De novo RNA synthesis and homology modeling of the classical swine fever virus RNA polymerase

Classical swine fever virus (CSFV) non-structural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase (RdRp), a key enzyme which initiates RNA replication by a de novo mechanism without a primer and is a potential target for anti-virus therapy. We expressed the NS5B protein in Escherichia coli. The rGTP can stimulate de novo initiation of RNA synthesis and mutation of the GDD motif to Gly-Asp-Asp (GAA) abolishes the RNA synthesis. To better understand the mechanism of viral RNA synthesis in CSFV, a three-dimensional model was built by homology modeling based on the alignment with several virus RdRps. The model contains 605 residues folded in the characteristic fingers, palm and thumb domains. The fingers domain contains an N-terminal region that plays an important role in conformational change. We propose that the experimentally observed promotion of polymerase efficiency by rGTP is probably due to the conformational changes of the polymerase caused by binding the rGTP. Mutation of the GDD to GAA interferes with the interaction between the residues at the polymerase active site and metal ions, and thus renders the polymerase inactive. (c) 2005 Elsevier B.V. All rights reserved.

Switch Region for Pathogenic Structural Change in Conformational Disease and Its Prediction

Many diseases are believed to be related to abnormal protein folding. In the first step of such pathogenic structural changes, misfolding occurs in regions important for the stability of the native structure. This destabilizes the normal protein conformation, while exposing the previously hidden aggregation-prone regions, leading to subsequent errors in the folding pathway. Sites involved in this first stage can be deemed switch regions of the protein, and can represent perfect binding targets for drugs to block the abnormal folding pathway and prevent pathogenic conformational changes. In this study, a prediction algorithm for the switch regions responsible for the start of pathogenic structural changes is introduced. With an accuracy of 94%, this algorithm can successfully find short segments covering sites significant in triggering conformational diseases (CDs) and is the first that can predict switch regions for various CDs. To illustrate its effectiveness in dealing with urgent public health problems, the reason of the increased pathogenicity of H5N1 influenza virus is analyzed; the mechanisms of the pandemic swine-origin 2009 A(H1N1) influenza virus in overcoming species barriers and in infecting large number of potential patients are also suggested. It is shown that the algorithm is a potential tool useful in the study of the pathology of CDs because: (1) it can identify the origin of pathogenic structural conversion with high sensitivity and specificity, and (2) it provides an ideal target for clinical treatment.

猪粪农用的生态风险——以东北黑土为例

集约化养殖大量使用铜(Cu)作为饲料添加剂会在养殖废物的排放和利用中导致一定的环境问题。随着东北老工业基地产业结构的调整，畜牧业作为东北地区二次振兴的主要产业得到迅速发展，由此会产生大量的畜禽粪便用于农业土壤施肥。而东北地区又是我国重要的农副产品生产基地，研究畜禽粪便农用的健康及环境风险就显得殊为必要。黑土是东北地区最主要的土壤类型，本文以东北黑土为供试材料，通过在盆栽实验中添加不同Cu浓度的猪粪来模拟施肥年限不同的菜园土，研究土壤中Cu累积对小白菜(Brassica chinensis L.)地上部分生物量与Cu含量、土壤微生物生物量和土壤酶(脱氢酶、脲酶、酸性磷酸酶)活性、微生物群落功能多样性以及对土壤功能微生物——氨氧化古菌(AOA)和氨氧化细菌(AOB)等的影响，从而实现含Cu猪粪农用对食物链途径的健康风险以及土壤生态系统的生态风险的综合评价。 研究结果表明，施用猪粪显著地改善了土壤的pH，增加了小白菜地上部分生物量，对其体内Cu浓度则没有显著影响;小白菜地上部分Cu浓度与土壤全Cu和水溶态Cu浓度显著正相关(P<0.01)，而地上生物量与土壤全Cu浓度显著负相关(P<0.05)。当土壤全Cu浓度达到300 mg kg-1后，小白菜地上部分生物量受到强烈抑制。 猪粪的施用在一定时期内显著地促进了脱氢酶和脲酶的活性，但随土壤Cu浓度的增加，脱氢酶、脲酶和酸性磷酸酶的活性逐渐受到抑制。当土壤全Cu浓度达到300 mg kg-1后，微生物生物量、脱氢酶、脲酶、酸性磷酸酶等均受到强烈抑制。 施加猪粪提高了土壤微生物群落功能多样性，当土壤Cu浓度达到170 mg kg-1后，显著地抑制了微生物群落功能多样性。种植小白菜后，各处理间微生物群落功能多样性没有显著差异，意味着植物能够维持微生物群落功能多样性。主成分分析(PCA)结果显示，当土壤全Cu浓度达到300 mg kg-1后，微生物群落功能多样性与其它处理显著不同。 猪粪显著地促进了土壤的硝化潜势速率(PNR)。随着Cu浓度的增加，硝化潜势速率逐渐受到抑制，当土壤全Cu浓度达到170 mg kg-1后，即与对照无显著差异。植物在维持硝化速率方面也起到重要作用。古菌amoA基因在每克干土中的拷贝数在9.4*106~2.59*107，细菌amoA基因在每克干土中的拷贝数约为1.48*106~1.41*107。猪粪在一定时期内显著地增加了AOA和AOB的数量，当土壤Cu浓度达到300 mg kg-1后，AOA和AOB的数量显著下降，并且AOA与AOB amoA基因拷贝数的比值增加，在小白菜栽种前(B1)和小白菜收获后(B2)分别为2.33和9.26，这意味着在高Cu浓度胁迫下，AOA的耐受性更强。古菌与细菌的amoA拷贝数与PNR之间则没有显著的相关关系，意味着AOA和AOB种群结构以及其中某些种群的活性可能发生了变化。 本文通过对东北黑土菜地土壤中Cu在作物体内的迁移积累及其对土壤微生物学指标(微生物生物量、酶活性、微生物群落功能多样性及功能微生物)的影响的综合研究，揭示了含Cu猪粪农用所造成的健康和生态风险，为建立适宜的土壤环境质量标准提供了一定的科学依据。通过本研究得出，黑土蔬菜种植中Cu浓度的阈值在170-300 mg kg-1之间。

SLA 的分型与检测

:传统的强调抗原多态性的SLA 分型方法主要是血清学、细胞学和生物化学的方法,随着分子生物学技术发 展,各具特点的SLA 分型技术不断涌现,如PCR2RFL P 法、PCR2SSCP 法、微卫星(MS) 法、DNA 序列的测定等,基于 强调SLA 的抗原多态性的分型和强调抗原保守性的功能学上的新分型技术(如SLA 超型和超基序) 进行了详细探 讨,比较了各类方法的优缺点,指明了未来SLA 分型的发展趋势。此外还指出了现行参考教材血清学SLA 单倍型 的编写错误以及重点强调了SL A2DQB 基因的准确分型技术必须借鉴于克隆测序。

Characterization of Sarcocystis species in domestic animals using a PCR-RFLP analysis of variation in the 18S rRNA gene: a cost-effective and simple technique for routine species identification

Thirteen restriction endonucleases were used to investigate nucleotide sequence variation in the 18S rRNA DNA of 88 individuals from ten Sarcocystis taxa collected as cysts from their intermediate hosts, swine, cattle and water buffalo. A DNA sequence of

Identification and characterization of a novel envelope protein in Rana grylio virus

Viral envelope proteins have been proposed to play significant roles in virus infection and assembly. In this study, an envelope protein gene, 53R, was cloned and characterized from Rana grylio virus (RGV), a member of the family Iridoviridae. Database searches found its homologues in all sequenced iricloviruses, and sequence alignment revealed several conserved structural features shared by virus capsid or envelope proteins: a myristoylation site, two predicted transmembrane domains and two invariant cysteine residues. Subsequently, RT-PCR and Western blot detection revealed that the transcripts encoding RGV 53R and the protein itself appeared late during infection of fathead minnow cells and that their appearance was blocked by viral DNA replication inhibitor, indicating that RGV 53R is a late expression gene. Moreover, immunofluorescence localization found an association of 53R with virus factories in RGV-infected cells, and this association was further confirmed by expressing a 53R-GFP fusion protein in pEGFP-N3/53R-transfected cells. Furthermore, detergent extraction and Western blot detection confirmed that RGV 53R was associated with virion membrane. Therefore, the current data suggest that RGV 53R is a novel viral envelope protein and that it may play an important role in virus assembly. This is thought to be the first report on a viral envelope protein that is conserved in all sequenced iridoviruses.

Genomic organization, gene duplication, and expression analysis of interleukin-1 beta in channel catfish (Ictalurus punctatus)

Interleukin-1 beta (IL-1 beta) is one of the pivotal early response pro-inflammatory cytokines that enables organisms to respond to infection and induces a cascade of reactions leading to inflammation. In spite of its importance and two decades of studies in the mammalian species, genes encoding IL-1 beta were not identified from non-mammalian species until recently. Recent research, particularly with genomic approaches, has led to sequencing of IL-1 beta from many species. Clinical studies also Suggested IL-1 beta as an immunoreagulatory molecule potentially useful for enhancing vaccination. However, no IL-1 beta genes have been identified from channel catfish, the primary aquaculture species from the United States. In this study, we identified two distinct cDNAs encoding catfish IL-1 beta. Their encoding genes were identified, sequenced, and characterized. The catfish IL-1 beta genes were assigned to bacterial artificial chromosome (BAC) clones. Genomic studies indicated that the IL-1 beta genes were tandemly duplicated on the same chromosome. Phylogenetic analysis of various IL-1 beta genes indicated the possibility of recent species-specific gene duplications in channel catfish, and perhaps also in swine and carp. Expression analysis indicated that both IL-1 beta genes were expressed, but exhibited distinct expression profiles in various catfish tissues, and after bacterial infection with Edwardsiella ictaluri. (c) 2005 Elsevier Ltd. All rights reserved.

Influence of Hypericum perforatum Extract on Piglet Infected with Porcine Respiratory and Reproductive Syndrome Virus

To study the influence of Hypericum perforatum extract (HPE) on piglets infected with porcine respiratory and reproductive syndrome virus (PRRSV), enzyme-labeled immunosorbent assay (ELISA) and cytopathic effect (CPE) were used to determine in vitro whether HPE could induce swine pulmonary alveolar macrophages (PAMs) to secrete IFN-gamma, and whether PRRSV titers in PAMs were affected by the levels of HPE-induced IFN-gamma. HPE (200 mg kg(-1)) was administrated by oral gavage to piglets infected with the PRRSV in vivo to observe whether HPE affected the viremia, lung viral titers, and weight gain of piglets infected with PRRSV. The results showed that HPE was capable of inducing PAMs to produce IFN-gamma in a dose dependent manner and HPE pretreatment was capable of significantly reducing PRRSV viral titers in PAMs (P<0.01). Administration of HPE to the PRRSV-infected animals significantly (P<0.05) reduced viremia over time as compared with the PRRSV-infected animals. But there was not significant decrease in lung viral titers at day 21 post-infection between the HPE-treated animals and the PRRSV-infected control piglets. There were no significant differences in weight gain over time among the HPE-treatment animals, the normal control, and the HPE control animals. The PRRSV-infected animals caused significant (P<0.01) growth retardation as compared with the HPE controls and the normal piglets. It suggested that HPE might be an effective novel therapeutic approach to diminish the PRRSV-induced disease in swine.

Reducing H2S production by O-2 feedback control during large-scale sewage sludge composting

Hydrogen sulfide (H2S) production patterns and the influence of oxygen (O-2) concentration were studied based on a well operated composting plant. A real-time, online multi-gas detection system was applied to monitor the concentrations of H2S and O-2 in the pile during composting. The results indicate that H2S was mainly produced during the early stage of composting, especially during the first 40 h. Lack of available O-2 was the main reason for H2S production. Maintaining the O-2 concentration higher than 14% in the pile could reduce H2S production. This study suggests that shortening the interval between aeration or aerating continuously to maintain a high O-2 concentration in the pile was an effective strategy for restraining H2S production in sewage sludge composting. (C) 2010 Elsevier Ltd. All rights reserved.