Masamichi Ishibashi

Empty virus‐like particle‐based enzyme‐linked immunosorbent assay for antibodies to hepatitis E virus

Hepatitis E, an enterically transmitted non‐A, non‐B hepatitis, is a serious viral infection that occasionally causes large epidemics in developing countries. In developed countries, the disease only appears sporadically due to the transmission routes, and it is considered to be less important. The hepatitis E virus (HEV) cannot grow in cultured cells and no reliable assay system has ever been developed. In addition, the present diagnostic are not perfect, and actual rates of HEV infection may be underestimated. Highly purified empty virus‐like particles (VLPs) of HEV have been produced by the use of a recombinant baculovirus vector in insect cells. Using these VLPs as an antigen, an enzyme‐linked immunosorbent assay (ELISA) for antibodies to HEV was developed. A panel of 164 sera that were randomized and coded, and sera collected periodically from three patients with hepatitis E were used for the evaluation. The sensitivity of the assay was shown to be equal to or better than that obtained in previous research that used the same serum panel. The ELISA demonstrated that the serum IgM level of the patients was highest at the onset of the clinical illness and then rapidly decreased. In contrast, a high level of circulating IgG antibody titers lasted for more than 4 years. In Japan, a non‐endemic country, the prevalence of the IgG class antibody to HEV in healthy individuals was found to range from 1.9% to 14.1%, depending on the geographical area. Only one out of 900 (0.1%) serum samples was IgM‐positive. The IgM class antibody to HEV was detected in 10.8% of non‐A, non‐B, and non‐C acute hepatitis patients in northeast China, whereas none of the patients in Korea had the IgM antibody. The ELISA utilizing the VLPs is sensitive and specific in its detection of the IgM and IgG antibodies to HEV. The ELISA is therefore useful for diagnosing HEV infection and for seroepidemiological study of hepatitis E. J. Med. Virol. 62:327–333, 2000.

Detection of Hepatitis G virus RNA in patients with Hepatitis B, Hepatitis C, and non-A-E Hepatitis by RT-PCR using multiple primer sets

Hepatitis G virus(HGV)/GB virus C(GBV‐C) is a newly identified virus associated with human hepatitis. The preliminary prevalence studies of HGV infection in Japan were entirely based on the detection of HGV RNA by RT‐PCR. However, the selection of the different primer sets in such assay may influence sensitivity of the test because of the extensive genetic heterogeneity of HGV, and influence the estimation of the prevalence of HGV. To address this potential problem, we designed two primer sets from well conserved domains in the 5′NC and NS5 regions of HGV genome, and tested them together with the NS3‐derived primer set in RT‐PCR for their ability to detect HGV RNA in serial dilution of synthetic viral RNA templates. Subsequently, we used these three primer sets to detect HGV RNA in the sera of 371 Japanese patients with hepatitis B, hepatitis C, and non‐A‐E hepatitis. The results indicated that the primer set derived from the 5′NC region appeared to be most effective in detecting HGV RNA. The results also showed that only two out of the 126 patients (1.6%) with non‐A‐E hepatitis were positive for HGV RNA although the RNA were detected more frequently in patients with hepatitis B (2/38; 5.3%) and hepatitis C (17/207; 8.2%), suggesting that HGV is not a common causative agent for non‐A‐E hepatitis in Japan. J. Med. Virol. 52:385–390, 1997.

Case Report: Interferon induced coma in Sheehan's syndrome

A 54‐year‐old woman who was being treated with 10 million units (mu) of natural interferon (IFN)‐α per day for chronic active hepatitis C at a local clinic, developed coma on the fourth day of treatment. On admission to Yamagata University Hospital, she was still in a state of semicoma with severe hyponatraemia (122 mEq/L) and hypochloraemia (89 mEq/L). After the administration of electrolytes, her condition improved remarkably. Endocrinological loading tests showed a hypofunction of the anterior pituitary gland. In consideration of these results, and her past experiences of haemorrhage during childbirth and subsequent amenorrhoea, we diagnosed her illness as a coma as a result of Sheehans syndrome which had become overt during IFN therapy. She recovered completely after treatment with hydrocortisone and 1‐thyroxine.

Epidemiological study and genetic analysis of GB virus C infection in general population from an area endemic for hepatitis C.

The aim of this work was to study the prevalence, potential risk factors, clinical and laboratory features of GB virus C (GBV‐C) infection in general population from an area endemic for hepatitis C. A reverse transcriptase‐polymerase chain reaction (RT‐PCR) for detection of GBV‐C RNA was used to examine the prevalence of GBV‐C RNA in both hepatitis C virus (HCV) endemic (R town) and nonendemic areas (M town) in Yamagata prefecture, Japan. In R town, GBV‐C RNA was detected in 23 (2.9%) out of the 800 residents, whereas anti‐HCV and HCV‐RNA were found in 226 (28.3%) and 163 (20.4%), respectively. The prevalence of GBV‐C RNA in R town (2.9%) was higher than that in M town (1.0%), although the difference was not statistically significant. The individuals with anti‐HCV had significantly higher frequency of active GBV‐C infection than those without anti‐HCV in both towns. No evidence indicating that GBV‐C infection affected the severity of hepatitis C was obtained. The multivariate analysis revealed that the young anti‐HCV positive individuals with a history of blood transfusion had higher incidence of active GBV‐C infection. The phylogenetic analysis showed that the GBV‐C isolates from both R and M towns were divided into two separate branch groups designated HG and Asia GB groups. J. Med. Virol. 54:237–242, 1998.

Electron microscopic study of hepatitis B virus-associated antigens on the infected liver cell membrane in relation to analysis of immune target antigens in chronic hepatitis B

SummaryTo clarify the characteristics of cell surface Pre-S2 Ag, HBcAg and HBeAg immunohistochemically and to explore their relationship with a cellular immune target antigen, 31 liver biopsy specimens from chronic HBV carriers were examined by immunoperoxidase staining. By immune light microscopy, Pre-S2 Ag was detected on the liver cell membrane in 18 (58%) of the 31 cases, HBcAg in 4 cases (13%) and HBeAg in 4 cases (13%). Pre-S2 Ag frequently showed a honeycomb-like membrane expression pattern which was present regardless of liver inflammation, whereas HBcAg and HBeAg exhibited a scattered membrane expression pattern detected in areas of marked inflammation. Of the 18 cases showing a honeycomb-like Pre-S2 Ag expression, 3 concomitantly showed a scattered membrane expression pattern. Immunoelectron microscopy revealed these two distinct membrane expression patterns. In areas showing a honeycomb-like membrane expression pattern, Pre-S2 Ag was demonstrated in the intercellular space and on the basolateral membranes of hepatocytes, but was not detected on the cell membranes in areas of the intercellular space lacking an immunoreaction. Cytoplasmic expression of Pre-S2 Ag was less extensive in these hepatocytes. These findings suggest that the honeycomb-like membrane expression of Pre-S2 Ag results from attachment of extracellular antigen to the liver cell membrane. In contrast, in areas showing a scattered membrane expression pattern, Pre-S2 Ag, HBcAg and HBeAg were each detected as single-layered linear deposits along the cell membrane, but were absent in the intercellular space. Each antigen was also expressed abundantly in the cytoplasm, and the immunoproducts appeared to fuse with the cell membrane. These findings suggest that the scattered membrane expression of these antigens results from intrahepatic transfer of antigen synthesized in the liver cell to the cell membrane, possibly serving as a target for the host immune-mediated response in connection with inflammation.

Quantitative Measurement of Fluorescent Intensity of the Rat Liver Surface Using Video Fluorescence Laparoscopy Following Intravenous Fluorescein Injection

Abstract: A new method for the quantitative measurement of fluorescent intensity of the rats liver surface after fluorescein injection is described. The measuring system of fluorescent intensity on the rat liver surface consisted of a video laparoscopy with a video densitometer. An excitation filter was placed in the light source and an absorbing filter was attached to the eyepiece of the magnifying scope in this fluorescence video laparoscopy system. Fluorescein was injected intravenously into the inferior vena cava. Video fluorescence images of the liver surface were recorded with a CCD TV camera which was connected to the scope. In measuring the mean intensity of video images, a region of interest was designated on the TV monitor. The fluorescent intensity in this region was measured by a video densitometer and expressed in volts. The time‐intensity curve was graphically presented using a pen recorder. In this way, the time course of the liver surface fluorescent intensity could be analyzed with data expressed in volts. In normal livers, the fluorescent intensity began to rise 4.0 ± 0.5 seconds after fluorescein injection and reached a peak at 14.4 ± 1.5 seconds. Thereafter, the intensity fell slightly but then rose again to a second peak 170±50 seconds after injection.