Transformation of African green monkey kidney cells with the RF strain of human papovavirus BKV.

Infection of semipermissive African green monkey kidney (AGMK) cells with human papovavirus RFV prodced a chronic infection with slow cytopathic effects. Prolonged maintenance of cell cultures in the presence of virus-neutralizing antibody resulted in the emergence of a stable cell line presently at the 100th generation intissue culture. The cell line MRF-325 has many of the properties of a tumor cell in culture, including increaed saturation density in monolayers, growth in soft agar and in suspension, and a decreased serum requirement for growth. Papovavirus T antigen and U antigen were detected in 100% of the MRF-325 nuclei by immunoperoxidase staining. No virion antigens were detected, and no infectious virions were isolated by cocultivation or fusion of MRF-325 with permissive human embryo kiney cells. Susceptibility of MRF-325 cells to SV40 infection was decreased compared to untransformed AGMK cells. MRF-325 cells supported high levels of replication of human adenovirus type 5 compared with nontransformed AGMK cells. Thus, RFV apparently provides a helper effect for adenovirus replication in AGMK cells similar to that observed for SV40.     

Efficiency of isolation of human rotavirus in primary African green monkey kidney cells

Out of 212 human rotavirus (HRV) containing fecal specimens, 173 (81.6%) yielded virus on first passage in primary African Green monkey kidney cells (AGMK), while additional 34 specimens, did not yield virus on first passage. However, following blind passages, 18 of the 34 yielded virus in passage levels 2-8, thus raising the overall isolation rate to 90.1%. The isolation rate of HRV strains obtained in embryonic Rhesus monkey kidney cell line (MA-104), was only 41.4%. ELISA tests performed on fluids from infected cell cultures proved to be an efficient tool to measure virus replication. No differences were encountered in the isolation rates between subgroup I and II strains, while viruses lacking the antigenic determinants of both subgroups did not grow at all. However, one of those unusual group A strains was isolated and grew well in AGMK cells. Primary AGMK and MA-104 cells supported the growth of tissue culture adapted virus most efficiently when compared with six human and primate cell types.     

Restricted replication of mouse adenovirus strain FL in mouse Balb/c cells transformed by Simian Adenovirus 7

Mouse kidney cells (Balb/c cells), in which mouse adenovirus strain FL (Ad FL) replicates, become restricted in their capacity to support Ad FL growth after transformation by simian adenovirus 7 (SA7). In transformed cells (Balb/c SA7 cells), the duration of the Ad FL cycle is significantly longer than in normal cells; some virus DNA replication and virus protein synthesis occur but the yield of infectious Ad FL particles is greatly reduced. Only a few cells produce virus as estimated by the number of infectious centers. However, no significant differences are noted in the adsorption and in the penetration of Ad FL in both cell types. Thus, the restrictive event in virus multiplication in Balb/c SA7 cells occurs probably after viral uncoating and before viral DNA synthesis.     

Host cell DNA synthesis as a possible factor in the enhancement of replication of human adenoviruses in simian cells by SV40

Temperature-sensitive mutants of SV40, ts-701 and ts-702, have been isolated after treatment of wild-type virus with nitrous acid and nitrosoguanidine, respectively. Although neither mutant produces infectious virus at the nonpermissive temperature, both can induce SV40 T and V antigens, virus DNA synthesis, and the stimulation of host cell DNA synthesis. Both are late mutants and appear to be in the same SV40 complementation group. Both mutants can also complement the replication of human adenovirus type 7 at the elevated temperature, as can another late mutant, pm-301, and an early mutant, tsA7. Therefore, the adenovirus complementation step appears to be prior to SV40 virus DNA synthesis. Since a BSC-1 clone in which SV40 infection stimulates host cell DNA synthesis allows the complementation of adenovirus type 7 by SV40 while parental BSC-1 cells neither support complementation of adenovirus type 7 by SV40 nor are stimulated to replicate their DNA after SV40 infection, the stimulation of host cell DNA synthesis appears to be critical to adenovirus enhancement. This stimulation appears to be specific since stimulation of DNA synthesis after nutrient deprivation does not induce the replication of human adenoviruses in simian cells.     

Primary isolation and serial passage of hepatitis A virus strains in primate cell cultures

Although several primate cell types have been reported to support replication of hepatitis A virus, optimal conditions for the isolation and production of quantities of virus have not been defined. We therefore examined seven different primate cell types for their ability to support replication of primate-passaged and wild-type virus as reflected by intracytoplasmic accumulation of viral antigen (direct immunofluorescence and radioimmunoassay) and propagation of cell culture-adapted virus. Of the cells tested, low-passage African green monkey kidney (AGMK) cells were most sensitive for initial isolation. Viral replication was documented after inoculation of AGMK cells with seven of nine hepatitis A virus antigen-positive fecal specimens (from seven epidemiologically distinct sources). With six inocula, virus was successfully passed in serial cultures. AGMK-adapted virus was readily propagated in continuous AGMK (BS-C-1) cells. The optimal temperature for the growth of virus in BS-C-1 cells was 35 degrees C. Viral release into supernatant fluids was documented in the absence of any cytopathic effect, and infectivity titers in supernatant fluids 21 days after inoculation (50% tissue culture infective does [TCID50], 10(6.0)/ml) equalled or exceeded those in the cell fraction (TCID50, 10(5.5)/ml). Cells maintained in serum-free media readily supported viral growth, with yields of virus (TCID50, 10(6.5)/ml) equal to or greater than those obtained with cells maintained in 2% fetal bovine serum.     

Comparative studies on nucleic acid synthesis and virus-induced RNA polymerase activity in mammalian cells infected with certain arboviruses

Summary Variations in the metabolism of African green monkey (AGMK) and stable porcine kidney (PS) cells infected with certain arboviruses were investigated. Virus-directed RNA synthesis and RNA polymerase activity were stimulated in actinomycin-treated infected cells. The rate of³H-uridine incorporation into RNA in Western equine encephalitis (WEE) virus-infected AGMK cells reached a maximum at approximately 5 hours after infection. In contrast, the viral RNA synthesis induced by Japanese encephalitis (JE) or dengue virus did not increase appreciably during the first 15 hours, and a maximum was reached from 24 to 30 hours after infection. Cytoplasmic large- and small-particle fractions from cells infected with WEEV or JEV were found to catalyze the incorporation of 4 nucleoside triphosphates into acid-insoluble products. In WEEV-infected AGMK cells, the enzyme activity was associated almost solely with the small-particle fraction, whereas nuclear and large-particle fractions of JEV-infeeted cells still contained 15 to 30% of the total enzyme activity 24 hours after infection. Rapid inhibition of cellular DNA synthesis was observed 4 hours after infection, with each of the three kinds of arbovirus used. Heat-inactivated WEEV was unable to suppress host cell DNA synthesis appreciably, whereas infection by UV-irradiated virus did result in a clear inhibition of DNA synthesis. However, the effect was apparently less marked than that of the active WEE virus.     

Helper function for adenovirus replication in monkey cells by BK human papovavirus.

CV-1 monkey kidney cells are semipermissive for BK human papovavirus at 37°. Although infected cells synthesize T antigen at this temperature, only a small percentage of the cells (less than 5%) produce viral antigen. However, when infected cells were incubated at 40°, characteristic CPE was observed with high virus yields. The inhibition of BKV growth in CV-1 cells was thus shown to be a temperature-dependent phenomenon. Experiments were then conducted to determine whether BKV provided a helper function for adenovirus growth in CV-1 cells at temperatures which were either permissive (40°) or semipermissive (37°) for BKV replication. At 37°, there was a low level of adenovirus enhancement of 0.5 to 1.0 log increase. However, at 40°, there was a 1.5 to 2.5 log increase in adenovirus yields, comparable to those obtained by coinfection with SV40 and adenovirus. These data provide additional information on common viral functions shared by BKV and SV40.     

表达GFP基因的溶瘤腺病毒对大肠癌细胞的体外杀伤作用

目的： 研究在端粒酶启动子驱动下表达绿色荧光蛋白（GFP）及 5型腺病毒早期基因（E1A）基因的腺病毒Ad/hTERT-GFP-E1对大肠癌细胞的杀伤作用及其可能的机制。方法:将不同滴度Ad/hTERT-GFP-E1感染大肠癌及正常细胞,以巨细胞病毒（CMV）启动子驱动下表达GFP基因的腺病毒Ad/CMV-GFP作为载体对照,通过半数组织培养感染量（TCID50）法测定病毒滴度及体外复制能力,然后用MTT法及细胞克隆形成试验评价该病毒在体外对肿瘤细胞及正常细胞的杀伤作用,并对病毒感染后的细胞进行原位凋亡检测。结果:Ad/hTERT-GFP-E1能够在DLD1细胞内持续复制,GFP的表达能够对病毒的感染和复制起到监测作用;MTT结果显示该病毒对于结直肠肿瘤细胞有显著杀伤作用,而对于正常细胞没有明显的杀伤作用;对病毒感染后的DLD1进行细胞凋亡检测发现Ad/hTERT-GFP-E1所引起的凋亡率显著高于对照组(P＜0.01)。结论:溶瘤腺病毒Ad/hTERT-GFP-E1能够选择性地在肿瘤细胞内复制并杀伤肿瘤细胞,其机制与细胞凋亡的途径有关。     

Vaccinia virus stimulates the growth of vesicular stomatitis virus at the level of protein synthesis in mouse L cells

Coinfection with vaccinia virus increases the growth of vesicular stomatitis virus (VSV) in mouse L cells by 10- to 20-fold. Although vaccinia has no significant effect on RNA synthesis by VSV, VSV protein synthesis is dramatically stimulated by double infection. The enhancement of VSV growth is correlated with the ability of vaccinia to inhibit the VSV-mediated damage to the host translational machinery. Coinfection with vaccinia fails to stimulate the growth of a VSV mutant which is deficient in its ability to shut off protein synthesis during infection.     

A conditionally replicating adenovirus with strict selectivity in killing cells expressing epidermal growth factor receptor

Virotherapy of cancer using oncolytic adenoviruses has shown promise in both preclinical and clinical settings. One important challenge to reach the full therapeutic potential of oncolytic adenoviruses is accomplishing efficient infection of cancer cells and avoiding uptake by normal tissue through tropism modification. Towards this goal, we constructed and characterized an oncolytic adenovirus, carrying mutated capsid proteins to abolish the promiscuous adenovirus native tropism and encoding a bispecific adapter molecule to target the virus to the epidermal growth factor receptor (EGFR). The new virus displayed a highly selective targeting profile, with reduced infection of EGFR-negative cells and efficient killing of EGFR-positive cancer cells including primary EGFR-positive osteosarcoma cells that are refractory to infection by conventional adenoviruses. Our method to modify adenovirus tropism might thus be useful to design new oncolytic adenoviruses for more effective treatment of cancer.     

Beta-Glucuronidase Response of Cells Infected with Adenovirus Types 5 and 12

Beta-glucuronidase activity was investigated during a 48-h period in which virus replication and changes in cell morphology occurred. Infection of an established line of chimpanzee liver cells with either nononcogenic adenovirus 5 or highly oncogenic adenovirus 12 under one-step growth conditions produced differing patterns of enzyme activity. There was an increase in total activity and also enhanced leakage of beta-glucuronidase from cells infected with adenovirus 12. In contrast, the enzymatic pattern of cells infected with adenovirus 5 was similar to that of uninfected cells. Hydrocortisone prevented the abnormal release of beta-glucuronidase from adenovirus 12-infected cells. The compound had no effect on total enzyme activity or on virus replication and the development of cytopathology.     

Programming of events in Epstein-Barr virus-activated cells induced by 5-iododeoxyuridine

Programming of the Epstein-Barr (EB) virus productive cycle was studied in synchronized nonproducer Raji cells and producer EB-3 cells after activation by 5-iododeoxy uridine (IdU) added for 60 min during the S-1 period of the cell cycle. The activated Raji cells synthesized only the virus-associated early antigen (EA) complex, while some activated EB-3 cells also synthesized virus structural antigens (VCA).Synthesis of EA occurred at a time which corresponded to the early G-2 period of the cell cycle. DNA synthesis after removal of the IdU was not required for subsequent synthesis of EA. The time of EA synthesis, including associated RNA and protein requirements, was the same in cells allowed to complete the S phase as in cells where DNA synthesis was inhibited by 1-B-d-arabino furanosylcytosine (ara-C) or hydroxyurea (HU) added at the time of removal of the IdU. The findings indicated that synthesis of EA was independent of the cells' S phase and suggested a possible association between EA synthesis and synthesis of cell G-2 proteins.Synthesis of EA in abortively infected Raji cells was followed by low levels of deoxycytidine (dCyt) incorporation which persisted for several hours. At least some EA-positive cells were also able to reach mitosis, indicating that synthesis of EA did not result in immediate cell death.Synthesis of VCA occurred 3–4 hr after synthesis of EA in approximately 50% of the activated EB-3 cells. Synthesis of VCA required a short period of DNA synthesis (approximately 60 min) after EA synthesis. The inability of approximately 50% of the activated EB-3 cells to synthesize VCA was ascribed to toxic effects of the incorporated IdU which precluded the cells from carrying out events required for initiation of the short period of DNA synthesis subsequent to completion of the S phase and EA synthesis. Those EB-3 cells which did progress to VCA synthesis showed productive replication of virus DNA which continued for several hours after the initial appearance of VCA.     

Nucleic acid synthesis during the focus formation by Shope fibroma virus on green monkey kidney cells

Summary Nucleic acid synthesis during the focus formation on Shope fibroma virus (SFV)-infected cells was studied. When African green monkey kidney (AGMK) cells were infected with SFV, the cell-focus was observed as a local piling up of cells at 3 to 5 days after infection. The number of foci increased in proportion to the size of the SFV inoculum. In the SFV-AGMK cell system, however, the growth rate of virus was comparatively low and no apparent cytopathic effect was demonstrated as a result of virus infection. The incorporation of³H-thymidine into the nuclear fraction of infected cells was suppressed early in the cycle of virus replication and before focus formation. On the other hand, the rate of DNA synthesis increased markedly in the infected cytoplasm. Cytoplasmic RNA synthesis also tended to increase gradually accompanying the induction of the viral DNA synthesis. In addition, the sedimentation properties of the newly synthesized DNA were investigated by sucrose density gradient centrifugation.     

T antigen synthesis and resistance to interferon in human adenovirus type 12 infected chick cells.

Tye specific T antigen formation has been demonstrated in primary and secondary chick embryo cells (CEC) infected with adenovirus type 12. The frequency of cells synthesizing T antigen was closely dependent on the multiplicity of infection (MOI). At a MOI of 2.85 TCD50 per cell, T antigen was formed in 50% of cells. Cycloheximide inhibited T antigen formation while cytosine arabinoside had no such effect. CEC infected with adenovirus 12 produced interferon and T antigen, both appearing early and at about the same time of infection Exogenous chick interferon had no inhibitory effect on the formation of T antigen. In adenovirus 12-infected CEC, virus specific structural antigen could not be detected.     

Evaluation of cell line 293 for virus isolation in routine viral diagnosis.

Cell line 293, a continuous line of transformed human embryonic kidney cells, has been recognized for its sensitivity in the isolation of adenoviruses, particularly the fastidious species 40 and 41, from stool specimens. To explore the possibility of using this cell line for the isolation of other viruses from clinical specimens, 293 cells were tested for their susceptibility to a variety of viruses including herpes simplex virus, parainfluenza viruses, respiratory syncytial virus, and the enteroviruses ECHO 11, coxsackie B5, and coxsackie B6. All of the viruses induced a cytopathic effect in 293 cells. Consequently, 293 cells were introduced into the diagnostic laboratory and used along with primary African green monkey kidney (AGMK) cell cultures for the inoculation of all respiratory and stool specimens. The study represents a retrospective analysis of the performance of 293 cells over a 22-month period. It was confirmed that 293 cells were more sensitive than AGMK cells for the isolation of adenoviruses from both respiratory and stool specimens. The 293 cells were also sensitive for the isolation of enteroviruses (untyped) but more so from stool specimens than from respiratory specimens. Parainfluenza virus and respiratory syncytial virus were only rarely isolated in 293 cells. Herpesvirus isolates were obtained with equal frequency in both 293 and AGMK cells. This retrospective analysis confirms the value of 293 cells for the isolation of adenoviruses and demonstrates that 293 cells are also useful for the isolation of certain enteroviruses from both respiratory and stool specimens.     

Effect of alkaline maintenance medium upon the growth of rabies virus in chick embryo cells

HEP Flury strain of rabies virus was propagated in chick embryo cells under maintenance media of different pH. It was found that viral growth was better and reached a markedly higher maximum titer when the initial pH of maintenance medium was 8.2 to 9.0 than when it was 7.4. The enhancement of viral growth was not ascribable to mere neutralization of acids produced from infected cells, because the different media became almost equally neutral within an early phase of growth curve. Serial passage of the virus in chick embryo cells using pH 8.2 maintenance medium resulted in altered growth characteristics of the progeny virus; first, the virus so passaged could now grow equally well under alkaline and neutral maintenance media, and, secondly, autointerference observable with the parent virus eventually lowered virus yield when neutral maintenance medium was used, but this effect of undiluted passage was eliminated by the use of pH 8.2 maintenance medium.     

Early cytoplasmic vacuolization of African green monkey kidney cells by SV40

Summary As early as 3–4 hours after infection with SV 40 at a high input multiplicity, African green monkey(Cercopithecus aethiops) kidney (AGMK) cells developed cytoplasmic vacuolization. At 10–20 hours after infection, the vacuolization reached its maximal level, then disappeared and SV 40 specific cytopathic change followed. This vacuolization developed before the synthesis of the specific T and V antigens. This early cytoplasmic vacuolization (ECV) was prevented by preincubating the virus with specific antiserum, or by heating the virus with MgCl₂. The ECV could be induced by UV-irradiated SV 40. Addition of metabolic inhibitors had no effect on the induction of the ECV. These results suggest that the capacity to induce the ECV resides in a structural component(s) of SV 40 virion and the vacuolization is not associated with the replication of SV 40.With 3 Figures     

Biological and physical properties of a virus (strain 127) associated with the egg drop syndrome 1976

The biological and physical properties of strain 127 virus, a haemagglutinating virus associated with the egg drop syndrome 1976, are described. Haematoxylin and eosin and immunofluorescent studies demonstrated that virus multiplication took place in the nucleus of cells with production of typical adenovirus inclusions. Thin section electron microscopy showed typical adenovirus particles and associated inclusions accumulating in nuclei. Strain 127 infectivity was stable in monovalent but not divalent cations, and stable to ether treatment and extremes of pH. IDU inhibition indicated presence of DNA. Growth of 127 as evidenced by HA production was better in duck kidney, fibroblast and liver cells, than in fowl cell cultures, while growth in turkey cells was limited to kidney and liver cultures. There was no evidence of growth in a range of mammalian cells. Strain 127 agglutinated erythrocytes from avian but not mammalian species and the haemagglutinin was stable to heating and freezing. There was no crossing in neutralisation or haemagglutination-inhibition tests between 127 and 11 fowl adenoviruses, and no cross-immunofluorescence between 127 and FAV-1. It is concluded that 127 is an avian adenovirus, possibly originating from ducks.