一种抗AIV共有独特型抗体具有AIV血凝素分子的内影象

用纯化的鸡抗禽流感病毒(AIV)IgG作免疫原,通过单克隆抗体技术制备出1株分泌针对鸡抗AIV和兔抗AIV共有独特型抗体的杂交瘤细胞。竞争抑制试验、特异性检验和诱导产生血凝抑制抗体的功能实验证明,此抗体具有AIV血凝素分子的内影象。     

一种抗AIV共有独特型抗体具有AIV血凝素分子的内影象

用纯化的鸡抗禽流感病毒(AIV)IgG作免疫原,通过单克隆抗体技术制备出1株分泌针对鸡抗AIV和兔抗AIV共有独特型抗体的杂交瘤细胞.竞争抑制试验、特异性检验和诱导产生血凝抑制抗体的功能实验证明,此抗体具有AIV血凝素分子的内影象.     

马铃薯卷叶病毒单克隆抗体的制备

采用杂交瘤技术,以马铃薯卷叶病毒(Potato Leafroll Virns,PLRV)为抗原,用直接将病毒注入脾脏和随后尾静脉注射的方法,免疫BALB/C小鼠。将免疫小鼠的脾细胞与小鼠骨髓瘤细胞SP2/0融合。用Dot-ELISA和间接血凝试验筛选分泌抗马铃薯卷叶病毒抗体的阳性克隆,建立了分泌抗PLRV单克隆抗体的杂交瘤细胞株。用微量玻片双扩散法测定单克隆抗体亚类为IgG_1和IgG2a,轻链为λ。注射杂交瘤细胞株A_1、A_3、C_3和D_3于小鼠腹腔,制备出含高效价单克隆抗体的腹水。用获得的四种单克隆抗体对马铃薯卷叶病毒15个分离物进行了鉴定。     

兔出血症病毒与细小病毒抗原相关性试验

用间接ELISA、ELISA交叉阻断法和交叉血凝抑制试验对兔出血症病毒(RHDV)与6种细小病毒进行抗原相关性试验。用间接ELISA证实,RFIDV与它们有轻度交叉关系,其抗原相关值分别为:小鼠细小病毒(MVM)5.59％;鹅细小病毒(GPV)3.54％;猪细小病毒(PPV)1.76％;水貂肠炎病毒0.7％。细小病毒间的抗原相关值:MEV与PPV为31.6％,MEV与MVM为35.36％;而CPV与MEM、PPV、MVM的相关值均为零,即无相关性。在ELISA交叉阻断法中证实:犬细小病毒(CPV)、猫泛白细胞减少症病毒(FPV)和MEV均不能阻断RHDV与其抗体结合,仅GPV有轻度阻断作用,其最大阻断率为40％。在血凝交叉抑制试验中,未发现RHDV与细小病毒及其相应抗体间存在交叉抑制现象。以上结果表明RHDV与细小病毒在血清学方面有轻度相关性。     

抗传染性犬肝炎病毒单克隆抗体的制备及鉴定

目的制备传染性犬肝炎病毒单克隆抗体。方法将用纯化的犬腺病毒1型（CAV-1）免疫的BALB／c小鼠脾细胞与SP2／0细胞在聚乙二醇作用下融合,通过酶联免疫吸附试验（ELISA）和免疫酶试验（1EA）筛选,以有限稀释法克隆3次,制备单克隆抗体,并对制备完成的单克隆抗体进行生物学鉴定。结果成功得到2株能稳定分泌抗CAV-1的单克隆抗体杂交瘤细胞,命名为1B、2H3,经鉴定其亚型分别为IgG2a和IgG2a,2株均为kappa链。腹水的ELISA效价可达10^7-10^8,IEA效价可达1：2560—1：5120。该单克隆抗体与CPV、CDV、FPV、CCV病毒无交叉反应。结论成功制备了抗CAV-1单克隆抗体,为进一步建立相关诊断方法奠定了基础。     

犬细小病毒的PCR诊断试剂盒的研制

目的　建立检测犬细小病毒的PCR诊断试剂盒。方法　通过在犬细小病毒 (CPV)的基因组中设计的特异性寡核苷酸引物 ,利用PCR技术研制犬细小病毒的PCR诊断试剂盒。结果　在特定的反应条件下 ,使用该试剂盒能特异地扩增含有犬细小病毒的样品 ,且能检出痕量 (10ng)的犬细小病毒DNA ,被测粪样只需进行简单煮沸就能进行PCR反应 ,该试剂盒能在常温下保存 1周以上、4℃保存 1年以上。同血凝试验 (HA〕及单克隆抗体ELISA方法比较 ,对 6 6份样品进行检测 ,显示该PCR试剂盒具有特异、灵敏等优点。结论　成功研制了犬细小病毒的PCR诊断试剂盒 ,利用该试剂盒能从DNA水平上对犬细小病毒进行监控     

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。     

四种动物病毒的细胞培养及血凝检测的比较研究

四种动物病毒的细胞培养及血凝检测的比较研究李天宪,赵林,罗怡珊,冯锋（中国科学院武汉病毒研究所,武汉４３００７１）关键词细小病毒,细胞培养,细胞病变,血凝试验云豹肠炎病毒（ＬＰＶ）、水貂肠炎病毒（ＭＥＶ）、犬肠炎病毒（ＣＰＶ）和猫泛白细胞减少症病毒（...     

汉坦病毒抗原性差异及其基因分型

用抗汉坦病毒单克隆抗体（Ｍｃ－Ａｂ）对汉坦病毒Ｂ７８株（来自病人血）和Ｒ１７８株（分离自褐家鼠）进行间接免疫荧光试验,以分析病毒的单克隆抗体反应谱,又用病毒免疫血清进行交叉中和试验,结果表明,两株病毒均具有双型（Ⅰ型／ＨＴＮ型和Ⅱ型／ＳＥＯ型）反应特征,但Ｒ１７８株基本上属于Ⅰ型。用汉坦病毒Ⅰ￣Ⅳ型型特异性引物进行ＰＣＲ扩增,Ｂ７８株用Ⅰ型和Ⅱ型引物均得到特异性扩增,而Ｒ１７８株虽经重复扩增均未见     

用核酸免疫技术制备抗丙肝病毒C区单克隆抗体

用丙肝病毒Ｃ＋Ｅ１区真核表达质粒ｐｃＤＮＡ－ＨＣＶ／Ｃ＋Ｅ１按４００ｕｇ／只剂量免疫ＢＡＬＢ／Ｃ小鼠,１４周后同一剂量再加强免疫一次。加强免疫后２周,在５０％ＰＥＧ１４５０介导下将脾细胞与ＳＰ２／０小鼠骨髓瘤细胞（５１）融合。实验结果融合率达５４．３％（３１３／５７６）。阳性率为５．４％（１７”３１３）。克隆化后得到６株稳定分泌抗丙肝病毒Ｃ区单克隆抗体杂交瘤细胞株。这６株杂交瘤均产生ＩｇＭ抗体,接种ＢＡＬＢ／Ｃ小鼠后产生腹水的效价为１６４－１３２０（ＥＬＩＳＡ）。     

B-cell epitopes of canine parvovirus: distribution on the primary structure and exposure on the viral surface.

Ten antigenic sites on canine parvovirus (CPV) were mapped with a complete set of overlapping nonapeptides of the capsid proteins VP1 and VP2: five of these sites were recognized by sera from CPV-infected dogs, three were recognized by a rabbit anti-CPV antiserum, and two were recognized by murine monoclonal anti-CPV antibodies. A region covering the first 21 amino-terminal amino acid residues of VP2 was recognized by three sera from infected dogs, one neutralizing rabbit antiserum, and one neutralizing murine monoclonal antibody. Immunoabsorption experiments with full virions indicated that at least 6 of the 10 antigenic sites are located on the surface. Of these six, three sites occur in the amino terminus of VP2. When superimposed on the three-dimensional structure of canine parvovirus (J. Tsao, M. S. Chapman, M. Agbandje, W. Keller, K. Smith, H. Wu, M. Luo, T. J. Smith, M. G. Rossmann, R. W. Compans, and C. R. Parrish, Science 251:1456-1464, 1991), the other three epitopes are located on two loops of VP2 which form the highly exposed "spike" around the threefold-symmetry axis of the virus. Thus, these regions (amino terminus and loops 1 and 3) are of interest as major target sites for induction of neutralizing antibodies.     

Self-assembly of virus-like particles of canine parvovirus capsid protein expressed from Escherichia coli and application as virus-like particle vaccine

Canine parvovirus disease is an acute infectious disease caused by canine parvovirus (CPV). Current commercial vaccines are mainly attenuated and inactivated; as such, problems concerning safety may occur. To resolve this problem, researchers developed virus-like particles (VLPs) as biological nanoparticles resembling natural virions and showing high bio-safety. This property allows the use of VLPs for vaccine development and mechanism studies of viral infections. Tissue-specific drug delivery also employs VLPs as biological nanomaterials. Therefore, VLPs derived from CPV have a great potential in medicine and diagnostics. In this study, small ubiquitin-like modifier (SUMO) fusion motif was utilized to express a whole, naturalVP2 protein of CPV in Escherichia coli. After the cleavage of the fusion motif, the CPV VP2 protein has self-assembled into VLPs. The VLPs had a size and shape that resembled the authentic virus capsid. However, the self-assembly efficiency of VLPs can be affected by different pH levels and ionic strengths. The mice vaccinated subcutaneously with CPV VLPs and CPV-specific immune responses were compared with those immunized with the natural virus. This result showed that VLPs can effectively induce anti-CPV specific antibody and lymphocyte proliferation as a whole virus. This result further suggested that the antigen epitope of CPV was correctly present on VLPs, thereby showing the potential application of a VLP-based CPV vaccine.     

犬、猫、貂细小病毒通用快速诊断盒的研制

本文报道,由犬细小病毒灭活抗原、特异抗体、戊二醛醛化猪红细胞及微量血凝板、稀释棒等组成的犬、猫、貂细小病毒通用快诊盒,可用于犬细小病毒性肠炎、猫泛白细胞减少症和水貂肠炎的特异诊断,适合于基层和野外应用,并可在接到病料后4小时内报告结果,有关试剂的有效期在一年以上。     

Recombinant vaccine for canine parvovirus in dogs.

VP2 is the major component of canine parvovirus (CPV) capsids. The VP2-coding gene was engineered to be expressed by a recombinant baculovirus under the control of the polyhedrin promoter. A transfer vector that contains the lacZ gene under the control of the p10 promoter was used in order to facilitate the selection of recombinants. The expressed VP2 was found to be structurally and immunologically indistinguishable from authentic VP2. The recombinant VP2 shows also the capability to self-assemble, forming viruslike particles similar in size and appearance to CPV virions. These viruslike particles have been used to immunize dogs in different doses and combinations of adjuvants, and the anti-CPV responses have been measured by enzyme-linked immunosorbent assay, monolayer protection assays, and an assay for the inhibition of hemagglutination. A dose of ca. 10 micrograms of VP2 was able to elicit a good protective response, higher than that obtained with a commercially available, inactivated vaccine. The results indicate that these viruslike particles can be used to protect dogs from CPV infection.     

Polarized entry of canine parvovirus in an epithelial cell line.

The binding and uptake of canine parvovirus (CPV) in polarized epithelial cells were investigated by growing the cells on a permeable support and inoculating with the virus either from the apical or basolateral surface. Binding of radiolabeled CPV occurred preferentially on the basolateral surface. In contrast, when a similar experiment was carried out on nonpolarized A72 cells, virus binding occurred regardless of the direction of virus input. Binding appeared to be specific for CPV and could not be competitively inhibited by either bovine or porcine parvovirus. Analysis of the binding data revealed a high-affinity receptor (10(5) per cell) for CPV on the basolateral surfaces of MDCK cells (Kd, 29 pM). In indirect immunofluorescence studies, virus entered only from the basolateral surfaces of MDCK cells. These results provide evidence for a functional CPV-specific receptor that is expressed only on the basolateral surfaces of polarized epithelial cells, a result that has interesting consequences for viral pathogenesis.     

Development of a polyclonal antibody-based AC-ELISA and its comparison with PCR for diagnosis of canine parvovirus infection

A polyclonal antibody-based antigen-capture ELISA (AC-ELISA) has been developed for detection of Canine parvovirus (CPV) antigens in faecal samples of dogs. The assay uses rabbit anti-CPV polyclonal antibody as the capture antibody, guinea pig anti-CPV polyclonal antibody as tracing antibody and anti-guinea pig HRPO conjugate as the detection system. The optimum dilution of the capture antibody and the tracing antibody capable of detecting the CPV-2 antigens was found to be 1:1 600 and 1:400, respectively, in the check-board titration. In this study, a total of 152 samples (129 faecal samples and 23 cell culture supernatant) were tested both by AC-ELISA and by polymerase chain reaction (PCR). Of the samples tested, 69 and 78 samples were found positive by AC-ELISA and PCR, respectively. The AC-ELISA had relative sensitivity, relative specificity and accuracy of 88.4%, 100.0% and 91.4% respectively. The analytical sensitivity of AC-ELISA was estimated to be 10^2.8 TCID₅₀/mL whereas PCR sensitivity was 10^0.8 TCID₅₀/mL. The AC-ELISA is a simple, quick and reliable method for screening large numbers of faecal samples of dogs suspected of CPV infection.     

The replication of a cytoplasmic polyhedrosis virus from Chrysodeixis eriosoma (Lepidoptera: Noctuidae) in Spodoptera frugiperda cells

A cytoplasmic polyhedrosis virus (CPV) from Chrysodeixis eriosoma (Lepidoptera: Noctuidae) replicated in Spodoptera frugiperda cells. Low rates of infection were achieved, even at high multiplicities of infection and TCID₅₀ assays showed that there was negligible release of virus particles from infected cells. In an infected focus assay, based on formation of PIB, the dose-response data demonstrated that a single particle could initiate infection. No loss of infectivity occurred in virus preparations stored at 4°, ?20°, or ?90°C, but infectivity of virus stored at 20°C declined sharply. A small isometric virus contaminant was present in some CPV preparations and its interaction with the CPV is discussed. Limited CPV infection was achieved in Trichoplusia ni cells, but attempts to infect Aedes aegypti cells were unsuccessful.     

Cellular uptake and infection by canine parvovirus involves rapid dynamin-regulated clathrin-mediated endocytosis, followed by slower intracellular trafficking

Canine parvovirus (CPV) is a small, nonenveloped virus that is a host range variant of a virus which infected cats and changes in the capsid protein control the ability of the virus to infect canine cells. We used a variety of approaches to define the early stages of cell entry by CPV. Electron microscopy showed that virus particles concentrated within clathrin-coated pits and vesicles early in the uptake process and that the infecting particles were rapidly removed from the cell surface. Overexpression of a dominant interfering mutant of dynamin in the cells altered the trafficking of capsid-containing vesicles. There was a 40% decrease in the number of CPV-infected cells in mutant dynamin-expressing cells, as well as a approximately 40% decrease in the number of cells in S phase of the cell cycle, which is required for virus replication. However, there was also up to 10-fold more binding of CPV to the surface of mutant dynamin-expressing cells than there was to uninduced cells, suggesting an increased receptor retention on the cell surface. In contrast, there was little difference in virus binding, virus infection rate, or cell cycle distribution between induced and uninduced cells expressing wild-type dynamin. CPV particles colocalized with transferrin in perinuclear endosomes but not with fluorescein isothiocyanate-dextran, a marker for fluid-phase endocytosis. Cells treated with nanomolar concentrations of bafilomycin A1 were largely resistant to infection when the drug was added either 30 min before or 90 min after inoculation, suggesting that there was a lag between virus entering the cell by clathrin-mediated endocytosis and escape of the virus from the endosome. High concentrations of CPV particles did not permeabilize canine A72 or mink lung cells to alpha-sarcin, but canine adenovirus type 1 particles permeabilized both cell lines. These data suggest that the CPV entry and infection pathway is complex and involves multiple vesicular components.     

A group B coxsackievirus/poliovirus 5' nontranslated region chimera can act as an attenuated vaccine strain in mice

The linear, single-stranded enterovirus RNA genome is flanked at either end with a nontranslated region (NTR). By replacing the entire 5' NTR of coxsackievirus B3 (CVB3) with that from type 1 poliovirus, a progeny virus was obtained following transfection of HeLa cells. The chimeric virus, CPV/49, replicates like the parental CVB3 strain in HeLa cells but is attenuated for replication and yield in primary human coronary artery endothelial cell cultures, in a human pancreas tumor cell line, and in primary murine heart fibroblast cultures. Western blotting analyses of CPV/49 replication in murine heart fibroblast cultures demonstrate that synthesis of CPV/49 proteins is significantly slower than that of the parental CVB3 strain. CPV/49 replicates in murine hearts and pancreata, causing no disease in hearts and a minor pancreatic inflammation in some mice that resolves by 28 days postinoculation. A single inoculation with CPV/49 induces protective anti-CVB3 neutralizing antibody titers that completely protect mice from both heart and pancreatic disease when mice are challenged 28 days p.i. with genetically diverse virulent strains of CVB3. That a chimeric CVB3 strain, created from sequences of two virulent viruses, is sufficiently attenuated to act as an avirulent, protective vaccine strain in mice suggests that chimeric genome technology merits further evaluation for the development of new nonpoliovirus enteroviral vectors.     

Activation of Caspases in Pig Kidney Cells Infected with Wild-Type and CrmA/SPI-2 Mutants of Cowpox and Rabbitpox Viruses

The cowpox virus (CPV) CrmA and the equivalent rabbitpox virus (RPV) SPI-2 proteins have anti-inflammatory and antiapoptosis activity by virtue of their ability to inhibit caspases, including the interleukin-1β-converting enzyme (ICE; caspase-1). Infection of LLC-PK1 pig kidney cells with a CPV CrmA mutant, but not with wild-type (wt) CPV, results in the induction of many of the morphological features of apoptosis (C. A. Ray and D. J. Pickup, Virology 217:384–391, 1996). In our study, LLC-PK1 cells infected with CPVΔcrmA, but not those infected with wt CPV, showed induction of poly(ADP-ribose) polymerase (PARP)- and lamin A-cleaving activities and processing of the CPP32 (caspase-3) precursor to a mature 18-kDa form. Surprisingly, infection of LLC-PK1 cells with either wt RPV (despite the presence of the SPI-2 protein) or RPVΔSPI-2 resulted in cleavage activity against PARP and lamin A and the appearance of the mature subunit of CPP32/caspase-3. The biotinylated specific peptide inhibitor Ac-Tyr-Val-Lys(biotinyl)-Asp-2,6-dimethylbenzoyloxymethylketone [AcYV(bio)KD-aomk] labeled active caspase subunits of 18, 19, and 21 kDa in extracts from LLC-PK1 cells infected with CPVΔcrmA, wt RPV, or RPVΔSPI-2 but not wt CPV. Mixed infection of LLC-PK1 cells with wt RPV and wt CPV gave no PARP-cleaving activity, and all PARP cleavage mediated by SPI-2 and CrmA mutants of RPV and CPV, respectively, could be eliminated by coinfection with wt CPV. These results suggest that the RPV SPI-2 and CPV CrmA proteins are not functionally equivalent and that CrmA, but not SPI-2 protein, can completely prevent apoptosis in LLC-PK1 cells under these conditions.