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.

The role of matrix protein p17 of the human immunodeficiency virus in the HIV morphogenesis has been demonstrated. In a pulse-chase experiment the gag p55 polyprotein was shown to be proteolytically processed 2 h after its synthesis. In so doing the p17 protein penetrated into the nuclei while the p24 protein remained in the cytoplasm. To elucidate the role of the p17 protein in the intracellular of viral genomic RNA, HIV-infected Jurkat-tat limphoblastoid cells were fractionated into nuclear, membraneous, and cytosolic fractions. Viral complexes were immunoprecipitated with monoclonal antibodies raised against p17 or other gag proteins, and then used for RNA isolation. This RNA was analyzed, like the supernatant RNA; by slot and blot hybridization. All the virus-specific RNA localized in the nuclei, including the full-size genomic RNA, proved to be associated with the p17 protein. It is suggested that p17 transfers the viral genomic RNA from the nucleus to the site of virus assembly, thus being a key factor in virus morphogenesis.

We have shown previously that HIV-1 matrix protein p17 is transported to the nucleus of Jurkat-tat and H9 cells soon after infection. As shown in this combination, gag polyprotein p55 synthesized 48 h after cell infection is cleaved in cytosol rapidly after its synthesis, and nascent p17 enters the nuclei and gradually accumulates there. Uncleaved p55 molecules and intermediate precursors are rapidly transported to the membranes and are also found in nuclei. Mature gag proteins are seen in membranes only after prolonged period of labelling or chase (4 or more hours later). 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 (MAbs) against gag proteins, and RNA was extracted and analyzed by slot and blot hybridization. MAb against p17 precipitated all the viral RNA from the nuclei including full-size genomic RNA and essential parts from membranes while MAb 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 raise the possibility that p17 may transfer viral nucleocapsids from the nuclei to plasma membranes, the site of virus assembly.

CV-1 cells were infected with two recombinant vaccinia viruses carrying the gag gene with deletion of 231 bp from 3' terminus (strain vC5) and env gene (strain vE234L) of human immunodeficiency virus type 1 (HIV-1). Both recombinant proteins synthesized in the cells (p50gag and gp160/120env) were localized predominantly in cell membranes; however, some amount of p50 was found in cell nuclei. Thin-section immunoelectron microscopy showed accumulation of viruslike particles undistinguished from immature HIV-1 virions in the culture medium of the cells infected with vC5. The similar particles containing gag and env proteins were produced into the culture medium when the cells were coinfected with vC5 and vE234L strains. The particles contained heterogeneous cellular RNA, but no virus-specific RNA as shown by Northern blot hybridization. Immunization of the rabbits with purified viruslike particles produced virus-specific antibodies against gag and env proteins. The titer of antibodies was significantly higher than after immunization with cell lysate or recombinant proteins purified from the infected cells. Highly immunogenic HIV-1-like particles containing gag and env proteins but no virus-specific RNA are good candidates for potential vaccine.

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.

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.

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).

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.

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.