Cloning full-length dengue type 4 viral DNA sequences: Analysis of genes coding for structural proteins

SUMMARYA mouse monoclonal antibody (MAb) to dengue 4 (DEN-4) virus reacted with the antigen in the nucleus as well as in the cytoplasm of DEN-4-infected mammalian and mosquito cells, as demonstrated by the peroxidase-antiperoxidase staining method. The intranuclear antigen appeared to accumulate at the nucleoli, forming spots, whereas the cytoplasmic antigen appeared to be localized mainly in large perinuclear loci in the infected cells. The MAb-reactive antigen was produced in the presence of actinomycin D, which caused the accumulation in the nucleus to be altered to a dispersed pattern. Radioimmunoprecipitation analysis of [35S]methionine-labelled purified virions and Western blot analysis of the antigens prepared from the infected mammalian and mosquito cells showed that the MAb was directed against the DEN-4 virus core protein (M~ 15-5K). These results indicated that the DEN-4 virus core protein was partially transported, soon after its synthesis in the cytoplasm, into the nucleus and accumulated at the nucleoli.

Section: Discussionmentioning

confidence: 60%

Detection of Dengue 4 Virus Core Protein in the Nucleus. I. A Monoclonal Antibody to Dengue 4 Virus Reacts with the Antigen in the Nucleus and Cytoplasm

Tadano

Makino

Fukunaga

et al. 1989

“…Alignment of the amino acid sequences of the E proteins of flaviviruses within the type-specific hypervariable domain proposed for TBE and dengue viruses. Sequences of LI (Shiu et al, 1991), TBE (Mandl et al, 1988;Ptetnev et al, 1990), LGT (Mandl et al, 1991), DEN1 (Mason et al, 1987), DEN2 (Deubel et al, 1986;Hahn et al, 1988), DEN3 (Osatomi & Sumiyoshi, 1990), DEN4 (Zhao et al, 1986), West Nile (WN) (Castle et al, 1985), Kunjin (KUN) (Coia et al, 1988), Japanese encephalitis (JE) (Sumiyoshi et al, 1987), Murray Valley encephalitis (MVE) (Dalgarno et al, 1986), St Louis encephalitis (SLE) (Trent et al, 1987) and yellow fever (Rice et al, 1985) viruses are aligned. The amino acids are numbered according to their positions in the respective E proteins.…”

Section: S S T ---A 232 Mve 228 P As T ---E 232 Sle 228 P At T ---D 2mentioning

confidence: 99%

Envelope protein sequences of dengue virus isolates TH-36 and TH-Sman, and identification of a type-specific genetic marker for dengue and tick-borne flaviviruses

Shiu

Jiang

Porterfield³

et al. 1992

Complementary DNAs were synthesized from the envelope protein genes of two isolates of dengue virus (TH-36 and TH-Sman, previously suggested as possible dengue virus type 5 and dengue virus type 6 respectively) and amplified by the polymerase chain reaction using sense and antisense primers designed from conserved dengue virus gene sequences. The amplified cDNA clones were sequenced in both directions by double-stranded dideoxynucleotide sequencing. Alignment with published dengue virus sequences enabled us to assign these viruses accurately to classified serotypes, confirming that TH-36 and THSman are strains of dengue virus type 2 and dengue virus type 1 respectively. Amino acid changes between the proteins encoded by these two isolates and strains of their respective serotypes may account for the significant antigenic differences observed during previous serological typing of these viruses. Moreover, sequence alignment of flavivirus envelope proteins revealed a hypervariable region, within which members of the dengue and tick-borne virus antigenic complexes show unique peptide sequences. This type-specific hypervariable domain may be useful as a genetic marker for typing dengue and tick-borne flaviviruses.Dengue viruses, mosquito-borne members of the family Flaviviridae (Westaway et al., 1985), are responsible for classic dengue fever and for the associated conditions dengue haemorrhagic fever (DHF) and dengue shock syndrome, each of which has a high level of morbidity and mortality in the tropics. There is evidence suggesting that the more severe forms of dengue fever occur when individuals (usually children) with cross-reactive but non-protective antibodies, acquired maternally or induced by prior infection with a different serotype, are naturally exposed to dengue virus infection (Halstead, 1981(Halstead, , 1988. Despite much research, effective dengue virus vaccines are not yet available, so the antigenic properties of dengue viruses have received much attention.Currently, four distinct dengue virus serotypes (types 1 to 4; DEN1 to DEN4) can be distinguished by complement fixation (CF), haemagglutination-inhibition and neutralization tests (Porterfield, 1980). Two Thai isolates are of particular interest. Strain TH-36 wasThe sequence data reported in this article have been deposited with the DDBJ, EMBL and GenBank databases under the accession numbers D01073 (TH-Sman) and D01074 (TH-36). isolated in 1958 by Hammon and co-workers from a patient with DHF in Bangkok, and TH-Sman was isolated from a similar patient by Dr Sman Vardhanabhuti. Strain TH-36 was initially identified as DEN2 and TH-Sman as DEN 1, but both were shown to differ to a significant degree from the respective prototype strains by CF, plaque neutralization, immunodiffusion and immunoelectrophoresis tests (Hammon et al., 1961; Hammon & Sather, 1964a, b; Ibrahim & Hammon, 1968a, b;Ibrahim et al., 1968). Tests using the soluble complement-fixing antigen also showed that strain THSman differed from the prototype DEN1 virus, but TH-36 was ind...

“…Sequence analysis of the genomes of flaviviruses has revealed considerable heterogeneity in the number and usage of glycosylation sites. Viruses having two potential N-linked glycosylation sites on the E glycoprotein are the MSI-7 strain of SLE (Trent et al, 1987), dengue type 1 , dengue type 2 (Deubel et al, 1986;Hahn et al, 1988;Gruenberg et aI., 1988), dengue type 3 (Osatomi et al, 1988;Osatomi & Sumioshi, 1990), dengue type 4 (Zhao et al, 1986) and American isolates of yellow fever (YF) virus (Ballinger-Crabtree & Miller, 1990). Other flaviviruses contain a single glycosylation site, including tick-borne encephalitis (TBE) virus (Mandl et al, 1988), Murray Valley encephalitis virus (Dalgarno et al, 1986), Japanese encephalitis (JE) virus (Sumiyoshi et al, 1987;McAda et aI., 1987) and an African isolate of YF virus (Rice et al, 1985;BallingerCrabtree & Miller, 1990).…”

Section: Introductionmentioning

confidence: 99%

Molecular and biological characterization of a non-glycosylated isolate of St Louis encephalitis virus

Vorndam¹,

Mathews

Barrett

et al. 1993

The glycosylation patterns of the envelope (E) glycoprotein of several naturally occurring strains of St Louis encephalitis (SLE) virus were investigated. SLE viruses were found that contained both glycosylated and non-glycosylated E proteins, and one isolate (Tr 9464) that lacks N-linked glycosylation sites on its E protein was identified. SLE virus monoclonal antibodies that define E protein B cell epitopes and demonstrate biological activities reacted essentially to the same extent with glycosylated and non-glycosylated virions. These results indicate that glycosylation is not essential for epitope conformation or recognition. However, failure to glycosylate the E protein was associated with possible morphogenetic differences as manifested by reduced virus yields and differences in specific infectivity.