Chuya Hamada

Molecular cloning of the hepatitis A virus receptor from a simian cell line.

Using a eukaryotic expression system in combination with a monoclonal antibody (MAb) capable of blocking hepatitis A virus (HAV) adsorption, a cDNA clone was selected from a library of S.la/Ve-1 cells, a cell line that is highly susceptible to the virus. Sequence analysis of the cDNA revealed a single open reading frame that encoded a protein consisting of 460 amino acids. The deduced primary structure of the protein included a signal sequence, a transmembrane domain, four sites for N-linked glycosylation, cysteine residues attributable to an immunoglobulin domain and threonine clusters characteristic of mucin-like protein. By employing a vaccinia virus expression vector, the cDNA was expressed in HeLa cells where it induced marked HAV attachment which was specifically blocked by the MAb. The cDNA obtained was thus assumed to encode a functional receptor for HAV.

Susceptibility of Ad12-Transformed S(+) and S(-) Mouse cells to Cell-Mediated Immunity In Vitro

Adenovirus type 12 (Ad 12)‐transformed mouse cells were examined for their susceptibility to cell‐mediated immunity in vitro, with respect to the activity of the virus‐specific surface (S) antigen in the cells. A transformed cell line, C57AT1, was established from embryonic cells of C57BL/6 mice by Ad 12 infection. In fluorescent antibody tests, the transformed cells were positive for the S antigen when the cells were maintained as cultures, whereas when the cells were grown as tumors in animals they became negative for the antigen (referred to as S(+) and S(—) cells, respectively). These S(+) and S(‐) cells were subjected to the 51Cr‐release test for cell lysis by immune spleen cells (ISC) raised in syngeneic mice by Ad 12 infection. When the S(+) cells at various passage levels were exposed to ISC, all of them were lysed extensively and to a similar extent irrespective of their passage history. In contrast, the S(‐) cells were consistently refractory to the action of ISC. In addition, the cytotoxic action of ISC was markedly impeded by pretreating the S(+) cells with antiserum to the S antigen, or the ISC with anti‐Thy‐1,2 serum plus complement. Taken these findings together, the S(+) cells were assumed to be injured by ISC through direct interaction of the S antigen with T‐lymphocytes.

Localization of hepatitis A virus in marmoset liver tissue during the acute phase of experimental infection

SummaryElectron microscopic and virological studies of marmoset liver tissue with acute infection of hepatitis A virus (HAV), especially in the earlier stages of infection, were carried out to characterize the maturation process of HAV. Four marmosets were inoculated intravenously with HAV suspension and sacrificed 1 week, 2 weeks, 3 weeks and 4 weeks after inoculation respectively. Hepatitis A antigen (HAAg) in 10% liver homogenates of marmosets was examined by radioimmunoassay and a large amount of HAAg was detected in the liver homogenate of two marmosets sacrificed 2 weeks and 3 weeks after inoculation respectively. The histodiagnosis of the marmoset sacrificed 2 weeks after HAV inoculation was normal. However, many clusters of virus-like particles about 27 nm in diameter, in both “solid” and “empty” forms were found, mainly in vesicles of Kupffer cells by electron microscopy. In the animal that developed mild hepatitis 3 weeks after inoculation HAV-like particles were found in vesicles of hepatocytes by electron microscopy. By immune electron microscopy using peroxidase-conjugated anti-hepatitis A antibody, HAAg was detected on the particles present within the cytoplasmic vesicles of Kupffer cells or hepatocytes and on the surrounding membrane of the vesicles which contained HAV-like particles.

Propagation of hepatitis A virus in hybrid cell lines derived from marmoset liver and Vero cells.

To establish monkey liver cell lines with a high susceptibility to hepatitis A virus (HAV), marmoset (Saguinus labiatus) liver cells were fused with Vero cells deficient in hypoxanthine-guanine phosphoribosyltransferase and the resulting hybrid cells were selected in HAT medium. Of four hybrid cell lines obtained (S. 1a/Ve-1 to -4), three (S. 1a/Ve-1, -3 and -4) were equally susceptible to HAV infection. When inoculated with a virus isolated from marmoset liver tissue (10% liver tissue extract) or a faecal virus (10% stool extract) from a human hepatitis A patient, all susceptible cell lines showed a significant elevation of viral antigen activity as seen in radioimmunoassay and/or immunofluorescent antibody assays, at 4 to 6 weeks post-infection (p.i.) with the liver-derived inoculum and at 6 to 8 weeks p.i. with the stool-derived inoculum. In S. 1a/Ve-1 cells, a representative of the susceptible hybrid cell lines, full adaptation of HAV (liver tissue virus concentrate) to cell culture was attained after four serial passages. Thereafter, the virus grew to a plateau titre of 10(8.5) TCID50/ml at 7 days p.i. in a growth experiment. The infected cells showed no cytopathic effects but eventually a persistent infection was established when a saturated level of virus growth was reached.

Close Association of Virus-Specific Cell Surface(S) and H-2 Antigens in Ad12-Infected and -Transformed Mouse Cells

A virus‐specific cell surface (S) antigen in adenovirus type 12 (Ad12)‐transformed mouse cells has been assumed to be a direct target for cytotoxic thymus‐derived lymphocytes (CTL). In this study, the spatial proximity between the S and H‐2 antigens was determined by three different methods, the proximity and co‐capping tests, and the test for blocking of CTL‐mediated lysis by anti‐H‐2 serum. In the proximity test with Ad12‐infected thymic and splenic lymphocytes, and an Ad12‐transformed line of C3H/He (H‐2k) mouse cells, anti‐H‐2k and anti‐S sera reciprocally inhibited fluorescent‐antibody staining of the opposite antigens. By contrast, anti‐Thy‐1, 2 serum as well as anti‐Ia and anti‐Ig sera failed to show any appreciable effect in this test, when paired with anti‐S serum. In addition, the S and H‐2 antigens co‐capped in the infected thymic lymphocytes, and CTL‐mediated lysis of the transformed cells was abrogated equally by treatment of cells with anti‐S and anti‐H‐2 sera. These results clearly demonstrate that there is a close proximity between the S and H‐2 antigens on the surface of Ad12‐infected and ‐transformed mouse cells.

Production of a low molecular weight growth inhibitory factor by adenovirus 12 — transformed cells

Malignant rodent cells transformed by human adenovirus 12 produce a potent cell growth inhibitory factor. The cell growth inhibitory factor inhibits the growth of and DNA synthesis in normal fibroblasts in vitro. Extent of the production of the cell growth inhibitory factor appears to be proportional to that of the malignancy of the transformed cells. C57AT1-AB cells, an adenovirus 12-transformant of C57BL/6 mouse origin, are highly tumorigenic in the syngeneic and allogeneic mice. The cell growth inhibitory factor produced by these cells was characterized for the physicochemical properties; the cell growth-inhibitory activity was quantitatively recovered in the filtrates of YM-2 membrane (M(r) less than 1,000), resistant to the heat treatments at 56 degrees C for 30 min and 100 degrees C for 5 min, and extractable by ethyl acetate under acid-condition. These results suggest that the cell growth inhibitory factor may be lipid or oligopeptides.

PAG stimulation does not affect primary antibody responses in rats

&NA; Adult male rats, which had electrodes chronically implanted in the periaqueductal gray (PAG), were immunized with sheep red blood cells (SRBCs). The number of direct and indirect plaque‐forming cells (PFCs) in the group receiving PAG stimulation after immunization did not differ significantly from that in the unstimulated group. Thus, the results indicate that short‐term PAG stimulation does not suppress antibody‐producing activity in the rat.

Roles of HTLV-I p19 and Natural Antibody to HTLV-I in Host Immune Responses

Since the discovery of human T cell leukemia/lymphoma virus type I (HTLV-I) [5], the immune status of HTLV-I- infected hosts, including patients with adult T cell leukemia (ATL), has not yet been sufficiently investigated [2–4]. As the first step in this kind of study, we have studied the nonspecific activity of peripheral mononuclear cells (PMNC) from ATL patients and seropositive HTLV-I carriers and therole of HTLV-I p19 antigen and HTLV-I-producing cell surface antigens in the host immune responses to HTLV-I.

Effector Cells Working for Rejection of Ad12 Tumors in Mice

Immune spleen cells (ISC) capable of inhibiting the growth of adenovirus type 12 (Ad12) tumors were raised in C57BL/6 mice by immunization with Ad12, fractionated according to their affinity for plastic and nylon‐wool substrates or treated with various antisera plus complement, and subjected to the tumor‐neutralization test (Winn) to define the effector cells for the cell species. Full antitumor activity of ISC was recovered in the cell fractions nonadherent to the two substrates; the antitumor activity of ISC was abrogated entirely by anti‐Thy‐1,2 serum and almost entirely by anti‐Lyt‐2.2 ascites fluid plus complement. These results clearly indicate that T‐lymphocytes, particularly those bearing Lyt‐2.2 antigen, are the principal effectors in ISC against Ad12 tumors in animals.

Adenovirus type 12 plaque formation in Vero cells.

The plaque technique is a fundamental and prerequisite method to develop quantitative and genetic studies of virus. The first report of plaque formation by human adenovirus was presented by Bonifas and Schlesinger in 1959, who found adenovirus type 2 to form plaques in KB cell monolayers when the cells were kept in arginine-enriched medium (1). Thereafter, a number of papers for plaque production accumulated eventually to cover all serotypes of the virus (2, 3, 6, 8, 9). However, the plaque technique for human adenovirus is still handicapped in certain virus serotypes including those of the highly oncogenic subgroup. Since these viruses produce small and slow-growing plaques (2), cells giving accurate and reproducible results are confined practically to human embryonic kidney (HEK) cells (6, 7), though KB (2) and FL (7) cells were claimed to be employable. This limitation is further narrowed by the situation that HEK cells are not necessarily available at ones convenience. Thus, it is desirable to select more conventional cells or cell lines that yield plaques with these slow-growing adenoviruses in an efficiency comparable to that in HEK cells. This paper describes an experimental result that Vero cells (10), a derivative of African green monkey kidney (AGMK) cells, can be used as an excellent system for plaguing human adenovirus type 12, a representative serotype of slow-growing adenoviruses. Human adenovirus type 12 (Ad12), Huie strain, was grown in KB cells. Vero cells used were of two sources : one cell line had been kept in this laboratory by serial subculture and another was recently purchased from Flow Laboratories (Md., U.S.A.). Vero cell monolayers prepared in 5 cm-petri dishes (Miharu Glass K.K., Tokyo) were washed once with PBS (phosphate buffered saline) and inoculated with 0.5 ml of virus dilutions per dish. After virus adsorption for 1 hr at 37 C in a CO2incubator, cells were overlaid with agar medium and incubated further in the same condition. Agar medium employed was based on Eagles minimum essential medium (MEM) enriched with twice the concentrations of amino acids and vitamines, and supplemented with 0.9% Bacto Agar (Difco Co., U.S.A.) and 10% fetal calf serum (Gibco, N.Y., U.S.A.). The amount of agar medium overlaid per dish was 5 ml on day 0 and 2.5 ml each on days 3, 6, and 9 post infection. At the last overlay on day 9, the medium contained 0.04% neutral red for staining dishes, and the results were read 24 hr thereafter. Since the results given by the two Vero cell lines, of our laboratory and of Flow Laboratories, were quite identical, those obtained with the former are described in