Masaru Nawa

Virus isolation as one of the diagnostic methods for dengue virus infection

Virus isolation is the most reliable evidence of infection. In the present study, we isolated virus from serum samples collected from confirmed dengue cases. When data were analyzed based on disease days, dengue viruses were isolated from 28 of 32 serum samples collected on disease day 5 or earlier. When analyzed based on fever days, dengue viruses were isolated from all the serum samples collected on fever day -3 or earlier, and from 10 of 13 samples collected on fever days -2 and -1. Viruses were isolated from one each of the serum samples collected on fever days 0, 1, 2 and 3, respectively. Virus was not, however, isolated from those collected on fever day 4 or later. The results of virus isolation and reverse transcriptase-polymerase chain reaction were consistent in 78 of 82 serum samples. These results suggest that virus isolation is a useful and sensitive technique for confirmation of dengue virus infection, especially when serum samples are collected before fever subsides.

Serotype-Cross-Reactive Immunoglobulin M Responses in Dengue Virus Infections Determined by Enzyme-Linked Immunosorbent Assay

ABSTRACT We developed immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assays (ELISAs) with four monovalent dengue virus antigens. We attempted to determine whether IgM responses in dengue virus infections are serotype specific or serotype cross-reactive. Serum samples from 14 confirmed dengue cases were examined. In these 14 cases, which consisted of 12 Japanese and 2 non-Japanese patients, infecting dengue virus serotypes were defined by reverse transcription-PCR. Thirteen of the 14 cases were IgM positive in ELISA. IgM responses were serotype cross-reactive in these 13 cases but were highest against infecting dengue virus serotype in 9 of the 13 cases. These results indicate that IgM responses are generally dengue serotype cross-reactive but that IgM levels are highest against the infecting serotype in most dengue cases.

Antibody Responses Determined for Japanese Dengue Fever Patients by Neutralization and Hemagglutination Inhibition Assays Demonstrate Cross-Reactivity between Dengue and Japanese Encephalitis Viruses

ABSTRACT Titers of antibodies to infecting dengue virus serotypes determined by serum neutralization assay were higher than those of antibody to Japanese encephalitis (JE) virus in Japanese dengue patients after disease day 8. Titers of antibody to dengue virus antigens determined by hemagglutination inhibition (HI) assay were higher in only 1 of 23 serum specimens after disease day 11. Thus, the neutralization test is more reliable than the HI test for serological diagnosis of dengue in countries where JE vaccination is widely used or JE is endemic.

Immunoglobulin A Antibody Responses in Dengue Patients: a Useful Marker for Serodiagnosis of Dengue Virus Infection

ABSTRACT We determined the usefulness of an immunoglobulin A (IgA) antibody-capture enzyme-linked immunosorbent assay for serodiagnosis of dengue virus infections. The results indicate that the presence of IgA and IgM in serum samples assures recent primary dengue virus infection even with a single serum sample.

Japanese encephalitis virus infection in Vero cells: the involvement of intracellular acidic vesicles in the early phase of viral infection was observed with the treatment of a specific vacuolar type H+-ATPase inhibitor, bafilomycin A1

The involvement of intracellular acidic vesicles in the early phase of Japanese encephalitis (JE) virus infection in Vero cells was observed by adding a specific vacuolar type H+ ‐ATPase (V‐ATPase) inhibitor (bafilomycin A1) in the cell culture medium. Studies with the detection of viral envelope (E) protein suggested that bafilomycin A1 inhibited virus infection in the cells. Subcellular distribution of incoming biotinylated virions and 3H‐uridine‐labeled viral RNA were observed in fractions of a Percoll density gradient. At 10 min of the chasing period, virions and viral RNA were found mainly in fractions with a mean density of 1.04 g/ml corresponding to the endosome both in the control and bafilomycin A1‐treated cells. At 60 min of the chasing period, the peak of biotin activity was detected in fractions with a mean density of 1.08 g/ml corresponding to the lysosome, whereas the peak of radioactivity did not run parallel with that of biotin and shifted to fractions with a mean density of 1.05 g/ml and higher than 1.084 g/ml, respectively. At 60 min of the chasing period in bafilomycin A1‐treated cells, the peak of biotin and radioactivity were still found mainly in the fraction with a density of 1.04 g/ml, representing the endosome. Subcellular fractionation by a Percoll density gradient revealed that bafilomycin A1 treatment resulted in the accumulation of virions in the endosome fraction and suggested the prevention of intracellular translocation of the virions which occurs during the early entry process of an infecting virus to the cells.

Stability of hemagglutinating activity of extracellular and intracellular forms of Japanese encephalitis virus exposed to acidic pH.

The hemagglutinating (HA) activity of extracellular and intracellular forms of Japanese encephalitis (JE) virus was comparatively titrated by exposure to acidic pH below 7.0. A pH‐dependent irreversible loss in titer was observed with the virus grown in both C6/36 and BHK 21 (BHK) cells maintained in the pH range of 5.8 to 7.0 for 10 min at 37 C. The HA activity of intracellular virus was relatively more stable than that of extracellular virus in the pH range of 5.8 to 6.4. Virion structural components, envelope glycoprotein (E), capsid (C), and membrane (M) proteins in extracellular virus and E, C, and the precursor form of M (prM) proteins in intracellular virus were detected by SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) and immunoblotting. A panel of monoclonal antibody (mAb) directed for nine antigenic epitopes on the JE virus E protein molecule was used for the analysis of antigenic reactivity of E protein after treatment at pH 6.0. The reaction between the extracellular virus and three HA‐inhibiting (HI) mAbs was significantly reduced after acid treatment; however, the antigenic reactivity of intracellular virus was much more stable with a 100‐ to 1,000‐fold difference. Infectivity titers of extracellular and intracellular viruses in Vero cells were reduced by 1/24,100 and 1/21,666 after acidic treatment at pH 6.0. In contrast, the infectivity of intracellular viruses was more stable, with residual infectivity of 1/182 and 1/340 for BHK and C6/36 cell‐grown virus, respectively. Acidic treatment of JE virus not only resulted in the irreversible loss of its HA activity but also affected the antigenic reactivity of HI epitopes on its E protein molecule.

An Enzyme-Linked Immunosorbent Assay Using a Chaotropic Agent (Sodium Thiocyanate) for Serotype Specific Reaction between Crude Dengue Viral Antigen and Anti-Dengue Mouse Antibody

An enzyme‐linked immunosorbent assay (ELISA) has been developed to detect serotype specific reaction between crude dengue viral antigen and anti‐dengue mouse hyperimmunized antibody under the stringent condition in the presence of a Chaotropic agent, sodium thiocyanate (NaSCN), in the reaction mixture of antigen and antibody. Rapidly sedimenting hemagglutinin (RHA) derived from type 2 dengue virus‐infected mosquito cell culture fluid reacted to the antibody for both type 2 and type 3 dengue viruses in the ELISA. In contrast, its reactivity was reduced after the addition of NaSCN in the ELISA. Soluble complement‐fixing antigen (SCF) derived from type 2 dengue virus‐infected mosquito cell culture fluid reacted serotype specifically to anti‐dengue type 2 antibody, and was relatively stable for the NaSCN treatment in the ELISA. Anti‐type 2 RHA mouse antibody reacted to both type 1 and type 2 dengue viral antigens and its reactivity was reduced after the addition of NaSCN in the ELISA. Anti‐type 2 SCF antibody reacted serotype specifically to type 2 dengue viral antigen with and without NaSCN in the ELISA.

Development of a new cell system for the infectivity assay of dengue viruses: plaque formation and virus growth of prototype and wild-type dengue virus strains in a newly established cell line, GK

A newly established cell line, GK, derived from the kidney tissue of Mongolian gerbils, produced plaques by infection of prototype and wild‐type dengue virus strains. Both prototype and wild strains of type 2 virus grew in GK cells and formed plaques at 35.5 C and at 31 C, while types 1, 3, and 4 wild strains grew and formed plaques only at 31 C. In GK cells, plaque formation and the growth of dengue viruses depended on the high (35.5 C) and low (31 C) incubation temperatures.