[PCR system for the detection of the RNA of hepatitis A virus]. / Razrabotka i ispytanie PTSR test-sistemy dlia detektsii RNK virusa gepatita A.

The detection of the causative agent of hepatitis A in the patient's body is the necessary element of the diagnostics of this disease. In this work the PCR system for the analysis of the RNA of hepatitis A virus in the patient's blood is presented and characterized. The method of the detection of the RNA of hepatitis A virus is based on the "nest" principle and consists of two consecutive reactions. In the first reaction the reverse transcription and amplification with the external pair of primers are carried out. The product thus obtained is used as material for the second reaction with the internal pair of primers. This method was used for the study of 44 blood samples from hepatitis A patients and 23 blood samples from healthy donors. The detection rate of the RNA of hepatitis A virus in blood samples from the patients was 82%. Viral RNA could be detected in the serum in 72% of cases, both in the serum and in mononuclear blood cells in 20% of cases, in mononuclear blood cells only in 8% of cases.

[Study of the effectiveness of the method of the nest polymerase chain reaction and the field of its use in hepatitis A diagnosis]. / Issledovanie éffektivnosti i oblasti primeneniia metoda gnezdovoi PTSR pri diagnostike gepatita A.

To determine the causative agent of hepatitis A in the blood of patients, we developed the method of the nest polymerase chain reaction (PCR) with reverse transcription. For the comparative evaluation of the diagnostic efficacy of the nest PCR and the enzyme immunoassay (EIA) serum samples and mononuclear blood cells obtained from 15 patients with diagnosed hepatitis A and from 33 patients having signs and carrying markers of other form of hepatitis were analyzed. On the whole, the method of PCR confirmed the results of EIA and in some cases exceeded it in sensitivity. In addition, hepatitis A virus RNA was detected in the blood of a clinically healthy person having had a contact with a hepatitis A patient. The PCR data were shown to correlate with the activity of liver amino transferases. The results obtained in this study indicate that the field of the use of PCR in the analysis of hepatitis A virus may include cases of mixed infection and virus carriership.

[Virus isolated in the human blood leukocyte culture and its interaction with hepatitis C and G viruses]. / Virus, vydelennyi na kul'ture leikotsitov krovi cheloveka, i ego vzaimootnoshenie s virusami gepatitov C i G.

Electrone-microscopic investigations are indicative of that the cultures of healthy donors, stimulated by phytohemagglutinin (PHA), can be successfully used to study the etiology of parenterally transmitted hepatitis. An electronic-microscopic study of a virus, isolated from the blood serum of a patient with hepatitis on the basis of the PHA-stimulated human leukocyte cultures and named a hepatitis leukocytic virus (HLV), enabled, by using the negative contrasting method, to detect viral particles of the hexagonal shape, sized 50-65 nm, with a coating divided by a 4-5 nm light space. Therefore, the HLV was described as belonging to the Flaviviridae family. RNA of the C hepatitis virus was detected in the K HLV strain stored, for 24 years, at the Museum of the Viruses Research Institute, Russian Academy of Medical Sciences, in a lyophilizated bed at -5 degrees C, however, an attempt to genotype the RNA failed. No RNA donor leukocytes were found in the materials of further passing of HLV by using the PHA-stimulated cultures, which can be explained by an inactivation of HLV at storage. No RNA of the C hepatitis virus was found in the above materials either, however, in 1999, DNA of the TT virus was detected at passing the strain, which indicates that the virus is widely spread in the population of healthy donors, whose lymphocytes are used preparing the blood leukocyte cultures.

[Adaptation of hepatitis A virus to nonprimate cells]. / Adaptatsiia virusa gepatita A k neprimatnym kletkam.

Hepatitis A virus (HAV) was adapted to nonprimate BHK-21 cell line (Syrian hamster kidney). Enzyme immunoassay, immunoblotting, and slot hybridization demonstrated the capacity of HM175 culture strain to stable reproduction in this cell line. More than 50 passages of adapted HAV were carried out, which showed no changes in the basic cultural characteristics of the virus. The data permit a conclusion on the possibility of HAV reproduction in nonprimate cells.

[HIV-1 p17 matrix protein is transported into the cell nucleus and binds with genomic viral RNA]. / Matriksnyi belok p17 VICh-1 transportiruetsia v kletochnoe iadro i sviazyvaetsia s genomnoi virusnoi RNK.

We have shown that gag polyprotein p55 is cleaved in cytosol rapidly after its synthesis, during 2 h, and p17 enters the nuclei while p24 resides in cytosol. To determine whether the nascent p17 is associated with viral genomic RNA in the nuclei, the cells were fractionated, the viral complexes were immunoprecipitated by monoclonal antibodies against gag proteins, and RNA was extracted and analyzed by slot and blot hybridization. Monoclonal antibodies against p17 precipitated all the viral RNA from the nuclei including full-size genomic RNA and essential part from membranes while monoclonal antibodies against p24 did not precipitate any viral RNA from the nuclei. These data suggest that matrix protein is linked to genomic RNA in the nuclei and rise the possibility that p17 may transfer viral nucleocapsids from the nuclei to plasma membranes, the site of virus assembly.

[Formation of virus-like particles by HIV-1 Gag proteins, expressed by a recombinant vaccinia virus]. / Obrazovanie virusopodobnykh chastits belkov Gag VICh-1, ékspressiruemym rekombinantnym virusom ospovaktsiny.

Monkey kidney cells CV-1 were infected with recombinant vaccinia virus carrying HIV-1 gag gene with a deletion of 230 nucleotide pairs from the 3'-terminus. The main gene product detected in the lysates of infected cells was the gag precursor rp50. The protein was accumulated on the cell membranes suggesting that it had a myristylated N-terminus, and was cleaved by a recombinant virus specific protease with the formation of two proteins, p17 and p24 corresponding in molecular masses to mature gag proteins. Virus-like particles similar to immature HIV virions were budding from the surface of infected cells. They look like the ring of optically dense material covered with a lipid bilayer, of the same size (100-120 nm) and of the same density in a sucrose gradient (1.16-1.18 g/ml) as HIV-1 virions. The particles contained rp50 and cellular heterogeneous RNA. Thus, the unprocessed gag precursor with deleted 77 amino acid residues from the C-terminus is able to form virus-like particles in the absence of env proteins and virus-specific RNA, and these particles are budding from the cell surface. The question about the use of extracellular Gag-particles for AIDS diagnostic work and construction of vaccines is discussed.

[Persistence of the influenza virus in children with congenital CNS pathology]. / Persistentsiia virusa grippa u detei s vrozhdennoi patologiei TsNS.

Sick infants born to mothers who experienced influenza during pregnancy were examined. The cerebrospinal fluid, serum and blood cells were collected from such children with signs of congenital immune deficiency and progressive pathology of the central nervous system. None of the specimens yielded infectious influenza virus, but by means of molecular hybridization virus-specific genetic sequences were found in small amounts in the cerebrospinal fluid and serum and in high concentrations in blood cells. Persistence of genes NP, M and H1 of influenza A/H1N1 virus was observed in the blood cells of one infant for 83 days (the observation period). At the same time, the lack of antibodies to viral M protein in serum of this baby was demonstrated by the immune blotting method.

[Heterologous system of immunoenzyme analysis for the diagnosis of human rotavirus infection and its comparison with RNA electrophoresis]. / Geterologichnaia sistema immunofermentnogo analiza dlia diagnostiki rotavirusnoi infektsii cheloveka i ee sopostavlenie s élektroforezom RNK.

On the basis of porcine rotavirus a heterologous EIA test system was worked out and tested for diagnosis of human rotavirus infection. A high sensitivity and specificity of the test system was demonstrated, its results were compared with those of electron microscopy, diffuse precipitation test, and RNA electrophoresis. Out of 201 specimens (fecal filtrates) collected from children ranging in ages from 14 days to 10 years, rotavirus antigen was detected in 68 (33.8%) RNA electrophoresis demonstrated the rotaviruses circulating in Moscow to have basically the same electrophoretic type, although rotaviruses with other electrophoretic types, including the "short" electrophoretic type, were also detected.

[Detection of HTLV-III-specific sequences in T-lymphocyte lines by molecular hybridization]. / Vyiavlenie HTLV-III-spetsificheskikh posledovatel'nostei v liniiakh T-limfotsitov metodom molekuliarnoi gibridizatsii.

The methods of molecular hybridization are described using 32P-labeled plasmid pBHIO carrying the cloned HTLV-III provirus employed for the detection of HTLV-III-specific sequences in lines of T-lymphocytes from patients with acquired immune deficiency syndrome. The presence of virus-specific sequences was demonstrated in 13 out of 20 lines examined by the dot-blot hybridization method. The blot-hybridization method revealed differences in sites of Hind III restriction in 2 out of 6 lines studied. The possibility of combined infection of line No. 17 with two different variants of HTLV-III is discussed.

[Rapid diagnosis of influenza: a comparative evaluation of different methods]. / Bystraia diagnostika grippa: sravnitel'naia otsenka razlichnykh metodov.

The results of influenza diagnosis during the outbreak of 1985 are presented. Nasopharyngeal secretions from 94 patients were examined by virus isolation in chick embryos, fluorescent antibody technique (FAT). enzyme-immunoassay (EIA), and dot-blot hybridization method (DBHM). The virus was isolated in 28%, FAT was positive in 22%, EIA in 47% of the cases. Among 94 secretion specimens 40 were tested by DBHM. In this instance, virus was isolated in 37%, EIA was positive in 65%, and DBHM in 85% of the cases. It seems advisable to use EIA based on the detection of the type-specific antigen (matrix protein) and DBHM which identifies the serovariant of influenza virus.

[Use of a method of molecular nucleic acid hybridization for the rapid diagnosis of influenza]. / Ispol'zovanie metoda molekuliarnoi gibridizatsii nukleinovykh kislot dlia ékspress-diagnostiki grippa.

A highly sensitive method of pinpoint hybridization of nucleic acids on nitrocellulose filters using 32P-labeled pHA plasmid carrying a DNA copy of hemagglutinin gene of influenza A/Udorn/307/72 (H3N2) was developed which permitted specific detection of minimal amounts of RNA (units of pikograms) of influenza A virus with H3 serotype hemagglutinin. The method of pinpoint hybridization was used for the detection of RNA of influenza A (H3 serotype) in nasopharyngeal washings of patients with acute respiratory diseases during the influenza outbreak of February-March, 1984. In parallel, the presence of viral antigen was determined by direct immunofluorescence using H3N2 antiserum, and the diagnosis of influenza was confirmed by the clinical picture of the disease. The results indicate that the pinpoint hybridization method may be used for rapid diagnosis of influenza as a highly sensitive and specific tool.

[Stimulating effect of cellular RNA on the in vitro polymerizing activity of influenza virus ribonucleoprotein]. / Stimuliruiushchee deistvie kletochnykh RNK na polimeraznuiu aktivnost' ribonukleoproteida virusa grippa in vitro.

The stimulating effect of RNAs isolated from noninfected and influenza virus-infected chick fibroblasts on the polymerase activity of influenza virus intracellular ribonucleoprotein (RNP) was studied in vitro. The infected cells were shown to contain two classes of RNAs which stimulated well the polymerase activity of influenza virus RNP. One class seemed to be represented by a heterogenous cellular 10-20 S mRNA since it contained poly (A)-sequences and was present in noninfected cells. The other RNA class was induced during the infection and differed in number of properties from the RNA isolated from noninfected cells. This class RNA was smaller (4-10 S) and appeared not to contain poly(A)-sequences. Treatment of both noninfected and infected cells with actinomycin D resulted in inhibition of synthesis of both classes of RNA-primers.

[Intracellular structures of influenza virus]. / Vnutrikletochnye struktury virusa grippa.

The intracellular influenza virus-containing structures involved in RNA synthesis in the cytoplasm and in the nucleoplasm of infected chicken fibroblasts were studied. Two approaches were used: (1) short pulse labeling of infected cell with [3H]uridine; (2) determination in vitro of polymerase activity of intracellular virus-specific structures. Both methods revealed functionally active virus-specific structures in the nucleoplasm and showed that a functionally active virus-specific structure was localized in the nucleoplasm of infected cells. This structure contained proteins of the viral ribonucleoprotein, but sedimented somewhat faster (at 60--90S in velocity sucrose and glycerol gradients). Meanwhile, polymerase-containing structures in the cytoplasm of infected cells sedimented in the position of viral ribonucleoproteins (25--60S).

[Parent structures of influenza virus in infected cells]. / Roditel'skie struktury virusa grippa v zarazhennykh kletkakh.

The fate of influenza virus (A/FPV/Weilbridge) parental structures was studied in permissive (chick fibroblasts) and nonpermissive (Ehrlich ascitic carcinoma cells) cell systems. The cells were infected with the virus labeled with 3H-precursors of RNA of the pulse label and by polymerase reaction in vitro. In the cytoplasm, the functionally active parental structures were found in the area of nucleocapsids (40-50 S). Upon recentrifugation in cesium chloride gradient, some of these structures showed nucleocapsid density of 1.34 g/cm3, and some a higher density (1.40-1.41 g/cm3). In the nucleoplasm, the functionally active structures 2 hours after infection sedimented in the zones of 20-35 S and 3 hours postinfection in the zone of 70-90 S. Both in the cytoplasm and nucleoplasm the parental structures were unstable, undergoing rapid deproteinization. Similar parental structures were found in permissive and nonpermissive cell systems.

[Unfragmented nucleocapsid of influenza virus in infected cells]. / Nefragmentirovannyi nukleokapsid virusa grippa v zarazhennykh kletkakh

In the cytoplasms of chick embryo fibroblast and Ehrlich ascitic carcinoma cells infected with influenza virus (fowl plague virus), in addition to fragmented virus nucleocapsid larger nucleocapsid structures were found which sedimented in the region of 90 -120S. The structures were detected upon short 3H-uridine label of the cells. Their buoyant density in cesium chloride was higher than that of the fragmented nucleocapsid (1.34 -1.39 g/cm3). In electron microscope, the structures were visualized as thin nonhelical filaments 3.5 nm in diameter, their morphology being no different from that of a similar rapidly sedimenting structure isolated from the nucleoplasms of the same cells. To determine the possibility of transfer of the rapidly sedimenting structure from the nucleus into the cytoplasm, a cell-free system was used containing nuclei from influenza virus-infected cells labeled with 3H-uridine for 5 min, as well as the cytoplasm from uninfected and unlabeled cells. The presence of a labeled rapidly sedimenting structure in the cytoplasm of the cell-free system suggests that the structure is synthesized in the nucleus and then transported into the cytoplasm. The relation of this structure to the fragmented nucleocapsid is unknown. It may be assumed to be its intracellular precursor.