Role of La Crosse virus glycoproteins in attachment of virus to host cells.

Data presented in this report demonstrate that the initial event of La Crosse virus (LACV) infection of cells is probably the interaction of viral glycoproteins with specific cellular receptor sites. We have shown that LACV glycoprotein G1 binds, in a dose-dependent manner, to continuous vertebrate and mosquito cell lines, but not to mosquito midguts isolated ex vivo. This binding can be inhibited by the pretreatment of cells with excess homologous glycoprotein but not with excess heterologous LACV glycoprotein. In contrast, we have shown that LACV glycoprotein G2 binds to the continuous mosquito cell line and vector midgut cells, but not to vertebrate cells. LACV infection of vertebrate cells can be inhibited by treatment of cells with purified G1, while infection in mosquito cells can be reduced by treatment of cells with a combination of G1 and G2. The results suggest that G1 is the viral attachment protein (VAP) for vertebrate cells, and that G2 serves the same purpose for mosquito midgut cells. We speculate that the protease-resistant G2 molecule may have evolved to serve as the VAP in the midgut under conditions in which G1 might be altered or removed from the virus envelope, and thus is essential to the evolution and maintenance of vertebrate-invertebrate transmission cycles.     

Enzyme processing of La Crosse virus glycoprotein G1: a bunyavirus-vector infection model

Efficient transmission, amplification, and dissemination of arboviruses require viral replication in vertebrate and invertebrate hosts. As a result, virions are exposed to two significantly different environments. Exposure of LaCrosse virus (LACV) to proteolytic enzymes, such as those that may be found in the mosquito midgut, increases virus affinity for mosquito cells. These enzymes remove the major envelope glycoprotein (G1) while leaving the second glycoprotein (G2) intact. Processing of LACV glycoproteins in the mosquito midgut may be necessary to expose attachment proteins on the virion surface before attachment to, and infection of, midgut cells can occur. This model may suggest answers to questions regarding the molecular basis for midgut infection barriers and species susceptibility to arbovirus infection in nature.     

Production and characterization of monoclonal antibodies directed against pseudorabies virus

Hybridoma cell lines producing monoclonal antibodies to pseudorabies virus (PRV) were established. The monoclonal antibodies were characterized with respect to their antigenic specifications and biological activities. Two monoclonal antibodies immunoprecipitated the 50 kDa PRV glycoprotein (gp50) and two immunoprecipitated the 82 kDa glycoprotein (gp82). The monoclonal antibodies were used to analyze the biological roles of these two glycoproteins. One monoclonal antibody directed against each glycoprotein did not require complement for in vitro viral neutralization while the other monoclonal antibody directed against the glycoprotein required complement for neutralization. The monoclonal antibodies against gp50 were shown to be directed against different epitopes within the glycoprotein. In contrast, the monoclonal antibodies against gp82 were shown to be directed against the same antigenic site on the glycoprotein. In vivo passive immunity studies in mice showed that monoclonal antibodies directed against either gp50 or gp82 could be protective.     

Monoclonal antibodies to Ebola virus: isolation, characteristics, and study of cross reactivity with Marburg virus

Thirteen hybridoma strains producing monoclonal antibodies (Mabs) to Ebola virus were prepared by fusion of NS-O mouse myeloma cells with splenocytes of BALB/c mice immunized with purified and inactivated Ebola virus (Mayinga strain). Mabs directed against viral proteins were selected and tested by ELISA. Protein specificity of 13 Mabs was determined by immunoblotting with SDS-PAGE proteins of Ebola virus. Of these, 11 hybridoma Mabs reacted with 116 kDa protein (NP) and 2 with Ebola virus VP35. Antigenic cross-reactivity between Ebola and Marburg viruses was examined in ELISA and immunoblotting with polyclonal and monoclonal antibodies. In ELISA, polyclonal antibodies of immune sera to Ebola or Marburg viruses reacted with heterologous filoviruses, and two anti-NP Ebola antibodies (Mabs 7E1 and 6G8) cross-reacted with both viruses. Target proteins for cross-reactivity, Ebola NP (116 kDa) and Marburg NP (96 kDa), and VP35 of both filoviruses were detected by immunoblotting with polyclonal and monoclonal antibodies (6G8) to Ebola virus.     

Immunoglobulin Fc binding activity is associated with the mouse hepatitis virus E2 peplomer protein

Antigenic variation among murine coronaviruses is associated primarily with the surface peplomer protein E2 (180,000 Da). E2 is responsible for attachment of the virus to the host cell, MHV-induced cell fusion, and eliciting neutralizing antibody. We report here the molecular mimicry between E2 and Fc gamma receptor (Fc gamma R). Molecular mimicry between E2 and Fc gamma R may allow the escape of virus-infected cells from destruction by immunological mechanisms. Rabbit IgG, monoclonal rat IgG1 and IgG2b, monoclonal mouse IgG2a and IgG2b, and the rat anti-mouse Fc gamma R monoclonal antibody 2.4G2 immunoprecipitated from MHV-JHM-infected cells a polypeptide with a molecular mass identical to that immunoprecipitated by anti-E2 antibodies. F(ab')2 fragments of rabbit IgG did not immunoprecipitate any proteins from MHV-infected cells. All of these antibodies did not immunoprecipitate any proteins from uninfected cells. The anti-mouse Fc gamma R monoclonal antibody 2.4G2 immunoprecipitated from MHV-JHM-, MHV-3-, or MHV-A59-infected L-2 cells and 17CL-1 cells, or MHV-JHM-infected cultures of neonatal BALB/c brain cells, a protein with a molecular weight identical to that of MHV-JHM E2. The anti-Fc gamma R monoclonal antibody did not immunoprecipitate any proteins from uninfected cells. Furthermore, the 2.4G2 monoclonal antibody (mab), unrelated rat and mouse monoclonal antibodies, and a goat antiserum against E2, but not normal goat serum, immunoprecipitated a 75,000- to 77,000-Da molecule from uninfected WEHI-3 cells, a Fc gamma R bearing cell line. Several lines of evidence demonstrated that the protein immunoprecipitated by the anti-Fc gamma R mab from MHV-JHM-infected cells is the E2 glycoprotein: (1) Partial proteolytic maps obtained by Staphylococcus aureus V-8 protease treatment of the 180,000-Da proteins immunoprecipitated by the anti Fc gamma R mab and the anti-E2 mab were identical. (2) Sequential immunoprecipitation experiments from MHV-JHM-infected cells revealed that the same polypeptide chain was recognized by the anti-E2 mab and by the anti-Fc gamma R mab 2.4G2, (3) Actinomycin D did not influence the induction and expression of the 180,000-Da polypeptide chain that was immunoprecipitated by the anti-Fc gamma R mab, demonstrating that this protein is of viral origin.     

Biological roles of the major capsid proteins and relationships between the two existing serotypes of infectious bursal disease virus

Summary Neutralizing monoclonal antibodies (n-MAbs) were produced against infectious bursal disease virus (IBDV) of serotypes 1 and 2. The n-MAbs recognizing the major antigenic proteins VP2 and VP3, were characterized using different strains of IBDV representing the existing two serotypes and a variant subtype of serotype 1. The biological properties of these viral antigens as defined by the MAbs in vitro, were studied utilizing post-adsorption virus neutralization tests and fluorescence-activated cell sorter analysis. The MAbs directed against the immunodominant epitopes on VP2 were capable of enhanced virus neutralization but did not inhibit the virus attachment to susceptible cells. These MAbs were able to neutralize the virus by interfering with an event subsequent to virus adsorption, possibly inhibiting virus penetration or uncoating. On the contrary, a MAb that immunoprecipitated the other capsid protein VP3 was able to prevent virus attachment although it possessed lower neutralization titers. Cross-immunoprecipitations of various virus strains by these MAbs and antisera revealed interrelationships between the two serotypes of IBDV.     

Identification and localization of conserved antigenic epitopes on the G2 proteins of California serogroup Bunyaviruses

California (CAL) serogroup Bunyaviruses are significant agents of arboviral encephalitis in humans. They are maintained and transmitted in nature by mosquitoes to preferred vertebrate amplifying hosts. The G2 envelope glycoprotein of La Crosse virus (LAC) was proposed by Ludwig et al. to be a determinant for virus attachment to mosquito midgut cells. Monoclonal antibodies to G2 neutralize the infectivity of pronase-treated virus for mosquito cells. We determined the location of antigenic sites on the LAC G2. We showed that antigenic areas present on the LAC G2 protein are conserved among viruses in the California encephalitis and Melao subgroups of the CAL serogroup, but not in trivatattus virus, nor within the BUN serogroup. A comparison of the G2 exodomain amino acid sequences of eight CAL and three BUN viruses with monoclonal antibodies (MAb) binding data predicted the possible location of the antigenic sites. We used in vitro mutagenesis of the LAC G2 gene to construct a set of G2 genes with replacement sequences in the coding regions for the suspected MAb binding sites. The native and mutated proteins were expressed in Hela cells and the ability of MAbs to bind to the expressed proteins was tested. Four discontinuous amino acid sequences, conserved among eight CAL serogroup viruses, were identified as contributing to two conformational binding domains for neutralizing LAC G2 MAbs.     

Monoclonal antibodies against African swine fever viral antigens

Monoclonal antibodies specific for African swine fever (ASF) viral proteins of 14, 32, 73, 174, and 240 kDa were produced and characterized. Immunoelectron microscopy detected the 73 kDa but not the 14-, 32-, or 240-kDa proteins at the surface of the virion. The 32-kDa protein was detected by radioimmunoassay 2 hr after infection of porcine monocytes and Vero cells, was detected in the seven widely divergent ASFV isolates tested, and stained brilliantly virus-infected cells in indirect immunofluorescence suggesting that monoclonal antibodies directed against this protein may be useful in ASFV diagnosis. Two monoclonal antibodies detected heterogeneity between ASF viruses.     

The use of immunodeficient male (CBA/N x BALB/c) F1 mice to produce monoclonal antibodies directed to proteins of Leptospira interrogans rather than to immunodominant lipopolysaccharides.

A method, using an immunodeficient mouse strain, for the production of monoclonal antibodies directed exclusively against the proteins in an antigen mixture also containing immunodominant LPS, is described. Male (CBA/N x BALB/c) F1 mice were immunized with an outer envelope antigen mixture from Leptospira interrogans strain Wijnberg containing both lipopolysaccharides and proteins. The immune response in these mice was shown to be predominantly directed against protein antigens. Hybridoma cell lines were generated by fusing spleen cells from a (CBA/N x BALB/c) F1 mouse with BALB/c Sp2/0 plasmacytoma cells. Hybridoma cell lines producing monoclonal antibodies reacting with the outer envelope preparation were identified by ELISA. All epitopes recognized by the monoclonal antibodies are sensitive to proteinase K degradation and resistant to oxidation by periodate indicating that they are located on proteins. All epitopes are located on a 35 kDa protein and specific for the pathogenic L. interrogans species.     

Serological and immunochemical characterization of monoclonal antibodies to Toxoplasma gondii.

The fusion of mouse NS-1 myeloma cells with spleen cells from mice chronically infected with Toxoplasma gondii resulted in eight clones of hybridomas producing monospecific antibodies against membrane or cytoplasmic antigens of Toxoplasma tachyzoites. One of the antibodies to a cytoplasmic determinant was an IgM; the others directed to membrane or cytoplasmic antigens belonged to the IgG2 or IgG3 isotypes. Antibodies of clones 1E11 (IgG3), 2G11, and 3E6 (IgG2) directed to membrane antigens, bound complement and were reactive in the complement-dependent cytotoxicity assay of Sabin-Feldman. These IgG2 antibodies were strongly agglutinating to parasites, whereas the IgG3 was relatively weak. Another IgG2 antibody (5B6), possibly recognizing a shared antigen of membrane and cytoplasm, exhibited a low titre in the cytotoxicity assay as well as in the agglutination assay. Two other antibodies to membrane antigens (2B7 and 2F8) as well as an antibody to a cytoplasmic antigen (3G3) did not bind complement and did not cause agglutination. The pattern of parasite staining produced by monoclonal antibodies to membrane antigens in an IFA test was different from that of polyvalent antisera. A strictly localized or 'beaded' staining was observed, as well as a smooth, rim fluorescence. Toxoplasma tachyzoites were surface radio-iodinated and the solubilized membrane proteins were immunoprecipitated with monoclonal antibodies and analysed by two-dimensional polyacrylamide gel electrophoresis. Two independently arising monoclonal antibodies to membrane antigens (2G11 and 3E6) consistently precipitated both the solubilized 35,000 and 14,000 mol. wt proteins, while 1E11 precipitated the 27,000 mol. wt protein.     

Human monoclonal antibodies to human cytomegalovirus derived from peripheral blood lymphocytes of healthy adults

Immunoglobulin G class human monoclonal antibodies to human cytomegalovirus (HCMV) were produced by the fusion of Epstein-Barr virus-transformed B cells and a murine myeloma cell line. The B cells were derived from the peripheral blood lymphocytes of healthy adult volunteers. Four hybridomas producing HCMV-specific monoclonal antibodies were established and each of four antibodies immunoprecipitated an HCMV-specific protein with a molecular weight of 68 kDa. However, the antibodies differed in some of their properties as characterized by indirect immunofluorescence assay and immunoblotting studies, due to the detection of different epitopes on the reacting antigen. None of the four antibodies had any virus neutralizing activity.     

Disulfide-linked surface molecules of monoclonal antigen-specific suppressor T cells: evidence for T cell receptor structures

By two-dimensional polyacrylamide gel electrophoresis analysis under nonreducing/reducing conditions, five proteins with interchain disulfide bridges are revealed on the surface of the suppressor T cell lymphoma line LH8-105 obtained by radiation leukemia virus-induced transformation of hen egg-white lysozyme-specific suppressor T lymphocytes. Two disulfide-linked surface proteins expressed by LH8-105 cells have been positively identified by immunoprecipitation with specific antisera. The major labeled membrane protein of LH8-105 cells is the murine leukemia virus env glycoprotein gp70. The second disulfide-linked molecule identified on LH8-105 cells has a molecular mass of 84 kDa under nonreducing conditions and 42 kDa after reduction, and is immunoprecipitated by an antiserum which recognizes the T cell receptor for antigen. A disulfide-linked molecule of a similar molecular mass is also immunoprecipitated from surface-labeled LH8-105 cells by a rabbit antiserum directed against a synthetic peptide predicted from the nucleotide sequence of a cDNA clone encoding the beta chain constant region of a helper T cell hybridoma. Therefore, a dimeric structure comparable to the T cell receptor expressed by cytotoxic and helper T cells is present on the cell surface of these monoclonal antigen-specific suppressor T cells.     

Two distinct P70 interleukin-2 receptors on a murine large granular lymphocyte clone Y479.

A continuous cloned cell line (Y479) was established by culturing normal mouse spleen cells in a high concentration of interleukin-2 (IL-2). Y479 cells showed morphological characteristics of large granular lymphocyte with the phenotypes of Thy-1.2+, T3+, Lyt-1-, Lyt-2-, L3T4-, B220-, AsGM1+, LFA-1+, and TcRV beta 8-. The Y479 cells required a high concentration of IL-2 for their growth but did not express detectable p55 IL-2 receptor (IL-2R) although they bound IL-2 with high and low affinities. Analysis of the IL-2 binding proteins on the Y479 cells revealed that both the high and low affinity receptors consisted only of 70 kDa protein. Analysis of the 70 kDa protein was performed using five monoclonal antibodies (L15, L20, L23, L34, and L61) against human recombinant IL-2. Although they recognized different epitopes, all monoclonal antibodies immunoprecipitated 70 kDa IL-2R that was cross-linked with radioiodinated IL-2. The supernatant after immunoprecipitation with L61 still contained IL-2/IL-2R complex that was L23-reactive, and the supernatant after immunoprecipitation with L23 contained L61-reactive IL-2/IL-2R complex, whereas L15 immunoprecipitated almost all the complex. Limited digestion of IL-2-cross-linked Y479 cells with trypsin caused the liberation of 45 kDa IL-2R fragment cross-linked with IL-2. This complex was immunoprecipitated by L15 or L61 but not by L23. These results suggest that there are at least two distinct 70 kDa IL-2R on the surface of Y479 cells.     

Analysis of the polypeptides synthesized in rinderpest virus-infected cells

We have identified, by [35S]methionine labeling, eight major induced proteins and a number of minor proteins in rinderpest virus-infected bovine kidney cells. The polypeptides ranged in molecular weight from 212 to 21.5 kDa. The majority of these polypeptides are virus specific, as demonstrated by immunoprecipitation with rabbit hyperimmune serum against rinderpest. Infected cells radiolabeled with glucosamine contained a 75-kDa polypeptide and a broad band migrating at 80 kDa, both identified as virus specific by immunoprecipitation. Phosphorylated virus-specific proteins of 65 kDa and a complex of polypeptides at 92.5 kDa were also identified. Monospecific and monoclonal antibodies against measles virus and canine distemper virus hemagglutinin, fusion protein, nucleocapsid protein, and phosphoproteins confirmed the identity of the corresponding rinderpest virus-specific polypeptides.     

Nucleocapsid protein N of Lelystad virus: expression by recombinant baculovirus, immunological properties, and suitability for detection of serum antibodies.

The ORF7 gene, encoding the nucleocapsid protein N of Lelystad virus (LV), was inserted downstream of the P10 promoter into Autographa californica nuclear polyhedrosis virus (baculovirus). The resulting recombinant baculovirus, designated bac-ORF7, expressed a 15-kDa protein in insect cells. This protein was similar in size to the N protein expressed by LV in CL2621 cells when it was analyzed on sodium dodecyl sulfate-polyacrylamide gels. The N protein expressed by bac-ORF7 was immunoprecipitated with anti-ORF7 was immunoprecipitated with anti-ORF7 peptide serum, porcine convalescent-phase anti-LV serum, and N protein-specific monoclonal antibodies, indicating that this N protein had retained its native antigenic structure. The recombinant N protein was immunogenic in pigs, and the porcine antibodies raised against this protein recognized LV in an immunoperoxidase monolayer assay. However, pigs vaccinated twice with approximately 20 micrograms of N protein were not protected against a challenge with 10(5) 50% tissue culture infective doses of LV. Experimental and field sera directed against various European and North American isolates reacted with the N protein expressed by bac-ORF7 in a blocking enzyme-linked immunosorbent assay. Therefore, the recombinant N protein may be useful for developing diagnostic assays for the detection of serum antibodies directed against different isolates of LV.     

The influenza c virus glycoprotein (HE) exhibits receptor-binding (hemagglutinin) and receptor-destroying (esterase) activities

A cDNA copy of RNA segment 4 of influenza C/Cal/78 virus was cloned into an SV40 vector and expressed in CV-1 cells. The gene product expressed from the SV40 recombinant virus was immunoprecipitated by monoclonal antibodies directed against the influenza C virus glycoprotein. Cells infected with the recombinant virus also exhibited C virus-specific hemagglutinin and O-acetylesterase activity. This suggests that the same C virus protein is associated with receptor-binding as well as receptor-destroying activity. The latter viral activity was measured using as substrates bovine submaxillary mucin or a low molecular weight compound p-nitrophenylacetate. In analogy to the parainfluenza virus HN protein, the influenza C virus glycoprotein was termed HE, because it possesses hemagglutinin and esterase (receptor-destroying) activity.     

Production and characterization of two monoclonal antibodies to human pancreatic trypsin 1

Two monoclonal antibodies (MAb) G6 and A8 directed against human trypsin 1 have been produced by hybridization of myeloma cells with spleen cells of OF1 immunized mice. Antibodies were screened by radioimmunoassay. Monoclonal antibodies were purified by affinity chromatography on Protein A-Sepharose, and we found that MAb G6 was of the IgG2b class and MAb A8 of the IgG2a class. Both MAbs had a high affinity for trypsin 1 with the respective affinity constants equal to 1.3 x 10(8) l/mol for G6 and 3.2 x 10(7) l/mol for A8. Epitope specificity was studied by western blotting, using human trypsinogens 1 and 2. Both MAbs gave a positive reaction with native trypsinogen 1 and no reaction with the same protein after reduction. Only MAb G6 reacted with trypsinogen 2 in the native form. Its affinity for trypsin 2 was found similar to that for trypsin 1 with a constant equal to 2.7 x 10(7) l/mol. Both antibodies appeared directed against conformational and not sequential epitopes.     

Characterization of rabies glycoprotein expressed in yeast

Summary The rabies virus surface glycoprotein was synthesized inSaccharomyces cerevisiae using an expression vector which contains an inducible promoter from the copper metallothionein gene. The rabies G protein was also expressed constitutively in yeast when cloned under control of the triose dehydrogenase promoter. Polypeptides of 65–68 kDa, which migrated at the same molecular weight as authentic viral rabies G protein species, were synthesized by yeast transformants as detected by immunoblotting with rabies specific antiserum. In addition, these polypeptides were immunoprecipitated with several rabies G-specific monoclonal antibodies which neutralize virus infectivity. The recombinant rabies G proteins were glycosylated and associated with membranes in yeast. When injected into guinea pigs, yeast extracts containing the rabies G protein protected animals from lethal rabies virus challenge when administered intramuscularly. However, the same material did not protect mice from a lethal rabies intracerebral challenge.     

Two Distinct P70 Interleukin-2 Receptors on a Murine Large Granular Lymphocyte Clone Y479

A continuous cloned cell line (Y479) was established by culturing normal mouse spleen cells in a high concentration of interleukin-2 (IL-2). Y479 cells showed morphological characteristics of large granular lymphocyte with the phenotypes of Thy-1.2⁺. T3⁺, Lyt-1^?, Lyt-2^?, L3T4^?, B220^?, AsGM₁⁺, LFA-1⁺, and TcRVβ8^?. The Y479 cells required a high concentration of IL-2 for their growth but did not express detectable p55 IL-2 receptor (IL-2R) although they bound IL-2 with high and low affinities. Analysis of the IL-2 binding proteins on the Y479 cells revealed that both the high and low affinity receptors consisted only of 70 kDa protein. Analysis of the 70 kDa protein was performed using five monoclonal antibodies (L15, L20, 1,23, L34, and L61) against human recombinant IL-2. Although they recognized different epitopes, all monoclonal antibodies immunoprecipitated 70 kDa IL-2R that was cross-linked with radioiodinated IL-2. The supernatant after immunoprecipitation with L61 still contained IL-2/IL-2R complex that was L23-reactive, and the supernatant after immunoprecipitation with L23 contained L61-reactive IL-2/IL-2R complex, whereas L15 immunoprecipitated almost all the complex. Limited digestion of IL-2-cross-linked Y479 cells with trypsin caused the liberation of 45 kDa IL-2R fragment cross-linked with IL-2. This complex was immunoprecipitated by L15 or L61 but not by L23. These results suggest that there are at least two distinct 70 kDa IL-2R on the surface of Y479 cells.