Identification of a 27.8 kDa protein from flounder gill cells involved in lymphocystis disease virus binding and infection

In vitro, lymphocystis disease virus (LCDV) infection of flounder gill (FG) cell cultures causes obvious cytopathic effect (CPE). We describe attempts to isolate and characterize the LCDV-binding molecule(s) on the plasma membrane of FG cells that were responsible for virus entry. The results showed that the co-immunoprecipitation assay detected a 27.8 kDa molecule from FG cells that bound to LCDV. In a blocking ELISA, pre-incubation of FG cell membrane proteins with the specific antiserum developed against the 27.8 kDa protein could block LCDV binding. Similarly, antiserum against 27.8 kDa protein could also inhibit LCDV infection of FG cells in vitro. Mass spectrometric analysis established that the 27.8 kDa protein and beta-actin had a strong association. These results strongly supported the possibility that the 27.8 kDa protein was the putative receptor specific for LCDV infection of FG cells.     

Relationship between Expression of Cellular Receptor-27.8kDa and Lymphocystis Disease Virus (LCDV) Infection

The 27.8kDa membrane protein from flounder (Paralichthys olivaceus) gill (FG) cells was previously identified as a putative cellular receptor involved in lymphocystis disease virus (LCDV) infection. In this paper, the expression of receptor-27.8kDa (27.8R) and LCDV loads in FG cells and hirame natural embryo (HINAE) cells were investigated upon LCDV infection and anti-27.8R monoclonal antibody (MAb) treatment. The results showed the 27.8R was expressed and co-localized with LCDV in both FG and HINAE cell surface. After LCDV infection, the expression of 27.8R exhibited a dose-dependent up-regulation with the increasing of LCDV titers, and demonstrated a tendency to increase firstly and then decrease during a time course up to 9 days; LCDV copies showed a similar variation trend to the 27.8R expression, however, it reached the highest level later than did the 27.8R expression. Additionally, the 27.8R expression and LCDV copies in FG cells were higher than those in HINAE cells. In the presence of increasing concentration of the anti-27.8R MAbs, the up-regulation of 27.8R expression and the copy numbers of LCDV significantly declined post LCDV infection, and the cytopathic effect induced by LCDV in the two cell lines was accordingly reduced, indicating anti-27.8R MAbs pre-incubation could inhibit the up-regulation of 27.8R expression and LCDV infection. These results suggested that LCDV infection could induce up-regulation of 27.8R expression, which in turn increased susceptibility and availability of FG and HINAE cells for LCDV entry, providing important new insights into the LCDV replication cycle and the interaction between this virus and the host cells.     

Characterization of monoclonal antibodies against heavy and light chains of flounder (Paralichthys olivaceus) immunoglobulin

Flounder (Paralichthys olivaceus) Immunoglobulins (Igs) were purified from the serum of mouse IgG-immunized flounder by using affinity chromatography. Under denaturing conditions in SDS-PAGE, the flounder Igs appeared to be composed of 2 heavy (H) chains (72 and 77 kDa) and two light (L) chains (26 and 28 kDa). Monoclonal antibodies (MAbs) were produced by the fusion of myeloma cells (SP2/0) with Balb/c mouse spleen cells that were previously sensitized against affinity-purified flounder Igs. In a Western blot analysis, the produced MAbs, FIM511, FIM519, and FIM562 recognized both the 72 and 77 kDa H chains, 26 kDa, and 28 kDa L chain, respectively. Mouse antiserum against flounder Igs reacted more strongly with the L chain of 28 kDa than with 26 kDa, suggesting that the 28 kDa molecule is more immunogenic than the 26 kDa L chain molecule. In a FACS analysis, the ratios of the Ig+ cell population in the flounder head kidney and spleen cells were 49% and 24%, respectively. Unexpectedly, however, the ratios of the Ig+ B-like cell population in the flounder were not significantly augmented, even after the immunization of an immunogenic antigen. This suggests that the humoral immune response in fish could be considerably different from that in mammals. The produced MAbs in this study would be useful in characterizing flounder Ig+ B-like cells and in developing flounder Ig detecting an immunoassay system.     

Whitespotted puffer Arothron hispidus, a new host for lymphocystis in Qingdao Aquarium of China

In April 2004 white nodular lesions were found on the fins of whitespotted puffer Arothron hispidus (Linnaeus). Diagnostic studies were carried out to confirm the disease using light and electron microscopy, histochemical methods and PCR. The results revealed that the nodules were composed of giant cells up to 400 microm in diameter. These cells were surrounded by a periodic acid-Schiff (PAS)-positive hyaline capsule containing dot-shaped, Feulgen-positive inclusion bodies in the cytoplasm and an irregular nucleus. Numerous virus particles 200 nm in diameter and with hexagonal profiles were observed in the cytoplasm. These features were consistent with those of lymphocystis disease. Additionally, based on the gene sequences of major capsid protein (MCP) of lymphocystis disease virus (LCDV) from Japanese flounder Paralichthys olivaceus, 2 pair primers were designed; after a nested PCR was performed for detection of LCDV in A. hispidus, a positive amplified product was obtained showing the presence of LCDV. Therefore, the white nodules were the lymphocystis lesions caused by LCDV infection and A. hispidus was demonstrated to be a new host for LCDV.     

Protein and glycoprotein content of lymphocystis disease virus (LCDV).

The polypeptide and glycoprotein composition of eight strains of the fish-pathogenic lymphocystis disease virus (LCDV) isolated from gilt-head seabream (Sparus aurata), blackspot seabream (Pagellus bogaraveo), and sole (Solea senegalensis) were determined. The protein electrophoretic patterns of all LCDV isolates were quite similar regardless of the host fish, showing two major proteins (79.9 and 55.6 kDa) and a variable number of minor proteins. Three groups of LCDV isolates were distinguished according to the number and molecular masses of the minor proteins. Eight glycoproteins were detected inside viral particles of LCDV 2, LCDV 3 and LCDV 5 isolates, but only seven glycoproteins were found inside viral particles of LCDV 1, LCDV 4, LCDV 6, LCDV 7, and LCDV 11 isolates and the reference virus ATCC VR 342 by using five lectins. LCDV glycoproteins were mainly composed of mannose and sialic acid. These glycoproteins could be part of an external viral envelope probably derived from the host cell membrane.     

Production of monoclonal antibodies against grouper nervous necrosis virus (GNNV) and development of an antigen capture ELISA

Five (2 IgG, 3 IgM) monoclonal antibodies (MAbs) against the G9508KS strain of grouper nervous necrosis virus (GNNV) were produced and characterized. All 5 MAbs showed positive signals in the retina of GNNV-infected grouper larvae and in the cytoplasm of GNNV-infected GF-1 cells using immunohistochemistry staining. Two MAbs reacted with the denatured capsid protein derived from GNNV-infected GF-1 cells in Western blot analysis, but did not react with the GNNV recombinant capsid protein expressed by E. coli in an indirect immnunosorbent assay (ELISA). All 5 MAbs were able to neutralize GNNV, tiger puffer NNV (TPNNV) and barfin flounder NNV (BFNNV), while only 2 of the MAbs neutralized striped jack NNV (SJNNV). A capture ELISA system based on the use of MAbs for capture and a rabbit polyclonal antibody for detection was developed. When absorbance values higher than 0.5 were judged to be positive, the sensitivity of the capture ELISA system was 2.5 ng per well of purified GNNV protein or 6.5 x 10(4) TCID50 per well of GNNV supernatant from culture cells. This capture ELISA system could become a more specific and sensitive tool for NNV diagnosis in the field and in routine laboratories.     

Fish Rhabdovirus Cell Entry Is Mediated by Fibronectin

Three monoclonal antibodies (MAbs) generated against rainbow trout gonad cells (RTG-2) have been selected for their ability to protect cells from the viral hemorrhagic septicemia virus (VHSV) infection, a salmonid rhabdovirus. Protection from infection was restricted to the salmonid-derived cell lines indicating species specificity of the blocking MAbs. Surprisingly, the blocking activity of these MAbs was also effective against other nonantigenically related fish rhabdoviruses. Indirect immunofluorescence and immunoelectron microscopy observations demonstrated that the three MAbs were all directed against an abundant cell plasma membrane component, and immunoprecipitation studies indicated that the target consisted of a heterodimeric complex with molecular masses of 200 and 44 kDa. Biochemical data provided the following evidence that fibronectin is part of this complex and that it could represent the main receptor for fish rhabdoviruses. (i) An antiserum generated against the 200-kDa protein reacted against the recombinant rainbow trout fibronectin expressed in Escherichia coli. (ii) The purified rainbow trout fibronectin was able to bind specifically to VHSV. To our knowledge, this is the first identification of a cellular component acting as a primary receptor for a virus replicating in lower vertebrates and, more interestingly, for viruses belonging to the Rhabdoviridae family.     

Neutralization of Chlamydia psittaci with Monoclonal Antibodies

Neutralization of Chlamydia (C.) psittaci avian strain P-1041 was examined in vitro using monoclonal antibodies (MAbs). Of the 10 MAbs used, 6 were found to exhibit neutralizing capability. These include 3 against major outer membrane protein (MOMP), 1 against lipopolysaccharide (LPS) and 2 against other protein molecules [90 kilodalton (kDa) and 90/50 kDa]. Most neutralizing MAbs were dependent on complement for efficient neutralization, while a strain-specific MAb (2B5) against the 90 kDa protein displayed a different requirement for complement and neutralized the infectivity of the P-1041 at high concentrations without complement. By competitive inhibition enzyme-linked immunosorbent assay (competitive inhibition ELISA), all 3 neutralizing anti-MOMP MAbs were demonstrated to recognize different epitopes found in very close proximity to each other on the outer membrane.     

Production of Monoclonal Antibodies against the Major Capsid Protein of the Lactococcus Bacteriophage ul36 and Development of an Enzyme-Linked Immunosorbent Assay for Direct Phage Detection in Whey and Milk

The only major structural protein (35 kDa) of the lactococcal small isometric-headed bacteriophage ul36, a member of the P335 species, was isolated from a preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Monoclonal antibodies (MAbs) were raised against the denatured 35-kDa protein. Six MAbs were selected and characterized. Western blots (immunoblots) showed that all MAbs recognized the 35 kDa but also a 45 kDa that is in lower concentration in the phage structure. Binding inhibition assays identified five families of MAbs that recognized nonoverlapping epitopes of the 35- and 45-kDa proteins. Immunoelectron microscopy showed that these two proteins are localized within the phage head, therefore indicating that the 35 kDa is a major capsid protein of ul36 and that the 45 kDa is a minor capsid protein. With two MAbs, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed for direct detection of lactococcal phages in whey and milk samples. Whey and milk components, however, interfered with the conduct of the assay. Partial denaturation of milk samples by heat treatment in the presence of SDS and β-mercaptoethanol removed the masking effect and increased the sensitivity of the assay by 100-fold. With the method used here, 10⁷ PFU/ml were detected by the ELISA within 2 h without any steps to enrich or isolate bacteriophages.     

登革2型病毒结合血管内皮细胞分子机制的初步研究

以ECV304细胞为对象分析登革病毒感染血管内皮细胞的机制。2型登革病毒(DEN2)吸附后微量蚀斑法测定ECV304细胞上清释放的病毒滴度,证实该细胞对DEN2感染有一定的敏感性。机械刮取或胰蛋白酶消化法收集ECV304细胞分离膜蛋白,SDS—PAGE见胰酶处理样品缺失一43 kDa的膜蛋白。将ECV304细胞膜蛋白与^35S—Met标记的DEN2进行病毒重叠蛋白结合试验(VOPBA),有29、34和43kDa的3种膜蛋白可与DV结合,其中29 kDa的蛋白对胰酶耐受。培养的ECV304细胞中加入重组E蛋白(rEgp)对DEN2吸附进行阻断试验,微量蚀斑法与间接免疫荧光表明rEgp抑制DEN2感染该细胞。VOPBA中rEgp可阻断病毒与细胞膜蛋白的结合。结果表明ECV304细胞表面可能存在29、34、43 kDa的3种与DEN2结合的相关蛋白,DEN2E蛋白可直接介导DV感染血管内皮细胞。     

Anti-viral activity of galectin-1 from flounder Paralichthys olivaceus

Galectins are a family of Ca²⁺-independent soluble lectins characterized by their affinity to β-galactosides. Mammalian galectins have been shown to play a defense role against certain bacteria, fungi and viruses. However, the immunological functions of galectins in fish is poorly characterized. Here we demonstrated that the expression of galectin-1 gene from the flounder Paralichthys olivaceus was decreased in the initial 8 h after challenge with poly I:C, then increased markedly from 24 h onwards, and the recombinant galectin-1 was able to neutralize the lymphocystis disease virus (LCDV), inhibiting the formation of cytopathic effects. In addition, the recombinant galectin had a potential anti-inflammatory activity against infection by LCDV, and was able to restrain the overexpression of the anti-viral protein gene mx against virus infection. These results indicate that flounder galectin-1 has an anti-viral activity, capable of reducing LCDV pathogenicity.     

Specific identification of pathogenic Yersinia enterocolitica by monoclonal antibodies generated against recombinant attachment invasion locus (rAil) protein

Classical pathogenic strains of Yersinia enterocolitica produce a 17 kDa outer membrane protein, Ail (attachment-invasion locus), which mediates bacterial attachment to some cultures epithelial cell lines and invasion of others. In the present study, hybridomas were developed for the production of monoclonal antibodies (MAbs) against Ail protein of Y. enterocolitica. A set of five stabilized hybridoma cell lines were generated, of which, two MAbs, YEA 302 and YEA 303, exhibited specific reaction to the native Ail protein (17 kDa) present in whole cell lysate of Y. enterocolitica strains beside having reaction with rAil. The other three MAbs, YEA 5, 17 and 32, had some cross reactions with proteins other than Ail. Two out of five MAbs were IgG1, two were IgG2b and one in IgM in nature. MAbs (YEA 302 and YEA 303) did not show any cross-reaction with whole cell lysate of Brucella abortus, Vibrio cholerae, Salmonella typhimurium and Escherichia coli and other species of Enterobacteriaceae including Y. pestis in ELISA and Western blot analysis. The presence of Ail protein among the strains recovered from pork and milk samples was evaluated by these sets of MAbs and the results were compared with the duplex PCR. Collectively, the data suggest that these MAbs may have the potential for their use in the detection of pathogenic Y. enterocolitica reliably, rapidly and at a relatively low cost.     

Development of molecular techniques for detection of lymphocystis disease virus in different marine fish species

AIMS: The development and evaluation of a protocol based on polymerase chain reaction (PCR) and nucleic acid hybridization techniques for the specific detection of lymphocystis disease virus (LCDV) in several marine fish species. METHODS AND RESULTS: The pair of primers for PCR, OBL3 and OBL4, was designed based on published nucleotide sequence (LCDV-1) and amplifies a fragment within the major capsid protein. The sensitivity was evaluated using DNA from purified viral particles, as well as from cells inoculated with several viral concentrations. The PCR combined with slot blot was the most sensitive methodology, detecting 2.5 ng of viral DNA. Using this methodology LCDV was detected at 5 days postinoculation from SAF-1 cells initially inoculated with 10(-5) TCID(50) ml(-1). The combination of PCR with membrane hybridization has also been proved to be adequate to detect LCDV from apparently healthy carriers by means of caudal fin sample analysis. This asymptomatic infection was also demonstrated by classical virological methods (cell culture and immunoblot). CONCLUSIONS: The protocol described in this study allows the specific detection of LCDV, both in cell cultures and in fin homogenates from asymptomatic fish. SIGNIFICANCE AND IMPACT OF THE STUDY: The detection of asymptomatic carriers by a rapid molecular method using caudal fin sampling, which does not imply animal killing, could be an important tool to control epizootics caused by LCDV, as fish could be analysed before their introduction and/or mobilization in farm facilities.     

A monoclonal antibody that recognizes a basolateral membrane protein in A6 epithelial cells

The A6 cell line is a model for tight epithelia and studies of epithelial polarity. Monoclonal antibodies (MAbs) were produced by immunization of mice with intact A6 cells and fusion of spleen cells to generate hybridomas. Hybridoma supernatants were screened by ELISA to select MAbs binding to the apical membrane of confluent A6 cells. Localization of MAb binding was examined by indirect immunofluorescence using cross sections of A6 monolayers grown on collagen coated filters. One MAb, designated 13F12, was positive by apical surface ELISA but localized specifically to the basolateral membrane of cross sections of A6 monolayers on filters. Immunofluorescence labeling of confluent A6 cells grown on glass cover slips revealed that MAb 13F12 does not bind to the apical membrane, but binds to basolateral determinants in the regions of domes, where it appears able to penetrate cellular junctions. Subconfluent A6 cells express the antigen all over the cell surface. Cells approaching confluency express the antigen on the apical membrane of some cells but not others, and as the cells reach confluency, the antigen disappears from the apical surface, and the cells become fully polarized. A6 cells at confluency on glass cover slips are equally polarized as cells grown on filters with respect to this antigen. The antigen has been identified by immunoprecipitation as a 22 kDa protein. High concentrations of MAb 13F12 did not inhibit cell plating, indicating that the antigenic site is not directly involved in cell adhesion to the substrate. MAb 13F12 should prove to be a useful tool to study many aspects of epithelial polarity, including the signals involved in sorting of proteins to specific membrane domains.     

番茄花叶病毒单克隆抗体的制备及检测应用

用番茄花叶病毒(ToMV)免疫的BAL B/c鼠脾细胞与SP2/0鼠骨髓瘤细胞融合,经筛选克隆,获得4株能稳定传代并分泌抗ToMV单克隆抗体(Mab)的杂交瘤细胞,其中2株能同时检测ToMV和烟草花叶病毒(TMV),各单克隆抗体腹水ELLSA效价在1∶32 000～1∶1 024 000之间。经TASELISA测定,4株单克隆抗体检测病汁液的稀释度均能达到1∶2 000倍以上。4株单克隆抗体与其他病毒无交叉反应。Westernblot分析表明,其中两株与ToMV176kD的外壳蛋白亚基有特异反应,而另两株无反应,推测它们是针对构象决定簇的抗体。     

Production, characterisation and applicability of monoclonal antibodies to immunoglobulin of Japanese flounder (Paralichthys olivaceus)

Immunoglobulin (Ig) of Japanese flounder (Paralichthys olivaceus) was purified by a combination of salting-out and DEAE Sepharose Column chromatography. The purified immunoglobulin had an apparent molecular weight of 74 kDa (heavy chain) and 24 kDa (light chain) in SDS-PAGE. Eighteen hybridomas secreting monoclonal antibodies (MAbs) against Japanese flounder Ig were obtained by immunisation of Balb/C mice with purified Ig preparations, which were selected on the basis of the double indirect enzyme-linked immunosorbent assay (D-ELISA). Two of them designated as 2D8 and 2H1 were cloned by limiting dilution and characterised with western blotting, indirect immunofluorescence assay test (IIFAT) and fluorescence-activated cell sorter (FACS) analysis. Under reducing conditions in western blotting, both MAb 2D8 and MAb 2H1 were specific for the heavy chain of Japanese flounder Ig. MAb 2D8 was used to identify surface Ig-positive lymphocytes in the peripheral blood, spleen and pronephros of healthy Japanese flounder by flow cytometry. FACS analysis revealed that 40.48% of lymphocytes in the peripheral blood, 17.32% in the spleen and 9.67% in the pronephros were reactive to 2D8.     

Cell fusion activities of Hantaan virus envelope glycoproteins

Hantaan virus (HTNV)-infected Vero E6 cells undergo cell fusion with both infected and uninfected cells under low-pH conditions. Flow cytometry and fluorescence microscopy of HTNV-infected Vero E6 cells showed that envelope glycoproteins (GPs) were located both on the cell surface and in the cytoplasm. Neutralizing monoclonal antibodies (MAbs) against the G1 and G2 envelope GPs inhibited cell fusion, whereas nonneutralizing MAbs against G1 or G2 and MAbs against the nucleocapsid protein (NP) did not. Transfected Vero E6 cells that expressed GPs but not those that expressed NP fused and formed syncytia. These results indicate that HTNV GPs act as fusogens at the cell surface. No fusion activity was observed either in infected Vero cells that were passaged more than 150 times or in BHK-21 cells, although GPs appeared to localize to the cell surface. This variability in fusion induction suggests the involvement of host cell factors in the process of cell membrane fusion.     

蚕豆萎蔫病毒单克隆抗体制备及检测应用

:用蚕豆萎蔫病毒(BBWV)免疫的BALB/C鼠脾细胞与SP2/0鼠骨髓瘤细胞融合,经筛选克隆,获得6株能稳定传代并分泌抗BBWV单克隆抗体(Mab)的杂交瘤细胞株,单抗腹水ELISA滴度为1:320000～1:640000,各单抗抗体类型均为IgG1。6株单抗与BBWV不同分离物均有反应,而与其它植物病毒无交叉反应。经Westernblot印迹分析表明,此6株单克隆抗体均是针对BBWV447kD的外壳蛋白大亚基的特异性抗体。这是国内外首次报道获得BBWV单克隆抗体     

Detection of white spot syndrome virus (WSSV) of shrimp by means of monoclonal antibodies (MAbs) specific to an envelope protein (28 kDa)

The vp28 gene encoding an envelope protein (28 kDa) of white spot syndrome virus (WSSV) was amplified from WSSV-infected tiger shrimp that originated from Malaysia. Recombinant VP28 protein (r-28) was expressed in Escherichia coli and used as an antigen for preparation of monoclonal antibodies (MAbs). Three murine MAbs (6F6, 6H4 and 9C10) that were screened by r-28 antigen-based enzyme-linked immunosorbent assay (ELISA) were also able to recognize viral VP28 protein as well as r-28 on Western blot. Three non-overlapping epitopes of VP28 protein were determined using the MAbs in competitive ELISA; thus, an antigen-capture ELISA (Ac-ELISA) was developed by virtue of these MAbs. Ac-ELISA can differentiate WSSV-infected shrimp from uninfected shrimp and was further confirmed by a polymerase chain reaction (PCR) and Western blot. Approximately 400 pg of purified WSSV sample and 20 pg of r-28 could be detected by Ac-ELISA, which is comparable in sensitivity to PCR assay but more sensitive than Western blot in the detection of purified virus. Hemolymph and tissue homogenate samples collected from a shrimp farm in Malaysia during December 2000 and July 2001 were also detected by Ac-ELISA and PCR with corroborating results.     

栉孔扇贝急性病毒性坏死症病毒单克隆抗体的制备及ELISA检测

利用蔗糖密度梯度离心技术从自然发病的栉孔扇贝 (Chlamysfarrreri)组织分离纯化急性病毒性坏死症(Acutevirusnecrobioticdisease,AVND)病毒 ,并以此为抗原免疫Balb c小鼠。将免疫小鼠的脾细胞与NS_1骨髓瘤细胞进行融合 ,最终筛选出 4株能稳定传代并分泌该病毒特异性单克隆抗体 (MonoclonalAntibody ,MAb)的杂交瘤细胞系。应用胶体金标记免疫电镜技术在超微水平上对这 4株MAbs进行测定表明 ,它们对AVND病毒均具有高度的特异性 ,并且所识别的特异性位点均位于病毒粒子囊膜的纤突上。应用该单克隆抗体对不同养殖季节的一龄贝进行间接ELISA检测发现 ,7月中旬至 7月底病毒感染率与感染强度均处于当年的最高峰 ,与这一时期栉孔扇贝所表现出的最高死亡率完全吻合