SUMMARYThe nucleotide sequence and the deduced amino acid sequence for the genes encoding the structural proteins of two strains of dengue virus type 2 (DEN-2) were determined from cDNA clones. The genes for C, prM(M) and E proteins were sequenced for the prototype DEN-2 virus, the New Guinea C strain. Also sequenced were the prM(M) and E genes of PUO-218. This strain of DEN-2 was isolated during 1980 in Bangkok and had received a limited number of laboratory passages.
SUMMARYThe antibody response to herpes simplex virus (HSV) is complex and involves antibody to at least 33 virus-induced polyeptides. Serum IgG contains four isotypes in mice and it is known that the isotypes differ in their biological functions and that individual antigenic proteins may preferentially elicit restricted isotype responses. We therefore examined the anti-polypeptide isotypes induced in immune mouse serum. By ELISA, we found that the total serum virus-specific antibody activity was 51% IgG1, 39% IgG2a, 11% IgG2b and 1% IgG3 in immune ICR strain mice and 51 ~o, 45%, 4% and 0.4% respectively in strain BALB/c mouse immune serum. These proportions are significantly different from those reported for other virus infections. Sepharose-Protein A affinity-purified isotypes were also studied and showed IgG 1 > IgG2a ~> IgG2b ~ IgG3 activity per ~tg of isotype, indicating that competition between isotypes present in high concentrations did not significantly alter the results. Immunoblotting studies of the purified isotypes showed that the major immunogenic HSV-1 proteins (VP155, gC, gB, pgB, gD and nucleocapsid proteins 42K and 44K) induced all isotypes. However, the isotype responses were not uniform among the glycoproteins and some other proteins. In addition neutralization assays of the purified isotypes indicated that IgG2a and IgG2b had significantly greater neutralizing capacity than IgG1, suggesting that less of the IgG1 was directed against neutralizing virion epitopes. These data are discussed with respect to the biological implications in host defence.
A part of the genome of dengue virus type 2 spanning the coding region from the carboxy terminus of the envelope protein E to the beginning of the NS3 protein was expressed using recombinant vaccinia virus. Additional constructs which contained open reading frames terminating within the NS1 or NS2A genes were also expressed. NS1 dimers were formed by extended NS1 molecules containing 61 amino acids of NS2A. No dimers were detected when NS1 was shortened by 79 amino acids at its carboxy terminus.
Summary.We developed an immunoblotting procedure to characterise the polyspecific immunoglobulin response to the proteins of herpes simplex virus (HSV)-1 . We found that 8-20% gradient polyacrylamide gels provided no advantage over fixed 8.5% gels for preparing Western blots for use in immunoblotting. The amount of protein loaded on the gels markedly influenced which proteins were detected by immune serum. The presence of Triton-X-100 0.5% in washes and buffers improved band clarity on immunoblots. In optimal conditions, immune mouse serum reacted with up to 33 HSV-1 lysate proteins. Six bands or regions appeared to be of major immunogenic reactivity, including the (122-130) x 103-mol. wt region, a 75 x 103-mol. wt protein, the gD region of approximately (56-64) x 103-mol. wt and two non-glycosylated bands at mol. wts( lo3) 42 and 44. Minor proteins, more weakly reactive, were detected at 27 other areas. The relative antibody titres in immune mouse serum to the different major regions showed antibody titre to gD > gB/gC > (42/44) x lo3 >> P75 >> VP154.Most human sera reacted with all of the major and many of the minor immunogenic proteins but individual sera varied markedly in the proteins recognised. We conclude that immunoblotting is valuable for evaluating immunoglobulin responses to major and minor immunogenic proteins of HSV-1.
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