Kaprelian, G.

Rusev, V.

Tzekova, F.

English

Varna Medical University Press: Journals

ATTEMPTS FOR CULTIVATION OF SOME DNA- AND RNA-VIRUSES ON C E L L C U L T U R E FROM HUMAN THYROID GLAND G. Kaprelian, F . Tzekova, V. Rusev The modern progress and successes of virology are almost entirely due to the application of s t i l l more various but easy for the experimental practice methods of cul t ivat ion of some D N A - and RNA-viruses . Many authors work over the invention of newer methods of reproduction of the viruses which cause various infectious diseases. ( 1 , 2, 5, 6) . Pulver ta l R . I . et a l . (1959) are the first to report cell culture from a thy j roid gland; later Betz A . et a l . (1963), Tzeipkin L . B . and S. A . Sorina (1965), Lev ina D . S. (1970) also represent their data concerning the same field. Our study has for an object to investigate some virological , cytological and immunofluorescent methods, also the reproduction of some D N A - and RNA-viruses on a model of a primary trypsinized cell culture from human thyroid gland. Material and methods The aforementioned cell culture was obtained by using material of hu­man thyroid gland adenoma removed during operation in the C l in i c of Surgery, Higher Institute of Medicine, Varna c i ty . The modified method of G . Kapre l ian et a l . (2, 3) was used for the trypsinization, cu l t iva t ion and contamination (infection) of the cell culture. The experiments were carried simultaneously wi th the contamination of monolayer trypsinized cell cultures Н Е К , as wel l as cell culture passages H e L a , F L , Har t , Hep 2 , Detr . 6 , etc. produced in the laboratories of Dr. Brad-varova (Insti tute of Infectious and Parasi t ic Diseases, Sofia). I n our experiments we used D N A - and RNA-viruses : Adenoviruses sero­types 1, 2, 3, 4, 5, 6, 7, 8 wi th titers^ 1 ( Г 2 — 1 ( Г 4 C P D 5 0 / 0 , 1 ml Vi rus vacc. variolae, wi th a preliminary titer 10 2 C P D 5 0 / 0 . 1 ml avid inhibitor-sensi­t ive Influenza strains A (Vn/535/72) and A (Vn/536/72) at 10 8 E I D 5 0 ; Parain-fluenza-virus type 1 ( P G i S ) wi th a titer 10 4 C P D 5 0 ; Coxsackie-virus type B 5 with a preliminary titer 10~ 2 C P D 5 0 / 0 . 1 m l ; A C H 0 6 and E C H 0 9 - v i r u s e s with titers 1СП2 C P D 5 0 / 0 . 1 ml and 1 ( Г 3 CPDJOA m l . The aforementioned viruses were stained wi th hemalaun-eosin as wel l as wi th the methods of Feulgen and Brachet for cytomorphological pur­poses. Lamellae wi th a monolayer of cells were used for fluorescent microscopic study. They were stained by using the standard direct method of immunofluo-rescency after the respective fixation and processing. Attempts for cultivation of some DNA- and RNA-viruses. 21 Result and discussion The results of the investigations show that the studied cell culture appears to be highly sensitive to a l l serotype adenoviruses in our experiments. A n ex­ception of these serotypes shows only type 2. The characteristic cytopathic effect ( C P E ) of this v i r a l group is available 12—24 hours after the contami­nation. Characteristic pil ing (aggregation) of the cells wi th a following total degeneration is established after 48—72 hours. The titer of the viruses on the thyroid gland cell culture is between 10 2 and 10 4 C P D 5 0 / 0 . 1 ml for a l l studied adenoviruses excluding serotype 2, which is not intensively reproduced on the cell culture. The control cell cultures H e L a and Н Е К show titers between 103 and 10 s C P D 5 0 / 0 . 1 ml at the 7 2 n d and 9 6 t h hour for a l l serotype adenoviruses. The reproduction on this model cell culture is established in dynamics and consecutively by using the method of immunofluorescence. Clearly ex­pressed fluorescence is present at the 6 t h hour after infection of the inve­stigated cell culture wi th a location around the nuclei. I t gradually increases with a maximum between the 2 4 t h and 7 2 n d hour when the infectious titer of the reproducing viruses is comparatively high. The vaccinating virus induces characteristic cytological changes on the experimental cell culture after the 12 t h , 2 4 t h , 4 8 t h hour of infection. The le­sions of the cell monolayer are represented as a local degeneration of the layer wi th rounding of the cells. After the 4 8 t h — 7 2 n d hour the degeneration totally affects the cell monolayer and there appear giant syncyt ia l cells. Meanwhile the vaccinating virus shows an infectious titer between 1СГ3 and 10~ 4 C P D 5 0 / 0 . 1 m l . The same titer but on the control cell cultures Н Е К , H e L a and H B B is between 10" 2 and 1 ( Г 3 CPD 5 < ( / 0 .1 m l . Green opalescent insertings against the orange background can be detec­ted 6 to 12 hours after the v i r a l reproduction. 24—48 hours later the lumi­nescence spreads over to the cell cytoplasm. The control cell cultures show s imilar changes but at later hours. The influenza-virus A / V n / 7 2 , as well as the parainfluenza-virus type 1 (Sendai) show a development and reproduction on the thyroid gland cell cul­ture. The contaminated wi th influenza-virus type A / V n / 7 2 cultures do not show any visible cytomorphological changes between the 4 8 t h and 9 6 t h hour. No changes are detected on the control cell cultures Н Е К . The infected wi th parainfluenza-virus type 1 (Sendai) cultures show some slight cytopathic changes on the first passages after the 7 2 n d hour: appearance of isolated giant cells, destruction of the monolayer, hypertrophy of the nuclei and vacuoli­zation of the cytoplasm. The infectious titer of influenza-virus type A / V n / 7 2 is between 1СГ 4 and 10~ 5 after 48—96 hours, while that of parainfuenza-virus type 1—between 1 ( T 3 and 1СГ 4. The results of our investigations show that the enteroviruses used in the experiments tend to an expressed reproduction on the suggested model of a cell culture. A characteristic cytopathic effect is observed 24 hours after the infection wi th E C H 0 6 and Coxsackie B 5 -viruses . Th i s includes rounding and wrinkl ing (curling) of the cells, while their nuclei show pycnotic changes. 72—96 hours later the degenerative changes spread over to the entire surface of the cell monolayer wi th a consecutive detachment. 22 G . Kaprelian, F . Tzekova, V. Rusev The infectious titer of A C H 0 6 and Coxsackie B 5 -viruses 72 hours after a reproduction on the experimental culture is between 10 3 and 1СГ 4 C P D 5 / 0 . 1 m l . The same titer on the control cell cultures Н Е К is 1СГ2 C P D 5 /0,1 m l . Characteristic opalescent agglomerations in the cytoplasm are detected by using immunofluorescent investigation of the v i r a l reproduction. 12—24 hours after the infection of the experimental ,cell cultures these opa­lescent agglomerations spread over to the entire cell cytoplasm. Conclusions 1) Adenovirus type 1, 3, 4, 5, 7, 8 can be cultivated on a model of cell culture of human thyroid gland. 2) Vi rus vacc. variolae can be cultivated and investigated on a model of cell culture of human thyroid gland. 3) Influenza-virus type A / V n / 7 2 and Parainfluenza-virus type 1 (Sen-dai) reproduce on experimental cell cultures with an expressed hemagglu-tinating, hemadsorbtive and infectious act ivi ty , but not clearly expresse d cytopathic changes. 4) The act ivi ty of glucose-6-phosphate-isomerase and aldolase is increased wi th the reproduction of the respiratory viruses. 5) Enteroviruses E C H 0 6 , A C H 0 9 and Coxsackie B 5 can be cultivated and investigated on a model of cell culture of human thyroid gland. 12—24 hours after the infection they cause characteristic cytological, cytochemical and fluorescent-microscopical changes. 6) A l l results received and reported in the present work probably have an important diagnostic and scientific-practical significance. R E F E R E N C E S 1. И p ж a H о в, С. Д . , Я- А. X е с и н. Вопр. вирусологии, 1972, 1, 30. — 2. К а-п р е л я н , Г., Ф л . Ц е к о в а , К р. Т у н ч е в а , Х р . Д и м и т р о в а . Ма­териали на I I I конгрес на микробиолозите в България , София, 1973, 19—22 ноември. — 3. К а п р е л я н , Г., Ф л . Ц е к о в а , К р. Т у н ч е в а . Материали на V научна сесия на МФ — Варна, 1973. — 4. Л е в и н а, Д . С., С. А. Д е м и д о в а, В. Н. Б о-л ь о м к и н, Э. И. С ч а с т н ы. Вопр. вирусологии, 1970, 4, 457. — 5. Л е н н е т а, Н . Ш м и д т . Лабораторная диагностика вирусных и рикетсиозных заболевании, Москва, 1974, 434—436. — 6. Ц ы п к и н, Л . Б . , С. А. С о р и н а. Труды инст. полиомиелита и вирусных энцефалитов АМН СССР, 6, М., 91965, 35. — 7. В е t z, A . , G i 1 g е n -k r a n z е t a 1. Ann. Inst. Pasteur, 1963, 105, 1047. — 8. P u l v e r t a l t , R . I . е t a l. Path. Bad., 1959, 77, 19. ПОПЫТКИ КУЛЬТИВАЦИИ НЕКОТОРЫХ ДНК И РНК-СОДЕРЖАЩИХ ВИРУСОВ НА КЛЕТОЧНОЙ КУЛЬТУРЕ ИЗ ЩИТОВИДНОЙ ЖЕЛЕЗЫ ЧЕЛОВЕКА Г. Капрелян, Фл. Цекова, В. Русев Р Е З Ю М Е При помощи вирусологических, цитологических и иммунофлюоресцентных ме­тодик, авторы изучают репродукцию некоторых Д Н К и Р Н К вирусов на модели из пер­вично трипсинизированной клеточной культуры из щитовидной железы человека. По­лученные результаты показывают, что использованные вирусы можно культивировать на клеточной культуре из щитовидной железы человека и имеют важное диагности­ческое и научно-практическое значение. 

ATTEMPTS FOR CULTIVATION OF SOME DNA- AND RNA-VIRUSES ON CELL CULTURE FROM HUMAN THYROID GLAND

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ATTEMPTS FOR CULTIVATION OF SOME DNA- AND RNA-VIRUSES ON CELL CULTURE FROM HUMAN THYROID GLAND

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