Final checkpoint in the drug-promoted and poliovirus-promoted apoptosis is under post-translational control by growth factors.

The treatment of HeLa subline (HeLa-B) cells with cycloheximide or Actinomycin D resulted in a rapid (approximately 1.5 h and approximately 2.5 h, respectively) development of morphological and biochemical signs of apoptosis. The addition of fetal bovine serum to the cycloheximide-treated or Actinomycin D-treated cells suppressed the apoptotic reaction, as evidenced by the postponement of the DNA fragmentation for at least 9 and 5 h, respectively. A similar suppressive effect was observed upon the serum addition to cells undergoing abortive infection with poliovirus, which died of apoptosis in the absence of the serum. The serum appeared to exert its anti-apoptotic effect without any appreciable lag and even immediately blocked further progress of ongoing DNA fragmentation. The epidermal growth factor also suppressed, although less efficiently and more transiently, the apoptotic reaction promoted by the metabolic inhibitors. It is concluded that growth factors may affect, without modulating either transcription or translation, the balance of pro-apoptotic and anti-apoptotic activities at a final checkpoint, just preceding the irreversible effector step of apoptosis.

[Strategy for choosing antibiotics for treating bacterial infections associated with chronic tick-borne encephalitis]. / Strategiia vybora antibiotikov dlia terapii bakterial'nykh infektsii, assotsiiruiushchikhsia s khronicheskim kleshchevym éntsefalitom.

The capacity of wide-spectrum antibiotics kefzol and ristomycin to activate the persisting tick-borne encephalitis (TBE) virus and cause an exacerbation of chronic process was investigated in Syrian hamsters in whom a prolonged (77 to 270 days) persistent TBE infection was induced by three TBE strains: Vasilchenko, V-383, and 205. The degree of antibiotic-induced activation was assessed using the criteria characterizing the reproduction and peculiarities of persisting TBE virus, immunodepression, and morphologic changes in the central nervous system. Effects of kefzol and ristomycin were compared with those of 8 antibiotics studied previously. Ristomycin, levomycetin (chloramphycin), penicillin, ampicillin (ampital), and levoridan were referred to drugs devoid of evident provoking effect. Kefzol (cefamezin), florimycin (viomycin), and kanamycin (kanamytrex) were characterized as weak activators and streptomycin and tetracycline as potent activators of the persisting TBE virus. These data may be used when selecting alternative agents for therapy of secondary bacterial infections concomitant with TBE.

Apoptosis-inducing and apoptosis-preventing functions of poliovirus.

Data showing that an apoptotic reaction (the exit into the cytoplasm and nucleolytic internucleosomal degradation of chromosomal DNA, compaction and fragmentation of chromatin, cellular shrinkage, and cytoplasmic blebbing) developed in a subline of HeLa-S3 cells upon nonpermissive poliovirus infection with either a guanidine-sensitive poliovirus in the presence of guanidine, a guanidine-dependent mutant in the absence of guanidine, or certain temperature-sensitive mutants at a restrictive temperature are presented. Essentially, no apoptotic reaction occurred upon permissive infection of these cells. Both permissive and nonpermissive infections resulted in the inhibition of host protein synthesis. Actinomycin D or cycloheximide also elicited a rapid apoptotic reaction in uninfected cells. However, preinfection or coinfection with poliovirus prevented the apoptotic response to the addition of actinomycin D, and preinfection blocked cycloheximide-induced apoptosis as well. These data fit a model in which the cells used are prepared to develop apoptosis, with their viability due to the presence of certain short-lived mRNA and protein species. Poliovirus infection turns on two oppositely directed sets of reactions. On the one hand, the balance is driven toward apoptosis, probably via the shutoff of host macromolecular synthesis. On the other hand, viral protein exhibits antiapoptotic activity, thereby preventing premature cell death. To our knowledge, this is the first description of an antiapoptotic function for an RNA virus.

Postinfection treatment with antiviral serum results in survival of neural cells productively infected with virulent poliovirus.

The death of human neuroblastoma cells undergoing productive infection with virulent poliovirus was prevented by addition of antiserum against the virus a few hours after the onset of infection; this treatment, however, did not prevent reproduction of the virus. Despite the presence of the viral antigen, the cells retained the ability to divide. Upon further cultivation in the absence of antiserum, the cells developed specific postinfection immunity or resistance to superinfection with poliovirus.

Restricted growth of attenuated poliovirus strains in cultured cells of a human neuroblastoma.

Cultured cells of a human neuroblastoma, SK-N-MC, were found to be highly resistant to Sabin attenuated poliovirus types 1 and 2 strains; no appreciable cytopathic effect was observed, and the total harvest was generally in the order of 1 PFU per cell or less. On the other hand, related neurovirulent strains of these antigenic types produced a relatively good (2 orders of magnitude higher) yield in a markedly protracted infectious cycle. The limited growth of the attenuated virus in the neuroblastoma cells appeared to be confined to a minor cell subpopulation. Experiments with intratypic (type 1) poliovirus recombinants suggested that the major genetic determinants limiting reproduction of the attenuated polioviruses in the neuroblastoma cells are located in the 5' half of the viral RNA, although the 3' half also appears to contribute somewhat to this phenotype. The possibility that neuroblastoma cells may represent an in vitro model for studying poliovirus neurovirulence is briefly discussed.

[Use of immunomorphologic methods in detecting antigens of the human immunodeficiency virus]. / Ispol'zovanie immunomorfologicheskikh metodov dlia obnaruzheniia antigenov virusa immunodefitsita cheloveka.

A comparative evaluation of the effectiveness of detection of human immunodeficiency virus (HIV) in persistently infected culture of T-lymphocytes by means of different morphological and immunomorphological methods: fluorescent antibody method (FAM), immune electron microscopy (IEM), negative staining, and ultrathin section technique, was carried out. The FAM was shown to detect virus antigen exclusively in the cytoplasm of large and multinuclear cells but not in the cytoplasm of small size cells. A marked cyclic pattern (3-passage interval) was established in expression of HIV antigen correlating with changes in the ratio of 3 types of cells in the culture and the intensity of HIV virions production detectable in electron microscopic preparations. The phenomenon of variability of HIV production associated with changes in the ratio of different cell types was used for selection of most productive cultures which were employed as the antigen for antibody determination in sera. The study showed good agreement in antibody detection by the FAM and immunoblotting method.

[Immunofluorescent study of the reproduction of human rotavirus in cell culture]. / Immunofliuorestsentnoe izuchenie reproduktsii rotavirusa cheloveka v kletochnoi kul'ture.

The mechanism of rotavirus antigen amplification in cell culture was studied by immunofluorescence. Reproduction of human rotavirus in cell culture was shown to begin 6 h after inoculation and to be accompanied by a regular increase in the number of antigen-containing cells. A good correlation with the results of electron microscopic studies was demonstrated. The possibility of reliable detection of rotavirus antigen in cell culture by the fluorescent antibody technique at relatively early intervals after infection has been established.

[Inhibiting effect of colcemid on the development of cytopathic changes due to the Sindbis virus]. / Ingibiruiushchee vliianie koltsemida na razvitie tsitopaticheskikh izmenenii, vyzvavaemykh virusom Sindbis.

The model of Chinese hamster (Cricetulus griseus) cell line infected with Sindbus virus was used to show that the presence in the maintenance medium of 0.01-0.1 micrograms/ml of colcemide inhibited the development of cytopathic changes, accumulation of viral antigens, and virus-specific complement-dependent cytolysis without affecting infectious virus production. The addition to the medium of 0.5% immune serum to Sindbis virus resulted not only in a similar inhibition but in a decrease of infectious virus titres. The latter effect was observed upon the addition of both colcemide and antiserum. Possible mechanisms of inhibition are discussed.

Chronic alphavirus infection of L cells.

Chronic infection of L cells with Sindbis virus, induced in the absence of antiviral antibody, was studied by virological methods in combination with light and electron microscopy. Both Sindbis virus and endogenous oncovirus antigens were revealed in the cells by immunofluorescence. Sindbis virus and interferon were detected in the culture fluid. Immunoelectron microscopy revealed aggregation of Sindbis virus particles by immune serum.

[Inhibitory effect of homologous antiviral antibodies on viral cell destruction]. / Ingibiruiushchee vliianie gomologichnykh protivovirusnykh antitel na virusnoe porazhenie kletok.

Experiments were performed on the model Chinese hamster cell culture -- Sindbis virus in which there develops an acute infection accompanied by active virus reproduction, cell rounding and detachment from the glass for 24--48 h. Addition in the cultural medium of a 0.5% immune serum to the Sindbis virus after its adsorption by the monolayer culture fully prevents the development of cytopathic changes and favours obtaining chronically infected cultures that produce the infection virus. The decreased production of intracellular and surface viral antigens in the infected cells seems likely to be accounted for by blocking the function of viral genome by homologous antibodies that join virus-induced antigens in the cellular plasmalemma.