Viruses and Bone Marrow Failure

S. J. Rosenfeld, N. S. Young SUMMA R Y. Many agents are associated with bone marrow failure, including toxins, inherited metabolic defects, ionizing radiation, and viral infection. In most cases, the etiologic agent is unknown. Many of these unclassified cases have symptomatic, immunologic, or epidemiologic similarities to viral infections. Viruses from different taxonomic families have been implicated in bone marrow failure syndromes, and they appear to cause hematosuppression by a variety of mechanisms. Some of the viruses involved in relatively well characterized suppressive interactions will be reviewed, including parvovirus B19, dengue, hepatitis viruses, Epstein-Barr virus, cytomegalovirus and the human immunodeficiency virus.

Several features and clinical associations of aplastic anemia suggest an etiologic role for viral infection. Bone marrow failure may follow closely on a discrete viral syndrome like hepatitis or mononucleosis.’ In circumstances that are more common but less well characterized, patients with newly diagnosed aplastic anemia will recall non-specific ‘flu-like’ symptoms several weeks to several months before presentation with the symptoms of bone marrow failure. Patients with aplastic anemia and those with viral infections share such immune system abnormalities as dysregulated lymphokine production (especially overproduction of gamma interferon), an inverted T4/T8 cell ratio, an abnormal polyclonal population of activated cytotoxic T-lymphocytes, and decreased NK cell activity.2 Animal models confirm that specific viruses can cause bone marrow depression; these include viruses such as feline leukemia virus and feline panleukopenia viruses with known similarities to human pathogens. Lastly, the incidence of aplastic anemia

S. J. Rosenfeld MD, N. S. Young MD, Cell Biology Section, Clinical Hematology Branch, National Heart, Lung and Blood Institute, Bethesda, Maryland, USA. Correspondence to Dr. Rosenfeld, 10/7C103, NIH, Bethesda, Maryland 20892, USA. Blood Reww (1991) 5. 71-77 CC, I99 I Longman Group UK Ltd

appears to be highest in the Orient.3 where many viral infections are endemic. In the last several years, a number of specific viral agents have been linked to bone marrow failure or suppression. These include parvovirus B19, EpsteinBarr virus (EBV), cytomegalovirus, the human immunodeficiency virus, and dengue. The association of hepatitis and aplastic anemia was first observed three and a half decades ago; the recent identification of the hepatitis C virus may soon allow its inclusion on this list. Human Parvovirus B19 Parvoviruses are small unenveloped DNA viruses. In addition to B19, the family contains adeno-associated virus, another virus that infects humans but is not known to cause disease. Most identified parvoviruses infect animals, including dogs, cats, pigs, cattle, rodents and birds; the family also includes insect viruses. Feline panleukopenia virus, minute virus of mice, and chicken anemia agent are all parvoviruses that are trophic for hematopoietic tissues. B19 was first identified in 1975 in asymptomatic blood donors.4 By seroepidemiologic studies the virus was shown subsequently to be the etiologic agent of

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fifth disease (erythema infectiosum), a common childhood exanthem.5 B19 parvovirus is the cause of transient aplastic crisis in patients with an underlying hemolytic state. This has been most frequently reported in sickle cell anemia,6v7 but can occur in any congenital or acquired disease characterized by shortened red cell survival such as hereditary spherocytosis,’ thalassemia,’ iron deficiency, acute blood loss, etc. In patients with immunodeficiency, B19 can persist in the bone marrow, and then the major clinical manifestation is pure red cell aplasia. Susceptible groups include children with leukemia receiving immunosuppressive chemotherapy, 11 patients with congenital immunodeficiency” or the acquired immunodeficiency syndrome (AIDS),13 and possibly other patients with iatrogenic immune suppression. The mechanism of B19 parvovirus-induced anemia is well understood. The virus productively infects cultures of human bone marrow,14,15 and failure to propagate virus in conventional continuous culture indicates the extraordinary tropism for human hematopoietic tissue. The target of the virus is an immature erythroid cell close to the CFU-E stage.16 Infection is directly cytotoxic to these erythroid progenitors. The development both in vitro and in vivo of characteristic ‘giant pronormoblasts’ is the early cytopathic marker of B19 infection. The mechanism of cytotoxicity is unknown, but expression of the single nonstructural protein has been implicated in transfection assays.17 In all patients, B19 parvovirus infection leads to arrest of red cell production. If red cell survival time is normal, the infection resolves before a significant decrease in peripheral red cell numbers occurs, but patients whose red cell survival is decreased develop a severe transient anemia. Immunocompromised patients cannot clear the infection, and anemia eventually develops despite normal red cell survival. These patients respond to treatment with commercial immunoglobulin,12~‘3 suggesting that antibody is a major and sufficient factor in the immune response, but the susceptibility of patients with primary T-cell defects implies that cell mediated immunity is necessary as well. The study of B19 has been hampered by a paucity of available antigen for serologic testing, until recently available only from infected serum. The production of B19 structural proteins in Chinese hamster ovary cellsls and in baculovirus systems’g*20 will provide a reliable source of purified antigen for these assays. In addition, the ability of structural proteins to self assemble into immunogenic and nonpathogenic empty capsids makes them an ideal vaccine material. Dengue Dengue is an enveloped, positive sense, single stranded RNA virus. It belongs to the flavivirus genus, which also includes yellow fever and the virus that causes St. Louis encephalitis. Like other patho-

genic flaviviruses, dengue is arthropod borne; it is transmitted primarily by feeding of the Aedes aegypti mosquito. The virus can be classified into four antigenitally distinct serotypes, antibodies to any one of which are only partially neutralizing for the others. The virus is endemic in South East Asia, and recent epidemics have occurred in South America” and the Caribbean basin.22 The disease was a major health hazard during construction of the Panama canal, and rare cases are still seen in the southern United States 23 The incidence of dengue is increasing in Africa,z4 possibly related to changing patterns of land use; Aedes aegypti mosquitos breed in pools of standing water, often present in pots and discarded tires around areas of human habitation. Although the areas of the world where the virus is endemic have an increased incidence of aplastic anemia, dengue is not likely to have a direct role in this association but may be a marker for other viruses with similar a ecology. Dengue causes two distinct clinical syndromes.25 The first is dengue fever (DF), which typically presents with fever, headache, myalgia and arthralgia, gastrointestinal symptoms and occasionally a petechial rash. These symptoms may be severe, but the disease is usually self-limited and rarely life-threatening in a previously healthy host. Exposure usually produces long term immunity to the initial serotype, but only short lived immunity to other serotypes. The second syndrome caused by the virus is dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). In addition to the symptoms described above, patients develop severe thrombocytopenia, hypotension and hemoconcentration. Mortality is significant, especially in infants and the elderly, but varies considerably with the level of medical care available. DHF/ DSS is probably caused by infection of cells bearing Fc receptors, typically monocytes/macrophages; their resulting release of soluble activators produces widespread and possibly dysregulated immune activation, ending in capillary leak and shock.26 DHF/DSS occurs when a second exposure to dengue involves a virus with a different serotype; antibodies produced to the first serotype bind but do not neutralize the second, and infectious virus is taken up more efficiently by the host cells’ Fc receptors. Hematopoiesis is affected in both syndromes.27 Neutropenia and thrombocytopenia occur early in the disease, coincident with the febrile stage. In the early stages of DF circulating lymphocytes are decreased, but in DHF/DSS lymphocyte number is persistently elevated, with prominent atypia, and activated lymphocytes probably play a major role in the shock syndrome. 28 Examination of bone marrow biopsies shows decreased cells of all lineages, most prominently megakaryocytes and myeloid cells, and ‘maturation arrest’ in all lineages has also been described. A study of serial changes in the bone marrow of a patient with DF showed a marked decrease in decreased granulopoiesis and megakaryocytes,

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slightly decreased erythropoiesis on day four of the infection, followed by development of a moderate lymphocytic infiltrate and prominent suppression of erythropoiesis characterized by a decrease from proerythroblasts to orthochromic normoblasts on day twelve.” The nature of bone marrow involvement with dengue is suggested by the results of in vitro studies. The virus is able to productively and often persistently infect all human hematopoietic cell lines.30y31Infected cells suffer a mild decrease in proliferation with little loss of viability. A significant portion of cellular RNA codes for viral proteins under these conditions, and decreased growth may be secondary to this redirection of the cellular machinery to virion production. In bone marrow culture in the absence of antibody, dengue is localized primarily to the erythroid lineage,30 and cell lines with erythroid characteristics are most susceptible to the establishment of persistent infection. The origin of the rapid and significant declines in vivo of circulating cells and progenitors is less clear. It is probable that host marrow cells are infected, and their subsequent loss may be caused by cumulative toxicity over time or by their destruction by an activated immune system. The latter hypothesis is supported by the observation of a lymphocytic infiltrate in the marrow.2g In addition, hematosuppression may be enhanced by the release of soluble inhibitors such as interferon gamma from activated T-cells2* and infected monocytes. Hepatitis C Virus (HCV)

Hepatitis C virus (HCV) is an enveloped, positive sense single stranded RNA virus the exact classification of which remains uncertain, although it appears to be most closely related to the flaviviruses or the pestiviruses by molecular character.32 Parenteral transmission is well documented, but the mode of transmission in the majority of cases remains unknown. There is no evidence for transmission by arthropod vectors, and the possibility of sexual transmission remains open. HCV was identified for the first time in 1989.33 Availability of a first generation serological test kit has established the virus as the major cause of post-transfusion and probably sporadic non-A non-B hepatitis, with a prevalence of seropositivity in blood donors of from 0.9-1.4% (United States)34 to 2% (Taiwan).35 The clinical association of bone marrow failure and hepatitis has been recognized since 1955.36 The later development of serologic tests for hepatitis A and B ruled out these viruses as etiologic agents in the majority of cases. 37 Post hepatitis aplastic anemia (PHAA) typically presents with the development of pancytopenia one to two months after an initial diagnosis of otherwise uncomplicated hepatitis,38-41 when the signs and symptoms of liver disease are usually resolved or improving. Bone marrow biopsies are generally not available from the hepatitic phase

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of the disease, and at the time of pancytopenia they do not differ from those of patients with aplastic anemia of other causes. The incidence of PHAA has been estimated at O.l-0.2% of all cases of viral hepatitis 42 but 2.5%43 to 8.70h3’ of all cases of aplastic anemia. The association appears to occur predominantly in young patients, especially males.44 The prognosis was originally felt to be poor, but recent studies of bone marrow transplantation and immunosuppressive therapy have demonstrated a response rate similar to other patients with aplastic anemia.45 A single study has reported no association of antibody to HCV and aplastic anemia,46 but this is not unexpected since some patients may take up to a year to develop antibodies detectable by the current test and others with demonstrated infectivity never become serologically positive.47 Transmission of the virus by transfusion further complicates analysis. Data from our laboratory using a highly sensitive and specific nested primer PCR method indicate the presence of hepatitis C nucleic acid sequences in a significant portion of aplastic anemia patients, some of whom received little or no transfusions. The remarkably high incidence of bone marrow failure in patients undergoing liver transplantation for fulminant non-A non-B hepatitis provides another strong link between this disease and aplastic anemia.42 The mechanism of aplasia in hepatitis C is unknown. Efforts to infect bone marrow or hematopoietic cell lines have thus far failed, probably due to inability to achieve an adequate multiplicity of infection with current virus stocks. One earlier study suggested that sera from chimpanzees infectious for non-A non-B hepatitis inhibited bone marrow colony formation in vitro, but the observed effect was small and not controlled for the presence of soluble inhibitors.48 Other Hepatitis Viruses Rare cases of aplastic anemia following acute hepatitis A and hepatitis B have been reported.4g~-51 The association with hepatitis B is complicated by a clinical association of this virus with HCV (when screening of blood products for hepatitis B virus was instituted, the incidence of post-transfusion hepatitis B and non-A non-B hepatitis both declined, suggesting that many infected units harbored both viruses), The addition of hepatitis B containing sera to bone marrow cultures from normal individuals has led to modest, dose dependent inhibition of colony formation that could be blocked by viral inactivation.52 Virus may infect and propagate in cells of both myeloid and erythroid lineages, but the relevance of this observation to PHAA is uncertain. Similar experiments with hepatitis A virus also showed a dose dependent suppression of colony formation with the addition of virus.53 A single case report of aplastic anemia in the setting of acute hepatitis A documented suppression of the patient’s in vitro colony formation

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FAILURE

by her own T-lymphocytes.” This effect has been observed in other aplastic patients with idiopathic disease and its relationship to hepatitis A is unclear. Epstein-Barr Virus (EBV) EBV is a member of the gamma herpesvirus family. It is an enveloped virus with a complex physical structure and a linear double stranded DNA genome encoding a large number of genes. EBV causes infectious mononucleosis and is associated with Burkitt’s lymphoma, nasopharyngeal carcinoma, and other lymphoproliferative diseases, especially in the immunocompromised host. The best recognized target cell for EBV is the B-lymphocyte, which is susceptible to viral transformation. Viral latency can also be established in these cells. Infectious mononucleosis is often associated with mild hematologic abnormalities, including neutropenia and hemolytic anemia. Development of aplastic anemia has also been described in a small group of patients.54-56 In general, the clinical course of these patients appears to be identical to those with idiopathic aplasia: case reports have documented complete hematologic recovery after treatment with steroids or ATG, as well as fatalities. In the most completely characterized group, 5 patients with aplastic anemia and a history suggestive of acute EBV infection were followed prospectively.” The presence of virus was documented in the bone marrow of all the patients by molecular or immunologic techniques. Three cases were fatal, and 2 recovered completely or partially after treatment with a combination of antithymocyte globulin and intravenous acyclovir. In addition, the same study reported the detection of EBV in only one out of forty bone marrow samples from previously treated patients. The association of EBV with aplastic anemia is strong in patients with congenital immune abnormalities, particularly the X-linked lymphoproliferative syndrome (XLP). ‘* This is a progressive combined variable immunodeficiency that becomes manifest when patients develop severe complications after infection with EBV. 75/100 patients in the XLP registry developed symptomatic mononucleosis, and 17 of these patients evolved to aplastic anemia. In this setting the prognosis was extremely poor, all patients having died within 1 week of developing pancytopenia. Another familial syndrome characterized by nasopharyngeal carcinoma in the index cases and associated with an increase family history of aplastic anemia and malignancies in a variety of sites has been described.5g All affected members of the family had serologic evidence of prior EBV infection. The mechanism of bone marrow suppression in patients with EBV infection is likely to be a combination of direct viral infection and immune mediated suppression/destruction. As noted above, viral antigen and nucleic acid sequences have been detected in the bone marrow of patients with aplastic anemia by

immunofluorescence, Southern blotting and in situ hybridization, 57 although the infected cell type could not be determined by these studies. The genome of the virus isolated from these patients had some of the characteristics of lytic rather than the more usual transforming strains of EBV, but the clinical significance of such intra-strain differences is unknown. The response to immunosuppressive therapy suggests the presence of an immune mediated mechanism of bone marrow suppression. A decrease in colony formation has been reported in vitro with the addition of T-lymphocytes from patients with heterophile positive mononucleosis to normal bone marrow cultures6’ and coculture experiments with the bone marrow from an infected patient and a normal donor showed significant inhibition of colony formation that resolved as the patient recovered.54 The immune system in EBV infection must actively suppress the uncontrolled expansion of the infected B-cells, and in some patients such suppression may have as a side effect the suppression of normal hematopoiesis. Cytomegalovirus (CMV) Cytomegalovirus (CMV) is also a member of the herpesvirus family. It is a large enveloped virus with a linear double stranded DNA genome of approximately 200 kilobases. Like EBV, CMV can become latent, and many clinical CMV infections probably represent reactivation. There is wide antigenic variation among strains, and secondary infections can also occur. Infected cells are characterized by large intranuclear inclusions, similar to those seen with other herpesviruses. CMV does not have a single well defined target cell, but is able to infect multiple cell types and organs both in vitro and in viv~.~l In the normal host, CMV is of low pathogenicity. Hematosuppression has been seen most frequently in the immunosuppressed transplant population. Pancytopenia has been reported with CMV infection in renal allograft recipients,62 as has delay of engraftment in patients undergoing bone marrow transplantation.63 Studies of the interaction of CMV and hematopoietic cells have yielded conflicting results. Most in vitro studies, performed with multiply-passaged strains of the virus, have failed to show direct infection of hematopoietic progenitors. Stromal fibroblasts have been easily infected, and the resultant changes in the hematopoietic microenvironment are reasonably hypothesized to cause the hematosuppression seen in vivo.64*6s One study was able to demonstrate a selective loss of transcripts for G-CSF from infected stromal cells, providing a mechanistic explanation for this loss of nurturing ability.66 In contrast to these results, a single study detected infection of hematopoietic progenitors by immunocytochemical staining and in situ hybridization. 67 Although less than 10% of progenitors were infected, the cultures lost their proliferative response to exogenous GM-CSF and G-CSF. Unlike passaged strains, some clinical isolates

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do appear able to directly infect both granulocyte and macrophage precursors.66,67 Thus bone marrow suppression in the immunocompromised host might be a result of both direct infection of hematopoietic precursors and loss of nurturing factors from infected stroma. Human Immunodeficiency Virus 1 (HIV)

Human Immunodeficiency Virus 1 (HIV-l), the etiologic agent of the acquired immunodeficiency syndrome, is an enveloped retrovirus with a single stranded RNA genome that replicates through a DNA intermediate. At the time of presentation with an AIDS related illness, most patients have anemia and about a third are and granulocytopenia, thrombocytopenic. These abnormalities are manifest before the clinically overt stages of the illness. A study of asymptomatic seropositive individuals documented anemia in 19%, granulocytopenia in 8%, and thrombocytopenia in 13%.68 The recognition of a direct relationship of the HIV-l virus to these diagnostic findings is complicated by the presence of concomitant infections, neoplasm, or drug effects in many of these patients. Indeed, many early studies of the bone marrow in AIDS patients are only descriptive, and patients were not categorized by treatment or disease manifestation. Probably the two most common confounding infections in HIV infected individuals are Mycobacterium avium intracellulare (MAI) and CMV. 69 MA1 can present with anemia and erythroid hypoplasia, while CMV produces nonspecific hematologic findings. Persistent B 19 parvovirus infection of erythroid progenitor cells causes chronic anemia in the setting of immunodeficiency, including AIDS. l3 Fungal infections, especially Cryptococcus neoformans and Histoplasma capsulatum, can also involve the bone marrow.” Drug toxicity is another major cause of marrow suppression, most commonly anemia caused by zidovudine, but decreased blood counts are also associated with trimethoprim pentamidine, sulfamethoxazole, pyrimethamine-sulfadiazine, acyclovir and ganciclovir. Lymphoma involving the bone marrow can present with pancytopenia as well. In contrast to most other hematosuppressive viruses, HIV-associated cytopenias occur in the presence of a hypercellular marrow.‘l Significant trilineage dysplasia has been described and is associated with both anemia and granulocytopenia,@’ and thrombocytopenia is associated with adequate numbers of megakaryocytes. In vitro data has suggested soluble inhibitors or an abnormal cytokine milieu as the mechanism for low peripheral blood counts. Some studies have demonstrated suppressive factors in patients’ sera,72,75 and peripherally normal myelopoiesis has been restored in patients given recombinant human GM-CSF.6* In anemic patients, serum immunoreactive erythropoietin levels are often decreased, and the erythropoietin response to anemia is

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blunted,75 although erythropoietin levels return to appropriate levels without resolution of anemia on treatment with zidovudine. Whether or not HIV directly infects hematopoietic progenitors remains problematic. In vitro attempts to infect CD34 + cells have been largely unsuccessful. Two studies have reported detection of HIV transcripts in immature myeloid cells73 and in CD34+ cells”j in bone marrow biopsies by in situ hybridization, but another study using PCR techniques failed to convincingly detect HIV sequences in CD34 + cells purified by FACS.” HIV-related thrombocytopenia appears to be a separate phenomenon; while anemia is associated with granulocytopenia, thrombocytopenia is independent of both. 68 Peripheral platelet destruction occurs; both antiplatelet antibodies and immune complexes associated with platelets have been demonstrated.” Particles resembling HIV-l have been demonstrated in megakaryocytes by electron microscopy after in vitro exposure to the virus, and infection may lead to defects in platelet production.78 While the direct relationship between HIV-l and hematologic disease remains unknown, feline leukemia virus (FeLV) provides a good model for possible pathologic interactions between retroviruses and the bone marrow. In addition to an AIDS-like illness, FeLV infection can lead to the development of leukemia, lymphoma, myelodysplastic syndromes, aplastic anemia, and pure red cell aplasia.79 The virus family is divided into three subgroups by virus interference and antibody neutralization: FeLV-A, B and C. Infection acquired outside the laboratory universally involves FeLV-A, but viruses of the other subgroups may be present as well. FeLV-C is the group associated with aplastic anemia and pure red cell aplasia.’ In vitro studies of FeLV-C show that it is not directly cytotoxic. Colony cultures from infected cats will produce BFU-E but not CFU-E colonies, suggesting that maturation in vivo is arrested between these stages but that cells can mature in vitro. Infected BFU-E’s are unusually sensitive to complement mediated lysis by the classical pathway, in the absence of virus specific antibodies. This defect produces a disease state analogous to paroxysmal nocturnal hemoglobinuria (PNH) in man, although the feline disease is not clonal and complement activation in PNH is via the alternate pathway. That the clinically observed anemia is caused by direct viral infection of hematopoietic progenitors is supported by the detection of viral envelope proteins on CFU-GM as well as CFU-E and BFU-E and the failure of other studies to show immune mediated suppression. Conclusion

The viruses discussed above were selected because their association with bone marrow suppression is relatively well supported; they are not meant to represent an exhaustive list. As a group, they illustrate

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the variety of pathogenic mechanisms that can lead to the clinical presentation of pancytopenia. One virus may be directly cytotoxic to hematopoietic progenitors, another may make the bone marrow susceptible to immune mediated destruction, still others may lead to the loss of the critical nurturing factors provided by the stromal microenvironment. These differences have major implications for treatment: immunosuppression would be inappropriate therapy for a lytic viral infection, while antivirals may have little effect on a hematopoietic compartment suppressed by a dysregulated immune system. We can reasonably expect that improved viral isolation and characterization will allow reclassification of many cases of so-called ‘idiopathic’ aplastic anemia. A better understanding of the mechanisms of virusinduced hematosuppression will lead to better diagnosis and more effective therapy for these patients.

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