Characterization of JC papovavirus adapted to growth in human embryonic kidney cells.

Human papovavirus JC virus was adapted to growth in human embryonic kidney (HEK) cells. After eight passages, the HEK-adapted JC virus produced high virus yields and was capable of forming plaques in HEK monolayer cultures. Eleven plaque-purified stocks were prepared and characterized. Biologically, the plaque-purified virus induced tumor and viral antigens in HEK cells earlier and in a higher percentage of cells than uncloned virus. Cytopathic changes were also evident sooner and were more extensive. The DNA from uncloned as well as plaque-purified isolates was analyzed by restriction endonuclease cleavage followed by gel electrophoresis. The DNA from uncloned HEK-adapted virus was heterogeneous. Plaque-purified virus isolates yielded DNA which, although much less heterogeneous than the uncloned stock, still consisted of two or more species of viral DNA.     

Isolation of cold-sensitive mutants of measles virus from persistently infected murine neuroblastoma cells.

Clone NS20Y of the mouse neuroblastoma C1300 was infected with wild-type Edmonston measles virus, and, after a transition to a carrier culture, became persistently infected. Persistently infected clones were derived and characterized morphologically by the appearance of multinucleate giant cells and nucleocapsid matrices in cytoplasm and nucleus, but very few budding virus particles. Antimeasles antibodies markedly suppressed the expression of viral antigens and giant cells, and the effect was totally reversible. When the cells were cultured at 33 degrees C, the number of giant cells began to diminish and ultimately disappeared; in contrast, when cultured at 39 degrees C, the cultures invariably lysed. Yields at 33 degrees C were ca. 2 logs lower than those at 39 degrees C. Cells cultured at 33 degrees C produced relatively high levels of interferon, whereas those at 39 degrees C produced little or no interferon. When the persistently infected cultures were exposed to anti-interferon alpha/beta serum at a nonpermissive temperature, there was a marked increase in multinucleate cells, suggesting that maintenance of the persistence state and its regulation by temperature may be related to the production of interferon. Viral isolates from cells cultured at 39 degrees C were obtained, and 90% of viral clones were found to be cold sensitive. Complementation studies with different viral clones indicated that the cold-sensitive defect was probably associated with the same genetic function. Western blot analysis of the persistently infected cells indicated a significant diminution and expression of all measles-specific proteins at a nonpermissive temperature. Infection of NS20Y neuroblastoma cells with the cold-sensitive virus isolates resulted in the development of an immediate persistent infection, whereas infection of Vero or HeLa cells resulted in a characteristic lytic infection, suggesting that the cold-sensitive mutants may be selected or adapted for persistent infection in cells of neural origin.     

Destruction of L Cells by Mengo Virus: Use of Interferon to Study the Mechanism

Interferon, when added to L cells, inhibited the synthesis of infectious Mengo viral ribonucleic acid, hemagglutinins, and infectious virus by 85 to 95%. Serum-blocking antigens were also reduced by the action of interferon, but threefold excess amounts of these antigens accumulated in interferon-treated cultures above the amounts expected for the quantity of infectious virus that was produced in these cultures. Radioautographic analysis showed that 28 to 36% of the cells of an interferon-treated population synthesized viral ribonucleic acid and 36 to 47% produced viral antigens as determined by an immunofluorescence technique. Despite the reductions in synthesis of viral components, all cells in an interferon-treated culture underwent cytopathic effects at the same time as cells in infected cultures which had not been treated with interferon. The results are compatible with the hypothesis that the cell destruction which results from the infection of L cells with Mengo virus is due to a protein which is coded for by the virus but is not a component of the mature virion.     

Effect of cell physiological state on infection by rat virus

Infection by rat virus has been studied in cultures of rat embryo cells to evaluate the Margolis-Kilham hypothesis that the virus preferentially infects tissues with actively dividing cells. An enhancement of infection was seen in cultures infected 10 hr after fresh medium was added as compared to infection of stationary cultures (infected before addition of fresh medium). Since addition of fresh medium stimulates deoxyribonucleic acid (DNA) synthesis, the number of cells per culture synthesizing DNA at the time of infection was compared with the proportion of cells which synthesized viral protein. Cells were infected before the medium change and 10 or 24 hr after the medium change and were pulse-labeled with ³H-thymidine at the time virus was added. The cells were allowed to initiate viral protein synthesis before they were fixed and stained with fluorescein-conjugated anti-rat virus serum. Fluorescence microscopy permitted both labels to be counted simultaneouly and showed that the greatest proportion of cells synthesizing viral protein were those which had incorporated ³H-thymidine at the time of infection.     

A human milk factor susceptible to cathepsin D inhibitors enhances human immunodeficiency virus type 1 infectivity and allows virus entry into a mammary epithelial cell line

Human immunodeficiency virus type 1 (HIV-1) growth in lymphocyte cultures was increased when the virus inoculum was incubated in breast milk. The enhancing effect of milk was abolished by anti-cathepsin D antibody or by pepstatin A, a cathepsin D inhibitor. The cathepsin D-producing CD4-negative MCF7 mammary cells supported the growth of some HIV-1 isolates. An MCF7 line chronically producing HIV-1 IIIb was obtained. Cathepsin D may induce conformational modification of viral gp120, allowing direct interaction with a coreceptor. We demonstrated the presence of CXCR4 mRNA in MCF7 cells.     

Defective interfering influenza viruses and host cells: establishment and maintenance of persistent influenza virus infection in MDBK and HeLa cells.

WSN (H0N1) influenza virus upon undiluted passages in different species of cells, namely, bovine kidney (MDBK), chicken embryo (CEF), and HeLa cells, produced a varying amount of defective interfering (DI) virus which correlated well with the ability of the species of cell to produce infectious virus. However, the nature of the influenza DI viral RNA produced from a single clonal stock was essentially identical in all three cells types, suggesting that these cells do not exert a great selective pressure in the amplification of specific DI viral RNAs either at early or late passages. DI viruses produced from one subtype (H0N1) could interfere with the replication of infectious viruses belonging to other subtypes (H1N1, H3N2). DI viral RNAs could also replicate with the helper function of other subtype viruses. The persistent infection of MDBK and HeLa cells could be initiated by coinfecting cells with both temperature-sensitive mutants (ts-) and DI influenza viruses. Persistently infected cultures cultures at early passages (up to passage 7) showed a cyclical pattern of cell lysis and virus production (crisis), whereas, at later passages (after passage 20), they produced little or no virus and were resistant to infection by homologous virus but not by heterologous virus. The majority of persistently infected cells, however, contained the complete viral genome since they expressed viral antigens and produced infectious centers. Selection of a slow-growing temperature-sensitive variant rather than the presence of DI virus or interferon appears to be critical in maintaining persistent influenza infection in these cells.     

Differential syncytium-inducing capacity of human immunodeficiency virus isolates: frequent detection of syncytium-inducing isolates in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex.

Human immunodeficiency virus isolates were studied with respect to syncytium-inducing capacity, replicative properties, and host range. Five of 10 isolates from patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex were able to induce syncytia in cultures of peripheral blood mononuclear cells (MNC). In contrast, only 2 of 12 isolates from asymptomatic individuals had syncytium-inducing capacity. Syncytium-inducing isolates were reproducibly obtained from the same MNC sample in over 90% of the cases, independent of the donor MNC used for propagation. Syncytium-inducing capacity was shown to be a stable property of an isolate, independent of viral replication rates. Evidence was obtained that the high replication rate of syncytium-inducing isolates observed during primary isolation may be due to higher infectivity of these isolates. The finding that only syncytium-inducing isolates could be transmitted to the H9 cell line is compatible with this higher infectivity. The frequent isolation of syncytium-inducing isolates from individuals with AIDS-related complex or AIDS and the apparent higher in vitro infectivity of these isolates suggest that syncytium-inducing isolates may unfavorably influence the course of human immunodeficiency virus infection.     

Accumulation of human immunodeficiency virus type 1 DNA in T cells: results of multiple infection events.

Human immunodeficiency virus type 1 DNA synthesis was followed in a CD4+ line of T cells (C8166) grown in the presence or absence of a monoclonal antibody to CD4 that blocks infection By 48 h after infection, cultures grown in the presence of the antibody contained approximately 4 copies of human immunodeficiency virus type 1 DNA per cell, whereas those grown in the absence of the antibody contained approximately 80 copies of viral DNA per cell. Most of the viral DNA in cultures grown in the absence of the antibody was present in a broad smear of apparently incomplete viral sequences. In cultures grown in the presence or absence of the antibody, the 9.6-kilobase linear duplex of viral DNA appeared to undergo integration within 24 h of its appearance. These results demonstrate that T cells accumulate unintegrated human immunodeficiency virus type 1 DNA as a result of multiple virions entering cells.     

Production of reovirus type-1 and type-3 from Vero cells grown on solid and macroporous microcarriers

Two strains of reovirus were propagated in Vero cells grown in stationary or microcarriers cultures. Vero cells grown as monolayers on T-flasks or in spinner cultures of Cytodex-1 or Cultispher-G microcarriers could be infected with reovirus serotype 1, strain Lang (T1L), and serotype 3, strain Dearing (T3D). A regime of intermittent low speed stirring at reduced culture volume was critical to ensure viral infection of cells in microcarrier cultures. The virus titre increased by 3 to 4 orders of magnitude over a culture period of 150 h. Titres of the T3D reovirus strain were higher (43%) compared to those of the T1L strain in all cultures. Titres were significantly higher in T-flask and Cytodex-1 microcarrier cultures compared to Cultispher-G cultures with respect to either reovirus type. The viral productivity in the microcarrier cultures was dependent upon the multiplicity of infection (MOI) and the cell/bead ratio at the point of infection. A combination of high MOI (5 pfu/cell) and high cell/bead loading (>400 for Cytodex-1 and >1,000 for Cultispher-G) resulted in a low virus productivity per cell. However, at low MOI (0.5 pfu/cell) the virus productivity per cell was significantly higher at high cell/bead loading in cultures of either microcarrier type. The maximum virus titre (8.5 x 10(9) pfu/mL) was obtained in Cytodex-1 cultures with a low MOI (0.5 pfu/cell) and a cell/bead loading of 1,000. The virus productivity per cell in these cultures was 4,000 pfu/cell. The lower viral yield in the Cultispher-G microcarrier cultures is attributed to a decreased accessibility of the entrapped cells to viral infection. The high viral productivity from the Vero cells in Cytodex-1 cultures suggests that this is a suitable system for the development of a vaccine production system for the Reoviridae viruses.     

High permissivity of the fish cell line SSN-1 for piscine nodaviruses.

Seventeen isolates of piscine nodavirus from larvae or juveniles of 13 marine fish species affected with viral nervous necrosis (VNN) were examined for their infectivity to a fish cell line SSN-1. Based on cytopathic effects (CPE) and virus antigen detection by fluorescent antibody technique (FAT) after incubation at 25 degrees C, the infectivity of these virus isolates was divided into 4 groups. Group 1, including 9 virus isolates from 4 species of grouper, 2 species of sea bass, barramundi, rock porgy, and Japanese flounder showed CPE characterized by rounded, granular cells with heavy cytoplasmic vacuoles within 3 d post-incubation (p.i.), and the monolayer partially or completely disintegrated over 3 to 6 d p.i. Scattered FAT-positive cells appeared at 3 h p.i. and spread through the cell sheet with an increasing fluorescence signal over 24 h p.i. Group 2, consisting of 3 virus isolates from striped jack, induced CPE with thin or rounded, granular, refractile cells without conspicuous vacuole formation, and extensive FAT-positive reaction was observed in a time course similar to that of Group 1. Cells inoculated with Group 3 (1 isolate from tiger puffer) developed no distinct CPE but viral infection was evidenced by localized FAT-positive cells. There were no FAT-positive cells in Group 4, which included 4 isolates from Japanese flounder, Pacific cod and Atlantic halibut. However, when incubation was performed at 20 degrees C, the SSN-1 cells inoculated with the Group 3 isolate showed CPE similar to that of Group 1 and extensive FAT-positive reaction. Evidence of virus proliferation at 20 degrees C was also obtained in Group 4 isolates. The virus titers in the infected fish varied from 10(11) to 10(16) tissue culture infectious dose (TCID50) g(-1) of fish. There is a good correlation between these infectivities to the SSN-1 cells and the coat protein gene genotypes of the isolates. The present results indicate that SSN-1 cells are useful for propagating and differentiating genotypic variants of piscine nodavirus.     

Relative inefficiency of soluble recombinant CD4 for inhibition of infection by monocyte-tropic HIV in monocytes and T cells

Macrophages are major viral reservoirs in the brain, lungs, and lymph nodes of HIV-infected patients. But not all HIV isolates infect macrophages. The molecular basis for this restrictive target cell tropism and the mechanisms by which HIV infects macrophages are not well understood: virus uptake by CD4-dependent and -independent pathways have both been proposed. Soluble rCD4 (sCD4) binds with high affinity to gp 120, the envelope glycoprotein of HIV, and at relatively low concentrations (less than 1 microgram/ml) completely inhibits infection of many HIV strains in T cells or T cell lines. HTLV-IIIB infection of the H9 T cell line was completely inhibited by prior treatment of virus with 10 micrograms/ml sCD4: no p24 Ag or HIV-induced T cell syncytia were detected in cultures of H9 cells exposed to 1 x 10(4) TCID50 HTLV-IIIB in the presence of sCD4. Under identical conditions and at a 100-fold lower viral inoculum, 10 micrograms/ml sCD4 had little or no effect on infection of monocytes by any of six different HIV isolates by three different criteria: p24 Ag release, virus-induced cytopathic effects, and the frequency of infected cells that express HIV-specific mRNA. At 10- to 100-fold higher concentrations of sCD4, however, infection was completely inhibited. Monoclonal anti-CD4 also prevented infection of these same viral isolates in monocytes. The relative inefficiency of sCD4 for inhibition of HIV infection in monocytes was a property of the virion, not the target cell: HIV isolates that infect both monocytes and T cells required similarly high levels of sCD4 (100 to 200 micrograms/ml) for inhibition of infection. These data suggest that the gp120 of progeny HIV derived from macrophages interacts with sCD4 differently than that of virions derived from T cells. For both variants of HIV, however, the predominant mechanism of virus entry for infection is CD4-dependent.     

Human immunodeficiency virus type 1 derived from cocultures of immature dendritic cells with autologous T cells carries T-cell-specific molecules on its surface and is highly infectious

During the budding process, human immunodeficiency virus type 1 (HIV-1) acquires cell surface molecules; thus, the viral surface of HIV-1 reflects the antigenic pattern of the host cell. To determine the source of HIV-1 released from cocultures of dendritic cells (DC) with T cells, immature DC (imDC), mature DC (mDC), T cells, and their cocultures were infected with different HIV-1 isolates. The macrophage-tropic HIV-1 isolate Ba-L allowed viral replication in both imDC and mDC, whereas the T-cell-line-tropic primary isolate PI21 replicated in mDC only. By a virus capture assay, HIV-1 was shown to carry a T-cell- or DC-specific cell surface pattern after production by T cells or DC, respectively. Upon cocultivation of HIV-1-pulsed DC with T cells, HIV-1 exclusively displayed a typical T-cell pattern. Additionally, functional analysis revealed that HIV-1 released from imDC-T-cell cocultures was more infectious than HIV-1 derived from mDC-T-cell cocultures and from cultures of DC, T cells, or peripheral blood mononuclear cells alone. Therefore, we conclude that the interaction of HIV-1-pulsed imDC with T cells in vivo might generate highly infectious virus which primarily originates from T cells.     

Infection of human cells by dengue virus is modulated by different cell types and viral strains

Although prior studies have investigated cellular infection by dengue virus (DV), many have used highly passaged strains. We have reassessed cellular infection by DV type 2 (DV2) using prototype and low-passage isolates representing genotypes from different geographic areas. We observed marked variation in the susceptibility to infection among cell types by different DV2 strains. HepG2 hepatoma cells were susceptible to infection by all DV2 strains assayed. Although the prototype strain generated higher titers of secreted virus than the low-passage isolates, this difference did not correspond to positive- or negative-strand viral RNA levels and thus may reflect variation in efficiency among DV2 isolates to translate viral proteins or package and/or secrete virus. In contrast, human foreskin fibroblasts were susceptible to the prototype and low-passage Thai isolates but not to five Nicaraguan strains tested, as reflected by the absence of accumulation of negative-strand viral RNA, viral antigen, and infectious virus. A similar pattern was observed with the antibody-dependent pathway of infection. U937 and THP-1 myeloid cells and peripheral blood monocytes were infected in the presence of enhancing antibodies by the prototype strain but not by low-passage Nicaraguan isolates. Again, the barrier appeared to be prior to negative-strand accumulation. Thus, depending on the cell type and viral isolate, blocks that limit the production of infectious virus in vitro may occur at distinct steps in the pathway of cellular infection.     

Avian reoviruses cause apoptosis in cultured cells: viral uncoating,but not viral gene expression,is required for apoptosis induction

The cytopathic effect evidenced by cells infected with avian reovirus S1133 suggests that this virus may induce apoptosis in primary cultures of chicken embryo fibroblasts. In this report we present evidence that avian reovirus infection of cultured cells causes activation of the intracellular apoptotic program and that this activation takes place during an early stage of the viral life cycle. The ability of avian reoviruses to induce apoptosis is not restricted to a particular virus strain or to a specific cell type, since different avian reovirus isolates were able to induce apoptosis in several avian and mammalian cell lines. Apoptosis was also provoked in ribavirin-treated avian reovirus-infected cells and in cells infected with UV-irradiated reovirions, indicating that viral mRNA synthesis and subsequent steps in viral replication are not needed for apoptosis induction in avian reovirus-infected cells and that the number of inoculated virus particles, not their infectivity, is the critical factor for apoptosis induction by avian reovirus. Our finding that apoptosis is no longer induced when intracellular viral uncoating is blocked indicates that intraendosomal virion disassembly is required for apoptosis induction and that attachment and uptake of parental reovirions are not sufficient to cause apoptosis. Taken together, our results suggest that apoptosis is triggered from within the infected cell by viral products generated after intraendosomal uncoating of parental reovirions.     

Natural and experimental infection of Caenorhabditis nematodes by novel viruses related to nodaviruses

An ideal model system to study antiviral immunity and host-pathogen co-evolution would combine a genetically tractable small animal with a virus capable of naturally infecting the host organism. The use of C. elegans as a model to define host-viral interactions has been limited by the lack of viruses known to infect nematodes. From wild isolates of C. elegans and C. briggsae with unusual morphological phenotypes in intestinal cells, we identified two novel RNA viruses distantly related to known nodaviruses, one infecting specifically C. elegans (Orsay virus), the other C. briggsae (Santeuil virus). Bleaching of embryos cured infected cultures demonstrating that the viruses are neither stably integrated in the host genome nor transmitted vertically. 0.2 μm filtrates of the infected cultures could infect cured animals. Infected animals continuously maintained viral infection for 6 mo (～50 generations), demonstrating that natural cycles of horizontal virus transmission were faithfully recapitulated in laboratory culture. In addition to infecting the natural C. elegans isolate, Orsay virus readily infected laboratory C. elegans mutants defective in RNAi and yielded higher levels of viral RNA and infection symptoms as compared to infection of the corresponding wild-type N2 strain. These results demonstrated a clear role for RNAi in the defense against this virus. Furthermore, different wild C. elegans isolates displayed differential susceptibility to infection by Orsay virus, thereby affording genetic approaches to defining antiviral loci. This discovery establishes a bona fide viral infection system to explore the natural ecology of nematodes, host-pathogen co-evolution, the evolution of small RNA responses, and innate antiviral mechanisms.     

Expression of baboon endogenous virus in exogenously infected baboon cells.

Strains of low-passage, fetal diploid, baboon (Papio cynocephalus) fibroblasts were susceptible to exogenous infection with three independent isolates of baboon endogenous virus, as measured by an immunofluorescence assay specific for viral p28. Infectivity of the M7 strain of baboon endogenous virus for baboon cells of fetal skin muscle origin was equivalent to that for human and dog cells in that similar, linear, single-hit titration patterns were obtained. The assay for supernatant RNA-dependent DNA polymerase, however, showed that baboon cells produced only low levels of virus after infection compared with the production by heterologous cells. The results showed that baboon endogenous virus was capable of penetrating baboon cells and that viral genes were expressed in infected cells. Replication of complete infectious virus was restricted, however, indicating that in this primate system homologous cells differentially regulated the expression of viral genes.     

Differential ability of human immunodeficiency virus isolates to productively infect human cells

Isolates of HIV showed distinct differences in the ability to replicate in continuous human hematopoietic cell lines. Moreover, although all PMC cultures obtained from healthy individuals could be infected with HIV, considerable variation in the amount of virus released from different PMC cultures was observed. These biological properties of HIV could not be correlated with clinical state, binding properties of the virus isolates to target cells, or differences in target cell CD4 antigen expression. Some isolates of HIV that could not directly infect the HUT-78 cell line showed productive infection when PMC infected with these viruses were added to this human T cell line. These observations emphasize the importance of cell to cell contact in the spread of virus. The results demonstrate for the first time the differences in the host range specificity of HIV isolates in several individual PMC cultures, and indicate that the optimal isolation of HIV is achieved with normal human PMC rather than established human cell lines.     

Heterologous interference in Aedes albopictus cells infected with alphaviruses.

Maximum amounts of 42S and 26S single-stranded viral RNA and viral structural proteins were synthesized in Aedes albopictus cells at 24 h after Sindbis virus infection. Thereafter, viral RNA and protein syntheses were inhibited. By 3 days postinfection, only small quantities of 42S RNA and no detectable 26S RNA or structural proteins were synthesized in infected cells. Superinfection of A. albopictus cells 3 days after Sindbis virus infection with Sindbis, Semliki Forest, Una, or Chikungunya alphavirus did not lead to the synthesis of intracellular 26S viral RNA. In contrast, infection with snowshoe hare virus, a bunyavirus, induced the synthesis of snowshoe hare virus RNA in both A. Ablpictus cells 3 days after Sindbis virus infection and previously uninfected mosquito cells. These results suggested that at 3 days after infection with Sindbis virus, mosquito cells restricted the replication of both homologous and heterologous alphaviruses but remained susceptible to infection with a bunyavirus. In superinfection experiments the the alphaviruses were differentiated on the basis of plaque morphology and the electrophoretic mobility of their intracellular 26S viral RNA species. Thus, it was shown that within 1 h after infection with eigher Sindbis or Chikungunya virus, A. albopictus cells were resistant to superinfection with Sindbis, Chikungunya, Una, and Semliki Forest viruses. Infected cultures were resistant to superinfection with the homologous virus indefinitely, but maximum resistance to superinfection with heterologous alphaviruses lasted for approximately 8 days. After that time, infected cultures supported the replication of heterologous alphaviruses to the same extent as did persistently infected cultures established months previously. However, the titer of heterologous alphavirus produced after superinfection of persistently infected cultures was 10- to 50-fold less than that produced by an equal number of previously uninfected A. albopictus cells. Only a small proportion (8 to 10%) of the cells in a persistently infected culture was capable of supporting the replication of a heterologous alphavirus.