Yuji Kono

Mutations occurring during serial passage of Japanese equine infectious anemia virus in primary horse macrophages.

An attenuated equine infectious anemia virus (EIAV), named V26, was previously obtained after 50 passages of the Japanese virulent strain V70 in primary macrophage culture. To clarify the differences between both viruses, their full-length sequences were determined. There were higher mutations in S2 (6.15% amino acid difference) and LTR (10.7% nucleotide difference). The presumed initiation codon of the S2 gene was absent from the sequence of V26. There was a large insertion within the long-terminal repeat (LTR) U3 hypervariable region of V26. In addition, there were minor mutations in gag (1.22% amino acid difference), pol (1.05% amino acid difference) and env (1. 65% amino acid difference) regions, but no mutation in tat region. No mutations were observed in the principal neutralizing domain in the gp90. Thus, the mutations in the S2 and LTR might be the major target sites of mutation in EIAV during serial passages in vitro.

Complement-mediated hemolysis of horse erythrocytes treated with equine infectious anemia virus

SummaryHorse erythrocytes treated with equine infectious anemia virus hemagglutinin were found to be lysed after incubation with fresh horse serum at 37° C. Fresh guinea pig serum induced more efficient hemolysis than horse serum. Direct immunofluorescence test revealed the adsorption of complement factors on the surface of the erythrocytes. Calcium and magnesium ions were necessary for the hemolysis to take place. Antibody against equine infectious anemia virus enhanced the virus-induced complement-mediated hemolysis. These observations indicated that the classical pathway of complement activation was responsible for this virus-induced hemolysis and suggest the possibility that virus antigen, anti-viral antibody and complement may play an important role in the genesis of the anemia of horses infected with the equine infectious anemia virus.

Establishment of bovine leukemia virus-producing and -nonproducing B-lymphoid cell lines and their proviral genomes

We have established four cell lines in vitro from peripheral leukemic cells of four independent enzootic bovine leukosis (EBL) cattle. All cell lines exhibited differentiated B-cell characters. Two of them produced infectious bovine leukemia virus (BLV), but the others did not. Nonproducer cell lines contained single copies of defective BLV proviral genomes with the same integration profiles as the uncultured cells. On the other hand, numerous proviral copies were detected in producer cell lines. One of the producer cell lines, BL407, whose original uncultured cell contained complete and defective proviral genomes retained the original two copies and had increased only complete genomes in different integration sites after long term culture. These findings suggest that the monoclonal leukemic cells from EBL cases are preferentially established in vitro irrespective of their proviral structures, and the producer B-lymphoid cells amplify their proviral copies by reinfection with viruses re-expressed from the cells during in vitro cultivation.

Expression of bovine herpesvirus 1 glycoprotein gIII by a recombinant baculovirus in insect cells.

Bovine herpesvirus 1 (BHV-1) glycoprotein gIII functions both as a major virus attachment protein and haemagglutinating protein. Here we constructed recombinant baculovirus incorporating the BHV-1 gIII coding sequence to characterize the expression, function and immunogenicity of the glycoprotein in insect cells. The recombinant gIII had an M(r) of 72K and seemed to form homodimers. The gIII was expressed on the surface of insect cells and a rosette formation assay demonstrated haemadsorbing activity of the glycoprotein. Antigenic authenticity of the recombinant gIII was confirmed by a panel of monoclonal antibodies specific for the glycoprotein produced in mammalian cells. Antisera raised to recombinant gIII neutralized the infectivity of BHV-1. These data suggest that recombinant gIII produced in insect cells may be a useful immunogen in a BHV-1 vaccine.

Haemagglutination by Bovine Leukaemia Virus

Bovine leukaemia virus (BLV) was found to agglutinate mouse erythrocytes. Under optimal conditions, including the use of neuraminidase-treated erythrocytes, 200 microgram/ml of BLV purified from the supernatant fluid of BLV-infected bat cells had haemagglutinating titres of about 512 units. BLV haemagglutination was drastically affected by pH and temperature; maximum agglutination occurred at pH 6 and 4 degrees C. That the BLV haemagglutinin is a glycoprotein was suggested by the fact that trypsin, potassium periodate or neuraminidase, but not lipid solvents or phospholipase C, significantly reduced the haemagglutinating (HA) activity of purified BLV. Furthermore, purified BLV glycoprotein of mol. wt. 51 000 (gp51) had HA activity. The receptors for BLV on mouse erythrocytes were inactivated by proteolytic enzymes but not by sodium deoxycholate or potassium periodate. Neuraminidase treatment of erythrocytes increase their agglutinability fourfold. Haemagglutination is a relatively sensitive test for detecting BLV glycoprotein because 0.4 microgram/ml of glycoprotein can be detected by this method. The pH and temperature sensitivity of the BLV HA reaction and specificity for mouse erythrocytes distinguish BLV from that of equine infectious anaemia virus and murine leukaemia virus, the other C type retroviruses known to have HA activity.

Studies on viral-induced anemia in horses infected with equine infectious anemia virus.

The mechanism of anemia of equine infectious anemia (EIA) was investigated using erythrocytes (RBCs) from five horses experimentally inoculated with the virus. The RBCs collected from these horses in pyrexial and viremic periods were found to have high titers of EIA virus. These RBCs were liable to be lysed in fresh guinea pig serum and to be phagocytized by cultivated horse leukocytes comparing with those collected in the preinoculation and incubation periods. All horses showed decrease in RBC counts followed by the first typical fever after inoculation though some of them did not have detectable antibody by virus neutralization, hemagglutination inhibition and immunodiffusion tests in these periods. Complement activity of horse sera were decreased in some degree after the peak of pyrexia. So it seems to be concluded that the interaction of RBCs with EIA virus, complement attraction on them without antiviral antibody and successive hemolysis and phagocytosis of them play important roles in the induction of anemia in the infected horses. However, complement coated RBCs were hardly detected by immunofluorescent test in the peripheral blood, probably because they were destructed and eliminated continuously from the circulatory system without accumulation.