病毒性出血性败血症病毒单克隆抗体的制备及其特性分析

目的 制备抗病毒性出血性败血症病毒(VHSV)的单克隆抗体(mAb).方法 以差速离心法提纯VHSV作为免疫原,免疫BALB/c小鼠.应用杂交瘤细胞技术进行细胞融合,间接ELISA进行筛选检测,且对mAb进行特性分析.结果 制备出1株抗VHSV mAb,命名为4A5.对其进行特性分析结果显示该抗体腹水效价104以上;属于IgG3型,K链;仅与VHSV本身反应,与其他病毒和细胞均不发生交叉反应;对应的抗原位点在VHSV蛋白的相对分子质量(Mr)70 000的条带处,该蛋白带是VHSV的G蛋白.结论 制备出的特异性好的抗VHSV mAb,为该病毒的快速诊断及流行病学监测提供了检测试剂.     

Stimulation of adherent cells by addition of purified proteins of viral hemorrhagic septicemia virus to trout kidney cell cultures

Purified proteins of the virus causing viral hemorrhagic septicemia in the trout were added to cultures on semisolid medium of leukocytes obtained from either healthy or immunized rainbow trout. Adherent cells were specifically stimulated by the glycoprotein of the viral spikes and, to a lesser extent, by the nucleoproteins. In contrast, a specific memory response was associated more with the nucleoproteins than with the glycoprotein when leukocytes from trout immunized with the virus were employed. These results suggest the necessity of employing both proteins in subunit vaccination trials and the possibility of using this assay to select the proper epitopes for genetically engineered proteins during subunit vaccine development.     

Susceptibility of trout kidney macrophages to viral hemorrhagic septicemia virus.

Viral hemorrhagic septicemia virus (VHSV) lysed the macrophages from rainbow trout kidney cultures either isolated by plastic adherence or stimulated with purified glycoprotein G from VHSV. The trout macrophages supported the replication of VHSV as tested by cell culture and by sandwich ELISA of the supernatants from infected cultures. VHSV-infected macrophages showed a decrease in both acridine-orange fluorescence and average size. Immunofluorescence studies with flow cytometry showed positive membrane staining with monoclonal antibodies (MAbs) anti-N and anti-G VHSV. These findings open the possibility of using trout macrophages as presenting cells to study the possible existence of helper or cytotoxic epitopes relevant to the protection of trout against VHSV.     

Rapid detection and quantitation of viral hemorrhagic septicemia virus in experimentally challenged rainbow trout by real-time RT-PCR

A quantitative real-time RT-PCR (Q-RT-PCR) was developed to detect and determine the amount of viral hemorrhagic septicemia virus (VHSV) in organs of experimentally infected rainbow trout. Primers and TaqMan probes targeting the glycoprotein (G) and the nucleoprotein (N) genes of the virus were designed. The efficiency, linear range and detection limit of the Q-RT-PCR were assessed on cell cultured virus samples. VHSV N gene amplification was more efficient and more sensitive than the VHSV G amplicon. On cell culture grown virus, samples could be accurately assayed over a range of seven logs of infectious particles per reaction. To demonstrate the utility of Q-RT-PCR in vivo, bath infection trials were carried out and samples from fish spleen, kidney, liver and blood were harvested and tested for VHSV. Q-RT-PCR was a more reliable method than either conventional RT-PCR or the cell culture assay for virus diagnosis. Results of VHSV RNA detection in fish shortly after infection as well as on asymptomatic fish several weeks after experimental challenge are presented here. This is the first report showing the utility of Q-RT-PCR for VHSV detection and quantitation both in vitro and in vivo. The suitability of this method to test the efficacy of antiviral treatments is also discussed.     

Temporal and subcellular localization of infectious pancreatic necrosis virus structural proteins

Summary.  The temporal subcellular localization of the structural proteins VP2 and VP3 of infectious pancreatic necrosis virus was analyzed with monoclonal antibodies conjugated with fluorochromes. Early in the infection both proteins were colocalized in the cytosol, at later times, VP2 was visualized as inclusion bodies around the nuclei of the cells and, sometimes, it was found in elongated tubular structures that might correspond to the type I tubules seen in cells infected with another Birnavirus. As VP2 is a glycosylated protein we determined its subcellular localization compared with that of ER and Golgi probes. These results suggest that VP2 is glycosylated freely in the cytoplasm. Received October 25, 1999/Accepted October 26, 1999     

Rescue of viral haemorrhagic septicaemia virus minigenomes by helper virus.

A mammalian expression vector containing the bacterial chloramphenicol acetyltransferase (CAT) gene was used to demonstrate that CAT could be successfully used as a reporter system in fish cells growing at low temperatures. We then constructed a viral haemorrhagic septicaemia virus (VHSV) minigenome by cloning the CAT reporter gene between the viral leader and trailer sequences. This construct was used in transfection experiments with helper VHSV to demonstrate that the minigenome can be encapsidated and transcribed by helper virus proteins. In addition, passaging of viruses collected from cells expressing the minigenome showed that the minigenome was being packaged and replicated in the presence of helper virus. These experiments provide the initiating steps for a reverse genetics system for VHSV.     

Enhanced detection of viral hemorrhagic septicemia virus (a salmonid rhabdovirus) by pretreatment of the virus with a combinatorial library-selected peptide

A 17-mer peptide (SAAEASAKATAEATAKG, p5) was selected by screening a combinatorial library for its ability to enhance in vitro the infectivity of viral hemorrhagic septicemia virus (VHSV), a salmonid rhabdovirus. Preincubation of VHSV samples with p5 at micromolar concentrations led to up to 5-fold increase of viral titers compared to untreated samples, as measured by a 1-day post-infection immunochemical focus assay. Treatment with p5 also increased VHSV titers when using the more traditional plaque and end-point dilution assays. Preincubation of p5 with infectious hematopoietic necrosis virus (another rhabdovirus of salmonids), but not with infectious pancreatic necrosis virus (birnavirus) also led to a similar increase in sensitivity. These results indicate that the addition of p5 may be used to improve the sensitivity of diagnostic tests for salmonid rhabdoviruses.     

Further analysis on the structural proteins of infectious pancreatic necrosis virus

The structural proteins of infectious pancreatic necrosis virus (IPNV) have been analyzed. Two-dimensional gel electrophoresis showed that IPNV proteins are slightly acidic with apparent pIs ranging from 5.8 to 6.6. To identify the IPNV surface-located proteins, purified virus was labelled either with fluorescein isothiocyanate (FITC) or with Na 125I. After analysis by SDS-PAGE, only the major viral protein, VP2, was labelled by either procedure. The accessibility of VP2 to these reagents suggests that this protein is externally located. In addition, using Concanavalin A conjugated with FITC and IPNV labelling with 3H-mannose, evidence is present that VP2 contains carbohydrate residues.     

Autophagy induced by infectious hematopoietic necrosis virus inhibits intracellular viral replication and extracellular viral yields in epithelioma papulosum cyprini cell line

Infectious hematopoietic necrosis virus (IHNV) is a common pathogen that causes severe disease in the salmonid aquaculture industry. Recent work demonstrated that autophagy plays an important role in pathogen invasion by activating innate and adaptive immunity. This study investigated the relationship between IHNV and autophagy in epithelioma papulosum cyprini cells. The electron microscopy results show that IHNV infection can induce typical autophagosomes which are representative structures of autophagy activation. The punctate accumulation of green fluorescence-tagged microtubule-associate protein 1 light chain 3 (LC3) and the protein conversion from LC3-I to LC3-II were respectively confirmed by confocal fluorescence microscopy and western blotting. Furthermore, the effects of autophagy on IHNV replication were also clarified by altering the autophagy pathway. The results showed that rapamycin induced autophagy can inhibit both intracellular viral replication and extracellular viral yields, while autophagy inhibitor produced the opposite results. These findings demonstrated that autophagy plays an antiviral role during IHNV infection.     

Vaccination of chickens against a Belgian nephropathogenic strain of infectious bronchitis virus B1648 using attenuated homologous and heterologous strains

White Leghorn chicks without and with maternally derived antibodies (MDA) to infectious bronchitis virus (IBV) and broiler chicks with MDA were vaccinated at 1 day of age either with H120 vaccine, combined H120 and D274 vaccines or with a non-commercial attenuated strain derived from the virulent Belgian nephropathogenic IBV strain, B1648. Protection following challenge with virulent B1648 was assessed 4 weeks later by virus isolation from the trachea, antigen detection in the kidney by immunofluorescence and mortality rates. Vaccination with either homologous or heterologous vaccines reduced the duration of virus replication in the trachea of all groups compared to unvaccinated controls. Homologous vaccination reduced the incidence of virus replication in the kidney. Heterologous vaccination (H120 to D274) did not reduce kidney infection in the MDA + groups; however, partial kidney-protection was found in the MDA - group. There was no correlation between serum antibody titres measured by ELISA and the degree of kidney protection.     

Toll样受体2、4对病毒囊膜蛋白的识别

TLR2、4是TLRs家族中重要的成员,通过识别囊膜病毒的PAMPs/DAMPs,活化依赖和非依赖于MyD88的信号通路,诱导促炎性细胞因子和趋化因子等的释放,介导先天性抗病毒免疫反应。本文就TLR2、4的基本概况、结构特征、抗病毒信号通路以及对不同囊膜病毒的天然免疫反应等相关研究进展进行简要综述。     

Purification of epizootic haematopoietic necrosis virus and its detection using ELISA

Epizootic haematopoietic necrosis virus (EHNV) was grown in Bluegill fry (BF-2) cells and purified using differential and gradient centrifugation. The lower band (B2) from 15-60% sucrose gradients contained infective EHNV but few contaminating cell components when assessed by electron microscopy, SDS-PAGE, and Western blotting using anti-BF-2 serum and anti-B2 serum. Both rabbit and sheep anti-B2 sera precipitated B2 in agarose gel immunodiffusion and detected EHNV in cell culture supernatant when used in an indirect antigen-capture ELISA. Rabbit anti-B2 serum was used as capture antibody while sheep anti-B2 serum was used to detect viral antigen. Pre-adsorption of diluted sheep anti-B2 serum using BF-2 cell lysate greatly improved the specificity and sensitivity of the technique.     

Replication of heterologous combinations of helper and defective RNA of citrus tristeza virus

Citrus tristeza virus (CTV) populations are among the more complex of plant RNA viruses with unusual mixtures of strains and defective RNAs (dRNAs). Citrus plants infected with different CTV isolates contain multiple dRNA molecules that differ in size and relative abundance within and between isolates. Additionally, we found mixtures of heterologous dRNAs in populations. To examine the replication of CTV dRNAs, the protoplast system had to be extended to support helper-assisted amplification of input dRNAs. The use of freshly extracted sap of CTV-infected tissue as inoculum increased the infection of Nicotiana benthamiana protoplasts sufficiently to result in accumulation of high levels of CTV RNAs as well as dRNAs within 2 or 3 days postinoculation. A series of dRNA-like molecules, each with a single large internal deletion, were created from an infectious cDNA clone of the CTV T36 isolate and examined for amplification in N. benthamiana protoplasts using a CTV deletion mutant as the helper virus. Of 12 synthetic dRNAs, only three with sizes of 3650, 3819, and 4460 nucleotides were efficiently replicated. CTV dRNA replication did not appreciably affect levels of accumulation of the genomic or the subgenomic RNAs of the helper virus. To investigate the maintenance of dRNAs in CTV populations, we examined heterologous interactions between dRNAs and helper viruses. Wild-type populations of heterologous strains T68 and T3, as well as the homologous T36, supported replication of synthetic T36 dRNAs. Replacement in the T36 dRNA of the 5' region, which is most variable among CTV strains, with the corresponding sequences from VT, T68, T3, or T30 resulted in chimeric dRNAs that failed to be replicated by the T36 helpers but were replicated to detectable levels by the T68 helper. The differential specificities of different CTV replicase complexes with dRNA replication signals is one possible factor that affects the maintenance of dRNA population structures.     

Specificity of response to viral proteins in horses infected with equine infectious anemia virus.

Three structural proteins of equine infectious anemia virus were purified, labeled with 125I, and utilized in radioimmunoassays with horse sera and antisera to heterologous retroviruses. Whereas radioimmunoassay titers for the major protein, p25, were 500- to 1,000-fold higher than titers in immunodiffusion, for clinical purposes these two procedures were equivalent. Antibodies to two low-molecular-weight proteins, p12 and p10, were also found in infected horses, but with a lower frequency and lower titers. As a rule, only sera positive for p25 also contained antibody to p12 and p10. Antisera to the major structural protein of other retroviruses did not precipitate equine infectious anemia virus p25. These sera include antibody to mammalian type C viruses, bovine leukemia virus, visna virus, mouse mammary tumor virus, squirrel monkey retrovirus, and Mason-Pfizer monkey virus.