Mutations within the 5' nontranslated RNA of cell culture-adapted hepatitis A virus which enhance cap-independent translation in cultured African green monkey kidney cells.

Mutations in the 5' nontranslated RNA (5'NTR) of an attenuated, cell culture-adapted hepatitis A virus (HAV), HM175/P16, enhance growth in cultured African green monkey kidney (BS-C-1) cells but not in fetal rhesus monkey kidney (FRhK-4) cells (S. P. Day, P. Murphy, E. A. Brown, and S. M. Lemon, J. Virol. 66: 6533-6540, 1992). To determine whether these mutations enhance cap-independent translation directed by the HAV internal ribosomal entry site (IRES), we compared the translational activities of the 5'NTRs of wild-type and HM175/P16 viruses in two stably transformed cell lines (BT7-H and FRhK-T7) which constitutively express cytoplasmic bacteriophage T7 RNA polymerase and which are derived from BS-C-1 and FRhK-4 cells, respectively. Translational activity was assessed by monitoring expression of a reporter protein, chloramphenicol acetyltransferase (CAT), following transfection with plasmid DNAs containing bicistronic T7 transcriptional units of the form luciferase-5'NTR-CAT. In both cell types, transcripts containing the 5'NTR of HM175/P16 expressed CAT at levels that were 50- to 100-fold lower than transcripts containing the IRES elements of Sabin type 1 poliovirus or encephalomyocarditis virus, confirming the low activity of the HAV IRES. However, in BT7-H cells, transcripts containing the 5'NTR of wild-type virus. This translational enhancement was due to additive effects of a UU deletion at nucleotides 203 and 204 and a U-to-G substitution at nucleotide 687 of HM175/P16. These mutations did not enhance translation in FRhK-T7 or Huh-T7 cells (a T7 polymerase-expressing cell line derived from human hepatoblastoma cells) or in vitro in rabbit reticulocyte lysates. These results demonstrate that mutations in the 5'NTR of a cell culture-adapted HAV enhance viral replication by facilitating cap-independent translation in a cell-type-specific fashion and support the concept that picornaviral host range is determined in part by differences in cellular translation initiation factors.

Analysis of hepatitis A virus translation in a T7 polymerase-expressing cell line.

Hepatitis A virus (HAV) exhibits several characteristics which distinguish it from other picornaviruses, including slow growth in cell culture even after adaptation, and lack of host-cell protein synthesis shut-down. Like other picornaviruses, HAV contains a long 5' nontranslated region (NTR) incorporating an internal ribosomal entry site (IRES), which directs cap-independent translation. We compared HAV IRES-initiated translation with translation initiated by the structurally similar encephalomyocarditis virus (EMCV) IRES, using plasmids in which each of the 5'NTRs is linked in-frame with the chloramphenicol acetyltransferase (CAT) gene. Translation was assessed in an HAV-permissive cell line which constitutively expresses T7 RNA polymerase and transcribes high levels of uncapped RNA from these plasmids following transfection. RNAs containing the EMCV IRES were efficiently translated in these cells, while those containing the HAV IRES were translated very poorly. Analysis of translation of these RNAs in the presence of poliovirus protein 2A, which shuts down cap-dependent translation, demonstrated that their translation was cap independent. Our results suggest that the HAV IRES may function poorly in these cells, and that inefficient translation may contribute to the exceptionally slow replication cycle characteristic of cell culture-adapted HAV.

Production and characterization of a monoclonal antibody recognizing a cytoplasmic antigen of equine mononuclear phagocytes.

An IgG1 mouse monoclonal antibody, designated 1.646, is described which recognizes a cytoplasmic antigen of equine mononuclear phagocytes. Indirect fluorescent antibody staining of peripheral blood leukocytes reveals a granular cytoplasmic staining, predominantly in adherent blood mononuclear cells. Indirect fluorescent antibody staining is positive for alveolar and peritoneal macrophages. In some horses, a few neutrophils are also stained. In equine tissue samples stained by immunohistochemistry, the distribution of positive cells is consistent with the distribution of tissue macrophages. The most intense and reliable staining occurs with splenic and lymph node macrophages. Hepatic Kupffer cells also stain with antibody 1.646, although the intensity of that staining is somewhat variable between horses. A granular pattern of staining typical of lipofuscin deposition is also seen in liver sections. There is also pale staining of some biliary and renal tubular epithelium. Equine erythrocytes, platelets and lymphocytes are not recognized by this antibody, and neither are monocyte/macrophages of human, canine or feline origin. Antibody 1.646 recognizes two proteins (150 and 30 kDa) of equine monocyte-derived macrophages when assayed by Western immunoblot. Because of the distribution of staining (tissue mononuclear phagocytes, lipofuscin-containing storage granules, biliary and renal tubular epithelium, and some neutrophils) we hypothesize that antibody 1.646 recognizes a cytoplasmic antigen that is closely associated with lysosomal membranes.

Immune-mediated thrombocytopenia in horses infected with equine infectious anemia virus.

An adult horse infected with a virulent, cell culture-adapted strain of equine infectious anemia virus (EIAV) developed cyclical thrombocytopenia in which the nadir of platelet counts coincided with peak febrile responses. In order to investigate the mechanism of thrombocytopenia during acute febrile episodes, four adult horses were experimentally infected with the wild-type Wyoming strain of EIAV. Platelet counts decreased from baseline as rectal temperature increased. Serum reverse transcriptase activity increased above background levels in all horses, coincident with increase in rectal temperature. All horses developed an EIAV-specific immune response detectable by Western immunoblot by postinfection day 10. Increases in platelet-associated immunoglobulins G and M were detectable by direct fluorescent-antibody test and flow cytometric assay. Viral replication in bone marrow megakaryocytes was not detectable by in situ hybridization. Results suggest an immune-mediated mechanism of thrombocytopenia in horses infected with EIAV. Despite an inability to identify virion particles in association with platelet-bound antibody, the cyclical nature of the thrombocytopenia and the occurrence of a marked cell-free viremia concomitant with fever and thrombocytopenia suggest immune complex deposition on platelets. We propose that clearance of virus and antibody-coated platelets from the peripheral circulation by hepatic Kupffer cells and splenic macrophages may target infectious virus particles, in the form of immune complexes, to host cells most permissive for in vivo viral replication.

Temporal appearance of structural and nonstructural bluetongue viral proteins in infected cells, as determined by immunofluorescence staining and flow cytometry.

The temporal appearance of 4 viral proteins was detected in bluetongue virus (BTV)-infected Vero cells by indirect immunofluorescence staining with monoclonal antibodies (MAb) to BTV structural proteins VP2 and VP7 and nonstructural proteins NS1 and NS2. Bluetongue viral proteins were detected at distinct intervals after inoculation of Vero cells; VP7 was first detected 3 hours after inoculation, NS1 and NS2 at 5 hours after inoculation, whereas VP2 was not detected until 8 hours after inoculation. Patterns of fluorescence varied with the fixative used, but each MAb induced a distinct pattern of fluorescence in infected cells. Flow cytometry, which was used with each of the 4 MAb, proved to be an accurate and sensitive method of detecting BTV-infected P3 mouse myeloma cells. The temporal appearance of each viral protein in BTV-infected P3 cells was similar to that detected in BTV-infected Vero cells. Advantages of flow cytometry over conventional immunofluorescence staining to detect BTV-infected cells included: (1) enumeration of the proportion of infected cells in a population; (2) further characterization of infected cells, including estimates of their viability; and (3) computer-assisted storage and analysis of data obtained.