Sequences of polycythemia-type Friend spleen focus-forming virus in clone-745-derived mouse erythroleukemia cells.

Friend leukemia virus complex consists of a replication-competent virus plus one of two replication-incompetent viruses, spleen focus-forming virus anemia virus or spleen focus-forming virus polycythemia virus. The replication-incompetent viruses induce rapid malignant transformation of erythroid precursor cells. Transformed cell lines from mice infected with the complex can be induced to undergo erythrodifferentiation in vitro. However, lines containing the anemia-type virus require erythropoietin and another agent such as dimethyl sulfoxide for optimal erythrodifferentiation, whereas those containing the polycythemia-type virus do not require or respond to erythropoietin. Mice infected with the original Friend virus isolates were anemic, so sub-lines derived from these mice should be erythropoietin-dependent for induction of erythrodifferentiation. However, many of the widely studied sub-lines are erythropoietin-independent. In order to clarify this apparent anomaly, the genomes of viruses present in two commonly used erythropoietin-independent sub-lines were sequenced. Sequence analysis demonstrates that they contain the polycythemia-type virus and not the anemia-type virus.

Role of erythropoietin receptor signaling in Friend virus-induced erythroblastosis and polycythemia.

Friend virus is an acutely oncogenic retrovirus that causes erythroblastosis and polycythemia in mice. Previous studies suggested that the Friend virus oncoprotein, gp55, constitutively activates the erythropoietin receptor (EPOR), causing uncontrolled erythroid proliferation. Those studies showed that gp55 confers growth factor independence on an interleukin-3 (IL-3)-dependent cell line (Ba/F3) when the EPOR is coexpressed. Subsequently, we showed that a truncated form of the stem-cell kinase receptor (sf-STK) is required for susceptibility to Friend disease. Given the requirement for sf-STK, we sought to establish the in vivo significance of gp55-mediated activation of the EPOR. We found that the cytoplasmic tyrosine residues of the EPOR, and signal transducer and activator of transcription-5 (STAT5), which acts through these sites, are not required for Friend virus-induced erythroblastosis. The EPOR itself was required for the development of erythroblastosis but not for gp55-mediated erythroid proliferation. Interestingly, the murine EPOR, which is required for gp55-mediated Ba/F3-cell proliferation, was dispensable for erythroblastosis in vivo. Finally, gp55-mediated activation of the EPOR and STAT5 are required for Friend virus-induced polycythemia. These results suggest that Friend virus activates both sf-STK and the EPOR to cause deregulated erythroid proliferation and differentiation.

Rapid presumptive diagnosis of hantavirus cardiopulmonary syndrome by peripheral blood smear review.

Hantavirus cardiopulmonary syndrome (HCPS) is a rare but frequently lethal acute zoonotic viral infection in rural North America. The rapidity of progression from febrile prodrome to cardiogenic shock and noncardiogenic pulmonary edema requiring intensive care creates high diagnostic urgency and a need for a rapid screening tool. In this retrospective cohort study, 2 pathologists scored blinded peripheral blood smears from 52 patients with HCPS and 128 seronegative patients referred for diagnosis of suspected hantavirus infection. During the prodromal phase, thrombocytopenia was the only consistent abnormality and could be used to indicate hantavirus serologic testing. After the onset of pulmonary edema detected radiographically, the presence of 4 of 5 findings (thrombocytopenia, myelocytosis, hemoconcentration, lack of significant toxic granulation in neutrophils, and more than 10% of lymphocytes with immunoblastic morphologic features) has a sensitivity for HCPS of 96% and a specificity of 99% and missed no patients with HCPS who required intensive care. While each abnormality is commonly seen, the combination of at least 4 of these CBC count data and peripheral blood smear findings can guide early treatment and patient transport decisions until rapid, specific, serologic testing becomes widely available.

Combined in vitro effect of marijuana and retrovirus on the activity of mouse natural killer cells.

Both marijuana and retroviruses impair natural killer (NK) cell functions. No data on their simulataneous effects are available. Similarities to human AIDS induced early by Friend leukemia complex (FLC) and its replication competent helper Rowson-Parr virus (RPV) provides a mouse model to study drug-virus action. Leukemia susceptible BALB/c and resistant C57BL/6 mice were infected, then at time intervals their nylon wool-separated splenocytes were exposed to tetrahydrocannabinol (THC) for 3h. Natural killer (NK) cell activity against Yac-1 cells was assayed by 51Cr-release for 4 and 18h. Recovery of splenocytes was found to be suppressed by FLC, but in BALB/c only by RPV. After a transient enhancement in C57BL/6 by FLC, NK cell activity of both mice became suppressed early (2 to 4 days), normalized subsequently and enhanced late (11 to 14 days) postinfection. A moderate increase in BALB/c, no change in C57BL/6 were induced by low (1-2.5 microgram/ml) THC doses. NK cell activity of BALB/c became suppressed exponentially by higher (5-10 microgrtam/ ml) THC doses in 18h as compared to 4h assays, while its proportional and moderate impairment was seen in C57BL/6. The magnitude of NK cell activity of infected mice was determined by THC: enhancement or impairment followed those of untreated, infected counterparts, but on the level of THC-treated cells. Low doses hardly, high doses additively influenced NK cells of infected BALB/c. THC hardly affected very early and late enhancement in NK cell activiy of FLC infected C57BL/6, but augmented RPV induced suppression late in 18h assays. Genetic factors similar to endotoxin resistance, altered cytokine profile might determine these effects. Similar phenomena in humans might result in earlier manifestation of AIDS.

Origin and rapid evolution of a novel murine erythroleukemia virus of the spleen focus-forming virus family.

The Friend spleen focus-forming virus (SFFV) env gene encodes a glycoprotein with apparent Mr of 55,000 that binds to erythropoietin receptors (EpoR) to stimulate erythroblastosis. A retroviral vector that does not encode any Env glycoprotein was packaged into retroviral particles and was coinjected into mice in the presence of a nonpathogenic helper virus. Although most mice remained healthy, one mouse developed splenomegaly and polycythemia at 67 days; the virus from this mouse reproducibly caused the same symptoms in secondary recipients by 2 to 3 weeks postinfection. This disease, which was characterized by extramedullary erythropoietin-independent erythropoiesis in the spleens and livers, was also reproduced in long-term bone marrow cultures. Viruses from the diseased primary mouse and from secondary recipients converted an erythropoietin-dependent cell line (BaF3/EpoR) into factor-independent derivatives but had no effect on the interleukin-3-dependent parental BaF3 cells. Most of these factor-independent cell clones contained a major Env-related glycoprotein with an Mr of 60,000. During further in vivo passaging, a virus that encodes an Mr-55,000 glycoprotein became predominant. Sequence analysis indicated that the ultimate virus is a new SFFV that encodes a glycoprotein of 410 amino acids with the hallmark features of classical gp55s. Our results suggest that SFFV-related viruses can form in mice by recombination of retroviruses with genomic and helper virus sequences and that these novel viruses then evolve to become increasingly pathogenic.

Both the polycythemia- and anemia-inducing strains of Friend spleen focus-forming virus induce constitutive activation of the Raf-1/mitogen-activated protein kinase signal transduction pathway.

The erythroleukemia-inducing Friend spleen focus-forming virus (SFFV) encodes a unique envelope glycoprotein which allows erythroid cells to proliferate and differentiate in the absence of erythropoietin (Epo). In an attempt to understand how the virus causes Epo independence, we have been studying signal transduction pathways activated by Epo to determine if SFFV exerts its biological effects by constitutively activating any of these pathways in the absence of Epo. We previously demonstrated that Stat proteins, the downstream components of the Epo-induced Jak-Stat pathway, are constitutively activated in SFFV-infected cells. In this study, we demonstrate that SFFV also activates Raf-1, MEK and mitogen-activated protein (MAP) kinase, the downstream components of the Raf-1/MAP kinase pathway. This pathway was activated in cells infected with the polycythemia-inducing strain of SFFV, which induces both proliferation and differentiation of erythroid cells in the absence of Epo, as well as in cells infected with the anemia-inducing strain of the virus, which still require Epo for differentiation. Inhibition of Raf-1 by using antisense oligonucleotides led to a partial inhibition of the Epo-independent proliferation of SFFV-infected cells. Expression of the transcription factors c-Jun and JunB, but not c-Fos, was induced in SFFV-infected cells in the absence of Epo, suggesting that constitutive activation of the Raf-1/MAP kinase pathway by the virus may result in deregulation of AP-1 activity. We conclude from our studies that infection of erythroid cells with SFFV leads to the constitutive activation of signal transduction molecules in both the Jak-Stat and Raf-1/MAP kinase pathways and that both of these pathways must be activated to achieve maximum proliferation and differentiation of erythroid cells in the absence of Epo.