Persistent infection with bovine herpesvirus-1 (infectious bovine rhinotracheitis virus) in cultured hamster cells.

Bovine herpesvirus-1 infection in hamster embryo cells was found to be dependent upon input multiplicity; productive infection was achieved at input multiplicities greater than one, while persistent infection was established when input multiplicities were about 0.5. This persistence was characterized by a noncyclic, minimal degree of cytopathic effect with a low level of released virus. Maintenance of the persistently infected cultures did not require external supportive measures. Subcultivation of the persistently infected cultures led to virus replication followed by CPE and then cell regrowth. With 3 to 4 weeks after subcultivation a persistent infection was re-established. The possible mechanism for the bovine herpesvirus persistence in hamster cells is discussed.     

Tyrosine phosphorylation of measles virus nucleocapsid protein in persistently infected neuroblastoma cells.

Subacute sclerosing panencephalitis is a slowly progressing fatal human disease of the central nervous system which is a delayed sequel of measles virus (MV) infection. A typical pathological feature of this disease is the presence of viral ribonucleocapsid structures in the form of inclusion bodies and the absence of infectious virus or budding viral particles. The mechanisms governing the establishment and maintenance of a persistent MV infection in brain cells are still largely unknown. To understand the mechanisms underlying MV persistence in neuronal cells, a tissue culture model was studied. Clone NS20Y/MS of the murine neuroblastoma C1300 persistently infected with the wild-type Edmonston strain of MV secretes relatively high levels of alpha/beta interferon (IFN). As shown previously, treatment of the persistently infected cultures with anti-IFN serum converted the persistent state into a productive infection indicated by the appearance of multinucleated giant cells. In this study, we have investigated whether alpha/beta IFN produced by NS20Y/MS cells activates cellular protein tyrosine kinases which will induce tyrosine phosphorylating activity specific to virus-infected cells. We present data to show augmented protein tyrosine kinase activity in the persistently infected cells. We demonstrate that the MV N protein is phosphorylated on tyrosine in addition to serine and threonine in the persistent state but not in NS20Y cells acutely infected with MV.     

Persistent infection of cultured cells with mouse hepatitis virus (MHV) results from the epigenetic expression of the MHV receptor.

The A59 strain of murine coronavirus mouse hepatitis virus (MHV) can cause persistent infection of 17C1-1 cells and other murine cell lines. Persistently infected cultures released large amounts of virus (10(7) to 10(8) PFU/ml) and were resistant to superinfection with MHV but not to infection with unrelated Semliki Forest and vesicular stomatitis viruses. The culture medium from persistently infected cultures did not contain a soluble inhibitor such as interferon that protected uninfected cells from infection by MHV or vesicular stomatitis virus. The persistent infection was cured if fewer than 100 cells were transferred during subculturing, and such cured cultures were susceptible to reinfection and the reestablishment of persistent infection. Cultures of 17C1-1 cells that had been newly cloned from single cells consisted of a mixture of MHV-resistant and -susceptible cells. 17C1-1/#97 cells, which were cured by subcloning after 97 passages of a persistently infected culture over a 1-year period, contained 5 to 10% of their population as susceptible cells, while 17C1-1/#402 cells, which were cured by subcloning after 402 passages over a 3-year period, had less than 1% susceptible cells. Susceptibility to infection correlated with the expression of MHV receptor glycoprotein (MHVR [Bgp1a]). Fluorescence-activated cell sorter analysis with antibody to MHVR showed that 17C1-1/#97 cells contained a small fraction of MHVR-expressing cells. These MHVR-expressing cells were selectively eliminated within 24 h after challenge with MHV-A59, and pretreatment of 17C1-1/#97 cells with monoclonal antibody CC1, which binds to the N-terminal domain of MHVR, blocked infection. We conclude that the subpopulation of MHVR-expressing cells were infected and killed in acutely or persistently infected cultures, while the subpopulation of MHVR-nonexpressing cells survived and proliferated. The subpopulation of MHVR-negative cells produced a small proportion of progeny cells that expressed MHVR and became infected, thereby maintaining the persistent infection as a steady-state carrier culture. Thus, in 17C1-1 cell cultures, the unstable or epigenetic expression of MHVR permitted the establishment of a persistent, chronic infection.     

Persistent infection with herpes simplex virus type 1 in an Ia antigen-positive murine macrophage cell line

The interaction of herpes simplex virus type 1 (HSV-1) with murine macrophage cell lines was examined. The cell lines appeared to be moderately permissive for HSV-1 replication, though the yield of the virus was limited compared with that in Vero cells. Furthermore, the murine macrophage cell line SL-1, bearing Ia antigen, was persistently infected with HSV-1 for over one year, and was designated SL-1/KOS. Persistent infection could not be established in an Ia antigen-negative macrophage cell line, SL-4. In the SL-1/KOS culture, there was a small number of infected cells as revealed by infectious center assay. Treatment with monoclonal antibody against HSV-1 cured the persistent infection. Therefore maintenance of the persistent infection is considered to be due to a carrier culture consisting of a minority of infected cells and a majority of uninfected cells. In the SL-1/KOS cultures a low level of interferon (IFN) was found. When a large amount of exogenous recombinant murine IFN-beta (10(5)-10(6) international units/ml) was added to the culture, virus production diminished to undetectable levels. These results suggest that IFN plays an important role in the maintenance of persistent infection. In long-term persistently infected cultures, syncytium formation appeared and the virus from such cultures had a different DNA structure from that of the virus originally used for infection as revealed by restriction endonuclease analysis.     

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.     

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.     

Antiapoptotic but Not Antiviral Function of Human bcl-2 Assists Establishment of Japanese Encephalitis Virus Persistence in Cultured Cells

Upon infection of Japanese encephalitis virus (JEV), baby hamster kidney (BHK-21) and Chinese hamster ovary (CHO) cells were killed by a mechanism involved in apoptosis. While readily established in a variety of cell lines, JEV persistence has never been successfully instituted in BHK-21 and CHO cells. Since stable expression of human bcl-2 in BHK-21 cells has been shown to delay JEV-induced apoptosis, in this study we investigated whether JEV persistence could be established in such cells. When constitutively expressing bcl-2, but not its closest homolog, bcl-X_L, following a primary lytic infection, approximately 5 to 10% of BHK-21 and CHO cells became persistently JEV infected during a long-term culture. From the persistent bulks, several independent clones were selected and expanded to form stable cell lines that continuously produced infectious virus without marked cytopathic effects (CPE). Among these stable cell lines, the truncated nonstructural protein 1 (NS1) was also detected and was indistinguishable from the NS1 truncations previously observed in JEV-persistent murine neuroblastoma N18 cells. However, the stable expression of NS1 alone, regardless of whether it was truncated or full length, failed to render the engineered cells persistently infected by JEV, implying that aberrant NS1 proteins were likely a consequence of, rather than a cause for, the viral persistence. Enforced bcl-2 expression, which did not affect virus replication and spread during the early phase of cytolytic infection, appeared to attain JEV persistence by restriction of virus-induced CPE. Our results suggest that it is the antiapoptotic, rather than the antiviral, effect of cellular bcl-2 which plays a role in the establishment of JEV persistence.     

Postnatal Persistent Infection with Classical Swine Fever Virus and Its Immunological Implications

It is well established that trans-placental transmission of classical swine fever virus (CSFV) during mid-gestation can lead to persistently infected offspring. The aim of the present study was to evaluate the ability of CSFV to induce viral persistence upon early postnatal infection. Two litters of 10 piglets each were infected intranasally on the day of birth with low and moderate virulence CSFV isolates, respectively. During six weeks after postnatal infection, most of the piglets remained clinically healthy, despite persistent high virus titres in the serum. Importantly, these animals were unable to mount any detectable humoral and cellular immune response. At necropsy, the most prominent gross pathological lesion was a severe thymus atrophy. Four weeks after infection, PBMCs from the persistently infected seronegative piglets were unresponsive to both, specific CSFV and non-specific PHA stimulation in terms of IFN-γ-producing cells. These results suggested the development of a state of immunosuppression in these postnatally persistently infected pigs. However, IL-10 was undetectable in the sera of the persistently infected animals. Interestingly, CSFV-stimulated PBMCs from the persistently infected piglets produced IL-10. Nevertheless, despite the addition of the anti-IL-10 antibody in the PBMC culture from persistently infected piglets, the response of the IFN-γ producing cells was not restored. Therefore, other factors than IL-10 may be involved in the general suppression of the T-cell responses upon CSFV and mitogen activation. Interestingly, bone marrow immature granulocytes were increased and targeted by the virus in persistently infected piglets. Taken together, we provided the first data demonstrating the feasibility of CSFV in generating a postnatal persistent disease, which has not been shown for other members of the Pestivirus genus yet. Since serological methods are routinely used in CSFV surveillance, persistently infected pigs might go unnoticed. In addition to the epidemiological and economic significance of persistent CSFV infection, this model could be useful for understanding the mechanisms of viral persistence.     

Persistent cyclic herpes simplex virus infection in vitro. 3. Asynchrony in the progression of infection and cell regrowth

Hampar, Berge (National Institute of Dental Research, Bethesda, Md.). Persistent cyclic herpes simplex virus infection in vitro. III. Asynchrony in the progression of infection and cell regrowth. J. Bacteriol. 91:1965-1970. 1966.-The progression of virus-induced cytopathic effects (CPE) and virus synthesis was studied in localized areas of Chinese hamster cell cultures persistently infected with herpes simplex virus (HSV). CPE was initially evidenced by the presence of small multinucleated giant cells, followed by expanding plaquelike lesions with an occasional uninfected cell remaining within the infected areas. Cell detachment rapidly followed the appearance of viral antigen in infected cells. The surviving cells which proliferated to re-establish the cell sheet arose from two sources. The first was from viable cells which remained attached after expansion of localized areas of CPE, and the second was from reattachment of viable cells in the medium. CPE in localized areas was initiated at various times during the cycle irrespective of the virus titer in the medium. Cell regrowth in some areas and CPE in other areas occurred simultaneously throughout the cycle in an asynchronous fashion. Consequently, during periods of rising virus titers, most areas showed CPE while few areas displayed cell regrowth. As the virus titers declined, more areas showed cell regrowth and fewer areas displayed new cycles of CPE. CPE in localized areas was not initiated until cell regrowth had occurred. It is proposed that the proliferating cells were temporarily resistant to HSV infection, and that this resistance was ultimately lost in their progeny cells.     

Persistent borna disease virus infection confers instability of HSP70 mRNA in glial cells during heat stress

Borna disease virus (BDV) is a highly neurotropic RNA virus that causes neurological disorders in many vertebrate species. Although BDV readily establishes lasting persistence, persistently infected cells maintain an apparently normal cell phenotype in terms of morphology, viability, and proliferation. In this study, to understand the regulation of stress responses in BDV infection, we investigated the expression of heat shock proteins (HSPs) in glial cells persistently infected with BDV. Interestingly, we found that BDV persistence did not upregulate HSP70 expression even in cells treated with heat stress. Furthermore, BDV-infected glial cells exhibited rapid rounding and detachment from the culture plate under various stressful conditions. Immunofluorescence analysis demonstrated that heat stress rapidly disrupts the cell cytoskeleton only in persistently infected cells, suggesting a lack of thermotolerance. Intriguingly, we found that although persistently infected glial cells expressed HSP70 mRNA after heat stress, its expression rapidly disappeared during the recovery period. These observations indicated that persistent BDV infection may affect the stability of HSP70 mRNA. Finally, we found that the double-stranded RNA-dependent protein kinase (PKR) is expressed at a constant level in persistently infected cells with or without heat shock. Considering the interrelationship between HSP70 and PKR production, our data suggest that BDV infection disturbs the cellular stress responses to abolish antiviral activities and maintain persistence.     

Selection of Temperature-Sensitive Mutants During Persistent Infection: Role in Maintenance of Persistent Newcastle Disease Virus Infections of L Cells

Virus mutants (NDV(pi)) recovered from L cells persistently infected with Newcastle disease virus (NDV, Herts strain) are temperature-sensitive (ts) at 43 C, although the wild-type virus (NDV(o)) which initiated the persistent infection replicates normally at that temperature. To study the relationship between the ts marker of NDV(pi) and the other properties which distinguish this virus from NDV(o), NDV(pi) ts(+) revertants were selected at the nonpermissive temperature and NDV(o) ts mutants were generated by treating NDV(o) with nitrous acid. Spontaneously-occurring ts mutants in the Herts NDV population were also isolated. The different virus populations were characterized with regard to plaque size, virulence for eggs, and thermal stability of infectivity, hemagglutinin, and neuraminidase. The NDV(pi) ts(+) revertants, although no longer temperature-sensitive, retained NDV(pi) properties, whereas both spontaneously-occurring and mutagen-induced ts mutants remained wild-type in their other properties. These findings showed that the properties which characterized NDV(pi) were independent of the ts marker. However, the ts marker and the other markers of NDV(pi) were coselected during the persistent infection, and the combination of those markers appeared to be important in the outcome of NDV infection of L cells. NDV(pi) replicated productively in L cells, whereas NDV(o), the NDV(pi) ts(+) revertants, and the spontaneously-occurring ts mutants all yielded covert infections in L cells. The role of the selection of ts mutants in persistent infection was confirmed as follows: L cells were persistently infected with NDV(pi) ts(+) revertants and NDV(o) ts mutants. Virus recovered from the persistently infected cultures after eight cell passages was always temperature-sensitive and of smaller plaque size than the parental virus in chicken embryo cell cultures. Similar results were obtained with virus recovered from L-cell cultures persistently infected with two other velogenic strains of NDV, the Texas-GB and Kansas-Man strains. These results strongly suggest that selection of ts mutants during the persistent infection was not random and played a role in establishment or maintenance of the persistent infection, or both.     

Virus-lymphocyte interaction: T cells of the helper subset are infected with lymphocytic choriomeningitis virus during persistent infection in vivo.

The lifelong persistence of lymphocytic choriomeningitis virus (LCMV) in neonatally or congenitally infected mice is accompanied by a suppression of virus-specific T-cell responses. In this study, we identified the subset of T cells infected with LCMV during persistent infection in vivo. Using specific monoclonal antibodies to separate the different lymphocyte cell populations and employing both an infectious center assay and immunofluorescence to detect the virus, we found that infection is confined primarily to T cells of the helper subset (L3T4+ Lyt2-), with minimal involvement of cytotoxic T cells (Lyt2+ L3T4-) and mature B cells. About 0.54 to 1.1% of L3T4+ T cells were producing the virus, as determined by the infectious center assay. In contrast, 9.1 to 12.2% of these L3T4+ T cells contained viral antigen, as shown by immunofluorescence studies. This finding suggested that, at any given time, a substantial number of infected T cells were not producing infectious virus. This infection of T helper cells may be involved in the suppression of LCMV-specific T-cell responses observed in persistently infected mice.     

Isolation of highly fusogenic variants of simian virus 5 from persistently infected cells that produce and respond to interferon.

A series of experiments were undertaken to examine how interferon and neutralizing antibodies influence the ability of simian virus 5 (SV5) (strain W3) to establish and maintain persistent infections in murine cells. In contrast to the rapid decline in SV5 protein synthesis observed in murine BALB/c fibroblasts (BF cells), which produce and respond to interferon, between 24 and 48 h postinfection there was no inhibition of virus protein synthesis in MSFI- cells, skin fibroblasts derived from alpha/beta-interferon receptor knockout BALB/c mice. Furthermore, the addition of anti-interferon antibodies to the culture medium of infected BF cells significantly reduced the observed decline in virus protein synthesis. Following infection of untreated BF cells, the majority replicated virus but survived the infection and eventually cleared the virus after 8 to 15 days. However, not all the cells were cured, and the cultures became persistently infected. Upon passage of persistently infected cultures, the virus fluxed between active and repressed states as a consequence of interferon production. This resulted in a balance being reached in which only 5 to 20% of the cells were infected at any one time. After 30 passages of the persistently infected cells, highly fusogenic virus variants arose (one of which was isolated and termed W3-f). W3-f remained as sensitive to interferon as the parental W3 isolate but, in the absence of interferon, spread much more rapidly than the parental W3 strain through BF cell monolayers. Sequence analysis revealed no deduced amino acid differences between the F proteins of W3 and W3-f. BF cell cultures persistently infected with W3-f were rapidly cleared of virus by the addition of virus-neutralizing antibodies to the culture medium. In contrast, neutralizing antibodies had little effect on the numbers of cells persistently infected with W3 over several passages. These results suggest that the ability of paramyxoviruses to cause cell-cell fusion may be selected for in vivo as a consequence of their adaptation to the interferon response rather than their need to escape from neutralizing antibodies. The significance of these observations with regard to persistent parainfluenza virus infections in vivo is further discussed.     

Persistent Infection of Human Fetal Endothelial Cells with Rubella Virus

Cardiovascular abnormalities are the leading cause of neonatal death among patients with congenital rubella syndrome (CRS). Although persistence of rubella virus (RV) in fetal endothelium has been repeatedly suggested as a possible cause of cardiovascular birth defects, evidence of the permissiveness of fetal endothelial cells to RV is lacking. In this study we evaluated the ability of RV to infect and persist in primary fetal endothelial cells derived from human umbilical vein (HUVEC). We found that wild type (wt) low passage clinical RV productively infected HUVEC cultures without producing cytopathology or ultrastructural changes. RV did not inhibit host cell protein synthesis, cell proliferation, or interfere with the cell cycle. Persistently infected cultures were easily established at low and high multiplicities of infection (MOI) with both laboratory and wt clinical RV strains. However, synchronous infections of entire HUVEC monolayers were only observed with clinical RV strains. The release of infectious virions into media remained at consistently high levels for several subcultures of infected HUVEC. The results indicate that macrovascular fetal endothelial cells are highly permissive to RV and allow slow persistent RV replication. The findings provide more evidence for the suggestion that vascular pathologies in CRS are triggered by persistent rubella virus infection of the endothelium.     

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.     

Effect of antiviral antibody on maintenance of long-term rubella virus persistent infection in Vero cells.

A Vero cell line with a long-term rubella virus persistent infection was maintained for 45 weeks in the presence of anti-rubella virus antibody of sufficient titer to completely neutralize the virus in the culture fluid to determine the effect of the presence of antibody on the maintenance of the persistent infection. Prior to antibody treatment, virus was continuously detected as plaque-forming units in the persistently infected culture fluid. Virus clones that were plaque purified from the persistently infected culture fluid were temperature sensitive and exhibited a reduced efficiency of replication and ability to induce cytopathic effects in Vero cells at the persistently infected culture temperature compared with the standard virus used to initiate the persistently infected culture. Defective interfering RNAs were the major intracellular virus-specific RNA species present in the persistently infected cells. Treatment with antibody failed to cure the persistently infected culture of virus, and the cells retained the ability to release virus after antibody treatment was discontinued. Interestingly, the presence of antibody led to the selection of a population of virus which was markedly less cytopathic for Vero cells than the virus population which was selected during persistent infection in the absence of antibody.     

Growth of measles virus in various human lymphoid cell lines.

Neurovirulent TYCSA strain and attenuated Schwarz strain of measles virus and Halle strain of subacute sclerosing panencephalitis (SSPE) virus replicated in cultures of human lymphoid cell lines of the T-cell type, MOLT-3, MOLT-4 and CCRF-CEM. TYCSA and Halle strains grew rapidly, but Schwarz strain grew slowly in these cell lines. Furthermore, these three strains established persistent infection in CCRF-CEM cells but not in the other cell lines. In these persistently infected cultures an almost entire population of cells were shown to be infected and infectious virus was produced constantly for over 100 days. Cells persistently infected with Schwarz strain contained nucleocapsid structures in both the nucleus and cytoplasm and produced low titered infectious virus, whereas nucleocapsid structures were observed only in the cytoplasm of cells persistently infected with either TYCSA or Halle strain and the titers of infectious virus produced from these cells were high.     

Acute and persistent infection of human neural cell lines by human coronavirus OC43

Human coronaviruses (HuCV) are recognized respiratory pathogens. Data accumulated by different laboratories suggest their neurotropic potential. For example, primary cultures of human astrocytes and microglia were shown to be susceptible to an infection by the OC43 strain of HuCV (A. Bonavia, N. Arbour, V. W. Yong, and P. J. Talbot, J. Virol. 71:800-806, 1997). We speculate that the neurotropism of HuCV will lead to persistence within the central nervous system, as was observed for murine coronaviruses. As a first step in the verification of our hypothesis, we have characterized the susceptibility of various human neural cell lines to infection by HuCV-OC43. Viral antigen, infectious virus progeny, and viral RNA were monitored during both acute and persistent infections. The astrocytoma cell lines U-87 MG, U-373 MG, and GL-15, as well as neuroblastoma SK-N-SH, neuroglioma H4, oligodendrocytic MO3.13, and the CHME-5 immortalized fetal microglial cell lines, were all susceptible to an acute infection by HuCV-OC43. Viral antigen and RNA and release of infectious virions were observed during persistent HuCV-OC43 infections ( approximately 130 days of culture) of U-87 MG, U-373 MG, MO3.13, and H4 cell lines. Nucleotide sequences of RNA encoding the putatively hypervariable viral S1 gene fragment obtained after 130 days of culture were compared to that of initial virus input. Point mutations leading to amino acid changes were observed in all persistently infected cell lines. Moreover, an in-frame deletion was also observed in persistently infected H4 cells. Some point mutations were observed in some molecular clones but not all, suggesting evolution of the viral population and the emergence of viral quasispecies during persistent infection of H4, U-87 MG, and MO3.13 cell lines. These results are consistent with the potential persistence of HuCV-OC43 in cells of the human nervous system, accompanied by the production of infectious virions and molecular variation of viral genomic RNA.     

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.