Ikuya Nagata

Growth of Newcastle disease virus in a HVJ carrier culture of HeLa cells

Abstract Growth of Newcastle disease virus (NDV) in a HVJ carrier culture of HeLa cells (HeLaHVJ) was investigated. Although most of the cells of the carrier culture contained HVJ antigen, its presence did not interfere with the growth of superinfecting NDV. When HeLaHVJ cells were infected with NDV at a low multiplicity of infection, the yield of progeny virus was higher, and the cytopathic changes were more extensive than those in normal HeLa cells. Plaques produced by NDV on HeLavHVJ cell monolayers were clearer and larger than those on HeLa cells. HeLaHVJ cells could be distinguished from normal HeLa cells both with respect to production of interferon and sensitivity to its action. Production of interferon which normally appeared in HeLa cell culture infected with NDV could not be demonstrated in HeLaHVJ cell culture. The cells of HeLaHVJ were less susceptible to the antiviral action of interferon than normal HeLa cells.

Temperature-sensitive phenomenon of viral maturation observed in BHK cells persistently infected with HVJ

Abstract A temperature-sensitive phenomenon in virus production was found in BHK-HVJ cells, a cell line of baby hamster kidney cells persistently infected with HVJ. BHK-HVJ cells continued to produce considerable amount of hemagglutinin in the medium at 31 C, while the cells released only restricted amount of hemagglutinin at 37 C. Temperature shift-up and shift-down experiments suggested that the step of incorporation of hemagglutinin into the outer membrane of the cells was temperature sensitive. No cell-associated hemagglutinin was induced when cycloheximide or puromycin was added at the time of temperature shift-down. It was also suggested that the hemagglutinin antigen detected in the cytoplasm by immunofluorescent staining acquired the hemagglutination and the hemadsorption activity only when it had been incorporated into cellular membrane. These results may show that BHK-HVJ cells would provide a convenient system for experiments to analyze the maturation steps in the growth of paramyxovirus.

Homologous interference induced by a temperature-sensitive mutant derived from an HVJ (Sendai virus) carrier culture.

Homologous interference between a temperature-sensitive small plaque mutant (HVJ-pB) derived from an HVJ (haemagglutinating virus of Japan - the Sendai strain of parainfluenza I virus) carrier culture of BHK cells and the original wild-type virus (HVJ-W) has been investigated. Prior infection of LLCMK2, HeLa, BHK or mouse L cells with HVJ-pB, both at permissive and non-permissive temperatures, for 24 h resulted in a reduced yield of superinfecting HVJ-W, reflecting a smaller number of cells capable of producing the superinfecting virus. However, HVJ-pB did not interfere with the replication of vesicular stomatitis virus, Sindbis virus or Newcastle disease virus. Interference in this system seems to be due to inhibition of the attachment of superinfecting HVJ-W as a result of intracellular mechanisms operating at a late stage in the replication of the interfering virus. There is also blocking or destruction of cellular receptors by extra-cellular particles of the interfering virus. Protein synthesis coded for by the complete virus genome is required to establish and maintain the interference, and treatment with actinomycin D has no effect on the interference phenomenon.

The mechanism of interferon induction in mouse spleen cells stimulated with HVJ

Abstract This study showed that functional viral RNA and the penetration of virus into the cell are needed for interferon induction in L cells, while simple contact of the viral glycoprotein with the cell surface appears to be sufficient for interferon induction by the same HVJ in mouse spleen cells. Thirty minutes of uv irradiation resulted in complete loss of the interferon-inducing ability of HVJ in mouse L cells. In contrast to this result, HVJ irradiated for 2 hr could induce interferon in mouse spleen cells as efficiently as untreated HVJ. These findings showed that the actual inducer of interferon in mouse spleen cells was not viral nucleic acid, but some other viral component. When HVJ was treated with potassium periodate at 37° for 1 hr, infectivity for eggs and the hemolytic and neuraminidase activities of the virus were not detectable, but a considerable portion of its hemagglutinating activity was retained. The binding to erythrocytes of this inactivated HVJ, which showed no interferon-inducing ability in both L and mouse spleen cells, was restored in mouse spleen cells but not in L cells. The results indicated that hemolytic and neuraminidase activities are not essential for interferon induction in mouse spleen cells and that hemagglutinating activity might be closely related to interferon induction in the cells, although the presence of hemagglutinating activity alone is not sufficient for interferon induction in the cells. It seems that structural integrity of hemagglutinin on the erythrocyte surface may be important for interferon induction. HeLa cell-grown HVJ, which is characterized by its inability to penetrate into tissue culture cells, was found to stimulate interferon production in mouse spleen cells but not in L cells. This suggests that the process of virus penetration may be essential for induction of interferon in L cells. Interferon was produced in mouse spleen cells incubated with membranous particles with HVJ glycoproteins, but interferon activity could not be detected in the culture fluids of L cells. Aggregation of the glycoproteins by an antibody enhanced its interferon-inducing ability in mouse spleen cells. These results showed that the actual inducer of interferon in mouse spleen cells is not viral nucleic acid, but viral glycoproteins of HVJ, and that the size of its membranous structure is related to interferon inducibility. The mechanism of interferon induction by influenza virus in mouse spleen cells is similar to that of interferon induction by HVJ.

Relationship between establishment of persistent infection of haemagglutinating virus of Japan and the properties of the virus.

The infectious virus (HVJ-pi) obtained from BHK cells persistently infected with haemagglutinating virus of Japan was found to be temperature-sensitive as well as causing little or no cytopathic effect (c.p.e.) and leading to establishment of carrier cultures in several cell lines at both permissive (32 degrees C) and non-permissive (38 degrees C) temperature. In order to obtain information about the role of HVJ-pi in the establishment of persistent infection, comparative studies were made of some phenotypic properties of HVJ-pi and HVJ-38 which was obtained by passing wild-type HVJ in eggs at 38 degrees C and was proved to be highly cytopathic. HVJ-pi differed from HVJ-38 in (1) temperature sensitivity in its ability to produce virus progeny, (2) infectivity for embryonated eggs, (3) neuraminidase activity, (4) the thermal stability of HA and neuraminidase activity, and (5) the polypeptide composition of BHK-grown viruses. B cells infected with HVJ-pi release haemagglutinin more efficiently, and less HA was accumulated on the cell membrane. In considering these results, it was concluded that the difference of envelope proteins might be involved in the striking difference in c.p.e. between HJV-pi and HVJ-38.

Polykaryocyte formation induced by VSV in mouse L cells.

Infection of mouse L cells with VSV leads to the formation of polykaryocytes about 4 to 12 h p.i. When anti-VSV immune serum was added during the course of infection, progression of cell fusion was soon suppressed. Cycloheximide completely suppressed the cell fusion when the drug was added within 1 h p.i., while the cell fusion was not suppressed at all when the drug was added at and after 3 h. Early polykaryocyte formation, fusion from without, was observed only at a low level in cells infected at very high multiplicities. The development of cell fusion induced by VSV was found to be different in several cell types, although all these cells produced a rather high yield of virus: L and C-243-3 mouse cell lines showed a high level of polykaryocytosis (80 to 100%), BHK and RK-13 cells responded at low level, and PS and Vero cells showed no cell fusion in response to VSV infection. In PS cells, however, cell fusion occurred when VSV-infected L cells were co-cultivated. From these observations, the mechanism of cell fusion induced by VSV was discussed.

Mechanism of endotoxin-type interferon production in mice

Abstract Splenectomized mice, when stimulated by endotoxin 3 days after operation, produced little or no circulating interferon, but the serum interferon-producing ability of these mice was restored by an intraperitoneal injection, prior to an endotoxin stimulus, of not only mouse (syngeneic), but also rat (xenogeneic) spleen cells. The serum interferon produced in the rat-to-mouse chimera exhibited the species specificity of mouse interferon. Interferon production-restoring capacity of rat spleen cells was attributed to glass-adherent cells (macrophages) and not to nonadherent cells (lymphocytes). Although spleen cells of mice pretreated with Bordetella pertussis vaccine produced far less interferon in vitro in response to endotoxin stimulus, they possessed the capacity to restore the interferon-producing ability of splenectomized mice. Culture fluid of spleen cells incubated with endotoxin was able to stimulate interferon production in splenectomized mice. From these observations, it is postulated that in vivo production of the endotoxin-type interferon is brought about through a two-step mechanism.

Production of interferon-like substance by mouse spleen cells through contact with BHK cells persistently infected with HVJ.

Abstract A virus inhibitor, an interferon-like substance, was found in the culture fluid of mouse spleen cells cocultivated with BHK-HVJ cells, the BHK cells persistently infected with HVJ, but not in the medium of cocultivation of mouse spleen cells and normal BHK cells. Neither the culture fluid of spleen cells alone nor that of BHK-HVJ cells alone was shown to contain the virus inhibitor. No virus inhibitory activity could be detected in the culture fluid of mouse spleen cells incubated with either the culture fluid of BHK-HVJ cells contained noninfectious HVJ particles or sonicated BHK-HVJ cell suspension. L cells or mouse liver cells, when cocultivated with BHK-HVJ cells, did not release a virus inhibitor. These findings suggest that mouse lymphoid cells have a capacity to produce a virus inhibitor when cocultivated with BHK-HVJ cells, and that nonlymphoid somatic cells may lack this capacity. Interposition of a Millipore filter between BHK-HVJ monolayer and mouse spleen cells or pretreatment of BHK-HVJ cells with anti-HVJ antiserum resulted in a blockade of virus inhibitor production. These findings suggest that the following sequence is necessary for the mouse spleen cells cocultivated with BHK-HVJ cells to produce the virus inhibitor: first, attachment of the spleen cells to BHK-HVJ cells and, second, recognition by the former of virus antigen(s) present on the surface of the latter. The BHK-HVJ cell membrane, isolated by sucrose density gradient centrifuge, was found to be an active inducer of the virus inhibitor. Moreover, some artificial membranous structures, such as HVJ-erythrocyte complex as well as HVJ spike-erythrocyte complex, exhibited a similar activity. This virus inhibitor induced in the present system appears to have all biologic attributes of interferon and its production might be initiated by membrane-membrane interaction between lymphoid cells and HVJ infected cells.

Effects of splenectomy on production of endotoxin-type interferon in mice

Abstract The present study was undertaken to examine the effects of omentumectomy on the production of interferon in response to an endotoxin stimulus and to compare them with those of splenectomy. Omentumectomized mice, administered endotoxin intraperitoneally, failed to produce any detectable amount of circulating interferon; if given endotoxin intravenously, they produced as much interferon as sham-operated controls. The role of the omentum in circulating interferon production in response to an endotoxin stimulus is discussed in comparison with that of the spleen.

Plaque formation by HVJ in calf kidney cells

The s tudy of HVJ** has been hampered by the lack of a satisfactory plaque method, the most accurate in vitro assay method. Plaque formation by H V J in chick embryo fihroblasts or chick kidney ceils has previously been reported; however, these methods have not found general acceptance because of the low efficiency of plating (1, 2). l~ecentIy the use of calf kidney cells (CK) for plaque assay of influenza viruses has been reported (3). Preliminary experiments showed tha t H V J could grow and cause extensive cytopathic changes in CK. This result suggested their use for the plaque assay. The present communication describes plaque formation by H V J in calf kidney cell cultures. An egg-passage line of the Nagoya/60 strain of H V J (4) which was carried through 50 allantoic passages was tested for capacity of plaque formation. Stocks were grown in the allantoic cavity of l l d a y chick embryos, and the harvested fluids were used as virus material. Monolayers of calf kidney ceils in two-ounce bottles were prepared as follows: Minced kidney was mixed with 0.25O/o trypsin (Difco 1:250, in Hanks solution) and stirred by magnetic mixer for 30 minutes at room temperature. The supernatant was removed and discarded. A fresh trypsin so]ution was added to the tissue, and stirred for further 3 hours. The released cells were collected and