Masayuki Tadano

Nuclear Localization of Japanese Encephalitis Virus Core Protein Enhances Viral Replication

ABSTRACT Japanese encephalitis virus (JEV) core protein was detected in both the nucleoli and cytoplasm of mammalian and insect cell lines infected with JEV or transfected with the expression plasmid of the core protein. Mutation analysis revealed that Gly42 and Pro43 in the core protein are essential for the nuclear and nucleolar localization. A mutant M4243 virus in which both Gly42 and Pro43 were replaced by Ala was recovered by plasmid-based reverse genetics. In C6/36 mosquito cells, the M4243 virus exhibited RNA replication and protein synthesis comparable to wild-type JEV, whereas propagation in Vero cells was impaired. The mutant core protein was detected in the cytoplasm but not in the nucleus of either C6/36 or Vero cell lines infected with the M4243 virus. The impaired propagation of M4243 in mammalian cells was recovered by the expression of wild-type core protein in trans but not by that of the mutant core protein. Although M4243 mutant virus exhibited a high level of neurovirulence comparable to wild-type JEV in spite of the approximately 100-fold-lower viral propagation after intracerebral inoculation to 3-week-old mice of strain Jcl:ICR, no virus was recovered from the brain after intraperitoneal inoculation of the mutant. These results indicate that nuclear localization of JEV core protein plays crucial roles not only in the replication in mammalian cells in vitro but also in the pathogenesis of encephalitis induced by JEV in vivo.

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.

Apoptosis induced by the histone deacetylase inhibitor FR901228 in human T-cell leukemia virus type 1-infected T-cell lines and primary adult T-cell leukemia cells.

ABSTRACT Inhibition of histone deacetylase (HDAC) activity induces growth arrest, differentiation, and, in certain cell types, apoptosis. FR901228, FK228, or depsipeptide, is an HDAC inhibitor effective in T-cell lymphomas. Adult T-cell leukemia (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1) and remains incurable. We examined whether FR901228 is effective for treatment of ATL by assessing its ability to induce apoptosis of HTLV-1-infected T-cell lines and primary leukemic cells from ATL patients. FR901228 induced apoptosis of Tax-expressing and -unexpressing HTLV-1-infected T-cell lines and selective apoptosis of primary ATL cells, especially those of patients with acute ATL. FR901228 also efficiently reduced the DNA binding of NF-κB and AP-1 in HTLV-1-infected T-cell lines and primary ATL cells and down-regulated the expression of Bcl-xL and cyclin D2, regulated by NF-κB. Although the viral protein Tax is an activator of NF-κB and AP-1, FR901228-induced apoptosis was not associated with reduced expression of Tax. In vivo use of FR901228 partly inhibited the growth of tumors of HTLV-1-infected T cells transplanted subcutaneously in SCID mice. Our results indicated that FR901228 could induce apoptosis of these cells and suppress the expression of NF-κB and AP-1 and suggest that FR901228 could be therapeutically effective in ATL.

Heteropentameric Cholera Toxin B Subunit Chimeric Molecules Genetically Fused to a Vaccine Antigen Induce Systemic and Mucosal Immune Responses: a Potential New Strategy To Target Recombinant Vaccine Antigens to Mucosal Immune Systems

ABSTRACT Noninvasive mucosal vaccines are attractive alternatives to parenteral vaccines. Although the conjugation of vaccine antigens with the B subunit of cholera toxin (CTB) is one of the most promising strategies for vaccine delivery to mucosal immune systems, the molecule cannot tolerate large-protein fusion, as it severely impairs pentamerization and loses affinity for GM1-ganglioside. Here we report a new strategy, in which steric hindrance between CTB-antigen fusion subunits is significantly reduced through the integration of unfused CTB “molecular buffers” into the pentamer unit, making them more efficiently self-assemble into biologically active pentamers. In addition, the chimeric protein took a compact configuration, becoming small enough to be secreted, and one-step affinity-purified proteins, when administered through a mucosal route, induced specific immune responses in mice. Since our results are not dependent on the use of a particular expression system or vaccine antigen, this strategy could be broadly applicable to bacterial enterotoxin-based vaccine design.

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.

Haemagglutination-inhibition Test for Haemorrhagic Fever with Renal Syndrome Using Virus Antigen Prepared from Infected Tissue Culture Fluid

Haemagglutinating (HA) antigens of four strains of virus related to that causing haemorrhagic fever with renal syndrome (HFRS) were prepared from infected tissue culture fluids by ultracentrifugation. The titres of the precipitated antigens were increased considerably by acetone extraction and sonication. Acetone extraction completely inactivated infectious virus in the antigen preparations. The antigens were pH-dependent, with pH optima at 5.8. Good correlations were observed in human and rat sera between the titres obtained by the haemagglutination-inhibition (HI) test and an indirect fluorescent antibody test. Moreover, strong cross-reactions among these strains were demonstrated by the HI test. The HI test has not been used previously with HFRS viruses because of the danger involved in preparing HA antigen, but these results indicate that a safe method is available for serological identification of HFRS.

Construction of Human Fab (Y1/K) Library and Identification of Human Monoclonal Fab Possessing Neutralizing Potency against Japanese Encephalitis Virus

A combinatorial human Fab library was constructed using RNAs from peripheral blood lymphocytes obtained from Japanese encephalitis virus hyper‐immune volunteers on pComb3H phagemid vector. The size of the constructed Fab library was 3.3 × 108 Escherichia coli transformants. The library was panned 3 times on the purified Japanese encephalitis virus (JEV) virion, and phage clones displaying JEV antigen‐specific Fab were enriched. The enriched phage pool was then screened for clones producing Fab molecule with JEV neutralizing activity by the focus reduction‐neutralizing test. Among 188 randomly selected clones, 9 Fab preparations revealed neutralizing activities against JEV strain Nakayama. An E. coli transformed with TJE12B02 clone, which produced human monoclonal Fab with the highest neutralizing activity was cultured in a large scale, and the Fab molecule was purified using affinity chromatography. The purified FabTJE12B02 showed the 50% focus reduction endpoint at the concentration of 50.2 μg/ml (ca. 1,000 nM) when JEV strain Nakayama was used. The FabTJE12B02 recognized E protein of JEV strain Nakayama, and the dissociation equilibrium constant (Kd) of the FabTJE12B02 against purified JEV antigen was calculated as 1.21 × 10–8 M. Sequence analysis demonstrated that TJE12B02 used a VH sequence homologous to the VH3 family showing 88.8% homology to germline VH3–23, and used a VK sequence homologous to the VκII subgroup showing 92.8% homology to germline A17.

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.

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.