Immunoaffinity purification of native dimer forms of the flavivirus non-structural glycoprotein, NS1.

The flavivirus non-structural glycoprotein, NS1 has been shown to elicit an immune response in animals which may confer protection from subsequent virus challenge (Schlesinger et al., 1985 and 1987). While previous reports have outlined methods for obtaining cell-associated NS1 in monomeric form for these studies, we describe here an efficient method for the immunoaffinity purification of both cell-associated and secreted NS1 in their native dimeric configuration. These dimer preparations were shown to be both more antigenic and immunogenic than their monomeric counterparts, a finding which may in part explain the reported failure to obtain solid protection of mice from homologous dengue virus challenge. In moderately sized virus growth experiments, greater than 1 mg quantities of purified NS1 were obtained.

[Antigen signature analysis of dengue-2 viruses strains in Hainan China].

In this paper, we try to define the extent of antigenic variation between dengue-2 viruses from Hainan province, which had been isolated over a period of 3 years (1985-1987). The dengue-2 viruses were compared with the prototype New Guinea B strain and were subjected to antigen signature analysis. Eight strains of dengue-2 virus were analyzed by three monoclonal antibodies: flavivirus group, subcomplex dengue-2 type-specific reactive epitopes, over a range of antigen concentration. Five out of eight dengue-2 virus strains showed them to be antigenically homogeneous, and the remaining three strains showed them to be heterogeneous. Signature analysis provides a rapid and simple means to different strains derived from different sources, thus permit monitoring changes or introductions of hew dengue virus populations in certain geographic region.

Definition of the carboxy termini of the three glycoproteins specified by dengue virus type 2.

The carboxy termini of the three glycoproteins (prM, E, and NS1) specified by dengue virus type 2 (DEN-2) were determined. The glycoproteins were radiolabeled with selected amino acids chosen following analysis of the deduced amino acid sequence of the polyprotein and then digested with carboxypeptidase A. The pattern of release of radioactive amino acids enabled definition of the carboxy termini. In addition, the amino terminus of NS2A was determined by Edman degradation of the radiolabeled protein. The results showed that no amino acids were lost at the carboxy termini of prM, E, and NS1 during their cleavage from the DEN-2 polyprotein. For each glycoprotein, the carboxy terminal amino acid immediately preceded the amino terminal acid of the following polypeptide.

Functional and antigenic domains of the dengue-2 virus nonstructural glycoprotein NS-1.

The gene coding for the nonstructural glycoprotein of dengue-2 virus was cloned, sequenced, and expressed in Escherichia coli. There was about 70% conservation at the amino acid level with dengue serotypes 1 and 4 suggesting an important common function for this protein. Conserved hydrophobic domains were found both before the amino-terminus and at the carboxy-terminus, consistent with transmembrane roles. Evidence for at least partial translocation of NS-1 through the inner membrane of E. coli was found. Also conserved were two signals for N-linked glycosylation located near the middle of NS-1. Various regions of NS-1 were tested for antigenicity with mouse and rabbit polyclonal and mouse monoclonal antibodies. The mouse polyclonal antibodies, made against a crude dengue-infected mouse brain immunogen, reacted most strongly with N-terminal regions of NS-1, whereas, the rabbit antiserum, made against purified NS-1 protein, reacted strongest with C-terminal regions. These findings suggest that immunogen presentation or species differences could be important. Although most of the monoclonals appeared to be unreactive in Western blots with expressed NS-1 proteins, two appeared to react strongly; the region from amino acid (a.a.) 273 to a.a. 346 was required for antibody binding. This region, located adjacent to the two conserved C-terminal hydrophobic domains, is highly charged and contains 5 of the 10 conserved cysteine residues of NS-1.

The influence of higher temperature on dengue-2 virus infected C6/36 mosquito cell line.

Dengue-2 virus infection of C6/36 cells was studied at 28 and 37 degrees C. In infected cells maintained at 28 degrees C, syncytial development was seen on day 4 postinfection, whereas at 37 degrees C, extensive syncytial development was seen by 32 h. Extracellular virus titre was found to correlate with the cytopathic changes. Nine Dengue-2 virus specified proteins were observed in polyacrylamide analyses of cytoplasmic extracts of C6/36 infected cells. All the proteins were observed, although in varied intensities by 32 h postinoculation at 37 degrees C and only on day 4 postinoculation at 28 degrees C. The GP60 glycoprotein appeared at 32 h postinfection when the cells were maintained at 37 degrees C and became prominent only on day 5 at 28 degrees C. The results revealed that a higher temperature accelerated the onset of cytopathic effects, hastened the development of virus specified proteins, and also enhanced the titre of extracellular infectious virus. The importance of the accumulation of the envelope protein GP60 for the development of CPE was indicated.

Topological mapping of unique epitopes on the dengue-2 virus NS1 protein using monoclonal antibodies.

Monoclonal antibodies were produced against two distinct Thai dengue-2 (DEN-2) virus strains isolated in 1980 from dengue haemorrhagic fever patients. Nine of 36 hybridomas produced monoclonal IgG antibodies which reacted in radioimmune precipitation assays with the NS1 non-structural protein (42,000 mol. wt.) from DEN-2-infected C6/36 (Aedes albopictus) cells. The virus specificity of NS1-reactive monoclonal antibodies was determined by indirect immunofluorescence assays using LLC-MK2 cells infected with either the Thai 1980 DEN-2 isolates, prototype DEN viruses (four serotypes), Japanese encephalitis (JE), Murray Valley encephalitis, West Nile, Wesselbron or Tembusu viruses. Eight of the monoclonal antibody preparations were DEN-2-serotype specific. One preparation defined a special serological relationship between DEN-2 and JE viruses. Four preparations had detectable complement fixation titres using Thai DEN-2 virus antigen. Six spatially unique epitopes were identified using competitive binding assays.

Variation in distribution of the three flavivirus-specified glycoproteins detected by immunofluorescence in infected Vero cells.

Indirect immunofluorescence with rabbit antisera was used to probe the intracellular locations of the antigens of envelope, prM (precursor to structural protein M) and the nonstructural glycoproteins NS 1 (formerly described as NV 3 or SCF) specified by the flaviviruses dengue-2 and Kunjin. Perinuclear staining in various types of foci was prominent for all antigens, and the distribution was influenced by whether cells were fixed with acetone or formaldehyde. Staining of Golgi-like masses or inclusions by anti-envelope sera occurred regularly and prominently in cells infected and stained with homologous anti-envelope antibodies; in the cross reactions, such staining was largely absent, especially in dengue-2 infected cells in which it was replaced by many small circular foci scattered throughout the cytoplasm. Anti-NS 1 also stained large perinuclear inclusions and small cytoplasmic foci, but the distribution of these was dissimilar to that observed with anti-envelope sera. Anti-prM appeared to contain a mixture of antibodies of different specificities, evident at different dilutions, possibly because of different cytoplasmic locations of prM and its cleavage products. All antisera produced small discontinuous foci on the plasma membrane of unfixed infected cells; antigens of NS 1 were sometimes prominent on the surface of acetone-fixed cells.

Characterization of the viral RNA species of prototype dengue viruses.

Dengue virus isolates have been grown in the Igarashi clone C6/36 Aedes albopictus cells in amounts suitable for biochemical studies. Oligonucleotide fingerprint analyses have been used to characterize the virion 40 S RNA species of the four prototype dengue (DEN) serotype viruses: DEN 1, Hawaiian strain; DEN 2, New Guinea C strain (both insect and mouse brain passaged derivatives); DEN 3, H-87 strain; and DEN 4, H-241 strain. Comparisons of the largest oligonucleotides derived by ribonuclease T1 digestion of these 40 S DEN virion RNA species indicate that ther are few, if any, large oligonucleotides that are homologous between any two fo the four dengue prototype strains. Preliminary analyses of the 40 S RNA 5' terminal sequence of DEN 3 virion RNA indicate that it has the composition of m7GpppAmpXp.

Isolation of the dengue virus envelope glycoprotein from membranes of infected cells by concanavalin A affinity chromatography.

The membranes isolated from type 2 dengue virus-infected BHK-21/15 cells contain three glycosylated virus-specified proteins; one corresponds to the virion envelope glycoprotein, V-3, whereas the other two are nonstructural virus-specified proteins, NV-2 and NV-3. A combination of two nonionic detergents, Nonidet P-40 and Triton X-305, solubilized greater than or equal to 80% of the membrane-bound protein and the majority of the type 2 dengue virus complement-fixing antigens. The soluble material was adsorbed by concanavalin A-Sepharose in the presence of the nonionic detergents, which were subsequently removed by washing with deoxycholate-containing buffer. Finally, the bound glycoprotein was eluted by the addition of alpha-methyl glucopyranoside. V-3 was the only virus-specified protein in the alpha-methyl glucopyranoside eluate. The V-3-containing fraction did not cross-react with antisera against other selected Flaviviruses in the complement fixation tests. The V-3 contained in the isolated fraction differed from the parent membrane-bound V-3 in two interesting, and as yet unexplained, ways: (i) it exhibited hemagglutinating activity similar to that of the infectious virus, but (ii) it did not block the action of neutralizing antibody.

Immunological and biophysical separation of dengue-2 antigens.

Antigenic compositions of slowly sedimenting dengue-2 hemagglutinin (SHA) and soluble complement-fixing antigen (SCF) were compared with the virion (rapidly sedimenting hemagglutinin, RHA) by radioimmune precipitation (RIP), RIP inhibition, kinetic neutralization, and neutralization blocking tests with the use of hyperimmune mouse ascitic fluids. RHA and SHA were unable to inhibit completely the RIP of each other by anti-RHA, and neutralization by anti-RHA was not blocked by SHA. This indicated that SHA is serologically related, but not identical, to RHA. SHA differed from RHA in that SHA lacked the "core" polypeptide but contained the two envelope polypeptides. In addition, SHA contained a polypeptide with a molecular weight of 16,500 daltons and a suggestion of several other proteins. These data, when considered with other evidence, suggest that SHA is a special form of "incomplete virus." SCF was unable to inhibit the RIP of SHA or RHA or to block neutralizing antibodies. Further, anti-SCF did not neutralize RHA or precipitate significant levels of SHA or RHA. Polyacrylamide gel electrophoresis separated SCF from structural polypeptides by molecular size. This evidence suggests that SCF is a nonstructural antigen.