No abstract

The four serotypes of dengue virus (a mosquito-borne flavivirus) cause an acute febrile illness (dengue fever) or a more prolonged illness with plasma leakage resulting in hypovolemia (dengue hemorrhagic fever). Hemorrhage may accompany either. Epidemiologic data suggest a role for dengue antibodies in pathogenesis. Computer analysis revealed a 20-residue region of similarity in amino acid sequence between the dengue type 4 envelope glycoprotein (E) and a family of clotting factors, including plasminogen, the prime mediator of fibrinolysis. By use of synthetic peptides in ELISA, E antibodies that potentially bind plasminogen were detected in 75% of 40 Thai patients acutely infected with dengue virus type 1, 2, 3, or 4. Plasminogen cross-reactivity of dengue antibodies was shown to be specific for the related sites in E and plasminogen. The dengue E sequence with similarity to plasminogen is largely conserved within the currently known flavivirus E sequences. However, 15 Thai patients hospitalized for illness caused by Japanese encephalitis virus (a flavivirus not associated with hemorrhage) did not develop plasminogen-cross-reactive antibodies, and this finding correlated with failure of Japanese encephalitis virus antibodies to bind to the plasminogen-cross-reactive site in E.

SUMMARYDengue type-2 viruses isolated in metropolitan Bangkok during 1980 (Bangkok/80) were characterized by oligonucleotide fingerprinting, restriction enzyme (RE) mapping and antigenic analysis using monoclonal antibody probes. Of 10 isolates analysed by oligonucleotide fingerprinting, nine were very closely related, showing 72-5 ~o to 91.4 ~ oligonucleotide homology. One isolate (D80-141) produced a distinctly different fingerprint (55.7~ to 58"0~o homology) and was less related to other Bangkok/80 dengue-2 virus isolates than to a 1964 Bangkok isolate (16681). RE mapping conducted on complementary dsDNA prepared from three Bangkok/80 isolates, strain 16681 and the prototype New Guinea C strain confirmed that D80-141 was genetically distinct. On antigenic analysis, only one of 22 monoclonal antibody probes produced against representative 1980 Bangkok dengue-2 isolates, D80-100 and D80-141, was able to distinguish between these virus strains. Monoclonal antibody 47-10/10, prepared using D80-100 virus and directed at the NS1 non-structural glycoprotein, had a significantly lower (100-fold) solid phase radioimmune assay endpoint titre for D80-141 antigen than for D80-100 antigen. By the indirect immunofluorescence assay, 47-10/10 had lower antibody endpoint titres against D80-141, the NGC strain and 13 (12~o) of 110 Bangkok/80 isolates than to a control antibody preparation. These results suggest that strain D80-141 represents a second minor topotype of dengue-2 which was circulating concurrently with the major endemic topotype in Bangkok in early 1980.

SUMMARYMonoclonal 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 (42000 mol. wt.) from DEN-2-infected C6/36 (Aedes albopictus) cells. The virus specificity of NS 1-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. INTRODUCTIONRecently, antibodies directed against a flavivirus non-structural glycoprotein (NS1) have been shown to confer passive protection against yellow fever virus (Schlesinger et al., 1985;Gould et al., 1986) or dengue (DEN) virus challenge (J. J. Schlesinger, personal communication) in the mouse model. In the human patient, large amounts of anti-NS1 antibodies appear in convalescent sera (Falker et al., 1973; W. E. Brandt, personal communication). Serotypespecific, dengue-complex, and flavivirus-group reactive antigenic determinants have been identified (Russell et al., 1970;Qureshi & Trent, 1973 ; Schlesinger et al., 1985). However, there has been some speculation that this soluble complement-fixing antigen (SCF) may stimulate an anamnestic response during human secondary heterologous dengue infections, contributing to the pathogenesis of dengue shock syndrome.With the advent of monoclonal antibody technology (K6hler & Milstein, 1976), it is now possible to identify important epitopes on viral antigens. We have previously identified discrete antigenic regions on the E glycoprotein that participate in neutralization, haemagglutination, and infection enhancement (Henchal et al., 1985). Moreover, these reagents have been used to identify serotype-specific and cross-reactive antigenic determinants . Here, we report the identification of unique epitopes on the NS1 non-structural protein.