Simple estimation of induced electric fields in nervous system tissues for human exposure to non-uniform electric fields at power frequency.

Most results regarding induced current in the human body related to electric field dosimetry have been calculated under uniform field conditions. We have found in previous work that a contact current is a more suitable way to evaluate induced electric fields, even in the case of exposure to non-uniform fields. If the relationship between induced currents and external non-uniform fields can be understood, induced electric fields in nervous system tissues may be able to be estimated from measurements of ambient non-uniform fields. In the present paper, we numerically calculated the induced electric fields and currents in a human model by considering non-uniform fields based on distortion by a cubic conductor under an unperturbed electric field of 1 kV m(-1) at 60 Hz. We investigated the relationship between a non-uniform external electric field with no human present and the induced current through the neck, and the relationship between the current through the neck and the induced electric fields in nervous system tissues such as the brain, heart, and spinal cord. The results showed that the current through the neck can be formulated by means of an external electric field at the central position of the human head, and the distance between the conductor and the human model. As expected, there is a strong correlation between the current through the neck and the induced electric fields in the nervous system tissues. The combination of these relationships indicates that induced electric fields in these tissues can be estimated solely by measurements of the external field at a point and the distance from the conductor.

A comparative epidemiological study of hantavirus infection in Japan and Far East Russia.

Hantaviruses are causative agents of some severe human illnesses, including hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). The viruses are maintained by rodent hosts, and humans acquire infection by inhaling virus-contaminated excreta from infected animals. To examine the epidemiology of hantavirus infections in Japan and Far East Russia, we conducted epidemiological surveys in these regions. In Japan, anti-hantavirus antibodies were found in four rodent species, Clethrionomys rufocanus, Rattus norvegicus, R. rattus, and Apodemus speciosus. Although no new HFRS cases have been officially reported over the past 20 years in Japan, one member of the Japan Ground Self-Defense Force did test positive for hantavirus antibody. Repeated surveys in Far East Russia have revealed that two distinct hantavirus types cause severe HFRS in this region. Hantavirus sequences identified from A. peninsulae, fetal HFRS cases in Vladivostok, and Amur virus are highly similar to each other (> 92% identity), but they are less similar (approximately 84% identity) to the prototypical Hantaan virus, which is carried by A. agrarius. Phylogenetic analysis also indicates that Amur and A. peninsulae-associated viruses are distinct from Hantaan virus, suggesting that A. peninsulae is the reservoir animal for Amur virus, which causes severe HFRS. From HFRS patients in the Khabarovsk region, we identified viruses with nucleotide sequences that are more similar to Far East virus (> 96%identity) than to the Hantaan (88-89% identity) or Amur (81-83% identity) viruses. Phylogenetic analysis also indicates that the viruses from Khabarovsk HFRS patients are closely related to the Far East virus, and distinct from Amur virus.

Critical role of PA28gamma in hepatitis C virus-associated steatogenesis and hepatocarcinogenesis.

Hepatitis C virus (HCV) is a major cause of chronic liver disease that frequently leads to steatosis, cirrhosis, and eventually hepatocellular carcinoma (HCC). HCV core protein is not only a component of viral particles but also a multifunctional protein because liver steatosis and HCC are developed in HCV core gene-transgenic (CoreTg) mice. Proteasome activator PA28gamma/REGgamma regulates host and viral proteins such as nuclear hormone receptors and HCV core protein. Here we show that a knockout of the PA28gamma gene induces the accumulation of HCV core protein in the nucleus of hepatocytes of CoreTg mice and disrupts development of both hepatic steatosis and HCC. Furthermore, the genes related to fatty acid biosynthesis and srebp-1c promoter activity were up-regulated by HCV core protein in the cell line and the mouse liver in a PA28gamma-dependent manner. Heterodimer composed of liver X receptor alpha (LXRalpha) and retinoid X receptor alpha (RXRalpha) is known to up-regulate srebp-1c promoter activity. Our data also show that HCV core protein enhances the binding of LXRalpha/RXRalpha to LXR-response element in the presence but not the absence of PA28gamma. These findings suggest that PA28gamma plays a crucial role in the development of liver pathology induced by HCV infection.

Involvement of the PA28gamma-dependent pathway in insulin resistance induced by hepatitis C virus core protein.

The hepatitis C virus (HCV) core protein is a component of nucleocapsids and a pathogenic factor for hepatitis C. Several epidemiological and experimental studies have suggested that HCV infection is associated with insulin resistance, leading to type 2 diabetes. We have previously reported that HCV core gene-transgenic (PA28gamma(+/+)CoreTg) mice develop marked insulin resistance and that the HCV core protein is degraded in the nucleus through a PA28gamma-dependent pathway. In this study, we examined whether PA28gamma is required for HCV core-induced insulin resistance in vivo. HCV core gene-transgenic mice lacking the PA28gamma gene (PA28gamma(-/-)CoreTg) were prepared by mating of PA28gamma(+/+)CoreTg with PA28gamma-knockout mice. Although there was no significant difference in the glucose tolerance test results among the mice, the insulin sensitivity in PA28gamma(-/-)CoreTg mice was recovered to a normal level in the insulin tolerance test. Tyrosine phosphorylation of insulin receptor substrate 1 (IRS1), production of IRS2, and phosphorylation of Akt were suppressed in the livers of PA28gamma(+/+)CoreTg mice in response to insulin stimulation, whereas they were restored in the livers of PA28gamma(-/-)CoreTg mice. Furthermore, activation of the tumor necrosis factor alpha promoter in human liver cell lines or mice by the HCV core protein was suppressed by the knockdown or knockout of the PA28gamma gene. These results suggest that the HCV core protein suppresses insulin signaling through a PA28gamma-dependent pathway.

Characterization of HCV-like particles produced in a human hepatoma cell line by a recombinant baculovirus.

Although processing of the hepatitis C virus (HCV) polyprotein and characterization of each of its viral proteins have been described in detail, analysis of the structure and assembly of HCV particles has been hampered by the lack of a robust cell culture system to support efficient replication of HCV. In this study, we generated HCV-like particles (HCV-LP) using a recombinant baculovirus encoding structural and a part of non-structural proteins in a human hepatoma cell line. The HCV-LP exhibited a buoyant density of 1.17 g/ml in CsCl equilibrium gradient and particles of 40 to 50 nm in diameter. Binding of the HCV-LP to human hepatoma cells was partially inhibited by the treatment with anti-hCD81 antibody, in contrast to the hCD81-independent binding of HCV-LP produced in insect cells. These results indicate that HCV-LP generated in different types of cells exhibit different cellular tropism for binding to target cells.

Involvement of the Toll-like receptor 9 signaling pathway in the induction of innate immunity by baculovirus.

We have previously shown that mice inoculated intranasally with a wild-type baculovirus (Autographa californica nuclear polyhedrosis virus [AcNPV]) are protected from a lethal challenge by influenza virus. However, the precise mechanism of induction of this protective immune response by the AcNPV treatment remained unclear. Here we show that AcNPV activates immune cells via the Toll-like receptor 9 (TLR9)/MyD88-dependent signaling pathway. The production of inflammatory cytokines was severely reduced in peritoneal macrophages (PECs) and splenic CD11c(+) dendritic cells (DCs) derived from mice deficient in MyD88 or TLR9 after cultivation with AcNPV. In contrast, a significant amount of alpha interferon (IFN-alpha) was still detectable in the PECs and DCs of these mice after stimulation with AcNPV, suggesting that a TLR9/MyD88-independent signaling pathway might also participate in the production of IFN-alpha by AcNPV. Since previous work showed that TLR9 ligands include bacterial DNA and certain oligonucleotides containing unmethylated CpG dinucleotides, we also examined the effect of baculoviral DNA on the induction of innate immunity. Transfection of the murine macrophage cell line RAW264.7 with baculoviral DNA resulted in the production of the inflammatory cytokine, while the removal of envelope glycoproteins from viral particles, UV irradiation of the virus, and pretreatment with purified baculovirus envelope proteins or endosomal maturation inhibitors diminished the induction of the immune response by AcNPV. Together, these results indicate that the internalization of viral DNA via membrane fusion mediated by the viral envelope glycoprotein, as well as endosomal maturation, which releases the viral genome into TLR9-expressing cellular compartments, is necessary for the induction of the innate immune response by AcNPV.

Genetic and antigenic characterization of the Amur virus associated with hemorrhagic fever with renal syndrome.

The genetic and antigenic characteristics of the Amur (AMR) and Far East (FE) virus lineages, which are both within the genus Hantavirus, were studied. Representative viruses, H5 and B78 for AMR and Bao 14 for FE, were used. The entire small (S) and medium (M) segments, except for the 3'- and 5'-ends, were sequenced. The deduced amino acid sequences of AMR had 96.7 and 92.0-92.2% identities with the Hantaan (HTN) virus in the S and M segments, respectively. The amino acid sequences of FE had 99.1 and 97.9% identities in the S and M segments, respectively. The three viral strains and HTN virus had similar binding patterns to a panel of monoclonal antibodies (MAbs), except that one MAb did not bind AMR. However, sera from Apodemus peninsulae, naturally infected with AMR virus, neutralized homologous viruses at 1:160 to 1:320 dilutions and HTN at 1:20 to 1:40 dilutions. The anti-AMR serum neutralized homologous viruses at a 1:80 dilution and HTN at a 1:40 dilution. The anti-HTN serum did not neutralize AMR (<1:40 dilution), although it had a high neutralizing titer (1:320) against the homologous virus. Therefore, we suggest that AMR virus may constitute a distinct serotype within the genus Hantavirus.

Development of an efficient method for recovery of Puumala and Puumala-related viruses by inoculation of Mongolian gerbils.

Puumala (PUU) virus and PUU-related viruses are difficult to isolate in cell culture. To determine whether animal inoculation would be a better alternative for virus recovery, the Sotkamo strain of PUU virus was inoculated into several animal species. Newborn Mongolian gerbils (MGs), mice, and rats were infected with the Sotkamo strain by intracerebral (ic), intraperitoneal (ip), and subcutaneous (sc) inoculation. Antibodies to PUU appeared in MGs at 30 days post-infection (dpi), and in mice and rats at 15 dpi. Interestingly, virus appeared at 7 dpi in lung and brain of MGs inoculated via ic and ip routes. Virus was detected in all tested tissues of MGs at 15 dpi, with a peak level of 1.36 x 10 (5) focus forming units (FFU)/g in brain tissue. The virus titer declined with the onset of the antibody response and became undetectable by 75 dpi, when the antibody titer reached the maximum level. The appearance of the virus in mice and rats was delayed as compared to MGs, and the virus titer was apparently lower, at approximately 4 to 8 x 10(3) FFU/g, at 15 dpi. In addition, lung homogenates of antibody-positive Clethrionomys (C.) rufocanus (captured in Tobetsu, Hokkaido, Japan) were inoculated into MGs by the ic route. PUU-related viral RNA was detected at 16 dpi in the brains of MG inoculated with the lung homogenate, and antibodies were detected at 45 dpi. These findings indicate that newborn MG inoculation is an efficient method to recover PUU and PUU-related viruses.