Yoshihiro Makino

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 dengue virus serotypes 2 and 3 in Paraguay during 2001-2006: the association of viral clade introductions with shifting serotype dominance.

To determine the genetic variability of dengue viruses (DENVs) in Paraguay, the complete envelope gene was sequenced for 4 DENV-2 and 22 DENV-3 strains isolated from 2001 to 2006. The sequence data were used in Bayesian phylogenetic analyses, which revealed that Paraguayan DENV-2 strains fell into two distinct clades within the American/Asian genotype, thus suggesting that the introduction of a new DENV-2 clade was likely associated with the shift of dominant serotype from DENV-3 to DENV-2 in 2005 and might have caused an outbreak of DENV-2. This study also indicated that DENV-3 strains fell into genotype III, of which, several 2006 isolates varied from the remaining isolates in their tree locations. The introduction of this new clade was likely associated with the shift of dominant serotype from DENV-2 to DENV-3 in 2006 and might have caused an epidemic of DENV-3. More data are needed to test this hypothesis.

Molecular evolution of Japanese encephalitis virus isolates from swine in Oita, Japan during 1980-2009.

In order to identify the patterns of genetic change of Japanese encephalitis virus (JEV) strains circulating in Oita, the complete envelope (E) gene has been sequenced for 35 isolates from swine in a 30-year span. Based on nucleotide and deduced amino acid sequences, the genetic variation was examined, phylogeny was estimated and selection pressures were also analyzed. This study demonstrated that the major genotype (G) of JEV isolates had shifted from GIII to GI in the mid-1990s in Oita. The intensities of selection acting on the Oita GIII and GI strains were found to be almost same. It suggests that the intensity of selection might not be the reason for such a genotype shift observed in Oita. Pairwise comparisons revealed the high conservation of the E gene at the protein level. Compared with the Oita GIII strains, all the Oita GI strains shared four amino acid changes at E129 (T-M), E222 (A-S), E327 (S-T) and E366 (A-S). Among all 70 JEV isolates involved in this paper, the GI strains shared only one amino acid change at E222 (A-S) in comparison with the GIII strains. No strong evidence for positive selection was found, the JEV evolution has generally been subject to strong purifying selection, but one ongoing evolutionary pathway was found to be under relaxed purifying selection in Oita. This study is a localized example of JEV molecular evolution in nature.

Molecular basis for adaptation of a chimeric dengue type-4/Japanese encephalitis virus to Vero cells.

The premembrane and envelope (E) genes of a full‐length cDNA clone of the dengue type‐4 (DEN4) virus 814669 strain were replaced with those of the Japanese encephalitis (JE) virus JaOH0566 strain. The in vitro‐synthesized RNA transcripts prepared from chimeric cDNA were used to transfect mosquito C6/36 cells. A viable chimeric virus (designated DEN4/JE) was recovered. Unexpectedly, DEN4/JE exhibited restricted growth in Vero cells. After a serial passage in Vero cells, the Vero‐adapted chimeras were obtained (two clones, designated Strain I and Strain II, respectively). The entire genomes of DEN4/JE, Strain I, and Strain II were sequenced and compared. There were multiple mutations, but amino acid substitutions occurred only in E and nonstructural (NS) protein NS4B. Our findings in this study indicate that the 5′ nontranslated region, E, and NS4B may be involved in Vero cell adaptation in this chimeric system.

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.

Trends in fatal coronary heart disease among people aged 25-74 years in Oita City, Japan, from 1987-1998

Serum cholesterol has been increasing in recent years in Japan. There is concern that risk of coronary heart disease (CHD) may be increasing too, but there is little information on validated fatal CHD trends in the Japanese population. We identified 1,056 deaths from heart disease and other deaths possibly hiding CHD from death certificates of residents aged 25-74 years in Oita City, Japan in 1987-1988, 1992-1993, and 1997-1998 (mean population, 273,000 in 1997-1998). We validated 994 of them by medical record review and physician interviews, classifying them into definite fatal acute myocardial infarction (AMI) and possible fatal AMI or CHD death based on Monitoring Trends and Determinants in Cardiovascular Disease projects criteria. Sudden death was defined to estimate the number of CHD sudden deaths. In men, age-adjusted mortality rates due to validated fatal CHD remained quite stable over 10 years (25.3 per 100,000 [95% CI, 15.0-35.5] in 1987-1988 to 24.2 per 100,000 [95% CI, 16.1-32.3] in 1997-1998). When 50% or all sudden deaths were included as fatal CHD, the rates for men tended to decline. This was due to decreasing out-of-hospital deaths in connection with a declining CHD death rate among men aged 65-74 years, whereas in-hospital CHD deaths were level. In women, the rate of validated fatal CHD was highest in 1992-1993, but the 1997-1998 rate was similar to the 1987-1988 rate. We did not find that fatal CHD rates increased in Oita men and women from 1987-1998. Rather, out-of-hospital fatal CHD tended to decline in Oita men.

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.