Isolation of Porcine Parvovirus from Commercial Trypsin

The isolation of porcine parvovirus from a lot of commercial 1:250 trypsin is reported and the method is described.     

猪细小病毒单克隆抗体的制备和鉴定

目的制备猪细小病毒（PPV）杂交瘤细胞株,并对其分泌的PPV单克隆抗体进行鉴定。方法按常规方法制备并获得2株杂交瘤细胞。用染色体分析对杂交瘤细胞进行鉴定,用间接ELISA、免疫过氧化物酶单层试验（IPMA）和间接免疫荧光试验（IFA）对其分泌的单克隆抗体进行效价测定、亚型鉴定和特异性鉴定。结果得到2株分泌单克隆抗体的杂交瘤细胞株2H9、1F9,染色体数目介于90～110之间。细胞上清效价均达1∶1×104,腹水效价均达1∶1×107,其亚型分别为IgG1、IgM,均为kappa链。2H9、1F9单抗与猪繁殖与呼吸综合征病毒（PRRSV）、猪瘟病毒（CSFV）、猪伪狂犬病毒（PRV）、猪圆环病毒I型（PCV-1）、猪圆环病毒Ⅱ型（PCV-2）、乙脑病毒（JEV）等均无交叉反应。IPMA和IFA检测结果显示2H9、1F9单抗均能与接种于PK-15细胞的PPV发生特异性反应。结论成功制备了2株抗PPV杂交瘤细胞株,证实其产生的单克隆抗体具有良好的特异性和敏感性。     

Complete Genome Sequence of a Novel Porcine Parvovirus in China

The porcine parvovirus JT strain (PPV-JT) was isolated from a piglet showing nonsuppurative myocarditis in Shandong, China, in 2010. The complete genomic sequence of PPV-JT, 4,941 bp long, was determined from clones made from replicative form (RF) DNA. The genomic analysis demonstrated that the PPV-JT might be involved in a recombination event, which will help us understand the molecular characteristics and evolutionary of PPV in China.     

猪圆环病毒2型、猪繁殖与呼吸综合征病毒及猪细小病毒混合感染的流行病学调查

根据GenBank上发表的PRRSV ORF7、PPV VP2及PCV的基因组序列设计合成引物,建立了分别用于检测PRRSV、PPV和PCV的RT-PCR、PCR及复合PCR方法.应用建立的复合PCR方法对送检的127份病料进行了PCV的检测,对鉴定为PCV2阳性的67份病料再分别进行PRRSV和PPV的检测,以确定猪群中PCV2与PRRSV和/或PPV混合感染情况,结果表明,35份样品表现为PRRSV与PCV2混合感染,占样品总数的52.3%;18份样品表现为PCV2与PPV混合感染,占26.9%.另外,还有一定比例的三重感染,共5个样品,占7.5%.由此可见,猪群中PCV2与PRRSV及PPV混合感染比较普遍.     

检测PCV2、PPV、PRV疫苗株与野毒株的多重PCR方法

本文建立了一种同时检测猪圆环病毒2型（PCV2）、细小病毒（PPV）、及伪狂犬病毒（PRV）疫苗株与野毒株的多重PCR方法.根据GenBank上发表的PCV2、PPV和PRV gB、gE基因序列,针对各自保守区各设计一对特异性引物,用这四对引物对同一样品中的PCV2、PPV和PRV gB、gE进行检测,结果可同时扩增出269bp（PCV2）、581bp（PPV）、372bP（PRV gB）及147bp（PRV gE）四条特异性片段.对JEV、PRRSRV、大肠杆菌和双蒸水的PCR扩增结果均为阴性;敏感性测定结果表明,该多重PCR能检出10pg PCV2、PPV和PRV gB、gE检测敏感度分别为10^-6.2、10^-3.8、10^-5.8TCID50的模板.该方法的建立对临床上进行这三种疾病的鉴别诊断和混合感染的检测具有重要意义.     

猪圆环病毒2型、猪繁殖与呼吸综合征病毒及猪细小病毒混合感染的流行病学调查

根据GenBank上发表的PRRSVORF7、PPVVP2及PCV的基因组序列设计合成引物,建立了分别用于检测PRRSV、PPV和PCV的RT-PCR、PCR及复合PCR方法。应用建立的复合PCR方法对送检的127份病料进行了PCV的检测,对鉴定为PCV2阳性的67份病料再分别进行PRRSV和PPV的检测,以确定猪群中PCV2与PRRSV和,或PPV混合感染情况,结果表明,35份样品表现为PRRSV与PCV2混合感染,占样品总数的52．3％;18份样品表现为PCV2与PPV混合感染,占26．9％。另外,还有一定比例的三重感染,共5个样品,占7．5％。由此可见,猪群中PCV2与PRRSV及PPV混合感染比较普遍。     

以CpG为佐剂的犬细小病毒基因疫苗的初步研究

目的研制犬细小病毒(CPV)基因疫苗。方法以CPV VP2基因为基因免疫的目的基因,以pcDNA3和pcDNAK质粒为基因免疫的载体,以非甲基化的胞嘧啶鸟嘌呤二核苷酸(CpG)为核心的免疫刺激序列为免疫佐剂,构建重组质粒并免疫BALB/c小鼠和毕格犬。结果经pcDNA3-VP2C1(含1个拷贝CpG基序)基因免疫的BALB/c小鼠能产生抗CPV血凝抑制抗体;对于经CPV灭活苗初次免疫的毕格犬,用pcDNAK-VP2C2(含2个拷贝CpG基序)质粒免疫产生的再次免疫应答优于pcDNA3-VP2C1。结论VP2基因、pcDNAK和犬源CpG可用于CPV基因疫苗的进一步研究。     

Characterization of a Novel Porcine Parvovirus Tentatively Designated PPV5

A new porcine parvovirus (PPV), provisionally designated as PPV5, was identified in U.S. pigs. Cloning and sequencing from a circular or head-to-tail concatemeric array revealed that the PPV5 possesses the typical genomic organization of parvoviruses with two major predicted open reading frames (ORF1 and ORF2), and is most closely related to PPV4 with overall genomic identities of 64.1–67.3%. The amino acid identities between PPV5 and PPV4 were 84.6%–85.1% for ORF1 and 54.0%–54.3% for ORF2. Unlike PPV4, but similar to bovine parvovirus 2 (BPV2), PPV5 lacks the additional ORF3 and has a much longer ORF2. Moreover, the amino acid sequences of ORF1 and ORF2 of BPV2 showed higher homologies to PPV5 than to PPV4. The conserved motifs of the Ca²⁺ binding loop (YXGXG) and the catalytic center (HDXXY) of phospholipase A₂ (PLA₂) were identified in VP1 (ORF2) of PPV5, as well as in BPV2, but were not present in PPV4. Phylogenetic analyses revealed that PPV5, PPV4 and BPV2 form a separate clade different from the genera Parvovirus and Bocavirus. Further epidemiologic investigations of PPV4 and PPV5 in U.S. pigs of different ages indicated a slightly higher prevalence for PPV5 (6.6%; 32/483) compared to PPV4 (4.1%; 20/483), with detection of concurrent PPV4 and PPV5 in 15.6% (7/45) of lungs of infected pigs. Evidence for potential vertical transmission or association with reproductive failure was minimal for both PPV4 and PPV5. The high similarity to PPV4 and the lack of ORF3 may suggest PPV5 is an intermediate of PPV4 during the evolution of parvoviruses in pigs.     

Study of Tissue-Specific CpG Methylation of DNA in Extended Genomic Loci

Modern approaches for studies on genome functioning include investigation of its epigenetic regulation. Methylation of cytosines in CpG dinucleotides is an inherited epigenetic modification that is responsible for both functional activity of certain genomic loci and total chromosomal stability. This review describes the main approaches for studies on DNA methylation. Under consideration are site-specific approaches based on bisulfite sequencing and methyl-sensitive PCR, whole-genome approaches aimed at searching for new methylation hot spots, and also mapping of unmethylated CpG sites in extended genomic loci.     

一氧化氮对猪细小病毒体外增殖的影响研究

本文研究了来源于不同前体物的一氧化氮(Nitro oxide,NO)对猪细小病毒(Porcine parvovirus,PPV)体外增殖的影响.结果表明,NO前体物S-硝基-N-乙酰青霉胺(SNAP)、L-精氨酸(L-Arg)均能够有效地诱导PK-15细胞产生NO,进而显著地抑制PPV在PK-15细胞上的复制,其效果与前体物的浓度呈正相关,在浓度为100μmol/L和200μmol/L时,SNAP产生NO的能力与抑制病毒复制的作用要强于L-Arg.在病毒感染前6 h和3 h添加SNAP或L-Arg对病毒复制的抑制作用比在病毒感染后3 h和6 h添加的作用强,表明NO的抗病毒作用主要发生在病毒感染的初始阶段.此外,添加具有抑制L-Arg产生NO作用的N-硝基-L-精氨酸(L-NNA)能抵消L-Arg体外抗病毒的作用.     

一氧化氮对猪细小病毒体外增殖的影响研究

本文研究了来源于不同前体物的一氧化氮(Nitro oxide,NO)对猪细小病毒(Porcine paruouirus,PPV)体外增殖的影响。结果表明,NO前体物S-硝基-N-乙酰青霉胺(SNAP)、L-精氨酸(L-Arg)均能够有效地诱导PK-5细胞产生NO,进而显著地抑制PPV在PK-5细胞上的复制,其效果与前体物的浓度呈正相关,在浓度为100μmol/L和200μmol／L时,SNAP产生NO的能力与抑制病毒复制的作用要强于L-Arg。在病毒感染前6h和3h添加SNAP或L-Arg对病毒复制的抑制作用比在病毒感染后3h和6h添加的作用强,表明NO的抗病毒作用主要发生在病毒感染的初始阶段。此外,添加具有抑制L-Arg产生NO作用的N-硝基-L-精氨酸(L-NNA)能抵消L-Arg体外抗病毒的作用。     

硒对猪细小病毒体外增殖抑制作用的研究

本文以PK-15细胞为模型,研究了亚硒酸钠、硒蛋氨酸和海藻硒多糖等三种硒化合物对猪细小病毒体外复制 的抑制作用,以及还原型谷胱甘肽、D-甘露醇等氧自由基清除剂对不同来源硒的抑制病毒作用的影响。结果表明: 三种硒化合物对猪细小病毒在PK-15中的复制呈现不同程度的抑制作用,在相同浓度时其强度依次为硒蛋氨酸、 亚硒酸钠、海藻硒多糖,随着浓度的增加,其抑制作用逐渐增强,呈剂量依赖性关系。还原型谷胱甘肽和甘露醇均 有增强硒的抑制病毒复制作用,两者同时添加时,协同增强硒的抑制病毒复制作用。     

Parvovirus b19 DNA CpG dinucleotide methylation and epigenetic regulation of viral expression

CpG DNA methylation is one of the main epigenetic modifications playing a role in the control of gene expression. For DNA viruses whose genome has the ability to integrate in the host genome or to maintain as a latent episome, a correlation has been found between the extent of DNA methylation and viral quiescence. No information is available for Parvovirus B19, a human pathogenic virus, which is capable of both lytic and persistent infections. Within Parvovirus B19 genome, the inverted terminal regions display all the characteristic signatures of a genomic CpG island; therefore we hypothesised a role of CpG dinucleotide methylation in the regulation of viral genome expression.The analysis of CpG dinucleotide methylation of Parvovirus B19 DNA was carried out by an aptly designed quantitative real-time PCR assay on bisulfite-modified DNA. The effects of CpG methylation on the regulation of viral genome expression were first investigated by transfection of either unmethylated or in vitro methylated viral DNA in a model cell line, showing that methylation of viral DNA was correlated to lower expression levels of the viral genome. Then, in the course of in vitro infections in different cellular environments, it was observed that absence of viral expression and genome replication were both correlated to increasing levels of CpG methylation of viral DNA. Finally, the presence of CpG methylation was documented in viral DNA present in bioptic samples, indicating the occurrence and a possible role of this epigenetic modification in the course of natural infections.The presence of an epigenetic level of regulation of viral genome expression, possibly correlated to the silencing of the viral genome and contributing to the maintenance of the virus in tissues, can be relevant to the balance and outcome of the different types of infection associated to Parvovirus B19.     

硒对猪细小病毒体外增殖抑制作用的研究

本文以PK-15细胞为模型,研究了亚硒酸钠、硒蛋氨酸和海藻硒多糖等三种硒化合物对猪细小病毒体外复制的抑制作用,以及还原型谷胱甘肽、D-甘露醇等氧自由基清除剂对不同来源硒的抑制病毒作用的影响.结果表明三种硒化合物对猪细小病毒在PK-15中的复制呈现不同程度的抑制作用,在相同浓度时其强度依次为硒蛋氨酸、亚硒酸钠、海藻硒多糖,随着浓度的增加,其抑制作用逐渐增强,呈剂量依赖性关系.还原型谷胱甘肽和甘露醇均有增强硒的抑制病毒复制作用,两者同时添加时,协同增强硒的抑制病毒复制作用.     

Methylation Pattern of Lambda Deoxyribonucleic Acid

Deoxyribonucleic acid (DNA) extracted from phage lambda grown on Escherichia coli K-12 strain W4032 had 113 +/- 10 5-methylcytosine residues and 215 +/- 20 6-methyl adenine residues per genome, as determined by three independent methods. These methylated nucleotides were distributed equally among the two strands of lambda DNA. Shearing of double-stranded DNA to half-length fragments revealed a slight deficiency of 5-methyl cytosine in the 55% guanine plus cytosine half. Shearing the DNA to fragments of smaller length showed that the distribution of methylated nucleotides along the double helix was uniform with the exception of an undermethylated fragment arising from the center of the lambda DNA molecule. The implication of these results for the function of methylated nucleotides in the lambda DNA molecule is discussed.     

Antiviral Effect of Diammonium Glycyrrhizinate on Cell Infection by Porcine Parvovirus

The goal of this study was to evaluate how two new hydrolysates from poultry by-products act on ten lactobacilli growth kinetics when supplemented to the growth medium. These effects were compared with ones induced by two most common commercial hydrolysates, i.e., tryptone and peptone. Growth medium, supplemented with one of new hydrolysates, 78T, as only nitrogen source, can sustain the maximum growth rate and the biomass yield in the same way of MRS, reach of different nitrogen sources. Moreover aminopeptidase activities (AA) of each strain were determined to investigate the effect of the growth condition on the modulation of aminopeptidase pattern. Five cell extracts of each ten strains, obtained from their cultivation in MRS and in the presence of the two common hydrolysates and the two new ones, were considered. AA was investigated against five different chromogenic substrates: β-naphthyl amide derivatives of l-anomers of leucine, lysine, proline, glycine–proline, and phenilalanine–proline. A great variability of AA was observed among the strains: also strains belonging to the same species showed peculiar AA profile.     

Prognostic Value of PLAGL1-Specific CpG Site Methylation in Soft-Tissue Sarcomas

Soft tissue sarcomas (STS) are rare, complex tumors with a poor prognosis. The identification of new prognostic biomarkers is needed to improve patient management. Our aim was to determine the methylation status of the 118 CpG sites in the PLAGL1 tumor-suppressor gene P1 CpG island promoter and study the potential prognostic impact of PLAGL1 promoter methylation CpG sites in STS. Training cohorts constituted of 28 undifferentiated sarcomas (US) and 35 leiomyosarcomas (LMS) were studied. PLAGL1 mRNA expression was investigated by microarray analysis and validated by RT-qPCR. Pyrosequencing was used to analyze quantitative methylation of the PLAGL1 promoter. Associations between global promoter or specific CpG site methylation and mRNA expression were evaluated using Pearson’s product moment correlation coefficient. Cox univariate and multivariate proportional hazard models were used to assess the predictive power of CpG site methylation status. Sixteen CpG sites associated with PLAGL1 mRNA expression were identified in US and 6 in LMS. Statistical analyses revealed an association between CpG107 methylation status and both overall and metastasis-free survival in US, which was confirmed in a validation cohort of 37 US. The exhaustive study of P1 PLAGL1 promoter methylation identified a specific CpG site methylation correlated with mRNA expression, which was predictive for both metastasis-free and overall survival and may constitute the first US-specific biomarker. Such a biomarker may be relevant for identifying patients likely to derive greater benefit from treatment.