNA sequence analysis. The results revealed that multidrug resistance was widely present in bacteria isolated from Giant pandas.

Molecular detection of Torque teno virus in different breeds of swine

BackgroundTorque teno virus (TTV), of the Anelloviridae family, Iotatorquevirus genus, is a non-enveloped, single-stranded, and negative sense DNA (ssDNA) virus infecting human and many domestic animals including swines. Very little information is known about the investigations of TTV prevalence in different swine breeds so far.MethodsIn this study, 208 serum samples collected from seven swine breeds (Rongchang pig, Chenghua pig, Zibet pig, Wild boar, Duroc, Landrace, Large Yorkshire) from two independent farms were detected to determine the prevalence of two swine TTV genogroups, TTV1 and TTV 2, by nested polymerase chain reaction methods, and to analyse prevalence difference among these breeds.ResultsThe results showed that the prevalence of TTV in the seven breeds was 92%-100%. No significant difference (p > 0.05) in TTV infection was observed between different breeds. Interestingly, significantly higher prevalence for TTV1 in Rongchang boars (90%) and for TTV2 in Rongchang sows (95%) were detected, while co-infection rate (43.8%) was lower than other breeds. Sequence analysis showed that the homology of TTV1 and TTV2 were over 90.9% and 86.4% in these breeds, respectively.ConclusionsThe results indicated that TTV was widely distributed in the seven swine breeds. The prevalence of both TTV genogroups associated with swine breeds and genders. This study also respented the first description of swine TTV prevalence in different swine breeds. It was vitally necessary to further study swine TTV pathogenicity.

Evaluation and Target Validation of Indole Derivatives as Inhibitors of the AcrAB-TolC Efflux Pump

Indole derivatives 3-amino-6-carboxyl-indole and 3-nitro-6-amino-indole were designed and synthesized based on the TolC structure. They proved to have potent synergistic antibacterial effects on chloramphenicol, tetracycline, erythromycin, and ciprofloxacin against Escherichia coli YD2 and FJ307 with decreased minimal inhibitory concentrations (MICs) at 2–64 folds. To research its functional site, Escherichia coli BL21(DE3)−3 expressing a target-site mutated TolC was constructed by red homologous recombination and the site-directed mutagenesis technique. They did not noticeably affect antimicrobial activity against BL21(DE3)−3. All the results indicate that these compounds match our design and can be developed as efflux pump inhibitors for the AcrAB-TolC efflux pump.

Expression, purification and characterization of a phyA(m)-phyCs fusion phytase.

The phyAm gene encoding acid phytase and optimized neutral phytase phyCs gene were inserted into expression vector pPIC9K in correct orientation and transformed into Pichia pastoris in order to expand the pH profile of phytase and decrease the cost of production. The fusion phytase phyAm-phyCs gene was successfully overexpressed in P. pastoris as an active and extracellular phytase. The yield of total extracellular fusion phytase activity is (25.4±0.53) U/ml at the flask scale and (159.1±2.92) U/ml for high cell-density fermentation, respectively. Purified fusion phytase exhibits an optimal temperature at 55 °C and an optimal pH at 5.5∼6.0 and its relative activity remains at a relatively high level of above 70% in the range of pH 2.0 to 7.0. About 51% to 63% of its original activity remains after incubation at 75 °C to 95 °C for 10 min. Due to heavy glycosylation, the expressed fusion phytase shows a broad and diffuse band in SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). After deglycosylation by endoglycosidase H (EndoHf), the enzyme has an apparent molecular size of 95 kDa. The characterization of the fusion phytase was compared with those of phyCs and phyAm.

Isolation, identification and complete genome sequence analysis of a strain of foot-and-mouth disease virus serotype Asia1 from pigs in southwest of China

BackgroudFoot-and-mouth disease virus (FMDV) serotype Asia1 generally infects cattle and sheep, while its infection of pigs is rarely reported. In 2005-2007, FMD outbreaks caused by Asia1 type occurred in many regions of China, as well as some parts of East Asia countries. During the outbreaks, there was not any report that pigs were found to be clinically infected.ResultsIn this study, a strain of FMDV that isolated from pigs was identified as serotype Asia1, and designated as Asia1/WHN/CHA/06. To investigate the genomic feature of the strain, complete genome of Asia1/WHN/CHA/06 was sequenced and compared with sequences of other FMDVs by phylogenetic and recombination analysis. The complete genome of Asia1/WHN/CHA/06 was 8161 nucleotides (nt) in length, and was closer to JS/CHA/05 than to all other strains. Potential recombination events associated with Asia1/WHN/CHA/06 were found between JS/CHA/05 and HNK/CHA/05 strains with partial 3B and 3C fragments.ConclusionThis is the first report of the isolation and identification of a strain of FMDV type Asia1 from naturally infected pigs. The Asia1/WHN/CHA/06 strain may evolve from the recombination of JS/CHA/05 and HNK/CHA/05 strains.

Prevalence of 16S rRNA methylase conferring high-level aminoglycoside resistance in Escherichia coli in China

entamicin, ciprofloxacin and/or third-generation cephalosporins ncreased from 7.8% at baseline to 49% after international travel. n 18% of individuals, prolonged colonisation for >6 months ccurred [5]. These individuals may act as reservoirs for infecion within the community or as sources of outbreak when hey are hospitalised [2,3,5]. In many countries there are no ational guidelines for admission screening of individuals who ave a history of medical treatment abroad. Second, the optimal reatment for invasive carbapenem-resistant Enterobacteriaceae nfections is uncertain and is complicated by the frequent occurence of co-resistance to many other antibiotics. The agents that re potentially active against carbapenem-resistant Enterobactericeae are colistin, tigecycline, fosfomycin and isepamicin. In solid rgan and bone marrow transplant recipients, infections by these rganisms represent a tremendous threat. In response to these hallenges, hospitals in Hong Kong have recently introduced active creening for carbapenem-resistant Enterobacteriaceae carriage t hospital admission. Under the arrangement, all newly admited patients with history of hospitalisation, surgery or dialysis n an overseas institution in the previous 12 months would be creened. Funding: This work was supported by research grants from the esearch Fund for the Control of Infectious Diseases (RFCID) of the ealth, Welfare and Food Bureau of the Hong Kong SAR Governent. Competing interests: None declared. Ethical approval: Not required.