Monoclonal antibodies specific to a Ca2(+)-bound form of lipocortin I distinguish its Ca2(+)-dependent phospholipid-binding ability from its ability to inhibit phospholipase A2.

Lipocortin I, a Ca2(+)-and phospholipid-binding protein without EF-hand structures, has many biological effects in vitro. Its actual role in vivo, however is unknown. We obtained and characterized five monoclonal antibodies to lipocortin I. Two of these monoclonal antibodies (L2 and L4-MAbs) reacted with the Ca(+)-bound form of lipocortin I, but not with the Ca2(+)-free form, both in vivo and in vitro. Lipocortin I required greater than or equal to 10 microM-Ca2+ to bind the two antibodies, and this Ca2+ requirement was not affected by phosphatidylserine. L2-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I and inhibited its binding to Escherichia coli membranes and to phosphatidylserine in vitro. L4-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I, but did not affect its binding to E. coli membranes or to phosphatidylserine. These findings indicated that the inhibition of phospholipase A2 by lipocortin I was not simply due to removal or capping of the substrates in E. coli membranes. Furthermore, an immunofluorescence study using L2-MAb showed the actual existence of Ca2(+)-bound form of lipocortin I in vivo.

Persistent infection of rats with haemorrhagic fever with renal syndrome virus and their antibody responses.

Newborn (within 24 h after birth), 1-week-old and 6-week-old (adult) rats were inoculated with a Hantaan-related virus (B-1) and attempts were made to isolate the virus from various organs. Virus-specific antigens were detected in various organs of newborn rats. Moreover virus could be isolated from almost all their organs even 25 weeks after infection. In contrast, in rats infected at 6 weeks of age the virus could be isolated from various organs but its concentration decreased progressively with time. The levels of haemagglutination-inhibiting (HI) and neutralizing antibodies to B-1 virus in the sera were measured. In adult rats, HI antibodies were first detected 2 weeks after infection and their titre rose to a maximum after 5 weeks. On the other hand, in newborn rats the levels of antibodies remained low until 5 or 6 weeks after infection and started to increase to a high level more than 9 weeks after infection. Furthermore, in rats infected soon after birth IgM antibodies predominated for a long time and these antibodies also neutralized infectivity at a high level. These data suggest that the induction of a high titre of neutralizing antibodies mainly of the IgM type does not result in virus clearance in newborn rats and that cellular immunity may be important for virus clearance in vivo.

Isolation of haemorrhagic fever with renal syndrome virus from leukocytes of rats and virus replication in cultures of rat and human macrophages.

Newborn rats were inoculated intraperitoneally with haemorrhagic fever with renal syndrome (HFRS)-related virus (B-1 strain), and virus isolation from their various organs was attempted between 1 and 25 weeks after inoculation. Virus could be isolated repeatedly from lung, brain, spleen and kidney and also from peripheral blood. When virus isolation was carried out on fractionated peripheral blood cells, virus was associated mainly with the macrophage fraction and to a lesser extent with granulocytes. Virus replicated well in peritoneal exudate cells of normal rats and it grew in the adherent mononuclear cells from normal human peripheral blood. These data suggest that macrophages, permissive for HFRS-related virus replication, might contribute to the spread of viral infection in vivo.

Antigenic differences between two viruses, isolated in Japan and Korea, that cause hemorrhagic fever with renal syndrome.

Hantaan virus (HV) 76-118, isolated from Apodemus agrarius coreae in Korea, and hemorrhagic fever with renal syndrome (HFRS) virus B-1, isolated from a rat in Japan, were examined for polypeptide compositions and for differences in immune responses in rats. In immunoprecipitation experiments, a major polypeptide of ca. 50 kilodaltons (K) was detected with antisera against HV 76-118 in cell extracts from Vero E6 cells infected with HFRS virus B-1, whereas three major polypeptides of 74 K (glycosylated), 57 K (glycosylated), and 50 K were detected with antisera against HFRS virus B-1. On the other hand, two polypeptides with molecular weights of 55,000 (glycosylated) and 50,000 were detected with either antiserum in cell extracts infected with HV 76-118. In neutralizing antibody tests with antisera prepared in rats, a remarkable difference in antibody titer (5 to 30 times higher to the homologous virus than to the heterologous virus) was observed between the two viruses. However, this difference was not so marked (1 to 4 times higher to the homologous virus) in the immunofluorescent antibody test. Twenty hybrid cell lines producing mouse monoclonal antibodies against HV 76-118 were isolated by fusion of spleen cells from BALB/c mice immunized against HV (strain 76-118) with mouse myeloma cells. The specificity of these monoclonal antibodies was established by immunofluorescent antibody, neutralizing antibody, and fluorescent antibody to membrane antigen tests and by analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These hybrid cell lines were classified into three groups based principally on the IF staining pattern of the HV-infected cells: (i) antibodies which showed a discrete patch pattern in the cytoplasm by the immunofluorescent antibody test, reacting with the membrane antigen of infected cells and immunoprecipitating a 55-K glycoprotein from HV 76-118-infected cell lysates and a 57-K glycoprotein from the heterologous (strain B-1) HFRS virus-infected cell lysates. Among these, depending on the neutralizing antibody activity and the reaction with the heterologous antigen, three subgroups designated I-A, I-B, and I-C were established; (ii) antibodies which showed large granular dots in the cytoplasm, neither having neutralizing antibody activity nor immunoprecipitating any antigen; (iii) antibodies which showed defined granular dots throughout the cytoplasm, reacting with a 50-K polypeptide of both virus strains. These antibodies also classified into two subgroups based on the reactivity with the B-1 strain.(ABSTRACT TRUNCATED AT 400 WORDS)

Experimental infection in newborn mice and rats by hemorrhagic fever with renal syndrome (HFRS) virus.

Newborn mice and rats were inoculated intracerebrally (ic) or intraperitoneally (ip) with Hantaan virus (76-118 strain) or HFRS-related virus (B-1 strain). The mortality and the influence on the increase of body weight in newborn mice were higher in the groups infected with the 76-118 strain than in the groups infected with the B-1 strain, while the B-1 strain was more virulent in rats than the 76-118 strain. Virus isolation from rats inoculated with either strain was attempted 7 and 11 weeks after inoculation. Virus could be isolated from various organs of rats infected with the B-1 strain, while it was recovered from only the brain and lungs of rats infected with the 76-118 strain. Viral antigen was readily detected in various organs of rats infected with the B-1 strain, but the amount and distribution of antigens were less in rats infected with the 76-118 strain. Our results suggest that the virulence of HFRS-related virus is variable, depending on the species of infected animals as well as on the virus strains. The virus also persists in the injected animals with high titers of antibodies for at least 11 weeks.

Isolation of hemorrhagic fever with renal syndrome (HFRS) virus from a tumor specimen in a rat.

A strain of hemorrhagic fever with renal syndrome (HFRS) virus was isolated in a cell culture from a tumor specimen in a rat kept in a medical institution in which there was a case of HFRS. Positive immunofluorescent reaction with sera from HFRS patients was recognized at the second passage and the number of cells containing antigen increased in the third passage. This virus, named B-1 strain, was identified as the HFRS virus by immunofluorescent tests with sera from patients.