Hepatitis C virus genotypes in Estonia.

Distribution of hepatitis C virus (HCV) geno(sub)types among 215 Estonian patients hospitalized with acute or chronic hepatitis and with HCV RNA-positive sera was investigated. For genotyping, both multiplex PCR with subtype-specific primers of the core region and RFLP analysis of cDNA of the 5' NCR region were used. These two methods permitted a correct characterization of genotypes, a more truthful characterization of mixed infections, and combined use of single-tube performances. They revealed, respectively, 200 and 202 (93.0% and 93.9%) HCV-positive samples of sera, subtype 1a- 0.9% and 0.9%, 1b- 56.3% and 64.2%, 3a- 13.9% and 22.3%, 2a- 6.5% and 5.6%, type 4 0.5% and 0%, mixed infections- 13.5% and 0%, and unidentified- 1.4% and 0.9%. In the majority of cases (84.7%) both methods gave completely or partially concordant results; in mixed infections, as determined by subtype-specific PCR, only one subtype was revealed by the RFLP method. In the remaining 15.3% of the cases (Ohno- 7.0%, RFLP- 8.3%) only one of the methods was positive. The epidemiological analysis of the dynamics of the subtypes' relative participation may indicate increasing 3a and decreasing 1b subtype infection during recent years.

[Effect of interferon on the cAMP system in cells]. / Vliianie interferona na sistemu cAMP v kletkakh.

The increase in cAMP concentration in CaOv cells affected by alpha-interferon has been found to have a two wave character with the maximums at 4 and 24 h after the effect. The waves are due to the increase in adenylate cyclase activity and to the decrease in the activity of cAMP phosphodiesterase. The described changes were characteristic of the native and partially purified interferon and depended on the concentration of interferon used (optimal effect at 1200 IU/ml-1). It suffices to notice that the described effects were more largely expressed when the preparations of the native alpha-interferon were used. The correlation was noticed between the increase in adenylate cyclase activity, the decrease in cAMP phosphodiesterase and the concentration of the cyclic nucleotide as well as the expression of antiproliferative effect. The correlation was less significant for antiviral effect.

[Effect of inhibitors that destroy cytoskeleton structures on the antiviral and antiproliferative activity of interferons]. / Vliianie ingibitorov, razrushaiushchikh struktury tsitoskeleta, na antivirusnuiu i antiproliferativnuiu aktivnost' interferonov.

The effect of a number of drugs (vincristine, vinblastine, cytochalasin B, colcemid, colchamine) destroying cytoskeleton structures (microtubules and microfilaments) in combination with homologous interferon was studied in L920 and CaOv cells. The above substances were shown to inhibit the antiviral and enhance (from additivity to synergism) the antiproliferative effect of interferons under study.

[Effect of human interferons on cAMP phosphodiesterase activity in a culture of human ovarian carcinoma cells (CaOv)]. / Vliianie chelovecheskikh interferonov na aktivnost' fosfodiésterazy cAMP v kul'ture kletok kartsinomy iaichnika cheloveka (CaOv).

Crude and purified human interferons of alpha type exerted 2 step inhibition of cAMP phosphodiesterase activity in CaOv cells: in 4 and 24 hours after cells treatment with interferon. The maximal inhibition was obtained in response to interferon doses 1200-2000 IU/ml. In contrast to natural interferons the human alpha 2 recombinant interferon (20-25000 IU/ml) did not inhibit the cAMP phosphodiesterase activity in CaOv cells.

[Antiviral and antiproliferative activity of human interferons and their induction of 2',5'-oligoadenylate synthetase]. / Antivirusnaia i antiproliferativnaia aktivnost' chelovecheskikh interferonov i induktsiia imi 2',5'-oligoadenilatsintetazy.

Native preparations of alpha, beta and gamma-interferons as well as recombinant beta-interferon and purified leukocyte alpha-interferon and purified leukocyte alpha-interferon exert antiviral and antiproliferative activity in CaOv cells. Native interferon preparations were shown to be more antiproliferative than purified interferons per unit of antiviral activity (with EMC as well as with less susceptible VSV used as test viruses). It was shown that level of 2'5' oligoadenylatesynthetase activity induction in general correlates with antiproliferative and pronounced antiviral activity of interferons, besides that, the earlier (by 11 hours) induction of the enzyme activity by beta-interferon correlates with more rapid expression of antiproliferative effects by this interferon in comparison with that of alpha-interferon, the latter inducing the peak of enzyme activity by 24 hours.

Antiviral activity of lambda-, beta-, and gamma-interferons in the presence of theophylline: involvement of 2',5'-oligo(A) synthetase.

Theophylline, an inhibitor of cAMP phosphodiesterase, induces in human ovary carcinoma cells (CaOv) a 2-2.5-fold elevation of intracellular cAMP. This rise in the cAMP level is followed by an increase of the activity of 2',5'-oligo(A) synthetase in CaOv cells -insignificant (1.5-fold) after 16 hr incubation, and substantial (3.7-fold) after 30 hr incubation, as well as the development of antiviral resistance. Once CaOv cells have been incubated with the mixtures containing theophylline (2 mM) and lambda-, beta-, and gamma-interferon preparations (0.5-13 IU/ml), the total antiviral effect of the mixtures exceeds that generated by interferon or theophylline separately; the action of the above agents being additive. These data agree with the previously obtained results and support the suggestion that cAMP phosphodiesterase inhibitors partially mimic the antiviral action of interferon.

[Effect of a number of antitumor preparations on RNA- and protein-synthesizing processes and viral interferon induction in Krebs 2 ascites carcinoma cells]. / Izuchenie vliianiia riada protivoopukholevykh preparatov na RNK- i beolksinteziruiushchie protsessy i virusnuiu induktsiiu interferona v kletkakh astsitnoi kartsinomy Krebs-II.

Antitumor drugs: adenosine cyclophosphane, adenosine thiophosphamide, colchamine thiophosphamide, and tribetamide inhibited RNA biosynthesis in Krebs-II ascitic carcinoma cells and in similar concentrations they were much less inhibiting for protein-synthesizing processes (in cells and cell-free protein-synthesizing system). The drugs could inhibit interferon synthesis, possibly after virus induction, in Krebs-II ascitic carcinoma cells only in concentrations significantly inhibiting the synthesis of cellular macromolecules.

[Sensitivity of a continuous cell culture of human ovarian carcinoma to interferon preparations of different origins]. / Izuchenie chuvstvitel'nosti perevivaemoi kul'tury kletok kartsinomy iaichnika cheloveka k preparatam inteferona razlichnogo proiskhozkdeniia.

A comparative study of homologous (human) and heterologous (porcine and bovine) leukocyte interferon preparations showed them to have antiviral and antiproliferative effects on CaOv tumor cells culture derived from human ovary carcinoma. These effects were lower in heterologous than in homologous interferon preparations.

[Effect of mouse interferons on the development of Ehrlich ascitic carcinoma]. / Vliianie myshinykh interferonov na razvitie astsitnoi kartsinomy Erlikha.

Intraperitoneal injection of various preparations of mouse interferons (L cell tissue culture interferons, concentrated or partly purified, and also serum interferon) significantly inhibited the development of Ehrlich's ascites carcinoma in randombred mice. In view of comparatively low activity of serum interferon, the effect of normal mouse serum on the tumour development and its action on L cell tissue culture interferon was investigated. It was shown that normal mouse serum inhibits the action of L cell tissue culture interferon and promotes the development of Ehrlich's ascites carcinoma.

[Initial stages of the interaction of murine interferon with L929 cells]. / Izuchenie nachal'nykh étapov vzaimodeistviia myshinogo interefona s kletkami L929.

After contact with mouse culture interferon in the cold, the resistance to viruses in L cells develops only after the cells are placed into a thermostate at 37 degrees C. The use of colcemide destroying microtubules prevents the development of this resistance at an early (actinomycin-sensitive) stage of interferon-cell interaction. In the actinomycin-resistant stage, however, colcemide practically exerts no influence on the interferon-induced resistance to viruses.

[Comparative study of the oncornavirus A and D proteins of human J-96 cells by the methods of isoelectric focusing and polyacrylamide gel electrophoresis]. / Sravnitel'noe izuchenie belkov onkornavirusov A i D v perevivaemykh kletok cheloveka J-96 metodami izoélektricheskogo fokusirovaniia i élektroforeza v poliakrilamidnom gele.

Three peaks of 14C-radioactivity with buoyant densities of 1.23--1.24, 1.26 and 1.29 g/ml were detected in a cytoplasmic extract of J-96 cells upon equilibrium centrifugation in sucrose gradient. Electron microscopy of the 1.23--1.24 g/ml buoyant density fraction revealed particles 60--80 nm in diameter showing morphology characteristic of oncornavirus A. Isoelectric focusing in polyacrylamide gel showed polypeptides of extracellular D virus and oncornavirus A to differ in isofocusing points (pI). Proteins of extracellular D virus were localized in zones with pH 3.7, 4.0, 4.4, 4.7, 5.6, 6.5, 8.1, 9.45, and 10.0; polypeptide of intracytoplasmic oncornavirus A had the following isofocusing points: 4.0, 4.9, 6.7, 7.3, 9.0, 9.45 and over 10.0. Electrophoresis of polypeptides of D virus and intracellular oncornavirus A revealed differences in the molecular weights of the components. No proteins with molecular weights of 10,000, 12,000, 15,000, and 27,000 dalton characteristic of the extracellular D virus were found in oncornavirus A virions. The analysis of protein patterns obtained in parallel experiments of isoelectric focusing and polyacrylamide gel electrophoresis suggests that oncornaviruses A and D of J-96 cells differ in the characteristics (pI and molecular weight) of the structural polypeptide components.

Mechanism of resistance to enteroviruses of some primate cells in tissue culture.

Two cell lines, M10-45-2 and L-41, were studied, each of which possessed specific resistance either to poliovirus or to coxsackievirus. Infection of M10-45-2 cells with poliovirus ribonucleic acid (RNA) and L-41 cells with infectious coxsackievirus RNA was accompanied by production of complete viruses in each of the resistant cell lines. During incubation of the cells with the virus to which they were resistant, the amount of infectious virus did not decrease. Treatment with glycine-HCl buffer solution (pH 2.5) of resistant M10-45-2 cells after incubation with poliovirus at 0 C did not result in recovery of infectious virus, although such release did take place after treatment of sensitive M10 cells. Infection of resistant cells with virus containing poliovirus RNA and coxsackievirus proteins resulted in production of poliovirus in M10-45-2 cells but not in L-41 cells. The resistant cells are apparently unable to adsorb the virus to which they are resistant.