Phylogenetic and phylodynamic analyses of hepatitis C virus subtype 1a in Okinawa, Japan.

Okinawa Island, located in Southern Japan, has a higher prevalence rate of hepatitis C virus subtype 1a (HCV-1a) infection than that in mainland Japan. Okinawa has a history of US military occupation after World War II. To elucidate the transmission history of HCV-1a in Okinawa, 26 whole-genome sequences were obtained from 29 patients during 2011-2016. Phylogenetic trees were reconstructed to identify the origin and characteristics of HCV-1a in Okinawa with epidemiological information. A phylogenetic tree based on whole-genome sequencing revealed that all of the samples were located below the US branches. Additionally, we identified one cluster comprised of 17 strains (Okinawa, n = 16; United States, n = 1). The majority of the patients in this cluster were people who inject drugs (PWID), indicating the presence of a people who inject drugs (PWID) cluster. Subsequently, Bayesian analyses were employed to reveal viral population dynamics. Intriguingly, a phylodynamic analysis uncovered a substantial increase in effective population size of HCV-1a from 1965 to 1980 and a slight increase in mid-2000, which were associated with an increase in illicit drug use in Okinawa. The estimated divergence time of the PWID cluster was 1967.6 (1964.2-1971.1). These findings suggest that HCV-1a was introduced into Okinawa from the United States in the late 1960s, coincident with the Vietnam War. Subsequently, HCV-1a might have spread among the Japanese population with the spread of injecting drug use. Our study provides an understanding of HCV transmission dynamics in Okinawa, as well as the key role of PWID in HCV transmission.

Inhibition of intracellular hepatitis C virus replication by nelfinavir and synergistic effect with interferon-alpha.

Liver diseases associated with hepatitis C virus (HCV) infection have become the major cause of mortality in patients with human immunodeficiency virus (HIV) infection since the introduction of highly active anti-retroviral therapy. HCV-related liver disease is more severe in HIV-infected patients than in non-HIV-infected patients, but the standard therapies used to treat chronic hepatitis C in HCV/HIV coinfected patients are the same as those for patients infected with HCV alone. HIV protease inhibitors might have potential to down-regulate HCV load of HCV/HIV coinfected patients. In this study, we evaluated the effects of nelfinavir on intracellular HCV replication using the HCV replicon system. We constructed an HCV replicon expressing a neomycin-selectable chimeric firefly luciferase reporter protein. Cytotoxicity and apoptosis induced by nelfinavir were assessed and synergism between nelfinavir and interferon (IFN) was calculated using CalcuSyn analysis. Nelfinavir dose-dependently repressed HCV replication at low concentrations (IC(50), 9.88 micromol/L). Nelfinavir failed to induce cytotoxicity or apoptosis at concentrations that inhibited HCV replication. Clinical concentrations of nelfinavir (5 micromol/L) combined with IFN showed synergistic inhibition of HCV replication in our replicon model. Our results suggest that the direct effects of nelfinavir on the HCV subgenome and its synergism with IFN could improve clinical responses to IFN therapy in HCV/HIV coinfected patients.

Potential use of a baculovirus-expressed dengue-4 E protein as a diagnostic antigen in regions endemic for dengue and Japanese encephalitis.

Truncated dengue-4 E protein was produced as a fusion protein in insect cells using a baculovirus expression vector to examine its usefulness as a diagnostic antigen. A peroxidase-anti-peroxidase (PAP) staining method was used to examine the immunoreactivity of the antigenic determinants in recombinant virus-infected Sf-9 cells with human sera obtained from dengue (DEN) and Japanese encephalitis (JE) endemic areas (41 sera from DEN patients and 39 sera from JE patients or individuals with high JE-antibody titers). The expressed E protein, in which one-third of the carboxy-terminal end was deleted, reacted with sera from DEN patients, but it failed to react or responded only faintly with sera from JE patients. The antibody titers obtained by the staining method correlated with those obtained by enzyme-linked immunosorbent assay (ELISA) (r = 0.64, P less than 0.01). Calculation of the ratio (R) of the titer obtained by the PAP staining method to the ELISA titer can clearly differentiate DEN antibody from JE antibody (high R values in DEN sera and low R values in JE sera). The recombinant protein would be especially useful for diagnostic purposes in regions where DEN and JE viruses co-circulate.

Comparative effect of casein and soybean protein isolate on body fat accumulation in adult rats.

The effect of dietary protein on the body fat accumulation was studied in rats. Adult rats weighing about 300 g were fed 21% protein (casein or soybean protein isolate) and 5% oil diets by pair-feeding for 65 days in Experiment 1. In Experiment 2, only protein and oil contents were changed, 25 and 10%, respectively. Final body weights of the two dietary groups were similar in both experiments, especially in Experiment 2. Total body fat was slightly lower in the soybean protein diet group than in the casein diet group in Experiment 2, only when it was expressed as the percentage against body weight. However, intra-abdominal fat was significantly lower in the soybean protein diet groups than in the casein diet groups in both experiments. Serum lipid levels were greatly lower in the soybean protein diet group than in the casein diet group in Experiment 2 (the data were not available in Experiment 1). The results suggest that dietary soybean protein has the effect to lower the intra-abdominal fat accumulation as compared with casein.

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 aspargine-proline-threonine sequence capable of serving as an attachment site of carbohydrate.

Immunocytochemical features of lens after cataract tissue--signalling molecules (growth factors, cytokines, other signalling molecules), cytoskeleton proteins, cellular and extracellular matrix proteins.

UNLABELLED: We examined the immunocytochemical features of after cataract tissue and cataractous lens epithelium prior to cataract surgery and at the time of surgery. METHODS: This study included 23 cases. Antibodies against the following antigens were used: epidermal growth factor (EGF), EGF receptor (EGF-R), fibroblast growth factor (FGF), FGF receptor (FGF-R), transforming growth factor (TGF)-beta, TGF-beta receptor II (TGF-beta -RII), insulin-like growth factor (IGF)II, platelet derived growth factor (PDGF)-AB, interleukin (IL)-6, IL-1 receptor II, tumor necrosis factor (TNF)-alpha, tissue plasminogen activator (TPA), prostaglandin (PG) E2, plasminogen activator inhibitor (PAI)-1, alpha smooth muscle actin (alpha-SMA), keratin, vimentin, myosin, fibronectin, laminin, types I, II, III, IV, V and VI collagen (Col), and lens epithelial cells (LECs). RESULTS: The following substances were present in posterior and anterior capsular fibrosis of after cataract: EGF, EGF-R, FGF, FGF-R, TGF-beta, TGF-beta-RII, IGF-II, PDGF-AB, IL-6, IL-1RII, TNF-alpha, PGE2, PAI-1, alpha-SMA, keratin, fibronectin, laminin, Type I col, Type II to VI col and LECs. Elschnig's pearls of after cataract expressed FGF-R, TNF-alpha, laminin and LECs. Soemmering's ring of after cataract tissue expressed EGF, FGF, FGF-R, IL-1RII, TPA, PAl-1, keratin and LECs. The following substances were present in cataractous lens epithelium prior to cataract surgery: EGF, EGF-R, FGF, FGF-R, TGF-beta-RII, IL-1-RII, TNF-alpha, PAI-1, keratin, laminin and LECs. The following substances were present in the lens epithelium of anterior capsules collected at the time of cataract surgery: EGF, EGF-R, FGF, FGF-R, TGF-beta, TGF-beta-RII, IGF-II, IL-6, IL-1-RII, TNF-alpha, PGE2, keratin, fibronectin, laminin, Type IV Col and LECs. CONCLUSIONS: These results prove that many kinds of signalling molecules and proteins exist in the process of after cataract formation.

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.

Immunization of rhesus monkeys with a recombinant of modified vaccinia virus Ankara expressing a truncated envelope glycoprotein of dengue type 2 virus induced resistance to dengue type 2 virus challenge.

Dengue epidemics increasingly pose a public health problem in most countries of the tropical and subtropical areas. Despite decades of research, development of a safe and effective live dengue virus vaccine is still at the experimental stage. To explore an alternative vaccine strategy, we employed the highly attenuated, replication-deficient modified vaccinia Ankara (MVA) as a vector to construct recombinants for expression of the major envelope glycoprotein of one or more dengue virus serotypes. MVA recombinants expressing the highly immunogenic C-terminally truncated dengue type 2 virus (DEN2) or dengue type 4 virus (DEN4) envelope protein (E), approx. 80% of the full-length, were evaluated for their protective immunity in animal models. Each of these recombinants elicited an elevated antibody response to DEN2 or DEN4 E in mice following the booster inoculation, as detected by radio-immunoprecipitation. Recombinant MVA-DEN2 80%E, but not MVA-DEN4 80%E, induced a neutralizing antibody response. The MVA-DEN2 80%E recombinant was chosen to further evaluate its ability to induce resistance to wild type DEN2 challenge in monkeys. Monkeys immunized twice with recombinant MVA-DEN2 80%E developed a low to moderate antibody response and were partially protected against DEN2 challenge, as determined by the viremia pattern. Importantly, the subsequent study showed that all four monkeys immunized with the recombinant in a three dose schedule developed an increased level of antibodies and were completely protected against DEN2 challenge. The potential efficacy of recombinant MVA-DEN2 80%E to protect primates against dengue infection suggests that construction and evaluation of MVA recombinants expressing other serotypes of dengue virus E for use in a tetravalent vaccine strategy might be warranted.