Induction of transformed phenotypes in sheep fibroblasts by culture fluids from cells persistently infected with bovine leukemia virus

FLK cells are fetal lamb kidney cells persistently infected with bovine leukemia virus (BLV). 3178 cells, originating from calf-form bovine lymphosarcoma, also showed persistent production of BLV and alteration of cell morphology, after treatment with 5'-iodo-2'-deoxyuridine. In the present paper, the first in vitro transformation of sheep fibroblasts by inoculation with BLV materials from these two cell lines is described. In a few passages after inoculation with these viral materials, morphological alteration occurred. The morphologically altered cells were grown as stable cultures and showed such transformed phenotypes as growth in soft agar medium, increased uptake of 2-deoxy-D-glucose and tumorigenicity in athymic nude mice. This result, together with our previous observation of simultaneous induction of BLV expression and morphological alteration of 3178 cells, suggests the presence of some transforming capacity in these BLV materials similar to that in, for example, murine or avian acute leukemia viruses. The possible acquisition of such capacity during the prolonged passage is discussed.     

Dissemination of bovine leukemia virus-infected cells from a newly infected sheep lymph node

To investigate the early establishment of bovine leukemia virus (BLV) infection, we injected BLV-infected or mock-infected allogeneic cells into the shoulder of sheep in which an efferent lymphatic duct of the draining prescapular lymph node had been cannulated. Rare mononuclear cells acting as centers of BLV infection in culture were present within 4 to 6 days in efferent lymph and within 6 to 10 days in blood. Soon after BLV injection, immunoglobulin M+ (IgM+) and CD8+ cells increased in efferent lymph and oscillated reciprocally in frequency. CD8+ blasts increased on days 4 to 6, when infectious centers increased 100-fold in lymph. On days 6 and 7, both lymph and blood were enriched with CD8+ cells that were labeled late on day 5 with an intravenous pulse of 5-bromo-2'-deoxyuridine (BrdU). Lymph, but not blood, was enriched with BrdU+ B cells on day 7. Capsid-specific antibodies became detectable in efferent lymph on days 6 to 8 and surface glycoprotein-specific antibodies on day 9, preceding their detection in serum by 9 to 14 days. Systemic dissemination of BLV-infected cells was thus accompanied by an increase in proliferating CD8+ cells and the onset of BLV-specific antibodies in lymph. Infectious centers reached maximum frequencies of 0.2% in lymph by days 11 to 13, and then their frequencies increased by 5- to 40-fold in blood cells, suggesting that many infected blood cells do not recirculate back into lymph. Beginning on days 10 to 13, a subpopulation of B cells having high levels of surface IgM increased sharply in peripheral blood. Such cells were not present in lymph. After a day 16 pulse of BrdU, recently proliferated cells that stained intensely for surface IgM appeared in blood within 15 h. Predominantly B lymphocytes contained the viral capsid protein when lymph and blood cells were cultured briefly to allow BLV expression. However, both early in lymph and later in blood, BrdU+ B cells greatly exceeded productively infected cells, indicating that new BLV infections stimulate proliferation of two different populations of B cells.     

Infection of peripheral blood lymphocytes in sheep with bovine leukemia virus in vitro

Continuous cultivation of peripheral blood lymphocytes from healthy sheep was carried out in vitro with the help of human recombinant interleukin-2. Lymphocytes were concurrently cultivated with the lethally X-rayed BLV-producing FLK culture cells. Electron microscopy and dot-blot hybridization established that sheep peripheral blood lymphocytes were infected with BLV and a full cycle of replication takes place in them. Infection of sheep leukocytes in vitro can be used to study the mechanisms of leukogenesis in vitro.     

Detection of px gene product of bovine leukemia virus in infected cells

Bovine leukemia virus (BLV), like its closest relatives human T-cell leukemia virus-I and II, contain a 'px' gene, between the 'env' gene and the 3' long terminal repeat in its genome. A monoclonal antibody prepared against a synthetic oligopeptide whose sequence was deduced from highly conserved region of 'px' gene of BLV, was used to detect the presence of 'px' gene product in chronically BLV infected synchronised cells. By immunoperoxidase staining the 'px' gene product was detected maximum after 6-9 hr after synchronization in the nucleus of the cells which demonstrated the close interaction of it with viral DNA which is integrated with host cell genome.     

Trypanosoma vivax: a novel method for purification from experimentally infected sheep blood

Trypanosoma vivax is the principal etiological agent of bovine trypanosomosis, a widely disseminated disease in tropical and subtropical regions. Here, we present a simple and reproducible method for the purification of T. vivax from experimentally infected and immunosuppressed sheep, using an isopycnic Percoll gradient, followed by DEAE-cellulose chromatography, with an estimated yield of 11-15%. This method could be used for the purification of T. vivax geographical isolates from various locations and from different natural hosts.     

Humoral immune response of asymptomatic cats naturally infected with feline leukemia virus

The humoral immune response of cats that were naturally infected with the feline leukemia virus (FeLV) was examined after antigenic stimulation with the synthetic antigen poly(L-Tyr, L-Glu)-poly(DL-Ala)-poly(L-Lys). The primary humoral antibody response in FeLV-infected cats was both delayed and greatly reduced, compared with that seen in uninfected control cats. A similar discordance was observed after secondary stimulation with the antigen, in the FeLV-infected cats had both a delayed response and a reduced response, compared with uninfected cats. The levels of total immunoglobulins of the immunoglobulin G and immunoglobulin M classes in the sera of FeLV-infected cats were significantly higher (two- and threefold, respectively) than were those of the uninfected control animals. The presence of an impaired humoral immune response to newly presented antigens in the presence of elevated immunoglobulin levels has been thoroughly documented in the case of people with the acquired immunodeficiency syndrome. This further emphasizes the potential value of FeLV-infected cats as a model for human acquired immunodeficiency syndrome.     

Transcriptional response of peripheral blood mononuclear cells from cattle infected with Mycobacterium bovis

Mycobacterium bovis is the causative agent of most cases of bovine tuberculosis. The identification of bTB biomarkers in specific stages of the disease will contribute to a better understanding of the immunopathology associated with tuberculosis and will enable their use in disease diagnosis and prognosis. The aim of this study was to evaluate the gene expression profile induced after specific stimulation of bovine peripheral blood mononuclear cells from cattle infected with M. bovis using the Affymetrix® GeneChip® Bovine Genome Array. A total of 172 genes showed differential expression profile that was statistically significant with log2-fold change >2.5 and <−2.5. Twenty-four out of these genes were upregulated and 148 were downregulated in bovine peripheral blood mononuclear cells of M. bovis-infected cattle. The highest differentially-expressed genes were related to immune and inflammatory responses, apoptosis, endocytosis, cellular trafficking and genes encoding proteins involved in cellular matrix degradation. Microarray results were confirmed in another group of infected cattle by RT-qPCR for the CD14, IL-1R, THBS1, MMP9 and FYVE genes. This study confirms previous findings that have shown that M. bovis infection in cattle results in the downregulation of immune response-related genes. Moreover, it validates the use of microarray platforms in combination with RT-qPCR to identify biomarkers of bovine tuberculosis. In addition, we propose CD14, IL-1R, THBS1, MMP9 and FYVE as potential biomarkers of bovine tuberculosis.     

Severe thrombocytopenia in young calves experimentally infected with noncytopathic bovine viral diarrhea virus.

Seven calves between 1 week and 2 months of age were infected with a noncytopathic field isolate of bovine viral diarrhea virus (BDV) in order to evaluate the effect of BDV infection on the concentration of circulating platelets in the blood. All calves were determined to be free of BDV and neutralizing antibodies to BDV before infection. Platelet counts were performed on a daily basis over a 30-day period beginning at the time of infection. By 2 weeks postinfection, all calves showed a significant drop in the number of circulating platelets and a marked hyperplasia of megakaryocytes in the bone marrow. In three of the seven calves, thrombocytopenia was severe (less than or equal to 5,000/microliters) for 1 to 6 days. In two of these three animals, extensive petechial and ecchymotic hemorrhages were observed on all mucosal surfaces and on various internal organs during the period of severe thrombocytopenia. BDV was consistently isolated from the platelets during the early phases of the infection, and viral antigen was occasionally detected on platelets by a fluorescent-antibody assay. The results demonstrate that BDV infection is associated with decreases in platelet numbers and suggest that platelets may serve as carriers of circulating virus.     

Membrane characteristics of old and Rauscher leukemia virus infected mouse red blood cells

Some membrane characteristics of normal and Rauscher leukemia virus (RLV)-infected mouse red blood cells (RBC) were compared, both with regard to total populations and young and old groups of cells. Osmotic fragility, density distribution of cells and agglutinability by poly--lysine (pLys), concanavalin A (ConA), phytohemagglutinin (PHA) and soybean agglutinin (SBA), were examined. RBC from RLV-infected mice were agglutinated at a higher rate and to a higher degree than normal mice RBC by pLys and by the lectins PHA and ConA. These RBC were generally osmotically more resistant and contained a young cell population of unusually high specific gravity. Comparison of RBC from RLV-infected mice with old RBC from normal mice showed some common membrane characteristics. Similarly to old RBC, RBC from RLV-infected mice have a high specific gravity and high agglutinability by pLys. However, they differ in that the RBC from RLV-infected mice are osmotically more resistant and are agglutinated by ConA; they are also agglutinated at a higher rate by PHA.     

Even attenuated bovine leukemia virus proviruses can be pathogenic in sheep

Based on a reverse genetics approach, we previously reported that bovine leukemia virus (BLV) mutants harboring deletions in the accessory R3 and G4 genes persist at very low proviral loads and are unable to induce leukemia or lymphoma in sheep, indicating that these R3 and G4 gene sequences are required for pathogenesis. We now show that lymphoma can occur, albeit infrequently (1 case of 20) and after extended periods of latency (7 years). Direct sequencing and reinfection experiments demonstrated that lymphomagenesis was not due to the reversion of the mutant to the wild type. Similar observations with another type of attenuated mutant impaired in the transmembrane protein (TM) YXXL signaling motifs were made. We conclude that the R3 and G4 genes and the TM YXXL motifs are not strictly required for pathogenesis but that their integrity contributes to disease frequency and latency.     

Apoptosis in bovine herpesvirus-1 infected bovine peripheral blood mononuclear cells

Apoptosis is a process whereby cells die in a controlled manner in response to various stimuli like cytotoxins, viral antigens and normal physiological signals during differentiation and development. Virus induced immunosuppression has been reported for various viral diseases including Bovine Herpesvirus-1 (BHV-1). In the present study, BHV-1 was found to cause apoptosis in ConA stimulated bovine peripheral blood mononuclear cells (PBMCs). Apoptotic index quantified by fluorescent dyes revealed a significant (P < 0.001) increase in percent apoptotic cells at 2, 24 and 48 hr post infection as compared to their respective non-infected controls. Apoptosis specific internucleosomal laddering in DNA from BHV-1 infected PBMCs was seen in agarose gel electrophoresis. No DNA fragmentation was observed in control non-infected PBMCs.     

Encephalomyelitis in mice experimentally infected with Akabane virus.

Lesions in the central nervous system of mice, induced by intracerebral injection of Akabane virus, were observed by the fluorescent antibody technique and histological method. Fluorescent antigens were recognized in the cytoplasm of nerve cells, but were not detected exactly in any other part. Fluoresced nerve cells were distributed almost all over the central nervous system, especially in medulla oblongata and spinal cord. The appearance of fluorescent antigens was followed by histological changes. So-called Nissl's acute severe degeneration was observed in nerve cells in the area where the fluorescent antigens were distributed. Spongy foci were seen in medulla oblongata and spinal cord. Virus was recovered from brain and spinal cord, but not from any other visceral organ or blood. Akabane virus showed an affinity to nerve cells and caused primary nonpurulent encephalomyelitis when inoculated intracerebrally to mice.     

Characterization of virus-specific RNA synthesized in bovine cells infected with bovine viral diarrhea virus.

Infection of bovine kidney cells with bovine viral diarrhea virus resulted in the synthesis of a single species of virus-specific RNA. Electrophoresis of this RNA on agarose-urea and agarose-formaldehyde gels indicated that it had a molecular weight of 2.9 X 10(6), corresponding to 8,200 bases (8.2 kilobases). This 8.2-kilobase RNA was resistant to RNase A treatment at 1 microgram/ml but was digested at higher concentrations of RNase (10 micrograms/ml). Sedimentation on neutral sucrose gradients indicated that the majority of this RNA (98%) sedimented at 21S, with a small amount sedimenting at 33S. Sedimentation on formaldehyde-containing sucrose gradients resulted in the conversion of all of the RNA to the faster-sedimenting form. At no time after infection were we able to detect virus-specific RNA species of lower molecular weight than the 8.2-kilobase RNA. The implications of these findings with respect to the means of replication of various togaviruses are discussed.     

In vivo infection of sheep by bovine leukemia virus mutants.

Direct inoculation of a cloned bovine leukemia virus (BLV) provirus into sheep has allowed study of the viral infectivity of genetic mutants in vivo. Three BLV variants cloned from BLV-induced tumors and 12 in vitro-modified proviruses were isolated and analyzed for viral expression in cell culture. The proviruses were then inoculated into sheep in order to assess viral infectivity in vivo. Of three variants cloned from BLV-induced tumors (344, 395, and 1345), one (344) was found infectious in vivo. This particular provirus was used to engineer 12 BLV mutants. A hybrid between the 5' region of the complete but noninfectious provirus 395 and the 3' end of mutant 344 was infectious in vivo, suggesting that the tax/rex sequences were altered in virus 395. As expected, several regions of the BLV genome appeared to be essential for viral infection: the protease, pol, and env genes. Even discrete modifications in the fusion peptide located at the NH2 end of the transmembrane gp30 glycoprotein destroyed the infectious potential. In contrast, mutations and deletions in the X3 region present between the env gene and the 3' tax/rex region did not interfere with viral infection in vivo. This region of unknown function could thus be used to introduce foreign sequences. A BLV recombinant carrying a ribozyme directed against the tax/rex sequences was still infectious in vivo. Cotransfection of two noninfectious mutants carrying deletions led to infection in two of four independent injections, the infectious virus being then a recombinant between the two deletants. The experimental approach described here should help to gain insight into essential mechanisms such as in vivo viral replication, cooperation between deletants for viral infectivity, and viral superinfections. The gene products in the X3 and X4 region which are dispensable for in vivo infection could be involved in leukemogenesis, and thus proviruses deleted in these sequences could constitute the basis for a live attenuated vaccine.     

In vivo leukocyte tropism of bovine leukemia virus in sheep and cattle.

Bovine leukemia virus (BLV), an oncovirus related to human T-cell leukemia virus type I, causes a B-cell lymphoproliferative syndrome in cattle, leading to an inversion of the T-cell/B-cell ratio and, more rarely, to a B-cell lymphosarcoma. Sheep are highly sensitive to BLV experimental infection and develop B-cell pathologies similar to those in cattle in 90% of the cases. BLV tropism for B cells has been well documented, but the infection of other cell populations may also be involved in the BLV-induced lymphoproliferative syndrome. We thus looked for BLV provirus in other leukocyte populations in sheep and cattle by using PCR. We found that while B cells harbor the highest proviral load, CD8+ T cells, monocytes, and granulocytes, but not CD4+ T cells, also bear BLV provirus. As previously described, we found that persistent lymphocytosis in cows is characterized by an expansion of the CD5+ B-cell subpopulation but we did not confirm this observation in sheep in which the expanded B-cell population expressed the CD11b marker. Nevertheless, BLV could be detected both in bovine CD5+ and CD5- B cells and in sheep CD11b+ and CD11b- B cells, indicating that the restricted BLV tropism for a specific B-cell subpopulation cannot explain its expansion encountered in BLV infection. Altogether, this work shows that BLV tropism in leukocytes is wider than previously thought. These results lead the way to further studies of cellular interactions among B cells and other leukocytes that may intervene in the development of the lymphoproliferative syndrome induced by BLV infection.