Abstract:[Background] Ansamitocins, macrocyclic lactam compounds related to maytansine, are synthesized by Actinosynnema pretiosum ssp. auranticun. Based on the differences of ester side chain moieties in C3 position, ansamitocins contain a series of derivatives. Currently, a novel anticancer drug named Kadcyla (T-DM1), formed from ansamitocin P-3 (AP-3), enters the market. T-DM1 had been approved by FDA as an antibody-drug for treating breast cancer. However, the low yield of ansamitocins limits its commercial application. [Objective] To improve AP-3 yield from A. pretiosum ssp. auranticum ATCC 31565, we applied adaptive evolution and addition of betaine. [Methods] The wild-type A. pretiosum ssp. auranticum was used as starting strain to the adverse stress streptomycin and paromomycin. The evolved mutants were screened with better production of AP-3. After 0.1% betaine was added into the fermentation broth of the evolved mutants, the production of AP-3 was further enhanced. Then, gene transcription levels related to AP-3 biosynthesis were analyzed in evolved mutants and starting strain, to investigate the reasons for enhancement of ansamitocin. [Results] Two evolved strains Str16-4-4 and Par16-2-1 have been obtained. After 7 days fermentation, their AP-3 contents were increased by 33.4% and 31.7%, respectively. Addition of 0.1% betaine ultimately resulted in an increase of 54.6% and 47.4% AP-3 production compared to the wild strain. [Conclusion] Through the adaptive evolution strategy, AP-3 production was improved in A. pretiosum ssp. auranticum. This study provides a new strategy to improve the production of AP-3, and also gives a new example for adaptive evolutionary strategy to promote the production of target products.

Abstract:[Background] Synergetic metabolism of multi-microbial strains is one of the essential feature of fermentation process for Chinese strong aroma Baijiu. [Objective] To study the relationship between functional microbial organisms in the fermentation process of multi-grain strong aroma Baijiu, which could provided theoretical references for optimization of fermentation process. [Methods] The typical yeasts and Lactobacillus acetotolerans in the fermentation process of multi-grain strong aroma Baijiu were isolated by culture-dependent method, and the relationship between these yeasts and L. acetotolerans strains and variation of major volatile metabolites were also discussed. [Results] Three typical yeast strains (Kazachstania humilis Z1, Pichia kudriavzevii Z2, Candida ethanolica Z3) and one Lactobacillus acetotolerans strain (L. acetotolerans W) were isolated. The number of L. acetotolerans W in the co-culture medium of K. humilis Z1 and L. acetotolerans W (Z1&W) was significantly less than that in the single culture medium of L. acetotolerans W. All of three yeast strains could inhibit the lactic acid production ability of L. acetotolerans W, and no lactic acid was produced in co-culture medium of Z1&W. The volatile profile of single Z1 was similar with that in Z1&W. Obvious difference in volatile profile was monitored between single Z2 and Z2&W, in which, concentrations of ethyl acetate and ethyl lactate in Z2&W were significantly increased. [Conclusion] Under the condition of co-culture system, ethanol-producing yeast had inhibition effect on lactic acid metabolism of L. acetotolerans, and L. acetotolerans had certain influence on ethanol metabolism of yeast. It was of great significance for the quality control of multi-grain strong aroma Baijiu and its relationship with the microflora.

Abstract:[Background] Organic sulfur desulfurization catalyzed by desulfurizing bacteria plays an important role in the biogeochemical cycle of sulfur and the desulfurization industry. [Objectives] In order to investigate the diversity of bacteria using the organic sulfur to produce hydrogen sulfide in marine sediments, this research focused on the isolation and characterization of desulfurizing bacteria. [Methods] The culturable desulfurizing bacteria were isolated from the marine sediments sampled from Beidaihe, which were subjected to the screening by the media containing both lead acetate and L-cysteine. Then, the 16S rRNA gene sequences of desulfurizing bacteria were retrieved against the GenBank database. All of the gene sequences with the reference sequences were used to construct the phylogenetic tree. Finally, the bacteria were tested for their capabilities for desulfurization and denitrification. [Results] 62 desulfurizing bacteria were screened out from a total of 97 bacterial strains, out of which, 12 desulfurizing bacteria were selected as the representatives to subject to phylogenetic analysis based on their 16S rRAN gene sequences. The results indicated that all the bacterial strains belong to the genus of Bacillus, Lysinibacillus, Planococcus and Rhodococcus, respectively. Five strains with strong desulfurization ability were affiliated to three genus of Lysinibacillus, Planococcus and Bacillus. Further test showed that the desulfurizing bacteria can produce cysteine desulfhydrase to catalyze the conversion of L-cysteine to pyruvate, hydrogen sulfide and ammonia, indicating they possess both of the capabilities of desulfurization and denitrification. [Conclusion] There are abundant L-cysteine desulfurization bacteria in marine sediments, which provide materials for further study of biogeochemical cycle of sulfur in the ocean.

Abstract:[Background] Quinoline is a nitrogen heterocyclic compound with high toxicity, carcinogenicity but low degradability. A lab-scale denitrifying quinoline-degrading bioreactor was developed and in operation for several years. [Objective] To isolate the potential quinoline-degrading bacteria from the influent pipeline of the bioreactor. [Methods] The media with quinoline as sole carbon source were adopted to enrich, screen and purify degrading strains. Phylogenetic analysis was performed through 16S rRNA gene sequencing. The characteristics of quinoline degradation by different strains were investigated under different pH and temperature conditions. [Results] Based on the phylogenetic analysis, four isolates Q1, Q3, Q7 and Q8 were identified as the genera of Sphingobium, Massilia, Rhodococcus and Dyadobacter, respectively. We demonstrated that 50 mg/L quinoline could be removed within 48 h by all of the strains with varied degradation characteristics. The accumulation of 2-hydroxyquinoline was detected during the cultivation of strain Q1, Q3 and Q8. Notably, the genera of Sphingobium, Massilia and Dyadobacter have not been reported yet on quinoline degradation. [Conclusion] The four quinoline-degrading bacteria isolated from the influent pipeline of bioreactor can provide novel strain resources for decontaminating industrial wastewater containing quinoline, which may contribute to further understanding of the mechanism of quinoline biodegradation.

Abstract:[Background] The Ebinur Lake wetland national nature reserve in Xinjiang is one of the most representative temperate arid regions wetland desert ecosystems in China, and plays an important role in maintaining regional ecological balance. At present, there has not been any report on the structure and abundance of nitrogen-fixing microorganisms in rhizosphere and non-rhizosphere soil at Ebinur Lake wetland. [Objective] To explore the characteristics of environmental heterogeneity of community structure and abundance of nitrogen fixation gene (nifH) in the soil rhizosphere and non-rhizosphere soil nitrogen-fixing microbial of Halocnemum strobilaceum in Ebinur Lake wetland in Xinjiang. Based on this, the potential forces of the microbial communities in the desertification and continuous salinization of the Ebinur Lake wetland ecosystem in the temperate arid regions are explored, providing the theoretical and data basis for the degradation and restoration of lake wetlands. [Methods] The correlations among soil physicochemical properties, microbial community structure and microbe abundance were investigated by using the methods of constructing clone library, q-PCR and redundant analysis (RDA). [Results] The results showed that the diversity of nifH gene in non-rhizosphere soil were higher than that in rhizosphere soil. The dominant species of nifH sequence were Azorhizobium and Desulfovibrio in the rhizosphere soil, Azoarcus, Heliobacterium modesticaldum and Desulfovibrio in non-rhizosphere soil. The number of nifH gene was 4.08×104 copies/g in rhizosphere soil and 5.52×103 copies/g in non-rhizosphere soil. The abundance of nifH in rhizosphere soil is higher than that in non-rhizosphere soil. Correlation analysis showed that the dominant groups and abundance of containing nifH bacteria in rhizosphere soil were significantly related with nitrate nitrogen (NO3?-N), available nitrogen (AN), total potassium (TK), soil moisture (SM) and other factors, and with nitrate nitrogen (NO3?-N), available nitrogen (AN), total phosphorus (TP), total potassium (TK) and total nitrogen (TN) in non-rhizosphere soil. [Conclusion] These results indicated that the abundance of nifH bacteria in rhizosphere soil was higher than that in non-rhizosphere soil, while the diversity was lower than that in non-rhizosphere soil. Nitrate nitrogen (NO3?-N), available nitrogen (AN), total phosphorus (TP) may affect the community structure and abundance of nitrogen-fixing microorganisms. These characteristics provide the theoretical and data basis for the degradation and restoration of lake wetlands.

Abstract:[Background] Banana wilt is a fungal devastating soil-borne disease caused by Fusarium oxysporum f. sp. cubense (Foc). In recent years, the application of biocontrol bacterium is an effective means of prevention and control. [Objective] Biocontrol bacteria with good control effect were isolated and screened from the rhizosphere soil of banana, and the number of biocontrol bacteria and antibacterial efficiency were improved by optimizing the medium and fermentation conditions. [Methods] The rhizosphere soil was collected from orchard in Zhangzhou, Fujian province. The pathogen of Foc Tropical Race 4 (Foc 4) was used as indicator fungus. An antagonistic strain named HQB-1 with strong antifungal activity was isolated by dilution coating and plate confrontation method. Strain HQB-1 was identified by morphological observation, physiological and biochemical experiments and 16S rRNA gene sequence analysis. The fermentation conditions were optimized by single factor experiment and orthogonal experiment design. [Results] Strain HQB-1 was identified as Burkholderia stagnalis, the optimized medium was beef extract 5.0 g/L, yeast extract 10.0 g/L, NaCl 5.0 g/L, and the optimal culture temperature at 27 °C, pH 7.0, rotation speed 200 r/min, inoculation amount 1%, and the culture time 36?h. [Conclusion] The number of effective viable cells and the inhibition rate were, improved significantly than before. The OD600 was increased from 1.251 to 1.881, and the inhibition rate was from 9.18% to 34.60%.

Abstract:[Background] Bailugu is an edible fungus isolated and domesticated from the wilderness of Sha county. As its fruit body is white and shaped like asparagus in the course from the formation of its primordium to the pick of fruit body, during which it experienced the rod-shaped period and the nail head period, it is called as “Bailugu” in local. [Objective] Identifying Bailugu and studying its characteristics in hyphal fermentation culture. [Methods] The morphological observation and rDNA-ITS analysis were used to identify Bailugu. Using the dry weight of mycelium biomass in shake flask fermentation as the main index, the optimum conditions and formula for the hyphae fermentation of Bailugu were obtained through the single factor screening and orthogonal comprehensive screening. [Results] Morphological observation and ITS analysis showed that Bailugu belongs to Pleurotus, Pleurotaceae, and its scientific name is Panus giganteus, its optimum conditions for mycelial shake flask fermentation were as follows: temperature of 25 °C, pH of 8.0 and protection from light, and the most suitable formula for fermentation of mycelial shake flask is corn flour of 25.0 g/L, soybean powder of 3.0 g/L, KH2PO4 of 1.5 g/L, MgSO4·7H2O of 1.0 g/L, thiamine of 50.0 mg/L. [Conclusion] This study conducted the research on the identification and mycelial fermentation culture of Bailugu, hoping to lay the foundation for the subsequent scientific promotion and development.

Abstract:[Background] In May 2016, an unprecedented outbreak of nodulous esophagitis occurred among the captive juvenile Siamese crocodiles (Crocodylus siamensis) in Xiamen Lonsun crocodile zoo, and diseased crocodiles manifested anorexia and some of them even died. [Objective] Pathogenic identification of crocodiles’ pathological materials was performed to explore its causes, to provide data for estimating this disease, and to make references for preventive treatment. [Methods] The bacteria isolated from crocodiles’ foci were identified by the physiological and biochemical characteristics, 16S rRNA gene sequence analysis, artificial infection test and sensitive drug test. [Results] Three bacterial strains isolated from esophagus, liver, and blood of diseased crocodiles, were identified as Proteus penneri by means of morphological, physiological and biochemical characteristics, and 16S rRNA gene sequence analysis. Besides, the P. penneri strain 2202 was confirmed as pathogen causing morbidity and mortality in C. siamensis after artificial infection test. The susceptibility test showed that the resistance rates of 3 isolated strains to 12 antimicrobial agents were all equal to 33%. The 3 pathogens were sensitive to 7 medicines, including enrofloxacin, sulfamethoxazole, cefotaxime, kanamycin, tetracycline, doxycycline, and nalidixic acid; resistant to rifampicin, penicillin G, erythromycin, and chloramphenicol; and intermediate resistant to streptomycin. [Conclusion] As a result, P. penneri was directly related to the death of diseased C. siamensis. To the best of our knowledge, this is the first report about P. penneri, the opportunistic pathogen mostly affected human, infected the captive crocodilians.

Abstract:[Background] Methicillin-resistant Staphylococcus aureus (MRSA) is a common conditional pathogenic bacterium. Mastitis in dairy cows caused by MRSA infection has brought significant economic losses to dairy farmers. [Objective] To understand the genomic sequence characteristic of MRSA epidemic strain in dairy cows in Ningxia province, and provide a theoretical basis for the prevention and treatment of MRSA infection. [Methods] The isolate ld11 was tested for antimicrobial susceptibility by agar diffusion method, and the high-throughput sequencing of genomic DNA of isolate ld11 was carried out based on Illumina high-throughput sequencing platform, and the obtained sequencing sequences were processed and analyzed through network databases. [Results] The susceptibility test showed that the isolate ld11 was resistant to ceftiofur, sulfisoxazole, ampicillin, erythromycin, gentamicin, oxacillin, clindamycin, tetracycline, and doxycycline, and data analysis showed isolate ld11 carried the drug resistance genes aadD, spc, str, blaZ, mecA, cat(pC194), erm(A), norA, tet(k), and tet(M), and there was a good correlation between the two; The isolate ld11 carried more resistance genes than the MRSA reference strains, and the relationship between isolate ld11 and MRSA252 was closer. The results of COG (Clusters of orthologous groups of proteins) functional analysis and GO (Gene ontology) annotation showed that the genes involved in maintaining the basic functions of the cells and the growth and proliferation of the strains predominated. The KEGG (Kyoto encyclopedia of genes and genomes) pathway analysis showed that the genes belonging to the metabolic pathway accounted for the most; four gene islands, nine questionable CRISPR sequences, and one complete prophage sequence were detected from the genome sequence. [Conclusion] This study revealed partial genomic sequence information of MRSA epidemic strain in dairy cows in Ningxia, which provided a reference for comparative analysis of genomic sequence information and prevention and control of MRSA infection among MRSA strains.

Abstract:[Background] Very long-chain fatty acids (VLCFA) are involved in the synthesis of membrane lipid sphingolipids and closely related to the metabolism of membrane lipid ergosterol and phospholipids. Defects in the synthesis of VLCFA in cells can damage the stability of cell membranes and thus enhance the fungal susceptibility to polyene-based drugs. However, the effect of the synthesis defects of VLCFA on the membrane stability and the sensitivity to polyene drugs is not entirely clear. [Objective] The role and function of the cell long-chain fatty acid elongases ELO1, ELO2 and ELO3. [Methods] The response of mutants with the synthesis defects of VLCFA, elo1?, elo2? and elo3?, to amphotericin B (AmB), one of the polyene drugs, and other drugs, such as nystatin (Ny) and econazole nitrate (Eco) was investigated. The ergosterol contents of different yeast cells were detected, and its response to Na+ and intracellular sodium and potassium levels was examined. [Results] We found that elo2? and elo3? mutants were highly sensitive to AmB. In addition, these two mutants were also sensitive to other drugs, such as Ny and Eco. Generally increased unsaturated fatty acids affect membrane stability. Our results showed that exogenous oleic acid (OLA) increased the sensitivity of elo2? and elo3? mutants to AmB. Compared with those of the wild-type and elo1? mutant, the ergosterol contents of the defective strains elo2? and elo3? decreased significantly. Sodium-potassium ion balance was a necessary condition for maintaining the normal physiology of cells, and was also an important parameter for measuring cell membrane stability. We found that defective strains were more sensitive to high concentrations of NaCl than wild-type strains. The levels of sodium and potassium in cells under different concentrations of AmB were detected by ICP-AES. The data showed that sodium levels were elevated in VLCFA synthesis-deficient strains compared with the wild-type strain. The potassium content in mutants was decreased significantly. [Conclusion] The synthetic defects of VLCFA could lead to the more fragile cell membrane and lower stability, thus increasing the sensitivity of fungi to polyenes. It suggests that fatty acid elongase could be the target of potential antifungal therapy.

Abstract:[Background] Sparassis latifolia is a valuable edible fungi. However, lignocellulose degradation mechanism is poorly understood. [Objective] To understand expression profiles of lignocellulose degradation associated genes cultivated with different carbon sources. [Methods] Based on RNA sequencing, we obtained the whole-genome expression profiles when the mycelia of S. latifolia were cultured with glucose, cellulose, cellulose/lignin and pine sawdust as the carbon source respectively. Using glucose sample as control, functional analysis of differentially expressed genes was carried out. [Results] Gene ontology enrichment analysis showed that, differently expressed genes which compared to glucose as the sole carbon source were mainly involved in polysaccharide catabolic process, carbohydrate catabolic process, polysaccharide metabolic process and carbohydrate metabolic process. Carbohydrate-active enzymes annotation showed that, transcript levels of genes encoding glycoside hydrolases, thought to be important for hydrolytic cleavage of hemicelluloses and cellulose were mainly influenced by the species of carbon source, and in which genes involved in hemicellulose degradation were mostly up-regulated. Several transcription factor genes up-regulated significantly when the carbon source was cellulose/lignin or pine sawdust respectively. [Conclusion] S. latifolia gene expression pattern is influenced substantially by the species of carbon source. Such adaptations to the carbon source may also reflect fundamental mechanisms by which S. latifolia attack plant cell walls. Our findings provide important information in exploring the potential genes responsible for lignocellulose degradation.

Abstract:[Background] MvaT protein belonging to the H-NS transcription factor family is involved in many important metabolic processes of Pseudomonas aeruginosa, such as phenazine synthesis pathway. However, up to now its regulation mode is still unclear. [Objective] The goal of this work was to determine if MvaT could directly regulate the two phenazine-1-carboxylic acid synthesis gene clusters (phzA1G1 and phzA2G2) and three transforming genes (phzH, phzS and phzM) of phenazine compounds, by detecting the binding capability of MvaT to the promoter regions of these genes. [Methods] Pseudomonas aeruginosa SJTD-1 was used as target and the mvaT-knockout strain SJTD-1(ΔmvaT) was constructed by homologous recombination. The yield of phenazine compounds of the two strains in different media was detected. Further the recombinant MvaT protein was obtained by the heterologous expression and affinity purification. The electrophoretic mobility shift assay (EMSA) was performed to determine if the recombinant MvaT protein could bind to the promoter regions of the five phenazine synthetic gene clusters/genes. [Results] The yield of phenazine compounds of strain SJTD-1(ΔmvaT) was significantly higher than that of the wild type SJTD-1 strain. The recombinant MvaT protein could be expressed and purified efficiently. In vitro EMSA results indicated that the recombinant MvaT protein could directly bind to the promoter regions of phenazine synthetic gene clusters/genes. The binding sites of MvaT to the promoter of phzA1G1 and phzA2G2 gene clusters were within the upstream 200 bp region, and that to the promoter of phzM, phzS and phzH genes were within its upstream 100 bp region. [Conclusion] MvaT protein can directly bind the upstream promoter regions of the five phenazine synthetic gene clusters/genes and regulate the synthesis of phenazine compounds.

Abstract:[Background] Vibrio parahaemolyticus is a seafood-borne pathogen. CalR is a global transcriptional regulator in V. parahaemolyticus. Type 3 secretion systems 2 (T3SS2) is one of the major virulence determinants of V. parahaemolyticus. VopB2 is a key effector protein of T3SS2. [Objective] To study the transcriptional regulation of vopB2 by CalR in Vibrio parahaemolyticus. [Methods] Primer extension assay was employed to detect the transcription start sites and the amount of primer extension porroducts of target genes in ΔcalR and WT. Quantitative RT-PCR was then carried out to calculate the transcriptional variation of target genes between ΔcalR and WT. LacZ assay was used to verify regulation relationship by measuring the β-galactosidase activities in cellular extracts using a β-Galactosidase Enzyme Assay System (Promega). Finally the electrophoretic mobility shift assay (EMSA) was applied to analyze the DNA-binding activity of His-CalR to target promoters in vitro. [Results] We detected one transcription start site for vopB2, which was located at 130 bp and 28 bp upstream of vopB2 and its transcribed activity was under the negative control of the CalR. We also found that His-CalR was bind to the promoter region of vopB2 directly. Besides, our data showed that CalR had no regulatory effect on vtrA transcription, a known regulator for vopB2. [Conclusion] V. parahaemolyticus CalR represses the transcription of vopB2 directly, which is not associated to vtrA.

Abstract:Saccharides and their derivatives play prominent roles in many primary and secondary metabolic processes. The diversity of their structures and the significance of sugars in diagnosis and treatment of diseases have promoted the fast development of glycobiology. D-sugars, especially D-hexoses have dominated the carbohydrates region, L-hexoses yet are also frequently observed in some important glycoprotein complexes, polysaccharides and antibiotics. Understanding the formation mechanisms of L-hexoses allow rational manipulation of sugar skeletons and further exploration of their applications. L-hexose is usually generated through epimerization of D-hexose by 3,5-epimerase or 5-epimerase. This transformation expands the diversity of sugar configurations and influences the bioactivities of many natural products. The functional and crystallographic studies of 3,5-epimerases and 5-epimerases have provided profound insights into the formation mechanisms of L-hexoses. In this paper, we summarize the catalytic mechanisms of different 3,5-epimerases and 5-epimerases for L-hexose formation. The physiological importance and biomedical prospects of L-hexoses are also discussed.

Abstract:People concern the ecological environment due to increasing shortage of land resources. The application of microbial fertilizers with natural soil microorganisms as the main component has attracted interests in farming on saline-alkali soil. This paper reviews the development and current status of microbial fertilizers worldwide. Moreover, we discuss the mechanisms how microbial fertilizers help plants resist salt stress and the effects on microbial communities in saline-alkali soils. We also propose two methods of immobilizing strains. These two methods can effectively solve the strains’ inactivation and prolong their action time in the soil. Finally, the problems and prospects of the current microbial fertilizers acting on saline-alkali soils are addresses.

Abstract:Lon protease is the first identified ATP-dependent protease that plays critical role in degrading misfolded proteins and maintaining intracellular protein balance in prokaryotes. Recent studies have suggested that, as a pressure-stress protein, Lon was involved in the degradation of a variety of transcription regulators and two-component regulatory system in bacteria, resulting in altered physiologically metabolic process of bacteria cells to adapt to the environment change. In this paper, the structure, function, and up and down-stream regulatory network of bacterial Lon protease are reviewed, aiming at enhancing understanding in the physiological function of Lon, as well as providing basis for elucidating the mechanism of intracellular regulation.

Abstract:In recent years, heavy metal contamination in China has greatly increased due to the discharge of industrial wastewater and daily human activities. People living in polluted areas suffer from serious health problems as a result of long-term ingestion of contaminated crops. Therefore, it is of utmost importance to repair heavy metal pollution in developing countries. Recent studies have mainly focused on the probiotics effectively combine with heavy metals in vitro and in vivo, and different mechanisms have been proposed to explain the functions of probiotics in heavy metals’ remediation. In view of the current situation, heavy metals such as cadmium, lead, and chromium have serious impacts on the environment and animal health, this review analyzes the sources of heavy metals pollution and the control of various heavy metals in the environment and animal intestines. Meanwhile, the mechanisms of heavy metal remediation processes by probiotics are summarized, and the utilization of probiotics in alleviating heavy metals’ toxicity on human and the environment in the future.

Abstract:Horizontal gene transfer (HGT) plays an important role in the evolution of bacteria. HGT occurs mainly through three mechanisms: transduction which is mediated by bacterial phage, conjugation and natural transformation. Natural transformation is the process by which natural competent bacteria can take up DNA from the environment and integrate it into their own genomes. Natural transformation was first found in Streptococcus pneumoniae in 1928. Until now, at least 83 bacterial species have been found to be natural competent. Among them, the natural transformation mechanism of Gram-positive bacteria Streptococcus pneumoniae and Gram-negative bacteria Neisseria were studied well, respectively. The biological functions of natural transformation have been hypothesized as follows: obtaining nutrition, repairing DNA damage, and biological evolution. However, there are still debates on this. In this paper, it will describe the molecular mechanism of bacterial natural transformation in details, and discuss the biological function of natural transformation. It will be helpful to further understand the bacterial natural transformation.

Abstract:Microbiology is an important part of the natural sciences and is essential for the cultivation of talents in the biological profession. The teaching content and teaching methods of microbiology in the new era need constant reform and innovation to meet the goal of cultivating talents with all-round development in moral, intellectual, and artistic development. By strengthening the construction of microbiology teaching resources, teaching forms and examine mechanisms, and actively explores the path of curriculum education around the goals of value shaping, capacity improvement and knowledge transfer, Huazhong Agricultural University realizes the improvement of students’ comprehensive quality, and provides services for cultivating first-class talent of agriculture and forestry.

Abstract:[Background] The probiotic functions of Bifidobacterium are widely recognized, and a large body of studies paid attention to the biodiversity of Bifidobacterium in the intestine. However, Bifidobacterium is a low abundance genus in the intestine, and it is difficult to study the bifidobacterial diversity by extant technologies in depth. [Objective] To screen a pair of specific primers for the analysis of diversity of Bifidobacterium with low abundance in fecal samples. [Methods] Initially, to obtain Bifidobacterium-specific primers with amplicons of over 800 bp, the primers were recombined and optimized according to the relative positions of the extant bifidobacterial primers and their matching rates with the Bifidobacterium 16S rRNA gene sequence. Then, the rest primers were screened by PCR amplification and agarose gel electrophoresis, and their specificity was verified. Finally, taking the universal bacterial primer (27f/1492r) as control, the DNA amplicons of bacterial microbiota in three fecal samples amplified by selected primers were sequenced by SMRT (Single-molecule real-time) sequencing technology, and different sequencing primers were compared and analyzed at the species level. [Results] Two pairs of primers, Bif164-f/Pbi R2 and Pbi F1/Pbi R2, were selected as the optimal Bifidobacterium primers theoretically with amplicon greater than 800 bp from 9 pairs of Bifidobacterium-specific primers collected from literature. The PCR amplification and agarose gel electrophoresis showed that the amplified bands of Bif164-f/Pbi R2 were bright and no tail. In addition, sequencing and analysis of DNA amplicons from bacterial microbiota in three fecal samples with primers 27f/1492r and Bif164-f/Pbi R2 using SMRT sequencing platform. The analysis of 27f/1492r amplicons showed that three samples contained 1, 3 and 4 bifidobacterial species respectively, and the average relative content of Bifidobacterium was 0.34%. The analysis of Bif164-f/Pbi R2 amplicons showed that three samples contained 2, 6 and 8 bifidobacterial species respectively, and the average relative content of Bifidobacterium was 98.72%. The above results indicate that Bif164-f/Pbi R2 can specifically detect bifidobacteria with low abundance in feces at species level, which realize the diversity analysis of Bifidobacterium in samples. [Conclusion] In summary, a pair of Bifidobacterium-specific primers Bif164-f/Pbi R2 were screened in the experiment, which can be used to analyze the diversity of low-abundance Bifidobacterium in fecal samples at species level. It was also confirmed that the combination of theory and experiment is feasible to perform primers screening.

Abstract:[Background] Volatile organic compounds (VOCs) produced by fungi have important biological functions. The high-throughput VOCs analysis method is required for the screening of VOCs producing fungi. [Objective] To establish a robust, simple, high-throughput analysis technique for VOCs produced in petri dishes by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS) analysis based on silicone tube sorptive extraction (STSE). [Methods] twenty-one representative VOCs including esters, ketones, alcohols, aldehydes and alkenes were selected as reference substances to establish the standard curve. TD-GC-MS analysis based on STSE was performed by dropping the reference substances into the PDA medium to determine the specificity and by inoculating Trichoderma strain T552 into the PDA medium to determine the repeatability. On this basis, the VOCs produced by 6 strains of Trichoderma were screened with the above method. [Results] The linear relationships of 21 reference substances were good in the range of 1.0?10.0 μg/mL and 0.1?10.0 μg/mL under the conditions of full scanning and extraction ion. TD-GC-MS analysis based on STSE showed that the reference substances dropped into the PDA separated well, and other substances in the PDA petri dish did not interfere with the determination of VOCs, indicating that the method had good specificity. Repeated determination of VOCs produced by Trichoderma strain T552 cultivated in the PDA medium was carried out, the relative standard deviation of the five VOCs identified were less than 10%, indicating that the method had good repeatability. A total of 37 VOCs including acid, alcohol, ester and alkene were identified from 6 Trichoderma strains were screened out by the established TD-GC-MS analysis based on STSE. [Conclusion] A robust, simple, high-throughput analysis technique for VOCs produced in petri dishes by TD-GC-MS analysis based on STSE was established, which can be used for the determination of fungal VOCs.

Abstract:动物病毒学与教育科研、社会生产、百姓生活等密切相关。我们在利用和享受动物病毒学研究成果的同时，也需要随时应对动物病毒性疫病新发和再现的严峻挑战。跟踪和展示动物病毒学领域最新研究成果有助于我们了解病毒这种最简单的生命形式，也有助于我们驯服、控制病毒并让其服务于社会需要。本专栏稿件包含了冠状病毒、非洲猪瘟病毒、圆环病毒、流感病毒、鸡马立克氏病毒、牛病毒性腹泻病毒等6种动物病毒病原体相关研究报告和综述，汇集了病原进化、表位筛选、病原与宿主互作等诸多研究内容，期望该专栏的出版有助于促进动物病毒性疫病相关研究领域的交流与进步。

Abstract:[Background] Porcine circovirus causes porcine circovirus associated disease, severe immunosuppression and clinical symptoms in pigs, causing substantial economic losses. [Objective] To study the role of porcine circovirus 3 (PCV3) capsid protein (Cap) in regulating host innate immune response, we constructed a eukaryotic plasmid expressing PCV3 protein Cap. [Methods] The expression of Cap was verified by Western blotting. The real-time quantification PCR (RT-PCR), dual luciferase gene reporting system and ELISA assay were used to study the effect of Cap on type I interferon pathway activation, and the Co-immunoprecipitation was done to study the involved antagonistic mechanism. [Results] The expression of Cap was verified, suggesting that Cap was highly expressed in the transfected cells, and Cap inhibited the activation of type I interferon signaling pathway stimulated by a synthetic DNA, poly(dA:dT). The interaction of Cap with the adaptor protein MITA of innate immune signaling pathway was identified. [Conclusion] These results indicate that Cap plays an important role in inhibiting the innate immune response of host cells by interacting with MITA. Our study provides a theoretical basis for clarification of the molecular mechanisms for PCV3-mediated immune suppression.

Abstract:[Background] Amino acid sequences alignment of the spike (S) proteins of porcine epidemic diarrhea virus (PEDV) classical and prevalent strains indicates that most of the variation maps to N-terminal region of the S protein. [Objective] The objective of this study is to identify linear B cell epitopes (BCE) containing peptides, especially prevalent strain specific linear BCE containing peptides, on the hypervariable region (S10A, 1?496 aa) of the S protein of PEDV prevalent strain. [Methods] The N-terminal hypervariable part of prevalent PEDV SD2014 S protein (S10ASD2014) was recombinantly expressed in Escherichia coli (E. coli). Polyclonal antibody was prepared by immunizing New Zealand white rabbits with S10ASD2014 purified by cutting out the aimed band from the SDS-PAGE. Serial Glutathione S-transferase (GST)-fusion expressed 16-mers with an overlap of 8 amino acids (aa) covering the entire S10ASD2014 sequence was produced in E. coli, and from which linear BCE containing 16-mers were identified by using western blot with prepared anti-S10ASD2014 serum as the primary antibody. The conserved linear BCE containing 16-mers on S10ASD2014 were identified with polyclonal serum against the counterpart of the S protein of PEDV CV777 vaccine strain (S10ACV777). [Results] The relative molecular mass of the expressed S10ASD2014 was about 72 kD. The purified S10ASD2014 could be recognized by clinical porcine anti-PEDV serum. The polyclonal serum prepared by immunizing the rabbits with the purified S10ASD2014 could react with PEDV DR13 in Vero cells. From 61 16-mers, twenty-eight linear BCE containing 16-mers were identified by western blot using the prepared anti-S10ASD2014 and anti-S10ACV777 serum. Thirteen out of the 28 linear BCE containing 16-mers could be recognized by both of the 2 serum. Homology analysis of the corresponding region of 24 typical strains from different PEDV subgroups showed that 2 positive 16-mers were conserved. [Conclusion] The identification of linear BCE containing peptides, especially the prevalent PEDV S protein-specific 16-mers laid a foundation for the understanding the S protein structure and function, as well as for developing epitope-based diagnostic methods.

Abstract:[Background] Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV) has caused significant economic losses in pig industry. PEDV S protein can induce the host to produce neutralizing antibodies. [Objective] To prokaryotically express the truncated S2 (aa: 961?1 382) of PEDV CV777 vaccine strain and prepare polyclonal antibodies with which to identify regions containing linear B cell epitopes on the truncated S2. [Methods] The codon-optimized DNA fragment encoding the truncated S2 of PEDV (s2t) was inserted into pET-28a. The resulting pET-28a-S2t was then transformed into Escherichia coli BL21(DE3) for expression under the induction of IPTG. Polyclonal antibody was prepared by immunizing New Zealand white rabbits with the truncated S2 purified by cutting out the aimed band from the SDS-PAGE. Serial 16-mers, overlapping 8 aa each other and covering the whole sequence of the truncated S2, were GST-fusion expressed in E. coli BL21. Positive 16-mers were identified by western blot (WB) using prepared polyclonal serum as the primary antibody. linear B cell epitopes containing regions were located. [Results] The truncated S2, about 50 kD, mainly existed in form of inclusion body, of which the expression level reached the maximum at 4 h post induction. The purified truncated S2 could be recognized by porcine anti-PEDV serum in WB analysis. The titer values of the polyclonal serum ranged from 1:25 600 to 1:102 400. Immunohistochemical and indirect immunofluorescence analyses indicated that the polyclonal antibodies showed specific reactivity with PEDV DR13 in Vero cells. Eleven positive 16-mers were identified from 52 GST-fused 16-mers by WB using the prepared serum. All the 11 16-mers could be recognized by porcine anti-PEDV serum in WB analysis. The identified positive 16-mers formed 4 linear B cell epitopes containing regions on the truncated S2 (aa: 969?984, 1 065?1 096, 1 225?1 280, and 1 361?1 382). [Conclusion] The preparation of high titer polyclonal antibodies against truncated S2 of PEDV and the identification of linear B cell epitopes containing sequences on it laid a foundation for understanding the structure and function of S protein, as well as developing effective PEDV diagnostic methods.

Abstract:[Background] Porcine circovirus can cause postweaning multisystemic wasting syndrome. [Objective] To understand the epidemiology and genetic variation of porcine circovirus 2 (PCV2) in Anhui province. [Methods] Total 31 tissue samples of pig suspected suffering PMWS were collected. PCR was employed to identify PCV2 and its genome. genetic evolution of the PCV2 virus strains were analyzed by DNAStar and other bioinformatics software. [Results] Nucleotide comparability of identified 8 strains with the strains published on GenBank was 92.8%?99.0%. The comparability of ORF2 and its derived amino acid sequences was 85.8%?99.6% and 82.5%?100%, respectively. The results of genetic evolution showed that there were one strain of PCV2a, two strains of PCV2b and five strains of PCV2d among the eight strains. Moreover, there were no genotypes of PCV2c, PCV2e and PCV2f. In addition, through the analysis of amino acid sequence, unique site was found on Cap protein with each genotype. [Conclusion] PCV2 infection in pigs was common in Anhui province. PCV2d was the dominant genotype. This study provides a basis for PCV2 prevention and control in Anhui province.

Abstract:[Background] H9N2 avian influenza viruses are widespread in chickens and cause great losses. [Objective] Elucidate the sequence characteristics and the variation of antigenicity of H9N2 subtype avian influenza virus (AIV) in Hebei province to understand heterologous protection of AIV. [Methods] seven strains of H9N2 subtype AIV were isolated and identified from farms in Hebei province in 2017. We sequenced the HA genome of 7 H9N2 isolated strains. Then, the phylogenetic trees, amino acid in key functional sites and antigenicity were analyzed. [Results] The homology of HA genes for 7 isolated strains was between 95.5% and 97.2%. Comparing with the epidemic strains before 2016, the cleavage sites and the glycosylation sites were not significantly different from those of the previous strains, except the receptor binding sites of the HA proteins. The antigenicity of the isolated strains changed and a new antigen group formed. The analysis of the HA amino acid sites related to the antigenicity shows that there was a significant mutation in 9 antigenic sites of the newly isolated strains, which may be the molecular basis of antigenic variation. [Conclusion] The prevalent strains of H9N2 subtype AIV in laying-hen farms in Hebei province have mutated in key functional areas and presented antigenic variation, reminding us the importance of continuous surveillance of the genetic evolution and antigenic variation of H9N2 AIV. Changing the candidate strain of vaccine in time is important for the prevention and control of H9N2 AIV.

Abstract:Marek’s disease viruses (MDV) is a group of important herpesviruses, which can induce acute or sub-acute immunosuppressive and neoplastic disease in chickens, namely Marek’s disease (MD). Based on a brief overview of early studies on the classical and molecular virology of MDV, the progresses in the last decade are reviewed in different aspects, such as the application of the technique of infectious bacterial chromosome clone (BAC) to study viral gene functions, the studies on proteomics of MDV protein-coding genes, the regulatory functions of virus-encoded non-coding RNAs, and the recombinant MDV vaccines. Furthermore, the expected subjects or aspects for the further study on MDV are also discussed.

Abstract:African swine fever (ASF) is an acute, hemorrhagic and severe infectious disease caused by African swine fever virus (ASFV) in domestic or wild pigs, which is characterized by short course, high fever and hemorrhagic lesions as well as 100% mortality rate of acute infection. So far, there is no effective vaccine available for ASF. ASFV is a large, double stranded DNA virus and the only member of the Asfarviridae family, Asfivirus genus, which replicates predominantly in the cytoplasm of macrophages. The genome of ASFV ranges in length from 170 to 193 kb depending on the isolate and contains 150 to 167 open reading frames (ORFs), encoding 150 to 200 proteins. However, only about 50 encoded proteins are functionally known and most of them are viral structure proteins. In addition to structure proteins, ASFV also have a full complement of enzymes and factors or other nonstructural proteins that are involved in regulation of the viral transcription, viral DNA repair, viral invasion of host cells, and viral-mediated modification of host cell function and evasion from host defense. In this review, we summarize the current knowledge of functions of ASFV-encoded proteins and anticipate to provide valuable information for future study and vaccine development.

Abstract:Since first isolated in America in 1937, avian infectious bronchitis virus causes serious economic loss to the poultry industry worldwide. Because of vast territory and climate diversity, the domestic epidemic situation of infectious bronchitis virus is very complex. The research and practice of isolation of pathogen, molecular epidemiology, detecting techniques, vaccine and integrated preventing & controlling techniques of infectious bronchitis in China is reviewed in this paper. At present, a variety of strains coexist in China, and the dominant epidemic strains are QX genotype. Moreover, the widely used vaccine belongs to Mass serotype such as H120, but 4/91 serotype vaccine and LDT3-A vaccine have also been used gradually. The combination immunization of attenuated live vaccine and inactivated vaccine can effectively control the economic loss of the poultry industry.

Abstract:The prevalence of bovine viral diarrhea (BVD) in China remains to be known due to the complicated BVDV infections , and its etiology BVDV (BVDV-1 and BVDV-2) is not limited to infect cattle, also goes to other animal infections including swine BVDV infection which could clinically confuse the classic swine fever monitoring and diagnosis, led to exacerbate the disease course. Due to persistent infection (PI) caused by bovine viral diarrhea virus (BVDV), the eradication of the disease is faced with great difficulty and poses a severe threat to the healthy development of the whole dairy farm. Considering currently there are 22 subtypes of BVDV-1 and 4 subtypes of BVDV-2 for the rapid antigen variation and evolution rate of BVDV, our effective prevention and control of this disease is far behind the viral mutation rate. Therefore, the epidemic status of BVDV-1 and BVDV-2 in pigs and cattle in China is regularly investigated and understood, which is the first and critical step of epidemic disease eradication. To be based on the successful experience of BVD eradication abroad, to comprehensively consider the national conditions, to take appropriate prevention and control strategies, and to gradually eradicate the pathogen infection and the related disease, which is conducive to promote the healthy development of domestic breeding industry.