Investigations on the RNA binding and phosphorylation of groundnut bud necrosis virus nucleocapsid protein

Groundnut bud necrosis virus belongs to the genus Tospovirus, infects a wide range of crop plants and causes severe losses. To understand the role of the nucleocapsid protein in the viral life cycle, the protein was overexpressed in E. coli and purified by Ni-NTA chromatography. The purified N protein was well folded and was predominantly alpha-helical. Deletion analysis revealed that the C-terminal unfolded region of the N protein was involved in RNA binding. Furthermore, the N protein could be phosphorylated in vitro by Nicotiana benthamiana plant sap and by purified recombinant kinases such as protein kinase CK2 and calcium-dependent protein kinase. This is the first report of phoshphorylation of a nucleocapsid protein in the family Bunyaviridae. The possible implications of the present findings for the viral life cycle are discussed.     

Immunodiagnosis of Prune dwarf virus using antiserum produced to its recombinant coat protein

Certification represents the first line of defense against fruit tree viruses. For certification or surveys dealing with large number of samples, ELISA is still considered the technique of choice and requires a continuous supply of good quality antibodies. Prune dwarf virus (PDV) is among the major viruses affecting stone fruits; it belongs to the genus Ilarvirus named so for its isometric labile particles. Recombinant DNA technology was investigated for production of PDV antiserum to avoid labile virus purification and virus maintenance problems. The PDV coat protein gene (CP) was cloned into a protein expression bacterial plasmid vector which allowed a good level of expression of up to 2mg native protein/L culture. The recombinant PDV CP was injected into rabbits and the crude antiserum was successfully used in indirect ELISA at dilutions of up to 1:5000 to detect PDV in infected leaf samples. Similar results were obtained in dot blot immunoassays (DBIA). The antibodies were used in double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and results were comparable to a reference commercial kit. The crude antiserum was efficiently used for coating ELISA plates, thereby reducing test costs.     

Serodiagnosis using recombinant nipah virus nucleocapsid protein expressed in Escherichia coli

Nipah virus nucleocapsid (NiV-N) protein was expressed in Escherichia coli and purified by histidine tag-based affinity chromatography. An indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for human and swine sera and an IgM capture ELISA for human sera were established using the recombinant NiV-N protein as an antigen. One hundred thirty-three suspected patient sera and 16 swine sera were used to evaluate the newly established ELISA systems in comparison with the CDC inactivated-virus-based ELISA systems. For the human sera, the NiV-N protein-based indirect IgG ELISA had a sensitivity of 98.6% and a specificity of 98.4%, and the NiV-N protein-based IgM capture ELISA had a sensitivity of 91.7% and a specificity of 91.8%, with reference to the CDC ELISA systems. The NiV-N-based IgM ELISA was found to be more sensitive than the inactivated-virus-based ELISA in that it captured eight additional cases. For the swine sera, the two test systems were in 100% concordance. Our data indicate that the Nipah virus nucleocapsid protein is a highly immunogenic protein in human and swine infections and a good target for serodiagnosis. Our NiV-N protein-based ELISA systems are useful, safe, and affordable tools for diagnosis of Nipah virus infection and are especially fit to be used in large-scale epidemiological investigations and to be applied in developing countries.     

Pathogen-derived resistance using a viral nucleocapsid gene confers only partial non-durable protection in peanut against peanut bud necrosis virus

Genetic engineering of peanut (Arachis hypogaea L.) using the gene encoding for the nucleocapsid protein (N gene) of peanut bud necrosis virus (PBNV; genus Tospovirus, family Bunyaviridae) was used to impart resistance to bud necrosis disease in peanut (PBND), a disease for which no durable resistance is available in the existing germplasm. Over 200 transgenic lines of peanut var. JL 24 were developed for which integration and expression of the transgenes was confirmed by PCR, Southern hybridization, RT-PCR and western blot analysis. The T₁ and T₂ generation transgenic plants were assayed through virus challenge in the greenhouse by using mechanical sap inoculation at 1:100 and 1:50 dilutions of PBNV, and they showed varying levels of disease incidence and intensity. Greenhouse and field evaluation with T₂ generation plants indicated somewhat superior performance of the three transgenic events that showed considerable reduction in disease incidence. However, only one of these events showed over 75 % reduction in disease incidence when compared to the untransformed control, indicating partial and non-durable resistance to PBND using the viral N-gene.     

Antigenic properties and diagnostic potential of recombinant dobrava virus nucleocapsid protein

Dobrava hantavirus (DOBV) causes severe hemorrhagic fever with renal syndrome in the Balkan region and has been detected recently also in Russia, Estonia, and Germany. DOBV nucleocapsid protein (N) was produced in insect cells, using the baculovirus expression system (bac-DOBV-N), and in E. coli as a truncated (aa 1-165) glutathione-S transferase fusion protein (DOBV-dN-GST). The antigenic properties of bac-DOBV-N were found identical to native DOBV-N when examined by a panel of hantavirus-specific monoclonal antibodies. Enzyme immunoassays for detection of IgM and IgG antibodies were set up using DOBV recombinant N proteins and compared with those based on recombinant Hantaan and Puumala virus N, using panels of sera collected from DOBV, Hantaan and Puumala virus-infected patients. Full-length N protein (bac-DOBV-N) was found to be a more sensitive antigen than DOBV-dN-GST. The sensitivity values for sera from DOBV-infected patients were 100% for bac-DOBV-N and 86% for DOBV-dN-GST by IgM assays, and 98% for bac-DOBV-N and 88% for DOBV-dN-GST by IgG assays. The specificity values were 100% for bac-DOBV-N and 99% for DOBV-dN-GST by IgM assays, and 100% for both antigens by IgG assays.     

Immunodiagnosis of AIDS using preparations of the recombinant antigen of the surface protein of the HTLV-III virus

A technological scheme has been developed for purification of recombinant antigen of surface protein (ASP) of the causative virus of AIDS from Escherichia coli cells carrying plasmid pL2 which codes for the synthesis of a hybrid polypeptide consisting of phage lambda N protein (59 amino acid residues) and a fragment of SP (env) of HIV virus (569 a.r.). Purification of ASP is based on two separation principles: fractionation of polypeptides of bacterial lysates by preparative isoelectrofocusing in a granulated gel layer and the method of preparative polyacrylamide gel electrophoresis in "Multiphor" apparatus (Pharmacia). The ASP purified by these methods was used for construction of an immunodiagnostic preparation for AIDS, employing for this purpose solid-phase enzyme-immunoassay in two modifications: competitive sandwich method and analysis of the tested sera with direct sorption of ASP on nitrocellulose filters.     

Immunodiagnosis of Citrus leprosis virus C using a polyclonal antibody to an expressed putative coat protein

Citrus leprosis virus C (CiLV-C), a causal agent for citrus leprosis disease, is present in South and Central America and is a threat for introduction into the U.S. citrus industry. A specific, inexpensive and reliable antibody based detection system is needed for the rapid identification of CiLV-C. The CiLV-C is very labile and has not been purified in sufficient amount for antibody production. The p29 gene of CiLV-C genome that codes for the putative coat protein (PCP) was codon optimized for expression in Escherichia coli and synthesized in vitro. The optimized gene was sub-cloned into the bacterial expression vector pDEST17 and transferred into E. coli BL21AI competent cells. The expression of PCP containing N-terminal His-tag was optimized by induction with l-arabinose. Induced cells were disrupted by sonication and expressed PCP was purified by affinity chromatography using Ni-NTA agarose. The purified expressed PCP was then used as an immunogen for injections into rabbits to produce polyclonal antibody (PAb). The PAb specific to the expressed PCP was identified using Western blotting. The antibody was successfully used to detect CiLV-C in the symptomatic CiLV-C infected tissues using double antibody sandwich-enzyme-linked-immunosorbent (DAS-ELISA), indirect ELISA and dot-blot immunoassay (DBIA) formats.     

Enzyme-linked immunosorbent assay for detection of antibodies to porcine reproductive and respiratory syndrome virus, by using recombinant nucleocapsid protein N

Recombinant nucleocapsid (rN) protein N of porcine reproductive and respiratory syndrome virus (PRRSV) was prepared, by using the E. coli expressiom system. Insertion of a polyhistidine marker into the structure of the protein allowed the latter to be purified by metal-chelate affinity chromatography. The purity of protein was confirmed by PAAG electrophoresis and its immunospecificity was verified by immunoblotting using rN-specific monoclonal antibodies. The protein was used as an antigen to develop indirect ELISA of PRRSV antibodies. ELISA was shown to be highly sensitive and specific.     

Primary structure of the membrane and nucleocapsid protein genes of feline infectious peritonitis virus and immunogenicity of recombinant vaccinia viruses in kittens.

Feline infectious peritonitis virus (FIPV) causes a mostly fatal, immunologically mediated disease in cats. Previously, we demonstrated that immunization with a recombinant vaccinia virus expressing the FIPV spike protein (S) induced early death after challenge with FIPV (Vennema et al., 1990, J. Virol. 64, 1407-1409). In this paper we describe similar immunizations with the FIPV membrane (M) and nucleocapsid (N) proteins. The genes encoding these proteins were cloned and sequenced. Comparison of the amino acid sequences with the corresponding sequences of porcine transmissible gastroenteritis virus revealed 84.7 and 77% identity for M and N, respectively. Vaccinia virus recombinants expressing the cloned genes induced antibodies in immunized kittens. Immunization with neither recombinant induced early death after challenge with FIPV, strongly suggesting that antibody-dependent enhancement is mediated by antibodies against S only. Immunization with the N protein recombinant had no apparent effect on the outcome of challenge. However, three of eight kittens immunized with the M protein recombinant survived the challenge, as compared to one of eight kittens of the control group.     

湖北地区汉滩病毒与普马拉病毒及多布拉伐病毒的血清学交叉反应研究

目的 研究湖北地区汉滩病毒(hantaan virus,HTNV)与普马拉病毒(Puumaa virus,PUUV)、多布拉伐病毒(Dobrava virus,DOBV)血清学交叉反应.方法 以酵母菌表达的PUUV、DOBV、HTNV重组核衣壳蛋白(recombinant nucleocapsidprotein,rNP)为包被抗原,ELISA法定性、定量分析湖北地区HTN型急性,恢复期血清中IgA、IgG、IgM抗体,比较疾病早晚期血清学交叉反应变化.结果 DOB-rNP与HTN型血清中IgA、IgG、IgM抗体交叉反应性较高,PUU-rNp对HTN型血清交叉反应很低;IgA、IgG抗体交叉反应在恢复期血清中均增高,且IgA抗体交叉反应更多更强.结论 湖北地区HTN型血清与DOBV存在广泛交叉反应,与PUUV交叉反应少.各交叉反应在恢复期增高对汉滩病毒血清学研究具重要意义.     

Competitive ELISA for detection of antibodies to porcine reproductive and respiratory syndrome virus using recombinant E. coli-expressed nucleocapsid protein as antigen

The 15 kDa nucleocapsid (N) protein is the most abundant protein of the porcine reproductive and respiratory syndrome virus (PRRSV), and is highly antigenic, which therefore makes it a suitable candidate for the detection of virus-specific antibodies and diagnosis of the disease. In this study, complementary DNA corresponding to the entire N gene of the IAF-Klop strain of PRRSV was cloned into the pGEX-4T-1 vector, and the N protein was expressed in Escherichia coli fused to the glutathione S-transferase (GST) protein. The resulting GST-N recombinant fusion protein was purified by affinity chromatography and used as antigen for serological testing by indirect enzyme-linked immunosorbent assay (ELISA). Two anti-N specific monoclonal antibodies (MAbs) (IAF-K8 and IAF-2B4), obtained following fusion experiments with spleen cells of BAlb/c mice that were immunized with the purified virus, were used in a competitive assay to increase the specificity of the ELISA. Both MAbs were found to be directed against highly conserved conformational epitopes of North American isolates of PRRSV. Optimal concentration of GST-N protein was determined by checkerboard titration, using hyperimmune pig antiserum to the homologous PRRSV strain, and corresponded to a range of 0.1-0.5 microg protein per well. When tested on 95 sera from pigs that were experimentally infected with the IAF-Klop strain, the competitive ELISA (K8-ELISA) was capable of detecting anti-PRRSV antibodies in 86.7% (65/75) and 92.6% (63/68) of pig sera known to be seropositive by indirect immunofluorescence (antibody titers >16) and a currently used commercial ELISA (HerdCheck(R); Idexx), with specificity values of 100 and 96.2%, respectively. When tested on clinical samples (542 sera) from 28 positive and 28 negative pig herds, the K8-ELISA performed in a similar way to HerdCheck(R) and immunofluorescence (IF) tests as shown by kappa values of 0.762 and 0.803. The sensitivity and specificity of K8-ELISA were 100% on a herd basis, whereas sensitivity values of 80 and 82% with a specificity of 98.7% were determined on an individual basis in comparison with HerdCheck(R) and IF tests.     

Selection and characterization of human respiratory syncytial virus escape mutants resistant to a polyclonal antiserum raised against the F protein

A human respiratory syncytial virus (HRSV) neutralization escape mutant was obtained after 56 serial passages in the presence of a polyclonal antiserum raised against the F protein. Nucleotide sequence analysis of this escape mutant virus revealed two amino acid substitutions: Asn268Ile and Val533Met. When this virus was allowed to grow in the absence of the anti-F polyclonal serum, only the mutation Asn268Ile was stably maintained. Both the double and single escape mutant viruses lost reactivity with mAbs belonging to antigenic site II of the fusion protein of RSV. Mutation Asn268Ile has already been reported in RS viruses that are resistant to mAbs 47F and 11 and palivizumab (PZ). We have thus identified a novel mutation (Val533Met) in the transmembrane domain of the F protein that was selected under immune pressure.     

Development of a polyclonal antibody specific to VP19 envelope protein of white spot syndrome virus (WSSV) using a recombinant protein preparation

The VP19 gene encoding a structural envelope protein of white spot syndrome virus was cloned into an expression vector and introduced into E. coli. The objective was to produce a recombinant VP19 structural protein. After induction, the recombinant VP19 protein (rVP19) was produced, purified by SDS-PAGE and used for immunization of Swiss mice for polyclonal antibody production. The mouse anti rVP19 antiserum had specific immunoreactivity to the viral antigen in WSSV infected Penaeus monodon as verified by immunohistochemistry and Western blot. The production of monoclonal antibodies against this rVP19 may be useful in order to combine with anti-VP28 monoclonal antibodies for enhancing the sensitivity of various WSSV serological assays.     

Immunoprecipitation of Epstein-Barr virus EBNA1 protein using human polyclonal serum

We have developed a method that permits the use of human polyclonal serum to immunoprecipitate BamH1-K EBNA(EBNA1) from EBV transformed cell lines and from cells transfected with an expression vector containing the Bam K region of EBV. Serum from healthy seropositive donors is preabsorbed once with lysate of EBV-negative Burkitt lymphoma cells, then fractionated by gel filtration. The main IgG fraction is then used for the immunoprecipitations. Immunoprecipitated material is visualized by immunoblotting using the same serum. Two proteins with apparent molecular weights of 74 and 62 kD are specifically precipitated from extracts of B95-8 cells. Several proteins are immunoprecipitated from cells transfected with the Bam K containing vector, the apparent molecular weights of the 4 major bands are 74, 68, 62 and 57 kD. Labelling of transfected cells with [3H]glycine and [32P]orthophosphate shows that the 74 and 62 kD proteins can be labelled with both isotopes.     

Detection of antibodies to the nucleocapsid protein of Hepatitis C virus by immunoenzyme analysis using synthetic peptides of nucleocapsid N-terminal part

Solid-phase synthesis of 7 linear peptides overlapping the immunodominant N-terminal region of hepatitis C virus (HCV) core protein (aa 7-75) was carried out. Their antigen reactivity was studied by non-competitive ELISA with sera from patients with chronic HCV infection. Three B-epitopes located within aa 7-19, 20-34, and 39-75 appeared to be the most immunoreactive. Use of the set of synthetic peptides in ELISA detected anti-HCV core antibodies with 93% efficiency in comparison with ELISA and commercial anti-HCV Recomblot based on the use of commercial recombinant HCV core protein.