Lymphocyte activators elicit bovine leukemia virus expression differently as asymptomatic infection progresses

Since bovine leukemia virus (BLV) replicates in B-lymphocytes, viral expression and production should respond to agents activating these cells. We asked whether synthesis of BLV capsid (CA) protein and production of infectious virus would increase when peripheral blood mononuclear cells (PBMCs) from infected sheep were stimulated in short-term culture with lipopolysaccharide (LPS), a polyclonal activator of B-cells, and compared its effects with those of phytohemagglutinin (PHA), a lymphocyte activator known to increase BLV expression. LPS treatment of PBMCs from asymptomatic sheep that had been infected for 1-4 years increased the number of cells synthesizing CA protein, the amount of CA protein per cell, and the number of PBMCs acting as infectious centers. LPS derived from several different microbes was effective. During the ensuing 4 years of asymptomatic infection, the number of PBMCs expressing virus under minimal stimulation increased for each animal. The ability of LPS to recruit additional cells to express CA protein remained constant or decreased in magnitude, yet at the same time, lower concentrations of LPS were required to elicit a maximal effect. This suggests that the cellular pathways affected by LPS are endogenously more activated as infection progresses. PHA initially stimulated fewer cells to synthesize BLV CA protein than LPS did although the amount of CA protein synthesized per cell was greater with PHA. As infection progressed, PHA surpassed LPS in the numbers of PBMCs induced to express CA protein. This suggests that the cellular pathways affected by PHA become more responsive to its effects as infection progresses. LPS increased CA expression early and transiently during culture whereas the PHA-mediated increase continued to develop for several days. Thus, LPS increases BLV expression but does so differently than PHA. Moreover, these longitudinal results show that the activation state of BLV-infected cells changes as asymptomatic infection progresses.     

Longitudinal studies of immune function in cattle experimentally infected with bovine immunodeficiency-like virus and/or bovine leukemia virus

The effects of single or dual infection with bovine immunodeficiency-like virus (BIV) and/or, bovine leukemia virus (BLV) on bovine immune function were examined over a 4 year period. Holstein calves were infected with BIV (four calves), BLV (five calves), BIV and BLV (five calves), or sham inoculated (three calves). Lymphocyte blastogenesis to mitogens, seven tests of neutrophil function, and mononuclear cell subset analysis by flow cytometry (BoCD4, BoCD8, BoCD2, BoWC1, sIgM+, and monocytes) were performed at regular intervals to 49 months post-infection. These data were analyzed for main effects of each virus and interaction as a 2 x 2 factorial. BIV infected cattle had lower neutrophil antibody-dependent cell-mediated cytotoxicity and iodination responses during 2 of the 4 years post-infection (P < 0.05). BIV infection was not associated with any long-term significant changes in lymphocyte blastogenesis to mitogens or changes in mononuclear cell subset numbers in blood. There was a tendency for animals infected with BIV alone to have decreased lymphocyte blastogenic responses to mitogens, but this was not statistically significant. BLV infection caused an increase in total mononuclear cells with no dramatic shift in the relative proportions of the various subsets. Co-infection with BIV and BLV did not consistently cause a different response than either virus did individually. One BIV infected animal died of non-BLV lymphosarcoma 7 months after infection. All other animals had no unusual clinical signs. In summary, infection with BIV caused a significant, temporary decrease in neutrophil function with no consistent statistically significant alteration in lymphocyte blastogenesis or mononuclear cell numbers during the first 4 years after infection. BLV infection caused an increase in lymphocyte numbers, and there appeared to be no synergism between the viruses.     

Macrophages infected with bovine leukaemia virus (BLV) induce humoral response in rabbits

BLV is a lymphotropic retrovirus which infects mainly B-cells. However, the possible infection of cells of the monocyte/macrophage lineage (M/M) might explain some aspects of the disease such as latency or disease progression. We infected sheep M/M with BLV either by culturing M/M with supernatant containing virus, or coculturing M/M with persistently infected cell lines. These BLV-infected M/M were inoculated into rabbits and the serological response was followed for two years. ELISA results using adsorbed sera showed a persistent production of specific antibodies from as early as the first week post inoculation. Two tests were used to detect the response against envelope glycoprotein gp51: Agar gel immunodiffusion (AGID) and a virus neutralization test read as syncytia inhibition (SI). Sera were positive by AGID after the second or third inoculation. Neutralizing titres (SI) were higher than those seen in control rabbits inoculated with persistently infected cell lines, suggesting that the virus may be expressed better in M/M. Gag-related proteins were analyzed by Western Blot (WB). Sera from rabbits inoculated with BLV-infected M/M recognized as many viral proteins as sera from BLV immunized control rabbits or infected cows, and this profile did not change with repeated inoculations. All these results suggest that BLV may infect M/M, where viral proteins are actively expressed to the point that they induce a humoral immune response in animals, and that animals get persistently infected.     

The YXXL sequences of a transmembrane protein of bovine leukemia virus are required for viral entry and incorporation of viral envelope protein into virions

The cytoplasmic domain of an envelope transmembrane glycoprotein (gp30) of bovine leukemia virus (BLV) has two overlapping copies of the (YXXL)2 motif. The N-terminal motif has been implicated in in vitro signal transduction pathways from the external to the intracellular compartment and is also involved in infection and maintenance of high viral loads in sheep that have been experimentally infected with BLV. To determine the role of YXXL sequences in the replication of BLV in vitro, we changed the tyrosine or leucine residues of the N-terminal motif in an infectious molecular clone of BLV, pBLV-IF, to alanine to produce mutated proviruses designated Y487A, L490A, Y498A, L501A, and Y487/498A. Transient transfection of African green monkey kidney COS-1 cells with proviral DNAs that encoded wild-type and mutant sequences revealed that all of the mutated proviral DNAs synthesized mature envelope proteins and released virus particles into the growth medium. However, serial passages of fetal lamb kidney (FLK) cells, which are sensitive to infection with BLV, after transient transfection revealed that mutation of a second tyrosine residue in the N-terminal motif completely prevented the propagation of the virus. Similarly, Y498A and Y487/498A mutant BLV that was produced by the stably transfected COS-1 cells exhibited significantly reduced levels of cell-free virion-mediated transmission. Analysis of the protein compositions of mutant viruses demonstrated that lower levels of envelope protein were incorporated by two of the mutant virions than by wild-type and other mutant virions. Furthermore, a mutation of a second tyrosine residue decreased the specific binding of BLV particles to FLK cells and the capacity for viral penetration. Our data indicate that the YXXL sequences play critical roles in both viral entry and the incorporation of viral envelope protein into the virion during the life cycle of BLV.     

Bovine leukemia virus-induced lymphocytosis and increased cell survival mainly involve the CD11b+ B-lymphocyte subset in sheep

In this study, we show that bovine leukemia virus (BLV)-induced persistent lymphocytosis (PL) results from the in vivo expansion of the CD11b+ B-lymphocyte population. This subset shares phenotypic characteristics with murine and human B-1 cells. BLV interactions with the sheep B-1-like subset were explored. We found that B-1- and B-2-like cells are initially infected to similar extents. However, in long-term-infected sheep, the viral load is higher in B-1-like cells and only B-1- and not B-2-like cells show increased ex vivo survival compared to that in uninfected sheep. Ex vivo viral expression was found in both B-1- and B-2-like cells, indicating that both cell types support viral replication. Finally, cycloheximide and a protein kinase C inhibitor (H7) that blocks the ex vivo activation of viral expression did not affect the increased survival in B-1-like cells, suggesting that resistance to apoptosis is acquired in vivo. Collectively, these results indicate a peculiar susceptibility of sheep B-1-like cells to BLV transforming effects and further support the involvement of increased survival in BLV pathogenesis.     

Conservative mutations in the immunosuppressive region of the bovine leukemia virus transmembrane protein affect fusion but not infectivity in vivo

Many retroviruses, including bovine leukemia virus (BLV), contain a highly conserved region located about 40 amino acids downstream from the fusion peptide within the sequence of the external domain of the transmembrane (TM) protein. This region is notably thought to be involved in the presentation of the NH2-terminal peptide to allow cell fusion. By using hydrophobic cluster analysis and by analogy with the influenza A hemagglutinin structures, the core of the TM structure including this particular region was predicted to consist, in the BLV and other retroviral envelope proteins, of an alpha-helix followed by a loop region, both docked against a subsequent alpha-helix that forms a triple-stranded coiled coil. The loop region could undergo, as in hemagglutinin, a major refolding into an alpha-helix integrating the coiled coil structure and putting the fusion peptide to one tip of the molecule. Based on this model, we have identified amino acids that may be essential to the BLV TM structure, and a series of mutations were introduced in the BLV env gene of an infectious molecular clone. A first series of mutations was designed to disturb the coiled coil structure (substitutions with proline residues), whereas others would maintain the general TM structure. When expressed by Semliki Forest virus recombinants, all the mutated envelope proteins were stable and efficiently synthesized in baby hamster kidney cells. Both proline-substituted and conservative mutants were strongly affected in their capacity to fuse to CC81 indicator cells. In addition, it appeared that the integrity of the TM coiled coil structure is essential for envelope protein multimerization, as analyzed by metrizamide gradient centrifugation. Finally, to gain insight into the role of this coiled coil in the infectious potential of BLV in vivo, the mutated TM genes were introduced in an infectious and pathogenic molecular clone and injected into sheep. It appeared that only the conservative mutations (A60V and A64S) allowed maintenance of viral infectivity in vivo. Since these mutations destroyed the ability to induce syncytia, we conclude that efficient fusion capacity of the recombinant envelopes is not a prerequisite for the infectious potential of BLV in vivo. Viral propagation of these mutants was strongly affected in some of the infected sheep. However, the proviral loads within half of the infected animals (2 out of 2 for A60V and 1 out of 4 for A64S) were close to the wild-type levels. In these sheep, it thus appears that the A60V and A64S mutants propagate efficiently despite being unable to induce syncytia in cell culture.     

Cell-associated viral RNA expression during acute infection with HIV type 1

The mechanism of decline in viremia following acute infection with HIV is unknown. To characterize this process virologically, the expression of viral RNAs was analyzed in samples of peripheral blood mononuclear cells (PBMCs) from a patient who experienced a 100-fold decline in plasma viremia over a 13-day period prior to the initiation of antiretroviral therapy. Cell-associated viral RNA declined in association with the decline in plasma virus. During the initial 7 days of observation, plasma viremia declined more than 10-fold with no change in the ratio of unspliced to multiply spliced mRNAs. The efficiency of viral gene expression did not decline during the study period and varied from 380 to 2800 unspliced RNA copies per productively infected cell. Together, these data indicate no change in the relative proportion of cells in late- and early-stage gene expression during the initial decline and provide evidence against shortening of the viral replication cycle by immune surveillance. However, the prevalence of productively infected cells declined markedly during the 13 days of observation, from 1 in 250 to 1 in 25,000 PBMCs. These data are compatible with depletion of available target cells during the initial decline in viremia. As the level of plasma virus stabilized after 8 days of observation, the ratio of unspliced to multiply spliced mRNAs rose; this rise was due to a relatively greater decline in multiply spliced mRNA. These data suggest the possible onset of a blockade to new infection events (for example, by neutralizing antibody or chemokines), causing an increase in the relative proportion of cells in late-stage gene expression. They may also be explained, however, by the persistence of cell-associated virions together with the near disappearance of productively infected cells from the circulation.     

Latero-cervical tumors: apropos of a case of paraganglioma of the vagus nerve]

Borna disease virus (BDV) is a neurotropic, as yet unclassified, non-segmented, negative-sense, single-strand RNA virus. Natural infection with this virus has been reported to occur in horses and sheep. In addition, antibodies to BDV in plasma or BDV RNA in peripheral blood mononuclear cells (PBMCs) were also found in patients with neuropsychiatric diseases. We describe here the possible link between the patients with chronic fatigue syndrome (CFS) and infection with BDV.     

The range and distribution of murine central nervous system cells infected with the gamma(1)34.5- mutant of herpes simplex virus 1

Wild-type herpes simplex virus 1 (HSV-1) multiplies, spreads, and rapidly destroys cells of the murine central nervous system (CNS). In contrast, mutants lacking both copies of the gamma(1)34.5- gene have been shown to be virtually lacking in virulence even after direct inoculation of high-titered virus into the CNS of susceptible mice (J. Chou, E. R. Kern, R. J. Whitley, and B. Roizman, Science 250:1262-1266, 1990). To investigate the host range and distribution of infected cells in the CNS of mice, 4- to 5-week-old mice were inoculated stereotaxically into the caudate/putamen with 3 x 10(5) PFU of the gamma(1)34.5- virus R3616. Four-micrometer-thick sections of mouse brains removed on day 3, 5, or 7 after infection were reacted with a polyclonal antibody directed primarily to structural proteins of the virus and with antibodies specific for neurons, astrocytes, or oligodendrocytes. This report shows the following: (i) most of the tissue damage caused by R3616 was at the site of injection, (ii) the virus spread by retrograde transport from the site of infection to neuronal cell nuclei at distant sites and to ependymal cells by cerebrospinal fluid, (iii) the virus infected neurons, astrocytes, oligodendrocytes, and ependymal cells and hence did not discriminate among CNS cells, (iv) viral replication in some neurons could be deduced from the observation of infected astrocytes and oligodendrocytes at distant sites, and (v) infected cells were being efficiently cleared from the nervous system by day 7 after infection. We conclude that the gamma(1)34.5- attenuation phenotype is reflected in a gross reduction in the ability of the virus to replicate and spread from cell to cell and is not due to a restricted host range. The block in viral replication appears to be a late event in viral replication.     

Cell-free HIV-1Zr6 vif mutants are defective in binding to peripheral blood mononuclear cells and in internalization

The vif gene of the human immunodeficiency virus (HIV-1) is required for productive virus infection of primary blood mononuclear cells (PBMCs) and macrophages in vitro. Replication of HIV-1 vif- mutants in T-lymphoid cell lines varies and is dependent on the cell line used for virus production. To further understand the role of Vif in HIV-1 infection, we constructed to vif deletion mutants from a molecular clone derived from an African patient (HIV-1Zr6). Cell-free Zr6 vif- virus pools made from transfected rhabdomyosarcoma (RD) cells do not replicate when added to cultures of stimulated PBMCs. However, vif mutants were able to spread from transfected RD cells to PBMCs if cell-to-cell contact was permitted. By Western blot analysis, viral structural proteins expressed after transfection of RD cells by wild-type or vif mutant proviruses were indistinguishable. However, binding of vif mutants to PBMCs or to purified CD4 and virus internalization were significantly reduced when compared with wild-type virus. The defects in cell-free infection, CD4 binding, and internalization were rescued by transcomplementation using a vif expression plasmid. Our results suggest a novel level at which the HIV-1 vif gene product acts to enhance cell-free infection and indicate that vif plays an important role in promoting HIV-1 binding and internalization. Combined with the previous reports of vif's effect at other steps in infection, this suggests that vif is a pleuripotent gene product that affects multiple stages of the infective process.     

Congenital diaphragmatic hernia--a review of pre- and postoperative chest radiology

Delayed-type hypersensitivity responses against bovine leukemia virus (BLV) envelope glycoprotein (gp60) were induced in the skin of sheep vaccinated with recombinant vaccinia virus (RVV) expressing BLV glycoprotein. The lesions were characterized by marked infiltration of lymphocytes, slight migration of neutrophils, eosinophils, and macrophages in the dermis to hypodermis, and partial intercellular edema in the reticular layer. Immunohistochemical analysis with monoclonal antibodies demonstrated that the lymphocytic infiltrates consisted mainly of CD8+ T cells (53.7-55.8% at 48 hours post-challenge of BLV), CD4+ T cells (24.7-26.7%), and B cells (11.5-16.9%). The role of CD4+ and CD8+ T cells in suppressing BLV growth in RVV-vaccinated animals is discussed.     

Progression to persistent lymphocytosis and tumor development in bovine leukemia virus (BLV)-infected cattle correlates with impaired proliferation of CD4+ T cells in response to gag- and env-encoded BLV proteins

The mechanism of leukemogenesis and persistent lymphocytosis (PL; benign expansion of B lymphocytes) in cattle infected with bovine leukemia virus (BLV; a retrovirus closely related to human T-cell leukemia virus type 1) is unknown; however, the immune system likely plays an important role in controlling the outcome of infection. In this study, we compared T-cell competence in serologically positive alymphocytotic (AL) animals with T-cell functions in animals with progressive stages of infection, PL and tumor bearing (TB). Dramatic differences were observed in lymphocyte proliferation to recombinant proteins encoded by BLV gag (p12, p15, and p24) and env (gp30, and gp51) genes in different disease stages. Lymphocytes from AL cattle recognized an average of three of five recombinant proteins per animal. Expansion of antigen pulsed lymphocytes in interleukin-2 increased protein recognition to almost five per animal. In contrast, lymphocytes from PL and TB animals failed to recognize any BLV recombinant proteins. Short-term T-cell cultures from the PL group expanded in interleukin-2, as well as the PL and TB cells cultured in indomethacin (3 to 6 microg/ml), increased the average of recognized proteins per animal to one. Cells proliferating to BLV antigens were CD4+ T lymphocytes, as shown by cell depletion studies. The positive effect of indomethacin suggests involvement of prostaglandin E2 as a negative regulatory factor in the later stages of disease. Thus, for the first time, advancing stages of BLV infection were correlated with decreased T-cell competence, providing deeper insight into pathogenesis of retroviral infections.     

Effect of genotype on the hormonal activity of the Leydig cells during stress in inbred strains of mice

Paraffin sections from various organs of sheep fetuses following transplacental infection with non-cytopathogenic (ncp) bovine viral diarrhoea virus (BVDV) or cytopathogenic (cp) BVDV were stained immunohistochemically with BVDV-specific monoclonal antibodies. Comparison of the distribution of viral antigen in sections from fetuses of experiment A revealed that in organs such as parotid, thyroid, thymus, lung, spleen, kidney, liver and skin from 20 days post inoculation (p.i.) onwards numerous antigen-containing cells were present. In organs of fetuses infected with cp BVDV, however, antigen-positive cells were only detectable until days 10 and 14 p.i. These findings suggest that the ncp BVDV used in experiment A replicated considerably faster and more efficient than the cp BVDV used in experiment B and that the two virus biotypes differ considerably concerning their tropism for fetal ovine organs.     

Detection of respiratory syncytial virus (RSV) antigen in the lungs of guinea pigs 6 weeks after experimental infection and despite of the production of neutralizing antibodies

Infections with respiratory syncytial virus (RSV) are characterized by frequently occurring reinfections and are regarded to be responsible for bronchial hyperreactivity. In this report we describe a small-animal model suited to study RSV-induced pathogenesis and immune response. Guinea pigs are infected by inhalation of an RSV-aerosol. Lungs of infected animals show signs of a bronchiolitis at 7 days after the initial infection. Although neutralizing serum antibodies are synthesized viral proteins are still detectable at 6 weeks post infection. Therefore, the presence of neutralizing antibodies is obviously not sufficient for rapid clearance of persistent RSV-proteins from the lungs of infected guinea pigs.     

Resistance of interleukin-1beta-deficient mice to fatal Sindbis virus encephalitis

Interleukin-1beta (IL-1beta) concentrations are frequently elevated in central nervous system (CNS) viral infections, but the pathophysiologic significance of such elevations is not known. To examine the role of IL-1beta in CNS viral pathogenesis, we compared the natural histories of IL-1beta-deficient and wild-type 129 SV(ev) mice infected with a neurovirulent viral strain, neuroadapted Sindbis virus (NSV). We found that the incidence of severe paralysis and death was markedly decreased in NSV-infected IL-1beta-/- mice compared to NSV-infected wild-type mice (4 versus 88%, P < 0.001). Despite this marked difference in clinical outcome, no differences in numbers of apoptotic cells or presence of histopathologic lesions in the brains of moribund wild-type mice and those of clinically healthy IL-1beta-/- mice could be detected. These results suggest that IL-1beta deficiency is protective against fatal Sindbis virus infection by a mechanism that does not involve resistance to CNS virus-induced apoptosis or histopathology.     

Influence of viral infection on expression of cell surface antigens in human retinal pigment epithelial cells

BACKGROUND: Subacute viral infection is known to change the phenotype of infected cells, thereby causing immune-mediated tissue damage. The aim of this study was to investigate the expression of different cell surface molecules on human retinal pigment epithelial cells (RPEC) following viral infection, with special emphasis on those having immune-regulatory functions. METHODS: Cultured RPEC were infected with cytomegalovirus (CMV), coxsackie-virus B3 (CVB) or herpes simplex virus type I (HSV). Double-staining fluorescence technique was used for visualization of virus infection and cell surface markers in the same cells by laser microscopy. RESULTS: CMV downregulated MHC class I antigens on RPEC, whereas CVB and HSV did not alter MHC class I antigen expression. No induction of class II antigens was observed in RPEC infected with CVB, HSV or CMV. The intercellular adhesion molecule ICAM-1 (CD54) was strongly expressed in uninfected RPEC, and a slight increase was observed after virus infection. Vascular cell adhesion molecule 1 (VCAM-1) was expressed in low amounts in both uninfected and infected RPEC. No expression of intercellular adhesion molecule 2 (ICAM-2), E-selectin ELAM-1 or lymphocyte-function-associated antigen 1 (LFA-1) was observed on RPEC before or after virus infection. CONCLUSION: Downmodulation of immune-regulating cell surface antigens has been suggested to provide a means of long-term survival of viruses in the infected cell, favoring establishment of persistent infection. Our observation in cultured human RPEC indicates that this mechanism might indeed contribute to the development of disease affecting retinal tissue.