Immune response in rabbits to dengue viral proteins.

Antisera to all types of dengue and Japanese encephalitis (JE) viruses were raised in rabbits. The first set of rabbits was immunized with crude antigens prepared by a sucrose acetone extraction. The first generation of antisera demonstrated antibody activity towards the group specific flaviviruses, without unwanted antibody activity towards the mouse brain material. Antibody activity towards E, NS3, NS5 and NS1 could be observed by western blot/immunoenzymatic assay. Another set of rabbits was immunized with the precipitin complexes formed between antisera raised against each type of dengue, or JE viruses, and their homologous antigens. Each rabbit serum was screened again by the western blot/immunoenzymatic method, and was absorbed with other types of dengue viruses until the specific activity towards the immunized viral antigen was obtained. The last 2 bands detected on the viral antigen strip were the doublet of protein bands at Mr 67 and 71 kDa, which are the NS3 protein. The specific polyclonal antisera obtained can be used with other tests as well as the monoclonal antibody. Since absorption can lead to type specific antisera, this NS3 protein must be quite unique, it processes a type specific determinant (s) and deserves further study as a target molecule at the polypeptide level as well as at the RNA level.

Dengue virus serotypic identification using suckling mouse and western blot technique.

The suckling mouse which is used in the classical method to detect and propagate dengue viruses was evaluated in conjunction with the western blot and immunoenzymatic methods to detect the infecting strains of dengue viruses. After intracerebral inoculation of patients' sera into the suckling mice for 7 days, the mice were examined for the presence of dengue proteins, even though the mice did not have the neurological symptoms which usually serve as an indicator for the presence of dengue infection in the mouse brain. With a blind study of a set of 12 specimens, the suckling mice could detect the virus with the same frequency as the mosquito system but in shorter time of incubation period. The whole process to identify the type of infection takes 9 days. Another important finding is the demonstration of the virion antigen in the liver. The quantity and quality of viral proteins in liver are comparable to those in the brain suggesting that the virus may replicate in the liver as well as in the brain.

Antibodies to dengue viral polypeptides. I. Sensitivity and specificity of the viral-antigen-strips/enzymeimmunoassay (VAS/EIA).

The authors applied polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), western blot, and enzymeimmunoassay (EIA) for the detection of antibodies toward individual dengue viral proteins or polypeptides. SDS-PAGE procedure as described by Laemmli et al., was applied and modified. The results can be observed by visualization. The scores, from 0 to 4, can be assigned by comparing the intensity of the color development. Besides being sensitive and rapid, this technique yields information of the polypeptides or molecular level. An increasing of intensities of positive reactions indicated rising in antibodies titers and all serotypes of dengue viruses (from type 1 to type 4) can be tested together allowing reliable comparison among serotypes. With hyperimmune human sera, at least 13 polypeptides reacted with sera while negative non-immune subject showed no reaction. It is highly possible to use this technique as a rapid quantitative and for qualitative analysis of antibodies to individual viral proteins as well.

Application of peroxidase-antiperoxidase (PAP) staining for detection and localization of dengue-2 viral antigen. II. Observations for the antibody enhancement activity in human monocytes.

Peroxidase-antiperoxidase (PAP) staining was applied to measure the antibody enhancement activity in human monocytes. Increasing in number of infected cells can be seen with increasing of staining intensity of the cells by ordinary light microscope. Shifting of the optimum enhancement activity was found in previously tritiated antiserum indicated that for titration of antibody enhancement activity several dilutions of antiserum should be included in each experiment. Validity of the PAP method was made by the comparison of the results with Infectious Center Assay (ICA). With this technique, titration for antibody enhancement for dengue virus infection can be done with non-expensive equipment and can be kept for comparison for months.

Precipitating antibodies to non-treated dengue type 2 viral antigens in sera of Thai hemorrhagic fever patients by crossed immunoelectrophoresis.

Crossed immunoelectrophoresis of dengue type 2 virus revealed at least two precipitating antigens which shared some antigenic determinants. Glycoprotein components of both antigens were detected by binding to concanavalin A. Sera from dengue hemorrhagic fever patients showed precipitating antibodies to both antigens which could be quantitated according to the precipitate patterns formed in the intermediate gel of crossed immunoelectrophoresis. All secondary dengue hemorrhagic fever patients demonstrated an increase in precipitin titers in convalescence sera. Most patients with mild illness contained precipitating antibodies in acute phase sera whereas severe cases did not. Convalescent sera from severe cases showed only low titers. These precipitating antibodies may be associated with protection since they were produced early only in those with mild form of illness.

Application of peroxidase-antiperoxidase (PAP) staining for detection and localization of dengue-2 antigen. I. In an endogenous peroxidase containing cell systems.

The unlabelled immunoperoxidase, peroxidase-anti-peroxidase (PAP), technique was used to detect dengue type-2 viral antigen in several cell systems including the endogenous peroxidase containing cells. These cells are the mosquito cell line (C6/36), continuous cell line of rhesus monkey kidney (LLC-MK2), human monocyte culture both cell suspension and monolayer, and human peripheral blood leukocytes. All of these specimens gave the same results that dengue-2 viral antigen presented in cytoplasm only and the patterns of marker presentation in positive cells varied depending on the duration after infection. The sensitivity of this method is extremely high since it can detect dengue-2 antigen after its attachment on mosquito cells (15 min) as seen in experiments with mosquito cell line, C6/36. False positive was not observed in all cell systems tested.