日本脑炎病毒囊膜蛋白单克隆抗体的制备及特性分析

将日本脑炎病毒(JEV)囊膜蛋白Cap-ED3重组蛋白免疫BALB/c雌鼠,应用细胞融合技术制备单克隆抗体,经ELISA筛选获得4株稳定分泌识别JEV ED3蛋白单抗的杂交瘤细胞,命名为1B10、3F5、4H1和5F9。这4株单抗在Western blot和间接免疫荧光试验中均能特异识别JEV NJ2008株。制备4株单抗的小鼠腹水,5F9效价最高,为1∶409 600。空斑减少中和试验结果表明:4株单抗均没有中和活性。相加ELISA试验表明:4株单抗中3F5和4H1识别相同抗原表位,1B10、5F9分别识别其他的抗原表位,且差异较大。应用鸭坦布苏病毒(DTMUV)特异性抗原进行ELISA检测的结果表明:5F9识别的表位与DTMUV有交叉,其他3株识别的抗原表位与DTMUV无交叉。本研究获得了3株JEV囊膜蛋白特异性的单抗和1株识别JEV和DTMUV交叉抗原表位的单抗,为JEV抗原和抗体检测试剂盒的研制提供了良好的物质基础。     

抗乙型脑炎病毒非结构蛋白NS1单克隆抗体的制备与鉴定

本研究利用原核表达的乙型脑炎病毒(JEV)SA14-14-2株非结构蛋白NS1作为免疫原.免疫8周龄BALB/c小鼠,采用淋巴细胞杂交瘤技术进行融合,共获得4株特异性针对JEV NS1的杂交瘤细胞,分别命名为1H6、2C3、3A7、4C8,经测定1H6单抗亚类属于IgG2b,其他3株为IgG1,轻链均为K链.4株杂交瘤细胞诱生小鼠腹水效价分别达1:20 480、1:2 560、1:20 480、1:10 240,western blot证实所得杂交瘤细胞分泌的抗体均可与JEVNS1蛋白发生特异性反应,间接免疫荧光试验表明1H6、3A7、4C8 3株单抗能够识别天然的JEV NS1蛋白.本研究为进一步探究JEV NS1蛋白结构及其功能奠定了基础.     

Antigenic differentiation of pestivirus strains with monoclonal antibodies against hog cholera virus

Thirty-one bovine viral diarrhoea virus (BVDV) or border disease virus (BDV) strains and 94 hog cholera virus (HCV) strains were grown in cell culture, and characterized by immunostaining with 13 monoclonal antibodies (MAbs) and one polyclonal serum (PAb) against HCV. All 125 strains were recognized by the PAb. None of the BVDV or BDV strains were detected by the 13 MAbs. Seven MAbs detected all 94 HCV strains. Six other MAbs detected heterogeneity among and within HCV strains. The MAbs are useful tools in differentiating between HCV and BVDV infections in pigs, and can also be used to differentiate infections induced by HCV field strains from infections induced by the "Chinese" strain of vaccine virus.     

Japanese encephalitis virus immunoglobulin M antibodies in porcine sera

A solid-phase enzyme-linked immunosorbent assay (ELISA) was developed for detection of porcine immunoglobulin (Ig)M antibodies to Japanese encephalitis virus (JEV). Antibodies in sera were captured onto the solid phase of Microtiter plates sensitized with mouse monoclonal antibodies to porcine mu heavy chain. Virus antigen binding to the lawn of IgM was quantitated by subsequent binding of peroxidase-labeled human hyperimmune anti-JEV IgG, which in the final step, catalyzed a substrate color change. In sucrose density-gradient fractionated sera from recently infected pigs, the peak of ELISA JEV IgM activity corresponded to the peak of 18-S, 2-mercaptoethanol-sensitive hemagglutination-inhibiting (HAI) antibody activity. Within 2 to 3 days, JEV-infected sentinel pigs developed high JEV IgM activity; this activity decreased within 2 weeks. Among specimens collected from 99 random swine at abattoirs in Thailand during a period of low JEV transmission, none of 25 JEV HAI-negative sera had JEV IgM activity, 7 of 74 JEV HAI-positive sera did have JEV IgM activity, and the remaining 67 sera had readily detectable JEV HAI antibodies, but lacked JEV IgM. The JEV IgM solid-phase ELISA was useful for rapidly diagnosing active or recent JEV infections in swine.     

Antigenic variation of porcine transmissible gastroenteritis virus detected by monoclonal antibodies

Mouse myeloma cells (SP2/O) were fused with spleen cells from BALB/c mice immunized with detergent-solubilized antigen of purified virus, and 21 monoclonal (MC) antibodies reactive in enzyme-linked immunosorbent assay with the TO-163 strain of porcine transmissible gastroenteritis (TGE) virus were obtained. Of these MC antibodies, 14, 6 and 1 were IgG1, IgG2a and IgM, respectively. All of the MC antibodies contained light chains of the kappa type. Of these MC antibodies, 8 were found to have neutralization (NT) activity against the TO-163 strain. Comparison of 7 strains of TGE virus by NT tests using our panel of MC antibodies confirmed their close antigenic relationships, but also revealed the occurrence of distinct antigenic differences. These results suggest that there may be at least 6 different epitopes involved in NT reaction on the virion of the TO-163 strain. This notion was confirmed by the competitive binding assay.     

Antigenic analysis of feline and bovine Chlamydia psittaci with monoclonal antibodies.

Monoclonal antibodies were established for antigenic analysis of feline and bovine Chlamydia psittaci. The monoclonal antibodies recognized lipopolysaccharide (LPS), 56-64, 84 or 86 kDa antigens. At least 5 antibody-binding sites were detected on LPS with the monoclonal antibodies. The 56-64 kDa antigen was suggested to have both polypeptide and carbohydrate antibody binding sites. Immunoblotting analysis of cat and cattle sera indicated that the 56-64 kDa antigen is an important antigen in host immune response. The monoclonal antibodies are extremely useful tools to analyse the structure and function of chlamydial antigens.     

Antigenic relationship among strains of ibaraki virus and epizootic haemorrhagic disease virus studied with monoclonal antibodies

Twelve monoclonal antibodies against ibaraki virus (IbV) were established and preliminarily characterised by indirect immunoperoxidase (IIP), haemagglutination inhibition (HI) and neutralisation (NT) tests. Five antibodies reacted in the IIP test with all IbV and epizootic haemorrhagic disease virus (EHDV) strains tested, and five antibodies reacted with IbV and Alberta strain (serotype 2) but not with New Jersey strain (serotype 1) of EHDV. Two of 12 antibodies showed both HI and NT activities. Viral proteins with molecular weights of about 24,000 daltons (24KD) and 78KD were determined by two monoclonal antibodies in Western blot analysis. One of two antibodies with the ability of both HI and NT recognised a viral protein with a molecular weight of about 78KD.     

Antigenic characterization of street rabies virus isolates from Nigeria using monoclonal antibodies

Twenty isolates of street rabies virus were recovered in mouse neutroblastoma cells from 84 rabies suspect brain specimens from Nigeria dogs and a cat. They were characterized with the Tübingen monoclonal antibody panels directed against nucleocapsid and glycoprotein antigens. Antigenic variations were detected both with antinucleocapsid (anti-NC) and antiglycoprotein (anti-GP) monoclonal antibodies (mabs). One isolate reacted positively with anti-NC mab P41 which hitherto has been known to react positively with polar rabies. Another isolate did not react with anti-NC mab 187.5; a reaction normally seen with ERA/SAD strains of rabies virus. With anti-GP mabs it was possible to group the isolates by their area of origin. Isolates from Plateau State were not neutralized by anti-GP mabs ERA 543 and P44.7.2. The isolates studied had glycoprotein antigenic patterns different from the pattern for low egg passage (LEP) Flury strain vaccine virus used in Nigeria for immunization of dogs.     

日本脑炎病毒及其疫苗的研究

乙型脑炎病毒又称日本脑炎病毒(Japanese encephalitis virus，JEV)，简称乙脑病毒，属于黄病毒科(Flaviridae)黄病毒属。由乙脑病毒引起的疾病乙型脑炎(简称乙脑)是严重威胁人畜健康的一种中枢神经系统的急性传染病。以蚊为媒介而传播，以高热和狂暴或沉郁等神经症状为特征。具有明显的季节性和一定的地理分布区，多发生于蚊虫较多的夏秋季节，属于自然疫源性疾病，病毒通常在蚊-猪-蚊等动物间循环。猪被认为是JEV最重要的自然增殖动物。怀孕母猪感染后导致流产和死胎，公猪感染后睾丸有急性炎症反应。     

Antigenic heterogeneity in Mycoplasma iowae demonstrated with monoclonal antibodies.

Western blots of proteins of 14 Mycoplasma iowae strains and isolates resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were probed with three monoclonal antibodies (MAbs), MI6, MI7, and MI8. MAb MI6 reacted with one or more antigens with apparent molecular weights of 60,000, 70,000, and 94,000. In three strains (N-PHN-D13, R-D2497, and K 1805), antigens located on a single peptide band were recognized, while in others additional epitopes at different molecular-weight positions were revealed. A similar pattern was observed with MAb MI7, although it reacted with fewer antigens than did MAb MI6 and failed to recognize antigens in strains N-PHN-D13 and R-D2497. MAb MI8 reacted with an antigen at an apparent molecular-weight position of 28,000 in four of the 14 strains and isolates. The diverse reaction patterns observed with the MAbs in the 14 M. iowae strains and isolates confirms the occurrence of antigenic variation within this species. Antigenic variation in M. iowae may be pivotal in determining host-parasite interactions, pathogenesis, and the outcome of disease.     

Antigenic characterization of bovine viral diarrhoea virus isolates from Spain with a panel of monoclonal antibodies

A group of 47 bovine viral diarrhoea virus (BVDV) strains isolated from a variety of bovine tissues from eight different geographical areas of Spain and two BVDV strains isolated from a cell line were characterized antigenically with a panel of 23 monoclonal antibodies (mAbs). The mAbs were directed at one of three viral proteins: E2, Erns and NS2-3. A peroxidase-linked assay was used to test the mAbs for reactivity against infected cell monolayers. The data were analysed by two computational methods: the Antigenic Distance Program (MAP) and the Phylogeny Inference Package (PHYLIP), and compared with those obtained previously using the same mAbs with other pestiviruses, including reference strains and UK field isolates. All the Spanish field strains studied appeared to be broadly similar to reference strains of BVDV and were included in the subgroup of classical BVDV, meanwhile the two strains isolated from a cell line were included in the subgroup of atypical pestiviruses.     

Mosquito collection in endemic areas of Japanese encephalitis in Hokkaido, Japan

A study was conducted during 1985 and 1986 to evaluate the roles of mosquito species as possible vectors of Japanese encephalitis (JE) virus in Hokkaido. The number of Culex tritaeniorhynchus was very low among the four pig farms where outbreaks of abortion caused by JE virus were observed in swine populations. At one farm near Sapporo, only one Cx. tritaeniorhynchus was found among a total of 510 mosquitoes collected during the survey period from July to October 1985, even when JE virus activity among sentinel pigs was revealed by seroconversion. At another farm in the south, no individuals of this mosquito species were found among 987 mosquitoes collected at the time of the outbreaks of abortion. Cx. pipiens pallens, Anopheles species, Aedes vexans nipponii, and Ae. japonicus were predominant over Cx. tritaeniorhynchus. Cx. tritaeniorhynchus, almost a solve vector species of JE virus in the southern part of Japan, is probably not a vector of the virus in Hokkaido. The collected mosquitoes (2,332 from 1985 and 1,403 from 1986) were processed for virus isolation but no JE virus was isolated. More extensive field studies are necessary to provide further information on the role of mosquito species other than Cx. tritaeniorhynchus in the transmission of JE virus in the northern limits of its range including Hokkaido.     

A serological survey of pigs in Hong Kong for antibodies to Japanese encephalitis virus

Summary A survey of 558 pigs for serological evidence of infection with Japanese encephalitis virus (JEV) performed during the summer of 1968 is recorded. Antibodies were detected by the goose red cell haemagglutination-inhibition test. A high incidence of infection was recorded among mature stock in the New Territories (75·4 per cent) with lower degrees of infection on Hong Kong Island (35·8 per cent) and in Kowloon (26·5 per cent). Of 57 pigs under one year of age only four showed evidence of infection. In view of the widely recorded incidence of JEV in Asia, its occurrence in Hong Kong was to be expected. The regional variation in level of infection within the Colony is probably due to differences in terrain and agricultural development, with consequent differences in availability of mosquito breeding sites. It is concluded that JEV is probably a significant pathogen of pigs and humans in the Colony.

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Resumen Se registra un estudio serológico de 558 cerdos para poner de manifiesto la infección con el virus de encefalitis japonesa (VEJ) llevado a cabo durante el verano de 1968. Fueron descubiertos anticuerpos por medio de la prueba de inhibición de la hemoaglutinación usando glóbulos rojos de ganso. Se registró alta incidencia de la infección entre los cerdos adultos en los Territorios Nuevos (75.4 por ciento) con niveles más bajos en la isla de Hong Kong (35.8 por ciento) y en Kowloon (26.5 por ciento). De 57 cerdos menores de un a?o solamente 4 mostraron evidencia de infección. En vista de la vasta incidencia registrada del VEJ en el Asia, su presencia en Hong Kong era de esperarse. La variación del nivel de infección dentro de la Colonia se debe probablemente a las diferencias en el campo, en el ambiente, y en el desarrollo agrícola con diferencias consecuentes en la disponibilidad de áreas favorables para la crianza del mosquito. Se concluye que el VEJ es probablemente un patógeno de significación para cerdos y humanos en la Colonia.

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Résumé Une enquête sérologique pour mettre en évidence l'infection par le virus de l'encéphalite japonaise a été effectuée sur 558 porcs pendant l'été de 1968. Les anticorps furent recherchés au moyen du test d'inhibition de l'hémagglutination des hématies d'oies. Un très haut taux d'infection fut signalé chez les porcs adultes des New Territories (75, 4 per cent) avec des degrés d'infection plus bas dans l'ile de Hong Kong (35,8 per cent) et dans Kowloon (26,5 per cent). Sur 57 porcs agés de moins d'un an, seulement 4 se révélèrent infectés. Etant donné la très large distribution du virus de l'encéphalite japonaise en Asie, on pouvait s'attendre à le retrouver à Hong Kong. Les différences régionales dans les taux d'infection à l'intérieur de la colonie sont probablement d?es aux différences de terrain et de degré de développement agricole, avec pour conséquence, des différences de possibilités de multiplication des moustiques. On en conclut que le virus de l'encéphalite japonaise est probablement un agent pathogène important pour les hommes et les porcs de la colonie.

     

日本脑炎病毒弱毒疫苗株全长cDNA的体外合成及恢复病毒的拯救

将病毒的全基因组分成3个重叠的区段分别扩增出来,把这3个片段连接到载体中。以这3个片段为模板,通过融合PCR方法,获得JEV的全长cDNA。以cDNA为体外转录的模板,体外转录获得病毒mRNA,转染BHK-21细胞,拯救JEV病毒。通过生物学特性、分子生物学、蛋白水平等几个方面对恢复病毒进行鉴定,并测定恢复病毒的生长曲线和LD50。结果显示,获得了全长cDNA,体外转录获得的病毒RNA转染BHK-21细胞后,二代恢复病毒可引起明显的细胞病变,间接免疫荧光试验和RT-PCR均为阳性。空斑试验表明,拯救病毒与原病毒空斑表型类似;恢复病毒与亲本毒相比在BHK-21细胞上生长更快;恢复病毒的LD50与亲本毒类似。     

Antigenic relationships of foot-and-mouth disease virus serotype Asia-1 isolates demonstrated by monoclonal antibodies.

A panel (26) of monoclonal antibodies (MAbs) was elicited against three distinct isolates of foot-and-mouth disease virus (FMDV) serotype Asia-1. Each MAb was characterized according to the location of its epitope: Class I, restricted to the intact virion (140S); Class II, restricted to 140S and the virion protein subunit (12Sps); Class III, available on 140S, 12Sps and virus protein 1; Class IV, restricted to 12Sps. In addition, the MAbs were further categorized by isotype, neutralization of viral infectivity, capacity to bind in radioimmunoassay and precipitation in the Ouchterlony reaction. Neutralization of FMDV infectivity by a MAb of the IgA isotype is reported for the first time. A minimum of seven distinct neutralization epitopes were described on FMDV Asia-1. Some of the neutralizing MAbs bound FMDVs in addition to those that they neutralized. The MAbs defined epitopes common to FMDV serotypes Asia-1, A, O1 and C but neutralizing capacity was restricted to serotype Asia-1. Class IV MAbs defined epitopes highly conserved throughout the FMDV serotypes. Identification of FMDV neutralization epitopes makes possible the direct selection of optimal FMDV strains for vaccine fabrication. In addition, these data are crucial to the design of future synthetic vaccines.     

Antigenic subtyping and epitopes' competition analysis of porcine circovirus type 2 using monoclonal antibodies

Porcine circovirus type 2 (PCV2) strains have been classified into two major genotypes (PCV2a and PCV2b) and 8 genetic clusters: PCV2b-1A to PCV2b-1C and PCV2a-2A to PCV2a-2E. To date, no studies have been performed to antigenically subtype PCV2 strains enclosing eight PCV2 clusters. The present study aimed to antigenically subtype PCV2 and perform epitopes' competition analysis using monoclonal antibodies (mAbs). Fourteen PCV2 strains representative for eight clusters were tested with 20 mAbs (fifteen of them were generated against PCV2a strain Stoon-1010 and 5 of them against PCV2b strain 1147) in immunoperoxidase monolayer assays. Four mAbs reacted to all 14 PCV2 strains and one mAb reacted with all strains except for a PCV2a-2C strain. One mAb reacted with all PCV2a strains, except for a PCV2a-2C strain and one mAb reacted with all PCV2b strains, except for a PCV2b-1C strain. Nine mAbs reacted with the strains of PCV2b-1A/1B, PCV2a-2A and PCV2a-2E. Three mAbs only reacted with the strains of PCV2a-2A and PCV2a-2E. One mAb reacted specifically with the strains of PCV2b-1A/1B. This suggests that discrete antigenic differences exist between different PCV2 genetic clusters and that these clusters can be discriminated by the use of a panel of universal and cluster-specific mAbs. Six mAbs were selected for cross-competition analysis by a competitive ELISA using PCV2 strain Stoon-1010. Six overlapping epitopes were identified on the PCV2 capsid protein. The universal mAbs recognized larger epitopes than the cluster-specific mAbs. These findings are helpful in the development of diagnostic tests and new generation vaccines against PCV2.     

乙型脑炎病毒E蛋白抗原表位E39特异性单克隆抗体的制备

为了制备乙型脑炎病毒E蛋白抗原表位特异性单克隆抗体,将编码乙型脑炎病毒E蛋白抗原表位E39的DNA序列进行人工合成,随后插入表达载体pGEX-6p-1的限制性酶切位点BamHⅠ与XhoⅠ之间,构建了表位肽与GST的融合表达质粒。该表住融合蛋白经亲和层析纯化后免疫BALB／c小鼠,按常规方法进行细胞融合。以化学合成的E39表位多肽为抗原,对融合细胞上清进行间接ELISA筛选。结果,筛选出1株分泌E39表位特异性抗体的杂交瘤细胞株,该杂交瘤细胞分泌抗体能力稳定。经鉴定,该单克隆抗体亚型为IgG2b,轻链类型为κ链。结果表明,用表位融合蛋白为抗原可以制备表位特异性单克隆抗体。