Production of cytopathology in FRhK-4 cells by BS-C-1-passaged hepatitis A virus

Cytopathic effects were produced in fetal rhesus monkey kidney (FRhK-4) cells 7 days postinfection by a serially BS-C-1-passaged strain of hepatitis A virus. Typical enterovirus cytopathology was produced by the HM-175 strain after 15 passages at 7-day intervals in BS-C-1 cells. No cytopathic effects were obtained after neutralization of virus with human anti-hepatitis A virus immunoglobulin G. Normal human serum had no effect on development of cytopathology. Maximum antigen and cDNA probe-based hybridization activity were associated with a CsCl gradient fraction having a density of 1.34 g/cm3. Large quantities of 27- to 30-nm virions typical of hepatitis A virus were associated with the same fraction. These data led to the conclusion that the observed cytopathology was caused by hepatitis A virus.     

Production of cytopathology in FRhK-4 cells by BS-C-1-passaged hepatitis A virus.

Cytopathic effects were produced in fetal rhesus monkey kidney (FRhK-4) cells 7 days postinfection by a serially BS-C-1-passaged strain of hepatitis A virus. Typical enterovirus cytopathology was produced by the HM-175 strain after 15 passages at 7-day intervals in BS-C-1 cells. No cytopathic effects were obtained after neutralization of virus with human anti-hepatitis A virus immunoglobulin G. Normal human serum had no effect on development of cytopathology. Maximum antigen and cDNA probe-based hybridization activity were associated with a CsCl gradient fraction having a density of 1.34 g/cm3. Large quantities of 27- to 30-nm virions typical of hepatitis A virus were associated with the same fraction. These data led to the conclusion that the observed cytopathology was caused by hepatitis A virus.     

In vitro infection of primary and retrovirus-infected human leukocytes by human foamy virus.

The infectivity of human foamy virus (HFV) was examined in primary and cultured human leukocytes. Cell-free infectious viral stocks of HFV were prepared from the human kidney cell line 293 transfected with an infectious molecular clone of HFV. HFV productively infects a variety of human myeloid and lymphoid cell lines. In addition, primary cell cultures enriched for human CD4+, monocytes and brain-derived microglial cells, were readily infected by HFV. Interestingly, while infected primary CD4+ lymphocytes and microglial cells showed marked cytopathology characteristic of foamy virus, HFV-infected monocyte-derived macrophages failed to show any cytopathology. In addition, marked cytotoxicity due to HFV infection was seen in both human T-cell leukemia virus type 1- and human immunodeficiency virus type 1-infected T-cell lines and in human immunodeficiency virus type 1-infected monocytoid cell lines. Thus, HFV infection produces differential cytopathology in a wide host range of primary human leukocytes and hematopoietic cell lines.     

Semliki Forest virus multiplication in clones of Aedes albopictus cells.

A total of 115 clones of Aedes albopictus cells were examined for their response to infection with Semliki Forest virus. Virus yield and cytopathology showed a bimodal distribution. More than 68% of the clones gave low yields of virus (between 8 x 10(6) and 2 x 10(8) PFU/ml) with no discernable cytopathology, and 30% gave high yields of virus (between 1 x 10(9) and 8 x 10(9) PFU/ml) and showed moderate to severe cytopathology. To determine the level at which restriction in virus growth occurs in the low-virus-producing clones, we compared the nature and extent of several virus-directed events in selected low-virus-producing clones with the same events in high-virus-producing clones. Specifically, we compared virus-specified polypeptide synthesis, positive- and negative-strand RNA synthesis, adsorption, uncoating, and transfection with virion 42S RNA. These studies showed that whereas events before negative-strand RNA synthesis and all subsequent virus-specified events were markedly reduced in the low-virus-producing lines, compared with the high-virus-producing lines. Thus, the restriction in virus growth in the low-virus-producing lines occurs at the level of synthesis of negative-strand RNA. The consequence of this restriction in an early step in the virus multiplication cycle is discussed in terms of the survival of invertebrate cells after alphavirus infection.     

Fate of microfilaments in vero cells infected with measles virus and herpes simplex virus type 1.

In herpes simplex virus type 1-infected Vero cells, reorganization of microfilaments was observed approximately 4 h postinfection. Conversion of F (filamentous) actin to G (globular) actin, as assessed by a DNase I inhibition assay, was continuous over the next 12 to 16 h, at which time a level of G actin of about twice that observed in uninfected cells was measured. Fluorescent localization of F actin, using 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin, demonstrated that microfilament fibers began to diminish at about 16 to 18 h postinfection, roughly corresponding to the time that G actin levels peaked and virus-induced cytopathology was first observable. In measles virus-infected cells, no such disassembly of microfilaments occurred. Rather, there was a modest decrease in G actin levels. Fluorescent localization of F actin showed that measles virus-infected Vero cells maintained a complex microfilament network characterized by fibers which spanned the entire length of the newly formed giant cells. Disruption of microfilaments with cytochalasin B, which inhibits measles virus-specific cytopathology, was not inhibitory to measles virus production at high multiplicities of infection (MOI) but was progressively inhibitory as the MOI was lowered. The carbobenzoxy tripeptide SV-4814, which inhibits the ability of Vero cells to fuse after measles virus infection, like cytochalasin B, inhibited measles virus production at low MOI but not at high MOI. Thus, it appears that agents which affect the ability of Vero cells to fuse after measles virus infection may be inhibitory to virus production and that the actin network is essential to this process.     

Cytopathology and release of an RNA virus from a strain of Trichomonas vaginalis

A strain of Trichomonas vaginalis infected with a double-stranded RNA virus showed pronounced cytopathology in the form of giant syncytia generated by the recruitment of single cells. The giant cells ultimately lysed, releasing virus into the culture medium. In the infected cells, clusters of electron-dense particles resembling viral structures were found in the cytoplasm. In addition, distinctive inclusions composed of similar particles were present in the nuclei of some cells. Double-stranded viral RNA of 5.5 kbp was demonstrated in both cytoplasmic and nuclear fractions from these cells. Viral particles collected from the cell-free culture supernatant were of the same shape and size as the RNA virus isolated from a strain of T. vaginalis described previously (Wang & Wang, Journal of Biological Chemistry, 260: 3697–3702, 1985; Wang & Wang, Proceedings of the National Academy of Sciences of the U.S.A. 83: 7956–7986) which does not show this cytopathology.     

Ovine cells: Their long-term cultivation and susceptibility to visna virus

Summary Sheep choroid plexus (SCP) cells have been subcultured more than 120 times and have undergone over 300 cell generations. These fibroblastic-appearing SCP II-B cells contain ovine-specific antigens, have an absolute plating efficiency of 23 to 28% and are as susceptible to visna virus infection and virus-induced cytopathology as their low passage level counterparts. Cultures of low, relatively high and high passage level SCP cells produced equivalent amounts of visna virus at similar rates when infected with equal amounts of visna virus. The passage level of the SCP II-B cells, their elapsed number of cell generations, their possession of ovine-specific antigens and their full susceptibility to visna virus allow these cells to be considered an established line of sheep cells. This work was partially supported by grants from the National Cancer Institute (CA12678) and the National Institute of Allergy and Infectious Diseases (AI12465).     

Superinfection exclusion of alphaviruses in three mosquito cell lines persistently infected with Sindbis virus.

Three Aedes albopictus (mosquito) cell lines persistently infected with Sindbis virus excluded the replication of both homologous (various strains of Sindbis) and heterologous (Aura, Semliki Forest, and Ross River) alphaviruses. In contrast, an unrelated flavivirus, yellow fever virus, replicated equally well in uninfected and persistently infected cells of each line. Sindbis virus and Semliki Forest virus are among the most distantly related alphaviruses, and our results thus indicate that mosquito cells persistently infected with Sindbis virus are broadly able to exclude other alphaviruses but that exclusion is restricted to members of the alphavirus genus. Superinfection exclusion occurred to the same extent in three biologically distinct cell clones, indicating that the expression of superinfection exclusion is conserved among A. albopictus cell types. Superinfection of persistently infected C7-10 cells, which show a severe cytopathic effect during primary Sindbis virus infection, by homologous virus does not produce cytopathology, consistent with the idea that cytopathology requires significant levels of viral replication. A possible model for the molecular basis of superinfection exclusion, which suggests a central role for the alphavirus trans-acting protease that processes the nonstructural proteins, is discussed in light of these results.     

Ovine cells: their long-term cultivation and susceptibility to visna virus.

Sheep choroid plexus (SCP) cells have been subcultured more than 120 times and have undergone over 300 cell generations. These fibroblastic-appearing SCP II-B cells contain ovine-specific antigens, have an absolute plating efficiency of 23 to 28% and are as susceptible to visna virus infection and virus-induced cytopathology as their low passage level counterparts. Cultures of low, relatively high and high passage level SCP cells produced equivalent amounts of visna virus at similar rates when infected with equal amounts of visna virus. The passage level of the SCP II-B cells, their elapsed number of cell generations, their possession of ovine-specific antigens and their full susceptibility to visna virus allow these cells to be considered an established line of sheep cells.     

A nonoccluded virus of the army cutworm

A nonoccluded virus was isolated from larvae of the army cutworm, Euxoa auxiliaris. Infected larvae became lethargic and shrunken, and death usually occurred 12–20 days after infection. The primary site of viral infection and replication appeared to be the nuclei of midgut epithelial cells; however, virus replication also occurred in cells of the tracheal matrix and in muscle. Nuclei in early stages of the infection contained large granular areas with the chromatin scattered near the nuclear membrane. These areas differentiated into viral particles that measured 24 nm and formed crystalline arrays, occasionally 10 μm long. Disruption of the nuclear membrane liberated these arrays of particles into the cytoplasm. Fluorescence microscopy studies indicated that the viral particles contained DNA. The crystalline arrays were Feulgen positive. The virus also infected larvae of the armyworm, Pseudaletia unipuncta, and corn carworm, Heliothis zea, in laboratory tests.     

Restricted replication of human hepatitis A virus in cell culture: intracellular biochemical studies.

When hepatitis A virus was inoculated into Vero cells, virus-specified protein and RNA synthesis was detected. Production of viral protein was detected by electrophoretic analysis in polyacrylamide gels by using a double-label coelectrophoresis and subtraction method which eliminated the contribution of host protein components from the profiles of virus-infected cytoplasm. Eleven virus-specified proteins were detected in the net electrophoretic profiles of hepatitis A virus-infected cells. The molecular weights of these proteins were very similar to those detected in cells infected with poliovirus type 1. Virus-specified protein synthesis could be detected at 3 to 6 h and continued for at least 48 h postinfection, but no significant effect on host-cell macromolecular synthesis was observed. Limited viral RNA replication occurred between 2 and 6 h postinfection. The genomic RNA of hepatitis A virus was extracted and shown to be capable of infecting cells and inducing the same set of proteins as intact virus, indicating that the RNA genome is positive stranded. Progeny virus was never detected in the supernatant fluids of infected cell cultures, and the cells showed no observable cytopathology, even though hepatitis A virus-specific proteins and antigens were being produced. The nature of the defect in the replicative cycle of hepatitis A virus in this system remains unknown.     

Electron microscope observations on granulosis virus replication in the fruit tree leaf roller, Archips argyrospila: Infection of the midgut

An electron microscope study was made of a granulosis virus causing epizootics in three consecutive generations of the fruit free leaf roller, Archips argyrospila. Replication of the virus in midgut epithelial cells was observed in only one insect, and the cytopathology indicated that viral replication at this site was partially blocked or inhibited.     

Growth, plaque assay and immunofluorescent studies on Tataguine virus in cell culture.

The growth characteristics of Tataguine virus were studied in Cercopithecus monkey kidney (Vero); rhesus monkey kidney (LLC-MK2), baby hamster kidney (BHK-21); porcine kidney (PK-15), mouse fibroblasts (L-929) and Aedes albopictus cell monolayers. The virus replicated without producing any cytopathology in Vero, BHK-21 and Aedes albopictus: but not in the other three cell culture systems. Two or three subsequent serial blind passages in those cultures supporting the growth of the virus did not produce any appreciable increase in virus titre. Immunofluorescent staining of inoculated Vero cells demonstrated the presence of Tataguine virus antigen in the cytoplasm of infected cells. Plaques 1--1.5 mm in diameter were produced only in Vero cell culture. In neutralization tests performed on Tataguine virus, immune mouse and hamster sera, higher antibody titres were obtained by plaque reduction than mouse protection tests.     

Characteristics ofHerpesvirus saimiri-induced lymphoma cells in tissue culture

Summary Lymphoblastoid cells were cultured from twoHerpesvirus saimiri (HVS) inoculated white-lipped marmosets and from one HVS-inoculated owl monkey. Cells from all three animals grew clumped in suspension. The cells from both species were diploid in chromo-some number and showed no unusual chromosomal abnormalities. The marmoset cell line examined was chimaeric. The marmoset cells lacked HSV-associated antigens as determined by immunofluorescence, and no evidence for the presence of virus was found by either infectivity assays or electron microscopy. Cocultivation of these cells with Vero cells resulted in cytopathology and the recovery of complete, infectious virus. The owl monkey lymphoid cells were positive to a small degree for both viral antigens and infectivity. The cells were resistant to rechallenge with HVS. Cocultivation of these cells with Vero cells led to the development of cytopathology and an increased yield of virus. This work was supported in part by Contract NIH-NCI-E-71-2025 from the Special Virus Cancer Program, National Cancer Institute, National Institutes of Health, United States Public Health Service.     

A new plaque system for canine distemper: characteristics of the green strain of canine distemper virus.

A Vero cell adapted Green strain of canine distemper virus (CDV) was tested for its plaque-forming capacity in different cell lines. Plaque formation was observed in HEp-2, BS-C-1, and HeLa cells but not in Vero or dog kidney cells even though replication and cytopathology were observed in the latter cell types. In the cells in which the virus was capable of producing plaques, the plaques were observed within 24 h post infection and continued to increase in size with subsequent cellular destruction such that by 72 h postinfection the size of the plaques approached 0.5 mm. With the use of the plaquing technique, it was possible to demonstrate the thermal lability of the virus as well as the kinetics of adsorption. Thus, it was shown that the half-life of the virus was 125 min at 25 degrees C, 75 min at 35 degrees C, and 65 min at 37 degrees C. The rate of adsorption of CDV to HEp-2 cells was 17.2% in 30 min at 37 degrees C and continued slowly for 4 h before completion. Application of this rapid plaque-forming assay to plaque-reduction tests for CDV antibody and for CDV-infected cells by the infectious center assay are described.     

Differential sensitivity of normal and transformed human cells to reovirus infection.

Normal and simian virus 40-transformed WI-38 cells exhibited a differential sensitivity to infection with type 3 reovirus. A progressive decrease in viability began 24 to 36 h after infection of transformed cells terminating in complete lysis of cultures by 96 h. Normal cells were productively infected and continued to produce and release virus for as long as 14 days after infection, but exhibited no detectable cytopathology. Inhibition of cellular DNA synthesis began 15 to 18 h after infection in transformed cells before development of cytopathology. No inhibition of DNA synthesis was detected in infected normal cells. No significant differences were noted in the adsorption or early replication characteristics of reovirus in normal and transformed cells. Virus replication and host cell DNA synthesis in normal and transformed human cells were compared to reovirus-infected L-929 mouse fibroblast cells.     

Cell surface effects of human immunodeficiency virus

Cell killing by human immunodeficiency virus (HIV) is thought to contribute to many of the defects of the acquired immunodeficiency syndrome (AIDS). Two types of cytopathology are observed in HIV-infected cultured cells: cell-cell fusion and killing of single cells. Both killing processes appear to involve cell surface effects of HIV. A model is proposed for the HIV-mediated cell surface processes which could result in cell-cell fusion and single cell killing. The purpose of this model is to define the potential roles of individual viral envelope and cell surface molecules in cell killing processes and to identify alternative routes to the establishment of persistently-infected cells. Elucidation of HIV-induced cell surface effects may provide the basis for a rational approach to the design of antiviral agents which are selective for HIV-infected cells.     

A comparison of Junin virus strains: Growth characteristics,cytopathogenicity and viral polypeptides

The growth characteristics, cytopathogenicity and viral polypeptides of the virulent strain XJ of Junin virus (JV), its attenuated derivative XJC13 and another naturally attenuated JV strain, IV4454, were comparatively studied. IV4454 and XJC13 viruses showed the highest and lowest cytopathology for Vero cells, respectively, as measured by plaque morphology, cell viability and inhibition of host cell protein synthesis. The kinetics and electrophoretic patterns of viral polypeptides in infected cell extracts were very similar among the three strains, whereas differences were detected in the surface glycoprotein GP38 by peptide mapping after limited proteolysis.