Production and use of monoclonal antibodies for the detection of apple chlorotic leaf spot virus

Stable hybridoma cell lines secreting antibodies specific for the apple chlorotic leaf spot virus (CLSV) were produced by fusing spleen cells of a Biozzi mouse immunized with CLSV P863 strain, with the non-secretory P3 X63 Ag8.653 myeloma cell line. Two hybridoma clones producing monoclonal antibodies of the IgG1 subclass were obtained. These monoclonal antibodies were used for virus detection by enzyme-linked immunosorbent assay (ELISA). In contrast to polyclonal antisera to CLSV, which always contain some antibodies to host components, monoclonal antibodies are highly specific for the virus. It was thus possible to develop a detection assay which is more sensitive and specific than the assays using polyclonal antibodies. Using monoclonal antibodies, it was possible to detect less than 0.1 ng/ml of purified virus. In addition, these two monoclonal antibodies recognize 17 strains or isolates maintained in our laboratory and representing most of the known CLSV strains.     

Soluble antigens associated with infection with apple chlorotic leaf spot virus

Virus-related soluble antigens, probably various polymeric forms of viral coat protein, were detected in crude extracts from plants infected with apple chlorotic leaf spot virus (CLSV), using highly specific antisera to two CLSV strains from apple and peach, respectively. The two antisera appeared to detect the soluble antigens differently, though with about the same sensitivity. Soluble antigen preparations were clarified by ultracentrifugation of crude sap, by acidification, freezing and thawing, and by ammonium sulfate precipitation. The last treatment allowed some concentration. Detection of soluble antigen in gel diffusion tests provided a specific diagnostic test for CLSV infection in Chenopodium quinoa, but not in apple tissue.     

Genetic variation analysis of apple chlorotic leaf spot virus coat protein reveals a new phylogenetic type and two recombinants in China

In this study, we generated sequences of the apple chlorotic leaf spot virus (ACLSV) coat protein (CP) gene. Genetic variation and phylogenetic analyses were carried out on these sequences along with others reported previously. ACLSV populations clustered into four types: in three of the four types, combinations of three amino acids at positions of 40, 75 and 79 were conserved. The fourth phylogenetic type, newly identified here, was characterized by co-variation of Ser⁴⁰-Tyr⁷⁵-Ser⁷⁹. Statistically significant genetic differentiation and infrequent gene flow were detected among the four types. Two natural recombinants were detected for the first time among ACLSV isolates/genotypes from China.     

Complete nucleotide sequences of the genomes of two isolates of apple chlorotic leaf spot virus from peach (Prunus persica) in China

The complete nucleotide sequences of two isolates of apple chlorotic leaf spot virus (Z1 and Z3) collected from peach in Henan Province, China, were determined. The genomes of both Z1 and Z3 were found to contain three open reading frames (ORFs). Sequence analysis showed that genomic sequences of Z1 and Z3 isolates shared 67.4%-82.9% and 67.2%-82.6% identity, respectively, with the other eight isolates of ACLSV that have been reported previously. Based on the putative amino acid sequences of the products of the three ORFs, Z1 and Z3 isolates showed the greatest identity to isolate PBM1 (GenBank accession number AJ243438) from plum and the least identity with isolate Ta Tao5 (GenBank Accession Number: EU223295) from peach. Considering the low level of sequence identity between Z1/Z3 isolate and Ta Tao5 isolate, two types of ACLSV may exist in peach.     

Detection of apple chlorotic leaf spot virus using a 5' nuclease assay with a fluorescent 3' minor groove binder-DNA probe

The development of a real-time 5' nuclease RT-PCR assay for the detection of apple chlorotic leaf spot virus (ACLSV) from infected plant material is described. A short fluorogenic 3' minor groove binder-DNA hydrolysis probe was used to circumvent genome variability between isolates and target a short conserved sequence. The covalent attachment of the minor groove binder moiety at the 3' end of the probe increased the probe/target duplex stability and raised the melting temperature to a range suitable for real-time analysis. The method is rapid, sensitive and takes place within a single tube without post-PCR handling of the amplification products.     

Use of monoclonal antibodies to lipopolysaccharide for antigenic analysis of Coxiella burnetii

Antigenic differences among Coxiella burnetii strains were analyzed. The monoclonal antibodies against the lipopolysaccharide outer core did not react with the strains containing a QpRS plasmid or with plasmidless strains, whereas they reacted with strains containing a QpH1 or QpDV plasmid. C. burnetii isolates could be divided into two groups immunologically.     

Topographical localization of distinct antigenic domains ofToxoplasma gondii with the aid of monoclonal antibodies

In the present study, a panel of six individual hybridoma derived antibodies produced against the BK strain ofToxoplasma gondii were evaluated for their reactivity in an immunofluorescence test. Each of the six monoclonal antibodies demonstrated a unique pattern of fluorescence localized to distinctive regions on the toxoplasmas. Although all of the six detected antigenic determinants which were shared by at least four differentT. gondii strains, monoclonal antibody TG-E₄A₁₇ has disclosed hitherto unrecognized population differences among the BK or RH strain parasites. The fact that some of the antigenic molecules are restricted to distinct regions of the toxoplasmas may have implications in the infections process/immune response.     

Complete nucleotide sequence of the genome of a severe cherry isolate of apple chlorotic leaf spot trichovirus (ACLSV)

Summary.  The genome of the Balaton1 severe cherry isolate of apple chlorotic leaf spot trichovirus (ACLSV-Bal1) has been cloned and sequenced. The genomic RNA is 7 549 nucleotide long, excluding the poly A tail. The genomic organization, with three overlapping open reading frames (ORF), is similar to that of the other sequenced ACLSV isolates. Sequence comparisons indicate a high variability between ACLSV isolates, with overall nucleotide sequence homology levels between 76 and 82%. The coat protein, encoded internally inside a larger ORF, is the most conserved protein (identity levels between 87 and 93%) while the central ORF, encoding the putative movement protein, is the most divergent (77 to 85% identity). Received August 5, 1996 Accepted October 21, 1996     

Structural domains of endogenous murine leukemia virus gp70s containing specific antigenic determinants defined by monoclonal antibodies

Eight distinct antigenic determinants, or epitopes (labeled a-h) were identified on endogenous ecotropic murine leukemia virus (MuLV) gp70s using a series of murine and rat monoclonal antibodies. These epitopes were characterized by their distribution patterns in a panel of cloned MuLVs, and by their localization in fragments of gp70 generated either by spontaneous breakdown of purified gp70, or by controlled proteolysis of gp70 in solubilized virions. A major 32K carboxy terminal fragment was formed which contained epitopes b, c, and f. This fragment also possessed the p15(E) disulfide linkage site, and contained approximately four complex (type 1) carbohydrate chains. An amino terminal 35K fragment contained epitopes a, d, g, and h, and possessed two glycosylated sites, including a site which occasionally retained an endoglycosidase H-sensitive oligosaccharide chain. A related 49K fragment was also obtained which included the entire 35K region and contained an additional sequence bearing epitope e. In a series of dual-tropic MCF-type viruses studied, only those epitopes located in the 32K fragment were ever retained, indicating that for these recombinant viruses at least a portion of that domain was derived from the ecotropic parent. A model is presented indicating the likely orientation of these fragments and their structural characteristics.     

Characterization of antigenic structures on arabis mosaic virus with monoclonal antibodies

Summary Seven different epitopes on arabis mosaic virus (ArMV) were discerned. Neo-, crypto-, and epitopes exposed on the virion and isolated coat protein were differentiated by their reactivity with monoclonal antibodies in an indirect enzyme-linked immunosorbent assay. The monoclonal antibodies, producedin vitro andin vivo, were of IgGl, IgM and IgA isotype. No epitope exclusively specific for one isolate was found. One epitope was specific for ArMV isolates only. With the common epitopes an operational antigenic map was devised. An immunological relationship between nepoviruses of different serological subgroups was demonstrated. Grapevine fanleaf virus, a member of the ArMV subgroup could not be shown to expose crossreactive epitopes. For serotyping ArMV isolates were discriminated by comparing the reactivity of two or more monoclonal antibodies specific for different epitopes.With 3 Figures     

Biophysical and biochemical characterization of apple chlorotic leafspot virus

The particles of apple chlorotic leafspot virus (CLSV) banded in isopycnic Cs₂SO₄ gradients at ? ? 1.27 g/cc and contained 5.2% nucleic acid. A single nucleic acid species of MW 2.3 × 10⁶ was isolated from the virus. The molecular weight and structure of CLSV particles are discussed.     

Use of monoclonal antibodies for studying the classical hog cholera virus]

Numerous monoclonal antibodies (MAb) to hog cholera virus are a highly specific and effective instrument for studies of this agent. Panels of MAb for differential diagnosis of Pestiviruses are characterized. International reference panel of 30 MAbs is a result of cooperation of European scientists; it was approved as the official reference for assessing all available and new diagnostic agents. MAb permit intraspecies differentiation between hog cholera virus strains and, which is particularly important, between vaccine and field strains. Study of antigenic structure and functional characteristics of surface proteins of the virus with the use of MAb panels helped single out and map the functions of four antigenic sites of surface glycoprotein E2 and detect a relationship between RNAse activity and structural component of another surface glycoprotein E0.     

An analysis of the antigenic structure of the hemagglutinins from different strains of the influenza B virus by using monoclonal antibodies

A comparative immunological analysis of the antigenic composition of hemagglutinin (HA) of influenza B virus drift-variants isolated during 46 years was carried out using monoclonal antibodies (MAb) to HA of B/Oregon 5/80 virus in HI test and solid-phase enzyme immunoassay. The presence of type- and group-specific antigenic determinants was demonstrated which agreed with our previous data obtained in studies with polyvalent sera. However, using Mab three more group-specific determinants were detected which characterize HA of influenza B viruses isolated in 1970-1979, 1970-1984, and 1970-1986.     

Use of hybridoma monoclonal antibodies in the detection of antigenic differences between rabies and rabies-related virus proteins. II. The glycoprotein

Twenty-five hybridoma cultures secreted monoclonal antibodies directed against the glycoprotein of rabies or rabies-related viruses. The antibodies had different specificities for the glycoproteins of eight rabies and rabies-related viruses. They could be classified into fourteen groups which probably correspond to different antigenic determinants on the glycoproteins. These hybridomas when used in either radioimmunoassay (RIA) or in neutralization tests allow differentiation of laboratory strains of rabies virus from each other as well as from the rabies-related viruses.     

Is bivalent binding of monoclonal antibodies to different antigenic areas on the hemagglutinin of influenza virus required for neutralization of viral infectivity?

Summary Biological activities of Fab fragments of monoclonal IgG antibodies to each of four nonoverlapping antigenic areas on the hemagglutinin molecule of A/seal/Massachusetts/1/80 (H7N7) influenza virus were examined. Fab fragments of the antibodies belonging to groups I and II neutralized viral infectivity. These Fab fragments inhibited hemagglutination of the virus and virus-induced hemolysis at pH 5.9. On the other hand, Fab fragments of groups III and IV antibodies showed neither neutralization nor hemolysis-inhibition activities, while intact IgG molecules of groups III and IV effectively neutralized viral infectivity and inhibited virus-induced hemolysis, as previously found. These IgG molecules scarcely or did not inhibit hemagglutination of the virus. Neutralization of viral infectivity, however, was observed when the virus was coated with Fab fragments of groups III and IV antibodies and then incubated with anti-Fab fragment antibodies. These findings suggest that bivalent binding of the IgG antibodies of groups III and IV is required for neutralization of viral infectivity through a proposed mechanism by which these antibodies interfere with a low pH-induced conformational change resulting in inhibition of the fusion step of the viral replication process (6).With 2 Figures     

Characterization of monoclonal antibodies against hepatitis C virus nonstructural protein 3: different antigenic determinants from human B cells

The nonstructural (NS3) region protein of hepatitis C virus (HCV) possesses major B-cell epitopes that induce antibodies after infection. To elucidate further the characteristics of these B cells and their role in the immune regulation of HCV infection, T9 (portion of NS3 region, amino acids [a.a.] 1188-1493)-specific monoclonal antibodies were derived and mapped for B-cell antigenic determinants with recombinant proteins. A total of 10 T9-specific hybridomas were generated and tested for B-cell antigenic determinants. To analyze the B-cell antigenic determinants, eight recombinant proteins including NS3-e (a.a. 1175-1334), NS3-a' (a.a. 1175-1250), NS3-a (a.a. 1251-1334), NS3-b (a.a. 1323-1412), NS3-c (a.a. 1407-1499), NS3-a/b (a.a. 1251-1412), NS3-bc (a.a. 1323-1499), and NS3-abc (a.a. 1251-1499) encoded by NS3-region internal clones were expressed and tested for immunoblotting. The data suggested IgG hybridomas recognized NS3-a, NS3-a', or NS3-b protein by immunoblotting. By contrast, the NS3-e protein bears the major antigenic determinant recognized by human sera. Half of the hybridomas were found to react with protein NS3-a', which is not a major B-cell antigenic determinant in humans. These data suggested that conformational epitopes in vivo may be important for B-cell recognition.