A Three-Dimensional Comparison of Tick-Borne Flavivirus Infection in Mammalian and Tick Cell Lines

Tick-borne flaviviruses (TBFV) are sustained in nature through cycling between mammalian and tick hosts. In this study, we used African green monkey kidney cells (Vero) and Ixodes scapularis tick cells (ISE6) to compare virus-induced changes in mammalian and arthropod cells. Using confocal microscopy, transmission electron microscopy (TEM), and electron tomography (ET), we examined viral protein distribution and the ultrastructural changes that occur during TBFV infection. Within host cells, flaviviruses cause complex rearrangement of cellular membranes for the purpose of virus replication. Virus infection was accompanied by a marked expansion in endoplasmic reticulum (ER) staining and markers for TBFV replication were localized mainly to the ER in both cell lines. TEM of Vero cells showed membrane-bound vesicles enclosed in a network of dilated, anastomosing ER cisternae. Virions were seen within the ER and were sometimes in paracrystalline arrays. Tubular structures or elongated vesicles were occasionally noted. In acutely and persistently infected ISE6 cells, membrane proliferation and vesicles were also noted; however, the extent of membrane expansion and the abundance of vesicles were lower and no viral particles were observed. Tubular profiles were far more prevalent in persistently infected ISE6 cells than in acutely infected cells. By ET, tubular profiles, in persistently infected tick cells, had a cross-sectional diameter of 60–100 nm, reached up to 800 nm in length, were closed at the ends, and were often arranged in fascicle-like bundles, shrouded with ER membrane. Our experiments provide analysis of viral protein localization within the context of both mammalian and arthropod cell lines as well as both acute and persistent arthropod cell infection. Additionally, we show for the first time 3D flavivirus infection in a vector cell line and the first ET of persistent flavivirus infection.     

Mouse hepatitis virus 3 replication in T and B lymphocytes correlate with viral pathogenicity

Viral pathogenicity may be regulated by host defense mechanisms at the virus-immune cell interaction level. The immune system plays an important role in the outcome of acute disease induced by the mouse hepatitis virus type 3 (MHV3) virus. The lymphoid cells act as effectors in the virus elimination as well as targets for viral replication. In order to demonstrate a correlation between MHV3 pathogenicity and viral replication in lymphocytes, genetically-determined resistant A/J and susceptible C57BL/6 mice were infected with pathogenic (L2-MHV3) or nonpathogenic (YAC-MHV3) viral strains. Pathogenicity and histopathologic studies have revealed that lymphoid organs such as thymus and spleen, showed injuries or atrophy in susceptible mice infected with L2-MHV3. No histopathologic lesions in the lymphoid organs occurred in C57BL/6 mice infected with YAC-MHV3 or A/J mice infected with both viruses. The mechanisms involved in the lymphoid injuries were studied regarding viral replication in the lymphoid organs and cells in infected mice. Results indicate that cell depletion in lymphoid organs is caused by a complete viral replication in lymphoid cells. Thy1.2+ and surface IgM+ lymphoid cells from susceptible C57BL/6 mice infected with L2-MHV3 were permissive to viral replication and to subsequent cell lysis. No cell lysis, however, occurred in lymphoid cells from C57BL/6 mice infected with YAC-MHV3 and A/J mice infected with both virus strains. In vitro studies, with purified T and B cell populations were performed to determine the mechanism effecting susceptibility or resistance to viral-induced cell lysis occurring in such cells. A blockade, probably occurring at the viral RNA polymerase activity level, prevents viral replication in resistant cells between the stages of fixation of the virus at the cell-surface receptor and the viral protein translation. These experiments indicate that an intrinsic virus-specific resistant mechanism occurs in lymphoid cells that plays a major role in the viral pathogenicity.     

Role of the host cell in persistent viral infection: Coevolution of L cells and reovirus during persistent infection

Mutant L cells, designated LR cells, were isolated after “curing” a persistently infected cell line (L/C) with antireovirus serum. The LR cells were shown to be virus-free; no reovirus was detectable by infectious center assays, plaque assays, presence of viral proteins, presence of viral dsRNA and immunofluorescence studies. Persistent infections were readily established in LR cells following infection with either cloned, low passage wild-type reovirus or cloned, low passage reovirus isolated from carrier cultures. Reovirus isolated from carrier cultures, however, grew much better than wild-type reovirus in LR cells and showed complete dominance over wild-type reovirus in coinfection experiments. Infection of LR cells with wild-type reovirus resulted in a low-level persistent infection with inefficient viral replication; these mutant L cells were partially resistant to infection with wild-type reovirus. In contrast, infection of the mutant L cells with virus isolated from the persistently infected cells resulted in a persistent infection accompanied with efficient viral replication. Infection of the original L cells with either wild-type reovirus or reovirus isolated from the persistently infected cells resulted in a lytic infection with no surviving cells. Thus the host cell plays a crucial role in the maintenance of persistent reovirus infection. Our results show that there is a coevolution of both mutant L cells and mutant reovirus during persistent infection.     

Effects of anti-E2 monoclonal antibody on sindbis virus replication in AT3 cells expressing bcl-2.

Antibodies directed to Sindbis virus (SV) envelope protein E2 are able to control virus replication in vivo and in persistently infected cultures of neurons in vitro. We investigated the mechanisms by which anti-E2 monoclonal antibody (MAb) alters virus replication by using AT3 rat prostatic carcinoma cells expressing the inhibitor of apoptosis bcl-2. Treatment of SV-infected AT3-bcl-2 cells with anti-E2 MAb G5 for 2 h decreased the rate of virus release for 6 to 8 h after removal of the antibody. Electron microscopic analysis of MAb-treated cells revealed that failure of virus release was linked to a defect in the budding process. The decrease in extracellular virus particles occurred despite continued formation of nucleocapsids and synthesis of envelope glycoproteins. MAb treatment delayed the inhibition of K+ influx and shutoff of host cell protein synthesis by SV infection in a dose-dependent manner. Synthesis of host cell factors and of nonstructural polyprotein precursors required for the formation of initial replication complexes was also prolonged, causing a slower shutdown of overall viral RNA synthesis. We conclude that one mechanism by which anti-E2 MAb treatment down-regulates SV replication is by reestablishing certain critical host cell functions in infected cells.     

Persistence of the cytomegalovirus genome in human cells.

A small percentage of human fibroblast cells survived high-multiplicity infection by cytomegalovirus and were isolated as persistently infected cultures. Approximately 30% of the cells were in the productive phase of infection, since virus-specific structural antigens and virions were associated with these cells. The remaining cells contained neither viral structural antigens nor particles. Nuclear DNA from these nonproductive cells contained approximately 120 genome equivalents of viral DNA per cell as determined by reassociation kinetics. In situ hybridization confirmed that nuclei from nonproductive cells contained a significant amount of viral DNA that was distributed in most of these cells. Early virus-induced proteins and antigens were also detected. Nonproductive cells continued to grow, and there was a slow, spontaneous transition of some of these cells to productive viral replication. The majority of the viral DNA in nonproductive cells persisted with restricted gene expression. When infectious virus production was eliminated by growing the persistently infected cultures in the presence of anticytomegalovirus serum, approximately 45 genome equivalents of the viral DNA persisted per cell. The reassociation reaction approached completion. After removal of the antiserum and subculturing, infectious virus production resumed. Therefore, it was assumed that all sequences of the viral genome remained associated with these cells. Restriction of cytomegalovirus gene expression in persistently infected cell cultures is discussed.     

Persistent infection of cells in culture by measles virus. I. Development and characteristics of HeLa sublines persistently infected with complete virus

Rustigian, Robert (Tufts University School of Medicine, Boston, Mass.). Persistent infection of cells in culture by measles virus. I. Development and characteristics of HeLa sublines persistently infected with complete virus. J. Bacteriol. 92:1792-1804. 1966.-After the development of marked cytopathic effects in HeLa cultures infected with the Edmonston strain of measles virus, renewed cell growth occurred, and HeLa sublines persistently infected with measles virus were obtained. Persistent infection has occurred in a large fraction of the cells of infected clonal lines for more than 300 to 500 cell generations during a period of 6 years. One mechanism by means of which infection was maintained in the clonal lines is transmission of virus or viral subunits from cell to cell at division. Continued subculture of the persistently infected populations resulted in the virtual disappearance of cytopathic effects, a marked decrease in the amount of extracellular virus, and alterations in the cytopathogenicity of virus recovered from persistently infected populations. The intracellular virus-host cell events in late passages of the infected clonal lines appeared to be similar to those in cells of primary infected cultures at early stages of infection, as judged by the pattern of viral immunofluorescence and the very low incidence of cells with intranuclear inclusion bodies. Cultures of the persistently infected clonal lines were highly resistant to super infection by parent Edmonston virus. Cultures of one of these clonal lines were just as susceptible as normal HeLa cultures to vaccinia, herpes simplex, and polio type 2 viruses, and a simian agent, with a possible low degree of resistance to the simian agent.     

Evolution of virus and defective-interfering RNAs in BHK cells persistently infected with Sindbis virus.

We analyzed a BHK cell line persistently infected with Sindbis virus for 16 months and a virus (Sin-16) cloned from these cells. Sin-16 virus was resistant to the defective interfering particles present in the original infection. We found that (i) cells infected with Sin-16 were impaired in the processing of a viral precursor glycoprotein, (ii) high-multiplicity passaging of Sin-16 gave rise to a variant that was able to generate and be inhibited by defective-interfering particles to which the original Sin-16 virus was resistant, and (iii) the persistently infected culture contained a heterogeneous mixture of defective Sindbis virus RNAs which were not packaged into extracellular particles. To determine whether these intracellular RNAs could interfere with the replication of Sin-16, we analyzed cells that were cloned from the persistently infected culture. One clone (A3) synthesized a single defective viral RNA which was lost with continued passaging in culture. Infection of A3 cells with Sin-16 showed that the presence of the defective RNA greatly enhanced cell survival and led to enrichment of this RNA. In contrast, cured cells were highly susceptible to killing by Sin-16, and survivors did not synthesize this RNA. Thus, A3 cells were not genetically altered in their response to Sin-16, but were protected from the cytopathic effects of infection by an RNA with the characteristics of a defective-interfering RNA.     

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.     

Persistent infection of cells in culture by measles virus. II. Effect of measles antibody on persistently infected HeLa sublines and recovery of a HeLa clonal line persistently infected with incomplete virus

Rustigian, Robert (Tufts University School of Medicine, Boston, Mass.). Persistent infection of cells in culture by measles virus. II. Effect of measles antibody on persistently infected HeLa clonal line persistently infected with incomplete virus. J. Bacteriol. 92:1805-1811. 1966.-The effect of viral antibody on persistent infection of HeLa cells by the Edmonston strain of measles virus was investigated by culturing cells from three persistently infected clones in medium supplemented with human immune globulin. The three infected HeLa clones were isolated from a persistently infected parent line. Two sublines which were grown in the presence of measles antibody developed a nonyielder state, wherein there is no detectable virus infectious for normal HeLa cultures. There is, however, continued synthesis of intracellular viral antigen and formation of viral intracytoplasmic inclusion bodies. The development of a nonyielder state was associated with a marked decrease in the degree of hemadsorption in cultures of both sublines. Further studies of the viral properties of non-yielder HeLa cell populations were made with a clone obtained from one of these sublines by plating under antibody. Persistent infection in this line was characterized by synthesis of incomplete virus even when the cells were cultured thereafter in anti-body-free medium. This was evidenced by (i) failure to recover infectious virus from the clonal population despite continued formation of intracellular viral antigen and viral intracytoplasmic inclusion bodies in a majority of the cells, (ii) the presence of only a few cells with surface viral antigen(s) including hemagglutinin, and (iii) the relatively weak antibody response to viral envelope antigen(s) after injection of cells into guinea pigs.     

Establishment of Vero E6 cell clones persistently infected with severe acute respiratory syndrome coronavirus

Little information is available on persistent infection of severe acute respiratory syndrome (SARS) coronavirus (CoV). In this study, we established persistent infection of SARS-CoV in the Vero E6 cell line. Acute infection of Vero E6 with SARS-CoV produced a lytic infection with characteristic rounding cytopathic effects (CPE) and the production of a large number of infectious particles in the culture fluid within 3 days post-infection. Upon subsequent culturing of the remaining adherent cells, the cells gradually proliferated and recovered normal morphology similar to that of the parental cells, and continued to produce large numbers of infectious viral particles during the observation period of 5 months. Among a total of 87 cell clones obtained from the persistently infected Vero E6, only four cell clones (named #13, #18, #21, and #34) were positive for viral RNA. Clones #13, #18, and #34 shifted to viral RNA-negative during subsequent cultures, while #21 continuously produced infectious particles at a high rate. The SARS-CoV receptor, angiotensin-converting enzyme 2, was almost completely down regulated from the cell surface of persistently infected cells. Western blot analysis as well as electron microscopy indicated that the ratios of spike to nucleocapsid protein in clone #21 as well as its parental persistently infected cells were lower than that in the cells in the acute phase of infection. These Vero E6 cells persistently infected with SARS-CoV may be useful for clarifying the mechanism of the persistent infection and also for elucidating the possible pathophysiologic significance of such long-term maintenance of this virus.     

Growth dynamics of a latent primate papovavirus.

The stumptailed macaque papovavirus strain HD was discovered in a persistently infected cell line of primate origin designated Vero 76 (K. Bosslet and G. Sauer, J. Virol. 25:596--607, 1978; W. Waldeck and G. Sauer, Nature [London] 269:171--173, 1977). In clonal derivatives of Vero 76 cells a minor and variable proportion of cells is engaged in the productive synthesis of the HD virus strain. A combination of immunofluorescence using simian virus 40 polyoma subgroup-specific antiserum and in situ hybridization with HD complementary RNA revealed that only those cells which harbor discernible amounts of HD DNA also contain the subgroup-specific antigen. Treatment with arabinofuranosylcytosine caused irreversible disappearance of the antigen, whereas actinomycin D, in contrast, reversibly inhibited both HD DNA replication and synthesis of the subgroup-specific antigen. The proportion of HD DNA and subgroup-specific antigen-synthesizing cells in Vero 76 clonal lines could be either decreased or increased by the mode of passaging of the cell cultures. When cell cultures were split every 3 to 7 days at a 1:4 ratio, the amount of HD DNA sequences as revealed by DNA-DNA reassociation and by the Southern blotting technique fell below the level of detection after only a few passages. Furthermore, expression of the viral subgroup-specific antigen was no longer discernible. However, viral DNA persists in such latently infected cells, because a change in the splitting protocol to a 2-week passaging rhythm led to reinitiation of both viral DNA replication and expression of the subgroup-specific antigen. The HD DNA is perpetuated in a restricted state in latently infected cells in an episomal, unintegrated form as shown by Southern blot analysis. This finding complies with the fact that HD DNA-free subclones could be derived from persistently infected clonal Vero 76 cells. Such subclones have lost the viral genomes, probably owing to segregation during cell division.     

Dual mechanisms of pestiviral superinfection exclusion at entry and RNA replication

For many viruses, primary infection has been shown to prevent superinfection by a homologous second virus. In this study, we investigated superinfection exclusion of bovine viral diarrhea virus (BVDV), a positive-sense RNA pestivirus. Cells acutely infected with BVDV were protected from superinfection by homologous BVDV but not with heterologous vesicular stomatitis virus. Superinfection exclusion was established within 30 to 60 min but was lost upon passaging of persistently infected cells. Superinfecting BVDV failed to deliver a translatable genome into acutely infected cells, indicating a block in viral entry. Deletion of structural protein E2 from primary infecting BVDV abolished this exclusion. Bypassing the entry block by RNA transfection revealed a second block at the level of replication but not translation. This exclusion did not require structural protein expression and was inversely correlated with the level of primary BVDV RNA replication. These findings suggest dual mechanisms of pestivirus superinfection exclusion, one at the level of viral entry that requires viral glycoprotein E2 and a second at the level of viral RNA replication.     

Inhibition of cellular protein secretion by poliovirus proteins 2B and 3A.

Poliovirus RNA replication occurs on the surface of membranous vesicles that proliferate throughout the cytoplasm of the infected cell. Since at least some of these vesicles are thought to originate within the secretory pathway of the host cell, we examined the effect of poliovirus infection on protein transport through the secretory pathway. We found that transport of both plasma membrane and secretory proteins was inhibited by poliovirus infection early in the infectious cycle. Transport inhibition did not require viral RNA replication or the inhibition of host cell translation by poliovirus. The viral proteins 2B and 3A were each sufficient to inhibit transport in the absence of viral infection. The intracellular localization of a secreted protein in the presence of 3A with the endoplasmic reticulum suggested that 3A directly blocks transport from the endoplasmic reticulum to the Golgi apparatus.     

Establishment of a persistent baculovirus infection in a lepidopteran cell line

The paper describes the first overt attempt to establish an insect cell line (Spodoptera frugiperda), persistently infected with its homologous baculovirus. The persistently infected cells were morphologically different and grew to a higher density than the noninfected parent line. The parent line, however, had a shorter doubling time. Persistently infected cells were passaged 40 times over 10 months; they still continued to produce infectious virus and polyhedral inclusion bodies. However, the infectious viral titer was ca. 100 times lower in the persistently infected line than in the parent line; also, the number of inclusion bodies was reduced ca. 98%. Interference with both homologous and heterologous baculoviruses was demonstrated in the persistently infected cell line. Sevently percent of the persistently infected cells contained antigens for S. frugiperda nuclear polyhedrosis virus, ca. 1% of the cells showed infectious viral centers, and ca. 3% of the cells contained inclusion bodies. Although the inclusion bodies from the persistently infected cells were infectious for S. frugiperda larvae, they were about 3 times less infectious than the inclusion bodies produced in the parent line.     

A proposed model to explain persistent infection of host cells with Coxiella burnetii

L929 mouse fibroblast cells and J774 macrophage-like cells are both susceptible to persistent infection with the Q fever agent Coxiella burnetti. Previously this laboratory has shown that persistently infected cell populations multiply with unaltered generation times or cell cycle progression. It has also been reported by others and us that highly infected cells typically exhibit one large parasite-containing vacuole. We now report that lightly and heavily infected cells are capable of division and in the process segregate the parasite-containing vacuole into one of the emerging daughter cells; the companion daughter cell emerges parasite-free. This asymmetric division of infected cells, revealed via photomicrography of stained cells, accounts for the appearance of uninfected cells within persistently infected host cell populations that were previously 100% infected. Some of the persistently infected L929 populations were maintained in culture for over two years without the addition of normal cells.     

Superinfection-induced apoptosis and its correlation with the reduction of viral progeny in cells persistently infected with Hz-1 baculovirus.

Differential induction of necrosis or apoptosis was found upon challenge of cells of the insect Spodoptera frugiperda productively or persistently infected with Hz-1 baculovirus, respectively. Unlike parental SF9 cells, which were essentially all killed by virally induced necrosis, persistently infected cells underwent a process of massive cell death by apoptosis; cells which were not killed by apoptosis then reestablished a cell monolayer. Upon viral challenge, the yield of viral progeny was reduced greatly in persistently virus-infected cells but not in parental cells. Immunolabelling of individual cells revealed that upon viral challenge, production of viral progeny was detectable only in necrotic cells and not in apoptotic cells. These results indicated that induction of apoptosis greatly reduces the yield of viral progeny in cells persistently infected with Hz-1 baculovirus. This is the first report of apoptosis induction in persistently infected cells upon viral superinfection.     

Target cell APOBEC3C can induce limited G-to-A mutation in HIV-1

The evolutionary success of primate lentiviruses reflects their high capacity to mutate and adapt to new host species, immune responses within individual hosts, and, in recent years, antiviral drugs. APOBEC3G (A3G) and APOBEC3F (A3F) are host cell DNA-editing enzymes that induce extensive HIV-1 mutation that severely attenuates viral replication. The HIV-1 virion infectivity factor (Vif), expressed in vivo, counteracts the antiviral activity of A3G and A3F by inducing their degradation. Other APOBECs may contribute more to viral diversity by inducing less extensive mutations allowing viral replication to persist. Here we show that in APOBEC3C (A3C)-expressing cells infected with the patient-derived HIV-1 molecular clones 210WW, 210WM, 210MW, and 210MM, and the lab-adapted molecular clone LAI, viral G-to-A mutations were detected in the presence of Vif expression. Mutations occurred primarily in the GA context and were relatively infrequent, thereby allowing for spreading infection. The mutations were absent in cells lacking A3C but were induced after transient expression of A3C in the infected target cell. Inhibiting endogenous A3C by RNA interference in Magi cells prevented the viral mutations. Thus, A3C is necessary and sufficient for G-to-A mutations in some HIV-1 strains. A3C-induced mutations occur at levels that allow replication to persist and may therefore contribute to viral diversity. Developing drugs that inhibit A3C may be a novel strategy for delaying viral escape from immune or antiretroviral inhibition.