Growth of poliovirus in HeLa cells persistently infected with HVJ (Sendai virus)

The growth of poliovirus in a HeLa cell culture persistently infected with the hemagglutinating virus of Japan (HVJ, the Sendai strain of parainfluenza 1 virus) (HeLaHVJ) was studied. Plaques produced by poliovirus on HeLaHVJ cell monolayers were hazier, smaller and fewer than those on HeLa cells. HeLaHVJ cells were indistinguishable from normal HeLa cells with respect to adsorption rate and penetration efficiency of poliovirus. Extracellular yields of poliovirus in HeLaHVJ cells were lower, and the cytopathic changes were less than those in normal HeLa cells, while cell-associated virus growth in HeLaHVJ cells was nearly equal to that in HeLa cells. HeLaHVJ cells responded more effectively to the action of magnesium chloride, which facilitates virus release from infected cells, resulting in an cytopathic effects. No reduction in poliovirus yield could be detected in HeLa cells acutely infected with HVJ. The relationship between the inhibition of the release of poliovirus from HeLaHVJ cells and the persistent infection of the cells with HVJ is discussed.     

HVJ infection in alveolar macrophages of various laboratory animals

Alveolar macrophages (AM) in bronchoalveolar lavage derived 6 species of laboratory animals (mouse, rat, Syrian hamster, Mongolian gerbil, guinea pig, and rabbit) were cultivated in vitro and were infected with HVJ, parainfluenza virus type 1, for the purpose of studying virus reproduction in these cells. Using infectivity assay, fluorescent antibodies and hemadsorption techniques, three categories were proposed depending on the potential of virus replication and antigen production. Category 1 (mouse, Syrian hamster, and guinea pig AM) was the most susceptible group to HVJ infection. All AM became antigen positive, and more than 10(6) PFU/ml of virus infectivity in the culture fluids was noted at 4 days after the virus infection. Rat and Mongolian gerbil AM were grouped into Category 2. Their AM showed a moderate virus yield (average 10(5) PFU/ml of infectivity) and antigen production. On the other hand, Category 3 (rabbit AM), exhibited no production of an infectious virus. It was likely that the virus penetrated into the AM but did not replicate complete virus. However, antigen positive cells ranging from 10 to 50% depended on the multiplicity of the infection to the AM. These findings suggest that HVJ infection in the rabbit AM was abortive (incomplete virus replication), since trypsin treatment of the culture fluids, which may contain incomplete HVJ particles, did not induce infectivity.     

The improved efficient method for introducing macromolecules into cells using HVJ (Sendai virus) liposomes with gangliosides

Macromolecules such as DNA and RNA can be entrapped within liposomes associated with gangliosides by reverse-phase evaporation. When these liposomes are incubated with HVJ2 (Sendai virus), they deliver their contents into cultured cells efficiently. More than 95% cells of a Ltk- cell line (thymidine kinase-deficient cells) transiently expressed thymidine kinase activity by thymidine kinase gene transfer using HVJ liposomes with gangliosides. Stable transformants could be obtained efficiently from various cell lines by use of HVJ liposomes containing the neoR gene. The neo+ transformants were obtained at frequencies of about 0.2-1.0, 0.06-0.25, and 0.06-0.1% in monolayers of L, CHO-Kl, and HeLa-S3 cells, respectively. Moreover, in Ehrlich ascites tumor cells which grow in suspension, the frequency was more than 0.01%. On introduction of plasmid pTK4 into Ltk- cells, about 0.5-1.0% TK+ transformants were obtained. Cosmid DNA containing the neoR gene (about 45 kbp) was also introduced into L cells by this method and neo+ transformants were obtained at a frequency of 0.1%. When rat liver mRNA was introduced into L cells by HVJ liposomes with gangliosides, immunoprecipitation studies showed that the L cells secreted rat albumin and some other proteins into the cultured medium. Moreover, using erythrocyte membrane vesicles containing IgM that had been incubated with HVJ empty liposomes with gangliosides, the IgM could be introduced into all the L cells.     

THE DEVELOPMENT OF VACCINIA VIRUS IN EARLE'S L STRAIN CELLS AS EXAMINED BY ELECTRON MICROSCOPY

A favorable system which is amenable to frequent and reproducible sampling, consisting of suspension cultures of strain L cells and vaccinia virus, was employed to study the animal virus-mammalian host cell relationship. The three principal aspects investigated concerned the adsorption and penetration of vaccinia into the host, the relationship between the sequence of virus development and the production of infectious particles, and the changes in the fine structure of the host cells. Experiments in which a very high multiplicity of infection was used revealed that vaccinia is phagocytized by L cells in less than 1 hour after being added to the culture, without any apparent loss of its outer limiting membranes. Regions of dense fibrous material, thought to be foci of presumptive virus multiplication, appear in the cytoplasm 2 hours after infection. A correlation between electron microscope studies and formation of infectious particles shows that although immature forms of the virus appear 4 hours after infection, infectious particles are produced 6 hours after infection of the culture, at the time when mature forms of vaccinia appear for the first time in thinly sectioned cells. Spread of the infection is gradual until eventually, after 24 hours, virus is being elaborated throughout the cytoplasm. Addition of vaccinia to monolayer cultures induced fusion of L cells and rapid formation of multinucleate giant forms. In both suspension and stationary cultures infected cells elaborate a variety of membranous structures not present in normal L cells. These take the form of tube-like lamellar and vesicular formations, or appear as complex reticular networks or as multi-laminar membranes within degenerating mitochondria.     

Formation of a host range mutant of the lymphotropic strain of minute virus of mice during persistent infection in mouse L cells

Minute virus of mice (i), the lymphotropic strain of minute virus of mice, established a persistent infection in normally restrictive L cells. The carrier state, which lasted 150 days, exhibited three clearly distinguishable stages. During the early stage (days 1 to 10 postinfection), small amounts of virus were formed. A "crisis" then developed that lasted 50 to 60 days and was characterized by massive cell lysis and high titers of virus. This was followed by a 70- to 80-day period in which small but stable quantities of virus were produced. Virus shed by the carrier culture during the latter phase had acquired an altered host range, namely, it had lost its ability to replicate in T-lymphocyte cell lines and had adapted to growth in L cells. Virus isolated at this time from a single plaque in L cells, designated hr301, was shown to possess similar host range properties. No differences, however, could be found between the DNAs of minute virus of mice (i) and of hr301 by restriction enzyme analysis, suggesting that the mutation that affected the viral host range did not involve an extensive region of the viral genome.     

Growth of an enveloped mycoplasmavirus and establishment of a carrier state.

The growth of an enveloped DNA-containing mycoplasmavirus (MVL2 obtained from R.N. Gourlay) has studied, by using the indicator host Acholeplasma laidlawii strain JA1. From virus one-step growth curves, artificial lysis experiments, and infected cell growth curves, it was found that virus infection is nonlytic. Newly infected cells grow slower and are osmotically more stable than uninfected cells. However, 4 to 6 h after infection, the cells reach a carrier state in which cell growth rate and osmotic fragility are indistinguishable from uninfected cells. Carrier cultures contain free virus. Every carrier culture cell gives rise to either a clone of carrier cells or a clone of MVL2-resistant cells.     

A LysM and SH3-domain containing region of the Listeria monocytogenes p60 protein stimulates accessory cells to promote activation of host NK cells

Listeria monocytogenes (Lm) infection induces rapid and robust activation of host natural killer (NK) cells. Here we define a region of the abundantly secreted Lm endopeptidase, p60, that potently but indirectly stimulates NK cell activation in vitro and in vivo. Lm expression of p60 resulted in increased IFNγ production by naïve NK cells co-cultured with treated dendritic cells (DCs). Moreover, recombinant p60 protein stimulated activation of naive NK cells when co-cultured with TLR or cytokine primed DCs in the absence of Lm. Intact p60 protein weakly digested bacterial peptidoglycan (PGN), but neither muropeptide recognition by RIP2 nor the catalytic activity of p60 was required for NK cell activation. Rather, the immune stimulating activity mapped to an N-terminal region of p60, termed L1S. Treatment of DCs with a recombinant L1S polypeptide stimulated them to activate naïve NK cells in a cell culture model. Further, L1S treatment activated NK cells in vivo and increased host resistance to infection with Francisella tularensis live vaccine strain (LVS). These studies demonstrate an immune stimulating function for a bacterial LysM domain-containing polypeptide and suggest that recombinant versions of L1S or other p60 derivatives can be used to promote NK cell activation in therapeutic contexts.     

Incorporation of sialoglycoprotein containing lacto-series oligosaccharides into chicken asialoerythrocyte membranes and restoration of receptor activity toward hemagglutinating virus of Japan (Sendai virus)

Bovine erythrocyte sialoglycoprotein (GP-2) (1) containing lactoseries oligosaccharide chains, which showed highly specific inhibition of hemagglutination by HVJ (Hemagglutinating virus of Japan, Sendai virus), was incorporated into neuraminidase-treated chicken erythrocytes which had lost their biological responsiveness to the virus. The GP-2-incorporated erythrocytes were agglutinated and lyzed again by the virus. Incorporation of 1,900 molecules of GP-2 per asialoerythrocyte restored fairly well the susceptibility of the cells to HVJ-mediated agglutination and hemolysis. Treatment of the erythrocytes with neuraminidase again resulted in the complete abolishment of the response to HVJ. The above observations are consistent with the view that exogenous sialoglycoprotein, GP-2, can be functionally integrated into the surface membrane of asialoerythrocytes and serve as the receptor for HVJ during the initial adsorption-fusion phase of the virus infection of the target cells.     

Mouse C2 myoblast cells resist HVJ (Sendai virus)-mediated cell fusion in the proliferating stage but become capable of fusion after differentiation

To investigate the mechanism of myoblast fusion, we attempted to prepare artificial myotubes of mouse C2 myoblast cells using the hemagglutinating virus of Japan (HVJ, Sendai virus). Proliferating C2 cells showed strong resistance to HVJ-mediated cell fusion and remained morphologically unchanged even though massive numbers of virions adsorbed onto their surface. They showed no membrane disruption, which occurs in the early stage of cell fusion induced by HVJ. These observations suggest that proliferating C2 cells are resistant to HVJ-mediated cell fusion. However, upon induction of differentiation, C2 cells gradually became capable of fusion induced by HVJ and then even generated heterokaryons with Ehrlich ascites tumor cells. When differentiated C2 cells that had become fusion-sensitive were treated with HVJ in the presence of EDTA, they did not fuse but degenerated, suggesting that their cell membranes were transiently disrupted by interaction with HVJ. These results suggest that the cell membranes of myoblasts change to a fusion-capable state during the process of differentiation.     

Changes in fused cells induced by hvj (sendai virus): redistribution of cytoplasmic organelles and cytoskeletal reorganization

To understand the relationship between the location of organelles and cellular function, we examined the dynamic state of cytoplasmic organelles and cytoskeleton in polynuclear Ehrlich ascites tumor (EAT) cells fused with hemagglutinating virus of Japan (HVJ; Sendai virus) by confocal laser scanning microscopy. Irregular fused cells gradually became spherical during culture, and nuclei and mitochondria were redistributed in the fused cell; nuclei formed a cluster surrounded by mitochondria. F-actin, vimentin, and microtubules were also reorganized with the redistribution of cell organelles. Further, when the morphological change was inhibited by L4-1, a chlorophyll-like substance derived from silkworm faeces, or pyropheophorbide-a, the arrangement of organelles and cytoskeleton remained disturbed, suggesting that the movement of the cytoskeleton is closely associated with cell shape and the distribution of cytoplasmic organelles.     

Spontaneous curing of a minute virus of mice carrier state by selection of cells with an intracellular block of viral replication.

We previously described a persistent infection established by the lymphotropic minute virus of mice in mouse L cells at the level of the cell population (D. Ron, P. Tattersall, and J. Tal, J. Virol. 52:63-69, 1984). This carrier state is maintained by a series of consecutive phenotypic changes which take place in both the cells and the virus and is cured spontaneously after 150 to 200 cell generations (D. Ron and J. Tal, J. Virol. 55:424-430, 1985). We show here that the cure was caused by the selection of virus-resistant cells in the culture. The resistance of these survivor cells to virus replication was due to an intracellular block. Infection of a spontaneously cured culture with the fibrotropic parental minute virus of mice resulted in a restrictive infection in which the viral replicative-form DNA was formed and amplified, but the synthesis of single-stranded progeny DNA was markedly reduced. The lymphotropic strain was blocked in these cells at an earlier stage, with little or no amplification of viral replicative-form DNA observed. These data indicate that the replication of minute virus of mice requires host-coded helper functions in at least two stages of its growth cycle.     

An electrophoretic and morphological analysis of Labiostrongylus (Labiosimplex) bancrofti (Johnston & Mawson, 1939) (Nematoda: Cloacinidae), from macropodid marsupials,with the description of two new species

Allozyme electrophoresis was used to compare specimens of Labiostrongylus (Labiosimplex) bancrofti from two species of Australian macropodids, Macropus dorsalis and M. parryi, with a related species, L. (Labiomultiplex) uncinatus which also infests M. dorsalis. Each nematode was characterised genetically at 18 enzyme loci. The level of fixed genetic differences detected between L. (Ls.) bancrofti from M. parryi and M. dorsalis (83%) is equivalent to that when each is compared to the morphologically distinct species L. (Lm.) uncinatus (89–94%), demonstrating unequivocally that the taxon L. (Ls.) bancrofti represents at least two species, one in M. parryi and one in M. dorsalis. In addition, morphological evidence from additional specimens collected from M. parryi suggested the existence of a third sibling species in this group. All three species differ in the shape of the spicule tips; L. (Ls.) bancrofti has longer spicules than either of the two new species. L. (Ls.) quasibancrofti n. sp. has smaller cephalic papillae, larger oesophago-intestinal diverticula, a larger genital cone and a longer female tail than L. (Ls.) turnbulli n. sp. The taxon L. (Ls.) bancrofti consists, therefore, of three species, L. (Ls.) turnbulli in M. parryi, L. (Ls.) quasibancrofti in M. dorsalis, and L. (Ls.) bancrofti found in both host species, as well as in four species of rock wallabies.     

Efficient introduction of contents of liposomes into cells using HVJ (Sendai virus)

The conditions for efficient introduction of the contents of liposomes into cells were examined using fragment A of diphtheria toxin (DA) as a marker; one molecule of DA can kill a cell when introduced into the cytoplasm. Liposomes containing DA (DA liposomes) were toxic to cells treated with HVJ (Sendai virus) at 4 degrees C just before exposure to DA liposomes at 37 degrees C, but were not toxic to untreated cells. This toxicity was temperature-dependent. DA outside of liposomes was not toxic to HVJ-treated cells. Results also showed that liposomes could fuse with HVJ at 37 degrees but not at 4 degrees C and that liposomes preincubated with HVJ at 37 degrees C could associate with cells. DA liposomes preincubated with HVJ at 37 degrees C were highly toxic to cells. This toxicity was dependent on the duration of preincubation with HVJ and the dose of HVJ. When plasmid DNA coded herpes simplex virus thymidine kinase was trapped in liposomes and fused with Ltk- cells with HVJ, the thymidine kinase activity was expressed in about 10% of the cells. These data show that naked liposomes fuse efficiently with cells with HVJ and that the contents of the liposomes can be introduced into the cytoplasm 100-10 000 times more efficiently by treatment of the cells or liposomes with HVJ.     

Improved methods using HVJ(Sendai virus) for introducing substances into cells.

When HVJ virions were sonicated in the presence of a given protein, about 0.2%–0.3% of total protein added was recovered in the virions. The protein molecules could be introduced effectively into cells. In this study, fragment A of diphtheria toxin was used as the test protein. More than 96% of L cells were killed after short exposure to the virus suspension containing fragment A diluted so as to contain only about 0.004 μg of fragment A per ml.     

AN ANALYSIS ON THE TROPISM IN HVJ (SENDAI VIRUS) INFECTION: MANIFESTATION OF HIGH VIRAL SUSCEPTIBILITY IN SPREADING CULTURES OF ISOLATED ALLANTOIC EPITHELIAL CELLS OF THE CHICK EMBRYO

By culturing pure allantoic epithelial cells separated from the chorioallantoic membrane of a 10-day-old chick embryo, sheets of monolayered epithelial cells were obtained and maintained for at least one week. These cells were highly susceptible to infection with HVJ (Sendai virus) in vitro . In similarly cultured mesenchymal cells of the chorioallantoic membrane, the susceptibility was found to be very low. It was concluded that the tropism observed in HVJ infection in vitro can be explained by differences in the susceptibility to HVJ among various cell types.     

Open reading frame S/L of varicella-zoster virus encodes a cytoplasmic protein expressed in infected cells

We report the discovery of a novel gene in the varicella-zoster virus (VZV) genome, designated open reading frame (ORF) S/L. This gene, located at the left end of the prototype VZV genome isomer, expresses a polyadenylated mRNA containing a splice within the 3' untranslated region in virus-infected cells. Sequence analysis reveals significant differences between the ORF S/Ls of wild-type and attenuated strains of VZV. Antisera raised to a bacterially expressed portion of ORF S/L reacted specifically with a 21-kDa protein synthesized in cells infected with a VZV clinical isolate and with the original vaccine strain of VZV (Oka-ATCC). Cells infected with other VZV strains, including a wild-type strain that has been extensively passaged in tissue culture and commercially produced vaccine strains of Oka, synthesize a family of proteins ranging in size from 21 to 30 kDa that react with the anti-ORF S/L antiserum. MeWO cells infected with recombinant VZV harboring mutations in the C-terminal region of the ORF S/L gene lost adherence to the stratum and adjacent cells, resulting in an altered plaque morphology. Immunohistochemical analysis of VZV-infected cells demonstrated that ORF S/L protein localizes to the cytoplasm. ORF S/L protein was present in skin lesions of individuals with primary or reactivated infection and in the neurons of a dorsal root ganglion during virus reactivation.     

Functional modification of Sendai virus by siRNA

Sendai virus (hemagglutinating virus of Japan; HVJ) is a negative-strand RNA virus with robust fusion activity, and has been utilized for gene transfer and drug delivery. Hemagglutinin-neuraminidase (HN) protein on the viral membrane is important for cell fusion, but causes agglutination of red blood cells. HN-depleted HVJ has been desired for in vivo transfection in order to improve safety. Here, we succeeded in producing HN-depleted HVJ using HN-specific short interfering RNA (siRNA). Viral production was not affected by the siRNA. HN protein was markedly decreased in the new HVJ, while other viral proteins were retained. Consequently, the hemagglutinating activity was substantially reduced and infection activity was suppressed. When the HN-depleted HVJ was mixed with cultured cells and the mixture was centrifuged for 10min at 2000xg, the modified HVJ recovered its infectivity to approximately 80% of wild HVJ. However, infectivity was abolished in the presence of anti-F antibody. Moreover, transfection of FITC-labeled oligodeoxynucleotides using the modified HVJ was also recovered by centrifugation. Thus, the HN-depleted HVJ produced using siRNA technology will be applicable to a delivery vector.     

Effect of double infection of cowpox virus-infected cells with paramyxovirus (Sendai virus) on formation of cowpox virus-specific cell surface antigen.

The formation of cowpox virus-specific cell surface antigen (CPV S-ag) was significantly enhanced by double infection with HVJ (Sendai virus). Simultaneous double infection, superinfection with HVJ and superinfection with CPV of cells persistently infected with HVJ similarly enhanced the formation of CPV S-ag, while pre-infection with HVJ was ineffective. To be effective, cells must be infected at a m.o.i. of greater than or equal to 1.0 and HVJ gene functions had to be expressed. The HVJ-infected cell extracts had an ability to accelerate uncoating (or degradation) of CPV, causing an early increase and a subsequent decrease in the infectivity of CPV. This activity reached a maximum 4--6 hr after HVJ infection, the increase paralleling enhancement of the total activity of several cellular enzymes. Addition of puromycin abolished the increase of these activities and the formation of CPV S-ag. Thus, the double infection with HVJ of CPV-infected cells induces an enhancement of CPV S-ag formation presumably as a consequence of activation of cellular enzymes which in turn accelerates uncoating of CPV.     

Modulation of the immune system by Listeria monocytogenes-mediated gene transfer into mammalian cells

In this study, we established a method for Listeria monocytogenes(Lm)-mediated gene transfer into mammalian cells to manipulate the immune response of the host during infection by pathogens. We used the Lm-mediated gene transfer method in an in vivo study to manipulate host immune responses against Leishmania major(L. major )-infection. The injection of Lm modulated the susceptible host into a resistant state against L. major-infection. A more efficient protective effect was obtained with the injection of IL-12-cDNA containing Lm, and the protective effect was stronger than that of the resistant strain. The protective mechanism of Lm-injection against L. major-infection observed here appeared to be a result of the activation of the local immune system by the Lm-mediated gene transfer method. The present study is the first demonstration that a gene introduced into a host by Lm works to modulate the murine host immune response against infections in vivo. Since this system strongly induces Th1 responses and suppresses Th2 responses in infected hosts, the system can be used for controlling infectious diseases and for protection against allergic responses in the future.