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。     

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.     

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.     

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。     

甘草酸对猪呼吸道冠状病毒的抗病毒作用

利用ST细胞研究了甘草酸对猪呼吸道冠状病毒(PRCV)体外复制过程中的抑制作用。通过RT-PCR和Western blot检测发现甘草酸能有效抑制PRCV的体外复制而对ST细胞不引起明显细胞毒作用。抑制效果与甘草酸的剂量成正比关系。PRCV病毒核衣壳蛋白的表达无论在mRNA水平还是在蛋白质水平均随着甘草酸添加量的增加而降低,提示甘草酸是一种有效的抗PRCV药物。     

PK-15细胞微载体悬浮培养生产猪细小病毒的工艺研究

通过对猪细小病毒接毒时间TOI、MOI和收毒时间进行优化,开发了一种基于PK-15细胞静置培养的猪细小病毒生产工艺,最大病毒滴度达到107.5TCID50/ml。通过进一步优化接毒时间,成功建立了基于PK-15细胞反应器微载体悬浮培养的猪细小病毒培养工艺,在5L反应器上最大病毒滴度达到107.2TCID50/ml。首次发现乳酸对葡萄糖得率与病毒滴度的正相关性,当猪细小病毒滴度处于最大值时,乳酸对葡萄糖得率也达到最大值,可作为指针病毒滴度及收毒时间的重要参数。     

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.     

Myeloid Cell Arg1 Inhibits Control of Arthritogenic Alphavirus Infection by Suppressing Antiviral T Cells

Arthritogenic alphaviruses, including Ross River virus (RRV) and chikungunya virus (CHIKV), are responsible for explosive epidemics involving millions of cases. These mosquito-transmitted viruses cause inflammation and injury in skeletal muscle and joint tissues that results in debilitating pain. We previously showed that arginase 1 (Arg1) was highly expressed in myeloid cells in the infected and inflamed musculoskeletal tissues of RRV- and CHIKV-infected mice, and specific deletion of Arg1 from myeloid cells resulted in enhanced viral control. Here, we show that Arg1, along with other genes associated with suppressive myeloid cells, is induced in PBMCs isolated from CHIKV-infected patients during the acute phase as well as the chronic phase, and that high Arg1 expression levels were associated with high viral loads and disease severity. Depletion of both CD4 and CD8 T cells from RRV-infected Arg1-deficient mice restored viral loads to levels detected in T cell-depleted wild-type mice. Moreover, Arg1-expressing myeloid cells inhibited virus-specific T cells in the inflamed and infected musculoskeletal tissues, but not lymphoid tissues, following RRV infection in mice, including suppression of interferon-γ and CD69 expression. Collectively, these data enhance our understanding of the immune response following arthritogenic alphavirus infection and suggest that immunosuppressive myeloid cells may contribute to the duration or severity of these debilitating infections.     

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.     

无环鸟苷抗乙型肝炎病毒复制长短程疗法随访观察

慢性乙型肝炎病毒感染s5例,随机分无环鸟苷长程组22例,短程组11例和对照组22例。经一年随访观察,无环鸟苷治疗组见血清HBeAg、DNA-P和HBV-DNA有规律性阴转,短程组于12个月又有部分指标阳转,长程组阴转较多,其血清HBeAg、DNA-P和HBV-DNA阴转率与短程和对照组比较有显著性差异。一年结果四项病毒复制指标全部阴转例数与对照组有显著性差异,结果表明,无环鸟苷长疗程是有较好疗效。     

猪细小病毒核酸疫苗的构建及其对小鼠免疫原性的研究

将猪细小病毒VP2基因克隆至pCI-neo真核表达载体中,构建了pCIneo-VP2重组质粒,转染至PK-15细胞中,利用免疫荧光方法检测在体外表达情况;并以小鼠为动物模型,将pCIneo-VP2、pCI-neo重组质粒、猪细小病毒活疫苗和对照组通过肌肉注射进行免疫,检测免疫小鼠的淋巴细胞转化功能,特异性CTL杀伤活性和血清抗体滴度。结果显示,pCIneo-VP2在体外能够诱导PK-15细胞表达VP2蛋白,小鼠注射pCIneo-VP2质粒 1周后能够诱导机体产生抗体,4周时达到峰值,与活疫苗对照组产生的抗体滴度、诱导T淋巴细胞增殖和诱导强的细胞毒性基本一致。试验表明,构建的pCIneo-VP2能够有效诱导机体产生体液免疫和细胞免疫,为研制出高效、新型猪细小病毒疫苗提供了科学依据和试验依据。     

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。     

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。     

猪流行性腹泻病毒与宿主抗病毒天然免疫抑制

猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)能引起猪腹泻等肠道疾病,属于α属冠状病毒,它的爆发给很多国家养猪业造成了严重的经济损失。2010年以来,PEDV感染在中国出现大规模爆发,一种突变型PEDV也于2013年在美国出现并迅速传播。 RNA病毒能够通过Toll样受体通路3（TLR3）和RIG-I样受体通路（RLR）诱导I型干扰素的产生。但以往的研究表明,PEDV感染能抑制I型干扰素的合成。近年来有关PEDV调节宿主天然免疫应答的研究取得了很大进展。PEDV主要通过编码作为干扰素拮抗剂的病毒蛋白以及隐藏病毒自身病原相关分子模式（PAMP）等两种方式逃逸宿主天然免疫应答。目前已报道,PEDV非结构蛋白1可通过降解CBP阻碍干扰素调节因子3(IRF-3)组装成增强子复合体;木瓜蛋白酶样蛋白酶可通过其去泛素化酶活性阻断天然免疫信号通路传递;3C样蛋白酶可通过剪切NEMO发挥干扰素拮抗剂活性;核衣壳蛋白通过结合TBK1抑制I型干扰素产生。PEDV也可通过合成加帽酶隐藏其病原相关分子dsRNA来避免激活天然免疫通路。PEDV抗病毒天然免疫机制阐明为研究PEDV感染免疫和致病机制提供了重要的理论依据,为研发抗PEDV新型疫苗和药物提供了基础。     

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.     

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.     

Effect of Poxvirus Infection on Host Cell Deoxyribonucleic Acid Synthesis

Deoxyribonucleic acid (DNA) synthesis was studied in poxvirus-infected cells by measuring (14)C-thymidine incorporation into viral and host cell DNA. A complete separation of the two species of DNA was achieved by combining the previously used "Dounce method" with a separation method based on different reannealing properties of viral and vertebrate DNA. Shortly after infection of HeLa cells with poxviruses, a burst of viral DNA synthesis occurred in the cytoplasm, but a rapid inhibition of host-cell DNA synthesis in the nucleus was observed. This inhibition of cellular DNA synthesis was also found if an accumulation of viral DNA was prevented. At high multiplicites, ultraviolet-irradiated virus inhibited host-cell DNA synthesis to the same extent as fully infectious poxvirus. Under the same conditions, heating at 60 C for 15 min caused a decrease in the ability of cowpox virus to inhibit host-cell DNA synthesis, but did not produce the same effect on vaccinia virus strain WR.