CD34+ hematopoietic stem cells support entry and replication of poliovirus: a potential new gene introduction route.

Pluripotent hematopoietic stem cells (HSC) are critical in sustaining and constantly renewing the blood and immune system. The ability to alter biological characteristics of HSC by introducing and expressing genes would have enormous therapeutic possibilities. Previous unpublished work suggested that human HSC co-express CD34 (cluster of differentiation 34; an HSC marker) and CD155 (poliovirus receptor; also called Necl-5/Tage4/PVR/CD155). In the present study, we demonstrate the co-expression of CD34 and CD155 in primary human HSC. In addition, we demonstrate that poliovirus infects and replicates in human hematopoietic progenitor cell lines. Finally, we show that poliovirus replicates in CD34+ enriched primary HSC. CD34+ enriched HSC co-express CD155 and support poliovirus replication. These data may help further understanding of poliovirus spread in vivo and also demonstrate that human HSC may be amenable for gene therapy via poliovirus-capsid-based vectors. They may also help elucidate the normal function of Necl-5/Tage4/PVR/CD155.

Overcoming the resistance of codling moth against conventional Cydia pomonella granulovirus (CpGV-M) by a new isolate CpGV-I12.

Recently, codling moth (CM, Cydia pomonella L.) populations with a significantly reduced susceptibility to C. pomonella granulovirus (CpGV) products have been observed in Germany. A novel CpGV isolate, designated CpGV-I12, is able to overcome the CpGV resistance. CpGV-I12 originated from Iran and showed superior efficacy in laboratory bioassays against a resistant CM strain (CpR), which has a 100-fold reduced susceptibility to commercially used isolate CpGV-M. Determination of the median lethal concentration (LC(50)) indicated that CpGV-I12 is nearly as efficient in resistant CpR as CpGV-M in a susceptible CM strain (CpS). Beyond, CpGV-I12 caused superior mortality in CpS. Infection experiments showed that the resistance breaking effect can be observed in all instars of CpR. CpGV-I12 is a promising alternative control agent of CM in orchards where conventional CpGV products fail. In addition, we demonstrate in bioassays with recombinant expressed Cry1Ab that cross-resistance to CpGV and Bacillus thuringiensis products is not likely.

Rapid emergence of baculovirus resistance in codling moth due to dominant, sex-linked inheritance.

Insect-specific baculoviruses are increasingly used as biological control agents of lepidopteran pests in agriculture and forestry, and they have been previously regarded as robust to resistance development by the insects. However, in more than a dozen cases of field resistance of the codling moth Cydia pomonella to commercially applied C. pomonella granulovirus (CpGV) in German orchards, resistance ratios exceed 1000. The rapid emergence of resistance is facilitated by sex-linkage and concentration-dependent dominance of the major resistance gene and genetic uniformity of the virus. When the gene is fixed, resistance levels approach 100,000-fold. Our findings highlight the need for development of resistance management strategies for baculoviruses.

Fluorescent poliovirus for flow cytometric cell surface binding studies.

Specific cell-surface binding is the essential first step for cellular invasion by viruses. To understand this process, various methods to evaluate binding properties of viruses to cells have been developed. However, many rely on radioactive labeling or indirect immunofluorescence. The development of a novel fluorescence binding assay for poliovirus is described. Poliovirus (type 1 Mahoney or Sabin) was labeled directly with fluorescein using a commercially available fluoresceination kit. Fluorescently labeled poliovirus was bound to its specific receptor on Hela or U937 cells and detected by flow cytometric analysis. Specific binding and infectivity was retained, although reduced, depending on the extent of fluoresceination. Therefore, depending on the users' requirements, the extent of fluoresceination must be titrated carefully to achieve maximal fluorescence and minimal functional destruction. It is likely that this method may be useful with other viruses.

Hematopoietic cells from CD155-transgenic mice express CD155 and support poliovirus replication ex vivo.

Despite identification of the poliovirus (PV) receptor (CD155), mechanisms by which this molecule mediates paralytic disease remain obscure. Unanswered questions include CD155 localization in human tissues, the nature of cells supporting the first round of replication, identity of nonneural replication sites, and route of entry into the CNS. In earlier work, we showed that CD155 is expressed on primary human monocytes and that these cells support low, but statistically significant, levels of PV replication ex vivo without prior culturing. We hypothesize that monocytes support PV replication in vivo and that they contribute to pathogenesis. In the current study, we tested whether CD155-transgenic mouse hematopoietic cells express cell surface CD155 and whether these cells support PV replication. We found that the majority of monocyte/macrophages from peritoneal washes express CD155. In addition, 26-32% of CD155-transgenic bone marrow and spleen cells express CD155 on monocyte/macrophages, T cells and hematopoietic precursor cells. Various tissues supported PV replication without pre-culturing, however, pre-culturing or pre-treatment of mice with thioglycollate increased virus yield. These results are consistent with those from human cells and suggest that the CD155 transgenic mouse model is useful to help understand the role of hematopoietic cells in PV pathogenesis.

Physical association between CD155 and CD44 in human monocytes.

Regulation of CD44-mediated binding to hyaluronan is critical in normal and diseased immune cell function. In earlier work by others (Shepley and Racaniello, J. Virol., 68, 1301 1309), anti-CD44 mAb blocked poliovirus binding to CD155 (the poliovirus receptor) in HeLa cells, suggesting that CD155 and CD44 may be physically associated. Here, we present evidence that CD155 and CD44 are physically associated in human monocytes. In co-modulation experiments in U937 monocytic cells, CD155 and CD44 reciprocally co-modulated. In primary human monocytes, CD 155 syn-capped with CD44. In immunofluorescence flow cytometric experiments, anti CD44 mAb inhibited up to 94% of binding by anti-CD155 mAb which blocks poliovirus binding to CD155. This inhibition was specific for CD155. Culturing monocytes increased the extent of inhibition. In addition, mAb against PRR2, a novel molecule that is related to CD 155, was inhibited by anti-CD44 in a dose-dependent manner, but not by anti-CD14. These data support the interpretation that CD155 (and related proteins) are physically associated with CD44 on monocyte cell surfaces. Although the current study does not address functional significance, we speculate that this interaction may have a role in regulating monocyte CD44 ligand binding which may be critical in pathological processes such as tumor metastasis and arthritis.

CD44 is not required for poliovirus replication in cultured cells and does not limit replication in monocytes.

Although poliovirus receptor is required to mediate poliovirus infection, its role in mediating the tissue specificity of poliovirus replication in natural infections remains unclear due to the presence of this receptor in nonsusceptible cells. It has been hypothesized that CD44 has a role in determining the susceptibility of cell to poliovirus. To test this hypothesis, we determined whether HepG2, a cultured cell line that lacks cell surface CD44, can support poliovirus replication. We found that PV(1) Mahoney, PV(2)W2, and PV(3)Leon strains of poliovirus can replicate in HepG2 cells. Jurkat cells, which also lack CD44, support PV replication. These results suggest that CD44 is not required for poliovirus replication in cultured cells. To examine this question further expression of CD44 in primary human monocytes was examined. Greater than 90% of monocytes express the poliovirus receptor but only 6% of these cells are susceptible to poliovirus infection, making this a good system to study blocks to poliovirus replication. 97% of primary human monocytes reacted with a monoclonal antibody against CD44 that has been shown to block poliovirus binding and replication. This finding suggests that CD44 is present on more cells that poliovirus infects, making CD44 unlikely to be the factor limiting poliovirus replication in human tissues.

Correlation between poliovirus type 1 Mahoney replication in blood cells and neurovirulence.

Poliovirus (PV) is not often described as a monocyte- or macrophage-tropic virus; however, previous work indicated that neurovirulent PV type 1 Mahoney [PV(1)Mahoney] can productively infect primary human monocytes. To determine whether this replication has a functional role in pathogenesis, primary human mononuclear blood cells were infected with pairs of attenuated and neurovirulent strains of PV. Two neurovirulent strains of PV, PV(1)Mahoney and PV(2)MEF-1, replicated faster and to higher titers than attenuated counterparts PV(1)Sabin and PV(2)W-2, respectively, in primary human monocytes, suggesting that this replication may contribute to pathogenesis. PV(3)Leon grew weakly, while PV(3)Sabin, PV(2)Sabin, and PV(2) P712 did not replicate in these cells, perhaps because of their slow replication cycle. In U937 cells, a monocytelike cell line, PV(1)Mahoney replicated but PV(1)Sabin did not, while both grew well in HeLa cells. When molecular recombinants of PV(1)Mahoney and PV(1)Sabin were assessed, a correlation between neurovirulence and the ability to replicate in primary human mononuclear blood cells was found. Surprisingly, infectious centers assays with primary human mononuclear blood cells and U937 cells indicated that despite the lower overall viral yield, more cells are initially infected with the attenuated viruses. These results indicate that there are virulence-specific differences in the ability of PV(1)Mahoney to replicate in monocytes and suggest that there may be factors in monocytes that virulent strains of PV require.

Role of poliovirus receptors in the spread of the infection.

Although the poliovirus receptor (PVR) has been cloned, lack of knowledge of its precise tissue distribution makes assessment of its role in mediating poliomyelitis difficult. Our recent work demonstrated that PVR is expressed on human monocytes and that primary human blood cells can support PV replication. In the current work, we demonstrate that CD14-positive cells (monocytes) support PV replication but that only a minority (< 10%) of the cells become infected. In other preliminary studies, immunocytochemical analyses of human brain tissue demonstrated the presence of PVR in the olfactory bulb, a tissue thought to not support PV replication. Thus, it appears that some apparently "ectopic" sites of PVR expression may in fact be sites for PV replication, whereas other sites may indeed be restricted. The ability of monocytes to replicate PV may pertain to some unexplained phenomena in PV pathogenesis, such as the specific cell type carrying out the initial round of replication in the gut, sites of extraneural replication and transport of the virus into the CNS. Preliminary studies with monocytes from post-polio syndrome patients showed no difference in the levels of PVR relative to control monocytes. In other preliminary work, PVR was shown to be phosphorylated and its expression on monocytes increased by treatment with gamma-interferon. The normal function of PVR is likely to be involved in monocyte function during immune activation.

Low levels of poliovirus replication in primary human monocytes: possible interactions with lymphocytes.

To investigate the molecular mediators of poliovirus tissue tropism, the correlation between poliovirus replication and poliovirus receptor expression was examined in a primary human tissue system. Earlier work [M. Freistadt, H. Fleit, and E. Wimmer, Virology 195: 798-803 (1993)] showed that the cellular receptor for poliovirus is present in 87% of primary human monocytes and that peripheral blood mononuclear cells support poliovirus replication. In the current work, monocytes, obtained by adherence or by a novel negative selection procedure using specific monoclonal antibodies to lymphocyte surface antigens, supported poliovirus replication. However, total virus yield was low and infectious centers assays revealed that a minority (6%) of monocytes become productively infected. Viral yield from monocytes was lower than from the heterogeneous mononuclear cells; however, when uninfected lymphocytes were added back to infected monocytes, the higher viral yield was restored. The purity of the cells did not significantly affect the number of cells infected. These results suggest that more poliovirus is produced per cell from activated rather than unactivated monocytes. Furthermore, poliovirus replication in monocytes may reflect genuine in vivo replication and comprise a system in which to determine molecular mediators of poliovirus tissue tropism.