Toshihiko Fukunaga

Studies on Serological Cross-Reaction in Sequential Flavivirus Infections

Acute‐ and convalescent‐phase sera from patients with dengue (DEN) hemorrhagic fever (DHF) and Japanese encephalitis (JE) that contained pre‐existing flavivirus antibodies were tested for cross‐reacting antibodies to DEN, JE and yellow fever (YF) viruses by a neutralization (N) test. A fourfold or greater rise in N antibody titer in the convalescent‐phase was considered significant. Of 39 DHF cases, obtained at Chiang Mai University Hospital, Thailand, 15 (38.5%) showed a rise in DEN antibody titer, while another 15 (38.5%) showed a significant rise in both DEN and JE N antibody titers. On the other hand, eight (61.5%) of 13 JE cases obtained at the same Hospital, showed a significant rise in JE antibody titer, while two (15.4%) showed a significant rise in both DEN and JE antibody titers. Sucrose gradient centrifugation and fractionation of these two cross‐reactive JE sera revealed that IgM class antibody was specific for JE, while IgG class antibody was cross‐reactive. Of three JE cases with pre‐existing YF antibody obtained in Okinawa, Japan, two showed a significant rise in YF and JE antibodies. Both IgM and IgG class antibodies to YF virus were elevated. These results indicate that the cross‐reactivity among flaviviruses in different subgroups (complexes), was observed quite often, even by the N test, in sequential flavivirus infection.

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

A 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 foci 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 (Mr 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.

Detection of dengue 4 virus core protein in the nucleus. II: Antibody against dengue 4 core protein produced by a recombinant baculovirus reacts with the antigen in the nucleus

The dengue 4 virus (DEN-4) core gene and part of the PreM genes were inserted into the baculovirus polyhedrin gene region. The recombinant baculovirus directed the synthesis of the DEN-4 core protein fused to a part of the polyhedrin protein (Mr 25K), as determined by Western blot analysis using DEN-4 core monoclonal antibody. A mouse polyclonal antibody prepared against the DEN-4 core fusion protein showed antigenic reactivity with the authentic DEN-4 core protein (Mr 15.5K) present in the nucleus as well as in the cytoplasm of DEN-4-infected Vero cells as demonstrated by the peroxidase-antiperoxidase staining method. This antibody did not react with cells infected with DEN-1, -2, -3 or Japanese encephalitis virus, or mock-infected cells.

Molecular Epidemiology of Japanese Encephalitis Virus in Okinawa

In order to elucidate the molecular characteristics of Japanese encephalitis (JE) virus in Okinawa, 23 strains of JE virus isolated in a 25‐year span were sequenced for the 240 nucleotides of the C‐preM junction region and 111 nucleotides of the E gene region and compared with those of reference strains isolated in mainland Japan. The results of phylogenic analysis showed that although all the Okinawan isolates showed more than 96% homology in the nucleotide sequence in each region, they were chronologically divided into two groups: the old group (nine strains) and a new group (14 strains). On the other hand, in a comparison with reference strains in mainland Japan, the Okinawan isolates showed more than 94% nucleotide sequence homology in both regions, indicating that the Okinawan strains belong to the same genotype as that of JE strains in mainland Japan. The nucleotide homology of the old group was relatively higher than that of the new group. Among the 14 strains in the new group, 13 strains were isolated from mosquitoes collected from a pig farm from 1986 through 1992. These strains showed higher nucleotide divergence than the old group strains, isolated from mosquitoes and swine sera collected at several sites, in both regions. A nucleotide substitution at the position 1920 in the E gene was identified in three isolates. This substitution generated an asparagine‐proline‐threonine sequence capable of serving as an attachment site of carbohydrate.

Epidemiological and Ecological Studies of Japanese Encephalitis in Okinawa, Subtropical Area in Japan. I. Investigations on Antibody Levels to Japanese Encephalitis Virus in Swine Sera and Vector Mosquito in Okinawa, Miyako and Ishigaki Islands

From 1985 to 1989, serum specimens of swine raised in the northern, central and southern areas in Okinawa island were examined for Japanese encephalitis (JE) virus antibody by ELISA and hemagglutination‐inhibition test. The antibody positive rate was found to be higher in the north and central than in the south. The 2‐mercaptoethanol sensitive antibody to JE was detected mostly in June and July, and occasionally in other months except February and March. There was no month when all specimens from three areas turned antibody‐negative simultaneously, indicating that JE virus transmission to swine lasted longer in Okinawa island than in other temperate areas in Japan. From 1986 to 1991, the vector mosquitoes (Culex tritaeniorhynchus) were collected in a pig farm in the south of Okinawa island. A total of 153 strains of JE virus was isolated from the vector mosquitoes mainly in June. In Miyako and Ishigaki islands, the antibody positive rate in swine sera was found to be extremely low, compared with that in Okinawa island. In Miyako island, where no paddy rice field is cultivated, a few adults as well as larvae of the vector mosquito were collected, while in Ishigaki island, where there are many watered rice fields, a lot of adults as well as larvae were collected. Although the environmental situation is quite different between the two islands, JE virus transmission appeared to be very low in both islands.

Electron Microscopic Observation of a Newly Isolated Flavivirus-like Virus from Field-caught Mosquitoes

Of many unidentified virus strains which were isolated from field-caught mosquitoes by using C6/36 cells (a virus-sensitive clone of Aedes albopictus cells), three strains which formed small size plaques (SP virus) in C6/36 cells were investigated by electron microscopy. Although the SP virus strains did not react with antisera against known arboviruses in serological tests, they closely resembled flaviviruses in morphology. However, when they were compared to Japanese encephalitis (JE) virus, several differences in morphogenesis were observed. Proliferating membranous structures and electron-dense amorphous areas involving precursors of the virus were observed only in cells infected with the SP virus strains. Enlarged areas of endoplasmic reticulum containing mature virions were often observed adjacent to these structures. Since the SP virus strains were isolated from wild mosquitoes and multiplied only in mosquito cells, it seems appropriate to classify them as insect viruses which resemble togaviruses morphologically.

Epidemiological and ecological studies of Japanese encephalitis in Okinawa, subtropical area in Japan. II: Prevalence of Japanese encephalitis antibody in residents in Okinawa, Miyako and Ishigaki Islands

During 1989 to 1990, human sera were collected by age groups in Okinawa (the northern, central and southern areas), Miyako and Ishigaki islands and examined for the neutralization (N) antibodies to two strains, Nakayama (vaccine strain) and C307 (Okinawan strain), of Japanese encephalitis (JE) virus. In Okinawa island, the N antibody positive rate to C307 was higher than that to Nakayama, while in Miyako and Ishigaki islands, the positive rate to Nakayama was higher than that to C307, suggesting that JE virus transmission rate was higher in Okinawa than in Miyako and Ishigaki islands. In Okinawa Prefecture, JE vaccine had not been administered to most of residents over 31 years of age at the time of serum collection. In residents over 31 years old, the positive rate to C307 was highest in the north of Okinawa (83.3%) and was lowest in Miyako (26.3%), with the second lowest in Ishigaki (33.3%). The distribution of N antibody titers to C307 gave hyperbolic patterns in the 0–5 age groups in Miyako and Ishigaki, and also in the 31–40, 41–50 age groups in Miyako and the 41–50 age group in Ishigaki, suggesting low rates of natural infection in these 4–5 decades in both islands. In residents of ages subjected to JE vaccine, a characteristic pattern was obtained, in which the curves to Nakayama shifted to higher titers than those to C307, suggesting that the first antigenic stimulation was caused by vaccine, not by natural infection of JE virus.