Chronic infection of HeLa cells with Japanese encephalitis virus. General characteristics of the system.

Chronic infection of HeLa cells was induced by an attenuated variant of Japanese encephalitis (JE) virus (HeLa-K3 cell line). The chronic infection was characterized by alternating phases of degeneration and recovery of the cell monolayer. JE virus was regularly released into the medium of chronically infected cell cultures and virus-specific antigen was regularly demonstrated in the cytoplasm of 15--25% of cells. JE virus persisting in HeLa-K3 cells was sensitive to pancreatic ribonuclease and resistant to treatment with 4 M urea. HeLa-K3 cells did not undergo cytological or karyological transformation; they were susceptible to superinfection with heterologous viruses but resistant to reinfection with homologous virus.

[The cytogenetic study of established Syrian hamster cell lines. I. A comparative analysis of the karyotypes of the BHK-21 (C-13) sublines]. / Tsitogeneticheskoe issledovanie postoiannykh kletochnykh linii siriiskogo khomiachka. I. Sravnitel'nyi analiz kariotipov kletochnykh sublinii BHK-21 (C-13).

Karyotypes of eight BHK-21 (C-13) cell sublines, obtained from a variety of sources and available in the cell culture collection of our Institute, have been studied using G- and C-banding, and silver staining of chromosomes. These sublines with similar modal numbers of chromosomes (40-44) are found to vary essentially in composition of both normal chromosomes and markers. In spite of the fact that many markers are constant in several cell sublines, it was impossible to determine the marker chromosomes typical of BHK-21 (C-13) cell by analogy with those typical of HeLa cells. Our studies have shown that BHK-21 (C-13) cells exhibit nonrandom chromosomal alteration involving heterochromatic regions, particularly long arms of X- and Y-chromosomes and centromeric regions of 1, 3, 6 and X-chromosomes. Deletions of Xq, Yq and heterochromatic short arms of autosomes have also been observed. The markers more commonly consist of the material of chromosomes 1, 2, 3, 4, 5, 8, 9, 16, 17, 18, 19, X and Y.

[The cytogenetic study of established Syrian hamster cell lines. II. A comparative analysis of the karyotypes of cell lines HaK, CER and BHK-21 (C-13)]. / Tsitogeneticheskoe issledovanie postoiannykh kletochnykh linii siriiskogo khomiachka. II. Sravnitel'nyi analiz kariotipov kletochnykh linii HaK, CER i BHK-21 (C-13).

A comparative cytogenetic investigation of the Syrian hamster HaK, CER cell lines and BHK-21 (C-13) sublines was undertaken using G- and C-banding, and silver staining of chromosomes. BHK-21 (C-13) cell sublines were found to have diploid associated chromosome sets, whereas HaK and CER cell lines are characterized with triploid and tetraploid associated chromosome sets, resp. In sum, in all the analysed cell lines 100 marker chromosomes were identified, with 85 ones having unique morphology. The finding indicates a nonaccidental appearance of lesions for normal chromosomes in forming markers. Another arrangement of "hot break-points" on chromosomes is more typical of HaK and CER cell lines, than of BHK-21 (C-13) sublines. The localization of "hot break-points" on chromosomes in BHK-21 (C-13) sublines is specific of the Syrian hamster cells transformed with oncogenic viruses.

[The cytogenetic study of established Syrian hamster cell lines. III. An analysis of the NOR-marker chromosomes in Syrian hamster cell cultures by using differential chromosome Ag-->G restaining]. / Tsitogeneticheskoe issledovanie postoiannykh kletochnykh linii siriiskogo khomiachka. III. Analiz iadryshkoobrazuiushchikh markernykh khromosom kul'tur kletok siriiskogo khomiachka s pomoshch'iu differentsial'nogo Ag-->G-pereokrashivaniia khromosom.

Using silver staining --> G-banding restaining procedure it was found that the Syrian hamster chromosomes had nucleolar organizers on subtelocentric chromosomes 2, 6, 9, 10 and 13, and on acrocentric chromosomes 16, 17 and 19. In this case, NORs may be recognized only on one of homologous chromosomes, and no more than 9 NO-chromosomes may be found in one metaphase plate. In HaK, BHK-21 (C-13) BKK and BHK-21 (C-13) C cell lines, chromosomes 6, 9, 16, 17 and 19 have NORs, and in each line there are specific marker chromosomes with NOR. The markers consist of the material of chromosomes 6, 9, 16 and 17, with only chromosomes 16 and 17 being incorporated into rearrangements by their NORs. The silver staining --> G-banding restaining procedure makes possible not only identification of NO-chromosomes, but also refinement of the marker origins.

[A karyotype study of the cells of the African green monkey kidney cell line 4647 cultured long term in media with different sera]. / Issledovanie kariotipa kletok pochki afrikanskoi zelenoi martyshki linii 4647, dlitel'no kul'tiviruemykh v sredakh s razlichnymi syvorotkami.

The karyological analysis of the cell line 4647 used for production of a killed vaccine to Hepatitis A virus was run in the 98th, 107th, 117th and 127th passages by the routine and C, G, and Ag methods of differential chromosome staining. A considerable balancing of the chromosome composition at 107-127 passage levels is shown. The cells of line 4647 present a significant heterogeneity, as to the number of chromosomes, and do not belong to any distinct modal class. The modal number of chromosomes ranged from 61 to 66 and from 121 to 125 for hyperdiploid and polyploid cells, respectively. The stable modal class of cells was established in the tetrasomic region, when culturing in the medium containing 10% CS from the 107th passage, and in the medium containing 10% FBS from the 117th passage, which conforms to one of the WHO requirements asserted to the substrate cells.

[Karyologic study of continuous cell lines. IV. Study of human cell lines using the C-method of staining chromosomes]. / Kariologicheskoe issledovanie perevivaemykh kletochnykh linii. IV. Izuchenie linii kletok cheloveka s pomoshch'iu C-metoda okraski khromosom.

With the aid of the C-method of chromosome staining marker chromosomes three classes of human continuous cell lines were studied: 1) HeLa and HeLa-like cell lines (HEp-2, U, KB); 2) non-HeLa cell lines, with type B mobility of glucose-6-phosphate-dehydrogenase (HOS, A-549, A-204); 3) lymphoblastoid cell lines (Raji, Namalva, L-101). Two C-marker chromosomes were observed in two investigated cell lines A-204 and KB, one C-marker chromosome was observed in HEp-2, HeLa, U, A-549, Namalva cell lines; C-markers were absent in HOS and L-101 cell lines. Y-chromosome was found in Raji, A-549 and L-101 cell lines. The C-method of chromosome staining is a simple method, promoting an intraspecific identification of human cell lines.

[A karyotype study of the Vero cell line cultured long term in a monolayer by static and roller methods]. / Issledovanie kariotipa kletok linii Vero, dlitel'no kul'tiviruemykh v monosloe staticheskim i rollernym sposobami.

A cytogenetic investigation of Vero cells, before and after adaptation to the medium containing a cattle serum, was carried out by methods of differential chromosome staining. Under these conditions, both the modal number of chromosomes (from 58 to 55) and the karyotype structure, namely the copy number of normal chromosomes and the marker composition were shown to change. The Vero cell karyotype stability was studied in the continued culture by the static (50 passages) and roll-bottle (37 passages) methods. The quantitative changes (the rising percentage of diploid cells, and the change of cell fraction involving the modal number of chromosomes) were shown to occur in spite of the chromosome composition stability, which limits the time of using Vero cells as a substrate for preparation of vaccines.

[Elaboration of live measles vaccine technology on human embryo lung diploid cell culture L-68]. / Razrabotka tekhnologii proizvodstva zhivoi korevoi vaktsiny na osnove kul'tury kletok émbriona cheloveka L-68.

Measles predominates among childhood droplet infections in many countries. Immunization of all human beings sensitive to this infection is the only radical measure in controlling measles. The quality of a vaccine is primarily determined by the properties of the virus strains and cell cultures and technology of production. Now live measles vaccine is produced in or country on the basis of fibroblasts from Japanese quail embryo. The production of live measles vaccine in the primary cell cultures has a number of drawbacks caused by the nonstandard pattern of the substrate and the probability of contamination. The use the certified human diploid cells deposited in liquid nitrogen in sufficient quantities is promising. The authors have elaborated a new technology of live measles vaccine production by using the Leningrad-16 virus strain on the basis of attested L-68 diploid cell culture from the human fetal lung. Experimental batches of vaccine were obtained and attested in accordance with the present requirements for immunobiological products.

[Morphological and karyologic analysis of primary and continuous lepidopteran cells]. / Morfologicheskii i kariologicheskii analiz pervichnykh i perevivaemykh kletok cheshuekrylykh nasekomykh.

Morphological basis of obtaining primary and continuous cell cultures from different tissues and organs of Lepidoptera has been developed. Peculiarities of transfer of primary cell cultures from these insects to continuous ones have been detected. Two new cell lines from Heliothis armigera pupa ovaries and from Agrothis segetum embryos were derived.

[A new cell line from Heliothis armigera (Hubn.) pupa ovaries]. / Novaia liniia kletok iaichnikov kukolok khlopkovoi sovki Heliothis armigera (Hubn.)

The new continuous cells line derived from insect cells is perspective for the production of nuclear polyhedrosis viruses. The culture was prepared by fragmentation and trypsin treatment of the ovaries of Heliothis armigera (Hubn.) pupa with subsequent inoculation of viable cells in nutrient medium. By the present time the culture has undergone more than 100 passages in vitro and is characterized by all parameters required in a present-day passport.

[Preparation of primary cultures of lepidopteran cells]. / Poluchenie pervichnykh kul'tur kletok cheshuekrylykh nasekomykh.

Presents data of more than 200 experiments with preparation of primary tissue cultures from various tissues and organs of different Lepidoptera species. Describes in detail the peculiarities of obtaining sterile cellular material from various tissues and organs, methods of tissue and organ dissociation, analyzes the behavior of the cells in culture, and discusses changes in their morphology. Offers the relevant methodologic recommendations.

[Several methodologic problems in the control of cell cultures]. / Nekotorye metodicheskie voprosy kontrolia kletochnykh kul'tur

Some human and animal continuous cell lines as well as primary cell cultures were examined by karyological, electron microscopial, virological and molecular biological methods and also by the electrophoretic motility of glucose-6-phosphate dehydrogenase (G-6-PDG) in polyacrylamide gel. All human and animal continuous cell lines were shown to contain mycoplasma, 17-to contain intracytoplasmic particles of type A oncornaviruses, 5 -- type B oncornaviruses similar to Mason-Pfizer virus, 8 -- paramyxoviruses, 2 --oncornaviruses type C. A high molecular RNA with sedimentation constant 64--70 S was found in oncornaviruses isolated from cell cultures. Intracellular virus or subviral structures were detected by association of the reverse transcriptase activity with high molecular RNA. The presence in the cell cultures of marker chromosomes of HeLa cells, the absence in these cultures of Y chromosomes, the presence of the G-6-PDG enzyme with type A motility indicate the possibility of contamination of human continuous cell lines with HeLa cells.

[Isoenzyme characteristics of glucose-6-phosphage dehydrogenase and lactate dehydrogenase of continuous cells from the collection of the Sverdlovsk Research Institute of Virus Infections]. / Izofermentnaia kharakteristika gliukozo-6-fosfatdegidrogenazy i laktatdegidrogenazy perevivaemykh kletok kollektsii Sverdlovskogo nauchno-issledovatel'skogo instituta virusnykh infektsii

An analysis of isoenzymes of glucose-6-phosphage dehydrogenase (G-6-P-DH) and lactage dehydrogenase (LDH) of continuous cells from the collection of Sverdlovsk Research Institute of Virus Infections was carried out. Seventeen continous human and animal cell lines were examined by electrophoresis in a vertical block of 7% polyacrylamide gel followed by histochemical detection of the enzymes. The HEp-2, HEp-2 clone No. 23, KB, RH, and FL cells were shown to have the electrophoretic motility of G-6-PDG characteristic of HeLa cell line. LEP and SOC cell lines were contaminated with mouse cells. A culture of rat fibroblasts had isoenzymes of G-6-PDG and LDG characteristic of HeLa cell line. The remaining cell lines had the isoenzymatic characteristics corresponding to their species appurtenance.

[Detection of oncornavirus type D in continuous breast cancer cells]. / Ubnaruzhenie onkornavirusa tipa D v perevivaemykh kletkakh raka grudnoi zhelezy.

An oncornavirus immunologically similar to oncornaviruses type D previously isolated from human continuous cells was detected in continuous cells of mammary cancer (SH3). The culture produces structures having densities of 1.18--1.19 and 1.22 g/ml which contain high molecular RNA (68S) and reverse-transcriptase activity. The similarity of this virus with other oncornaviruses was also demonstrated in molecular hybridization experiments.

[Oncornavirus D associated antigen in cultured human and animal cells]. / Assotsiirovannyi s onkornavirusom D antigen v kul'tiviruemykh kletkakh cheloveka i zhivotnykh.

A large number of long-term continuous (LTC) and primarily trypsinized (PT) human and animal cell cultures were examined for the presence of a new oncornavirus D-associated antigen. All the PT cultures were found to be free from this antigen as also human diploid cells and LTC cells of man, rabbit, mouse, dog, swine and bat. LTC cells of monkey, bull, and Syrian hamster, however, were found to contain oncornavirus D-associated antigen identical to that previously found in HeLa, HEp-2 and J-96 cells. The detection of oncornavirus D-associated antigen in cells may indicate their infection with this virus.

[Alphavirus reproduction in cultures of transplantable human lymphoblastic cells in acute infection]. / Reproduktsiia al'favirusov v kul'turakh perevivaemykh limfoblastoidnykh kletok cheloveka pri ostroi infektsii.

The results of investigations of acute infection of continuous human B- and T-cells with typical members of the alphavirus group--Semliki Forest, Sindbis, and Venezuelan equine encephalomyelitis viruses, are presented. Virus amplification was shown to pass through the typical phases: eclipse, logarithmic growth, plateau. Infectious virus production per one cell was from 10 to 10,000 PFU in various cultures. Cell infection results in interferon production. Replication of the viruses under study in lymphoblastoid cell cultures is not accompanied by the active cytocidal effect. The regularities determining the sensitivity of lymphoblastoid cells to viruses in general and alphaviruses in particular are discussed. Proceeding from the results of the study of alphavirus replication in human continuous B- and T-cells it is suggested that this system be used as a model for the analysis of antiviral activity of interferon, its inducers, and chemopreparations in special cells. Lymphoblastoid and fibroblast interferon are as active in B-cells and show no antiviral activity in continuous T-cells, as interferon inducer.

[Alphavirus reproduction in cultures of transplantable human lymphoblastoid cells during persistent infections]. / Reproduktsiia al'favirusov v kul'turakh perevivaemykh limfoblastoidnykh kletok cheloveka pri persistiruiushchei infektsii.

The results of virological investigations of alphavirus persistence in human B-cell lines Raji and L-101 are presented. The formation of persisting infection was shown to depend both on the cell line and on the virus type. Productive persistent infection of Raji cells with Venezuelan equine encephalitis virus was followed for 11 months. The presence of the virus was confirmed by electron microscopic and immunofluorescent examinations. Infectious virus production varied from 0.001 to dozens PFU/cell, and the content of viable cells from 100,000 to 300,000 in 1 ml of the culture fluid. Virus infectivity in the culture medium varied within the range of 5-7 lg PFU/ml. Human lymphoblastoid cells Raji persistently infected with Venezuelan equine encephalitis virus were examined cytologically, karyologically, and electron microscopically. The long-term presence of the virus resulted in profound alterations in the cell population. Morphology of the cells and processes of division were changed, the mitotic index decreased, the rate of 3H-thymidine incorporation into cellular DNA increased. The mechanisms of persistence are discussed.

[Contamination of continuous cell lines]. / Kontaminatsiia kletok perevivaemykh linii.

During 1976--1979 approximately 150 lines and variants of human cells (derivatives of HeLa cells, tumor lines not contaminated with HeLa cells, lymphoblastoid lines) and cells from 16 animal species were examined. The karyotype analysis, determination of mobility of isoenzymes and immunological method of mixed hemadsorption were used for cell identification. Among the lines examined 73 lines were selected which comprised the cell culture collection of the Laboratory of tissue cultures of the D. I. Ivanovskiy Institute of Virology of the USSR AMS. The results of the detection of cell contamination are presented: about 20% of the cultures were found to be contaminated with cells of other origin.

[Morphological, cytogenetic and proliferative characteristics of Syrian hamster HTC-2 and HTC-1 cells and of the HTCT tumor cell line transformed by herpes simplex type 2 virus]. / Morfologicheskie, tsitogeneticheskie i proliferativnye osobennosti transformirovannykh virusom prostogo gerpesa tipa 2 kletok siriiskogo khomiachka KhTK-2, KhTK-1 i linii opukholevykh kletok KhTKO.

Morphological, caryological, cytoproliferative, and isoenzyme studies of Syrian hamster cells HTC-2 and HTC-1 transformed by herpes simplex virus type 2 and a HTCT line of tumor cells were carried out. The hamster origin of these cell lines was established by chromosomal analysis and electrophoretic mobility of lactate dehydrogenase and glucose-6-phosphate-dehydrogenase isoenzymes. The transformed and tumor cell lines differ from normal cells by altered morphology, an aneuploid chromosome set and increased proliferative activity. Electron microscopic examination of transformed cells revealed increased amounts of filamentous and tubular structures in the cytoplasm. No retroviruses were found.

[Cytomegalovirus isolation in chronic salivary gland diseases of adults]. / Vydelenie tsitomegalovirusa pri khronicheskikh zabolevaniiakh u vzroslykh.

The results of virological investigation of a rare clinical instance of familial disease with a localized form of cytomegalovirus infection which, as a rule, is not diagnosed in life are presented. The infection localized in the salivary glands was the source of generalization of the process with a high fever, eruptions, lymphadenopathy, inflammatory changes in the excretory ducts of the salivary glands. Exacerbation of the chronic process was accompanied with high antibody titres (1:256--1:1024) with inhibition of the cellular immunity reactions in the mother. The latent form of infection in the father and two daughters ran without clinical manifestations, with low antibody titres and moderate inhibition of cellular reactions. The isolated strains of cytomegalovirus were identical in all the subjects under study and antigenically close to the Ad-169 strain.